Water deficit improves reproductive fitness in Nicotiana benthamiana plants infected by Cucumber mosaic virus

Citation: Moreno, M.; Ojeda, B.; Hernández-Walias, F.J.; Sanz-García, E.; Canto, T.; Tenllado, F. Water Deficit Improves Reproductive Fitness in Nicotiana benthamiana Plants Infected by Cucumber mosaic virus.Plants 2022,11, 1240. https://doi.org/10.3390/ plants11091240 Academic Editors: Manuella van Munster and Denis Vile Received: 22 March 2022 Accepted: 27 April 2022 Published: 4 May 2022 Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations. Copyright: © 2022 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/). plants Article Water Deficit Improves Reproductive Fitness in Nicotiana benthamiana Plants Infected by Cucumber mosaic virus Marina Moreno, Belén Ojeda, Francisco J. Hernández-Walias, Eugenio Sanz-García , Tomás Canto and Francisco Tenllado * Departamento de Biotecnología Microbiana y de Plantas, Centro de Investigaciones Biológicas Margarita Salas, CSIC, 28040 Madrid, Spain; [email protected] (M.M.); [email protected] (B.O.); [email protected] (F.J.H.-W.); [email protected] (E.S.-G.); [email protected] (T.C.) *Correspondence: [email protected] Abstract: Plants are concurrently exposed to biotic and abiotic stresses, including infection by viruses and drought. Combined stresses result in plant responses that are different from those observed for each individual stress. We investigated compensatory effects induced by virus infection on the fitness of hosts grown under water deficit, and the hypothesis that water deficit improves tolerance, estimated as reproductive fitness, to virus infection. Our results show that infection by Turnip mosaic virus (TuMV) or Cucumber mosaic virus (CMV) promotes drought tolerance in Arabidopsis thaliana and Nicotiana benthamiana. However, neither CMV nor TuMV had a positive impact on host reproductive fitness following withdrawal of water, as determined by measuring the number of individuals producing seeds, seed grains, and seed germination rates. Importantly, infection by CMV but not by TuMV improved the reproductive fitness of N. benthamiana plants when exposed to drought compared to watered, virus-infected plants. However, no such conditional phenotype was found in Arabidopsis plants infected with CMV. Water deficit did not affect the capacity of infected plants to transmit CMV through seeds. These findings highlight a conditional improvement in biological efficacy of N. benthamiana plants infected with CMV under water deficit, and lead to the prediction that plants can exhibit increased tolerance to specific viruses under some of the projected climate change scenarios. Keywords: water deficit; virus infection; combined abiotic and biotic stresses; reproductive fitness; tolerance to drought; tolerance to virus; climate changes 1. Introduction Plants are exposed to a diverse range of biotic and abiotic stresses that do not occur separately in time but are commonly present simultaneously [ 1 , 2 ]. The outcomes of multiple stresses can provide either tolerance or increased susceptibility to any of the stresses depending on the plant species, developmental stage and stress severity [ 3 , 4 ]. Although plant viruses give rise to many important diseases in crops worldwide, losses in agriculture due to abiotic stresses such as cold, salinity, heat and drought generally exceed those caused by viruses [ 5 ]. In particular, stress caused by water deficit is one of the major threats that affect plant physiology and growth, particularly owing to the increase of drought episodes caused by global warming [ 6 ]. Plants have developed a range of approaches to buffer the negative impact of drought on their physiology [ 7 ]. At an early stage of water stress, water content is kept within relatively narrow limits by increasing water capture and by limiting water loss from evapotranspiration by partially closing stomata. Stomatal closure reduces the entry of CO 2 , consequently decreasing photosynthesis and productive capacity. The effect of water deficit on plants leads to profound changes in hormones and secondary metabolites involved in plant defenses [8,9], the outcomes of which for plant resistance to pathogens are largely unexplored. Plants 2022,11, 1240. https://doi.org/10.3390/plants11091240 https://www.mdpi.com/journal/plants Plants 2022,11, 1240 2 of 16 Several lines of evidence show that certain combinations of biotic and abiotic stresses could confer a positive effect on plant performance by increasing the tolerance to abiotic stresses. In particular, mechanisms and processes operating in compatible plants–virus interactions between might offer plants a better performance under abiotic stresses. For instance, it has been reported that infection by viruses can enhance the tolerance of host plants to cold and drought [ 10 – 15 ]. In addition, infection of Arabidopsis by different viruses rendered seeds with improved tolerance to deterioration by elevated temperature [ 16 ]. The reason for this increased tolerance to abiotic stresses