Respuestas fúngicas a señales ambientales: Funciones de las proteínas CarO, OpsA, CutA y AcyA en Fusarium fujikuroi

PORTADA Respuestas fúngicas a señales ambientales: Funciones de las proteínas CarO, CutA y AcyA en Fusarium fujikuroi INSTITUCIONAL AGRADECIMIENTOS ÍNDICE DE CONTENIDOS INTRODUCCIÓN .................................................................................. 19 1. Introducción ................................................................................ 21 1.1 El género Fusarium ...................................................................... 21 1.2 Ciclo de vida de F. fujikuroi ........................................................... 23 2. Metabolismo secundario .................................................................... 25 2.1 Metabolismo secundario en hongos filamentosos ................................... 25 2.2 Un regulador global: la adenilato ciclasa ............................................ 27 2.2.1 Clases de adenilato ciclasas ....................................................... 28 2.2.2 Regulación de las adenilato ciclasas ............................................. 30 2.2.3 Factores que modifican la concentración intracelular de AMPc ............. 31 2.2.4 Dianas del AMPc ..................................................................... 33 2.2.5 Adenilato ciclasas en hongos ...................................................... 34 2.3 Metabolismo secundario en F. fujikuroi .............................................. 36 2.3.1 Policétidos: fusarinas y bikaverina ............................................... 38 2.3.1.1 Bikaverina ....................................................................... 38 2.3.1.2 Fusarinas ........................................................................ 41 2.3.2 Terpenoides: giberelinas y carotenoides ....................................... 43 2.3.2.1 Giberelinas...................................................................... 44 2.3.2.2 Carotenoides ................................................................... 47 2.3.2.2.1 Los carotenoides en la naturaleza ..................................... 48 2.3.2.2.2 Los carotenoides en F. fujikuroi ....................................... 50 2.3.2.2.3 Fenotipos asociados a defectos en la carotenogénesis en hongos: los fenotipos carS y ovc. ............................................................. 54 3. Opsinas ........................................................................................ 58 3.1 Opsinas en hongos ....................................................................... 60 3.2 Opsinas en F. fujikuroi .................................................................. 64 3.3 Usos aplicados de las rodopsinas microbianas ....................................... 64 OBJETIVOS/AIMS ................................................................................. 67 CAPÍTULO 1/CHAPTER 1: ADENYLYL CYCLASE PLAYS A REGULATORY ROLE IN DEVELOPMENT, STRESS RESISTANCE AND SECONDARY METABOLISM IN Fusarium fujikuroi .......................................................................................... 71 1. Introduction ................................................................................... 73 2. Results ......................................................................................... 76 2.1 Generation of F. fujikuroi ΔacyA mutants: .......................................... 76 2.2 The ΔacyA mutants show alterations in growth, morphology and conidiation: . 79 2.3 The ΔacyA mutants are affected in stress sensitivity: .............................. 81 2.4 The ΔacyA mutants overproduce a red pigment: .................................... 84 2.5 The ΔacyA mutants exhibit changes in gibberellin and carotenoid production: 86 8 3. Discussion: .................................................................................... 89 CAPÍTULO 2/CHAPTER 2: THE GENE cutA OF Fusarium fujikuroi, ENCODING A PROTEIN OF THE HALOACID DEHALOGENASE FAMILY, IS INVOLVED IN OSMOTIC STRESS AND GLYCEROL METABOLISM .................................................................. 95 1. Introduction ................................................................................... 97 2. Results ......................................................................................... 99 2.1 Identification of cutA, a gene of Fusarium orthologous to cut-1 of N. crassa: . 99 2.2 Targeted ΔcutA mutants are partially sensitive to osmotic stress: ............. 101 2.3 cutA expression is regulated by osmotic stress and is needed for glycerol accumulation: ............................................................................... 105 2.4 cutA is linked and co-regulated with a gene encoding a putative glycerol dehydrogenase: ............................................................................. 107 2.5 cutA and gldB are upregulated by heat shock and light: ......................... 109 2.6 The HOG pathway gene hogA is regulated by light: .............................. 111 3. Discussion: .................................................................................. 111 CAPÍTULO 3/CHAPTER 3: BIOLOGICAL ROLE OF FUNGAL RHODOPSINS: THE LIGHT DRIVEN, PROTON-PUMPING RHODOPSIN CarO OF Fusarium fujikuroi RETARDS GERMINATION OF LIGHT-EXPOSED CONIDIA................................................ 119 1. Introduction ................................................................................ 121 2. Results ...................................................................................... 125 2.1. Pumping activity of CarO ............................................................ 125 2.2 Effect of light wavelength and intensity on CarO pump activity ............ 129 2.3 Ion specificity of CarO pump activity ............................................... 131 2.4 Effect of gluconate on CarO pump activity ........................................ 132 2.5 CarO localisation in F. fujikuroi ..................................................... 135 2.6 Physiological function of CarO ....................................................... 139 3. Discussion ................................................................................. 145 4. Conclusion ................................................................................ 152 DISCUSIÓN GENERAL/GENERAL DISCUSSION ............................................... 155 CONCLUSIONES/CONCLUSIONS .............................................................. 169 MATERIALES Y MÉTODOS ..................................................................... 173 1. Estirpes .................................................................................... 175 1.1 Otros organismos utilizados en esta tesis ....................................... 176 1.1.1 Escherichia coli ................................................................ 176 1.1.2 Saccharomyces cerevisiae ................................................... 176 1.1.3 Células de mamífero .......................................................... 176 9 2. Vectores de clonación y plásmidos .................................................... 177 3. Oligonucleótidos ......................................................................... 181 4. Soluciones, medios y condiciones de cultivo ........................................ 182 4.1 Medios de cultivo para E. coli .................................................... 182 4.2 Medios de cultivo para S. cerevisiae ............................................. 183 4.3 Medios de cultivo para F. fujikuroi .............................................. 183 4.4 Medios de cultivo para células de mamífero ................................... 185 5. Condiciones de cultivo de F. fujikuroi ................................................ 185 5.1 Iluminación ............................................................................... 185 5.2 Cultivos para análisis de carotenoides .......................................... 186 5.3 Cultivos para análisis de bikaverinas ............................................ 186 5.4 Cultivos para análisis de giberelinas ............................................. 186 5.5 Cultivos para análisis de expresión de los genes car .......................... 186 5.6 Cultivos para análisis de la expresión del gen gibB............................ 186 5.7 Cultivos para el estudio de la respuesta a choque térmico de la expresión de los genes cutA y gldB ...................................................................... 187 5.8 Cultivos para el estudio de la respuesta a choque osmótico de la expresión de los genes cutA y gldB ................................................................... 187 5.9 Cultivos para estudios de acidificación del medio por el hongo ............. 188 6. Cuantificaciones de crecimiento y conidiación ...................................... 188 6.1 Crecimiento en “tubos de carrera” .............................................. 188 6.2 Crecimiento en medio líquido .................................................... 188 6.3 Crecimiento en cajas de Petri .................................................... 189 6.4 Ensayos de conidiación ............................................................ 189 7. Ensayos de crecimiento sobre materia vegetal ...................................... 189 8. Tratamientos de estrés ................................................................. 190 8.1 Estrés térmico ...................................................................... 190 8.2 Estrés por H2O2 ...................................................................... 191 8.3 Estrés por metales pesados ....................................................... 191 9. Determinación del contenido de glicerol ............................................. 191 10. Métodos de transformación de células................................................ 192 10.1 Transformación de E. coli ........................................................... 192 10.1.1 Preparación de células competentes ......................................... 192 10.1.2 Transformación de células competentes de E. coli ........................ 193 10.2 Transformación de S. cerevisiae ................................................... 193 10.3 Transformación de Fusarium ....................................................... 194 ÍNDICE DE TABLAS Tabla 1. Grupos de cruzamiento del complejo de especies Gibberella fujikuroi 22 Tabla 2. Clasificación de las ciclasas por dominios conservados 29 Tabla 3. Características de los genes que constituyen el agrupamiento génico pks 42 Tabla 4. RDs y ORPs encontradas en distintos genomas de hongos 61 Tabla 5. Fungi with ORP-like rhodopsins and their role in phytopathology 151 Tabla 6. Estirpes fúngicas usadas en la presente Tesis 175 Tabla 7. Estirpes de E. coli usadas en la presente Tesis 176 Tabla 8. Líneas celulares de mamífero usadas en la presente Tesis 176 Tabla 9. Oligonucleótidos usados como cebadores en reacciones de PCR estándar 181 Tabla 10. Oligonucleótidos usados como cebadores en reacciones de RTPCR 182 INTRODUCCIÓN Introducción 21 1. Introducción Para el estudio de los problemas biológicos que aborda esta Tesis Doctoral se han empleado dos hongos filamentosos del género Fusarium, Fusarium fujikuroi y Fusarium proliferatum. Los hongos filamentosos constituyen un grupo de organismos de gran interés en investigación. Algunos son modelos extensamente empleados en la actualidad para estudiar problemas biológicos básicos. Entre ellos destaca Neurospora crassa, ascomiceto haploide especialmente útil en estudios de desarrollo o de fotobiología (Perkins and Davis 2000). Sin embargo, el principal interés de los hogos filamentosos radica en su impacto sobre la especie humana, tanto como fuentes de metabolitos valiosos para las industrias química o alimentaria (Lomascolo, Stentelaire et al. 1999) (Mapari, Nielsen et al. 2005), como por su capacidad para producir metabolitos tóxicos (micotoxinas) o de causar enfermedades, especialmente en pacientes inmunocomprometidos (Nelson, Dignani et al. 1994). Los mecanismos que controlan la producción de micotoxinas por parte de algunos de estos hongos o su actividad fitopatógena son campos de investigación potencialmente importantes. Los hongos del género Fusarium destacan en todos estos aspectos 1.1 El género Fusarium El género Fusarium fue definido por Link en 1809 (Booth 1971) como un grupo de numerosos hongos imperfectos cuya característica común es la producción de esporas fusiformes y frecuentemente septadas, denominadas conidios. Una de las principales dificultades a la hora de clasificar las especies dentro del género Fusarium ha sido la falta de ciclo sexual reproducible en el laboratorio y la carencia de estructuras morfológicas identificables. Durante muchos años se han denominado hongos imperfectos a aquellos que carecen de ciclo sexual, y que por tanto no producen las estructuras sexuales características de cada grupo taxonómico (ascas en ascomicetos, basidios en basidiomicetos, oosporas en oomicetos y zigosporas en zigomicetos). Introducción 22 El cultivo de diferentes estirpes de Fusarium permitió identificar ciclo sexual en muchos casos. Sin embargo, lo que debió ser un aspecto fenotípico adicional que contribuyera a asentar la nomenclatura, solo sirvió para complicarla debido al mal hábito de los micólogos de cambiar la denominación de las especies anamorfas o imperfectas una vez identificadas como teleomorfas o perfectas. Éste fue el caso de Fusarium moniliforme, cuyo teleomorfo se llamó Gibberella fujikuroi (orden Hypocreales, subclase Ascomycota, clase Euascomycetes) (Nelson, Toussoun et al. 1983). Con el paso del tiempo se observó que diferentes estirpes de esta supuesta especie formaban grupos de cruzamiento independientes, hablándose por tal motivo del “complejo de especies” Gibberella fujikuroi. Las estirpes de cada grupo de cruzamiento son generalmente heterotálicas, con sexos separados determinados por un locus sexual con dos alelos, MAT-1 y MAT-2 (Steenkamp, Wingfield et al. 2000) que definen el tipo sexual de cada hongo (+ o -). El complejo comprende al menos 11 grupos de cruzamiento, cada uno correspondiente a una especie diferente. A partir de los años 90 el empleo de criterios moleculares asentó la distinción de las especies y se recuperó la terminología tradicional, renombrándose cada grupo de cruzamiento como una especie diferente del género Fusarium (Tabla 1). Especie Grupo de cruzamiento Referencia verticillioides sacchari fujikuroi proliferatum subglutinans thapsinum nygamai circinatum konzum gaditjirrii xylaroides A B C D E F G H I J K O´Donnell et al., 1998 O´Donnell et al., 1998 O´Donnell et al., 1998 O´Donnell et al., 1998 O´Donnell et al., 1998 O´Donnell et al., 1998 O´Donnell et al., 1998 Britz et al., 1999 Zeller et al., 2003 Phan et al., 2004 Lepoint et al., 2005 Tabla 1. Grupos de cruzamiento del complejo de especies Gibberella fujikuroi. Introducción 23 En condiciones de laboratorio, el cruzamiento se realiza añadiendo esporas de la estirpe de sexo (+) sobre micelio de la estirpe (-). Tras periodos de incubación en condiciones específicas de iluminación que abarcan generalmente varias semanas, se desarrollan los peritecios, estructuras muy pigmentadas en cuyo interior tienen lugar las meiosis y la formación tétradas de ascosporas desordenadas (Sidhu 1983) (Leslie, Summerell et al. 2005). Ciertas especies de Fusarium, como F. oxysporum, no han podido ser cruzadas hasta ahora en laboratorio, por lo que han quedado excluidas de estas confusiones taxonómicas. Como es habitual en el género Fusarium, las especies pertenecientes al complejo Gibberella fujikuroi son fitopatógenas, normalmente específicas de único hospedador vegetal (Kuhlman 1983) (Leslie 1991) (Voigt, Schafer et al. 2005). En Estados Unidos, las pérdidas generadas por la reducción en la cantidad y calidad del grano en cultivos de cereales atacados por Fusarium superaron ampliamente los 1.000 millones de dólares a finales del siglo pasado (McMullen, Jones et al. 1997) (Arnold, Maynard et al. 2001). A pesar de ser eminentemente fitopatógenas, la presencia de especies del género Fusarium en hospitales supone graves riesgos para la salud de personas con enfermedades inmunodeprimidas o transplantadas (Austwick 1984), figurando entre los agentes causales de infecciones por hongos oportunistas, como la onicomicosis o la queratomicosis (Guarro and Gené 1992). 