微丝结合蛋白GhVLN4调控棉花抗黄萎病的分子机制研究

Actin cytoskeleton is a highly structured and dynamic system, and involved in plant immune responses. Dynamics of the actin cytoskeleton is the underlying of its various functional roles, and this process requires the activity of actin-binding proteins. In previous studies, we found that expression of GhVLN4 in cotton plants was induced by Verticillium dahliae infection and responded to the application of defense signaling molecules, including salicylic acid, ethylene, and abscisic acid. Moreover, our results also showed that the bundling of actin filaments was regulated by actin-binding protein GhVLN4 and rearrangement of actin cytoskeleton in GhVLN4-overexpressing Arabidopsis plants conferred enhanced tolerance to V. dahliae, while knockdown of GhVLN4 expression led to increased susceptibility of cotton plants to the pathogen. In this project, we attempt to elucidate the expression patterns of GhVLN4 using qRT-PCR and functional analysis of the promoter, and further clarify the functional role of GhVLN4 through identifying the tolerance to V. dahliae in transgenic cotton lines. The effect of actin cytoskeleton reorganization on V. dahliae infection will be detected by employing the fluorescence-labeled technique. Meanwhile, we will investigate the molecular mechanism of GhVLN4 in defense against V. dahliae by screening and identification the interaction proteins of GhVLN4, transcriptome sequencing, content determining of endogenous hormone, and examining the expression changes of the genes related to phytohormonal signaling pathways. This research is aimed at providing evidence in the coordination of actin cytoskeleton and phytohormone signaling involved with plants against pathogens, clarifying the resistance mechanism of cotton against V. dahliae from a new angle, and generating novel ideas of controlling Verticillium wilt and molecular breeding for cotton resistance to V. dahliae.

微丝骨架是一个高度结构化和动态化的系统，参与植物免疫反应。微丝骨架的动态变化是其行使功能的基础，而该过程需要微丝结合蛋白的调控。前期工作中，我们发现微丝结合蛋白基因GhVLN4的表达受激素和黄萎病菌的胁迫诱导，微丝成束度受GhVLN4的调控，微丝骨架重排导致过表达拟南芥植株对黄萎病的抗性增强，而VIGS沉默棉花植株对黄萎病菌更敏感。本项目拟利用qRT-PCR及启动子功能分析，阐明GhVLN4的表达模式；通过转基因棉花黄萎病抗性鉴定，进一步明确其生物学功能；利用荧光标记技术，分析微丝骨架重排对黄萎病菌侵染的影响；通过互作蛋白筛选、鉴定，转录组测序，内源激素含量测定和激素信号途径中相关基因的差异表达分析，解析GhVLN4参与抗黄萎病的分子通路。本研究将完善微丝骨架系统与激素信号途径协调参与植物抗病原菌的调控网络，有望从全新角度阐明棉花抗黄萎病机制，为该病防治和棉花抗黄萎病分子育种提供新思路。

微丝骨架是细胞结构和信号转导的平台，受多种微丝结合蛋白（actin-binding proteins，ABPs）的调控。villin（VLN）是一种负责微丝成束的主要ABP，在植物生长发育中起着重要作用。然而，villin在逆境胁迫响应中的功能知之甚少。本项目通过细胞生物学、分子生物学、生物信息学、代谢组学、基因工程等手段，揭示了GhVLN4在黄萎病防御应答以及干旱和盐胁迫响应中的功能。qRT-PCR分析表明，GhVLN4在包括根、茎和伸长期纤维在内的伸长器官中优势表达，并且受赤霉素、乙烯、脱落酸、水杨酸、茉莉酸、盐、干旱和黄萎病菌（大丽轮枝菌Vd991）诱导而上调表达；VIGS沉默GhVLN4，沉默植株更易受Vd991侵染，真菌在沉默植株受感染茎的韧皮部和木质部中优先定殖和快速生长；农杆菌介导的遗传转化法过表达GhVLN4，与野生型相比，过表达植株表现出更强的黄萎病抗性，以及对盐、干旱非生物逆境胁迫具有更高的耐性；激素防御信号途径相关基因的表达分析表明，GhVLN4可能是通过增强SA介导的系统获得性抗性来增强过表达植株对Vd991的抗性；在盐和/或干旱胁迫下，GhVLN4通过调节气孔孔径以及 ABA依赖性和 ABA 非依赖性信号通路增强植株的耐受性。除此之外，本项目鉴定并分析了陆地棉基因组中的14个villin家族基因。这14个GhVLNs具有高度保守的基因结构和保守结构域分布；GhVLNs表现出多种表达模式，一些在特定组织如根、花瓣、雄蕊或伸长的纤维中积累，一些受环境变化诱导，特别是GhVLN3和GhVLN10在伸长的纤维中高度优势表达，并且在非生物（盐、PEG、冷和热）和生物（Vd991）胁迫下明显上调表达。本项目为优质、多抗棉花种质资源的创制提供基因资源。