Anatomy and functions of LTP interactomes and their relationship to small RNA signals in systemic acquired resistance

这项建议侧重于一种独特的全植物免疫形式，称为系统获得性抗性(SAR)。当微生物病原体的局部初次感染导致系统传输的移动信号的产生时，SAR被诱导(S)，该信号为植物未受感染的部分做好准备，以抵御未来广泛范围的病原体的感染。SAR的机制非常复杂，虽然早期的研究集中在SAR上，免疫挑战引发了经典的基因对基因的相互作用，但也有报道称，SAR是在非致病或致病的病原体挑战之后进行的。不同的病理系统和生长条件进一步混淆了比较研究。基于SAR受通用关键信号起始过程调控的假设，该建议解决了令人兴奋的新发现，并部署了新的工具来揭开经典植物抗病蛋白识别诱导SAR的早期机制。SAR信号的产生和传输发生在初侵染后3-6h这一非常短的早期时间段内，尽管受感染的叶片似乎随着时间的推移继续产生和传输信号。这种嫁接传递的信号(S)在整个植物中传递，可能以顶瓣为主的方式通过韧皮部。此外，移动信号要么包含蛋白质成分(S)，要么需要它们来运动/功能。综上所述，要符合移动信号(S)的要求，生物分子必须是合成孔径雷达所必需的，必须在初次感染后4小时内被物理运输到远端组织，并且在局部应用时必须诱导全身抵抗。虽然特定移动信号的身份仍未解决，但它似乎在单子叶植物和双子叶植物之间是保守的，重要的是，它诱导了对各种病原体和害虫的免疫力。到目前为止，已经确定了许多导致SAR的化学品，其中一些具有物理移动性，或者由于其挥发性而被认为是移动性的。这些化合物包括水杨酸(SA)及其衍生物甲基SA(MESA)、氮杂环丁酸(Aza)、3-磷酸甘油(G3P)、脱氢松香酸(DA)、活性氮和氧物种以及N-羟基吡喃甲酸(NHP)等。当外源使用时，这些化学物质具有系统抵抗力，是病原体诱导的SAR所必需的。在最近的突破中，并在这一合作的基础上，我们(I)开发了一种新型的SAR荧光素酶报告，为SAR的建立提供了时空背景；(Ii)确定了来自反式小干扰RNA3a(TAS3a)的两个阶段性21核苷酸RNA(TASI-RNA)对SAR是必需的。基于它们的合成时间框架(3HPI)、后时间框架(POST)和系统运动时间框架(4HPI)，我们认为Tasi-RNAs是难以捉摸的早期移动SAR信号。TASI-RNAs正向调节先前发现的脂转移蛋白(LTP)样SAR调节因子AZI1(杜鹃酸诱导的1)以及LTP3、LTP4和关键的A70的基因的表达。LTPS调节TASI-RNAs的系统转运，根据观察到的LTPS的系统流动性，它们与AZI1的相互作用，检测到含有AZI1的高分子量(HMW)复合体，以及AZI1免疫沉淀物中TASI-RNA的存在，我们认为SAR的诱导与含有LTP的HMW蛋白复合体介导的TASI-RNAs的系统转运有关。该提案将使用计算建模和功能分析来表征LTP-RNA互动组及其与A70的关系。最后，利用这些洞察力，我们将对支撑早期SAR事件的代谢重组进行第一次全面分析。在这里获得的知识将对发展对这种独特的免疫形式的基本了解非常重要，并有助于将其用于制定可持续和环境友好的作物保护战略。

This proposal focuses on a unique form of whole-plant immunity called systemic acquired resistance (SAR). SAR is induced when localized primary infection by microbial pathogens results in the generation of systemically transported mobile signal(s), which prepare the uninfected parts of the plant against future infections by a broad spectrum of pathogens. The mechanisms underlying SAR are remarkably complex and while early studies focused on SAR where the immunizing challenge initiated a classical gene-for-gene interaction, SAR has also been reported following challenges with non-pathogenic or virulent pathogens. Comparative studies are further confounded by different pathosystems and growth conditions. Founded on the assumption that SAR is regulated by universal key signal initiation processes, this proposal addresses exciting new discoveries and deploys new tools to unravel the early mechanisms of SAR induction by classical plant disease resistance protein recognition. The SAR signal generation and transport occur within a very short and early time frame of 3-6 h after primary infection although the infected leaf appears to continue generating and transmitting the signal over time. This graft transmissible signal(s) moves throughout the plant, probalby through the phloem in a predominantly acropetal manner. Additionally, the mobile signal either comprises proteinaceous component(s) or requires them for movement/functionality. In summary, to qualify as the mobile signal(s), a biomolecule must be essential for SAR, must be physically transported to distal tissue within 4 h of primary infection, and must induce systemic resistance when applied in a localized manner. While the identity of a specific mobile signal remains unresolved, it appears to be conserved between monocots and dicots and, importantly, induces immunity to a diverse collection of pathogens and pests. To date numerous SAR-inducing chemicals, some of which exhibit physical mobility or are considered mobile due to their volatile nature, have been identified. These include, salicylic acid (SA), and its derivative methyl SA (MeSA) azelaic acid (AzA), [glycerol-3-phosphate (G3P), dehydroabietinal (DA), reactive nitrogen and oxygen species, and N-hydroxy pipecolic acid (NHP) amongst others. These chemicals confer systemic resistance when applied exogenously and are required for pathogen-induced SAR. In recent breakthroughs, and underpinning this collaboration we have (i) developed a novel SAR luciferase reporter to provide spatial-temporal context to SAR establishment and (ii) identified two phased 21 nucleotide RNA (tasi-RNA) derived from Trans-Acting Small Interfering RNA3a (TAS3a) as essential for SAR. Based on their time frame of synthesis (3 hpi) post and systemic movement (4 hpi) we propose that tasi-RNAs function as the elusive early mobile SAR signal. Tasi-RNAs positively regulates the expression of genes encoding the previously identified lipid transfer protein (LTP)-like SAR regulator AZI1 (azelaic acid induced 1), as well as the LTP3, LTP4 and critically A70. LTPs regulate systemic transport of tasi-RNAs and based on the observed systemic mobility of LTPs, their interactions with AZI1, the detection of high molecular weight (HMW) complexes comprising AZI1 and presence of tasi-RNA in AZI1 immunoprecipitates we propose that SAR induction is associated with LTP-containing HMW protein complex-mediated systemic transport of tasi-RNAs. This proposal will characterize the LTP-RNA interactome and its relationship with A70 using computational modeling and functional analyses. Finally, using these insights we will generate the first comprehensive analysis of the metabolic reconfiguraiton underpinning early SAR events.The knowledge gained here will be important for developing a basic understanding of this unique form of immunity and facilitate its use in developing sustainable and environmentally friendly crop protection strategies.