Molecular characterization of proteolytic cleavage sites of the Pseudomonas syringae effector AvrRpt2
Molecular characterization of proteolytic cleavage sites of the Pseudomonas syringae effector AvrRpt2
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DOI：
10.1073/pnas.0409468102
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发表时间：
2005-02-08
影响因子：
11.1
通讯作者：
Staskawicz, BJ
中科院分区：
文献类型：
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作者：
Chisholm, ST;Dahlbeck, D;Staskawicz, BJ
在拟南芥感染期间，细菌假单胞菌番茄致病变种将效应蛋白AvrRpt 2递送到植物细胞胞质溶胶中。在植物细胞内，AvrRpt 2经历N-末端加工并引起拟南芥RIN 4的消除。先前的工作确定AvrRpt 2是推定的半胱氨酸蛋白酶，并且AvrRpt 2加工和RIN 4消除需要AvrRpt 2中的完整预测催化三联体。在这项工作中，依赖于AvrRpt 2活性的蛋白水解事件的特征。围绕AvrRpt 2加工位点的氨基酸序列和来自RIN 4的两个相关序列触发合成底物的AvrRpt 2依赖性蛋白水解切割，表明这些序列是AvrRpt 2活性的切割识别位点。加工缺陷型AvrRpt 2突变体被鉴定并显示出保留其消除野生型RIN 4的能力。在两个RIN 4切割位点中的每一个中进行单个氨基酸取代，并且两个位点的突变导致抗切割RIN 4。在拟南芥rin 4/rps 2突变体植物上，表达AvrRpt 2的假单胞菌的生长显著高于催化失活突变体，这表明存在AvrRpt 2的另外的蛋白质靶标，其解释了该效应子的毒力活性。生物信息学分析确定了推定的拟南芥蛋白含有类似于AvrRpt 2和RIN 4中保守的蛋白水解切割位点的序列。这些蛋白质中的几种在瞬时植物表达系统中以AvrRpt 2依赖性方式消除。这些结果鉴定了对AvrRpt 2底物识别和切割重要的氨基酸，并且证明了AvrRpt 2蛋白酶活性消除了瞬时表达系统中的多种拟南芥蛋白。
During infection of Arabidopsis thaliana, the bacterium Pseudomonas syringae pv tomato delivers the effector protein AvrRpt2 into the plant cell cytosol. Within the plant cell, AvrRpt2 undergoes N-terminal processing and causes elimination of Arabidopsis RIN4. Previous work established that AvrRpt2 is a putative cysteine protease, and AvrRpt2 processing and RIN4 elimination require an in-tact predicted catalytic triad in that AvrRpt2. In this work, proteolytic events that depend on AvrRpt2 activity were characterized. The amino acid sequence surrounding the processing site of AvrRpt2 and two related sequences from RIN4 triggered AvrRpt2-dependent proteolytic cleavage of a synthetic substrate, demonstrating that these sequences are cleavage recognition sites for AvrRpt2 activity. Processing-deficient AvrRpt2 mutants were identified and shown to retain their ability to eliminate wild-type RIN4. Single amino acid substitutions were made in each of the two RIN4 cleavage sites, and mutation of both sites resulted in cleavage-resistant RIN4. Growth of Pseudomonas expressing AvrRpt2 was significantly higher than catalytically inactive mutants on Arabidopsis rin4/rps2 mutant plants, suggesting there are additional protein targets of AvrRpt2 that account for the virulence activity of this effector. Bioinformatics analysis identified putative Arabidopsis proteins containing sequences similar to the proteolytic cleavage sites conserved in AvrRpt2 and RIN4. Several of these proteins were eliminated in an AvrRpt2-dependent manner in a transient in planta expression system. These results identify amino acids important for AvrRpt2 substrate recognition and cleavage as well as demonstrate AvrRpt2 protease activity eliminates multiple Arabidopsis proteins in a transient expression system.