Abstract
Downy mildew, caused by the oomycete Plasmopara viticola, is one of the diseases causing the most severe economic losses to grapevine (Vitis vinifera) production. To date, the application of fungicides is the most efficient method to control the pathogen and the implementation of novel and sustainable disease control methods is a major challenge. RNA interference (RNAi) represents a novel biotechnological tool with a great potential for controlling fungal pathogens. Recently, a candidate susceptibility gene (VviLBDIf7) to downy mildew has been identified in V. vinifera. In this work, the efficacy of RNAi triggered by exogenous double-stranded RNA (dsRNA) in controlling P. viticola infections has been assessed in a highly susceptible grapevine cultivar (Pinot noir) by knocking down VviLBDIf7 gene. The effects of dsRNA treatment on this target gene were assessed by evaluating gene expression, disease severity, and development of vegetative and reproductive structures of P. viticola in the leaf tissues. Furthermore, the effects of dsRNA treatment on off-target (EF1α, GAPDH, PEPC, and PEPCK) and jasmonic acid metabolism (COI1) genes have been evaluated. Exogenous application of dsRNA led to significant reductions both in VviLBDIf7 gene expression, 5 days after the treatment, and in the disease severity when artificial inoculation was carried out 7 days after dsRNA treatments. The pathogen showed clear alterations to both vegetative (hyphae and haustoria) and reproductive structures (sporangiophores) that resulted in stunted growth and reduced sporulation. Treatment with dsRNA showed signatures of systemic activity and no deleterious off-target effects. These results demonstrated the potential of RNAi for silencing susceptibility factors in grapevine as a sustainable strategy for pathogen control, underlying the possibility to adopt this promising biotechnological tool in disease management strategies.
Highlights
Since the late 1800s, Vitis vinifera has suffered damage from downy mildew, a disease caused by the oomycete Plasmopara viticola, originating from Northern America
Results highlighted that the VviLBDIf7 gene is constitutively expressed in leaves of Pinot noir plants grown in the glasshouse: there was no significant variation in basal expression level in the examined time points (0.45 ± 0.11 versus 0.83 ± 0.23, for first and second sampling, respectively, p value > 0.05), suggesting a relatively constant expression of the candidate susceptibility genes (S-genes)
The phenotypic characterization of quantitative and qualitative P. viticola traits allowed us to establish the efficacy of exogenous double-stranded RNA (dsRNA) application in silencing the VviLBDIf7 gene, which led to a reduced pathogen growth and sporulation rate in Pinot noir, a cultivar that is normally highly susceptible to the pathogen
Summary
Since the late 1800s, Vitis vinifera has suffered damage from downy mildew, a disease caused by the oomycete Plasmopara viticola, originating from Northern America This biotrophic, obligate parasite can infect all the green parts of grapevine plants, causing quantitative and qualitative damage and leading to extensive yield losses (Yu et al, 2012). Silencing Downy Mildew Susceptibility in Grapevine with center of origin in North America and Asia, are resistant to P. viticola due to coevolution with the pathogen. This resistance is mediated by different mechanisms that at first detect the pathogen and initiate a proper defense response (Gessler et al, 2011). Activation of ETI leads to disease resistance and is often associated with localized apoptosis at the infection site (hypersensitive response) (Jones and Dangl, 2006)
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