1.2 Ciclo de vida de F. fujikuroi F. fujikuroi corresponde al grupo C de cruzamiento del complejo Gibberella fujikuroi. Como ya se ha indicado, se reproduce asexualmente mediante la formación de esporas fusiformes que reciben el nombre de conidios. Este hongo produce dos tipos de conidios: microconidos, pequeños y uninucleados (Ávalos et al., 1985), y macroconidos, más alargados, de forma arqueada, tabicados y multinucleados (Kuhlman 1983) (Figura 1). F. fujikuroi generalmente produce microconidos, aunque bajo ciertas condiciones de cultivo también forma macroconidios. Su producción mayoritaria de microconidos uninucleados, junto con su naturaleza haploide, facilita enormememente el aislamiento en laboratorio de estirpes con mutaciones Introducción 24 recesivas mediante exposición a agentes mutagénicos físicos o químicos (Puhalla and Spieth 1983), (Avalos, Casadesús et al. 1985) (Koelblin, Brückner et al. 1990). En cuanto a su reproducción sexual, F. fujikuroi es una especie heterotálica. En condiciones de laboratorio, la adición de conidios de sexo (-) a micelio de una estirpe de sexo (+) previamente incubada en un medio basado en un sustrato vegetal (habitualmente zanahoria) y la incubación posterior bajo un régimen especial de iluminación da lugar a la aparición de peritecios. La micromanipulación de las ascosporas contenidas en los peritecios (Figura 2) hace potencialmente posible el análisis genético por recombinación en este hongo. Se ha descrito también la generación de heterocariontes (Ming, Lin et al. 1966) (Adams, Johnson et al. 1987) (Correll, Klittich et al. 1987), aunque en muchos casos lo impiden fenómenos de incompatibilidad vegetativa o autoincompatibilidad (Puhalla and Spieth 1985) (Correll, Klittich et al. 1987). Figura 1. Formas de reproducción asexual en Fusarium fujikuroi: microconidos (A) y mezcla de macro y microconidos (B). A B Figura 2. Estructuras sexuales en F. fujikuroi: conjunto de peritecios maduros de Fusarium (A) y asca conteniendo cuatro ascosporas desordenadas (B, marcado con una flecha). Las ascosporas son notablemente mayores que los microconidos, menos refringentes y azulados en la imagen. A B A B Introducción 25 2. Metabolismo secundario 2.1 Metabolismo secundario en hongos filamentosos Los metabolitos secundarios se pueden definir como moléculas producidas en ciertas etapas del ciclo de vida de un hongo, generalmente de bajo peso molecular y frecuentemente bioactivos, que se caracterizan por no desempeñar ninguna función esencial para la vida del hongo. El término secundario fue acuñado a finales del siglo XIX (Mothes 1980) para definir moléculas sin función conocida en el hongo, típicamente subproductos de rutas metabólicas primarias. El estudio sistemático del metabolismo secundario en hongos filamentosos se inicia en 1922 con el hallazgo de más de 200 metabolitos (Howard and Raistrick 1954). Sin embargo, no será hasta la década de los años 50 del pasado siglo cuando el estudio de los metabolitos secundarios reciba un fuerte impulso de la mano de la industria farmacéutica. El papel de la penicilina, descubierta por Fleming en 1929 (Hobby 1985) (Bennett and Chung 2001) y usada masivamente en la Segunda Guerra Mundial, junto con la constatación de que numerosos metabolitos secundarios de hongos poseían actividades antibióticas, hizo avanzar con rapidez el estudio de estos compuestos. Existen numerosas clasificaciones de metabolitos secundarios, pero las más comunes se basan en el tipo de enzima que cataliza los primeros pasos de la ruta biosintética (Keller, Turner et al. 2005). La clasificación incluye al menos cuatro grandes grupos: péptidos no ribosomales, alcaloides, policétidos y terpenoides. Los péptidos no ribosomales son sintetizados por enzimas multidominio conocidas como NRPs, de “Nonribosomal Peptide Synthetases” (Finking and Marahiel 2004). La penicilina o la cefalosporina son antibióticos pertenecientes a este grupo. Su síntesis la lleva a cabo la enzima NRPs descrita, conocida como ACVS (Smith, Earl et al. 1990). Por su parte, los alcaloides generalmente derivan del triptófano y el dimetilalil pirofostato, Introducción 32 de AMPc están implicados en la percepción de los sabores salado y dulce (Margolskee 2002). c) AMPc: como hemos visto anteriormente, los niveles de AMPc están sujetos a regulación por producto final. En E. coli existe retroalimentación negativa mediada por la proteína CRP, mientras que en eucariotas superiores se han descrito ejemplos de retroalimentación positiva. En este último caso, altos niveles de AMPc activan la PKA que lleva a cabo la fosforilación inhibitoria de la PDE (Jackson, Mi et al. 2007). d) Calcio: los niveles de calcio modulan la concentración de AMPc a través de distintas rutas. Por ejemplo, en células de retina, iones Ca++ libres activan AC solubles e inhiben AC transmembranales mientras que el complejo calcio-calmodulina activa la PDE1 (Dunn, Wang et al. 2006). e) CO2, bicarbonato y otros estímulos: en eucariotas, principalmente en mitocondrias, y en algunas bacterias se han descrito respuestas mediadas por ACs ante cambios en la concentración de CO2/bicarbonato (Kobayashi, Buck et al. 2004) (Townsend, Holliday et al. 2009). En el caso de las mitocondrias, el ciclo de los ácidos tricarboxílicos eleva los niveles de CO2, lo cual actúa como señal activadora para la producción de AMPc, que incrementará la eficiencia de la fosforilación oxidativa (Acín-Pérez, Salazar et al. 2009). También se han descrito respuestas a niveles de NaCl en Pseudomonas aeruginosa (Rietsch and Mekalanos 2006) o respuestas a luz azul en medusas (Koyanagi, Takano et al. 2008) o en el flagelado unicelular Euglena gracilis (Yoshikawa, Suzuki et al. 2005). f) Nutrientes: la presencia de nutrientes en el medio quizás sea uno de los factores mejor estudiados. En células de mamífero, los niveles de AMPc son dependientes de la presencia de glucosa en una ruta mediada por glucagón (Lavine, Voyles et al. 1975). Este descubrimiento llevó a postular que el AMPc actua como una “señal de hambre”. Esta hipótesis que está aún en discusión (Gancedo 2013), ya que en otros organismos la glucosa afecta de distinto modo. Así, P. aeruginosa no varía sus niveles de AMPc en presencia de glucosa (Phillips and Mulfinger 1981), mientras que E. coli Introducción 33 reduce sus niveles intracelulares si el medio está suplementado con este azúcar (Ullmann and Danchin 1983). En Saccaromyces cerevisiae, la presencia de glucosa activa la AC a través de la proteína G Gpa2 y del complejo Ras1/Ras2 (Yun, Tamaki et al. 1997), mientras que en Candida albicans la disponibilidad de esta fuente de carbono en el medio eleva los niveles de AMPc intracelular por mecanismos aún no dilucidados (Miwa, Takagi et al. 2004) (Maidan, Thevelein et al. 2005). 2.2.4 Dianas del AMPc Como se ha visto en apartados anteriores, el AMPc tiene un papel relevante en multitud de organismos y rutas de transducción de señales. A continuación se describen algunas de las principales dianas de esta molécula y el mecanismo de acción de estas interacciones. a) PKA: estas cinasas contienen dos dominios de unión a AMPc, llamados CDB- A y CDB-B. Una molécula de AMPc puede unirse al dominio B promoviendo un cambio conformacional en la proteína y haciendo que el dominio A quede expuesto. La exposición del dominio A posibilitaría la entrada de una segunda molécula de AMPc, activándose la PKA mediante la disociación de sus unidades catalíticas y reguladoras (Das, Esposito et al. 2007) (Kim, Cheng et al. 2007). Una vez activada, la cinasa fosforila diversas dianas (Gancedo 2013) mediante el reconocimiento de secuencias proximales y distales al sitio de fosforilación R/K-R/K-X-S/T. Las PKA activas son capaces de reconocer y fosforilar numerosos sustratos, tales como factores de transcripción (Görner, Durchschlag et al. 2002), enzimas metabólicas (Portela, Moreno et al. 2006) o canales iónicos (Liu, Shi et al. 2004). A su vez, la propia PKA es capaz de modular su propia actividad a través de dos vías: fosforilación activadora de la PDE o autofosforilación inhibidora de las subunidades reguladoras, fenómeno muy conservado en eucariotas (Zakhary, Fink et al. 2000). b) Proteínas Epac: presentes en eucariotas superiores (de Rooij, Zwartkruis et al. 1998) (Kawasaki, Springett et al. 1998) se diferencian en dos familias en función del número de sitios de unión a AMPc. Cuando las proteínas Epac son activadas por su unión con una o dos moléculas de AMPc, se Introducción 34 localizan en la membrana plasmática donde actúan como factores intercambiadores de nucleótidos de guanina asociados a proteínas Rap (Bos 2006) (Ponsioen, Gloerich et al. 2009). c) Canales iónicos activados por nucleótidos cíclicos: distintos canales que comparten la presencia de dominios CNB (cyclic nucleotide binding domains) en su región C-terminal expuesta en el citosol (Craven and Zagotta 2006), que unen AMPc disparando distintas respuestas. Estos canalesno han sido encontrados hasta la fecha en hongos (Kaplan, Sherman et al. 2007). d) Factores de transcripción: la unión directa del AMPc a factores de transcripción es bien conocida en procariotas, siendo E. coli donde se descubrió por primera vez la interacción ya mencionada con la proteína CRP (Zubay, Schwartz et al. 1970). La unión de CRP con AMPc provoca cambios conformacionales que posibilitan la unión del complejo al ADN, habiéndose descrito más de 300 regiones intergénicas a las que es capaz de asociarse (Shimada, Fujita et al. 2011). En P. aeruginosa existe una proteína similar llamada Vfr (Suh, Runyen-Janecky et al. 2002). 2.2.5 Adenilato ciclasas en hongos Las adenilato ciclasas de hongos juegan un papel esencial en la biología de estos organismos, habiéndose descrito una enorme variedad de fenotipos asociados a sus pérdidas de función (Lee, D'Souza et al. 2003). La mutación del gen cr-1 de N. crassa produce gran variedad de cambios fenotípicos, incluyendo defectos en el patrón de conidiación en medios líquido o sólido, crecimiento anormalmente colonial y acortamiento de las hifas aéreas. De igual manera, los mutantes cr-1 muestran defectos en la formación de peritecios al cruzar con una estirpe silvestre del sexo opuesto. Sorprendentemente, los mutantes de N. crassa carentes de AC funcional muestran mayor tolerancia a estrés térmico u oxidativo (Ivey, Kays et al. 2002). La participación del AMPc en rutas de transducción de señales de hongos ha recibido especial atención en algunas especies por su papel en Introducción 35 patogénesis. La capacidad invasiva del patógeno humano C. albicans requiere una transición morfológica de su estado de levadura a su forma filamentosa. Los mutantes carentes de AC son avirulentos y más susceptibles de ser fagocitados (Rocha, Schroppel et al. 2001). La presencia de AC funcional también es necesaria para la virulencia del patógeno oportunista Aspergillus fumigatus, cuya carencia da lugar a la inhibición de la expresión del gen pksP, necesario para la patogénesis (Liebmann, Muller et al. 2004). Los estudios en hongos fitopatógenos también son abundantes en la literatura. En el basidiomiceto patógeno del maíz Ustilago maydis, de manera similar a lo descrito en C. albicans, la expresión del gen de la AC (uac1) afecta a la transición de su estado levaduriforme a su forma filamentosa, necesaria en las primeras etapas de la invasión de la planta (Gold, Duncan et al. 1994). Mutantes carentes de AC muestran un crecimiento filamentoso constitutivo. En otros hongos, la mutación del gen de la AC impide la formación de apresorios, generando estirpes avirulentas. Este fenómeno se ha estudiado en profundidad en Magnaporthe grisea (Choi and Dean 1997). Un último ejemplo es proporcionado por Botrytis cinerea, cuyo mutante carente de AC es capaz de penetrar las hojas de la vid, si bien el tamaño de las lesiones es menor en comparación con las que produce la estirpe silvestre y la producción de esporas in planta es nula (Klimpel, Gronover et al. 2002). Las ACs de hongos dependen de un gen de copia única (Lee, D'Souza et al. 2003) y son las únicas que poseen dominios ricos en leucina (LRR) (Ivey and Hoffman 2005). Los dominios LRR son motivos estructurales formados por 20- 30 residuos de aminoácidos que adoptan un plegamiento en herradura α/β (Kobe and Deisenhofer 1994). Estos dominios forman parte de numerosas familias de receptores en casi todos los reinos eucariotas, pero son sin embargo más infrecuentes en hongos. Tanto ascomicetos como zigomicetos y basidiomicetos poseen ACs con dominios LRR y un dominio adenilato/guanilato ciclasa en la misma cadena polipeptídica, una combinación única entre las proteínas eucariotas (Soanes and Talbot 2010). Introducción 36 La estructura típica de una AC de hongos incluye varios dominios (Figura 3), algunos de ellos muy conservados. Todas las ACs estudiadas poseen dominios LRR y los dominios catalíticos adenilato ciclasa y fosfatasa 2C. La mayoría de las AC contienen también un dominio de unión a proteínas Gα y otro de unión a proteínas Ras (Soanes and Talbot 2010). 2.3 Metabolismo secundario en F. fujikuroi Las especies del género Fusarium tienen un enorme interés aplicado debido a la riqueza de su metabolismo secundario. Entre los numerosos metabolitos secundarios producidos por estos ascomicetos figuran micotoxinas producidas durante la infección (Gil-Serna, Mateo et al. 2013) y pueden resultar dañinas tanto para los seres humanos como para los animales que se alimentan de vegetales contaminados (Conková, Laciakova et al. 2003). Entre las micotoxinas más estudiadas figuran las fusarinas (Barrero, Sánchez et al. 1991), los tricotecenos (Desjardins, Hohn et al. 1993), las fumonisinas (Marasas 1995), el ácido fusárico (Bacon, Porter et al. 1996) o la bikaverina (Giordano, Avalos et al. 1999a). Figura 3. Comparación de adenilato ciclasas del género Fusarium. Se muestran las adenilato ciclasas de F. fujikuro (Ff), F. oxysporum (Fo), F. verticillioides (Fv) y F. proliferatum (Fp). Se muestran los dominios de unión a proteínas Gα (verde), de unión a proteínas Ras (rojo), repeticiones ricas en leucina (violeta), fosfatasa 2C (naranja) y catalítico (azul). En el caso de F. proliferatum, aparecen codones de fin de lectura antes del dominio fosfatasa 2C, debido a errores de anotación de la secuencia. Introducción 37 Otros metabolitos secundarios producidos por Fusarium no son perjudiciales y en algunos casos tienen interés biotecnológico. Es el caso de dos grupos de terpenoides: los carotenoides (Avalos and Cerdá-Olmedo 1987) y las giberelinas (Fernández-Martín, Reyes et al. 1995) (Figura 4). En las secciones siguientes se detallan las síntesis de algunos metabolitos relevantes en este trabajo. Figura 4. Metabolismo secundario en F. fujikuroi: se muestran dos grupos mayoritarios de metabolitos secundarios en F. fujikuroi. Tanto policétidos como terpenoides parten de un precursor común, el acetil-CoA. En color aparecen algunos de los genes implicados en la ruta biosintética de los distintos compuestos. Introducción 38 2.3.1 Policétidos: fusarinas y bikaverina Los policétidos se sintetizan a partir de acetil-CoA mediante la actividad enzimática de las sintetasas de policétidos (PKSs). En bacterias se han descrito tres tipos de PKS: las de tipo I, enzimas multifuncionales de gran tamaño, de tipo II, complejos formados por enzimas monofuncionales, y de tipo III, como las de tipo I pero carentes del dominio transportador de grupos acilo. Las PKSs de tipo I son parecidas a las sintetasas de ácidos grasos eucariotas. Estas últimas llevan a cabo un paso de reducción completa del carbono β de sus sustratos, mientras que en el caso de las PKSs esta reducción puede ocurrir o no. En hongos sólo han sido descritas PKSs pertenecientes a al tipo I (Staunton and Weissman 2001) (Menzella, Reid et al. 2005). El gran tamaño de estas enzimas, superior a 1000 aminoácidos, se debe a la presencia de diversos dominios. Entre ellos destacan los dominios transportador de grupos acilo (ACP), transferasa de grupos acilo (AT), de síntesis de cetoacil-CoA (KS), cetorreductasa (KR), reductasa de enoílo (ER) y deshidratasa (DH). Los tres últimos pueden estar o no presentes en las distintas PKSs, mientras que los tres primeros son indispensables para la actividad enzimática de todas ellas. La combinatoria de módulos en estas enzimas multifuncionales explica la enorme variedad de policétidos producidos por distintos hongos. F. fujikuroi produce diferentes policétidos, entre ellos las fusarinas (Barrero, Sánchez et al. 1991), la bikaverina (Giordano, Avalos et al. 1999a) y la fusarubina (Studt, Wiemann et al. 2012). 2.3.1.1 Bikaverina La bikaverina es un pigmento rojizo-violáceo (Figura 5) con propiedades antibióticas frente a distintos organismos. Así, se ha descrito toxicidad de este compuesto frente a nematodos (Kwon, Son et al. 2007), hongos filamentosos (Son, Kim et al. 2008) y protozoos (Balan, Fuska et al. 1970). Su actividad le proporciona a la bikaverina interés biotecnológico. Introducción 39 La síntesis de bikaverina a partir de acetil-CoA la lleva a cabo la PKS de tipo I Bik1 (Wiemann, Willmann et al. 2009), identificada en un escrutinio de genes regulados por nitrógeno y denominada anteriormente Pks4 (Linnemannstöns, Schulte et al. 2002a). La producción de mutantes carentes de copia funcional del gen bik1, junto con la expresión heteróloga de dicha proteína en E. coli (Ma, Zhan et al. 2007) demostró la función de Bik1 en la ruta de síntesis de este compuesto. Como ya sucedió con los genes para otros metabolitos secundarios en hongos, el análisis de las secuencias génicas adyacentes a bik1 llevó a la identificación de otros genes corregulados que constituyen un agrupamiento génico responsable de la síntesis de éste policétido (Wiemann, Willmann et al. 2009) (Figura 6). El producto del gen bik1 es suficiente para la síntesis de pre-bikaverina (Wiemann, Willmann et al. 2009) mientras que las enzimas cifradas por bik2 y bik3 llevan a cabo pasos oxidativos y de metilación sobre este sustrato, respectivamente. El agrupamiento génico incluye bik4 y bik5, dos genes reguladores, y bik6, que cifra una proteína transportadora presumiblemente responsable de excretar la bikaverina al medio de cultivo. Así, mutantes carentes de Bik6 presentan menor cantidad de bikaverina en el medio de cultivo que la estirpe silvestre (Wiemann, Willmann et al. 2009). Figura 5. A: Estructura química de la bikaverina. B: Cultivos en medio sólido: F. fujikuroi cultivado en medio DG con (izquierda) y sin (derecha) nitrógeno. Puede apreciarse la coloración rojiza debido a la bikaverina sólo en el medio carente de nitrógeno. C y D. Filtrados de cultivo líquido: los filtrados provienen de cultivos de F. fujikuroi en medio líquido con (C) y sin (D) nitrógeno. Introducción 40 Como es habitual en la síntesis de metabolitos secundarios, la síntesis de bikaverinas en F. fujikuroi es regulada por distintos factores ambientales. Entre ellos destacan la disponibilidad de nutrientes y el pH. La síntesis de bikaverina requiere una relación nitrógeno/carbono, actuando el exceso de nitrógeno en el medio de cultivo como una señal represora (Giordano, Avalos et al. 1999a). La carencia de fósforo y azufre en el medio, así como la presencia de sacarosa (Rodríguez-Ortiz, Mehta et al. 2010) o de calcio (Bell, Wheeler et al. 2003) ejercen un efecto estimulador sobre la síntesis. Respecto al pH, la síntesis de bikaverinas requiere pH ácido (Balan, Fuska et al. 1970) (Giordano, Avalos et al. 1999a). Respecto a las proteínas que controlan la ruta, se ha atribuido a AreA y PacC funciones en la regulación por nitrógeno y por pH, respectivamente. El mecanismo de acción de AreA es confuso, ya que aunque inicialmente se observó que su mutación aumenta la expresión del gen bik1 (Linnemannstöns, Schulte et al. 2002a), este resultado no fue confirmado posteriormente (Wiemann, Willmann et al. 2009), por lo que deben participar también otras proteínas reguladoras. Por su parte, PacC actúa de regulador negativo, ya que su ausencia aumenta la expresión de los genes bik (Wiemann, Albermann et al. 2012). Esta regulación la ejerce a nivel de unión directa a los promotores de dichos genes, como indica la presencia en sus secuencias promotoras de sitios de unión similares a los previamente descritos para PacC en otros organismos (Linnemannstöns, Schulte et al. 2002a). Además de estas dos proteínas, los mutantes carentes de adenilato ciclasa F. proliferatum (Kohut, Olah et al. 2010) mostraron altos niveles de producción de bikaverina, lo que implica a la molécula de AMPc en la regulación de la síntesis de éste pigmento. Figura 6. Agrupamiento de los genes bik (adaptado de (Wiemann, Willmann et al. 2009)) Introducción 41 2.3.1.2 Fusarinas Las fusarinas son producidas por distintas especies del género Fusarium, como F. verticillioides, F. graminearum y F. fujikuroi (Barrero, Sánchez et al. 1991) (Song, Cox et al. 2004) a partir de la PKS FusA/FgPks10 (Gaffoor, Brown et al. 2005) (ver Figura 4). Además de los dominios habituales, esta PKS de tipo I contiene un módulo de síntesis de péptidos no ribosómicos (NRPS) adicional en su extremo C terminal, que participa en la síntesis de la pre-fusarina C (Song, Cox et al. 2004). La presencia de dominios NRPS es muy inusual en las PKSs, aunque hay otros ejemplos. Así, la mixobacteria Melittagium lichenicola Me 146 posee una enzima híbrida PKS/NRPS implicada en la síntesis de melitiazol. El término fusarinas engloba una familia de compuestos con diversas variaciones estructurales (Figura 7). F. fujikuroi produce mayoritariamente Figura 7. Compuestos químicos pertenecientes a la familia de las fusarinas. A: fusarinas A, C y D. B: estereoisómeros de la fusarina C. C: Intermediarios de la fusarina C detectados en F. fujikuroi (modificado de (Niehaus, Kleigrewe et al. 2013). Introducción 48 ciclados. El término carotenoides engloba a dos tipos de moléculas, los carotenos y las xantofilas. Los primeros poseen tan solo carbono y nitrógeno en su estructura, mientras que las xantofilas además presentan átomos de oxígeno. El licopeno o el -caroteno son ejemplos típicos de carotenos, mientras que la luteína o la astaxantina lo pueden ser para las xantofilas. 