甘蔗镰孢菌碳水化合物结合模块效应基因Fs11724的分离鉴定及功能分析

Identification and Functional Analysis of Carbohydrate Binding Module Effector Gene Fs11724 of Fusarium sacchari

甘蔗镰孢菌(Fusarium sacchari)是引起毁灭性病害甘蔗梢腐病(sugarcane pokkah boeng disease, PBD)的主要病原菌之一。病原菌分泌的效应蛋白在病原与植物互作中发挥至关重要的作用。本研究基于课题组前期甘蔗镰孢菌全基因测序数据,设计基因特异性引物,通过RT-PCR扩增得到一个新的含CBM4;结构域的效应蛋白基因,并对其功能进行了初步探索。序列分析表明Fs11724基因开放阅读框822 bp,编码273个氨基酸,分子量为28.24 kDa,理论pI值为4.29,亲水性平均系数–0.072,属于亲水性分泌蛋白;通过农杆菌介导的烟草瞬时表达系统证实Fs11724抑制由BAX诱导的细胞程序性死亡,表明Fs11724具有潜在抑制寄主防御反应的毒性功能;同时,利用酵母分泌系统对其编码蛋白信号肽的分泌功能进行验证,确定该基因编码蛋白为典型分泌蛋白;qRT-PCR结果分析表明Fs11724在甘蔗镰孢菌侵染前、后期上调表达,表达量在168 hpi达到峰值。该研究表明Fs11724编码蛋白为甘蔗镰孢菌F. sacchari的一个效应蛋白,其信号肽具有分泌功能,并且可以在非寄主植物烟草上抑制BAX诱导的细胞坏死,在真菌侵染植物过程中显著表达,可能在调控植物免疫反应中发挥作用。研究结果为进一步研究病原–甘蔗互作,揭示甘蔗镰孢菌发病机制奠定基础。

Fusarium sacchari is one of the main pathogens causing the devastating disease(sugarcane pokkah boeng disease, PBD). Effector proteins secreted by pathogens play an important role in the interaction between pathogens and plants. In this study, the gene-specific primer was designed based on the previous sequencing data of the whole genome of F. sacchari. A new effector protein gene containing the CBM4_9 domain(carbohydrate-binding module, CBM) was amplified by reverse transcription PCR(RT-PCR). Subsequently, we conducted a preliminary exploration of its function.Sequence analysis showed that Fs11724 contained a 822 bp open reading frame(ORF) encoding a protein of 273 amino acids residues with predicted molecular mass of 28.24 kDa and theoretical isoelectric point(pI) of 4.29. Through hydrophobicity analysis by ProtParam, the grand average of hydropathicity(GRAVY) of the protein encoded by Fs11724 was –0.072, which was less than zero, indicating that the protein encoded by Fs11724 might be a hydrophilic secreted protein. The toxic function of Fs11724 gene was identified by Agrobacterium-mediated transient expression system in Nicotiana benthamiana, The result showed that Fs11724 suppressed BAX-triggered programmed cell death in N. benthamiana leaves, indicating its potential toxic function on inhibiting host plant defense response. At the same time, the secretory function of the predicted signal peptide of Fs11724 was verified by yeast secretory system, as a result, the protein encoded by Fs11724 was identified as a typical secretory protein. In addition, the gene expression level was monitored by quantitative real-time PCR(qRT-PCR) experiments. The results and analysis of qRT-PCR indicated that the expression of Fs11724 showed the overall upregulated trend not only at the early phase of F. sacchari infestation but also at the late phase of F. sacchari infestation. And the gene expression reached the highest expression level at 168 hours post inoculation(hpi). In conclusion, our results in this study suggested that the protein encoded by Fs11724 was an extracellular effector protein of F. sacchari. The signal peptide of this protein had secretory function. In addition,Fs11724 could inhibit BAX-induced programmed cell death in non-host plant N. benthamiana and significantly expressed in the process of pathogenic fungi infecting plants. Therefore, it was speculated that Fs11724 might play a role in regulation of plant immune response. The results would lay a foundation for further research on pathogen-sugarcane interaction and revealing the molecular pathogenesis of F. sacchari.