Domain/domain interactions in RPS4/RRS1 immune complex activation by bacterial effectors

植物病害会造成巨大的作物损失，这对农民来说代价高昂。例如，控制马铃薯晚疫病每年要花费英国农民6000万英镑。抗性(R)基因使植物能够检测和抵抗病原体，但人们对R蛋白的工作原理知之甚少。植物病原体将一种叫做效应物的分子传递到宿主细胞中，干扰宿主的免疫机制。R基因使植物能够识别这些效应物，然后激活免疫。为了克服R基因，病原体必须通过识别的效应物的突变来逃避检测。我们的目标是详细了解R蛋白的分子机制，以便设计新的R基因来识别来自任何病原体的效应物。短期目标是生成智能设计的R蛋白变体文库，可以筛选识别以前未识别的效应物。在我们可以做到这两点之前，我们需要彻底了解R蛋白如何将对效应物的识别转化为防御的激活。我们研究了模式植物拟南芥中的一个R基因位点，该基因对两种不同的细菌和一种真菌具有抗性。该位点由两个R基因RPS4和RRS1组成，它们编码两种蛋白，结合形成一个受体复合体，识别AvrRps4和PopP2细菌效应物。一个相似的，连接的R基因对，RPS4B和RRS1B，也识别AvrRps4，但不识别PopP2。虽然RPS4和RPS4B、RRS1和RRS1B亲缘关系密切（每对相同约60%），但RPS4和RRS1B相互关联但不发挥作用；RPS4B和RRS1也是如此。我们的目标是确定RPS4和RRS1，或RPS4B和RRS1B蛋白结构域之间的特定相互作用，这种相互作用能够形成一个功能复合物，该复合物能够(i)感知效应物，（ii）然后激活信号传导。我们还旨在了解为什么不真实的组合不起作用。RPS4和RPS4B包含4个域（AAAA或BBBB）， RRS1和RRS1B包含5个域（AAAAA或BBBBB）。我们将在同源物之间交换结构域，测试RPS4/RPS4B结构域是否与RRS1结合后交换BAAA、ABAA、AABA或AAAB功能，是否与RRS1B结合后交换ABBB、BABB、BABB和BBBA功能。我们还将测试RRS1/RRS1B互换，如BAAAA， ABAAA等结合RPS4。我们可以利用农杆菌（agroinfiltration）在烟草叶片组织中瞬时表达RPS4和RRS1结构域交换变异体后3天内检测它们的功能。我们还将通过表达这4种蛋白的每个结构域来研究结构域/结构域相互作用，并在酵母和植物共表达和共免疫沉淀中寻找结构域/结构域相互作用。通过移除RRS1末端的一个结构域，或将突变引入该结构域，我们创建了一个RPS4/RRS1复合体，该复合体可以组成性地激活防御。我们将研究这个本构活动的领域要求。野生型RRS1抑制由RPS4和RRS1的本构形式触发的这种本构防御激活；我们将研究这种抑制的机制。RRS1可以与自身形成更高阶的关联，因此我们将研究RRS1结构域/结构域之间的相互作用，这可能解释了这种多元化。此外，RRS1的两端似乎能够相互作用；我们将对此进行更详细的研究。我们可以使用分裂荧光蛋白来监测活细胞中蛋白质/蛋白质或结构域/结构域的相互作用。它们不会单独发出荧光。当它们附着在感兴趣的蛋白质上时，只有当它们所附着的结构域的蛋白质/蛋白质相互作用将它们聚集在一起时，它们才会变得活跃。使用这种方法，我们发现RPS4和RRS1的N端结构域仅在感知AvrRps4或PopP2效应物时才变得密切相关。我们将研究这种关联的时间和遗传要求，我们认为这是导致激活的蛋白质复合物重新配置的关键

Plant disease can cause big crop losses, which is costly to farmers. For example, control of potato late blight costs UK farmers ~ £60M/year. Resistance (R) genes enable plants to detect and resist pathogens, but how R proteins work is very poorly understood. Plant pathogens deliver molecules called effectors into host cells to interfere with host immune mechanisms. R genes enable plants to recognize such effectors and then activate immunity. To overcome R genes, pathogens must evade detection by mutations in recognized effectors. We aim to understand R protein molecular mechanisms in such detail that we can design new R genes to recognize effectors from any pathogen. A shorter-term goal is generate intelligently designed libraries of R protein variants that can be screened for recognition of previously unrecognized effectors. Before we can do either, we need to thoroughly understand how R proteins convert recognition of effectors into activation of defense.We study an R gene locus in the model plant Arabidopsis that confers resistance to two different bacteria, and to a fungus. The locus comprises two R genes, RPS4 and RRS1, which encode two proteins that associate to form a receptor complex that recognizes AvrRps4 and PopP2 bacterial effectors. A similar, linked R gene pair, RPS4B and RRS1B, also recognizes AvrRps4, but not PopP2. Although RPS4 and RPS4B, and RRS1 and RRS1B, are closely related (each pair ~ 60% identical), RPS4 and RRS1B associate with each other but do not function; this also true of RPS4B and RRS1. We aim to define the specific interactions between protein domains of RPS4 and RRS1, or of RPS4B and RRS1B, that enable formation of a functional complex that (i) perceives effectors and (ii) then activates signaling. We also aim to understand why non-authentic combinations fail to function.RPS4 & RPS4B comprise 4 domains (AAAA or BBBB), and RRS1 & RRS1B comprise 5 domains (AAAAA or BBBBB). We will exchange domains between homologs, and test, for example, whether RPS4/RPS4B domain swaps BAAA, ABAA, AABA or AAAB function in combination with RRS1, and ABBB, BABB, BBAB and BBBA function in combination with RRS1B. We will also test RRS1/RRS1B swaps such as BAAAA, ABAAA etc in combination with RPS4. We can test function of RPS4 and RRS1 domain swap variants, within 3 days after transient expression of these genes in tobacco leaf tissue using Agrobacterium ("agroinfiltration"). We will also investigate domain/domain interactions by expressing each of the domains of these 4 proteins and looking for domain/domain interactions, both in yeast and after co-expression in plants and coimmunoprecipitation.By removing a domain from the end of RRS1, or by introducing mutations into this domain, we create an RPS4/RRS1 complex that constitutively activates defense. We will investigate the domain requirements of this constitutive activity. Wild type RRS1 suppresses this constitutive defence activation triggered by RPS4 and constitutive forms of RRS1; we will investigate the mechanisms of this suppression.RRS1 can form higher order associations with itself, so we will investigate RRS1 domain/domain interactions that might explain this multimerisation. Furthermore, the two ends of RRS1 appear to be able to interact with each other; we will investigate this in more detail.We can monitor protein/protein or domain/domain interactions in living cells using split fluorescent proteins. These do not fluoresce separately. When attached to proteins of interest, they only become active when brought together by protein/protein interactions of the domains to which they are attached. Using this method, we have found that the N terminal domains of RPS4 and RRS1 become closely associated only upon perception of AvrRps4 or PopP2 effectors. We will investigate the temporal and genetic requirements for this association, which we believe to be key to the reconfiguration of the protein complex that leads to activation