in plants infected with viruses might reside in that plants made use of interlinked signaling pathways to respond to different environmental stresses, and that several of these responses are common in tolerance against virus and abiotic stresses [8,17]. On the other hand, abiotic stresses (some of them associated with global warming, i.e., elevated O 3 , CO 2 and temperature, drought) can modulate plant tolerance toward pathogens by mechanisms that include changes in the response of plants to changing environmental conditions [ 18 , 19 ]. For instance, a lower incidence of virus diseases and severity of symptoms was accompanied by modulated defenses in plants grown at elevated CO 2 [ 20 – 22 ]. Bilgin et al. [ 23 ] reported that ozone stress enhanced soybean tolerance to Soybean mosaic virus. Elevated temperatures have been described to cause a weakening and even a masking of symptoms in some compatible plant–virus interactions [ 24 ]. With regard to drought, it has been reported that the dynamics of symptoms and virus spread can be altered by water deficit in Arabidopsis infected with Cauliflower mosaic virus (CaMV) [ 25 , 26 ]. Indeed, survival of plants infected by CaMV grown under water deficit was increased when compared to plants grown under normal irrigation. However, the vegetative performance of CaMV-infected plants under water deficit could not be correlated with a better performance during the reproductive phase, since none of the infected plants developed seeds. Heat and drought tolerance mechanisms mediated by an increase in osmolytes may be common themes in plant tolerance to heat and drought [1]. The preferential survival of virus-infected plants under water deficit represents an advantage in biological efficacy (fitness) only if the survivors can subsequently produce offspring at higher rates than infected plants grown under watered conditions [ 12 , 27 ]. In this sense, it has been reported that virus-induced tolerance to drought was not always correlated with an increase in fecundity, with virulence being detrimental to reproductive fitness (defined as the relative success of an individual to pass on its genes to the subsequent generations) [ 10 , 28 ]. In addition, enhanced survival of the infected plants under water deficit could represent an advantage for the virus through increased opportunities to spread in the plant population, either by an increased duration of the vegetative phase or via propagation by seeds [ 26 ]. In this study, we utilized Cucumber mosaic virus (CMV) and Turnip mosaic virus (TuMV), and Arabidopsis thaliana and Nicotiana benthamiana. Both viruses are commonly found in wild populations of Arabidopsis, indicating that the Arabidopsis– TuMV and Arabidopsis–CMV pathosystems are significant in nature [ 29 ]. N. benthamiana has been adopted as a model plant by virologists due to its general susceptibility to virus infection. It is an allotetraploid species from the Suaveolentes section, resulting from the hybridization of a maternal progenitor of section Noctiflora, and a member of section Sylvestres as a paternal subgenome donor [ 30 ]. Transmission of CMV through seeds in N. benthamiana and Arabidopsis and of TuMV in Arabidopsis has been well established [ 31 ], but no data have been reported on seed transmission of TuMV in N. benthamiana . The aim of this work was to investigate compensatory effects induced by virus infection on host fitness when grown under water deficit, and to test the hypothesis that water deficit improves tolerance, estimated as reproductive fitness, to virus infection. In addition, we also analyzed the effect of water deficit on virus transmission through seeds. Our results show that infection by TuMV or CMV promoted drought tolerance in Arabidopsis and N. benthamiana . More importantly, infection by CMV but not by TuMV improved the reproductive fitness of N. benthamiana when exposed to drought compared to watered, virus-infected plants. However, no such conditional phenotype was observed in Arabidopsis plants infected Plants 2022,11, 1240 3 of 16 with CMV. Water deficit did not affect seed transmission of CMV in N. benthamiana and Arabidopsis plants. 2. Results 2.1. Tolerance to Drought in Virus-Infected N. benthamiana Plants N. benthamiana plants were mock-inoculated or inoculated with TuMV and CMV. Virus-infected and mock-inoculated plants were normally irrigated or deprived of watering at 12 days after inoculation (dai) resulting in stress caused by water deficit. The damaging effect caused by viral infection on the biomass of watered plants was higher for TuMV than for CMV at 19 dai (Figure 1A). After withholding water, drought symptoms in mockinoculated plants first appeared as drooped, curled, or wilted leaves. The prolonged water deficit eventually led to plant collapse and death (Figure 1B). In plants infected with TuMV and CMV, the onset of drought symptoms was delayed by several days and they clearly maintained a reduced wilting appearance compared to mock-inoculated plants throughout the experiment. Water