大豆快生根瘤菌SMH12效应蛋白NopP在共生固氮过程中的功能

Functional characterization of a T3SS effector protein NopP of Sinorhizobium fredii SMH12 in the symbiotic nitrogen fixation and interaction with soybean

摘要:

[目的] 研究Sinorhizobium fredii SMH12中的nopP在共生固氮过程中的功能，为深入解析根瘤菌效应蛋白的菌植互作机理提供线索，进而为大豆高效根瘤菌的遗传改良提供一定的科学依据。[方法] 利用生物信息学分析nopP的结构特征，构建nopP缺失、过表达和互补菌株，并对其进行共生表型分析；通过qRT-PCR分析nopP在共生过程中的时空表达特征，测定在接野生型和突变体的冀豆17中NIN、ENOD40、PR1、PR2和PR5的表达量；采用激光共聚焦显微镜观察NopP的亚细胞定位。[结果] 根瘤菌的NopP不包含任何已知功能域，与病原体的任何Avr效应物没有同源性。nopP缺失之后对冀豆17和中黄13的根瘤固氮酶活均有显著影响，在瘤数上对冀豆17有显著增加，表明nopP突变后促进其与冀豆17和中黄13的共生固氮。qRT-PCR显示，nopP在自生条件下少量表达，在共生条件下表达量显著升高，尤其在接菌2 d后表达量达到最高，显示该基因可能与根瘤菌早期侵染相关。此外，发现NopP在烟草叶片和大豆根中均定位于细胞膜和细胞核。接种突变体的冀豆17根中NIN的表达量升高1.2倍，PR5的表达量降低3.6倍。[结论] 效应蛋白NopP在与大豆共生过程中，参与根瘤菌的早期侵染以及在根瘤菌与豆科宿主植物之间的免疫防御反应中发挥重要功能。

关键词:共生固氮;大豆快生根瘤菌SMH12;效应蛋白NopP;共生表型

Abstract:

[Objective] To understand the molecular mechanism of the effector nopP in the nodulation and nitrogen fixation of Sinorhizobium fredii SMH12, and to provide foundation for effective genetic improvement of rhizobium-soybean symbiosis, we studied the function of nopP in symbiotic nitrogen fixation between S. fredii SMH12 and soybean. [Methods] The phylogenetics of the NopP was analyzed by bioinformatics, and the deletion mutant (ΔnopP), overexpression (OE-nopP) and complementation strains (CM-nopP) were constructed by genetic operation. We further analyzed the symbiotic phenotypes. Meanwhile, the expression pattern of nopP under free-living and symbiotic conditions and the expression of NIN, ENOD40, PR1, PR2 and PR5 in ΔnopP mutant in roots of Glycine max cv. JD17 inoculated with SMH12 and SMH12 ΔnopP were detected by quantitative real-time PCR (qRT-PCR). The subcellular localization of NopP was observed by laser confocal microscopy. [Results] The NopP protein of S. fredii SMH12 does not contain any functional domains and has no homologues with Avr effectors in the genome of several pathogens. Deletion of nopP in SMH12 significantly increased the nitrogen fixation activity of JD 17 and Zhong Huang 13 compared with the wild type strain SMH12, and the nodule number significantly increased in JD17 inoculated with the mutant ΔnopP. It is indicated that the deletion of nopP promoted the symbiotic nitrogen fixation with JD17 or Zhong Huang 13. qRT-PCR showed that the expression level of nopP under symbiosis was significantly up-regulated compared to that under free-living condition, and the nopP was highly expressed at a 2-day post inoculation (dpi), indicating that the nopP gene plays important role in the rhizobia early infection and the symbiotic nitrogen fixation. In addition, the NopP protein localizes at the cytoplasm membrane and nucleus in both tobacco leaves and soybean roots. Moreover, the expression of NIN in the roots of JD17 inoculated with the mutant ΔnopP was up-regulated by 1.2 times, whereas the expression level of PR5 was down-regulated by 3.6 times compared to the wild-type SMH12 inoculation. [Conclusion] The effector protein NopP of SMH12 plays important roles in the early infection of rhizobia and the defense response regulation in soybean under symbiosis.

Key words:symbiotic nitrogen fixation;Sinorhizobium fredii SMH12;effector protein NopP;symbiotic phenotypes