2.3.2.2.1 Los carotenoides en la naturaleza Los carotenoides se encuentran ampliamente distribuidos en la naturaleza (Figura 11), hallándose también en organismos que no son capaces de sintetizarlos, y que los ingieren en la dieta (Maiani, Castón et al. 2009). Los organismos capaces de sintetizar carotenoides incluyen las plantas y algas (Edge, McGarvey et al. 1997), así como numerosas bacterias (Armstrong 1997) y hongos filamentosos (Sandmann and Misawa 2002). Los carotenoides son pigmentos muy importantes en las plantas, ya que juegan un papel esencial en numerosos procesos básicos de su biología. Por ejemplo, los carotenoides son componentes esenciales de los centros de reacción fotosintéticos (Edge, McGarvey et al. 1997) y actúan además como señales en el desarrollo. Además, son responsables de una extensa gama de Figura 11. Carotenoides en la naturaleza: los carotenoides se encuentran presentes en una enorme variedad de organismos, siendo parte imprescindible de nuestra dieta. Introducción 49 colores en flores, y frutos, que va desde los tonos amarillentos a los rojizos. En las estructuras verdes estos colores son enmascarados por la clorofila, pero se ponen en evidencie en las hojas en senescencia en el otoño. La pigmentación en flores y frutos constituye un importante aspecto evolutivo, ya que contribuye a mejorar las posibilidades de dispersión de las semillas por mamíferos, pájaros e insectos. Igualmente, los carotenoides contribuyen al sabor y olor de flores y frutos (Auldridge, McCarty et al. 2006). Con una única excepción conocida en áfidos, explicada por trasferencia génica horizontal desde hongos (Moran and Jarvik 2010), los animales no son capaces de sintetizar carotenoides. Sin embargo, los carotenoides o sus derivados pueden desempeñar funciones muy importantes en estos organismos. Así, el retinol (vitamina A), cuyo principal precursor es el -caroteno, desempeña un papel fundamental en la visión a través de su papel como cromóforo de las opsinas. Un derivado de éste, el ácido retinoico, es un morfógeno necesario para el correcto desarrollo embriológico e inmunológico de cordados (Rhinn and Dolle 2012). En seres humanos, el déficit de carotenoides en la dieta es causante de ceguera por un incorrecto desarrollo de la retina (West and Darnton-Hill 2008). Además, la carencia en la dieta de xantofilas como la zeaxantina y la luteína aumenta el riesgo de degeneración de la mácula del ojo, un hecho asociado a la mayoría de casos de ceguera en la tercera edad (Krinsky, Landrum et al. 2003). En la actualidad está muy aceptado que la presencia de carotenoides en la dieta, especialmente por sus propiedades antioxidantes, es beneficiosa para la salud (Fraser and Bramley 2004). La ingesta de ciertos carotenoides, como el licopeno, abundante en el tomate o en la sandía, potencian la capacidad de respuesta inmunológica (Jyonouchi, Zhang et al. 1993) y reducen el riesgo de padecer ciertos tipos de cáncer y enfermedades cardiovasculares (Nishino 1998) (Wang, Gaziano et al. 2008). La importancia de los carotenoides no solamente se debe a sus propiedades saludables para el ser humano, sino a que se trata de pigmentos relevantes para la industria. De esta manera, los carotenoides son utilizados como aditivos precursores de vitamina A y como colorantes naturales Introducción 50 bioactivos en la industria alimentaria (Castillo, Fernández et al. 2005) (Mapari, Nielsen et al. 2005). En la industria cosmética es destacable el uso de los mismos como ingredientes protectores frente al daño oxidativo provocado en la piel por los radicales libres (Lademann, Schanzer et al. 2011). La capacidad de sintetizar carotenoides es un carácter frecuente en los hongos y algunos de ellos, como N. crassa, Phycomyces blakesleeanus, Mucor circinelloides, Xanthophyllomyces dendrorhous o F. fujikuroi han sido modelos ampliamente usados para su estudio (Avalos, Díaz-Sánchez et al. 2014). Sin embargo, los carotenoides no parecen jugar un papel importante en la biología de estos organismos, como se deduce de la existencia de hongos que carecen de ellos o de mutantes albinos en los hongos productores, perfectamente en su crecimiento. Existe actualmente cierta controversia sobre el posible papel de los carotenoides en los hongos como protectores frente a estrés oxidativo. Sin embargo, numerosos ejemplos parecen indicarlo. Por citar algunos, el hongo Sclerotium rolfsii produce altos niveles de -caroteno en respuesta a extrés oxidativo, lo cual es suficiente por sí mismo para inducir la formación de esclerocios (Georgiou, Zervoudakis et al. 2001), y la adición de compuestos generadores de especies reactivas de oxígeno a cultivos de X. dendrorhous provoca un incremento de los niveles de producción de astaxantina (Liu and Wu 2006). En ciertos zigomicetos, como Blakeslea trispora o P. blakesleeanus, el -caroteno actúa como precursor de sus hormonas sexuales, los ácidos trispóricos (Austin, Bu'Lock et al. 1970) (Polaino, Herrador et al. 2010). Así, los mutantes bloqueados en pasos previos de la síntesis de -caroteno son sexualemente incompetentes (Sutter 1975). 2.3.2.2.2 Los carotenoides en F. fujikuroi El carotenoide mayoritario en F. fujikuroi es la neurosporaxantina (ácido -apo-4’-carotenoico), una xantofila originariamente identificada en N. crassa (Zalokar 1957). Los primeros pasos de su ruta biosintética (Figura 12A), similares a los de otros hongos, parten de la condensación de dos moléculas de GGPP para formar un caroteno incoloro, el fitoeno. La reacción es llevada a cabo por la enzima CarRA (Linnemannstöns, Prado et al. 2002b). La Introducción 51 molécula de fitoeno es objeto de cuatro desaturaciones, catalizadas por la desaturasa CarB, y de una ciclación terminal llevada a cabo por el dominio ciclasa de la enzima CarRA, que es una enzima bifuncional. El producto de la ciclación, el -caroteno (Prado-Cabrero, Schaub et al. 2009), es desaturado en una quinta posición por CarB para producir toruleno, que será cortado por la oxigenasa CarT (Prado-Cabrero, Estrada et al. 2007a) para producir -apo-4’ carotenal. Una última reacción enzimática llevada a cabo por la deshidrogensa de aldehído CarD (Díaz-Sanchez, Estrada et al. 2011) genera el grupo carboxilo para producir la neurosporaxantina. En una rama paralela a esta ruta, el -caroteno es objeto de una segunda ciclación por CarRA para producir -caroteno (Avalos and Cerdá- Olmedo 1987). Este caroteno es sustrato de la oxigenasa CarX, que lo escinde en dos moléculas de retinal (Prado-Cabrero, Scherzinger et al. 2007b). La producción de retinal puede tener especial relevancia biológica en F. fujikuroi ya que es el cromóforo potencial de las rodopsinas que posee este ascomiceto, CarO y OpsA. El -caroteno se encuentra en concentraciones relativamente bajas en el hongo, pero el retinal no es visible a simple vista y no se han podido determinar sus concentraciones. De los genes car citados arriba, carRA y carB son necesarios para la síntesis tanto de -caroteno como de neurosporaxantina, mientras que carT carD y solo son necesarios para la síntesis de esta última. Es interesante hacer notar que carRA y carB se encuentran en el genoma en un agrupamiento génico junto a carX y el gen de una de las dos opsinas, carO. Por tanto, este agrupamiento contiene la información necesaria para la síntesis de opsina CarO fotoactiva. Sin embargo, los genes carD y carT se encuentran localizados en distintas regiones del genoma del hongo (Figura 12B). Introducción 52 La regulación de la síntesis de carotenoides en F. fujikuroi ha sido objeto de especial atención en el laboratorio donde se ha desarrollado esta Tesis. En los medios sintéticos empleados habitualmente en el laboratorio, la estirpe silvestre produce en oscuridad cantidades muy bajas de carotenoides. En la luz, sin embargo presenta una pigmentación anaranjada debido a la acumulación de neurosporaxantina y cantidades menores de carotenoides precursores, como el toruleno (Avalos and Cerdá-Olmedo 1987). La exposición del hongo a la luz desencadena una rápida activación transcripcional de los genes car (revisado por (Avalos and Estrada 2010), con la excepción de carD ((Díaz-Sanchez, Estrada et al. 2011). En las condiciones estudiadas, tras unos minutos de exposición a luz se aprecia una subida en los niveles de ARNm, que alcanzan su máximo tras unos 60 minutos de iluminación (Avalos, Bejarano et Figura 12. Síntesis de carotenoides en F. fujikuroi. A: Ruta biosintética. Se indican los nombres de los genes responsables de cada paso y se resaltan en gris los lugares donde se producen las reacciones químicas. B: Organización genética de los genes car. Los huecos dentro de los genes indican intrones. Introducción 53 al. 1993) (Saelices, Youssar et al. 2007) (Estrada and Avalos 2008a) (Figura 13). Posteriormente, los niveles de ARNm disminuyen, fenómeno conocido como fotoadaptación. La búsqueda del sistema fotorreceptor encargado de la fotoinducción de los genes car de Fusarium se basó en resultados previos en N. crassa, donde el complejo White Collar (WC), formado por los fotorreceptores WC-1 y WC-2, es responsable de la fotoinducción de la carotenogénesis. En este hongo, los mutantes carentes de un complejo WC completo presentan micelio completamente albino en la luz (Degli-Innocenti and Russo 1984), mientras que los conidios producidos por las hifas aéreas muestran un color anaranjado debido a poseer una regulación independiente por desarrollo. El complejo WC controla también otras fotorrespuestas investigadas en N. crassa, como el ritmo circadiano (Russo 1988) o el desarrollo de protoperitecios (Degli- Innocenti and Russo 1984). F. fujikuroi posee genes homólogos de wc-1 y wc-2 de N. crassa, por lo que se deduce que posee un complejo WC similar. Sin embargo, los mutantes carentes de una copia funcional del homólogo de wc-1, wcoA, siguen mostrando fotoinducción de la carotenogénesis (Estrada and Avalos 2008a), y un resultado similar se encontró en F. oxysporum (Ruiz-Roldán, Garre et al. 2008). Esto indica que el complejo WC no es el único fotorreceptor Figura 13. Efecto de la luz sobre los niveles de ARNm de los genes car: Niveles de ARNm de distintos genes car en oscuridad (tiempo 0’) y a distintos tiempos de iluminación. Puede apreciarse el fenómeno de la fotoinducción, alcanzándose el máximo tras iluminar 1 hora. En el panel inferior, control de carga correspondiente a RNAr. Introducción 54 responsable de la fotoinducción de la carotenogénesis en Fusarium, por lo que recientemente se ha extendido el estudio a otros posibles fotorreceptores . Así, se ha estudiado el efecto de las mutaciones de los genes cryD y vvdA, que cifran un criptocromo de tipo DASH (Castrillo, García-Martínez et al. 2013) y la flavoproteína VvdA (Castrillo and Avalos 2014), respectivamente. Los mutantes carentes de estas fotoproteínas siguen mostrando fotoinducción de la síntesis de carotenoides, pero sus cinéticas de acumulación están alteradas, mostrando además los mutantes wcoA una respuesta más lenta (Castrillo 2014). Los resultados conducen a un modelo en el que participan varios fotorreceptores de forma cooperativa, con distintos papeles en fases tempranas o tardías del proceso, con un papel central para WcoA como principal inductor transcripcional. El caso del gen vvdA es especialmente interesante, ya que es homólogo de vvd de N. crassa, que juega un papel relevante en el mecanismo de fotoadaptación (Schwerdtfeger and Linden 2003). Los resultados indican que en F. fujikuroi VvdA no participa en la fotoadaptación, pero controla posiblemente la actividad de los otros fotorreceptores, además de ejercer influencia en el desarrollo del micelio en la luz (Castrillo and Avalos 2014). Además de la luz, otros factores ambientales modulan la producción de carotenoides en F. fujikuroi. Por ejemplo, la estirpe silvestre IMI58289 acumula más carotenoides si se incuba a 30ºC que cuando se hace a 22ºC (Avalos and Cerdá-Olmedo 1987). De igual manera, la escasez de nitrógeno en el medio ejerce de señal inductora de manera independiente y aditiva al efecto de la luz (Rodríguez-Ortiz, Limón et al. 2009). 2.3.2.2.3 Fenotipos asociados a defectos en la carotenogénesis en hongos: los fenotipos carS y ovc. En muchos hongos se han descrito mutantes desregulados para la síntesis de carotenoides (Avalos, Díaz-Sánchez et al. 2014). Ese es el caso de los mutantes carS (superproductor de carotenoides) de F. fujikuroi, que presentan una coloración intensamente anaranjada en cualquier condición de cultivo (Figura 14A). El análisis de los genes car en estos mutantes indica que Introducción 55 se transcriben a altos niveles tanto en luz como en oscuridad (Figura 14B) (Linnemannstöns, Prado et al. 2002b) (Prado, Prado-Cabrero et al. 2004) (Thewes, Prado-Cabrero et al. 2005). Recientemente se ha identificado el gen carS, cuya mutación produce el fenotipo aludido tanto en F. fujikuroi (Rodríguez-Ortiz, Limón et al. 2013) como en F. oxysporum (Rodríguez-Ortiz, Michielse et al. 2012). El gen CarS cifra una proteína homóloga a CrgA de M. circinelloides, para la que se ha descrito actividad ligasa de ubiquitina (Nicolas-Molina, Navarro et al. 2008). En N. crassa no existen mutantes con el nivel de desregulación de la carotenogénesis independiente de la luz mostrado por los mutantes carS de Fusarium. Sin embargo, algunos mutantes muestran más carotenoides que la estirpe silvestre en la luz, como es el caso del mutante ovc (de Figura 14. Fenotipo mutantes CarS. A: Mutantes CarS y WT incubados en oscuridad. El color anaranjado de los mutantes CarS se debe a la acumulación de carotenoides, mientras que la estirpe WT no los sintetiza en oscuridad. B: Niveles de ARNm de distintos genes car en oscuridad. Tanto carRA como carX y carO se encuentran reprimidos en oscuridad en la estirpe WT, pero inducidos en tres mutantes CarS distintos. En el panel inferior, control de carga correspondiente a RNAr. Introducción 56 overaccumulation of carotenoids), si bien los niveles en oscuridad no se ven afectados (Harding, Philip et al. 1984). Además de este fenotipo, el mutante ovc muestra una inhibición parcial de su crecimiento en condiciones de alta presión osmótica (obtenidos suplementando el medio con 0,65 M de NaCl, KCl, NaNO3 o, KNO3, así como 1,3 M de sorbitol, (Youssar, Schmidhauser et al. 2005)), y una alteración en el desarrollo en tubos de agar inclinado, consistente en una compactación del micelio en la parte alta del cultivo. Los dos últimos fenotipos son mostrados también por otro mutante, llamado cut (Kuwana 1953) (Figura 15), pero éste manifiesta una fotoinducción normal de la carotenogénesis. La identificación del gen cut, llamado cut-1, puso en evidencia que su mutación causa simultáneamente la sensibilidad a condiciones hiperosmóticas y la compactación micelial. Datos genéticos previos indicaron que las mutaciones cut y ovc eran alélicas (Banks, Bennett et al. 1997). Este hecho Figura 15. Comparación fenotípica entre las estirpes WT y cut de N. crassa: puede apreciarse la formación de un “tapón” de micelio aterciopelado en la parte superior del tubo correspondiente al mutante cut. Introducción 57 quedó confirmado por el hallazgo en el mutante ovc de una deleción de 77 kb que incluye el gen cut-1, y la constatación de que el fenotipo superproductor de carotenoides en la luz se debe al efecto sinérgico de la pérdida de otro de los genes delecionados (Youssar and Avalos 2007). El gen cut-1 cifra una proteína de la familia HAD (haloacid dehalogenases), que incluye enzimas con diferentes actividades, normalmente asociadas a reacciones de defosfatación. El estudio del gen cut-1 reveló que su expresión se induce por alta presión osmótica, mientras que otras condiciones de estrés, como la adición al medio de cultivo de agentes oxidantes o del antifúngico fludioxonil (dispara la ruta HOG de resistencia a estrés osmótico) no produce cambios importantes en los niveles de ARNm cut-1 (Youssar and Avalos 2006). Además de por estrés osmótico, el gen cut-1 es regulado por luz: sus niveles de ARNm en oscuridad caen drásticamente al iluminar al hongo. Interesantemente, los mutantes de la proteína VIVID y el complejo WC, entre otros, mantienen la fotorepresión pero los niveles globales de ARNm del gen son más elevados (Youssar and Avalos 2006) (Figura 16). Figura 16. Efecto de la iluminación sobre los niveles de ARNm de cut-1 en la estirpe silvestre (WT) y en los mutantes vvd, wc-1 y wc-2 de N. crassa. En la estirpe silvestre los niveles de ARNm son relativamente bajos en oscuridad (D), pero desaparecen totalmente al iluminar el cultivo Tiempos de iluminación indicados en minutos). En la regulación de la expresión del gen cut-1 parecen participar VVD y WC-1 y WC-2, como indica los mayores niveles de ARNm en las mismas membranas. En el panel inferior, control de carga correspondiente a RNAr [adaptado de (Youssar and Avalos 2006)]. Introducción 64 3.2 Opsinas en F. fujikuroi En el caso de F. fujikuroi, se han descrito en su genoma tres genes que cifran posibles opsinas. De estas tres proteínas, dos poseen el residuo de lisina en posición 216 necesario para la unión del retinal, por lo que podrían ser presumiblemente activas, mientras que la tercera carece de él (Figura 18). Entre las opsinas presumiblemente fotoactivas se encuentran CarO y OpsA. La primera es cifrada por el gen carO, mencionado en apartados anteriores y situado en el agrupamiento génico de los genes car. El gen opsA, por el contrario, se encuentra en una región distinta del genoma del hongo. La mutación dirigida de ambos genes no ha mostrado ningún fenotipo aparente bajo condiciones de laboratorio en F. fujikuroi (Prado, Prado-Cabrero et al. 2004) (Estrada and Avalos 2009), por lo que el papel de ambas proteínas permanece sin aclarar. El estudio del papel de CarO y OpsA en la biología de Fusarium es uno de los objetivos de esta tesis. La tercera opsina de Fusarium, HspO, carece del residuo de lisina al que se une covalentemente el retinal, por lo que no parece ser fotoactiva. HSpO es homóloga a YRO2 de N. crassa y a HSP30 de S. cerevisiae, por lo que se sospecha una posible función como chaperona (Zhai, Heijne et al. 2001). 