content, stomatal conductance and the relative soil water content (RSWC) are valuable tools for providing information about plant responses to water deficit. The water content of virus-infected and mock-inoculated plants growing under watered and drought conditions was compared at 19 dai, i.e., 7 days after the water was withdrawn (daww). Under drought conditions, average water content was higher in plants infected with TuMV and CMV compared to mock-inoculated plants, indicating that infections by TuMV and CMV promote drought tolerance in N. benthamiana (Figure 1C). A similar water content was found in watered plants infected with TuMV and CMV compared to mockinoculated plants (Figure 1C). Comparative analysis of virus accumulation before and after the water was withdrawn by western blot revealed that the level of CMV CP and TuMV CP in plants grown under drought was similar to that in plants grown under normal irrigation at 19 dai (Figure 1D). Because a decrease in transpiration rate is an important trait of plant tolerance to drought, measurements of stomatal conductance were calculated in both watered and drought-stressed plants at 15 dai (3 daww) (Figure 1E). Under normal irrigation, infection by CMV and TuMV caused a decrease in conductance compared to mock-inoculated plants. After several days without watering, leaves of mock-inoculated plants showed a sharp drop in stomatal conductance compared to watered plants, whereas leaves of plants infected with either CMV or TuMV exhibited only a smaller reduction in conductance. To determine whether water is similarly depleted in pots of virus-infected and mock-inoculated plants under drought stress, the water content of the soil was measured in both watered and drought-stressed plants at the end of the water deficit period (7 daww; 19 dai). An obvious difference in the RSWC was observed under the two water regimes assayed (Figure 1F). Moreover, a small but statistically significant higher level of RSWC was observed in virusinfected plants compared with mock-inoculated plants under drought stress. Thus, relative differences in soil moisture were correlated with differences in stomatal conductance before the water was withdrawn and tolerance to drought in virus-infected N. benthamiana plants. 2.2. CMV-Infected N. benthamiana Plants Improved Their Reproductive Fitness When Exposed to Drought The effect of virus infection on the reproductive fitness of both drought-stressed and watered N. benthamiana plants was determined by the number of infected plants producing seeds and the seed grains per plant, and by comparing them with those produced by mockinoculated plants. Under watering conditions, infection by TuMV and CMV conferred a severe detrimental effect on the number of seed-producing plants and on seed grain per plant compared to mock-inoculated plants (Figure 2A,B). Under drought conditions, infection by CMV led to a decrease in the number of plants producing seeds compared to mock-inoculated controls, albeit less severe than under the watered regime. Remarkably, the number of CMV-infected plants grown under drought that produced seeds was statistically higher than that observed in plants infected with CMV under normal irrigation (Figure 2A). Plants 2022,11, 1240 4 of 16 By contrast, no statistical differences were observed in the number of TuMV-infected plants producing seeds when grown under watered or drought conditions (Figure 2A). On the other hand, infection by either TuMV or CMV caused a statistical significant reduction on seed grain per plant when compared to mock-inoculated plants, under drought conditions (Figure 2B). However, no significant differences in seed grains were observed between CMV-infected plants that were irrigated or deprived of irrigation (Figure 2B). Plants 2022, 11, x FOR PEER REVIEW 4 of 18 Figure 1. Comparison of tolerance to drought in mock-inoculated, Cucumber mosaic virus (CMV)- and Turnip mosaic virus (TuMV)-infected Nicotiana benthamiana plants. (A) Biomass of watered (W) plants at 19 days after inoculation (dai). (B) Seven days after the water was withheld (daww) (upper panel), representative plants were photographed together with their watered counterparts (bottom panel). (C) Water content percentage in virus-infected and mock-inoculated plants grown under W or drought (D) conditions at 19 dai (7 daww). (D) Western blot analysis of protein extracts from plants infected with CMV (upper panels) or TuMV (lower panels) grown under W or D conditions at 12 or 19 dai, using antibodies against CMV CP or TuMV CP. Two independent pooled samples were analyzed for each inoculum. The lower panels show the Ponceau S-stained membrane after blotting, as a control of loading. (E) Effect of drought on stomatal conductance in virus-infected and mock-inoculated plants grown under W or D conditions at 3 daww. (F) The relative soil water content (RSWC) was measured in each pot of plants grown under W or D conditions. Data represent the means ± standard errors of 18 plants that received the same treatment. Statistical comparisons between means were made among treatments (i.e., Mock, CMV and TuMV) within each watering condition (i.e., watered, drought) by employing Scheffé’s multiple range test (A,E,F) and a Mann–Whitney U test with a Bonferroni correction for multiple comparisons of α to α = 0.016 (C). Different letters indicate significant differences (p < 0.05). For pairwise comparisons, an asterisk indicates the statistical significance of drought-stressed plants compared to watered plants (p < 0.05). 