Nucleoside decoys - metabolic interference in plant defence

植物抗病(R)基因被广泛应用于植物育种中，以帮助减轻全球农作物对病虫害的损失，这些损失超过30%。不幸的是，随着病原体进化出越来越复杂的方法来部署多功能“效应器”--病原体军械的关键成分--以避免被发现并抑制宿主免疫，抗药性经常在现场被克服。尽管我们在25年前就克隆了R蛋白，但直到12个月前，我们对这些蛋白的功能知之甚少。R蛋白分为TnL和CNL两种类型，它们都含有共同的关键功能结构域，即中央核苷酸结合(N)区域和羧基末端富含亮氨酸重复序列(L)区域。最近的重大研究突破表明，“T”NL抗病蛋白的氨基末端TIR(Toll IL 1)结构域二聚生成能够切割细胞关键能量来源NADH或NADPH的复合体。关键的是，这种“NADase”活性对于激活抗病能力是必不可少的。值得注意的是，尽管动物和细菌的TIR结构域具有相似的酶活性，但产物似乎不同。植物和细菌产生一种名为v-cADPR(变体环状ADP核糖)的化合物。多年来，我们一直在研究从防御到疾病的代谢转变，特别是观察植物细菌病原体丁香假单胞菌如何克服宿主防御。对受感染组织进行全面的非靶向代谢产物图谱的最令人兴奋的发现是鉴定出一种全新的分子，该分子在感染假单胞菌的叶片中迅速积累，该分子将导致疾病，而不是非致病突变。值得注意的是，我们最近证实了这种分子，我们根据其分子质量将其称为“540”，它与由激活的TNL抗病蛋白形成的v-cADPR具有相同的分子质量。关键的是，它有不同的保留时间，细菌TIR产生的v-cADPR也是如此，这表明一些非常细微的结构变化可能赋予了相当不同的特异性。到目前为止，还不清楚tnl产生的植物或细菌v-cADPR是否具有任何生物活性。重要的是，我们之前公布了两个关键证据。首先，致病细菌，而不是解除武装的细菌，会诱导一个由6个截断的TNL基因(TTN)组成的非常特异的基因座。这乍一看是有违常理的。为什么要诱导R基因？其次，我们知道致病细菌会迅速抑制叶绿体中的一种防御反应--称为活性氧爆发。这对于疾病的发展是必要的。其结果是NADase底物NADP+升高。这些都是非常快速的事件，发生在TNL蛋白被激活之前。将这些证据放在一起；540的快速积累与氧化猝发的抑制和tTN的诱导同时发生，我们推测细菌效应器既抑制了氧化猝发，又同时诱导tTN清除积累的NADP+，否则这将激活TNL。我们也不能排除tTns也可以结合并干扰阻止激活的功能性TnL TIR结构域。我们以前的工作将540描述为高度不稳定的“环状磷苷”。在与澳大利亚研究小组的合作下，我们阐明了原核生物v-cADPR的结构，它与540略有不同。我们最近开发了一种稳定540的方法，并将确定核磁共振结构，并与美国研究人员在植物v-cADPR结构上合作。随之而来的是，我们的工作计划将使用遗传学、基因编辑、生物化学和结构方法来全面表征tTN。最后，我们将使用最先进的基因编码记者来确定疾病和防御发展过程中NAD/P积累/损失的动态。这个多学科的项目得益于澳大利亚、香港和美国的合作。

Plant disease resistance (R) genes are widely deployed in plant breeding to help mitigate global crop losses to pests and pathogens which exceed 30%. Unfortunately, resistance is often overcome in the field as pathogens evolve ever sophisticated methods of deploying multi-functional "effectors" - key elements of the pathogens armoury the work collectively to avoid detection and suppress host immunity. Despite having cloned R proteins more than 25 years ago, until 12 months ago we had little idea how these functioned. R proteins come in two flavours, TNLs and CNLs, both containing common key functional domains, the central nucleotide binding (N) domain and carboxyl terminal "leucine rich repeat" (L) region.The recent major research breakthrough showed that the amino terminal TIR, (Toll Interleukin 1) domain of "T"NL disease resistance proteins dimerises to generate a complex capable of cleaving NADH or NADPH, key energy sources for cells. Critically, this "NADase" activity was essential to activate disease resistance. Notably, although animal and bacteria TIR domains have similar enzymatic activities, the products appear to differ. Plants and bacteria produce a compound called v-cADPR (variant cyclic ADP Ribose).For a number of years we have studied the metabolic transition from defence to disease, specifically looking at how the bacterial plant pathogen Pseudomonas syringae overcomes host defences. The most exciting finding of comprehensive untargeted metabolite profiling of infected tissue was the identification of a totally novel molecule that accumulated rapidly in leaves that were infected with Pseudomonas that was going to cause disease but not a non-disease causing mutant. Remarkably, we have recently confirmed that this molecule, which we call "540" based on its molecular mass, is of identical molecular mass to v-cADPR formed by activated TNL disease resistance proteins. Critically, it has a different retention time, as also does the bacterial TIR produced v-cADPR suggesting some very subtle structural variations probably confer quite different specificities. To date, it is unclear whether either the TNL produced plant or bacterial v-cADPR have any biological activity. Importantly, we had previously published two key pieces of evidence. First, disease causing bacteria, but not disarmed bacteria induce a very specific locus of 6 truncated TNL genes (tTNs). This is at first counterintuitive. Why induce R genes?Secondly, we know disease causing bacteria rapidly suppress a defense response - called a reactive oxygen burst - in the chloroplast. This is necessary for disease progression. A consequence of this is elevated NADP+ - an NADase substrate. These are really rapid events, occurring before TNL proteins are activated.Putting this evidence together; the rapid accumulation of 540 co-incident with suppression of the oxidative burst and induction of the tTNs, we theorise that bacterial effectors both suppress the oxidative burst and simultaneously induce the tTNs to mop up accumulating NADP+, which would otherwise activate TNLs. We also cannot rule out the tTNs can also bind to, and interfere with, functional TNL TIR domains preventing activation.Our previous work characterised 540 as a highly labile "cyclic phosphoriboside". In collaboration with an Australian group we elucidated the structure of a prokaryotic v-cADPR, which is subtly different from 540. We have recently developed a method to stabilize 540 and will determine the NMR structure and collaborate on the plant v-cADPR structure with US researchers.Concomitantly, our work programme will fully characterise the tTNs using genetics, gene-editing, biochemistry and structural approaches. Finally we will determine the dynamics of NAD/P accumulation/loss during disease and defence development using state-of-the-art genetically encoded reporters. This multidisciplinary project benefits from collaborations in Australia, Hong Kong and the USA.