3.3 Usos aplicados de las rodopsinas microbianas El estudio de las rodopsinas microbianas ha cobrado especial relevancia en las últimas décadas. Las características electrofisiológicas de estas proteínas las hacen especialmente útiles en diversos campos. Por ejemplo, la capacidad de crear voltaje dependiente de energía lumínica por parte de las membranas que contienen rodopsinas puede ser aprovechada para construir células fotoeléctricas o retinas artificiales (Chen and Birge 1993). Por otro lado, la posibilidad de usar rodopsinas microbianas capaces de crear un gradiente de protones a través de una membrana es una característica muy interesante para procesos tan diversos como la desalinización de agua marina (Oesterhelt 1976) (Eisenbach, Weissman et al. 1977) o la producción de biofuel (Walter, Greenfield et al. 2010). Introducción 65 Estas posibles aplicaciones se engloban en un campo de investigación actualmente pujante, la optogenética, consistente en el uso de la tecnología del ADN recombinante para llevar a cabo transfecciones de neuronas animales con vectores virales con genes para rodopsinas microbianas (Scanziani and Hausser 2009). La expresión heteróloga de estas proteínas en neuronas animales ofrece la posibilidad de reprimir o disparar potenciales de acción a través de estímulos lumínicos. La rodopsina 2 del alga Chlamydomonas reinhardtii, un canal iónico, ha sido usada para disparar potenciales de acción (Nagel, Szellas et al. 2003), mientras que la halorrodopsina de Natromonas pharaonis es capaz de importar iones cloruro y reprimir potenciales de acción en neuronas (Han and Boyden 2007). El uso de rodopsinas microbianans para el tratamiento de desórdenes neurológicos asociados a enfermedades de la locomoción y el aprendizaje (Szobota and Isacoff 2010) es un campo con gran futuro en la investigación biomédica, si bien existen numerosos problemas bioéticos que han de ser resueltos, tales como la necesidad de experimentación con humanos o el uso de vectores virales como herramienta para la transfección neuronal. OBJETIVOS AIMS Objetivos/Aims 69 The aim of this study is to contribute to the current knowledge on the regulatory mechanisms that control the responses of fungi to environmental signals, using the model Fusarium fujikuroi. In particular, this Thesis has investigated the following subjects: 1. The role of adenylyl cyclase AcyA of F. fujikuroi on the regulation of secondary metabolism, pathogenicity and stress responses. 2. The biological function of the Cut-1 ortholog CutA and its relation with stress responses. 3. The elecrophysiological features of the CarO rhodopsin, its in vivo localization and its biological function. CHAPTER 1 ADENYLYL CYCLASE PLAYS A REGULATORY ROLE IN DEVELOPMENT, STRESS RESISTANCE AND SECONDARY METABOLISM IN Fusarium fujikuroi Jorge García-Martínez, Attila L. Ádám, Javier Avalos Published in PlosOne (2012) Capítulo 1: Adenylyl cyclase plays a regulatory role in development, stress resistance and secondary metabolism in Fusarium fujikuroi 73 1. Introduction Some species of the ascomycete fungus Fusarium are useful research models for secondary metabolite production. A representative example is Fusarium fujikuroi, able to produce gibberellins, carotenoids or bikaverin, among other compounds of potential biotechnological interest (Avalos, Cerdá- Olmedo et al. 2007). The gibberellins are growth-promoting plant hormones with applications in agriculture (Rademacher 1997). Among them stands out for its physiological effects gibberellic acid (GA3), the major gibberellin product in this fungus. Carotenoids are fatsoluble pigments produced by photosynthetic organisms and many heterotrophic bacteria and fungi (Britton, Liaaen-Jensen et al. 1998). Both groups of compounds are called terpenoids (Fig. 20). Figure 20. Main terpenoids in F. fujikuroi: both groups share the same precursor and possess biotechnological interest. Capítulo 1: Adenylyl cyclase plays a regulatory role in development, stress resistance and secondary metabolism in Fusarium fujikuroi 80 Upon aging, the ΔacyA mutants exhibited a reddish pigmentation in the race tubes (Fig. 22A), a trait described in a later section. Additionally, surface cultures of the mutants produced fewer conidia (Fig. 22C). This difference cannot be attributed to the slower mycelial development, since these analyses were carried out on surface colonies that stopped their growth approximately four days before conidia determination. In a former experiment in carboxymethyl cellulose supplemented medium (Cappellini and Peterson 1965), the acy1 mutants of F. proliferatum produced more conidia than the wild type (Kohut, Olah et al. 2010). However, incubation of the same strains under the culture conditions tested for F.fujikuroi revealed the same conidiation pattern in both species (data not shown). Thus, the acyA gene plays a central role in mycelial development, including conidia formation. However, in contrast to F. proliferatum (Kohut, Olah et al. 2010), the reduced growth capacity did not affect the ability of the mutants to colonize tomato fruit tissue (inset picture in Fig. 22B). Addition of exogenous cAMP to agar cultures of a ΔacyA mutant did not fully restore normal growth (Fig. 23). However, growth and morphology of the wild Figure 23. Effect of cAMP addition on growth and pigmentation of the wild type and an ΔacyA strain. A: Colonies of the wild typeand the ΔacyA mutant T9 after eight days incubation at 30ºC on DGasn minimal agar (5.5 cm Ø Petri dishes) supplemented with the indicated concentration of the cAMP analogue dibutyryladenosine 39,59-cyclic monophosphate. Middle pictures: detail of colony edges from the same cultures. Below: pigmentation of the back of the colonies. Capítulo 1: Adenylyl cyclase plays a regulatory role in development, stress resistance and secondary metabolism in Fusarium fujikuroi 81 type was visibly altered at the higher cAMP concentration tested, resembling those of the ΔacyA mutant under the same conditions. Therefore, the phenotypic differences between wild type and ΔacyA mutant were attenuated by cAMP addition. This was particularly manifest at the colony edges, sparse in the wild type and dense in the compact ΔacyA colonies in control plates, but with a similar aspect for both strains in the presence of 2.5 mM cAMP (Fig. 23). Furthermore, an orange pigmentation unrelated to carotenoids, manifest on the back sides of wild type colonies grown in the dark at 30ºC, was much weaker in the ΔacyA mutant but it was restored in 2.5 mM cAMP supplemented medium (Fig. 23). These observations strongly suggest that the phenotypic alterations exhibited by the ΔacyA mutants are due to lack of cAMP synthesizing activity. 2.3 The ΔacyA mutants are affected in stress sensitivity: The adenylyl cyclase mutants of F. proliferatum and F.verticillioides also exhibited alterations in their responses to different stress conditions, such as heat-shock, oxidative agents, or heavy metals (Choi and Xu 2010; Kohut, Olah et al. 2010). We examined the effect of these stresses on the F. fujikuroi mutants compared to the control strains. First, we tested the ability of the ΔacyA and acyA+ strains to grow under toxic concentrations of two heavy metals, Cd and Cu. Growth inhibition, referred to control plates without the metal, was similar irrespective of the presence of a functional acyA gene in the tested strain (Figure 24). Capítulo 1: Adenylyl cyclase plays a regulatory role in development, stress resistance and secondary metabolism in Fusarium fujikuroi 82 One hour exposure to 42ºC, 45ºC or 50ºC did not affect significantly the growth capacity of the ΔacyA mutants compared to the acyA+ strains in conidia-dilution tests on DGasn agar plates. However, one hour exposure to 60ºC completely abolished the growth of the control strains, while the ΔacyA mutants were still able to grow with 105 conidia (Fig. 25A). In this experiment the cells were exposed to 60ºC immediately after inoculation of the conidia drops, but similar results were obtained if the 60ºC treatment was done 4 hours after inoculation, i.e., when the germination process was already triggered (data not shown). Hence, cAMP probably participates in the tolerance mechanism against heat shock in a similar manner than in other Fusarium species. Former studies in F. verticillioides and F. proliferatum revealed 49ºC and 45ºC as effective heat shock temperatures in liquid media assays, respectively. The comparison of heat shock sensitivity of wild type F. fujikuroi and F. proliferatum in conidia-dilution assays on DG agar plates (both species were sensitive to heat stress only at 55–60ºC in a conidial concentration dependent fashion) suggest that this disparity is due to differences in culture conditions rather than to higher heat tolerance of F. fujikuroi (data not shown). Figure 24. Lack of effect of the ΔacyA mutation on growth in the presence of metals. Effect of 1 mM Cd or 3 mM Cu on radial growth of the acyA and ΔacyA strains on DGasn agar plates. Capítulo 1: Adenylyl cyclase plays a regulatory role in development, stress resistance and secondary metabolism in Fusarium fujikuroi 83 Similar conidia-dilution tests achieved with a low (1 mM) H2O2 concentration showed a reduction in the growing capacity of the five F. fujikuroi strains, but the inhibition was apparently more pronounced in the ΔacyA mutants (Fig. 25A). Therefore, we decided to investigate in more detail the effect of this oxidative agent. Incubation of the strains on DGasn agar plates supplemented with higher H2O2 concentrations confirmed a greater sensitivity of the DacyA mutants: they were not able to grow at either 50 or 100 mM H2O2, while the strains with the functional acyA allele still showed a significant growing capacity (Fig. 25B). Figure 25. Effect of the ΔacyA mutation on growth or viability under heat or oxidative stress. A: Serial conidia-dilution tests of the effect of heat stress (60ºC/1 h) and oxidative stress (1 mM H2O2) on DGasn agar plates. Each spot was inoculated with the conidia amounts indicated on the left. B: Effect of H2O2 concentration (50–100 mM) on radial growth of the acyA+ and ΔacyA strains on DGasn agar plates. Overlapping symbols were separated for better visualization. C: Survival of conidia following 2 h incubation in liquid DGasn medium supplemented with 5 mM H2O2. Capítulo 1: Adenylyl cyclase plays a regulatory role in development, stress resistance and secondary metabolism in Fusarium fujikuroi 84 In another approach, conidia from the same strains were exposed for two hours to 5 mM H2O2 in DGasn minimal liquid medium and tested for their ability to develop colonies after plating on CM agar. As aresult, the ΔacyA mutants produced about 30% less colonies tan the control strains, which were not significantly affected by this treatment (Fig. 25C). We conclude that lack of adenylyl cyclase in F. fujikuroi results in a weakened response to H2O2- induced oxidative damage. 2.4 The ΔacyA mutants overproduce a red pigment: As mentioned above (Fig. 22A), the ΔacyA mutants exhibited a reddish pigmentation, absent in the control strain. This color was more This color was more apparent under nitrogen starvation, and it diffused to the culture medium (Fig. 26A), indicating that the pigment is soluble and secreted out of the cell. The enhanced reddish pigmentation fits the derepressed bikaverin production formerly found in the adenylyl cyclase mutants of F. proliferatum and F. verticillioides (Choi and Xu 2010; Kohut, Olah et al. 2010). The production of this pigment was investigated in the five strains under analysis, grown on minimal agar with either high or low nitrogen content (Fig. 26B). According to absorption data, the two control strains produced low pigment amounts in high nitrogen agar, while the production by the three ΔacyA mutants increased about three-fold. The difference was more noteworthy in low nitrogen agar, where the ΔacyA mutants produced high pigment amounts while only traces were found in the cultures of the control strains. The accumulation of the pigment was drastically reduced in the present of exogenous cAMP (Fig. 26E), providing further evidence for the lack of cAMP as the major cause for the phenotypic alterations of the ΔacyA mutants. Capítulo 1: Adenylyl cyclase plays a regulatory role in development, stress resistance and secondary metabolism in Fusarium fujikuroi 85 The absorption spectra of the red pigments extracted from the ΔacyA mutants differed from that of bikaverin, as deduced from comparison with a bikaverin sample produced by the strain SG1 (Rodríguez-Ortiz, Limón et al. 2009). While the latter coincided with the published bikaverin spectrum (Cornforth, Ryback et al. 1971), including maximal absorption wavelength (521 nm), the spectrum of the red pigment produced by the ΔacyA mutants had a maximal absorption at ca. 500 nm (Fig. 26C). Figure 26. Production of red pigments by the acyA+ and ΔacyA strains. A: Agar cultures of the acyA+ and ΔacyA strains grown for 14 days at 30ºC on high or low nitrogen medium. B: Concentration of red pigments in the mycelia from the plates displayed on the left picture. The data show absorption units corrected by dry weight of the samples, and referred to the absorption of the wild type grown in high nitrogen medium, which was taken as 1. C: Absorption spectra of the red pigments isolated from the T2 mutant grown in high nitrogen medium compared to the spectrum of our bikaverin standard (Bk, dashed line). Chemical structure of bikaverin is shown above. D: TLC separation of the bikaverin standard (Bk) and the red pigments produced by the wild type on high nitrogen medium and by the T2 mutant on high or low nitrogen medium. E: Effect of addition of 0.5 mM cAMP (dibutyryladenosine 39,59-cyclic monophosphate) on pigmentation in agar cultures of the wild type and the ΔacyA strain T9 grown for 14 days at 30ºC on low nitrogen medium. Capítulo 1: Adenylyl cyclase plays a regulatory role in development, stress resistance and secondary metabolism in Fusarium fujikuroi 86 Absorption spectra of the pigments from the control strains, produced in much lower concentrations, were more similar to that of our bikaverin standard (result not shown). To gain more insights on the chemical difference between the pigments accumulated by control and mutant strains, the samples obtained in these analyses were separated on TLC and compared to SG1-produced bikaverin. The pigment from the ΔacyA cultures, either in high or low nitrogen agar, showed two bands of different chromatographic properties than bikaverin (Fig. 26D). However, the absorption spectrum and chromatographic separation of the pigment produced by the wild type strain were more similar to those of bikaverin, although it run at a slightly lower position on the TLC plate and it was hardly visible because of its low concentration. The chemical nature of the red pigments produced by the ΔacyA mutants is currently under investigation. 