Functional Analysis of the Kinome of the Wheat Scab Fungus Fusarium graminearum

Chenfang Wang,Shijie Zhang,Qian Zheng,Jin-Rong Xu,Zhensheng Kang,Dawei Zheng,H Corby Kistler,Zhongtao Zhao,Guanghui Wang,Huiquan Liu,Xuli Gao,Ji-Wen Ma,Qijun Xu,Rui Hou

doi:10.1371/journal.ppat.1002460

Abstract

As in other eukaryotes, protein kinases play major regulatory roles in filamentous fungi. Although the genomes of many plant pathogenic fungi have been sequenced, systematic characterization of their kinomes has not been reported. The wheat scab fungus Fusarium graminearum has 116 protein kinases (PK) genes. Although twenty of them appeared to be essential, we generated deletion mutants for the other 96 PK genes, including 12 orthologs of essential genes in yeast. All of the PK mutants were assayed for changes in 17 phenotypes, including growth, conidiation, pathogenesis, stress responses, and sexual reproduction. Overall, deletion of 64 PK genes resulted in at least one of the phenotypes examined, including three mutants blocked in conidiation and five mutants with increased tolerance to hyperosmotic stress. In total, 42 PK mutants were significantly reduced in virulence or non-pathogenic, including mutants deleted of key components of the cAMP signaling and three MAPK pathways. A number of these PK genes, including Fg03146 and Fg04770 that are unique to filamentous fungi, are dispensable for hyphal growth and likely encode novel fungal virulence factors. Ascospores play a critical role in the initiation of wheat scab. Twenty-six PK mutants were blocked in perithecia formation or aborted in ascosporogenesis. Additional 19 mutants were defective in ascospore release or morphology. Interestingly, F. graminearum contains two aurora kinase genes with distinct functions, which has not been reported in fungi. In addition, we used the interlog approach to predict the PK-PK and PK-protein interaction networks of F. graminearum. Several predicted interactions were verified with yeast two-hybrid or co-immunoprecipitation assays. To our knowledge, this is the first functional characterization of the kinome in plant pathogenic fungi. Protein kinase genes important for various aspects of growth, developmental, and infection processes in F. graminearum were identified in this study.

Highlights

In eukaryotic organisms, reversible protein phosphorylation by protein kinase (PK) is involved in the regulation of various growth and developmental processes and responses to environmental stimuli
F. graminearum has two orthologs of IPL1 (Fg06959 and Fg02399) and CDC28 (Fg08468 and Fg03132), which are single copy genes in yeast. It contains 28 putative PK genes, including Fg01058, Fg02488, Fg00792, and Fg01559, that have no distinct orthologs in S. cerevisiae (Table S1)
Mutants deleted of the Fg00408, Fg08691, and Fg09612 genes that are orthologous to yeast SSK22, PBS2, and HOG1 [30] were blocked in perithecium formation (Table 3). These results indicate that all three MAP kinase (MAPK) pathways are important for sexual reproduction in F. graminearum

Summary

Introduction

Reversible protein phosphorylation by protein kinase (PK) is involved in the regulation of various growth and developmental processes and responses to environmental stimuli. The eukaryotic PK superfamily consists of conventional and atypical protein kinases. Conventional PKs (ePKs) have been classified into eight groups, AGC, CAMK, CK1, CMGC, RGC, STE, TK, and TKL, based on their similarities in amino acid sequences, domain structures, and modes of regulation [2,3]. Protein kinases with a conserved kinase domain (PF00069) but not classified into these eight groups are categorized as the ‘other’ group of ePKs. Atypical PKs (aPKs) lack significant sequence similarity with ePKs. Four groups of aPKs, Alpha, PIKK, PDHK, and RIO, are known to possess protein kinase activity [2,3]

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Results

Conclusion