Abstract：Rhizobium type Ⅲ secretion system (T3SS) effector proteins play important roles in nodulation, nitrogen fixation and decision of host range. In order to express and analysis the type Ⅲ-like secretion system effector proteins of Azorhizobium caulinodans ORS571, the whole proteome of ORS571 was analyzed by bioinformatics software BPBAac and Effective T3 and related literature reports. Seven candidate effector proteins of ORS571 were obtained, and they were AZC_0308, AZC_0649, AZC_0667, AZC_2699, AZC_2928, AZC_3165 and AZC_4087, respectively. Specific amplification primers with restriction enzyme cutting sites were designed and synthesized, and whole gene sequences of the 7 effector proteins were obtained by PCR. Than the 7 gene fragments were cloned into the prokaryotic expression vector pMAL-c4x, respectively. Through optimizing the expression conditions, a large amount of soluble fusion proteins were obtained. Amylose Resin affinity chromatography was used to purify fusion proteins, and the results indicated that purification effect was better. Detection of fusion proteins expression were completed by Western blot using anti- maltose binding protein (MBP) monoclonal antibody (horse radish peroxidase conjugated), and the results implied that these 7 fusion proteins expressed correctly and the effect of protein expression was better. AZC_2928 gene deletion mutant was constructed and infected wheat (Triticum aestivum) XiaoYan 22, and the results showed that AZC_2928 gene had inhibitory effect on the growth of wheat. In conclusion, this research lays a foundation for the application of the Azorhizobium caulinodans ORS571 type Ⅲ-like secretion system effector proteins in symbiotic nitrogen fixation of non-leguminous plants.
[1]Amann E, Brosius J.ATG vectors’ for regulated high-level expression of cloned genes in Escherichia coli[J].Gene, 1985, 40(2):183-190[2]Arnold R, Brandmaier S, Kleine F, et al.Sequence-based prediction of type III secreted proteins[J].PLoS Pathog, 2009, 5(4):e1000376-[3]Büttner D, Bonas U.Common infection strategies of plant and animal pathogenic bacteria[J].Current opinion in plant biology, 2003, 6(4):312-319[4]Bartsev A V, Boukli N M, Deakin W J, et al.Purification and phosphorylation of the effector protein NopL from Rhizobium spNGR234[J].FEBS letters, 2003, 554(3):271-274[5]Bartsev A V, Deakin W J, Boukli N M, et al.NopL,an effector protein of Rhizobium spNGR234,thwarts activation of plant defense reactions[J].Plant physiology, 2004, 134(2):871-879[6]Büttner D, Nennstiel D, Klüsener B, et al.Functional analysis of HrpF,a putative type III translocon protein from Xanthomonas campestris pvvesicatoria[J].Journal of Bacteriology, 2002, 184(9):2389-2398[7]Cleenwerck I, Vandemeulebroecke K, Janssens D, et al.Re-examination of the genus Acetobacter,with descriptions of Acetobacter cerevisiae spnov. and Acetobacter malorum sp. nov[J].International Journal of Systematic and Evolutionary Microbiology, 2002, 52(5):1551-1558[8]Cornelis G R, Van Gijsegem F.Assembly and function of type III secretory systems[J].Annual Reviews in Microbiology, 2000, 54(1):735-774[9]Deakin W J, Broughton W J.Symbiotic use of pathogenic strategies: rhizobial protein secretion systems[J].Nature Reviews Microbiology, 2009, 7(4):312-320[10]Deakin W J, Marie C, Saad M M, et al.NopA is associated with cell surface appendages produced by the type III secretion system of Rhizobium spstrain NGR234[J].Molecular plant-microbe interactions, 2005, 18(5):499-507[11]di Guan C, Li P, Riggs P, et al.An Escherichia coli vector to express and purify foreign proteins by fusion to and separation from maltose-binding protein. [J].Gene, 1987, 67:21-30[12]Dreyfus B, Garcia J L, Gillis M.Characterization of Azorhizobium caulinodans gennov.,sp. nov.,a stem-nodulating nitrogen-fixing bacterium isolated from Sesbania rostrata[J].International Journal of Systematic and Evolutionary Microbiology, 1988, 