新型毒性分泌蛋白基因的鉴定及其在香蕉与枯萎病菌互作中的作用

The secreted proteins from fungal pathogens especially effectors play crucial roles in plant-fungal pathogen interactions. Recently, several novel secreted proteins from fungal phytopathogens were found to be virulence proteins. In previous works, we identified 26 genes encoding novel, putative secreted proteins(FoSP1-26), and found that their expression were up-regulated in the early stage of banana (Musa spp.cv.Brazilian) infection by Fusarium oxysporum f. sp. cubense race 4 B2 strain (Foc4). These imply that the 26 genes may be involved in the early interaction between Foc4 and banana. To test the hypothesis, we will identify the genes encoding secreted virulence proteins from those 26 candidate genes by the gene-deletion assays and pathogenicity tests. After that, gene complementary assays will be performed to further confirm their fuctions. We also will investigate their temporal and spatial expression patterns to know when and where they function. Additionally, we will try to find out conserved sites in their peptide sequences, and create amino acid substitutions to analyze their contributions to functions of the secreted virulence proteins. Finally, we will use digital gene expression sequencing techonology to study the global gene expression changes in banana roots during the host infected by the wild type and gene-deletion strains. These data may provide a great insight into the changes in some biosynthesis, metabolism pathways and defense responses in host roots. After completing the research mentioned above, roles of the genes encoding novel secreted virulence protein in the banana-Foc4 interactions and mechanism involved in would be elucidated. These will contribute to molecular design breeding program of banana against Fusarium wilt and development of some new disease control strategies.

在植物病原真菌中,以效应物为代表的分泌蛋白在植物与病原菌互作中具有重要作用。最近研究发现一些不同于已知效应物的新型分泌蛋白也是真菌中重要的毒性蛋白。在前期研究中,我们从尖孢镰刀菌古巴专化型(Foc4)中鉴定了26个推定的新型分泌蛋白基因(FoSP1-26）,它们在侵染香蕉初期显著上调表达,暗示它们可能参与Foc4与香蕉的早期互作。为验证上述假设,本项目拟通过基因删除和致病性测定等实验，从中鉴定毒性分泌蛋白基因；在此基础上，拟分析它们的时空表达模式，以明确其发生作用时期和作用部位；解析蛋白序列中保守位点，阐明这些位点对毒性分泌蛋白功能的影响；用数字基因表达谱测序分析香蕉受新型毒性蛋白基因突变体侵染后基因表达变化，以从转录水平上了解香蕉根系一些合成代谢途径和防御反应的变化。通过上述研究揭示毒性分泌蛋白在枯萎病菌与香蕉互作中的作用及机制，为今后香蕉抗病分子设计育种提供靶标，为枯萎病防治提供新策略

本项目采用基因敲除方法成功构建了假定分泌蛋白基因（fosp1-26）的敲除载体，并获得了其中24个基因敲除突变体。通过生理生化分析和形态显微观察，发现其中绝大部分假定分泌蛋白基因敲除突变体在生长、发育、产孢、抗氧化和高渗透压胁迫等方面没有发生明显变化，表明敲除这些基因不影响香蕉枯萎病菌的生长、发育和抗氧化和高渗透压胁迫能力。致病性测定结果显示，共有 8个（fosp3、fosp9、fosp10、fosp18、fosp20、fosp22、fosp23、fosp24）假定分泌蛋白基因敲除突变体的致病性显著减弱，其中fosp9敲除突变体减弱最为显著。对fosp9敲除突变体进行互补分析，发现互补菌株的致病性可恢复至野生型水平，这进一步验证fosp9基因是一个致病相关的假定分泌蛋白基因。再者，构建了fosp9自身启动子驱动的fosp9::GFP融合表达载体，并进行了遗传转化和时空表达分析，发现转化菌株在PDA平板上培养5d后，Fosp9::GFP融合蛋白在菌丝体和分生孢子均可大量表达并积累，而在PDB液体中培养5d，融合蛋白在菌丝体和分生孢子中积累显著减少。这表明fosp9是一个分泌蛋白，可分泌至胞外，因而出现在液体中培养时Fosp9::GFP融合蛋白在菌丝和分生孢子中积累量少的现象。对fosp9编码的蛋白质进行点突变，将第80和151位的半胱氨酸残基均突变为丙氨酸，并用此突变基因与GFP基因组成的表达盒互补fosp9敲除突变体，发现在转化菌株中fosp9::gfp融合蛋白表达分泌没有发生明显变化，致病性也与野生型菌株相当。这表明第80和151位的半胱氨酸残基突变不影响Fosp9蛋白的分泌和毒性功能。对fosp9敲除突变体的转录组进行分析，与野生型菌株相比，共有2100多个差异表达基因（DEGs）。对这些DEGs进行GO分析，发现其在细胞组分水平上主要在其中3项富集。在分子功能水平上，DEGs在其中2项富集。进行KEGG分析，发现DEGs主要富集在氨基酸、淀粉和糖代谢等途径中。这些结果表明，Fosp9可能影响细胞组分及跨膜转运蛋白的表达，进而影响氨基酸、淀粉和糖类物质等物质的代谢。这些代谢途径的异常，可能是导致fosp9敲除突变体在侵染过程中表现出毒性减弱的原因。本项目研究为揭示分泌蛋白在香蕉枯萎病菌致病过程中的作用奠定了基础，同时为抗病育种和防治策略制定提供理论指导。