A Glutamic Acid-Rich Protein Identified in Verticillium dahliae from an Insertional Mutagenesis Affects Microsclerotial Formation and Pathogenicity

Feng Gao,Pei-Song Jia,Bang-Jun Zhou,Yun-Long Zhao,Hui-Shan Guo,Hui Li,Pan Zhao,Guo-Ying Li,Gui-Xian Xia,Alexander Idnurm

doi:10.1371/journal.pone.0015319

Abstract

Verticillium dahliae Kleb. is a phytopathogenic fungus that causes wilt disease in a wide range of crops, including cotton. The life cycle of V. dahliae includes three vegetative phases: parasitic, saprophytic and dormant. The dormant microsclerotia are the primary infectious propagules, which germinate when they are stimulated by root exudates. In this study, we report the first application of Agrobacterium tumefaciens-mediated transformation (ATMT) for construction of insertional mutants from a virulent defoliating isolate of V. dahliae (V592). Changes in morphology, especially a lack of melanized microsclerotia or pigmentation traits, were observed in mutants. Together with the established laboratory unimpaired root dip-inoculation approach, we found insertional mutants to be affected in their pathogenicities in cotton. One of the genes tagged in a pathogenicity mutant encoded a glutamic acid-rich protein (VdGARP1), which shared no significant similarity to any known annotated gene. The vdgarp1 mutant showed vigorous mycelium growth with a significant delay in melanized microsclerotial formation. The expression of VdGARP1 in the wild type V529 was organ-specific and differentially regulated by different stress agencies and conditions, in addition to being stimulated by cotton root extract in liquid culture medium. Under extreme infertile nutrient conditions, VdGARP1 was not necessary for melanized microsclerotial formation. Taken together, our data suggest that VdGARP1 plays an important role in sensing infertile nutrient conditions in infected cells to promote a transfer from saprophytic to dormant microsclerotia for long-term survival. Overall, our findings indicate that insertional mutagenesis by ATMT is a valuable tool for the genome-wide analysis of gene function and identification of pathogenicity genes in this important cotton pathogen.

Highlights

Cotton wilt disease, caused by the phytopathogenic fungus Verticillium dahliae Kleb., is one of the most widespread, damaging diseases in most cotton-growing countries, including China [1], the Americas [2,3] and Mediterranean regions [4]
The multiple and various degrees of mutant traits indicated that mutant phenotypes resulted from V. dahliae genes affected by T-DNA random insertion
To examine the severe inhibition of melanized microsclerotial development and defective pathogenicity that resulted from the loss of VdGARP1 function, we introduced a copy of the VdGARP1 genomic sequence of wild type V592, including the upstream 1600-bp putative promoter region, into the mutant vdgarp1 by Agrobacterium tumefaciens-mediated transformation (ATMT)

Summary

Introduction

Cotton wilt disease, caused by the phytopathogenic fungus Verticillium dahliae Kleb., is one of the most widespread, damaging diseases in most cotton-growing countries, including China [1], the Americas [2,3] and Mediterranean regions [4]. Colonization of cotton roots by V. dahliae in soil naturally leads to colonization of vascular tissues in cotton [6,7]. Fungal hyphae grow from the root surface toward the cortical tissue that is adjacent to the stele [7] and subsequently attack the aerial parts of the plant. Vascular discoloration is a key diagnostic symptom of V. dahliae infection. V. dahliae-infected cotton plants show symptoms including leaf vein browning and chlorosis, wilting, premature defoliation, and most severely, plant death [1,8]

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Conclusion