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首页> 外文期刊>Journal of general plant pathology >Members of 14-3-3 protein isoforms interacting with the resistance gene product N and the elicitor of Tobacco mosaic virus
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Members of 14-3-3 protein isoforms interacting with the resistance gene product N and the elicitor of Tobacco mosaic virus

机译：14-3-3蛋白同工型的成员与抗性基因产物N和烟草花叶病毒的引发剂相互作用

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摘要

A functional cDNA for the resistance gene N from Nicotiana tabacum cv. Samsun NN to Tobacco mosaic virus (TMV) was obtained by reverse transcription polymerase chain reaction. Recombinant proteins containing various domains of the N protein were used in far-Western screening for the N-interacting protein from tobacco cDNA expression libraries. Nucleotide sequence analysis revealed that 199 cDNAs of 217 positive clones screened from 1.6 × 10~6 phage plaques were related to the 14-3-3 gene family. Phylogenetic analysis indicated that the 14-3-3 proteins were divided into 17 different 14-3-3 isoforms, including hitherto unidentified novel isoforms i-1 and i-2. In vitro binding assays showed that the probes TIR and LRR domains of N protein and helicase (HEL) domain of TMV replicase bound significantly to 14-3-3 isoforms of groups and ψ. These domains barely bound to those of group κ and did not bind to that of group ε. The same probes did not bind to any domains of N protein or the viral HEL domain. Northern blotting showed an increase in 14-3-3 isoform h transcripts coinciding with PR-1a induction after TMV inoculation. The 14-3-3 isoform h as a probe had significant binding to the LRR domain of N protein and the viral HEL domain, but there was only minor binding to the TIR domain. These results raise the possibility that 14-3-3 acts as a scaffolding or adaptor between the N protein and the helicase domain to support the interaction between the R gene product and the viral elicitor.
机译：来自烟草（Nicotiana tabacum）cv的抗性基因N的功能性cDNA。通过逆转录聚合酶链反应获得了Samsun NN的烟草花叶病毒（TMV）。含有N蛋白各个结构域的重组蛋白被用于从烟草cDNA表达文库中进行N相互作用蛋白的远西筛选。核苷酸序列分析表明，从1.6×10〜6噬菌斑中筛选的217个阳性克隆的199个cDNA与14-3-3基因家族有关。系统发育分析表明，将14-3-3蛋白分为17种不同的14-3-3亚型，包括迄今未鉴定的新亚型i-1和i-2。体外结合试验显示，N蛋白的探针TIR和LRR结构域以及TMV复制酶的解旋酶（HEL）结构域与14-3-3亚型和ψ显着结合。这些结构域几乎不与κ组结合，而与ε组不结合。相同的探针不与N蛋白的任何结构域或病毒HEL结构域结合。 Northern印迹显示TMV接种后与PR-1a诱导一致的14-3-3同工型h转录物增加。作为探针的14-3-3同工型h与N蛋白的LRR结构域和病毒HEL结构域具有显着结合，但与TIR结构域的结合很小。这些结果提高了14-3-3充当N蛋白和解旋酶结构域之间的支架或衔接子的可能性，以支持R基因产物与病毒引发剂之间的相互作用。

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