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

该建议侧重于一种称为系统性获得性抗性（SAR）的独特形式的全植物免疫。当微生物病原体的局部原发感染导致产生系统传输的移动信号时，SAR就会被诱导，这些信号使植物未感染的部分准备好抵抗未来广泛病原体的感染。SAR的机制非常复杂，虽然早期的研究集中在SAR上，其中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是在非致病性或毒性病原体的挑战下发生的。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受通用关键信号启动过程调控的假设，本研究提出了令人兴奋的新发现，并部署了新的工具来揭示经典植物抗病蛋白识别诱导SAR的早期机制。虽然随着时间的推移，被感染的叶片似乎继续产生和传递信号，但SAR信号的产生和传输发生在初次感染后3-6小时的很短和早期的时间框架内。这种移植物传递信号在整个植株中移动，可能主要通过韧皮部以顶尖的方式移动。此外，移动信号要么包含蛋白质成分，要么需要它们来运动/功能。总之，要符合移动信号的条件，生物分子必须是SAR所必需的，必须在原发性感染后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确定，但它似乎在单子花和双子花之间是保守的，重要的是，它诱导对多种病原体和害虫的免疫。迄今为止，已经确定了许多诱发sar的化学物质，其中一些化学物质具有物理流动性，或者由于其挥发性而被认为具有流动性。这些物质包括水杨酸（SA）及其衍生物甲基SA （MeSA）、壬二酸（AzA）、甘油-3-磷酸（G3P）、脱氢枞醇（DA）、活性氮和活性氧以及n -羟基胡椒酸（NHP）等。这些化学物质在外源应用时赋予系统抗性，并且需要病原体诱导的SAR。为支持这一合作，我们(i)开发了一种新的SAR荧光素酶报告基因，为SAR的建立提供时空背景；（ii）鉴定了两种由反式作用小干扰RNA3a （TAS3a）衍生的21相核苷酸RNA (tasi-RNA)，它们对SAR至关重要。基于它们的合成（3 hpi）后和系统运动（4 hpi）的时间框架，我们提出tasi-RNA作为难以捉摸的早期移动SAR信号。Tasi-RNAs正调控先前鉴定的脂质转移蛋白（LTP）样SAR调节因子AZI1 （azelaic acid induced 1）以及LTP3、LTP4和criticala70的基因表达。LTPs调节tasi-RNA的全身转运，基于观察到的LTPs的全身移动性、它们与AZI1的相互作用、含有AZI1的高分子量（HMW）复合物的检测以及AZI1免疫沉淀物中tasi-RNA的存在，我们提出SAR诱导与含有ltp的HMW蛋白复合物介导的tasi-RNA的全身转运有关。本研究将利用计算模型和功能分析来描述LTP-RNA相互作用组及其与A70的关系。最后，利用这些见解，我们将生成支持早期SAR事件的代谢重构的第一个综合分析。在此获得的知识对于发展对这种独特免疫形式的基本了解并促进将其用于制定可持续和环境友好型作物保护战略具有重要意义。

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.