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

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.