Genome Sequencing Of Mesorhizobium Amorphae CCNWGS0123 And Functions Of The Two Type ? Secretion Systems

Due to its fast-growing and high N₂-fixing capacity,Robinia pseudoacacia can be used for recultivation of devastated soil.R.pseudoacacia could nodulate with Mesorhizobium and Sinorhizobium strains in China,the mutualistic symbiosis established between legume plants and rhizobia was highly specific.Nodulation factors,exopolysaccharides and type ?secretion system?T3SS?effectors derived from rhizobia played important roles in the symbiosis specificity.In previous work,M.amorphae CCNWGS0123?GS0123?was isolated from R.pseudoacacia nodules,and it could establish effective symbiosis with R.pseudoacacia.In the present study,the complete genome sequence of GS0123 was obtained by using single molecular real time?SMRT?technology.GS0123 was the unique strain who contained two clusters of T3SS when compared with the other nine Mesorhizobium strains.In order to reveal the role which T3SS played in symbiosis process established between R.pseudoacacia and GS0123,single and double T3SS deletion mutants were constructed.In the early stage of symbiosis process,the infection events were observed;the content of signal molecules related with plant immunity was measured and the expression of plant symbiosis genes was analyzed.In the late stage of nodulation,the number of effective nodules was counted and the morphology of nodule cells was observed by using optical microscope and transmission electron microscopy.Results are listed as follows:?1?The genome of GS0123 was consisted of one circular chromosome with a 6,268,270bp length and four plasmids-pM0123a?102,093 bp?,pM0123b?17,414 bp?,pM0123c?7,607bp?and pM0123d?948,568 bp?,and the pM0123d was the symbiotic plasmid.The genome had an average G+C content of 62.87%,and 7,134 coding sequences were identified in the genome.GS0123 and the other nine Mesorhizobium strains shared 7,134 orthologous genes.GS0123 had 1,758 specific genes.According to KEGG?Kyoto Encyclopedia of Genes and Genomes?annotation,83/1,758 genes participate in Transporter pathway?ko02000?.Four T3SS core genes–rhc U,rhcV,rhcT and rhcS were included.According to COG?Cluster of Orthologous Groups of proteins?classification,the 10 rhizobia had high proportion of functional genes participating in amino acid transport and metabolism,transcription,carbohydrate transport and metabolism.GS0123 had similar nod gene composition with M.loti MAFF303099,and similar nif/fix gene composition with M.ciceri CC1192.T3SS clusters were identified in GS0123?M.loti MAFF303099?M.huakuii 7653R and M.ciceri CC1192genomes with core protein similarities of 60%⁸5%.?2?GS0123 contained two clusters of T3SSs in its genome,they were named as T3SS_? and T3SS-? based on the SctU protein classification,respectively.T3SS_? located in symbiotic plasmid,and contained all basal components for a functional T3SS.T3SS-? located in chromosome,and it lacked the primary extracellular element?needle?for a functional T3SS.T3SS-? was specific for GS0123 genome.The expression of T3SS_? core genes were up-regulated by flavonoid?plant symbiosis signal molecule?induction,while the expression of T3SS-? core genes were down-regulated by flavonoid induction.?3?GS0123 T3SS-? deletion mutant-GS0123?rhcN1?GS0123?T1?triggered plant defense response in H₂O₂ pathway and salicylic acid?SA?pathway.Compared with plants inoculated with wild type strain GS0123,plants inoculated with GS0123?T1 displayed lower shoots,yellow leaves and obvious nitrogen deficiency phenotypes;formed fewer infection threads,nodule primordium,effective nodules,infected nodule cells and bacteroids.The deletion of T3SS_? from GS0123 decreased the rhizobia compatibility in nodulating with R.pseudoacacia.In addition,in the infected nodule cells,single or a few bacteroid were enclosed in“vesicles”.?4?GS0123 T3SS-? deletion mutant-GS0123?rhcN2?GS0123?T2?triggered plant defense response in H₂O₂ pathway,but suppressed the plant defense response in SA pathway.Plants inoculated with GS0123?T2 had similar growth status with that of plants inoculated with the wild type strain GS0123.The deletion of T3SS-? from GS0123 did not affect the rhizobia compatibility in nodulating with R.pseudoacacia.In addition,in the infected nodule cells,multiple bacteroids were enclosed in“vesicles”,and the phenomena were frequently observed.?5?In most sampling time points,the double T3SS deletion mutant–GS0123?rhcN1?rhcN2?GS0123?S?did not trigger or suppress plant immunity.Compared with plants inoculated with wild type strain GS0123,plants inoculated with GS0123?S displayed lower shoots,yellow leaves and obvious nitrogen deficiency phenotypes.Almost no infection threads or effective nodules were observed in plants inoculated with GS0123?S.Hardly any infected cells were observed in nodules.The deletion of both T3SSs from GS0123dramatically decreased the rhizobia compatibility in nodulating with R.pseudoacacia.Compared with plants inoculated with GS0123?T1,plants inoculated with GS0123?S displayed fewer infection threads or effective nodules.The deletion of T3SS-? from GS0123?T1 further decreased the rhizobia compatibility in nodulating with R.pseudoacacia.It was indicated that single T3SS-? from GS0123 played positive roles in the symbiosis establishment between R.pseudoacacia and GS0123;coexistence of T3SS_? and T3SS-? promoted infection by suppressing host plant defense response in H₂O₂ pathway.This study extended our understanding of the role which rhizobia T3SS played in the symbiosis establishment between legume and rhizobia.It provided the base for studying the symbiosis interaction between R.pseudoacacia and GS0123.