2.2. CMV-Infected N. Benthamiana Plants Improved Their Reproductive Fitness When Exposed to Drought The effect of virus infection on the reproductive fitness of both drought-stressed and watered N. benthamiana plants was determined by the number of infected plants producing seeds and the seed grains per plant, and by comparing them with those Figure 1. Comparison of tolerance to drought in mock-inoculated, Cucumber mosaic virus (CMV)- and Turnip mosaic virus (TuMV)-infected Nicotiana benthamiana plants. ( A ) Biomass of watered (W) plants at 19 days after inoculation (dai). ( B ) Seven days after the water was withheld (daww) (upper panel), representative plants were photographed together with their watered counterparts (bottom panel). ( C ) Water content percentage in virus-infected and mock-inoculated plants grown under W or drought (D) conditions at 19 dai (7 daww). ( D ) Western blot analysis of protein extracts from plants infected with CMV (upper panels) or TuMV (lower panels) grown under W or D conditions at 12 or 19 dai, using antibodies against CMV CP or TuMV CP. Two independent pooled samples were analyzed for each inoculum. The lower panels show the Ponceau S-stained membrane after blotting, as a control of loading. ( E ) Effect of drought on stomatal conductance in virus-infected and mock-inoculated plants grown under W or D conditions at 3 daww. ( F ) The relative soil water content (RSWC) was measured in each pot of plants grown under W or D conditions. Data represent the means ± standard errors of 18 plants that received the same treatment. Statistical comparisons between means were made among treatments (i.e., Mock, CMV and TuMV) within each watering condition (i.e., watered, drought) by employing Scheffé’s multiple range test ( A , E , F ) and a Mann– Whitney U test with a Bonferroni correction for multiple comparisons of α to α = 0.016 ( C ). Different letters indicate significant differences (p< 0.05). For pairwise comparisons, an asterisk indicates the statistical significance of drought-stressed plants compared to watered plants (p< 0.05). Plants 2022,11, 1240 5 of 16 Plants 2022, 11, x. https://doi.org/10.3390/xxxxx www.mdpi.com/journal/plants Figure 2. Effect of TuMV and CMV infection on the number of Nicotiana benthamiana plants producing seeds, seed grain, seed weight and seed viability. ( A ) Virus-infected and mock-inoculated plants were grown under watered and drought conditions, and the number of plants producing seeds was recorded. ( B ) Seeds were weighted separately after threshing and recorded as seed grain per plant. ( C ) Effect of virus infection on the weight of individual seeds. Seed weight was estimated after determining the weight of 80 seeds derived from each of four to seven plants per treatment. ( D ) Effects of virus infection on seed viability. Seed viability was measured as the germination percentage of approximately 100 seeds per plant, using 3 to 6 individuals per treatment. Statistical comparisons between means were made among treatments within each watering condition by employing Fisher’s exact test with a Bonferroni correction for multiple comparisons of α to α= 0.016 ( A ) and Scheffé’s multiple range test ( B – D ). Different letters indicate significant differences. For pairwise comparisons, asterisks indicate significant differences between treatments (Student’s t-test, p< 0.05); NS: not significative. Next, we examined the effect of drought on the weight of individual seeds and seed viability of plants infected with TuMV and CMV compared to mock-inoculated plants (Figure 2C,D). The water regime did not affect the seed weight or rate of germination, regardless if seeds were derived from mock-inoculated or virus-infected plants. Although infection by CMV reduced seed germination by ca. 40% relative to non-infected plants, the viability of seeds and the weight of individual seeds derived from virus-infected plants grown under drought conditions was not differentially affected when compared to those grown under normal irrigation. Altogether, water deficit improved the reproductive fitness of CMV-infected N. benthamiana plants, but not that of plants infected with TuMV, when compared to watered, virus-infected plants. 