38(1):89-98[13]Duplay P, Bedouelle H, Fowler A, et al.Sequences of the malE gene and of its product,the maltose-binding protein of Escherichia coli K12[J].Journal of Biological Chemistry, 1984, 259(16):10606-10613[14]Gopalaswamy G, Kannaiyan S, O'Callaghan K J, et al.The xylem of rice (Oryza sativa) is colonized by Azorhizobium caulinodans[J].Proceedings of the Royal Society of London B: Biological Sciences, 2000, 267(1439):103-107[15]Kouchi H, Imaizumi-Anraku H, Hayashi M, et al.How many peas in a pod? Legume genes responsible for mutualistic symbioses underground[J].Plant and Cell Physiology, 2010, 51(9):1381-1397[16]Krause A, Doerfel A, G?ttfert M.Mutational and transcriptional analysis of the type III secretion system of Bradyrhizobium japonicum[J].Molecular plant-microbe interactions, 2002, 15(12):1228-1235[17]Lee K B, De Backer P, Aono T, et al.The genome of the versatile nitrogen fixer Azorhizobium caulinodans ORS571[J].BMC genomics, 2008, 9(1):1-[18]Margaret I, Becker A, Blom J, et al.Symbiotic properties and first analyses of the genomic sequence of the fast growing model strain Sinorhizobium fredii HH103 nodulating soybean[J].Journal of biotechnology, 2011, 155(1):11-19[19]Marie C, Broughton W J, Deakin W J.Rhizobium type III secretion systems: legume charmers or alarmers?[J].Current opinion in plant biology, 2001, 4(4):336-342[20]Nelson M S, Sadowsky M J.Secretion systems and signal exchange between nitrogen-fixing rhizobia and legumes. [J].Frontiers in plant science, 2015, 6:-[21]Okazaki S, Kaneko T, Sato S, et al.Hijacking of leguminous nodulation signaling by the rhizobial type III secretion system[J].Proceedings of the National Academy of Sciences, 2013, 110(42):17131-17136[22]Okazaki S, Okabe S, Higashi M, et al.Identification and functional analysis of type III effector proteins in Mesorhizobium loti[J].Molecular plant-microbe interactions, 2010, 23(2):223-234[23]Oldroyd G E D, Downie J A.Coordinating nodule morphogenesis with rhizobial infection in legumes. [J].Annual Review of Plant Biology, 2008, 59:519-546[24]Oldroyd G E D, Murray J D, Poole P S, et al.The rules of engagement in the legume-rhizobial symbiosis.[J].Annual review of genetics, 2011, 45:119-144[25]Saad M M, Kobayashi H, Marie C, et al.NopB,a type III secreted protein of Rhizobium spstrain NGR234,is associated with pilus-like surface appendages[J].Journal of bacteriology, 2005, 187(3):1173-1181[26]Sabry S R, Saleh S A, Batchelor C A, et al.Endophytic establishment of Azorhizobium caulinodans in wheat[J].Proceedings of the Royal Society of London B: Biological Sciences, 1997, 264(1380):341-346[27]Senthilkumar M, Madhaiyan M, Sundaram S P, et al.Induction of endophytic colonization in rice (Oryza sativa L) tissue culture plants by Azorhizobium caulinodans[J].Biotechnology letters, 2008, 30(8):1477-1487[28]Skorpil P, Saad M M, Boukli N M, et al.NopP,a phosphorylated effector of Rhizobium spstrain NGR234,is a major determinant of nodulation of the tropical legumes Flemingia congesta and Tephrosia vogelii[J].Molecular microbiology, 2005, 57(5):1304-1317[29]Spaink H P.The molecular basis of infection and nodulation by rhizobia: the ins and outs of sympathogenesis[J].Annual review of phytopathology, 1995, 33(1):345-368[30]Sugawara M, Epstein B, Badgley B D, et al.Comparative genomics of the core and accessory genomes of 48 Sinorhizobium strains comprising five genospecies[J].Genome biology, 2013, 14(2):1-[31]Tampakaki A P.Commonalities and differences of T3SSs in rhizobia and plant pathogenic bacteria.[J].Frontiers in plant science, 2014, 5:114-[32]Wang Y, Zhang Q, Sun M, et al.High-accuracy prediction of bacterial type III secreted effectors based on position-specific amino acid composition profiles[J].Bioinformatics, 2011, 27(6):777-784[33]Wassem R, Kobayashi H, Kambara K, et al.TtsI regulates symbiotic genes in Rhizobium species NGR234 by binding to tts boxes[J].Molecular microbiology, 2008, 68(3):736-748
