Gene Mining for Conserved, Non-Annotated Proteins of Podosphaera xanthii Identifies Novel Target Candidates for Controlling Powdery Mildews by Spray-Induced Gene Silencing

Abstract

The powdery mildew fungus Podosphaera xanthii is one of the most important limiting factors for cucurbit production worldwide. Despite the significant efforts made by breeding and chemical companies, effective control of this pathogen remains elusive to growers. In this work, we examined the suitability of RNAi technology called spray-induced gene silencing (SIGS) for controlling cucurbit powdery mildew. Using leaf disc and cotyledon infiltration assays, we tested the efficacy of dsRNA applications to induce gene silencing in P. xanthii. Furthermore, to identify new target candidate genes, we analyzed sixty conserved and non-annotated proteins (CNAPs) deduced from the P. xanthii transcriptome in silico. Six proteins presumably involved in essential functions, specifically respiration (CNAP8878, CNAP9066, CNAP10905 and CNAP30520), glycosylation (CNAP1048) and efflux transport (CNAP948), were identified. Functional analysis of these CNAP coding genes by dsRNA-induced gene silencing resulted in strong silencing phenotypes with large reductions in fungal growth and disease symptoms. Due to their important contributions to fungal development, the CNAP1048, CNAP10905 and CNAP30520 genes were selected as targets to conduct SIGS assays under plant growth chamber conditions. The spray application of these dsRNAs induced high levels of disease control, supporting that SIGS could be a sustainable approach to combat powdery mildew diseases.

RNAi Technology: A New Path for the Research and Management of Obligate Biotrophic Phytopathogenic Fungi

“…This process of RNA trafficking from plant host cells to interacting pathogens has also been described in a variety of plant pathogenic fungi and oomycetes, such as Botrytis cinerea, Cochliobolus sativus, Fusarium graminearum, Plasmopara viticola, Podosphaera xanthii, Sclerotinia sclerotiorum and Venturia inaequalis [23][24][25][26][27][28][29][30]. Currently, the mechanisms of the transfer of sRNAs from plants to pathogenic fungi are unknown; however, the discovery that these eukaryotic pathogens are inhibited by sRNAs targeting their essential and/or pathogenicity genes has raised the possibility that plants could be protected by a new generation of environmentally friendly RNA-based fungicides that can be extremely specific and easily adaptable to control multiple diseases at the same time [31].…”

Exogenous double‐stranded RNA inhibits the infection physiology of rust fungi to reduce symptoms in planta

“…The most effective inhibition of A. psidii and C. plumeriae urediniospore germination was observed at dsRNA concentrations of 100 ng/μl. The significant decrease in spore germination with increased dsRNA concentration suggests that the optimal working concentration is higher than in other RNAi studies that have used a concentration of 20 ng/μl (Hu et al, 2020;Koch et al, 2016;Ruiz-Jiménez et al, 2021). Based on the successful inhibition of disease in our assays, the ideal dsRNA concentration (to maximize impact and minimize cost) is closer to 100 ng/μl, which we plan to optimize for applied uses in agriculture and conservation.…”

Spray‐induced gene silencing to identify powdery mildew gene targets and processes for powdery mildew control

“…Because PM fungi are obligate biotrophs that cannot be cultured, it has been difficult to interrogate gene function. Recently, Ruiz‐Jiménez et al ( 2021 ) showed SIGS against the melon PM fungus, Podosphaera xanthii , can be used to identify conserved proteins with no annotated functional domain that are important to PM fungal proliferation; SIGS against three such targets dramatically reduced PM coverage on melon leaves. We showed SIGS can rapidly identify genes and processes that contribute to PM growth and reproduction.…”