Shantharaj Deepak, Römer Patrick, Figueiredo Jose F L, Minsavage Gerald V, Krönauer Christina, Stall Robert E, Moore Gloria A, Fisher Latanya C, Hu Yang, Horvath Diana M, Lahaye Thomas, Jones Jeffrey B
Plant Pathology Department, University of Florida, Gainesville, FL 32611, USA.
Genetics, Department of Biology, Ludwig-Maximilians-University Munich, Martinsried, D-82152, Germany.
Mol Plant Pathol. 2017 Sep;18(7):976-989. doi: 10.1111/mpp.12454. Epub 2016 Sep 7.
Xanthomonas citri ssp. citri (X. citri), causal agent of citrus canker, uses transcription activator-like effectors (TALEs) as major pathogenicity factors. TALEs, which are delivered into plant cells through the type III secretion system (T3SS), interact with effector binding elements (EBEs) in host genomes to activate the expression of downstream susceptibility genes to promote disease. Predictably, TALEs bind EBEs in host promoters via known combinations of TALE amino acids to DNA bases, known as the TALE code. We introduced 14 EBEs, matching distinct X. citri TALEs, into the promoter of the pepper Bs3 gene (ProBs3 ), and fused this engineered promoter with multiple EBEs (ProBs3 ) to either the β-glucuronidase (GUS) reporter gene or the coding sequence (cds) of the pepper gene, Bs3. TALE-induced expression of the Bs3 cds in citrus leaves resulted in no visible hypersensitive response (HR). Therefore, we utilized a different approach in which ProBs3 and ProBs3 were fused to the Xanthomonas gene, avrGf1, which encodes a bacterial effector that elicits an HR in grapefruit and sweet orange. We demonstrated, in transient assays, that activation of ProBs3 by X. citri TALEs is T3SS dependent, and that the expression of AvrGf1 triggers HR and correlates with reduced bacterial growth. We further demonstrated that all tested virulent X. citri strains from diverse geographical locations activate ProBs3 . TALEs are essential for the virulence of X. citri strains and, because the engineered promoter traps are activated by multiple TALEs, this concept has the potential to confer broad-spectrum, durable resistance to citrus canker in stably transformed plants.
柑橘溃疡病菌（Xanthomonas citri ssp. citri，简称X. citri）是柑橘溃疡病的病原菌，它利用转录激活样效应因子（TALEs）作为主要致病因子。TALEs通过III型分泌系统（T3SS）被递送到植物细胞中，与宿主基因组中的效应因子结合元件（EBEs）相互作用，激活下游感病基因的表达以促进病害发生。可以预测，TALEs通过TALE氨基酸与DNA碱基的已知组合（即TALE密码）与宿主启动子中的EBEs结合。我们将14个与不同X. citri TALEs匹配的EBEs导入辣椒Bs3基因（ProBs3）的启动子中，并将这个带有多个EBEs的工程化启动子（ProBs3）与β-葡萄糖醛酸酶（GUS）报告基因或辣椒基因Bs3的编码序列（cds）融合。TALE诱导的Bs3 cds在柑橘叶片中的表达未导致可见的超敏反应（HR）。因此，我们采用了一种不同的方法，将ProBs3和ProBs3与柑橘黄单胞菌基因avrGf1融合，该基因编码一种能在葡萄柚和甜橙中引发HR的细菌效应因子。我们在瞬时试验中证明，X. citri TALEs对ProBs3的激活依赖于T3SS，并且AvrGf1的表达引发HR并与细菌生长减少相关。我们进一步证明，来自不同地理位置的所有测试致病X. citri菌株都能激活ProBs3。TALEs对X. citri菌株的毒性至关重要，并且由于工程化启动子陷阱可被多种TALEs激活，这一概念有可能赋予稳定转化植物对柑橘溃疡病的广谱、持久抗性。
