RNAi of a Putative Grapevine Susceptibility Gene as a Possible Downy Mildew Control Strategy

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.

Keywords: Vitis vinifera; disease resistance; dsRNA; gene silencing; obligate parasite; susceptibility gene.

FIGURE 1

Scheme of the treatment and sampling carried out in the first (A) and second experiment (B). Numbers indicate the number of days after treatments (dat) at which the leaves were collected. S7 indicates the untreated leaves that were sampled at 7 dat for the evaluation of systemic activity of dsRNA in the second experiment.

FIGURE 2

Effect of dsRNA treatment on VviLBDIf7 gene expression. VviLBDIf7 gene expression analysis determined based on the 2^–ΔΔCt method at 3, 5, 7, and 15 days after treatment (dat) on Pinot noir plants grafted onto SO4 (A), and at 5 and 7 dat on self-rooted Pinot noir plants (B). Bars represent standard errors. Asterisks indicate statistically significant differences among the dsRNA- and water-treated conditions at each time point (*p value = 0.05).

FIGURE 3

Effect of dsRNA treatment on percentage of sporulating area (PSA) evaluated 7 days after Plasmopara viticola inoculation. PSA evaluated on Pinot noir plants grafted onto SO4 at 3, 5, 7, and 15 days after treatment (dat) (A), and on self-rooted Pinot noir plants at 5 and 7 dat (B). Bars represent standard error. Asterisks indicate statistically significant differences among the dsRNA- and water-treated conditions at each time point (*p value < 0.036).

FIGURE 4

Production of sporangia (number of sporangia cm^–2) evaluated 7 days after Plasmopara viticola inoculation on dsRNA- and water-treated leaves at 3, 5, 7, and 15 days after treatment of Pinot noir plants grafted onto SO4. Bars represent standard error. Asterisks indicate statistically significant differences among the dsRNA- and water-treated conditions at each time point (**p value 0.005).

FIGURE 5

Development of Plasmopara viticola structures inside untreated (A–C) and treated (D–G) leaf tissues inoculated at 7 dat. (A) Hyphae with haustoria developing in the mesophyll cells and sporangiophores emerging from the stomata. (B) Mycelium with haustoria; (C) detail of sporangiophores emerging from a stoma; (D) degenerating hyphae, slightly colored and vacuolized, with no visible haustoria, and hyperbranched sporangiophores emerging from the stoma; (E) short, hyperbranched and sterile sporangiophores; (F) light-colored haustoria; (G) hypha developing from the substomatal vesicle with no visible haustoria. S = stoma; H = hypha; HA = haustorium; SP = sporangiophore; SPO = sporangium; SV = substomatal vesicle. Scale bar, 20 μm.

FIGURE 6

Effect of dsRNA treatment on EF1α [elongation factor 1α; (A)], GAPHD (glyceraldehyde-3-phosphate dehydrogenase; B), PEPC [phosphoenolpyruvate carboxylases; (C)], PEPCK [PEP carboxykinases; (D)], and COI1 [coronatine insensitive 1; (E)] gene expression at 3, 5, 7, and 15 days after treatment (dat) on Pinot noir plants grafted onto SO4. Gene expression analysis determined based on the 2^–ΔΔCt method. Bars represent standard errors. Asterisks indicate statistically significant differences among the dsRNA- and water-treated conditions at each time point (*p value = 0.05).