Existing, emerging, and reemerging strains of phytopathogenic fungi pose a significant threat to agricultural productivity globally. This risk is further exacerbated by the lack of resistance source(s) in plants or a breakdown of resistance by pathogens through co-evolution. In recent years, attenuation of essential pathogen gene(s) via double-stranded (ds) RNA-mediated RNA interference (RNAi) in host plants, a phenomenon known as host-induced gene silencing, has gained significant attention as a way to combat pathogen attack. Yet, due to biosafety concerns regarding transgenics, country-specific GMO legislation has limited the practical application of desirable attributes in plants. The topical application of dsRNA/siRNA targeting essential fungal gene(s) through spray-induced gene silencing (SIGS) on host plants has opened up a transgene-free avenue for crop protection. However, several factors influence the outcome of RNAi, including but not limited to RNAi mechanism in plant/fungi, dsRNA/siRNA uptake efficiency, dsRNA/siRNA design parameters, dsRNA stability and delivery strategy, off-target effects, etc. This review emphasizes the significance of these factors and suggests appropriate measures to consider while designing in silico and in vitro experiments for successful RNAi in open-field conditions. We also highlight prospective nanoparticles as smart delivery vehicles for deploying RNAi molecules in plant systems for long-term crop protection and ecosystem compatibility. Lastly, we provide specific directions for future investigations that focus on blending nanotechnology and RNAi-based fungal control for practical applications.

Summary: Pea powdery mildew (PM) is an important fungal disease caused by an obligate biotroph, Erysiphe pisi ( Ep ), which significantly impacts pea production worldwide. The phytopathogen secretes a plethora of effectors, primarily through specialized infection structures termed haustoria, to establish a dynamic relationship with its host. To identify Ep effector candidates, a cDNA library of enriched haustoria from Ep ‐infected pea leaves was sequenced. The Ep transcriptome encodes 622 Ep candidate secreted proteins (CSPs), of which 167 were predicted to be candidate secreted effector proteins (CSEPs). Phylogenetic analysis indicates that Ep CSEPs are highly diverse, but, unlike cereal PM CSEPs, exhibit extensive sequence similarity with effectors from other PMs. Quantitative real‐time PCR of a subset of EpCSEP / CSPs revealed that the majority are preferentially expressed in haustoria and exhibit infection stage–specific expression patterns. The functional roles of EpCSEP001 , EpCSEP009 and EpCSP083 were probed by host‐induced gene silencing (HIGS) via a double‐stranded (ds) RNA‐mediated RNAi approach. Foliar application of individual EpCSEP / CSP dsRNAs resulted in a marked reduction in PM disease symptoms. These findings were consistent with microscopic and molecular studies, suggesting that these Ep CSEP/CSPs play important roles in pea PM pathogenesis. Homology modelling revealed that Ep CSEP001 and Ep CSEP009 are analogous to fungal ribonucleases and belong to the RALPH family of effectors. This is the first study to identify and functionally validate candidate effectors from the agriculturally relevant pea PM, and highlights the utility of transcriptomics and HIGS to elucidate the key proteins associated with Ep pathogenesis.

Powdery mildew (PM) caused by the obligate biotrophic fungal pathogen Erysiphe pisi is an economically important disease of legumes. Legumes are rich in isoflavonoids, a class of secondary metabolites whose role in PM resistance is ambiguous. Here we show that the pterocarpan medicarpin accumulates at fungal infection sites, as analysed by fluorescein‐tagged medicarpin, and provides penetration and post‐penetration resistance against E. pisi in Medicago truncatula in part through the activation of the salicylic acid (SA) signalling pathway. Comparative gene expression and metabolite analyses revealed an early induction of isoflavonoid biosynthesis and accumulation of the defence phytohormones SA and jasmonic acid (JA) in the highly resistant M. truncatula genotype A17 but not in moderately susceptible R108 in response to PM infection. Pretreatment of R108 leaves with medicarpin increased SA levels, SA‐associated gene expression, and accumulation of hydrogen peroxide at PM infection sites, and reduced fungal penetration and colony formation. Strong parallels in the levels of medicarpin and SA, but not JA, were observed on medicarpin/SA treatment pre‐ or post‐PM infection. Collectively, our results suggest that medicarpin and SA may act in concert to restrict E. pisi growth, providing new insights into the metabolic and signalling pathways required for PM resistance in legumes.

Obligate biotrophic pathogens like the pea powdery mildew© (PM) Erysiphe pisi establish long-term feeding relationships with their host, during which they siphon sugars from host cells through haustoria. Plants in turn deploy sugar transporters to restrict carbon allocation toward pathogens, as a defense mechanism. Studies in Arabidopsis have shown that sugar transport protein 13 (STP13), a proton-hexose symporter involved in apoplasmic hexose retrieval, contributes to bacterial and necrotrophic fungal resistance by limiting sugar flux toward these pathogens. By contrast, expression of Lr67res,a transport-deficient wheat STP13 variant harboring two amino acid substitutions (G144R and V387L), conferred resistance against biotrophic fungi in wheat and barley, indicating its broad applicability in disease management. Here, we investigated the role of STP13 and STP13^G144R in legume–PM interactions. We show that Medicago truncatula STP13.1 is a proton-hexose symporter involved in basal resistance against PM and indirectly show that Lr67res-mediated PM resistance, so far reported only in monocots, is transferable to legumes. Among the 30 MtSTPs, STP13.1 exhibited the highest fold induction in PM-challenged leaves and was also responsive to chitosan, ABA and sugar treatment. Functional assays in yeast showed that introduction of the G144R mutation but not V388L abolished MtSTP13.1’s hexose uptake ability. Virus-induced gene silencing of MtSTP13 repressed pathogenesis-related (PR) gene expression and enhanced PM susceptibility in M. truncatula whereas transient overexpression of MtSTP13.1 or MtSTP13.1^G144R in pea induced PR and isoflavonoid pathway genes and enhanced PM resistance. We propose a model in which STP13.1-mediated sugar signaling triggers defense responses against PM in legumes.