2.5 The ΔacyA mutants exhibit changes in gibberellin and carotenoid production: The effect of the ΔacyA mutation on gibberellin and carotenoid biosyntheses was investigated under appropriate culture conditions for each pathway. For gibberellin production, secreted GA3 levels were assayed by TLC and HPLC in shake cultures of the five strains grown in low nitrogen broth. As expected, significant GA3 amounts (about 42 mg l-1 after 15 days incubation) were detected in the cultures of control acyA+ strains, but GA3 concentration was severely reduced in the ΔacyA cultures (Fig. 27A). To test if the ΔacyA mutation affects negatively the induction of the gib genes by nitrogen starvation, mycelia of the five strains grown in high nitrogen broth were washed and transferred to a nitrogen-free solution. Upon these controlled inducing conditions, the mRNA levels of gene gibB, coding for the key enzyme in GA biosynthesis ent-kaurene synthase/ent-copalyl diphosphate synthase (cps/ks (Tudzynski and Hölter 1998)), showed a rapid and transient induction. This result, and differences in time-course patterns of gibberellin Capítulo 1: Adenylyl cyclase plays a regulatory role in development, stress resistance and secondary metabolism in Fusarium fujikuroi 87 accumulation (Oller-López, Avalos et al. 2003), suggests regulatory differences with another wild type strain previously investigated, IMI58289, in which the mRNA induction under the same experimental conditions was slower and sustained (Rodríguez-Ortiz, Limón et al. 2009). In agreement with the lower GA3 production, gibB mRNA induction was significantly reduced in the ΔacyA mutants compared to the acyA+ control strains (Fig. 27B), pointing to cAMP as a positive signal in the regulation of GA biosynthesis. Because of the regulation by light of the carotenoid pathway, the production of these pigments was analyzed on surface cultures, grown either in the dark or under continuous illumination. As expected, the control strains Figure 27. Production of gibberellins by the acyA+ and ΔacyA strains. A: Relative GA3 contents in the filtrates of the cultures of the indicated strains after 9 days (pale front bars) and 15 days (dark back bars) incubation in low nitrogen broth in the dark. The data show average and standard deviation from two independent experiments. Inner picture: TLC separation of gibberellin samples from the five strains in a representative experiment. The fluorescent spot is GA3 (chemical formula shown above). B: Quantitative RT-PCR analyses of gibB mRNA levels in the five strains grown in high nitrogen medium for 3 days and incubated afterwards in 2 g l-1 glucose for the time indicated in abscissas. Capítulo 1: Adenylyl cyclase plays a regulatory role in development, stress resistance and secondary metabolism in Fusarium fujikuroi 88 exhibited a marked photoinduction, with a 15-fold increase in the amount of carotenoids in illuminated cultures compared to the dark (Fig. 28A). However, the carotenoid content of the ΔacyA mutants was increased about 3-fold in the dark and it was reduced by 50% in the light compared to the control strains. Consequently, the photoinduction rate was markedly reduced in the ΔacyA mutants. The ratio of polar (neurosporaxanthin) to neutral carotenoids (precursors) was not significantly modified in the ΔacyA mutants, indicating that all the enzymatic activities of the carotenoid pathway were similarly affected by the loss of functional adenylyl cyclase. Figure 28. Production of carotenoids and carRA/carB gene expression in the acyA+ and ΔacyA strains. A: Carotenoid content in mycelia grown for 7 days on minimal medium in the dark or under continuous illumination. The data show average and standard deviations for the amounts of polar (upper bars) and neutral (lower bars) carotenoids in two independent experiments. The chemical formula of neurosporaxanthin (Nx) is shown above. B: mRNA levels of the enzymatic genes for the carotenoid pathway carRA and carB in the same strains grown in the dark or illuminated for 30 or 60 min. Data show average and standard deviation from two independent experiments. Relative mRNA levels are referred to the value of the wild type in the dark. Capítulo 1: Adenylyl cyclase plays a regulatory role in development, stress resistance and secondary metabolism in Fusarium fujikuroi 89 For a better understanding of the regulatory basis for the altered carotenoid accumulation, the mRNA levels of two key enzymatic genes of the carotenoid pathway, carRA and carB (Avalos, Cerdá-Olmedo et al. 2007), were determined in the acyA+ and ΔacyA strains grown in the dark and upon illumination. The dark levels were induced about 4-fold for carRA and 2-fold for carB in the ΔacyA mutants compared to the wild type (Fig. 28B), fitting their corresponding carotenoid contents in vivo (Fig. 28A). In the control transformant T1, carRA and carB mRNA levels in the dark were even lower than in the wild type (not shown in the graph). However, mRNA levels in the light were similar in any of the strains tested, either after 30 min or 1 hour illumination. Therefore, in contrast to the partial deregulation in the dark, the lower carotenoid content of the ΔacyA mutants in the light cannot be attributed to changes in carB or carRA expression. 3. Discussion: The product of adenylyl cyclase, cAMP, is a key regulatory signal in all taxonomic groups, from bacteria (Lory, Wolfgang et al. 2004) to mammals (Hanoune and Defer 2001). Many reports have attributed biological functions to cAMP in filamentous fungi. The recent finding of the partial deregulation of bikaverin biosynthesis in adenylyl cyclase mutants of F.proliferatum and F. verticillioides led us to undertake the investigation of a possible regulatory effect on gibberellin and carotenoid production in F. fujikuroi. As expected, the targeted mutation of the acyA gene in this species also resulted in reduced growth and enhanced reddish pigmentation. However, at least under the experimental conditions tested, the red pigments produced by the ΔacyA mutants of F. fujikuroi are not bikaverin, as indicated by the significant shift in the maximal absorption wavelength and the different behavior on thin layer chromatography. The different characteristics of these red pigments suggest a novel class of secondary metabolites, whose chemical nature remains to be elucidated. Capítulo 2: The gene cutA of Fusarium fujikuroi, encoding a protein of the haloacid dehalogenase family, is involved in osmotic stress and glycerol metabolism 97 1. Introduction In nature, survival of fungi relies on their ability to deal with a changing environment. Appropriate regulatory networks are necessary to overcome variations in different physical agents such as temperature, pH or osmotic pressure. Changes in water activity in fungal ecological niches determine their capacity for growth and colonization. Fungal growth requires the maintenance of appropriate turgor pressure in their hyphae (Lew 2011), a parameter governed by an osmotic mitogen-activated protein kinase(MAPK) cascade. This regulatory mechanism controls the internal pressure through ion uptake and synthesis of osmolytes such as glycerol (Ellis, Grindle et al. 1991) and is also used to adapt the fungal cells to hyper-osmotic conditions. The components of the high osmolarity glycerol (HOG) regulatory cascade have been thoroughly investigated in Saccharomyces cerevisiae (O'Rourke, Herskowitz et al. 2002) and are widespread in filamentous fungi (Krantz, Becit et al. 2006). The genes of the HOG pathway were first identified through the study of osmosensitive mutants in Neurospora crassa (Mays 1969). Genetic analyses of a collection of 25 mutants with reduced ability to grow on 0.68 M NaCl led to the identification of six genes. Mutants of genes os-1, os-2, os-4 and os-5, which are unable to grow on 0.68 M NaCl,were found to be affected in different components of the HOG cascade: a histidine kinase, and MAP, MAPK and MAPKK kinases (Schumacher, Enderlin et al. 1997; Zhang, Lamm et al. 2002; Fujimura, Ochiai et al. 2003). Two other mutants, cut and flm-2, were partially sensitive to hyper-osmotic conditions.The flm-2 mutation was later identified as an os-4 allele (Perkins and Davis 2000) and the cut mutation affects a protein of the haloacid dehalogenase (HAD) family, called CUT-1 (Youssar, Schmidhauser et al. 2005), with no apparent function in the HOG regulatory cascade. Sensitivity of the cut and os mutants to hyper-osmotic conditions is due to their inability to accumulate glycerol to counteract this Capítulo 2: The gene cutA of Fusarium fujikuroi, encoding a protein of the haloacid dehalogenase family, is involved in osmotic stress and glycerol metabolism 98 stress. However, the cut mutant differs from the os mutants in several aspects: (i) slant cultures of the cut strain exhibit a sharp interruption of aerial development, the origin of the mutant denomination. This unusual morphological alteration is absent in os mutants. (ii) Some dicarboximide antifungals, such as fludioxonil, trigger the synthesis of glycerol in the wildtype through activation of the HOG pathway in the absence of hyper-osmotic conditions. The os mutants are resistant to this antibiotic (Zhang, Lamm et al. 2002; Fujimura, Ochiai et al. 2003), but the cut mutant is sensitive (Fujimura, Ochiai et al. 2000), suggesting that it does not participate in the regulatory HOG cascade. The gene cut-1 is implicated in a different mutant phenotype, called Ovc from overaccumulation of carotenoids (Harding, Philip et al. 1984). Induction of carotenogenesis by light is a widespread phenotypic trait in filamentous fungi, including Neurospora spp. (Avalos and Corrochano 2013). The ovc mutant was identified because of its higher accumulation of carotenoids in response to light, although it is similar to the wild-type in the dark (Harding, Philip et al. 1984). The only ovc mutant described so far contains a large DNA deletion, covering cut-1 and 20 additional genes (Youssar and Avalos 2007); cut-1 plays a central role in the Ovc phenotype, since reintroduction of a wild-type cut-1 allele restores the normal carotenoid photoresponse. However, the single cut mutant exhibits normal carotenogenesis, indicating that the Ovc phenotype is due to the simultaneous loss of cut-1 and at least a second unidentified gene (Youssar and Avalos 2007). Intriguingly, cut-1 gene expression is repressed by light and is controlled by proteins involved in light regulation in N. crassa (Youssar and Avalos 2006), suggesting a possible link with the light-dependent phenotype of the ovc mutant. The phytopathogenic fungus Fusarium fujikuroi is a model for the production of secondary metabolites, such as gibberellins, carotenoids, bikaverin and fusarins (Avalos, Cerdá-Olmedo et al. 2007). The biosynthesis of these compounds is subject to complex regulation involving different Capítulo 2: The gene cutA of Fusarium fujikuroi, encoding a protein of the haloacid dehalogenase family, is involved in osmotic stress and glycerol metabolism 99 environmental signals, such as nitrogen availability, pH or light (Tudzynski 2005; Avalos and Estrada 2010; Limón, Rodríguez-Ortiz et al. 2010; Díaz- Sanchez, Avalos et al. 2012). These regulatory networks are frequently interconnected and may share common elements with stress-control mechanisms, such as those involving the key regulatory signal cAMP (García- Martínez, Ádám et al. 2012; Studt, Humpf et al. 2013). Because of the down regulation of N. crassa cut-1 expression by light and the possible regulatory connections of this gene with carotenoid biosynthesis, we analysed the function of the cut-1 orthologue in F. fujikuroi, here called cutA. Deletion of this gene resulted in normal morphology and pigmentation but reduced tolerance to osmotic stress. cutA mRNA levels correlated with osmotic conditions and, in contrast to cut-1, were induced by light. ΔcutA mutants did not show the increase in intracellular glycerol levels exhibited by the wildtype under hyper-osmotic conditions expected from the HOG response. Interestingly, cutA is linked and co-regulated with gldB, which encodes a putative glycerol dehydrogenase, suggesting that both genes participate in enzymic steps of glycerol metabolism. 2. Results 2.1 Identification of cutA, a gene of Fusarium orthologous to cut-1 of N. crassa: The Fusarium orthologues for the N. crassa gene cut-1, previously associated with the osmotic stress response (Youssar, Schmidhauser et al. 2005), were identified through BLAST analyses against the Comparative Fusarium genome Database (BroadInstitute). This gene, here called cutA, consists in Fusarium verticillioides of a 1485 bp continuous ORF encoding apredicted 494 aa polypeptide (FVEG_12092), a size slightly smaller than the Capítulo 2: The gene cutA of Fusarium fujikuroi, encoding a protein of the haloacid dehalogenase family, is involved in osmotic stress and glycerol metabolism 100 526 aa CUT-1 protein. Very similar cutA genes were found in the genomes of Fusarium oxysporum(FOXG_03768) and Fusarium graminearum (FVEG_12092).The F. fujikuroi counterpart was identified afterwards in a sequence of the genome of this fungus (Wiemann, Sieber et al. 2013), corresponding to the locus FFUJ_14511 in EMBL accession number HF679026.1. Figure 29. Sequence features of CutA. A: Schematic representation of the CLUSTAL alignment between the CutA polypeptides from F. graminearum (Fg, FGSG_07907), those from F. verticillioides (FVEG_12092), F. oxysporum (FOXG_03768) and F. fujikuroi (Ff; identical patterns, only Ff shown), and the CUT-1 polypeptide of N. crassa (Nc). The HAD domain is marked in color. Breaks between the boxes represent gaps introduced by the CLUSTAL program to facilitate alignment. Residues conserved in the five proteins are indicated below using bars. B: Detailed representation of the alignment of the amino segment preceding the HAD domain in F. graminearum (Fg), F. oxysporum (Fo), F. verticillioides (Fv), F. fujikuroi (Ff) and N. crassa (Nc). The putative PEST (HTPPESPTETQI) and polyglutamine (QQQQEQPQQ) domains of CUT-1 are indicated; a highly conserved 52 aa segment is boxed. C: CLUSTAL comparison of the highly conserved segment highlighted above and the corresponding segments in the predicted CutA orthologues from Trichoderma reesei (Tr, EGR47034), Botryotinia fuckeliana (Bf, XP_00155677), Podospora anserina (Pa, XP_001909058) and Aspergillus nidulans (An, XP_663168). Capítulo 2: The gene cutA of Fusarium fujikuroi, encoding a protein of the haloacid dehalogenase family, is involved in osmotic stress and glycerol metabolism 101 CutA of F. fujikuroi exhibits high similarity to CUT-1 of N.crassa, with 66% coincident positions, rising to 84% in the HAD domain (Fig. 29A). The amino extension of CUT-1, unusual for the proteins of the HAD family, contains presumed regulatory segments, such as a PEST domain, a polyglutamine tract, and two predicted nuclear localization signals (Youssar, Schmidhauser et al. 2005). Such elements were not found in this region of the Fusarium spp. CutA predicted proteins, which however did contain a 52 aa segment with 37 positions coincident with the same CUT-1 segment (Fig. 29B). This sequence is highly conserved in orthologues from other ascomycete fungi, such as Aspergillus nidulans, Trichoderma reesei, Botryotinia fuckeliana and Podospora anserina (Fig. 29C), suggesting a role in the function of this protein family. 2.2 Targeted ΔcutA mutants are partially sensitive to osmotic stress: The cut-1 mutation results in enhanced sensitivity to hyper-osmotic conditions (Youssar, Schmidhauser et al. 2005). To analyse the possible biological role of cutA in Fusarium spp., we obtained targeted knockout mutants for this gene by transformation with a plasmid in which a HygR cassette replaced the cutA coding sequence (Fig. 30A). After incubation of protoplasts of the wild-type strain FKMC1995 with this plasmid, 26 transformants were obtained and subcultured from uninucleate microconidia to ensure homokaryosis. Culture in medium supplemented with 0.68 M NaCl showed retarded growth of some of the transformants. Three of them (T1, T2 and T3), and one with wild-type growth, T4, were chosen for detailed molecular and phenotypic analysis. Genomic DNA samples from the wild-type and the four transformants were digested with EcoRV or HindIII and hybridized in a Southern blot with a cutA probe (Fig. 30C). The wild-type and the T4 samples showed a hybridizing band of the expected size but no bands could be detected in the T1, T2 and T3 samples. To confirm the replacement of cutA A C Capítulo 2: The gene cutA of Fusarium fujikuroi, encoding a protein of the haloacid dehalogenase family, is involved in osmotic stress and glycerol metabolism 102 with the HygR cassette, PCR assays were carried out combining a primer from the cutA DNA region not affected by the gene replacement and a primer from an internal sequence of the HygR cassette (Fig. 30B). Amplification products of the expected size were obtained only from T1, T2 and T3,indicating the occurrence of the expected cutA deletion in these transformants. No amplification was detected with T4 DNA, suggesting that this transformant resulted from integration of the vector through ectopic recombination. The wild-type and the four transformants exhibited similar pigmentation in the light or in the dark on DGagar, indicating that the ΔcutA mutation does not affect the regulation of carotenogenesis. The strains then were grown on DG agar with two different nitrogen sources and checked for the effect of hyper-osmotic conditions (Fig. 31A). The colonies were slightly larger with nitrate than with asparagine in control plates, and the diameter of the ΔcutA mutants (T1, T2 and T3) was similar to that ofthe cutA+ strains (wild-type and T4) on each medium, indicating that the ΔcutA mutation does not alter the growth of the fungus under normal culture conditions. Supplementation of the medium with 0.68 M NaCl resulted in visible diameter reductions of the ΔcutA mutant colonies compared to those of the control strains. This growth reduction was compensated totally (in DGmedium) or partially (in DGasn medium) by the presence of 36 mM glycerol, suggesting that the mutants are affected in their mechanism of glycerol production. The sensitivity of the ΔcutA mutants to NaCl is due to osmotic stress and not to ionic stress, as indicated by similar growth inhibition resulting from supplementation with 1.2 M sorbitol. Experiments in race tubes with DGasn medium with 0.68 M NaCl showed Capítulo 2: The gene cutA of Fusarium fujikuroi, encoding a protein of the haloacid dehalogenase family, is involved in osmotic stress and glycerol metabolism 103 Experiments in race tubes with DGasn medium with 0.68 M NaCl showed Figure 30. Generation of ΔcutA mutants of F. fujikuroi. A: Schematic representation of the gene replacement event leading to the ΔcutA mutation. The black arrow represents the cutA gene. Segments shaded in grey in the background, support the homologous recombinations leading to the replacement. The green box is the HygR cassette, containing the hph gene. The PstI-delimited segment (P, orange box), absent in the mutants, was used as a probe in the Southern blot experiment shown below (C). Small arrowheads indicate the position and orientation of primers used in the PCR experiment displayed below (B). B: Agarose gel electrophoresis of amplification products obtained by PCR from DNA samples of the same strains with the primer sets indicated (see upper scheme for primer locations). Amplification of the DNA segment with primers reveals the presence of a hygR-interrupted cutA gene. C: Southern blot of genomic DNA from the wild type and four transformants digested with EcoRV or HindIII and hybridized with an internal cutA DNA segment (orange box). Probe locations and relevant EcoRV (E) or PstI (P) sites are indicated on the upper scheme. The represented DNA segments lack HindIII sites. Capítulo 2: The gene cutA of Fusarium fujikuroi, encoding a protein of the haloacid dehalogenase family, is involved in osmotic stress and glycerol metabolism 104 only a 20% reduction in the linear growth of the ΔcutA mutants compared to the cutA+ strains (Fig. 31C). Similar results were obtained in submerged cultures (Fig. 31B). Figure. 