Functional Analysis Of2C Ser/Thr Phosphatases(PTCs) And Tyrosine Phosphatases (PTPs) In Botrytis Cinerea

Protein phosphorylation and dephosphorylation executed by protein kinases and protein phosphatases, respectively, is a major mechanism regulating many cellular processes. Most phosphorylation events in eukaryotes occur at serine, threonine and tyrosine residues. Accordingly, removal of the phosphates is catalyzed by protein Ser/Thr phosphatases or tyrosine phosphatases. Type2C Ser/Thr phosphatases (PP2Cs) and tyrosine phosphatases (PTPs) are involved in a large variety of regulatory processes in many eukaryotes, but little has been known about their functions in filamentous fungi.Botrytis cinerea contains five putative PP2C genes, named BcPTCl,-3,-5,-6and-7, and two putative protein tyrosine phosphatase (PTP) genes (BcPTPA and BcPTPB). Biological functions of these genes were analyzed by gene deletion and complementation. While no phenotypes aberrant from the wild type were observed with mutants of BcPtc5, BcPtc6and BcPtc7, mutants of BcPtc1, BcPtc3, BcPtpA and BcPtpB showed significant phenotype changes, indicating that these four genes play important roles in B. cinerea. Results of this study showed that1) BcPtc1, BcPtc3, BcPtpA and BcPtpB regulate vegetative differentiation. Mutants of all the four genes could not produce any sclerotium. All the mutants had reduced hyphal growth rate except BcPtpB.2) In addition, increased conidiation were observed in BcPtcl, BcPtc3, and BcPtpB mutants, while mutant of BcPtpA had reduced conidiation.3) BcPtc1, BcPtc3, BcPtpA and BcPtpB were involved in regulation of hypal melanization. Disruption of the four genes all led to increased pigmentation and the pigment was confirmed as melanin.4) They exhibited increased sensitivity to osmotic stress mediated by NaCl and KC1, to oxidative stress generated by H2O2, to DCFs fungicides (iprodione) and to cell wall damaging agents (Congo red, caffeine and the cell wall degrading enzyme).5) In S. cerevisiae, Ptc1, Ptc3, Ptp2and Ptp3negatively regulate the high-osmolarity glycerol (HOG) pathway and the cell wall integrity (CWI) pathway. In B. cinerea, however, BcPtc3, but not BcPtcl, negatively regulates phosphorylation of BcSakl (the homologue of Hog1) although both BcPTCl and BcPTC3were able to rescue the growth defects of a yeast PTC1deletion mutant under various stress conditions. Moreover, deletion of BcPTCl or BcPTC3led to decreased phosphorylation of the mitogen-activated protein (MAP) kinase BcBmp3(a homologue of Saccharomyces cerevisiae Mpkl/Slt2in the CWI pathway). In contrast to S. cerevisiae, BcPtpA and BcPtpB positively regulate phosphorylation of BcSak1(the homologue of Hog1) and BcBmp3in B. cinerea.6) All the mutants exhibited dramatically decreased virulence on cucumber, rapeseed or tomato leaves, apples and grapes. These results demonstrated that BcPtc1, BcPtc3, BcPtpA and BcPtpB play important roles in the regulation of hyphal growth, vegetative differentiation, virulence and multi-stress tolerance in Botrytis cinerea.