The transportome of the endophyte Serendipita indica in free life and symbiosis with Arabidopsis and its expression in moderate salinity.

Serendipita indica is an endophytic root symbiont fungus that enhances the growth of various plants under different stress conditions, including salinity. Here, the functional characterization of two fungal Na+/H+ antiporters, SiNHA1 and SiNHX1 has been carried out to study their putative role in saline tolerance. Although their gene expression does not respond specifically to saline conditions, they could contribute, together with the previously characterized Na+ efflux systems SiENA1 and SiENA5, to relieve Na+ from the S. indica cytosol under this stressed condition. In parallel, an in-silico study has been carried out to define its complete transportome. To further investigate the repertoire of transporters expressed in free-living cells of S. indica and during plant infection under saline conditions, a comprehensive RNA-seq approach was taken. Interestingly, SiENA5 was the only gene significantly induced under free-living conditions in response to moderate salinity at all the tested time points, revealing that it is one of the main salt-responsive genes of S. indica. In addition, the symbiosis with Arabidopsis thaliana also induced SiENA5 gene expression, but significant changes were only detected after long periods of infection, indicating that the association with the plant somehow buffers and protects the fungus against the external stress. Moreover, the significant and strongest induction of the homologous gene SiENA1 occurred during symbiosis, regardless the exposure to salinity. The obtained results suggest a novel and relevant role of these two proteins during the establishment and maintenance of fungus-plant interaction.

Transgenerational Tolerance to Salt and Osmotic Stresses Induced by Plant Virus Infection.

Following pathogen infection, plants have developed diverse mechanisms that direct their immune systems towards more robust induction of defense responses against recurrent environmental stresses. The induced resistances could be inherited by the progenies, rendering them more tolerant to stressful events. Although within-generational induction of tolerance to abiotic stress is a well-documented phenomenon in virus-infected plants, the transgenerational inheritance of tolerance to abiotic stresses in their progenies has not been explored. Here, we show that infection of Nicotiana benthamiana plants by Potato virus X (PVX) and by a chimeric Plum pox virus (PPV) expressing the P25 pathogenicity protein of PVX (PPV-P25), but not by PPV, conferred tolerance to both salt and osmotic stresses to the progeny, which correlated with the level of virulence of the pathogen. This transgenerational tolerance to abiotic stresses in the progeny was partially sustained even if the plants experience a virus-free generation. Moreover, progenies from a Dicer-like3 mutant mimicked the enhanced tolerance to abiotic stress observed in progenies of PVX-infected wild-type plants. This phenotype was shown irrespective of whether Dicer-like3 parents were infected, suggesting the involvement of 24-nt small interfering RNAs in the transgenerational tolerance to abiotic stress induced by virus infection. RNAseq analysis supported the upregulation of genes related to protein folding and response to stress in the progeny of PVX-infected plants. From an environmental point of view, the significance of virus-induced transgenerational tolerance to abiotic stress could be questionable, as its induction was offset by major reproductive costs arising from a detrimental effect on seed production.

Water Deficit Improves Reproductive Fitness in Nicotiana benthamiana Plants Infected by Cucumber mosaic virus.

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.

Topical Application of Escherichia coli-Encapsulated dsRNA Induces Resistance in Nicotiana benthamiana to Potato Viruses and Involves RDR6 and Combined Activities of DCL2 and DCL4.

Exogenous application of double-stranded RNAs (dsRNAs) for inducing virus resistance in plants represents an attractive alternative to transgene-based silencing approaches. However, improvement of dsRNA stability in natural conditions is required in order to provide long-term protection against the targeted virus. Here, we tested the protective effect of topical application of Escherichia coli-encapsulated dsRNA compared to naked dsRNA against single and dual infection by Potato virus X expressing the green fluorescent protein (PVX-GFP) and Potato virus Y (PVY) in Nicotiana benthamiana. We found that, in our conditions, the effectiveness of E. coli-encapsulated dsRNA in providing RNAi-mediated protection did not differ from that of naked dsRNA. dsRNA vaccination was partly effective against a dual infection by PVX-GFP and PVY, manifested by a delay in the expression of the synergistic symptoms at early times after inoculation. Using PVX-GFP as a reporter virus together with a suite of RNAi knockdown transgenic lines, we have also shown that RNA-directed RNA polymerase 6 and the combined activities of DICER-like 2 (DCL2) and DCL4 act to promote efficient resistance to virus infection conferred by topical application of dsRNA in N. benthamiana. Our results provide evidence that exogenous dsRNA molecules are processed by the RNA silencing pathways commonly used by the host in response to virus infection.