Tomato TFT1 is required for PAMP-triggered immunity and mutations that prevent T3S effector XopN from binding to TFT1 attenuate Xanthomonas virulence.
Tomato TFT1 is required for PAMP-triggered immunity and mutations that prevent T3S effector XopN from binding to TFT1 attenuate Xanthomonas virulence.
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DOI：
10.1371/journal.ppat.1002768
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发表时间：
2012
期刊：
影响因子：
6.7
通讯作者：
Mudgett MB
中科院分区：
文献类型：
--
作者：
Taylor KW;Kim JG;Su XB;Aakre CD;Roden JA;Adams CM;Mudgett MB
XopN是野油菜黄单胞菌致病变种水泡菌的III型效应蛋白，可抑制番茄的PAMP触发免疫(PTI)。以前的工作报道了XopN与番茄14-3-3亚型TFT1相互作用，但TFT1‘S在PTI和/或XopN毒力中的作用尚未确定。在这里，我们证明了TFT1在PTI中发挥作用，是XopN毒力靶标。病毒诱导的TFT1mRNA沉默导致番茄XcvΔxopN和XcvΔhrpF的生长增加，表明TFT1是抑制XcV增殖所必需的。XCV诱导番茄PTI5、GRAS4、WRKY28和LRR22mRNAs的积累需要TFT1的表达。缺失分析表明，XopN C-末端结构域(氨基酸344-733)足以与TFT1结合。去除605-733个氨基酸会破坏植物提取物中XopN与TFT1的结合，并抑制依赖于XopN的番茄毒力，表明这些残基是XopN/TFT1相互作用所必需的。PHOS-Tag凝胶分析和质谱分析表明，XopN在植物提取物中的丝氨酸688位处被磷酸化，推测为14-3-3识别基序。S688突变降低了XopN的磷酸化状态，但不足以抑制与TFT1的结合或降低XopN的毒力。然而，S688和XopN中的两个亮氨酸(L64、L65)的突变消除了XopN与植物提取物中TFT1的结合和XopN的毒力。XopN需要L64和L65才能与TARK1结合，TARK1是PTI所需的番茄非典型受体激酶。这表明TFT1与XopN的C-末端结构域的结合可能通过TARK1/XopN相互作用而稳定。Pull-Down和BIFC分析表明，XopN通过作为分子支架在体外和植物体内促进TARK1/TFT1复合体的形成。这是第一个显示III型效应器靶向参与PTI的宿主14-3-3以促进细菌致病的报告。植物和动物的细菌病原体利用III型分泌系统分泌效应蛋白并将其转移到宿主细胞中，以抑制防御反应。生化分析表明，一些效应蛋白模拟宿主酶的活性，直接干扰病原菌的感知、防御信号转导和/或抗菌化合物的分泌。野油菜黄单胞菌的XopN效应物是番茄和辣椒上细菌性斑点病的病原菌，它能抑制PAMP触发的免疫(PTI)，但结构模型预测它编码一种独特的支架样蛋白，没有明显的酶折叠。我们推测，XopN通过与调节防御的宿主蛋白结合来介导其毒力功能。XopN先前被证明在植物中与番茄14-3-3蛋白TFT1物理上相互作用。TFT1在植物免疫信号中的意义以及XopN/TFT1相互作用的相关性尚未确定。在这里，我们证明了TFT1是番茄PTI的正调控因子，需要抑制XCV的生长。此外，我们提供了TFT1是XopN的真正靶标的证据，因为破坏XopN/TFT1结合的突变也消除了依赖于XopN的番茄毒力。这是第一个针对与宿主免疫相关的14-3-3的细菌效应器的例子。
XopN is a type III effector protein from Xanthomonas campestris pathovar vesicatoria that suppresses PAMP-triggered immunity (PTI) in tomato. Previous work reported that XopN interacts with the tomato 14-3-3 isoform TFT1; however, TFT1's role in PTI and/or XopN virulence was not determined. Here we show that TFT1 functions in PTI and is a XopN virulence target. Virus-induced gene silencing of TFT1 mRNA in tomato leaves resulted in increased growth of Xcv ΔxopN and Xcv ΔhrpF demonstrating that TFT1 is required to inhibit Xcv multiplication. TFT1 expression was required for Xcv-induced accumulation of PTI5, GRAS4, WRKY28, and LRR22 mRNAs, four PTI marker genes in tomato. Deletion analysis revealed that the XopN C-terminal domain (amino acids 344–733) is sufficient to bind TFT1. Removal of amino acids 605–733 disrupts XopN binding to TFT1 in plant extracts and inhibits XopN-dependent virulence in tomato, demonstrating that these residues are necessary for the XopN/TFT1 interaction. Phos-tag gel analysis and mass spectrometry showed that XopN is phosphorylated in plant extracts at serine 688 in a putative 14-3-3 recognition motif. Mutation of S688 reduced XopN's phosphorylation state but was not sufficient to inhibit binding to TFT1 or reduce XopN virulence. Mutation of S688 and two leucines (L64,L65) in XopN, however, eliminated XopN binding to TFT1 in plant extracts and XopN virulence. L64 and L65 are required for XopN to bind TARK1, a tomato atypical receptor kinase required for PTI. This suggested that TFT1 binding to XopN's C-terminal domain might be stabilized via TARK1/XopN interaction. Pull-down and BiFC analyses show that XopN promotes TARK1/TFT1 complex formation in vitro and in planta by functioning as a molecular scaffold. This is the first report showing that a type III effector targets a host 14-3-3 involved in PTI to promote bacterial pathogenesis. Bacterial pathogens of plants and animals employ the type III secretion system to secrete and translocate effector proteins into host cells to suppress defense responses. Biochemical analyses have revealed that several effector proteins mimic host enzyme activities to directly interfere with pathogen perception, defense signal transduction, and/or secretion of antimicrobial compounds. The XopN effector from Xanthomonas campestris pathovar vesicatoria, the causal agent of bacterial spot on tomato and pepper plants, suppresses PAMP-triggered immunity (PTI) but structural modeling predicts it encodes a unique scaffolding-like protein with no obvious enzymatic fold. We postulate that XopN mediates its virulence function by associating with host proteins regulating defense. XopN was previously shown to physically interact with the tomato 14-3-3 protein TFT1 in planta. The significance of TFT1 in plant immune signaling and the relevance of the XopN/TFT1 interaction were not determined. Here we show that TFT1 is a positive regulator of PTI in tomato required to inhibit Xcv growth. Moreover, we provide evidence that TFT1 is a bona fide target of XopN because mutations that disrupt XopN/TFT1 binding also eliminate XopN-dependent virulence in tomato. This is the first example of a bacterial effector targeting a 14-3-3 associated with host immunity.
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DOI：
10.1073/pnas.90.6.2320
发表时间：
1993-03-15
影响因子：
11.1
作者：
FU, H;COBURN, J;COLLIER, RJ
通讯作者：
COLLIER, RJ
影响因子：
3.5
作者：
BONAS, U;SCHULTE, R;STALL, RE
通讯作者：
STALL, RE
影响因子：
64.8
作者：
Fu, Zheng Qing;Guo, Ming;Alfano, James R.
通讯作者：
Alfano, James R.
影响因子：
3.6
作者：
Axtell, MJ;Chisholm, ST;Staskawicz, BJ
通讯作者：
Staskawicz, BJ
影响因子：
64.8
作者：
Asai, T;Tena, G;Sheen, J
通讯作者：
Sheen, J