Gopalan S, Bauer D W, Alfano J R, Loniello A O, He S Y, Collmer A
Department of Energy, Michigan State University, East Lansing 48824-1312, USA.
Plant Cell. 1996 Jul;8(7):1095-105. doi: 10.1105/tpc.8.7.1095.
The nonpathogenic bacteria Pseudomonas fluorescens and Escherichia coli can elicit a genotype-specific hypersensitive response (HR) in plants if they express both the HR and pathogenesis (Hrp) protein secretion system and the HrpZ harpin from P. syringae pv syringae 61 and a P. syringae avirulence (avr) gene whose presence is recognized by a corresponding disease resistance gene in the plant. We have found that the recognition event appears to require transfer of the Avr protein into the plant cell. Elicitation of a genotype-specific HR was observed with avrB+ P. fluorescens in soybean and Arabidopsis plants carrying resistance genes RPG1 and RPM1, respectively, and with avrPto+ E. coll in tomato plants carrying resistance gene PTO, but only if the Hrp secretion system, HrpZ, and the appropriate Avr proteins were produced in the same bacterial cell. The failure of avrB hyperexpression and exogenous AvrB or HrpZ to alleviate these requirements in soybean and Arabidopsis suggests that the site of AvrB action is not in the bacterial cell or plant apoplast. An Arabidopsis rps3 (rpm1) glabrous1 mutant was transformed with constructs expressing avrB and was crossed with an Arabidopsis ecotype Columbia (RPM1 GLABROUS1) plant. F1 seedlings (identified by their kanamycin-resistant, pubescent phenotype) exhibited extensive necrosis on cotyledon leaves 10 days postgermination. Ecotype Columbia and rps3-1 leaves biolistically cobombarded with plasmids expressing the beta-glucuronidase (GUS) gene and avrB failed to produce GUS activity (indicative of cell death) only when RPM1 and avrB were present in the leaf. Thus, both stable and transient expression of avrB in Arabidopsis resulted in RPM1-dependent necrosis, and the only demonstrable site of action for AvrB was inside plant cells.
如果非致病性细菌荧光假单胞菌和大肠杆菌表达丁香假单胞菌丁香致病变种61的过敏反应（HR）和致病（Hrp）蛋白分泌系统以及HrpZ激发子蛋白，并且表达一种能被植物中相应抗病基因识别的丁香假单胞菌无毒（avr）基因，那么它们就能在植物中引发基因型特异性过敏反应。我们发现，识别事件似乎需要将Avr蛋白转移到植物细胞中。分别在携带抗性基因RPG1和RPM1的大豆和拟南芥植株中，观察到携带avrB⁺的荧光假单胞菌引发了基因型特异性HR；在携带抗性基因PTO的番茄植株中，观察到携带avrPto⁺的大肠杆菌引发了该反应，但前提是Hrp分泌系统、HrpZ和相应的Avr蛋白在同一细菌细胞中产生。在大豆和拟南芥中，avrB过表达以及外源性AvrB或HrpZ未能满足这些条件，这表明AvrB的作用位点不在细菌细胞或植物质外体中。用表达avrB的构建体转化拟南芥rps3（rpm1）无毛1突变体，并与拟南芥生态型哥伦比亚（RPM1无毛1）植株杂交。F1代幼苗（通过其对卡那霉素抗性、有毛的表型鉴定）在萌发10天后子叶上出现广泛坏死。当叶片中同时存在RPM1和avrB时，用表达β-葡萄糖醛酸酶（GUS）基因和avrB的质粒对生态型哥伦比亚和rps3 - 1叶片进行生物弹轰击，未能产生GUS活性（表明细胞死亡）。因此，avrB在拟南芥中的稳定和瞬时表达均导致依赖RPM1的坏死，并且AvrB唯一可证明的作用位点在植物细胞内。