31. Effect of ΔcutA mutations on growth and conidiation. A: Colonies of the ΔcutA mutants (transformants T1, T2 and T3) and the cutA+ strains (wild-type, WT, and transformant, T4) grown for 7 days at 30 ºC in the dark on DG minimal medium with either NaNO3 (DGNO3–) or asparagine (DGasn) as a nitrogen source. Labels in the top left image indicate positions of the strains in all plates. The medium was supplemented with 0.68 M NaCl, 0.68 M NaCl+36 mM glycerol, 36 mM glycerol or 1.2 M sorbitol, as indicated. White numbers below cutA+ strains indicate the ratio between mean diameter of the WT and T4colonies in the supplemented plate and mean diameter in the control plate. Numbers below each picture (Growth ratio) indicate ratio between mean diameter of ΔcutA transformants (T1, T2 and T3) and mean diameter of cutA+ strains (WT and T4) in the same culture conditions. B: Mycelial mass (left) and conidia density (right) in submerged cultures (open bars, cutA+; filled bars, ΔcutA) incubated for 1 week in DGasn medium, alone (Control) or supplemented with either 0.68 M NaCl or 1.2 M sorbitol (Sorb.). Values are means ±SD from two independent biological replicates. C: Experiments in race tubes with DGasn medium with 0.68 M NaCl showed only a 20% reduction in the linear growth of the ΔcutA mutants compared to the cutA+ strains Capítulo 2: The gene cutA of Fusarium fujikuroi, encoding a protein of the haloacid dehalogenase family, is involved in osmotic stress and glycerol metabolism 105 Mycelia grown in DGasn medium, supplemented with either 0.68 M NaCl or 1.2 M sorbitol, were filtered and lyophilized. The mycelial dry mass of ΔcutA was slightly lower under hyper-osmotic conditions compared to the cutA+ strains; all strains were more sensitive to NaCl than to sorbitol. The wild-type strain FKMC1995 produces conidia in submerged conditions. Conidiation was enhanced at least three fold in the cutA+ strains under high NaCl concentration compared to DGasn medium alone (control), but not under high sorbitol concentration, suggesting that conidiation is stimulated by ionic stress but not by osmotic stress. Conidiation was however enhanced upon high concentrationof either NaCl or sorbitol in the ΔcutA strains compared to control values. 2.3 cutA expression is regulated by osmotic stress and is needed for glycerol accumulation: The phenotype of the ΔcutA mutants indicates the participation of cutA in the hyper-osmotic resistance mechanism of F. fujikuroi. We investigated the effect of osmotic changes on the regulation of cutA using real-time RTPCR. Addition of 0.68 M NaCl resulted in a rapid increase in cutA mRNA levels, by over ten fold 30 min after the onset of hyper-osmotic shock (Fig. 32A), followed by a subsequent return to initial levels 2 h later. To investigate the effect of hypo-osmotic stress, the fungus was grown in medium supplemented with 0.68 M NaCl and transferred to standard non-supplemented medium. Following the transfer,a rapid five fold decrease of cutA mRNA levels was produced in the first hour; these low levels were maintained for at least 4 h. These results show that cutA mRNA levels are regulated in response to external osmotic conditions. Capítulo 2: The gene cutA of Fusarium fujikuroi, encoding a protein of the haloacid dehalogenase family, is involved in osmotic stress and glycerol metabolism 112 with numerous examples in all taxonomic groups, from E. coli (Kuznetsova, Proudfoot et al. 2006) to humans (Seifried, Schultz et al. 2013). In addition to their highly conserved HAD-like catalytic domain, CutA and CUT-1 exhibit an amino extension usually absent in other proteins from the HAD family (Youssar, Schmidhauser et al. 2005). A similar extension is found in Cut-like proteins encoded in the genomes of other ascomycetes, but not in representative zygomycete and basidiomycete species, such as Mucor circinelloides and Ustilago maydis (closest CutA relatives 87690 and UM04682 in the respective annotated genomes).The widespread occurrence of CUT-1/CutA orthologues in ascomycetes indicates a conserved functional role for this HAD subfamily in this fungal group. Figure 36. Effect of light on hogA mRNA levels in F. fujikuroi. Results of RT-PCR of total RNA samples from the wild-type ( ) and ΔwcoA ( ) and ΔcryD ( ) mutants grown for 3 days in the dark followed by exposure to 15 min, 30 min, 1 h, 2 h or 4 h to 7 W white light m-2. Data show means ±SD from three independent biological replicates for the wild-type, and from two independent biological replicates of two independent mutants (ΔwcoA: SF225 and SF226; ΔcryD: SF236 and SF237). Values are expressed relative to the value obtained with mycelia from the same strain collected before illumination (time zero). Capítulo 2: The gene cutA of Fusarium fujikuroi, encoding a protein of the haloacid dehalogenase family, is involved in osmotic stress and glycerol metabolism 113 The regulation of the genes cut-1 and cutA of N. crassa and F. fujikuroi by heat shock and hyper-osmotic shock and the phenotype of the respective mutants (Youssar, Schmidhauser et al. 2005) indicate a similar role of both genes in the osmotic stress response. This is further supported by the rapid reduction of cutA mRNA content after hypo-osmotic shock, and its transient induction by illumination. However, photoinduction of cutA mRNA differs strikingly from the downregulation of cut-1 mRNA levels by light in N. crassa (Youssar and Avalos 2006). Moreover, cut-1 mRNA content increases notably in the dark in different regulatory mutants involved in the light response, including those of the WC complex, an effect independent of the downregulation by light. By contrast, mutants of the orthologous wc-1 gene of F. fujikuroi, wcoA (Estrada and Avalos 2008a), contain lower cutA mRNA levels in the dark than the wild-type, and have apparently lost their photoinduction. Thus, our results show different light-dependent regulations for cut-1 in N. crassa and cutA in F. fujikuroi, probably reflecting different survival requirements in their respective natural habitats. The HOG response, mediated by a signalling transduction pathway through a MAPK cascade, triggers the accumulation of glycerol inside the cell in response to external hyper-osmotic conditions. This regulatory mechanism has been investigated in detail in S. cerevisiae (Nevoigt and Stahl 1997), and seems to be ubiquitous in fungi (Krantz, Becit et al. 2006), including Fusarium species, as indicated by the occurrence of the expected HOG pathway genes in their genomes. Accordingly, exposure to 0.68 M NaCl results in a rapid increase of glycerol content in wild-type F. fujikuroi mycelia. However, the increase was modest compared to the proportionally higher glycerol accumulation in response to hyper-osmotic stress found in S. cerevisiae (Reed, Chudek et al. 1987), suggesting that glycerol is not the major osmolyte in Fusarium spp. The ΔcutA mutants do not exhibit this response, and their glycerol content is very low even without osmotic stress, pointing to an enzymic role of CutA in the synthesis of glycerol. A similar reduction in glycerol content was previously found in the cut mutant of N. crassa (Lew and Levina 2007). The incapacity of these mutants to accumulate glycerol, in Capítulo 2: The gene cutA of Fusarium fujikuroi, encoding a protein of the haloacid dehalogenase family, is involved in osmotic stress and glycerol metabolism 114 either N. crassa or F. fujikuroi, had no apparent consequences on their ability to grow under standard laboratory conditions, probably because ion uptake mechanisms are sufficient to maintain appropriate turgor values (Lew and Levina 2007). The ability of the ΔcutA mutants to grow in hyper-osmotic conditions suggests that F. fujikuroi possess other mechanisms to counteract hyperosmotic stress, such as ion uptake (Lew 2011) or production of alternative osmolytes. In A.nidulans and Aspergillus oryzae, growth under high osmotic pressure results in the accumulation of arabitol and erythritol in addition to glycerol (de Vries, Flitter et al. 2003; Ruijter, Visser et al. 2004). The Fusarium spp. genomes contain orthologous genes for the enzymes responsible for the last enzymic steps in the synthesis of arabitol and erythritol (Fig. 37B): FFUJ_05626, FVEG_0255, FGSG_04036 and FOXG_03675 (protein codes in the F. fujikuroi, F. verticillioides, F. graminearum and F. oxysporum genomes, respectively) exhibit approximately 40% identity with erythrose reductase of Yarrowia lipolytica (accession number AGG15954), the enzyme producing erythritol; on the other hand, FFUJ_00595, FVEG_00541, FGSG_00655 and FOXG_00973 exhibit approximately 52% identity with L-arabitol dehydrogenase from Aspergillus niger (XP001397484). The occurrence of these genes suggests that Fusarium spp. Are able to produce these polyalcohols in addition to glycerol. Likewise, the synthesis of other classes of osmoprotectants, such as trehalose, cannot be discounted. The Fusarium spp. genomes also contain orthologues for tpsA and orlA of A.nidulans, encoding the trehalose-6-phosphate synthase and phosphatase needed for trehalose production from glucose-6P (F. fujikuroi orthologues: FFUJ_13242 and FFUJ_13709, with approximately 77% and 43% identical positions with TpsA and OrlA, respectively). However, trehalose is not accumulated in response to osmotic stress in A. oryzae (Ruijter, Visser et al. 2004), and in A. nidulans it is rather accumulated in sexual and asexual spores (Ni and Yu 2007), pointing to arabitol and erythritol as more likely osmotic stress-induced osmolytes in F. fujikuroi. Capítulo 2: The gene cutA of Fusarium fujikuroi, encoding a protein of the haloacid dehalogenase family, is involved in osmotic stress and glycerol metabolism 115 The biochemical basis of glycerol production has been investigated in detail in S. cerevisiae, where this polyalcohol is synthesized from dihydroxyacetone phosphate through its conversion to glycerol-3-phosphate by glycerol-3-phosphate dehydrogenase, and the subsequent dephosphorylation of this product by glycerol-3-phosphatase to yield glycerol (Fig.37B). S. cerevisiae contains two glycerol-3-phosphatase (GPP)isoforms, with 92.4% identical positions and encoded bygenes gpp1 and gpp2 (Pahlman, Granath et al. 2001). The Fusarium spp. genomes contain a single gene putatively orthologous to gpp1 and gpp2, encoding a protein with only 32% and33% coincident positions, respectively. Glycerol also may be produced by a parallel pathway, in which dihydroxyacetonephosphate is first dephosphorylated to dihydroxyacetone by a putative GPP, and then converted to glycerol by glyceroldehydrogenase. Figure 37. Late steps of osmolyte production in fungi. Glycerol, erythritol and arabitol biosynthetic steps adapted from (Diano, Bekker-Jensen et al. 2006). Trehalose biosynthetic steps adapted from Fillinger et al. (Fillinger, Chaveroche et al. 2001a). Numbers indicate enzymaticc activities mentioned in the text: 1, L-arabitol dehydrogenase; 2, erythrose reductase; 3, trehalose-6-phosphate synthase; 4, trehalose-6-phosphate phosphatase.The enzymatic reactions catalysed by CutA (tentative assignment) and GldB are indicated. Capítulo 2: The gene cutA of Fusarium fujikuroi, encoding a protein of the haloacid dehalogenase family, is involved in osmotic stress and glycerol metabolism 116 This alternative pathway seems to function in A. nidulans, as indicated by the persistence of glycerol accumulation in mutants of the glycerol-3- phosphatedehydrogenase gene gfdA (Fillinger, Ruijter et al. 2001b). Moreover,the null gfdA mutants exhibit growth defects under standard growth conditions, but not under hyper-osmotic stress, and a role of this enzymic activity in cell wall integrity has been proposed. In F. fujikuroi, cutA is linked and co-regulated with gldB,encoding the putative NADP+-dependent glycerol dehydrogenase, i.e, the enzyme converting dihydroxyacetone into glycerol. The gene gldB was previously investigated in A. nidulans, where the null mutants were totally devoid of glycerol dehydrogenase activity and exhibited impaired growth under hyperosmotic conditions (de Vries, Flitter et al. 2003). Moreover, glycerol dehydrogenase activity was markedly enhanced under hyper-osmotic stress in the wild-type, but not that of glycerol-3-phosphate dehydrogenase, explaining the incapacity of the gldB mutant to grow under hyper-osmotic conditions. Taken together, these results are consistent with the synthesis of glycerol through dihydroxyacetone instead of glycerol-3P, suggesting that A. nidulans and S. cerevisiae have diverged in their biochemical mechanisms to produce this osmolyte. The similar regulation by osmotic stress of gldB in F.fujikuroi suggests the same biochemical scenario in this fungus. If glycerol biosynthesis proceeds via dihydroxyacetone in filamentous fungi, the enzyme producing this precursor from dihydroxyacetone-3P remains to be identified. Recently, a HAD enzyme of Corynebacterium glutamicum(cgR_2128 or HdpA) was found to catalyse the dephosphorylation of dihydroxyacetone phosphate to produce dihydroxyacetone (Jojima, Igari et al. 2012). HdpA is much shorter than CutA (274 and 494 amino acids, respectively), and as typically found in HAD enzymes, exhibits low similarity (60 coincident positions, Fig. S3). We propose that CutA catalyses the same enzymic reaction to produce dihydroxyacetone in Fusarium spp. (Fig. 7). Supporting this hypothesis: (i) many enzymes from the HAD family exhibit Capítulo 2: The gene cutA of Fusarium fujikuroi, encoding a protein of the haloacid dehalogenase family, is involved in osmotic stress and glycerol metabolism 117 phosphatase activities on a large variety of substrates, including among them dihydroxyacetone phosphate, and (ii) cutA is divergently transcribed and coregulated with gldB, encoding the predicted dihydroxyacetone-metabolizing enzyme. Fitting this hypothesis, and as found for the ΔcutA mutant of F. fujikuroi, intracellular glycerol levels were strongly decreased in the gldB mutants of A. nidulans compared to the wild-type (de Vries, Flitter et al. 2003). In summary, our results are consistent with cutA/gldB in F.fujikuroi as a coordinated transcriptional set in charge of dihydroxyacetone-mediated glycerol biosynthesis, responding to different environmental cues associated to osmotic stress and desiccation, such as sun exposure and elevated temperatures. The HOG regulatory cascade is likely to play a central role in this regulation, including the induction by light. Moreover, the faster WcoA- dependent photoinduction exhibited by the hogA gene compared to cutA and gldB is consistent with a sequential regulatory mechanism. Thus, hogA could be activated by light by WcoA, and hogA could in turn activate cutA/gldB. Confirmation of this hypothesis will require a deeper investigation of this regulatory system. The same genetic organization of cutA and gldB orthologues in other ascomycetes suggests a common origin from an early ancestor of this biochemical and regulatory pattern for glycerol biosynthesis. CHAPTER 3 BIOLOGICAL ROLE OF FUNGAL RHODOPSINS: THE LIGHT DRIVEN, PROTON-PUMPING RHODOPSIN CarO OF Fusarium fujikuroi RETARDS GERMINATION OF LIGHTEXPOSED CONIDIA Jorge García-Martínez, Michael Brunk, Javier Avalos, Ulrich Terpitz Submitted to Nature Scientific Reports (2014) Biological role of fungal rhodopsins: The light driven, proton-pumping rhodopsin CarO of Fusarium fujikuroi retards germination of light exposed conidia 121 1. Introduction Light is an important environmental factor, strongly influencing the behavior of microorganisms in their natural habitats. A diversity of light sensors, able to detect blue, green, or red wavelengths in the visible spectrum (Corrochano 2007) (Bayram, Braus et al. 2010) provides filamentous fungi the ability to sense light and adapt accordingly. Among them stand out blue-light photoreceptors, such as the proteins of the White collar, Vivid and cryptochrome families (Avalos and Estrada 2010) (Idnurm, Verma et al. 2010), which absorb light by flavin chromophores. In contrast, phytochromes are red light-sensing proteins that use the chromophore billiverdin (Avalos and Estrada 2010). Analysis of mutants has revealed the participation of these photoreceptors in the modulation of a variety of physiological and morphological responses (Corrochano 2007) (Herrera-Estrella and Horwitz 2007) (Corrochano and Avalos 2010). A further