病毒致病因子C4在细胞质膜和叶绿体中毒力功能差异的分子机制研究

Plant viruses cause serious crop losses worldwide, currently posing a threat to food security. Geminiviruses are a family of plant viruses that infect some of the most economically important cultivated plant species at a global scale; among them, Tomato yellow leaf curl virus (TYLCV) is one of the top ten viruses in molecular plant pathology, and the main virus affecting tomato production in China. C4 is the smallest of the six proteins encoded by TYLCV. Notably, C4 is essential for the viral infection and determines the development of symptoms, but its molecular function in the plant cell is unclear. In our group, we have found that C4 shows a dual localization in the plant cell, at the plasma membrane and in chloroplasts. This dual localization has never been described for a pathogen protein before. This dual targeting is consistent with the presence in the protein sequence of two target signals, namely a myristoylation motif and a chloroplast transit peptide. Strikingly, the presence of these two signals is conserved in C4 homologues of other geminiviruses, suggesting functional relevance. Our preliminary data indicate that the C4 protein has distinct virulence functions in these two subcellular compartments. The specific goal of this project is to determine the effect and function of C4 both at the plasma membrane and in chloroplasts, as well as unravel the regulation underpinning this dual targeting. Considering the relevance of the C4 protein for the infection by TYLCV and its determining role in symptom development, gaining insight into the molecular mechanism(s) underlying the virulence activity of C4 will contribute to acquiring a comprehensive view of the viral infection at the molecular level, gaining a better understanding of the interplay between viral infection and alterations in plant development, and assist in the design of effective, sustainable anti-viral strategies for crop protection.

植物病毒引起的作物减产对全球粮食安全造成重大威胁。双生病毒中的番茄黄叶卷曲病毒（TYLCV）是十大植物病原体之一，是影响中国区番茄产量的主要病毒，它编码6个蛋白，C4蛋白是最小的一个，且是病毒侵染所必须的。C4蛋白可以调控植物发育，但其在植物细胞中的分子功能未知。我们发现C4蛋白亚细胞定位在植物质膜和叶绿体中，至今从未发现其他病毒蛋白有这种双亚细胞定位现象。这一定位结果与C4蛋白序列中豆蔻酰化结构域和叶绿体转运肽这两个靶标信号域吻合，而它们在与C4同源的双生病毒中高度保守，表明信号域与功能相关。初步数据显示C4亚定位不同时功能不同，致病毒力有明显区别。我们将采用点突变的方法研究C4蛋白在细胞质膜和叶绿体中双重亚细胞定位的分子调控机制及蛋白功能。研究C4毒力活性的分子机制有助于我们在分子水平全面了解病毒侵染机制，理解病毒侵染和植物发育改变之间的关联，并以此设计高效可持续的抗病毒作物保护策略。

病原体需要通过抑制防御反应从而成功建立入侵感染。RNA干扰(RNAi)被认为是抗病毒防御的主要机制，然而越来越清楚的是，植物有其他防御病毒入侵的策略，包括产生防御激素，如水杨酸(SA)。C4是由双生病毒编码的多功能小蛋白。我们的研究表明，来自番茄黄曲叶病毒和其他双生病毒的C4定位于两个不同的亚细胞室，即质膜(PM)/胞间连丝(PD)和叶绿体。这种双重定位与C4中存在的两个重叠的靶向信号相关：PM定位所需的N -肉豆蔻酰化基序和叶绿体定位所需的叶绿体转运肽。我们发现PM定位的C4可以通过靶向两种植物受体样激酶BAM1和BAM2来抑制RNAi的细胞间移动；另一方面，叶绿体定位的C4通过与钙敏感受体(CAS)的相互作用，干扰细胞器到细胞核的逆行信号，抑制病原体感知时SA生物合成的激活。因此，C4在抑制植物防御中发挥双重作用，提高病毒性能。有趣的是，这种双重定位信号引导的在质膜和叶绿体的双重定位也出现在许多病原体效应因子以及一系列防御相关的植物蛋白中，这表明植物可能存在一条内在的途径，将蛋白质从PM传递到叶绿体，并被植物病原体劫持，以抑制下游的防御反应。综上所述，我们的研究说明了病原体效应因子的亚细胞分隔是如何影响其功能多样性，从而提高致病性能。