大豆斑疹病菌羧基蛋白酶前体基因的功能分析

Functional analysis of xanthomonappepsin precursor from soybean pathogen Xanthomonas axonopodis pv. glycines

大豆斑疹病菌(Xanthomonas axonopodis pv. glycines,Xag)具有强烈的胞外蛋白酶活性,然而编码其羧基蛋白酶前体(xanthomonappepsin precursor,Xpp)的xpp基因是否参与Xag的蛋白酶活性与致病性,并不清楚。为了阐明Xpp的作用,通过同源重组技术获得了Xag的xpp基因缺失突变体(NΔxpp),并对其功能进行研究。研究表明:该突变体在寄主大豆上的生长能力和致病力相对于野生型均显著降低;功能互补恢复NΔxpp的植物表型至野生型水平。蛋白酶实验表明,NΔxpp胞外蛋白酶活性相对于野生型并无变化;而且xpp基因异源表达也不具有蛋白酶活性。qRT-PCR结果显示,DSF(diffusible signal factor)信号通路及Clp(Crp-like protein)显著负调控xpp基因的转录表达;与大豆汁液互作时xpp基因受诱导表达,并受HrpX和HrpG正调控。这些结果说明:xpp基因不参与Xag的蛋白酶活性,但其是病原菌在寄主大豆上具有致病力所需的。

Intense extracellular protease activity,usually contributed to the virulence of Xanthomonas axonopodis pv.glycines(Xag),is unclear whether xanthomonappepsin precursor(Xpp)encoded by xpp is involved in.To elucidate Xpp biological functions,we generated the xpp deletion mutant(NΔxpp)by allelic exchange.Compared with the wild-type,the resulting xpp mutant exhibited significantly reduced on reproduction or growth and virulence in soybean host.The lost properties in NΔxpp were completely restored by the wild-type xpp.Protease assays showed that NΔxpp displayed the protease activity similarly to that of the wild-type,and the heterologous expression of xpp also did not take on the protease activity.qRT-PCRs confirmed that the transcription of xpp was significantly negatively regulated by DSF(diffusible signal factor)signals pathway and Clp(Crp-like protein).The expression of xpp was also inducible in soybean juice rather than in NYG medium,and positively regulated by Hrp G and Hrp X.Our results suggest that xpp is required for pathogenicity to soybean independent of the protease activity.