The LmSNF1 Gene Is Required for Pathogenicity in the Canola Blackleg Pathogen Leptosphaeria maculans

Jie Feng,Hui Zhang,Sheau-Fang Hwang,Stephen E Strelkov,Yin-Won Lee

doi:10.1371/journal.pone.0092503

Abstract

Leptosphaeria maculans is a fungal pathogen causing blackleg in canola. Its virulence has been attributed, among other factors, to the activity of hydrolytic cell wall degrading enzymes (CWDEs). Studies on the pathogenicity function of CWDEs in plant pathogenic fungi have been difficult due to gene redundancy. In microorganisms many CWDE genes are repressed by glucose and derepressed by the function of the sucrose non-fermenting protein kinase 1 gene (SNF1). To address the molecular function of SNF1 in L. maculans, the ortholog of SNF1 (LmSNF1) was cloned and functionally characterized using a gene knockout strategy. Growth of the LmSNF1 knockout strains was severely disrupted, as was sporulation, spore germination and the ability to attach on the plant surface. When inoculated on canola cotyledons, the LmSNF1 knockout strains could not cause any symptoms, indicating the loss of pathogenicity. The expression of 11 selected CWDE genes and a pathogenicity gene (LopB) was significantly down-regulated in the LmSNF1 knockout strains. In conclusion, knockout of LmSNF1 prevents L. maculans from properly derepressing the production of CWDEs, compromises the utilization of certain carbon sources, and impairs fungal pathogenicity on canola.

Highlights

Plant pathogenic fungi secrete an array of cell wall degrading enzymes (CWDEs) capable of depolymerizing the polysaccharides of primary cell walls [1,2,3]
Sequencing of the LmSNF1 Polymerase chain reactions (PCR) fragments revealed an open reading frame (ORF) of 2,782 bp, which is identical to the sucrose non-fermenting protein kinase 1 gene (SNF1) gene (XM_003844721) in the whole-genome sequenced L. maculans strain JN3
Targeted gene knockout in L. maculans Successful introduction of foreign DNA into a fungal genome is dependent on the DNA double-strand break (DSB) repair mechanisms

Summary

Introduction

Plant pathogenic fungi secrete an array of cell wall degrading enzymes (CWDEs) capable of depolymerizing the polysaccharides of primary cell walls [1,2,3]. These enzymes received special attention from researchers and many of them have been demonstrated to be important for the pathogenicity of various fungi. To test the role of CWDEs as a whole in fungal pathogenicity, disruption of the elements that control the derepression mechanism could be a more reliable approach than working on single CWDE genes [10]. Repression of CWDE genes by creA has been studied in a number of plant pathogenic fungi [13,14,15,16]

Methods

Results

Conclusion