Elucidating the molecular basis of effector-triggered host susceptibility mediated by the TAL-like effector Brg11 from the bacterial pathogen Ralstonia solanacearum

植物病原体对全球粮食安全构成了永久的威胁。为了入侵植物，植物病原微生物依赖于它们注入宿主细胞的效应蛋白来促进宿主的易感性。了解宿主细胞内的效应靶点为基于知识的抗虫作物育种提供了可能。因此，阐明效应物引发的寄主易感性的分子基础仍然是植物病理学基础和应用的主要目标。我们研究的Ralstonia solanacearum是一个毁灭性的细菌病原体，引发细菌性枯萎病在许多作物物种。我们鉴定了一种与黄单胞菌（Xanthomonas）的转录激活因子样效应物（transcription activator-like effector, TALEs）高度相似的Brg11。TALEs与宿主启动子中的效应结合元件（effector binding elements, EBEs）结合，通过转录激活下游宿主易感基因（susceptibility， S）来促进疾病。考虑到Brg11与TALEs的相关性，可以想象Brg11通过激活宿主S基因的转录来促进疾病。因此，我们的目标是鉴定被Brg11转录激活的宿主基因，以阐明它如何促进疾病。为了鉴定Brg11宿主靶基因，我们确定了Brg11的首选靶基因序列（Brg11- ebe）。在茄青霉宿主基因组中所有精氨酸脱羧酶（ADC）基因的上游都发现了一个与Brg11-EBE相似的18bp基序，并将其命名为ADC-box。结合RNA-Seq数据分析和靶位预测表明，ADC1和ADC2是Brg11在番茄中仅有的直接靶基因。对brg11诱导的ADC转录本和内在ADC转录本的检测分别显示了短（100 bps）和长（450 bps）的5 ' utr。初步数据表明，它们的5 ' utr的差异导致内在转录本的翻译受到代谢物的反馈调节，而不是brg11诱导的转录本，代谢物是由于ADC酶的活性而积累的。因此，brg11诱导的ADC转录物可能逃避了限制内在转录物翻译的翻译控制机制。综上所述，我们的研究结果表明，R. solanacearum Brg11通过转录激活宿主ADC基因来促进细菌性枯萎病。在本提案的框架内，我们的目标是：-研究内在的与Brg11诱导的ADC转录物的翻译调节-建立由于ADC蛋白水平升高而变化的代谢物目录-分析Brg11诱导的代谢物对植物相关微生物的生长抑制/促进活性-建立细胞报告以可视化Brg11靶细胞和Brg11诱导的变化-研究Brg11诱导的转录和翻译水平的变化总之，这些研究将提供brg11诱导的ADC基因激活如何促进细菌性枯萎病的见解。

Plant pathogens pose a permanent thread to global food security. To invade plants, phytopathogenic microbes rely on effector proteins that they inject into host cells to promote host susceptibility. Knowledge of effector targets inside host cells provides a possibility for knowledge-based breeding of pest-resistant crops. Thus, elucidation of the molecular basis of effector-triggered host susceptibility remains a major aim of fundamental and applied plant pathology.We study Ralstonia solanacearum a devastating bacterial pathogen that triggers bacterial wilt disease on numerous crop species. We have characterized Brg11, a R. solanacearum effector with high similarity to transcription activator-like effectors (TALEs) from the bacterial genus Xanthomonas. TALEs bind to effector binding elements (EBEs) present in host promoters and transcriptionally activate downstream host susceptibility (S) genes to promote disease. Given its relatedness to TALEs it’s conceivable that Brg11 promotes disease by transcriptional activation of host S genes. Thus, we aimed to identify host genes that are transcriptionally activated by Brg11 to clarify how it promotes disease.To identify Brg11 host target genes we determined a preferred target sequence of Brg11 (Brg11-EBE). A 18bp-motif resembling Brg11-EBE was identified upstream of all arginine decarboxylase (ADC) genes in R. solanacearum host genomes and accordingly this motif was designated as ADC-box. Combined analysis of RNA-Seq data and target site prediction suggests that ADC1 and ADC2 are the only direct target genes of Brg11 in tomato. Inspection of Brg11-induced versus intrinsic ADC transcripts revealed short (100 bps) and long (450 bps) 5’UTRs, respectively. Preliminary data suggest that differences in their 5’UTRs cause that translation of intrinsic but not Brg11-induced transcripts is feedback regulated by metabolites that accumulate due to activity of ADC enzymes. Thus, Brg11-induced ADC transcripts likely evade translational control mechanisms that limit translation from intrinsic transcripts. In summary, our findings suggest that R. solanacearum Brg11 promotes bacterial wilt disease by transcriptional activation of host ADC genes.In the framework of this proposal we aim to:- study translational regulation of intrinsic versus Brg11-induced ADC transcripts- establish a catalogue of metabolites that change as consequence of elevated ADC protein levels- Analyze growth-inhibiting/promoting activity of Brg11-induced metabolites on plant associated microbes- Establish cellular reporters to visualize Brg11 target cells and Brg11-induced changes- Investigate Brg11-induced changes at the transcriptional and translational levelIn summary, these studies shall provide insights into how Brg11-induced activation of ADC genes promotes bacterial wilt disease.