假禾谷镰孢转录因子FpAPSES的鉴定与功能分析

[目的]假禾谷镰孢(Fusarium pseudograminearum)侵染小麦引起的小麦茎基腐病严重影响中国小麦的安全生产.查找假禾谷镰孢中的APSES转录因子,分析其在病原菌致病过程中的作用,为解析假禾谷镰孢的致病机制及小麦茎基腐病的防治提供理论依据.[方法]从GenBank获得物种中已知的APSES氨基酸序列,...

Identification and Functional Analysis of Transcription Factors FpAPSES in Fusarium pseudograminearum

【Objective】Fusarium crown rot (FCR) caused by Fusarium pseudograminearum seriously affects wheat production in China. The objective of this study is to find and analyze the functions of APSES transcription factors in F. pseudograminearum, and to provide theoretical basis for revealing the pathogenic mechanism of F. pseudograminearum and prevention and treatment of FCR.【Method】The known APSES proteins were obtained from GenBank, and APSES candidates in F. pseudograminearum were found by BLASTP. Domains of FpAPSES proteins were determined using Pfam. The phylogenetic tree of APSES proteins was constructed using MEGA 5.05. The relative expression levels of FpAPSES1 and FpAPSES4 during infection were examined by quantitative RT-PCR (qRT-PCR). FpAPSES1 and FpAPSES4 deletion mutants were generated by polyethylene glycol (PEG)-mediated protoplast fungal transformation, and screened by PCR. The wild type, Δfpapses1 and Δfpapses4 strains were activated on PDA medium, and cultured on PDA for mycelial growth and morphology assays; mycelial blocks were introduced into liquid CMC medium to assess conidiation and conidia morphology; conidia were cultured in sterile distilled water to explore conidia germination; mycelial blocks or conidia suspension were prepared and inoculated wheat coleoptiles and barely leaves to explore pathogenicity. The pot-culture experiment was also used for virulence assay. ELISA was used to detect deoxynivalenol (DON) in inoculation millet. 【Result】 Four APSES candidates were found in F. pseudograminearum, which all contained the conserved DNA binding domain HTH. The phylogenetic tree analysis showed that FpAPSES1, FpAPSES2 and FpAPSES4 were divided into A group, while FpAPSES3 belonged to the C group. The qRT-PCR analysis revealed that FpAPSES1 and FpAPSES4 were up-regulated at infection stages, which suggested that FpAPSES1 and FpAPSES4 might play important roles in infection. Two FpAPSES1 deletion mutants (Δfpapses1-T10 and Δfpapses1-T27) and two FpAPSES4 deletion mutants (Δfpapses4-T1 and Δfpapses4-T2) were obtained. Compared with the wild type strain, both FpAPSES1- and FpAPSES4-deleted mutants exhibited significantly reduced colony growth rates. FpAPSES1-deleted mutants had normal hyphal branches, while FpAPSES4-deleted mutants exhibited curved and more branched hypha. Both FpAPSES1- and FpAPSES4-deleted mutants exhibited significantly reduced conidiation in CMC, and the conidia numbers were respectively reduced by 99.5% and 97.4% comparing with that of the wild type. Furthermore, conidia of FpAPSES1- and FpAPSES4-deleted mutants were shorter, less septa and lower germination rates. The wheat coleoptiles were point-inoculated with mycelial blocks or conidia, and the virulence of FpAPSES1- and FpAPSES4-deleted mutants was significantly reduced comparing to the wild type. Reduced pathogenicity was further observed by barely leaves inoculation and pot culture experiment. The DON levels in millet upon infection with FpAPSES1- and FpAPSES4-deleted mutants were reduced by 78% and 44% comparing to the wild type, respectively.【Conclusion】Both A group APSES homologs FpAPSES1 and FpAPSES4 play important roles in growth, conidiation and pathogenicity of F. pseudograminearum.