甘蔗梢腐病病原菌Fusarium sacchari Nep1-like蛋白的筛选鉴定及功能分析

Identification and functional analysis of Nep1-like proteins of Fusarium sacchari,the pathogen of sugarcane pokkah boeng disease

甘蔗镰孢菌Fusarium sacchari(F.sacchari)引起的甘蔗梢腐病严重影响了甘蔗的产量和质量。解析病原菌的致病机理,对指导甘蔗抗病育种和绿色防控具有重要意义。研究发现NLPs(Nep1-like proteins)类基因在真菌侵染及定殖过程中发挥重要作用。本实验在对梢腐病病原菌基因组测序(尚未公布)基础上,通过BLASTP分析得到甘蔗梢腐病病原菌F.sacchari中4个NLP家族基因Fs_00548、Fs_03159、Fs_06646、Fs_11062。将克隆到的3个基因(Fs_00548、Fs_03159、Fs_11062;Fs_06646未克隆到)构建至PVX载体,利用农杆菌介导的烟草叶片瞬时表达系统,发现只有Fs_00548可以诱导细胞产生与Bax相似的坏死,Fs_03159和Fs_11062则不能。利用酵母信号肽分泌功能验证方法,发现Fs_00548和Fs_03159的信号肽具有分泌活性。并且Fs_00548的信号肽区域对其发挥功能具有重要作用。qRT-PCR结果显示该基因在侵染过程中均有表达,其中72 h表达量最高,是菌丝中该基因表达量的48倍。以上研究结果表明,Fs_00548在病原菌侵染过程中发挥重要作用,研究结果为进一步探究病原菌与甘蔗互作提供参考。

Pokkah boeng disease,causing agent Fusarium sacchari,is a serious disease on sugarcane and causes substantial reduction in the yield and the quality.It is of great significance to elucidate the pathogenic mechanism,which can provide insights for sugarcane disease resistance breeding.Our studies have found that NLP(Nep1-like protein)genes play important roles in fungal infection and colonization.Based on genome sequence of the pathogen,four NLP genes(Fs_00548,Fs_03159,Fs_06646,and Fs_11062)were selected out through sequence similarity using BLASTP.Among these genes,only Fs_00548 induce cell death in Nicotiana tabacum leaves by transient expression,as consistent with the Bax-induced apoptotic cell death.Moreover,yeast invertase secretion assay showed that Fs_00548 was a secreted protein with a signal peptide that played a key role in the pathogenesis.Infectivity rate of Fs_00548 was further confirmed through qRT-PCR analyses,which reported the maximum expression of Fs_00548 at 72 hours post infection.Altogether,these results suggest that Fs_00548 plays an important role in the infection process of F.sacchari and provide understanding for further research on the interaction between sugarcane and F.sacchari.