class of fungal photoreceptors comprises the rhodopsins, members of the homonymous group of green light sensing microbial proteins firstly discovered in archaea (Spudich and Jung 2005) (Klare, Chizhov et al. 2008) (Grote, Engelhard et al. 2014) (Ernst, Lodowski et al. 2014). Microbial rhodopsins consist of seven transmembrane helices (TM) forming an interior pocket for the chromophore all-trans-retinal, which is covalently bound via a protonated Schiff’s base to a lysine residue, located in the TM7. Some residues of the binding pocket are well conserved across kingdoms, whereas conservation outside the pocket is more sparse (Sharma, Spudich et al. 2006). In light-driven rhodopsin pumps a single ion is translocated per photocycle, which is triggered by light-induced all-trans to 13-cis retinal isomerization and characterized by a sequence of photointermediates responsible for ion-uptake, -transfer, -release, and chromophore-recycling. Genes for putative rhodopsins are ubiquitous in the genomes of fungal species (Brown 2004) and they are frequently up-regulated by light (Estrada and Avalos 2009) (Estrada, Brefort et al. 2009) (Kihara, Tanaka et al. 2009). Some fungal rhodopsins have been purified and photochemically analysed, but Biological role of fungal rhodopsins: The light driven, proton-pumping rhodopsin CarO of Fusarium fujikuroi retards germination of light exposed conidia 128 decreased to a positive stationary value. Upon switching off the light the pump current decayed in a biexponential manner exhibiting a fast (off-1 1.4±0.26 ms SD, n=10) and a slower time constant (off-2 20±3.8 ms SD, n=10). While in our system determination of the on constant was limited by the opening time of the fast shutter, the closing constants off-1 and off-2 were voltage-dependent (Fig. 42C). Figure 42. Electrophysiological analysis of CarO::YFP expressed in HEK293 cells. A: Typical current traces of the ion pump CarO recorded at pH 7.4 at membrane potentials of +40 mV, 0 mV, and -100 mV, respectively, as indicated. Cells were illuminated by a 561 nm diode pumped laser. The green bar represents illumination time. B: I-V-relationship of the light-dependent normalized pump current of CarO::YFP measured at pH 7.4 corresponding to data shown in A. Note, that activity of CarO is voltagedependent. C: Time constants on, off-1 and off-2. on determination is limited by the opening time of the fast shutter (300 µs), while closing constants are voltage-dependent. Photocurrent shows a fast rise, followed by a slower biphasic decay. Biological role of fungal rhodopsins: The light driven, proton-pumping rhodopsin CarO of Fusarium fujikuroi retards germination of light exposed conidia 129 The photocurrent showed a clear voltage-dependency (Fig. 42B), with larger pump activity at higher membrane potentials. The outward direction of charge transfer remained even when the electrochemical gradient was contrary to the charge-transfer direction (Fig. 42B). Accordingly, the values for the reversal potentials estimated by nonlinear fit of the I-V-plot (Fig. 43) were much more negative than those calculated by Nernst equation at intracellular pH 7.4 and extracellular pH 5 (-150 mV vs. 139 mV), pH 7.4 (-222 mV vs. 0 mV), and pH 9 (-243 mV vs. -9,3 mV). 2.2 Effect of light wavelength and intensity on CarO pump activity The pump activity of fungal rhodopsins depends on the lightwavelength, and according to former studies maximal efficiency is expected after activation with green light. We recorded the pump current at different wavelengths and plotted the data (action spectrum, Fig. 44A). The light emitted by a 150W-xenon lamp was filtered through narrow-band pass filters. At every wavelength the same dose of photons per time and area (2 x 1015 Figure 43. pH-depen- dent shift of the reversal potentials. I-V blot of normalized CarO pump current at different extracellular pHs. The reversal potential was obtained by fitting the data with an exponential growth function, which was extrapolated to give the point of intersection with the abscissae axis. Biological role of fungal rhodopsins: The light driven, proton-pumping rhodopsin CarO of Fusarium fujikuroi retards germination of light exposed conidia 130 photons s-1 mm-2) was used for pump activation. Values were corrected for eventually occurring loss of intensity (reference wavelength 510 nm). The action spectrum was essentially the same at different intracellular proton concentrations (pH 5, 7.4, and 9, Fig. 44A); therefore, for better signal to noise-ratio, pH of internal pipette solution was set to 5. As expected, the pumping activity of CarO::YFP was highly wavelength dependent, with highest activity at around 520 nm (green light). Figure 44. Light dependency of CarO::YFP. A: Action spectrum. Effect of light-wavelength on CarO::YFP activity (mean value and standard deviation of 7 cells). The number of photons used for excitation was set to 2 x 1015 photons s-1 mm-2. In order to enhance signal-to- noise ratio, internal pH 5 was used. B: Dependency of pump current amplitude on photon density. Mean normalized photocurrent and corresponding standard deviation (5 cells, 3 recordings each). Data were fitted by a Hill function (red curve). Biological role of fungal rhodopsins: The light driven, proton-pumping rhodopsin CarO of Fusarium fujikuroi retards germination of light exposed conidia 131 Beside the light wavelength the pump activity also depended on the light intensity. We measured the pump current at 0 mV voltage potential (1 ms illumination time) while varying the photon-density emitted by a 561 nm- DPSS-laser (Fig. 44B). The pump exhibited typical saturation behavior, which could be fitted by means of a standard Hill equation. Half maximal activity was reached at a photon density of 1.6 x 1017 photons s-1 mm-2. For patchclamp experiments photon density was adjusted to 2.3 x 1017 photons s-1 mm-2 providing >60% pump-activity. 2.3 Ion specificity of CarO pump activity Sequence features of CarO suggested a pump function for protons (Fig. 38A, (Prado, Prado-Cabrero et al. 2004; Avalos and Estrada 2010)). If CarO was an outward directed proton pump, we would expect an increase or decrease in pump activity when the amount of intracellular protons is heightened or diminished, respectively. Therefore, we varied the pH of bath (Fig. 45A) and pipette (Fig. 45B) solution, and investigated the influence of the proton gradient on the pump intensity. When the pH of the bath-solution was reduced from 9 to 5 the mean photocurrent of CarO decreased. This circumstance was more heavily pronounced when the extracellular pH was set to pH 7.4 and the intracellular pH was increased from 5 to 9. As a consequence a remarkable loss in pump activity was observed (Fig. 45B). Some microbial rhodopsins were reported to pump chloride (Bamberg, Tittor et al. 1993) or even sodium (Inoue, Ono et al. 2013) ions instead of protons. To test the possibility of such activities of CarO, we changed the ion composition of either bath or pipette solution and measured the corresponding pump current. When sodium chloride was replaced extracellularly by sodium sulfate or intracellularly by potassium chloride, we could not detect any ion species induced difference in the pump activity (Fig. 45C). Biological role of fungal rhodopsins: The light driven, proton-pumping rhodopsin CarO of Fusarium fujikuroi retards germination of light exposed conidia 132 Thus, neither extracellular chloride nor intracellular sodium is required for pump activity, providing further support to CarO as a proton pump. 2.4 Effect of gluconate on CarO pump activity Gluconate is one of the standard compounds used in electrophysiological experiments when chloride ions have to be substituted (Walz 2007). Unexpectedly, CarO pump activity was strongly influenced by gluconate (Fig. Figure 45. Pump-activity of CarO::YFP at various pHs and other ion concentrations. Cells were illuminated by a 561 nm-laser. A: Influence of external pH on pump-activity. pH of bath solution varied from pH 5 (black squares, 10 cells), to pH 7.4 (red circles, 25 cells) and pH 9 (blue triangles, 13 cells); intracellular pH was kept at 7.4. B: Influence of internal pH on pump activity. Extracellular pH was maintained at pH 7.4. Mean area specific photocurrent at intracellular pH 9 (3 cells), 7.4 (8 cells), and 5 (10 cells), respectively. C: Analysis of pump activity in absence of extracellular chloride or intracellular sodium. Pump current to membrane voltage relationship of CarO is given. Intra- and extracellular standard solutions (sodium chloride, red circles, 14 cells) were replaced either intracellularly by potassium (black squares, 5 cells) or extracellularly by sulfate (blue triangles, 7 cells), without any remarkable effect on pump activity. Biological role of fungal rhodopsins: The light driven, proton-pumping rhodopsin CarO of Fusarium fujikuroi retards germination of light exposed conidia 133 46A). Compared to the pump activity in sodium chloride at neutral pH (7.4), in sodium gluconate at extracellular pH 5 the pump activity was about 3 fold enhanced. When the pH was suddenly increased from 5 to 9 a further transient increase (up to ten fold compared to neutral sodium chloride) in pump activity could be observed, which relaxed within 10-15 min. After relaxation the I-V-curve for gluconate pH 9 was similar as for NaCl pH 7.4. In contrast, this transient pH-dependent increase in pump activity was not observed when sodium chloride in the pipette solution was exchanged for sodium gluconate, while at pH 5 and pH 7.4 the pump activity was about the same as with sodium chloride (Fig. 46C). In addition to gluconate we also tested the activity of CarO in the presence of other weak organic acids. With glutamate (Fig. 46B) the pump activity was noticeably increased at pH 5 compared to pH 7.4. However, no further increase was observed at pH 9; actually, the activity at pH 9 was even reduced compared to neutral pH. In contrast, the behavior of CarO in galacturonic acid (Fig. 46D) did not significantly differ from its behavior in sodium chloride. Biological role of fungal rhodopsins: The light driven, proton-pumping rhodopsin CarO of Fusarium fujikuroi retards germination of light exposed conidia 134 Figure 46. Influence of weak organic acids on the pump-activity of CarO. I-V blot of the normalized light-driven pump current (linear fit of mean values and standard deviation of measurements recorded from at least 5 different cells) at pH 5 (black squares), pH 7.4 (red circles) and pH 9 (blue triangles), respectively in extracellular presence of the weak organic acids gluconate (A), glutamate (B), and galacturonate (D). Note that in the presence of gluconate and glutamate at pH 5 the pump activity is enhanced, though the contra-directed gradient is increased. In case of gluconate at pH 9 a transient enhanced pump current (half triangles, upper part of the graph) is observed, which disappears in a time scale of over ten minutes (closed triangles). C: Similar conditions as in A, B and D were chosen, but sodium in pipette solution was substituted by sodium gluconate to test the effect of intracellular gluconate on the pump activity. For all conditions the pump showed similar behavior as before, but the transiently enhanced pump current, which was observed with intracellular sodium chloride (A), was absent. Biological role of fungal rhodopsins: The light driven, proton-pumping rhodopsin CarO of Fusarium fujikuroi retards germination of light exposed conidia 135 2.5 CarO localisation in F. fujikuroi As a rhodopsin, CarO is supposed to be membrane-located. To gain more insights on its possible biological function, we aimed to find out in which membranes of the F. fujikuroi conidia and hyphae CarO is mainly localized. For that reason we generated a F. fujikuroi strain expressing the fluorescent carO::yfp construct under control of its native light-regulated promoter. Successful transformation of the plasmid by protoplast electroporation and integration of the fusion gene into the F. fujikuroi genome was proofed by Southern blot analysis: a single band specific for carO was observed in wild type genomic DNA, while an additional band was present in the genomic DNA of the carO:yfp transformant (Fig. 47A). A hybridizing band was also found in the carO::yfp transformant in a Southern blot with a yfp-probe, absent in the wild type sample. Taken together, these results indicate that the carO::yfp construct was incorporated into the F. fujikuroi genome, but it did not replace the native carO gene. Fluorescence analysis of F. fujikuroi carO::yfp conidia and hyphae by confocal laser scanning microscopy confirmed the expression of CarO::YFP. The carO::yfp gene expression was controlled by the carO promoter, which is upregulated by light (Prado, Prado-Cabrero et al. 2004). Accordingly, conidia produced by light-grown mycelia (abbreviated hereafter as LG conidia) exhibited strong YFD-mediated fluorescence (Fig. 47B.i), whereas the fluorescence of conidia from dark-grown mycelia (DG conidia) was notably weaker (Fig. 47B.ii). In contrast, only very weak background fluorescence was observed in wild type LG or DG conidia (light- and dark-exposed mycelia; Fig. 47B.iii). CarO::YFP was mainly located in the plasma membrane but it was also present in membranes of inner organelles, possibly corresponding to vacuoles, endoplasmic reticulum, and nuclear envelope (Figs. 47 and 48). Similar distribution was observed when carO::yfp cDNA was expressed in other fungal organism, as S. cerevisiae (Fig. 47D). Biological role of fungal rhodopsins: The light driven, proton-pumping rhodopsin CarO of Fusarium fujikuroi retards germination of light exposed conidia 136 Figure 47. Localisation of CarO::YFP in F. fujikuroi. A: Southern blot analysis of gDNA of F. fujikuroi FKMC1995 transformed with pHJA2-carO::YFP and selected for hygromycin resistance. DNA samples were digested with NcoI or BclI. The carO::yfp gene was introduced into the fungal genome and the native carO gene was not replaced. B: Localization of CarO::YFP in F. fujikuroi conidia. The CarO::YFP fusion protein is mainly located in the plasma membrane but also in the membranes of inner organelles (vacuole, nucleus, ER). i-iii. Photomicrographs of conidia obtained by transmitted light microscopy (upper raw) and corresponding confocal laser scanning microscopy (cLSM; lower raw). The expression of carO::yfp in the conidia is induced by light. i) Light exposed (left: bicellular, right, monocellular), ii) dark exposed, iii) control (light exposed wild type). White bars represent 5 µm (i) and 2 µm (ii, iii), respectively. C: Localization of CarO::YFP in light-exposed F. fujikuroi hyphae 20 h after germination. The CarO::YFP fusion protein is heterogeneously distributed in the mycelia, reflecting a dynamic process. White bar represents 5 µm. Biological role of fungal rhodopsins: The light driven, proton-pumping rhodopsin CarO of Fusarium fujikuroi retards germination of light exposed conidia 137 The expression of CarO::YFP was also analyzed in young F. fujikuroi hyphae (<24h after germination) developed in light or dark, and germinated either from LG or DG Overall, fluorescence intensity was lower in young mycelia (<24h after germination) than in the conidia (Fig. 49). In general, YFP-mediated fluorescence was more marked in light-grown than in darkgrown hyphae. In dark-grown hyphae the fluorescence was mainly observed in germlings resulting from LG conidia. On the other hand, light-exposed hyphae derived from the germination of DG conidia did also exhibit strong fluorescence. Figure 48. Localization of CarO::YFP in hyphae of F. fujikuroi. Wide-field (B,C) and confocal fluorescence (A,D-G) images with corresponding bright-field images of F .fujikuroi expressing CarO::YFP. A: Overlay of fluorescence and bright-field image showing the heterologous distribution of the fungal rhodopsin in young hyphae. B-C: Widefield fluorescence images of regions with high concentrations of CarO::YFP. D-F: Confocal laser scanning micrographs of regions with concentrated CarO::YFP expression. G: cLSM picture of macro- and microconidium showing localisation of CarO in almost every membrane in the spore. White bars represent 25 µm (A), 10 µm (B), and 5 µm (C-G), respectively. Biological role of fungal rhodopsins: The light driven, proton-pumping rhodopsin CarO of Fusarium fujikuroi retards germination of light exposed conidia 144 We also analyzed the spore germination rate after 9 hours and found that the percentage of germinated conidia (>10µm) was almost double for CarO- strain (64.9%) than for CarO+ strain (34.2%). Taking all data together, the results suggest a faster germination of CarO- conidia under light compared to those of the control CarO+ strain. Another possible role for a proton pump as CarO is contributing to the acidification of fungal environment. It is well known that pH is regulated during plant invasion by many fungi (Prusky and Yakoby 2003). We decided to check whether CarO- strains were impaired in its capability to invade plant fruit tissues. For that purpose, we obtained fresh conidia from CarO-, CarO+, FKMC1995 (wild type), SF134 (CarS mutant, carotenoids overproducer) and SF223 (OpsA-). In sterility conditions, apple slices were cut, inoculated with 2.5x105 conidia from each strain and incubated during 4 days (Fig. 53). No differences were observed between the different strains, suggesting that rhodopsins are not involved in plant tissue invasion in F. fujikuroi. Figure 53. Plant tissue colonization. Apples were cleaned with soap and water, and washed with distilled water. Slices (0.8 cm width) were cut with a sterile scalpel and placed in sterile Petri dishes. For inoculation, 5 µl of a 5x107 conidia ml-1 suspension were drop on the surface of the slice. Slices were incubated for 4 days under 100% humidity at 30ºC in the dark or light. No obvious difference in growth was observed between the different strains. SF223 strain [obtained by Estrada and Avalos (Estrada and Avalos 2009)] was