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

细菌效应子激活 RPS4/RRS1 免疫复合物中的结构域/结构域相互作用

• 批准号: BB/M008193/1
• 负责人: Jonathan Jones
• 金额: $ 42.01万
• 依托单位: University of East Anglia
• 依托单位国家: 英国
• 项目类别: Research Grant
• 财政年份: 2015
• 资助国家: 英国
• 起止时间: 2015 至 无数据
• 项目状态: 已结题
• 来源: https://gtr.ukri.org/projects?ref=BB%2FM008193%2F1
• 关键词: Domain; domain; interactions; RPS4; RRS1

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

植物病害可能会造成农作物的巨大损失，这对农民来说代价高昂。例如，控制马铃薯晚疫病每年花费英国农民约6000万英镑。抗性(R)基因使植物能够检测和抵抗病原体，但R蛋白是如何工作的还知之甚少。植物病原体将称为效应器的分子输送到宿主细胞中，以干扰宿主的免疫机制。R基因使植物能够识别这种效应器，然后激活免疫力。为了克服R基因，病原体必须通过识别效应器的突变来逃避检测。我们的目标是详细了解R蛋白的分子机制，以便我们能够设计新的R基因来识别来自任何病原体的效应器。一个较短期的目标是开发智能设计的R蛋白变异体文库，这些文库可以被筛选出来，用于识别以前未识别的效应器。在我们做这两件事之前，我们需要彻底了解R蛋白是如何将对效应器的识别转化为防御的激活的。我们研究了模式植物拟南芥中的一个R基因位点，它对两种不同的细菌和一种真菌具有抗性。该基因座包括两个R基因，RPS4和RRS1，它们编码两种蛋白质，它们相互关联，形成识别AvrRps4和PopP2细菌效应物的受体复合体。一对相似的连锁R基因，RPS4B和RRS1B，也识别AvrRps4，但不识别PopP2。虽然RPS4和RPS4B以及RRS1和RRS1B密切相关(每对~60%相同)，但RPS4和RRS1B彼此关联但不起作用；RPS4B和RRS1也是如此。我们的目标是定义RPS4和RRS1或RPS4B和RRS1B蛋白结构域之间的特定相互作用，使形成一个功能复合体，该复合体(I)感知效应器，(Ii)然后激活信号。RPS4和RPS4B由4个结构域(AAAA或BBBB)组成，RRS1和RRS1B由5个结构域(AAAAA或bbbb)组成。我们将在同源基因之间交换结构域，并测试例如RPS4/RPS4B结构域是否与RRS1结合交换BaaA、ABAA、AABA或AAAB功能，以及ABBB、BABB、BBAB和BBBA功能与RRS1B结合。我们还将结合RPS4测试RRS1/RRS1B掉期，如BAAA、ABAAA等。利用农杆菌对RPS4和RRS1结构域交换变异体基因在烟草叶片组织中瞬时表达后的3天内进行功能检测。我们还将通过表达这4种蛋白质的每个结构域，寻找在酵母中和在植物中共表达后的结构域/结构域相互作用，以及免疫共沉淀来研究结构域/结构域的相互作用。通过从RRS1末端移除一个结构域，或通过在这个结构域中引入突变，我们创建了一个RPS4/RRS1复合体，它可以结构性地激活防御。我们将研究此构成活动的域要求。野生型RRS1抑制由RPS4和RRS1的构成形式触发的结构性防御激活；我们将研究这种抑制的机制。RRS1可以与自己形成更高的顺序关联，所以我们将研究RRS1结构域/结构域的相互作用，这可能解释这种多聚化。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。我们可以使用分离的荧光蛋白来监测活细胞中的蛋白质/蛋白质或结构域/结构域相互作用。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，它们只有在它们所附着的结构域的蛋白质/蛋白质相互作用的情况下才会变得活跃。使用这种方法，我们发现RPS4和RRS1的N个末端结构域只有在感受到AvrRps4或PopP2效应器时才变得紧密相关。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体重新配置的关键

项目成果

Deadlier than the malate

• DOI: 10.1038/s41422-018-0042-6
• 发表时间: 2018-05
• 期刊: Cell Research
• 影响因子: 44.1
• 作者: P. Ding;Hailong Guo;Jonathan D. G. Jones

High-resolution Expression Profiling of Selected Gene Sets during Plant Immune Activation

植物免疫激活过程中选定基因集的高分辨率表达谱

• DOI: 10.1101/775973
• 发表时间: 2019
• 作者: Ding P