Elucidating in planta target genes of Ralstonia solanacearum TALE-like proteins

在美国和欧盟，青枯病病原菌是一种检疫性微生物，是导致青枯病的病原菌。我们的目标是在分子水平上研究茄青霉与其寄主植物之间的相互作用，并期望我们的研究结果能够使我们在长期内制定有效的对策来对付这种毁灭性的病原体。病原菌将效应蛋白注入宿主细胞，或促进疾病，或触发植物免疫。因此，它们是植物与微生物相互作用结果的关键决定因素。在本提案的框架内，我们将研究riptal，这是一类广泛存在于R. solanacearum中的效应物，与细菌属Xanthomonas的转录激活因子样效应物（Transcription Activator Like effector, TALEs）具有同源性。TALE代码描述了给定的TALE DNA结合域中定义的氨基酸与其效应结合元件（EBE）中的特定DNA碱基的相关性。这允许在计算机上预测已知序列的故事的ebe。在初步研究中，我们发现，像TALE一样，RipTAL ebe可以用TALE代码来预测。此外，像TALEs一样，riptal被注射到宿主细胞中，能够转录激活具有预测EBE的启动子。因此可以想象，riptal和TALEs一样，通过转录激活寄主基因来促进寄主植物的易感性。考虑到茄枯病菌是一种根病原体，而黄单胞菌通常感染地面组织，可以想象它们通过激活宿主易感基因(S)来促进疾病，这些基因编码功能不同的蛋白质。我们的目标是确定RipTAL激活的宿主S基因，当被给定的RipTAL转录上调时，促进细菌疾病。一旦宿主S基因被分离出来并在功能上得到验证，我们想要阐明S基因产物是如何在分子水平上促进茄枯病的。从世界各地分离的茄属真菌可分为4个系型，并与它们的地理来源有关。值得注意的是，龙葵I、II和IV种型在其N端和c端区域及其预测的ebe上含有不同的riptal。到目前为止，riptal的分子研究几乎都是针对i种型的riptal进行的。在本提案的框架内，我们的目标是对II和IV种型菌株的riptal进行表征，长期目标是鉴定和表征相应的宿主S基因和基因产物。综上所述，我们的目的是鉴定和表征来自不同种型的番茄红霉中被riptal激活的宿主S基因。我们期望我们的研究结果将有助于了解茄青霉的病害策略，帮助制定有效的对策来保护作物。

Ralstonia solanacearum, the causal agent of bacterial wilt disease, is a quarantine organism in the USA and EU that ranks among the most devastating microbial pathogens in crop plants. We aim to study the interaction between R. solanacearum and its host plants at the molecular level and anticipate that our findings will enable us, in the long run, to develop effective countermeasures against this devastating pathogen. The effector proteins injected by bacterial pathogens into host cells either promote disease or trigger plant immunity. They are thus key determinants for the outcome of plant-microbe interactions. In the framework of this proposal we will study RipTALs, one class of effectors that are widespread in R. solanacearum and that share homology with Transcription Activator Like Effectors (TALEs) from the bacterial genus Xanthomonas.The TALE code describes the correlation of defined amino acids in a given TALE DNA binding domain with specific DNA bases in its effector binding element (EBE). This allows in silico prediction of EBEs for TALEs of known sequence. In preliminary studies we showed that, like for TALEs, RipTAL EBEs can be predicted with the TALE code. Furthermore RipTALs, like TALEs are injected into host cells and are capable of transcriptionally activating promoters bearing a predicted EBE. It is thus conceivable that RipTALs, like TALEs, transcriptionally activate host genes to promote susceptibility in host plants. Given that R. solanacearum is a root pathogen, while xanthomonads typically infect aerial tissues, it is conceivable that they promote disease by activation of host susceptibility (S) genes that encode functionally distinct proteins.We aim to identify RipTAL activated host S genes that promote bacterial disease when being transcriptionally upregulated by a given RipTAL. Once host S genes have been isolated and functionally validated we want to clarify how S gene products promote R. solanacearum disease at the molecular level.R. solanacearum strains isolated from all over the world can be subdivided into four phylotypes, which correlate with their geographical origin. Notably R. solanacearum phylotypes I, II and IV contain RipTALs that differ in their N- and C-terminal regions and their predicted EBEs. Till now molecular studies of RipTALs have been conducted almost exclusively on RipTALs from phylotype I. In the framework of this proposal we aim to also characterize RipTALs from phylotype II and IV strains with the long term goal to identify and characterize corresponding host S genes and gene products.In summary we aim to identify and characterize host S genes that are activated by RipTALs from diverse R. solanacearum phylotypes. We anticipate that our findings will provide insights into the disease strategies of R. solanacearum, aiding the development of efficient countermeasures to protect crop plants.