Comprehensive analysis of Verticillium nonalfalfae in silico secretome uncovers putative effector proteins expressed during hop invasion.

Kristina Marton,Katarina Košmelj,Branka Javornik,Marko Flajšman,Jernej Jakše,Sebastjan Radišek,Sabina Berne,Zhengguang Zhang

doi:10.1371/journal.pone.0198971

Kristina Marton, Katarina Košmelj + Show 6 more

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https://doi.org/10.1371/journal.pone.0198971

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Abstract

The vascular plant pathogen Verticillium nonalfalfae causes Verticillium wilt in several important crops. VnaSSP4.2 was recently discovered as a V. nonalfalfae virulence effector protein in the xylem sap of infected hop. Here, we expanded our search for candidate secreted effector proteins (CSEPs) in the V. nonalfalfae predicted secretome using a bioinformatic pipeline built on V. nonalfalfae genome data, RNA-Seq and proteomic studies of the interaction with hop. The secretome, rich in carbohydrate active enzymes, proteases, redox proteins and proteins involved in secondary metabolism, cellular processing and signaling, includes 263 CSEPs. Several homologs of known fungal effectors (LysM, NLPs, Hce2, Cerato-platanins, Cyanovirin-N lectins, hydrophobins and CFEM domain containing proteins) and avirulence determinants in the PHI database (Avr-Pita1 and MgSM1) were found. The majority of CSEPs were non-annotated and were narrowed down to 44 top priority candidates based on their likelihood of being effectors. These were examined by spatio-temporal gene expression profiling of infected hop. Among the highest in planta expressed CSEPs, five deletion mutants were tested in pathogenicity assays. A deletion mutant of VnaUn.279, a lethal pathotype specific gene with sequence similarity to SAM-dependent methyltransferase (LaeA), had lower infectivity and showed highly reduced virulence, but no changes in morphology, fungal growth or conidiation were observed. Several putative secreted effector proteins that probably contribute to V. nonalfalfae colonization of hop were identified in this study. Among them, LaeA gene homolog was found to act as a potential novel virulence effector of V. nonalfalfae. The combined results will serve for future characterization of V. nonalfalfae effectors, which will advance our understanding of Verticillium wilt disease.

Highlights

The vascular plant pathogen Verticillium nonalfalfae causes Verticillium wilt in several important crops
Several putative secreted effector proteins that probably contribute to V. nonalfalfae colonization of hop were identified in this study
LaeA gene homolog was found to act as a potential novel virulence effector of V. nonalfalfae

Summary

Introduction

The vascular plant pathogen Verticillium nonalfalfae causes Verticillium wilt in several important crops. The prediction of fungal effectors, mainly small secreted proteins, which typically lack conserved sequence motifs and structural folds, is challenging and largely based on broad criteria, such as the presence of a secretion signal, no similarities with other protein domains, relatively small size, high cysteine content and species-specificity [8,9,10]. Using these features to mine predicted secretomes for candidate effectors has been valuable, but has not produced a one-size-fits-all solution [11]. The EffectorP application has recently been presented as the first machine learning approach to predicting fungal effectors with over 80% sensitivity and specificity [16]

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