Host-Induced Gene Silencing of a Sclerotinia sclerotiorum oxaloacetate acetylhydrolase Using Bean Pod Mottle Virus as a Vehicle Reduces Disease on Soybean.

Megan Mccaghey,Ashish Ranjan,Steven A Whitham,Shawn P Conley,Dandan Shao,Ally Lindstrom,Damon L Smith,Brett Williams,Mehdi Kabbage,Jake Kurcezewski

doi:10.3389/fpls.2021.677631

Abstract

A lack of complete resistance in the current germplasm complicates the management of Sclerotinia stem rot (SSR) caused by Sclerotinia sclerotiorum in soybean. In this study, we used bean pod mottle virus (BPMV) as a vehicle to down-regulate expression of a key enzyme in the production of an important virulence factor in S. sclerotiorum, oxalic acid (OA). Specifically, we targeted a gene encoding oxaloacetate acetylhydrolase (Ssoah1), because Ssoah1 deletion mutants are OA deficient and non-pathogenic on soybean. We first established that S. sclerotiorum can uptake environmental RNAs by monitoring the translocation of Cy3-labeled double-stranded and small interfering RNA (ds/siRNAs) into fungal hyphae using fluorescent confocal microscopy. This translocation led to a significant decrease in Ssoah1 transcript levels in vitro. Inoculation of soybean plants with BPMV vectors targeting Ssoah1 (pBPMV-OA) also led to decreased expression of Ssoah1. Importantly, pBPMV-OA inoculated plants showed enhanced resistance to S. sclerotiorum compared to empty-vector control plants. Our combined results provide evidence supporting the use of HIGS and exogenous applications of ds/siRNAs targeting virulence factors such as OA as viable strategies for the control of SSR in soybean and as discovery tools that can be used to identify previously unknown virulence factors.

Highlights

Sclerotinia sclerotiorum is a broad host range fungal pathogen that infects many dicotyledonous plants worldwide
Fluorescent signals were detected in S. sclerotiorum hyphae, after a two-day growth period in potato dextrose broth (PDB) supplemented with Cy3-labeled Ssoah1-Double-stranded RNAs (dsRNAs)/small interfering RNAs (siRNAs)
Fluorescent signals were detected in Cy3-Ssoah1-dsRNA/siRNA treated samples using fluorescent confocal microscopy, but not in the negative control (Figure 1)

Summary

Introduction

Sclerotinia sclerotiorum is a broad host range fungal pathogen that infects many dicotyledonous plants worldwide. It is the causal agent of Sclerotinia stem rot (SSR; or white mold) on soybean, a challenging and significant yield-limiting disease (Allen et al, 2017). HIGS Against Sclerotinia sclerotiorum globally (Wrather et al, 2010), and between 2010 and 2014 SSR resulted in yield losses between 150 million and 1.1 billion kgs in the United States, annually (Allen et al, 2017). Current SSR management strategies have limited efficacy and comprise of integrated cultural, chemical, and biological control practices (Peltier et al, 2012; Willbur et al, 2019). Disease control is complicated by the lack of complete genetic resistance to SSR, breeding efforts have identified partially resistant soybean genotypes in the laboratory and through field trials (Boland and Hall, 1986; Chun et al, 1987; Kim and Diers, 2000; Iquira et al, 2015; McCaghey et al, 2017)

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Conclusion