水稻条斑病菌hrpD5基因在致病性中的功能分析

Functional analysis of the hrpD5 gene in pathogenesis of rice pathogen Xanthomonas oryzae pv. oryzicola

水稻条斑病菌(Xanthomonas oryzae pv.oryzicola,Xoc)成功侵染水稻主要依靠其III型分泌系统(Type III secretion system,T3SS)分泌的效应蛋白。T3SS由hrp-hrc-hpa基因编码,其中主要的hrp和hrc基因突变,病原菌将丧失在寄主水稻上的致病性和非寄主烟草上的过敏性反应(hypersensitive response,HR)。hrpD5基因位于hrp基因簇hrpD操纵单元的第5个基因,在致病性中的功能未知。本研究构建了Xoc的hrpD5缺失突变体RΔhrpD5。植物接种试验显示,RΔhrpD5丧失了对寄主水稻的致病性和在非寄主烟草上激发HR反应的能力;功能互补子虽然能够恢复这2种表型,但是,与野生型菌株相比,其在感病水稻上的毒性显著降低,在非寄主烟草上形成延迟的HR反应;荧光定量PCR结果显示:hrpD5缺失影响其下游hrpD操纵单元基因hrpD6、hrpE和hpaB以及HrpX操纵单元基因hrpF和hrpB1的表达;同时,hrpD5缺失降低了hrpX的mRNA水平,但是不影响hrpX的启动子活性;酵母双杂交结果显示,HrpD5蛋白能够与HrpF蛋白的N端互作。这些结果暗示在Xoc中hrpD5不仅为主要的致病相关基因,而且调控主要的hrp调节基因hrpX的表达。HrpD5新功能的鉴定将为解析T3SS在致病性中的功能提供线索。

Xanthomonas oryzae pv. oryzicola depends on Type III secretion system（ T3 SS） to cause disease in host plant rice. T3 SS is encoded by hrp（ hypersensitive response and pathogenicity）,hrc（ hrp conserved） and hpa（ hrp associated） genes. Mutations of essential hrp or hrc genes usually abolish the bacterial ability to cause pathogenicity in host rice and hypersensitive response（ HR） in non-host tobacco. The hrpD5 gene is the fifth gene with unknown function located in the hrpD operon of the hrp gene cluster. In this work,we constructed a deletion mutant of hrpD5 in Xoc strain RS105（ named as RΔhrpD5）. Inoculation assays showed that RΔhrpD5 was not able to cause pathogenicity in host rice and failed to trigger HR in non-host tobacco. The lost functions were partially restored in the complementary strain CRΔhrpD5 which had reduced virulence on the susceptible rice and caused delayed HR on non-host tobacco compared to the wild type. Transcriptional analysis showed that mutation of hrpD5 affected the expression of downstream genes hrpD6,hrpE and hpaB in the hrpD operon and genes hrpF and hrpB1 in the HrpX operon,and led to decreased level of hrpX mRNA,but didn’t affect the promoter activity of hrpX. The yeast two-hybrid analysis revealed that HrpD5 interacted with the N terminal ofHrpF. These results indicate that hrpD5 not only is essential for the pathogenicity of Xoc,but also regulates the expression of hrpX,the major hrp regulatory gene. Further functional investigation of HrpD5 may provide some clues to elucidate the roles of T3 SS in pathogenicity of Xoc.