2.3. Tolerance to Drought in Virus-Infected Arabidopsis Plants Arabidopsis seedlings were either mock-inoculated or inoculated with TuMV or CMV. Virus-infected and mock-inoculated plants were normally watered or subjected to water stress resulting from water withholding at 16 dai. The damaging effect of viral infection on the biomass of watered plants was higher for TuMV than for CMV at 30 dai (Figure 3A). After water deprivation, TuMVand CMV-infected plants wilted more slowly and displayed milder drought-related symptoms than mock-inoculated plants (Figure 3B). At 30 dai (14 daww), the water content of virus-infected and mock-inoculated plants was Plants 2022,11, 1240 6 of 16 compared (Figure 3C). The average water content was higher in TuMVand CMV-infected plants than in mock-inoculated plants under non-watered growth conditions, indicating that infection by TuMV and CMV promotes tolerance to drought in Arabidopsis. Indeed, the water content in plants infected with TuMV grown under water deficit was similar to that observed in TuMV-infected plants grown under watered conditions. Plants 2022, 11, x FOR PEER REVIEW 7 of 18 Figure 3. Comparison of tolerance to drought in mock-inoculated, CMVand TuMV-infected Arabidopsis plants. (A) Biomass of watered plants at 30 days after inoculation. (B) Twelve daww (upper panel), representative plants were photographed next to their watered counterparts (bottom panel). (C) Water content percentage in virus-infected and mock-inoculated plants at 14 daww. Data represent the means ± standard errors of at least 18 plants that received the same treatment. Statistical comparisons between means were made among treatments within each watering condition by employing a Mann–Whitney U test with a Bonferroni correction for multiple comparisons of α to α = 0.016. For pairwise comparisons, asterisks indicate the statistical significance of drought-stressed plants compared to watered plants (Mann–Whitney U test, p < 0.05). Figure 3. Comparison of tolerance to drought in mock-inoculated, CMVand TuMV-infected Arabidopsis plants. ( A ) Biomass of watered plants at 30 days after inoculation. ( B ) Twelve daww (upper panel), representative plants were photographed next to their watered counterparts (bottom panel). ( C ) Water content percentage in virus-infected and mock-inoculated plants at 14 daww. Data represent the means ± standard errors of at least 18 plants that received the same treatment. Statistical comparisons between means were made among treatments within each watering condition by employing a Mann–Whitney U test with a Bonferroni correction for multiple comparisons of α to α = 0.016. For pairwise comparisons, asterisks indicate the statistical significance of drought-stressed plants compared to watered plants (Mann–Whitney U test, p< 0.05). Plants 2022,11, 1240 7 of 16 In our growth conditions, infection by TuMV in both watered and drought-stressed Arabidopsis plants affected several plant developmental traits, including flower and silique viability, which eventually led to sterility. Thus, the effect of CMV infection on the reproductive fitness of both watered and drought-stressed plants was studied. Infection by CMV did not affect the number of Arabidopsis plants producing seeds compared to mock-inoculated controls, regardless of whether plants were grown under drought or under watered conditions (Figure 4A). By contrast, seed grains per plant were reduced in plants infected with CMV compared to those of mock-inoculated plants when grown under watered conditions. However, negligible differences were observed in seed grains between CMVand mock-inoculated plants when grown under drought (Figure 4B). Infection by CMV did not affect the viability of Arabidopsis seeds regardless of the water regime ( Figure 4C ). Thus, infection by CMV did not improve the reproductive fitness of Arabidopsis plants exposed to drought when compared to that of plants infected by CMV grown under watered conditions. 2.4. The Effect of Drought on the Transmission of CMV through N. benthamiana and Arabidopsis Seeds To analyze the effect of water deficit on CMV transmission through seeds, progenies derived from infected plants grown under watered or drought treatments were germinated in vitro . Seeds were sterilized to ensure that any positive detection was not the result of virus contamination on the seed coat, but rather the existence of embryonic infection. Four pools of seedlings derived from each of three CMV-infected N. benthamiana plants grown under watered and drought conditions were analyzed by real-time quantitative reverse transcription (RT-qPCR), using seedlings derived from non-infected plants as a negative control. CMV was detected in all of the pools of seedlings analyzed, regardless of whether the progenitors were grown under watered or drought conditions (Figure 5A). On average, the relative level of CMV in progenies derived from watered plants tended to be higher than in drought-stressed plants, although there was no statistical significance due to the variability of the samples (Figure 5B). To confirm the above results, pools of seedlings derived from a subset of the progenies analyzed above were assayed by western blot analysis. The CMV CP was detected in most of the progeny pools derived from infected plants grown under watered or drought conditions (Figure 5C, arrows). We also measured the efficiency of seed