OpsA- and SF134 was a CarS mutant (Estrada and Avalos 2009). Biological role of fungal rhodopsins: The light driven, proton-pumping rhodopsin CarO of Fusarium fujikuroi retards germination of light exposed conidia 145 3. Discussion Fungal rhodopsins are widespread in the fungal kingdom (Brown 2004; Idnurm, Verma et al. 2010), but their biological functions, particularly how their light-dependent functions affect fungal growth and physiology, remain unknown. In this chapter we investigate the possible biological role of the auxiliary ORP-like rhodopsin CarO of the filamentous fungus F. fujikuroi. For this purpose, we used three different experimental approaches (a) the study of pump activity of heterologously expressed CarO through patch-clamp experiments, (b) the identification of in vivo location of YFP-tagged CarO, and (c) the phenotypic comparison of a targeted CarO- mutant and an isogenic CarO+ strain. Taken together, the results provide solid clues on a possible role of CarO as a proton pump associated to germination of conidia. Beside this role, CarO may also participate in the maintenance of the proton gradient, which is used for nutrient uptake. Typically, an H+-ATPase is responsible for the maintenance of this gradient (Hesse, Ruijter et al. 2002). Thus, a lightdriven pump would save energy contributing to this task. A first goal was to determine if CarO is an ion pump rather than a photosensor, as strongly suggested by the conservation of residues needed for proton pumping in other rhodopsins (Fig. 38). While pumps must have rather fast photochemical cycles to maintain transmembrane ion gradients, the photosensors are characterized by their relatively slow turnover-rates, which allow the signaling states for efficient interaction with their transducers. The photocycle of CarO has not been investigated, but recently the photocycle of another auxiliary ORP-like rhodopsin, PhaeoRD2 from Phaeosphaeria nodorum, was analysed in detail (Fan, Solomon et al. 2011). This fungal rhodopsin exhibits a very fast photocycle, hence providing support to a role of auxiliary ORP-like rhodopsins as proton pumps. However, to our knowledge, no electrophysiology studies are available on auxiliary ORP-like rhodopsins. To facilitate the generation of reliable electrophysiological data, we carried out patch-clamp experiments in mammalian cells expressing Biological role of fungal rhodopsins: The light driven, proton-pumping rhodopsin CarO of Fusarium fujikuroi retards germination of light exposed conidia 146 CarO::YFP. This method has advantages on the use of fungal protoplasts. F. fujikuroi protoplasts are only obtained in a narrow time window, about 12 hours after inoculation, and digestion efficiency strongly depends on quality of cell wall lytic enzymes and growth rate, while patch-clamp experiments require clean membrane surfaces, special perfusion chambers, and highly experienced experimenters (Garrill and Davies 1994; Terpitz, Raimunda et al. 2008; Terpitz, Letschert et al. 2012). In contrast, the use of a stable CarO::YFP-expressing mammal cell line allowed us to record abundant and reproducible data. The fusion protein was sufficiently expressed, targeted to plasma membranes, and therefore accessible by the patch-clamp pipette. Considering that the expression pattern was similar in F. fujikuroi hyphae, baker yeast, and HEK293 cells (Fig. 40 and fig. 47), we expect the same biochemical activity for the protein in different eukaryotic host systems, as was shown in former reports on other microbial rhodopsins (Nagel, Mockel et al. 1995; Terpitz, Raimunda et al. 2008; Ernst, Lodowski et al. 2014). Our results provide solid support to the activity of the ORP-like rhodopsin CarO as an outwardly directed, light-driven proton pump. CarO was activated by green light and exhibited maximal activity at around 520 nm (Fig. 44A). The action spectrum showed a bell shape, similar to that found for other microbial rhodopsins in previous electrophysiological investigations (Nagel, Szellas et al. 2003; Kleinlogel, Terpitz et al. 2011). The reversal potentials, estimated by nonlinear fit of the respective I-V-curves, were clearly shifted to negative values when compared with the values calculated for passive transport by Nernst equation, and therefore an active ion transport occurred against the electrochemical gradient. Outward directed light-induced pump current rose with increasing proton gradient, a result consistent with the fact of protons as the transported ion species (Fig. 45A and B). Furthermore, neither chloride nor sodium ions were required for pump activity (Fig.45C), thus narrowing down transportable ion species to protons. In general, CarO pump characteristics were similar to those described for BR. As observed for BR and other microbial proton pumps (Nagel, Mockel Biological role of fungal rhodopsins: The light driven, proton-pumping rhodopsin CarO of Fusarium fujikuroi retards germination of light exposed conidia 147 et al. 1995), pump activity increased with rising membrane potentials. In BR this voltage-dependent behavior is due to the M decay, which is slowed down at negative membrane potentials, and represents the rate-limiting step of the pump cycle (Geibel, Lorinczi et al. 2013). Our data on CarO reflect a similar situation, as the photocurrent of CarO decayed in a biexponential manner upon switching off the light. While the fast closing time constant off-1 of CarO at 0 mV (1.4 ± 0.26 ms, 13 cells) was in the same range with BR [1-2 ms (Geibel, Lorinczi et al. 2013)], the slow closing time constant (off-2) was smaller in CarO (20 ± 3.8 ms) than in BR (27.2 ± 2.2 ms). Furthermore, we observed that the off-2 was voltage-dependent with highest values at negative membrane potentials. In our system the rise time constant (0.3 ± 0.1 ms) was limited by the opening time of the fast shutter (around 300 µs). Thus we could not determine if CarO is activated more rapidly than BR, but the whole CarO photocycle was faster than that of BR, a result that fits flash-photolysis data from the auxiliary ORP-like Rhodopsin PhaeoRD2 (Fan, Solomon et al. 2011). In PhaeoRD2 deprotonation of the Schiff base occurred very fast, resulting in reduced accumulation of the late red-shifted intermediate, and thus in a very fast photocycle. Fast photocycling of CarO is also supported by the observation that, in comparison to other rhodopsins, pump function saturated at very high light intensities (half maximal activity at 1.6 x 1017 photons s-1 mm-2; 561 nm; Fig. 44B). While CarO behaved as a typical proton pump when the bath solution was based on the anions chloride, sulfate, or galacturonate, atypical behavior was observed in the presence of the weak organic acids (WOAs) gluconate or glutamate (Fig. 46). In this case the outward directed current was increased at pH 5 compared to neutral pH. At first glance, this is a very surprising result, as the pump worked against the proton gradient; hence, we would expect better pump function at pH 7.4. This may suggest that CarO possesses additional anion pump functions besides its proton pump ability, i.e. gluconate and glutamate could be transferred into the cell. However, the pump activity was similar independently of the intracellular presence of Biological role of fungal rhodopsins: The light driven, proton-pumping rhodopsin CarO of Fusarium fujikuroi retards germination of light exposed conidia 148 gluconate (Fig. 46C), though the gradient was considerably changed, therefore contradicting this hypothesis. A more reliable explanation could be inferred from the proton transfer mechanism of BR and other microbial rhodopsins. Once the proton has been transferred from the Schiff base to the proton acceptor BR-D85, it is guided to the extracellular space via a cascade of proton acceptors with different pKas to the well conserved proton releasing site (Dencher, Sass et al. 2000; Lanyi 2006). A fast pumping activity is expected to generate a “proton traffic jam”, which occurs when many other protons surround the releasing site and thus slow down proton liberation to the extracellular medium (Kikukawa, Toamogami et al. 2012). The substances producing enhanced CarO pump activity exhibit pKa values around 4 (sodium gluconate pKa 3.86, sodium glutamate second pKa 4.07). Thus, at pH 5 those WOAs may act as buffers, supporting the release of protons from the pumping CarO protein. This would avoid proton traffic jam, and would lead to a faster photocycle and thus to more efficient pump activity, as indicated by smaller time constants (Fig. 54) and higher current amplitudes (Fig. 46). Among the investigated WOAs, gluconate exhibited an additional extraordinary effect in response to pH change. When the protein had been subjected to gluconate solution at pH 5 and the pH gradient was turned to pH 9, the pump activity transiently increased to about 10-fold of the pump intensity in standard bath solution. Seemingly the supporting activity of gluconate transiently remained at pH 9, which in turn resulted – now in combination with the driving force of the outward directed proton gradient – in a temporary faster photocycle (Fig. 54). Within a time scale of about 10 minutes the supporting effect of gluconate disappeared, as the pump intensity was in a similar range as observed in standard bath solution. Biological role of fungal rhodopsins: The light driven, proton-pumping rhodopsin CarO of Fusarium fujikuroi retards germination of light exposed conidia 149 Strong pH-dependency of the photocycle was also observed in the related auxiliary ORP-like rhodopsin PhaeoRD2, where the photocycle was considerably faster at pH 5 than at neutral or basic pH (Fan, Solomon et al. 2011). Auxiliary ORP-like rhodopsins are presumed to interact with transducer proteins (Fan, Solomon et al. 2011). So, we may also speculate that gluconate could affect CarO conformation, or its interaction capacity. Interestingly gluconate does not only influence the pump activity of CarO: its presence results in a remarkable stimulation of mycelial growth of F. fujikuroi on solid medium (Fig.51B). This behavior is independent of the presence of a functional CarO protein and it is also noticeable in the dark, and therefore has no connection with the stimulatory effect of gluconate on CarO pump activity. The ecological relevance of gluconate in the F. fujikuroi natural habitat is not Figure 54. Pump characteristics of CarO in presence of gluconate. A: Typical whole cell measurement of CarO::YFP at 0 mV in whole cell configuration. Note that the pump current in presence of extracellular sodium gluconate pH5 is enhanced 4-5 fold compared to standard conditions, though the proton gradient is acting against the pump activity. B: Mean and standard deviation of the time constants describing the bi-exponential current decay after switching off the light for NaCl pH 7.4 (n=11), gluconate pH 5 (n=6), gluconate pH 7.4 (n=7), gluconate pH 9 directly after pH jump (*, n=5) and 10 min after (n=4) the pH-jump. Small time constants result in increased pump activity. At pH 9 we observed a transient increase in pump activity, which is accompanied by small time constants at pH 9*. After several minutes the pump current relaxes to less intense currents, which is also seen in the increase of the time constants. Biological role of fungal rhodopsins: The light driven, proton-pumping rhodopsin CarO of Fusarium fujikuroi retards germination of light exposed conidia 150 clear. The bakanae disease is seed born and it is known that root associated bacteria produce gluconic acid (Rodríguez, González et al. 2004). Such bacteria were present in ten different rice root samples (Hardoim, Andreote et al. 2011), thus this compound might play a role as a chemical attractant supporting fungal growth. Under standard laboratory conditions no visible phenotype was observed for the CarO- mutant (Prado, Prado-Cabrero et al. 2004). Our fluorescence assays (Fig. 47, fig. 48 and fig. 49) with a CarO::YFP fusion protein expressed under control of the carO promoter showed that the protein is accumulated mainly in conidia produced by light-exposed mycelia and predominantly in the cytoplasmic membrane. In young hyphae the expression was rather irregular, with higher amounts in particular locations, and highly dynamic, with fluorescence appearing transiently in the cytoplasmic membrane in regions behind the hyphal tip and septae, or in the vacuoles. Since the protein is located not only in plasma membrane but also in inner organelles, CarO might be involved in pH dependent signaling and pH homeostasis (Bagar, Altenbach et al. 2009). So, CarO activity could provoke external and internal changes in pH, predictably affecting hyphal development. Proton-gradients were reported to be involved in cell polarity pH-gradient based cell polarity (Hesse, Ruijter et al. 2002; Bagar, Altenbach et al. 2009; Valkonen, Penttila et al. 2014). However, the prevalence of CarO in the conidia suggests that it might play a role in germination, possibly through the generation of a proton gradient. In support to this hypothesis, under certain culture conditions the formation of mycelia from conidial spots on the agar surface was delayed by illumination in minimal medium with ammonium chloride; this effect was mitigated in the absence of a functional CarO protein (Fig. 51A and B). It is known that ambient pH stress reduces spore germination rates and germ tube lengths in the phytopathogen Penicillium expansum (Li, Lai et al. 2010). It seems plausible that CarO may play a role in fungal pathogenesis and/or plant interaction. Biological role of fungal rhodopsins: The light driven, proton-pumping rhodopsin CarO of Fusarium fujikuroi retards germination of light exposed conidia 151 Table 5. Fungi with ORP-like rhodopsins and their role in phytopathology. SPECIES DATABASE ENTRY PHYTOPHAT./ENDOPHYT Aureobasidium melanogenum keq66642, keq73330,keq61603 (+) Aureobasidium pullans keq89333,keq89333 (+) Aureobasidium subglaciale keq96811 (+) Baudoinia compniacensis xp007674357 Saprophytic Bipolaris maydis emd90083 + Bipolaris oryzae xp007685632 + Bipolaris sorokiniana xp007698250 + Bipolaris victoriae eun31652 + Bipolaris zeicola xp007712526 + Botrytis cinerea xp001547284 + Capronia coronata xp007719813 (+) Cladophialophora yegresii xp007758501 + Colletotrichum fioriniae xp007591714 + Colletotrichum gloeosporioides xp007287120 + Coniosporium apollinis eon66253 rock inhabiting Cyphellophora europaea ETN45976 + Dothistroma septosporum eme38958, eme42627 + Eutypa lata xp007798926 + Exophiala aquamarina KEF61142 fish path Exophiala dermatitidis EHY58312, ehy54696 human path Fusarium fujikuroi CAD97459 + Fusarium graminearum ESU10217 + Fusarium oxysporum FOVG17056 + Fusarium pseudograminearum EKJ77664 + Fusarium verticilloides FVEG10716 + Glarea lozoyensis xp008088280, ehk97458 ? (+) Leptospaeria maculans xp003838475 + Marssonina brunnea xp007288546 + Nectria haematococca xp003042779 + Neofusicoccum parvum xp007586513 + Penicillium oxalicum EPS29498 + Pestalotiopsis fici xp007841943 + Phaeosphaeria nodorum xp001791031 + Pseudocercospora fijiensis xp 007922601, xp007927292 + Pyrenophora teres xp003299029 + Pyrenophora tritici-repentis xp001937696 + Sclerotinia borealis ESZ89976 + Sclerotinia sclerotiorum xp001594532 + Setosphaeria turcica xp008030750 + Spaerulina musiva emf08052 + Verticillium dahliae EGY21409 + Biological role of fungal rhodopsins: The light driven, proton-pumping rhodopsin CarO of Fusarium fujikuroi retards germination of light exposed conidia 152 It is suggestive that green light should be predominant in internal tissues of thin aerial plant structures, filtered by chlorophyll. Furthermore, phytophatogenic ascomycetes frequently possess several fungal rhodopsins including a CarO counterpart, with highly conserved pumping residues, while some well-known non-pathogenic fungal models, as Neurospora crassa, do not (Table 5). Moreover, the vast majority of fungi with ORP-like rhodopsin orthologues in their genome are either plant pathogens or endophytes. Our plant tissue invasion assays in apple (Fig. 53) revealed no significant difference between the CarO+ and CarO- strains, but situation might be different in rice, its natural host. The use of xylose and other sugars for the drop experiments shown in figure 51A was due to the fact that these compounds are part of the plant cell wall. CarO activity is not stimulated in the presence of these compounds, suggesting that CarO is not playing a role in host sensing by the fungus. A pH regulatory network mediated by the specific transcription factor PacC plays a decisive role in phytopathogenesis by Fusarium oxysporum (Caracuel, Roncero et al. 2003), and a similar PacC protein is involved in pH control of transcriptional regulation in F. fujikuroi (Wiemann, Willmann et al. 2009). A possible connection between CarO activity and PacC regulation and/or phytopathogenesis cannot be discarded, and should be object of future investigation in more suitable phytopathogenic models, e.g. F. oxysporum. 4. Conclusion The ORP-like fungal rhodopsin CarO from the filamentous fungus F. fujikuroi is a functional green-light driven proton pump, which is highly accumulated in the cytoplasmic membrane of conidia. In the presence of ammonium chloride, the CarO deficient strain exhibited a faster germination under light compared to the control strain. This represents the first relevant phenotypic alteration observed for a rhodopsin mutant in a fungus. CarO orthologues are predominant in phytopathogenic fungi, which inhabit niches Biological role of fungal rhodopsins: The light driven, proton-pumping rhodopsin CarO of Fusarium fujikuroi retards germination of light exposed conidia 153 where green light might be particularly abundant. CarO provides a fast photocycle, saturates at high light intensities and can enhance the proton gradient used for nutrient supply, thus provides optimal features to save energy in the light. A potential role of this protein in phytopathogenesis remains to be investigated.