小麦ALP蛋白通过与FgSUN1互作抑制禾谷镰刀菌生长的分子机制

Fusarium graminearum is the major casual agent of fusarium head blight (FHB), a severe wheat disease worldwide. Avenin-like proteins (ALPs) are a type of wheat seed storage proteins, and show positive effects on dough strength. Interestingly, exogenous application of ALPs can significantly reduce the spread of mycelia of F. graminearum on plates. However, the underlying mechanisms remain unclear. We identified a F. graminearum gene FgSUN1 that interacts with ALP, by screening a yeast two-hybrid library for F. graminearum. The physical interaction between FgSUN1 and ALP proteins were confirmed by yeast two-hybrid and bimolecular fluorescence complementation experiments. On the basis of the previous results, this project aims to clarify the molecular mechanisms on the inhibiting effect of wheat ALP proteins on mycelial growth by analyzing the interaction between ALP and FgSUN1 with the following experiments: 1) creating knockout, complementation and overexpression mutants for FgSUN1; 2) in vitro expression, purification and renaturation of FgSUN1 protein; 3) enzyme-linked immunosorbent assay; 4) subcellular localization; 5) wheat transformation; 6) wheat inoculation and virulence assay; 7) microsectile observation and electron microscope observation; 8) gene expression analysis. Based on these experiments, we can characterize the function of FgSUN1 in F. graminearum, and clarify how ALP proteins reduce fungal mycelial growth by interacting with FgSUN1 both on plates and on wheat. This research would deepen our understanding of the molecular mechanisms of wheat seeds in response to the infection of F. graminearum, and provide reference information for the reduction of yield loss and mycotoxin contamination in seeds.

赤霉病危害严重，是小麦的重要病害之一，其主要致病菌是禾谷镰刀菌。ALP蛋白是一种小麦种子储藏蛋白，与加工品质密切相关。前期研究表明ALP蛋白可显著抑制禾谷镰刀菌菌丝的生长，但其作用机制不明。本项目通过筛选禾谷镰刀菌酵母文库，获得与ALP互作的FgSUN1基因。酵母双杂和双分子荧光互补实验证明ALP与FgSUN1存在互作。本项目拟在前期工作基础上，综合利用禾谷镰刀菌基因敲除、回复和过表达技术、体外表达纯化和蛋白复性技术、酶联免疫技术、亚细胞定位技术、小麦转基因、小麦接种实验、显微切片观察和电镜观察、基因表达分析等技术，鉴定FgSUN1的功能，明确ALP蛋白通过与FgSUN1互作抑制禾谷镰刀菌生长的分子机制。本研究将有助于了解小麦种子应对禾谷镰刀菌侵染的分子机制，为降低籽粒的真菌毒素累积量和产量损失提供有益参考。

赤霉病严重危害小麦的产量和品质，是小麦的重要病害之一，其主要致病菌是禾谷镰刀菌。种子储藏蛋白对小麦的产量和品质均有重要贡献，也影响小麦的赤霉病抗性。本项目系统鉴定了小麦ALP（avenin-like protein）类种子储藏蛋白通过与FgSUN1互作抑制禾谷镰刀菌菌丝生长的分子机制。发现,ALP蛋白的表达受禾谷镰刀菌诱导；ALP仅在小麦胚、糊粉层和传递细胞中表达；ALP蛋白直接且显著抑制禾谷镰刀菌菌丝的生长;ALP与禾谷镰刀菌FgSUN1蛋白物理互作，且亚细胞定位存在重叠；FgSUN1蛋白具有β-葡萄糖苷酶活性，而ALP蛋白显著抑制其酶活；FgSUN1基因缺失导致禾谷镰刀菌孢子变短，隔膜数减少，菌丝分泌物增多，菌丝生长受到抑制，且敲除突变体的致病力显著降低；小麦接种实验表明相比野生型小麦，过表达ALP后小麦籽粒的赤霉病抗性显著增强，下调表达则相反。综上，ALP蛋白通过抑制禾谷镰刀菌FgSUN1蛋白的β-葡萄糖苷酶活性提高小麦籽粒对禾谷镰刀菌的抗性，可进一步挖掘优异ALP基因，应用于小麦育种。