Functional Analysis Of The Type Three Secretion Protein(NOPT) Of Mesorhizobium Amorphae CCNWGS0123

Molecular communication between legume and rhizobia resulting in symbiosis formation are subtle and complex. It had shown that the type three secretion system(T3SS) of Mesorhizobium amorphae CCNWGS0123 played an important role in symbiotic interactions between rhizobia and its host legumes by previous experiments in our laboratory. This research made a whole genome screening based on the conserved tts-box sequence to predict possible effectors and one ttx-box was found in the promoter region of the 5221 gene, which was tcGTCAGcTtgtcGtcAGttcgactctctA. Then the phylogeny of NopT was analysed by bioinformatics methods. Furthermore, we knocked out the gene encoding NopT and obtained the mutant. To research the role of NopT in the process of the nodule symbiosis between rhizobia and legume, the mutant and wild type strains were used for the inoculation experiment and we observed the plant phenotypic characteristics.Main results for this study were as follows:(1) The analysis of amino acid sequence indicated that the NopT protein encoded by 5221 gene in M. amorphae CCNWGS0123 was different from the type three effector protein NopT in Rhizobium sp.strain NGR234 and it might be belong to a new effector family;(2) The results of qRT-PCR showed that the 5221 gene expression increased nder flavonoid induction, this data indicted that the 5221 gene responsed to the lavonoid signal;(3) The inoculation experiment revealed that the nodules formation between the host egume Robinia Pseudoacacia with wild type strain or mutant strain had no much ifferences. The nodules were all pink. The paraffin section results showed that the rganizational structure of the nodules were alike. But the nodules amount of the utant was much less than the wild type strains, NopT mainly affected the number of odules.We speculated that the mutant strains of 5221 gene may affect the symbiotic nodulation ability with R. Pseudoacacia through the type three secretion system. But what and how did it work during the formation of nodules?More researches still need to do in future.