transmission of CMV in progenies derived from infected Arabidopsis plants grown under watered or drought conditions. Pools of progenies derived from each of three CMV-infected plants grown under watered and drought conditions were analyzed by western blot. Two out of three progeny pools in each group of plants accumulated CMV CP (Figure 5D, arrows). Altogether, our data showed that water deficit did not significantly affect seed transmission of CMV in N. benthamiana and Arabidopsis plants. After several attempts to detect TuMV in progenies derived from watered and droughtstressed N. benthamiana plants, we could not detect seed transmission of TuMV by RT-qPCR and western blot analyses (Figure S1). Plants 2022,11, 1240 8 of 16 Plants 2022, 11, x FOR PEER REVIEW 8 of 18 Figure 4. Effect of CMV infection on the number of Arabidopsis plants producing seeds, on seed grain and on seed viability. (A) Virus-infected and mock-inoculated plants were grown under watered and drought conditions, and the number of plants producing seeds was recorded. (B) Seeds were weighted separately after threshing and recorded as seed grain per plant. (C) Effect of CMV infection on seed viability. Seed viability was measured as the germination percentage of approximately 100 seeds per plant, using six individuals per treatment. Statistical comparisons between means were made among treatments within each watering condition by employing Student’s t-test (B,C, p < 0.05) and Fisher’s exact test with a Bonferroni correction for multiple comparisons of α to α = 0.025 (A). Different letters indicate significant differences. For pairwise comparisons, asterisks indicate significant differences between treatments (Student’s t-test, p < 0.05); NS: not significative. Figure 4. Effect of CMV infection on the number of Arabidopsis plants producing seeds, on seed grain and on seed viability. ( A ) Virus-infected and mock-inoculated plants were grown under watered and drought conditions, and the number of plants producing seeds was recorded. ( B ) Seeds were weighted separately after threshing and recorded as seed grain per plant. ( C ) Effect of CMV infection on seed viability. Seed viability was measured as the germination percentage of approximately 100 seeds per plant, using six individuals per treatment. Statistical comparisons between means were made among treatments within each watering condition by employing Student’s t-test ( B,C,p< 0.05 ) and Fisher’s exact test with a Bonferroni correction for multiple comparisons of α to α = 0.025 ( A ). Different letters indicate significant differences. For pairwise comparisons, asterisks indicate significant differences between treatments (Student’s t-test, p< 0.05); NS: not significative. Plants 2022,11, 1240 9 of 16 Plants 2022, 11, x FOR PEER REVIEW 10 of 18 Figure 5. Effect of drought on CMV seed transmission. (A) CMV relative levels estimated by RT-qPCR in four pools of Nicotiana benthamiana seedlings derived from each of three progenitor plants grown under W and D conditions. (B) Average level of CMV estimated by RT-qPCR in progenies derived from watered and drought-stressed N. benthamiana plants. NS: not significative (Student’s t-test, p < 0.05). Western blot analysis of protein extracts derived from pools of N. benthamiana (C) and Arabidopsis (D) seedlings, using antibodies against CMV CP. The lower panel shows the Ponceau S-stained membrane after blotting, as a control of loading. Lane C+ corresponds to an extract from a CMV-infected plant diluted 1:500. Lane Ccorresponds to an extract from non-infected seedlings. We also measured the efficiency of seed transmission of CMV in progenies derived from infected Arabidopsis plants grown under watered or drought conditions. Pools of progenies derived from each of three CMV-infected plants grown under watered and drought conditions were analyzed by western blot. Two out of three progeny pools in each group of plants accumulated CMV CP (Figure 5D, arrows). Altogether, our data Figure 5. Effect of drought on CMV seed transmission. ( A ) CMV relative levels estimated by RTqPCR in four pools of Nicotiana benthamiana seedlings derived from each of three progenitor plants grown under W and D conditions. ( B ) Average level of CMV estimated by RT-qPCR in progenies derived from watered and drought-stressed N. benthamiana plants. NS: not significative (Student’s t-test, p< 0.05). Western blot analysis of protein extracts derived from pools of N. benthamiana ( C ) and Arabidopsis ( D ) seedlings, using antibodies against CMV CP. The lower panel shows the Ponceau S-stained membrane after blotting, as a control of loading. Lane C+ corresponds to an extract from a CMV-infected plant diluted 1:500. Lane Ccorresponds to an extract from non-infected seedlings. Plants 2022,11, 1240 16 of 16 31. Montes, N.; Pagán, I. Light intensity modulates the efficiency of virus seed transmission through modifications of plant tolerance. Plants 2019,8, 304. [CrossRef] 32. González, R.; Butkovi´c, A.; Elena, S.F. 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