Functional Analysis Of Type ? Secretion Effectors NopT And NopJ Of Mesorhizobium Amorphae CCNWGS0123 On The Symbiotic Nodulation

Type 3 secretion effectors(T3SEs)are directly injected into the eukaryotic cells to function through type 3 secretion system(T3SS).T3 SEs are not only virulence factors in pathogen-plant interaction,but also have an effect in symbiosis between rhizobia and leguminous plants.Some T3 SEs in rhizobia are homologous to T3 SEs in pathogenic bacteria,but others are unique to rhizobia.T3 SEs in rhizobia are also named nodulation outer proteins(Nops).Compared with pathogenic bacteria,there are few researches about T3 SEs in rhizobia at present,whose function during the establishment of symbiosis remains to be further elucidated.Previous studies found that Robinia pseudoacacia could establish the effective symbiosis with Mesorhizobium amorphae CCNWGS0123 which contains two clusters of T3 SS.Among them,T3SS-1 has a positive effect on symbiosis of M.amorphae CCNWGS0123 and R.pseudoacacia.The T3SS-1 gene cluster located on the p M0123 d plasmid.NopT and NopJ that are respectively homologous to YopT and Yop J in pathogenic bacteria are found on p M0123 d through sequence alignment.In order to study the function of NopT and NopJ on symbiotic nodulation of M.amorphae CCNWGS0123 and R.pseudoacacia,the following works are carried out in this study: The bioinformatic analysis of the amino acid sequences of NopT and NopJ were performed;Genes encoding NopT and NopJ were respectively knocked-out to obtain mutants which were then inoculated on the original host R.pseudoacacia roots for analyzing the symbiotic nodulation;That whether NopT and NopJ caused plant immune response were studied by the Agrobacterium-mediated transformation on tobacco system.Simultaneously,the response of R.pseudoacacia to NopT and NopJ were analyzed through comparative transcriptomics;The target proteins of NopT and NopJ were respectively searched by screening the c DNA library of R.pseudoacacia roots using the yeast two-hybrid assay,and their interaction was verified by bimolecular fluorescence complementation and co-immunoprecipitation assays;The subcellular localizations of NopT,NopJ,and their target proteins,were detected.The main results are as follows:1.NopT of M.amorphae CCNWGS0123 belongs to the YopT/Avr Pph B family,which contained the conserved catalytic triad C97/H210/D225.A conserved tts box was present in the upstream from the start codon of nopT.NopT expressed in Escherichia coli showed autoproteolytic activity.NopJ belongs to the Yop J family.NopJ contained the conserved catalytic triad H171/E190/C232,a consensus myristoylation site G2,and an autoacetylation site K301.2.The function-loss mutants of NopT and NopJ(?nopT and ?nop J)were respectively inoculated on R.pseudoacacia.At 30 days post-inoculation,samples were collected for statistics analysis of nodulation.Compared with plants inoculated with the wild-type strains,the indexes(shoot and root length,fresh and dry weight,nodule number,and nitrogenase activity)of plants inoculated with ?nopT were slightly decreased but there was not significant difference.The shoot and root length,fresh weight and dry weight of plants inoculated with ?nop J were all increased.Additionally,the nodule numbers were significantly increased.These results indicated that the symbiosis between M.amorphae CCNWGS0123 and R.pseudoacacia was neutrally affected by NopT and negatively affected by NopJ.3.In tobacco plants,NopT caused a hypersensitive response(HR)and NopJ caused a rapid HR.In the injected leaves sites,HR resulted in a decrease in the chlorophyll content,an increase in electrolyte leakage,the accumulation of reactive oxygen species(ROS),and locally yellowing and necrosis symptom.At the early stage of symbiosis,the phytohormones(salicylic acid and jasmonic acid)and ROS(hydrogen peroxide)levels in R.pseudoacacia plants inoculated with mutant strains were higher than those plants inoculated with wild type(WT)strains.These results suggested the plant immune response of R.pseudoacacia was inhibited by NopT and NopJ.The results of transcriptome analysis showed that the disease resistance genes in R.pseudoacacia roots inoculated with WT strains were up-regulated,compared with that inoculated mutant strains.The results indicated that the inhibition might be recognized by the disease resistance genes in plants and triggered ETI.Compared with R.pseudoacacia inoculated with WT strains,most genes of plants inoculated with ?nopT were down-regulated at 36 hours post inoculation(hpi)and up-regulated at 72 hpi;most genes of plants inoculated with ?nop J were down-regulated at 36 hpi and the numbers of genes up-regulated and down-regulated were basically the same at 72 hpi.Many differentially expressed genes were involved in the metabolic pathways.4.In the yeast and tobacco plant cells,the target proteins of NopT and NopJ were respectively screened.The interactions of NopT and ATP-CSACP2,NopT and HIRP,and NopJ and transcription factor WRKY69,were verified.In the tobacco plant cells,NopT,ATP-CSACP2,and HIRP,were all localized to the plasma membrane(PM)and nucleus when all of them were expressed alone.The subcellular localization of NopT and its target proteins were the same when they were co-expressed in the cells.Interestingly,NopJ was localized to PM and nucleus and WRKY69 was localized to the nucleus when NopJ and WRKY69 were expressed alone.However,NopJ was only localized to the PM when it was co-expressed with WRKY69 and it was not observed on the nucleus.The research laid an important foundation for revealing the specific functions of T3 SEs during the symbiosis establishment,which will contribute to study how employ T3 SS and T3 SEs to promote the rhizobia infection of leguminous plants and then improve the efficiency of symbiotic nitrogen fixation.