BcCFEM1, a CFEM Domain-Containing Protein with Putative GPI-Anchored Site, Is Involved in Pathogenicity, Conidial Production, and Stress Tolerance in Botrytis cinerea.

Wenjun Zhu,Fang Peng,Xiaowen Xu,Wei Wei,Shaopeng Zhang,Yang Zhou,Ping Chen,Yayun Wu

doi:10.3389/fmicb.2017.01807

Abstract

We experimentally isolated and characterized a CFEM protein with putative GPI-anchored site BcCFEM1 in Botrytis cinerea. BcCFEM1 contains a CFEM (common in several fungal extracellular membrane proteins) domain with the characteristic eight cysteine residues at N terminus, and a predicted GPI modification site at C terminus. BcCFEM1 was significantly up-regulated during early stage of infection on bean leaves and induced chlorosis in Nicotiana benthamiana leaves using Agrobacterium infiltration method. Targeted deletion of BcCFEM1 in B. cinerea affected virulence, conidial production and stress tolerance, but not growth rate, conidial germination, colony morphology, and sclerotial formation. However, over expression of BcCFEM1 did not make any observable phenotype change. Therefore, our data suggested that BcCFEM1 contributes to virulence, conidial production, and stress tolerance. These findings further enhance our understanding on the sophisticated pathogenicity of B. cinerea beyond necrotrophic stage, highlighting the importance of CFEM protein to B. cinerea and other broad-host-range necrotrophic pathogens.

Highlights

The ascomycetous fungus Botrytis cinerea is an economically important and destructive necrotrophic fungal pathogen with the capability of causing gray mold disease on more than 200 plant species (Williamson et al, 2007)
Results demonstrated that the pathogenicity of BcCFEM1 was decreased compared to wild type (WT), BcCFEM1-Com and OEBcCFEM1 strains, but over expression of BcCFEM1 had no visible effect on virulence (Figure 6A)
To investigate whether BcCFEM1 is involved in external stress tolerance, growth assays of indicated strains on PDA media supplemented with salt stress (1 M NaCl), osmotic stress (1 M Sorbitol), H2O2 (20 mM) and cell wall stress (0.3 mg/ml Calcofluor White (CFW), 0.02% SDS, and 0.5 mg/ml Congo Red (CR)) were respectively analyzed as previously described (PérezHernández et al, 2017)

Summary

Introduction

The ascomycetous fungus Botrytis cinerea is an economically important and destructive necrotrophic fungal pathogen with the capability of causing gray mold disease on more than 200 plant species (Williamson et al, 2007). B. cinerea is responsible for leading to serious economic losses each year in harvested fruits and vegetables during storage stage (van Kan, 2006; Williamson et al, 2007), and has been ranked as the second most important plant pathogenic fungus (Dean et al, 2012). Necrotrophic pathogens kill host plant cells prior to or during colonization, and feed on dead plant tissue. Hemibiotrophic pathogens initially establish an biotrophic phase with living cells and subsequently switch into the necrotrophic phase and kill plant cells for nutrient acquisition

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Discussion

Conclusion