Functional analysis of the type III effectors AvrRpt2 and AvrRpm1 of Pseudomonas syringae with the use of a single-copy genomic integration system
Functional analysis of the type III effectors AvrRpt2 and AvrRpm1 of Pseudomonas syringae with the use of a single-copy genomic integration system
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DOI：
10.1094/mpmi.2001.14.2.145
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发表时间：
2001-02-01
影响因子：
3.5
通讯作者：
Greenberg, JT
中科院分区：
文献类型：
--
作者：
Guttman, DS;Greenberg, JT
革兰氏阴性植物病原细菌需要III型分泌装置用于致病，推测是将Avr效应蛋白直接递送到植物细胞中。为了扩展以前的研究Avr效应器，采用质粒编码Avr蛋白，我们开发了一个系统，允许任何基因整合到假单胞菌基因组中的单拷贝。利用该系统，我们证实了早期的发现，该发现显示表达AvrRpt 2效应子的灰斑潜蝇致病变种菌株PsmES 4326在具有同源R基因RPS 2的植物中诱导抗性应答。然而，位于染色体上的avrRpt 2引起了比在RPS 2（+）植物中用质粒表达的AvrRpt 2观察到的更强的抗性反应。此外，AvrRpt 2的染色体表达赋予了在rps 2（-）植物中生长的烟草赤星病菌的适应性优势，有助于在叶内生长和逃逸到叶表面，这是难以用质粒携带的avrRpt 2检测的。最后，利用基因组整合系统，我们发现一个嵌合蛋白组成的N末端的异源AvrRpm 1效应和C-末端效应区的AvrRptZ被传递到植物细胞。由于AvrRpt 2的C末端不能自行易位到植物细胞中，这表明N末端区域可以在感染期间指导分泌和易位，这支持Avr蛋白具有模块化设计的观点。这项工作建立了一个易于操作的系统，在生物现实的背景下研究III型效应器。
Gram-negative phytopathogenic bacteria require a type III secretion apparatus for pathogenesis, presumably to deliver Avr effector proteins directly into plant cells. To extend previous studies of Avr effecters that employed plasmids encoding Avr proteins, we developed a system that permits the integration of any gene into the Pseudomonas syringae genome in single copy. With this system, we confirmed earlier findings showing that P. syringae pv, maculicola strain PsmES4326 expressing the AvrRpt2 effector induces a resistance response in plants with the cognate R gene, RPS2. Chromosomally located avrRpt2, however, provoked a stronger resistance response than that observed with plasmid-expressed AvrRpt2 in RPS2(+) plants. Additionally, chromosomal expression of AvrRpt2 conferred a fitness advantage on P, syringae grown in rps2(-) plants, aiding in growth within leaves and escape to leaf surfaces that was difficult to detect with plasmid-borne avrRpt2. Finally, with the use of the genomic integration system, we found that a chimeric protein composed of the N terminus of the heterologous AvrRpm1 effector and the C-terminal effector region of AvrRptZ was delivered to plant cells. Because the C terminus of AvrRpt2 cannot translocate into plant cells on its own, this indicates that the N-terminal region can direct secretion and translocation during an infection, which supports the view that Avr proteins have a modular design. This work establishes a readily manipulatable system to study type III effecters in a biologically realistic context.