Studies On The Pathogenicity-Associated Gene Hpa2 In Xanthomonas Oryzae

The phytopathogenic bacteria Xanthomonas oryzae(X.oryzae) causes wide-spread diseases in rice including leaf blight and streak.The leaf blight caused by X.o.pathovar oryzae(Xoo) is one of the most serious diseases.The leaf streak caused by X.o.pathovar oryzicola(Xooc) is emerging as an important disease.As an important pathogenic bacterium,the mechanism of the bacterial invasion in host is poorly understood.Interaction between the pathogen and host associates with effector proteins through secretion systems. Existed in many species of phytopathogenic bacteria,typeâ…¢secretion system(TTSS) is one of such pivotal systems,which involves pathogenicity,hypersensitive response(HR) and the secretion of effector protein.Hrp proteins encoded by HR and pathogenicity(hrp) genes in a cluster consist of Hrc, Hrp and Hpa proteins in phytopathogenic Xanthomonas.The hrp-conserved(hrc) consists of eleven genes hrcC,T,N,L,J,U,V,Q,R,S and D.Corresponding Hrc proteins are required by the components of the TTSS.The hrp consists of nine genes hrpB7,B5,B4, B2,B1,D5,D6,E,and F.The hrp-associated(hpa) consists of eight genes hpa2,1,P,A,B, 4,3,and F.The plant-inducible promoter(PIP,conserved sequence TTCGC-N₁₅-TTCGC), including perfect and imperfect PIP boxes,has been identified in some operons of the hrp cluster.To clarify the function of hpa2 gene,we cloned five hpa2 genes from five bacterial strains Xoo PXO99,Xoo ZHE173,Xoo JXOâ…¢,Xooc HAN1 and Xooc RS105,and analyzed the gene and amino acid sequences.The result suggests that the conserved hpa2 gene contained 712 bp of genomic sequence including a promoter of 148 bp and an ORF of 564 bp.An imperfect PIP box(TTCGC-N₁₅-TTCGT) was identified in upstream of the promoter region.Several bases change in the nucleotide sequences.In promoter region,the base A₈₀ substitutes for G₈₀ in strain Xooc HAN1.In ORF region,the base T₄₃₀ substitutes for C₄₃₀,C₅₉₂ for T₅₉₂ and C₆₇₃ for T₆₇₃ in strain Xooc RS105;the base T₆₀₁ substitutes for A₆₀₁ and the G₆₁₈ for A₆₁₈ in strain Xoo PXO99.Because of degeneracy,all the five hpa2 ORF encodes a consensus amino acid sequence.The result of protein database research indicates that the Hpa2 may contain a Soluble Lytic Transglycosylase(SLT) domain.In enzyme,there are seven residues(Glu,Asp,Lys,Arg,Tyr,Cys and His)of general acid-base catalysis potential.The residue content(30.5%) of the Hpa2 was close to that(28.6%) of the soluble 70 kDa lytic transglycosylase (SLT70)or that(32.3%) of Enterobacteria phage T4 lysozyme(T4L),respectively.An Hpa2 protein from the strain Xoo PXO99 was successfully expressed in vector pET-30a(+).The protein consisted of a putative 187 amino acids,with a molecular weight of 20.8 kDa.Its isoelectric point(pI) was calculated to be 9.62.Consistent with this,we found that in vitro-expressed Hpa2 was a strongly basic protein,and a higher concentration of protein could be obtained in pH 10.1 TE buffer.To observe the secretion activity of the Hpa2,we co-cultured the over-expressed strains 564-6 and Micrococcus luteus(negative control),and investigate the changed OD₆₀₀ value.Compared with M.luteus alone,the OD₆₀₀ value of the co-cultured solution could not change significantly,but done when compared with the two positive controls including Hpa2 protein.This suggests that the Hpa2 cannot be secreted from the over-expressed strain E.coli cells.Six strains M.luteus,Clavibacter michiganense subsp.sepedonicum,Bacillus subtilis,Escherichia coli,Xoo PXO99 and Xooc RS105 were treated with different solutions,such as Hpa2 and pET-30a(+) supematants,lysozyme and TE buffer.All OD₆₀₀ values of treatments were measured in 2,4,6,8,10 and 12 hours.The result indicates that the OD₆₀₀ of all treatment solutions changed with the time.Compared with two negative controls [pET-30a(+) and TE buffer],treatments of the lysozyme and Hpa2 changed obviously.But, the change trend was different between the two treatments except the strain M.luteus.For the strains C.m.subsp.sepedonicum,E.coli and Xoo PXO99,the OD₆₀₀ value of the Hpa2 was lower than that of lysozyme in 2 hours while,for the former two strains,that of the Hpa2 was higher than the lysozyme in 8 hours.This suggests that the Hpa2 lytic activity may have faster kinetics and lower activity than lysozyme.Compared with the five others, the OD₆₀₀ value change of the strain M.luteus did not fluctuate.The OD₆₀₀ value of lysozyme was obviously lower than that of the Hpa2 in all treatments.Before 8 hours,the OD₆₀₀ values of lysozyme and Hpa2 were lower than the pET-30a(+) and TE buffer. However,after 10 hours the OD₆₀₀ value of the Hpa2 was not different from the two negatives. Together with above data,we conclude that the Hpa2 has lytic effect on the six resulting strains,but the activity was different among different bacteria,the strains M.luteus,C.m.subsp.sepedonicum and B.Subtilis are gram-positive,while the strains E. coli,Xoo PXO99 and Xooc RS105 are gram-negative.This suggests that the Hpa2 has lytic activity to diverse bacteria.To furtherly determine the site of the cell lysed by the Hpa2,based on the OD₆₀₀ measurement we selected the strain M.luteus for transmission electron microscope(TEM) observation.Treated with the Hpa2,cell wall was obviously waved,even disrupted and damaged.This indicates that the Hpa2 can possess a lytic activity against bacterial cell wall.For the elucidation of the interaction between hpa2 gene and TTSS,with the suicide vector pKNOCK-Cm and cosmid pUFR034 we furtherly made vector constrctions,and screened a loss-of-function mutant and a complemental transformant.After rice inoculumn, the leaf blight of the complement was smaller than that of the wild-type strain,but larger than that of the mutant.In 2 days,the wild-type cell growth was about 1000 fold,the mutant 10,and the complement 100,in rice leaves.After nonhost plant injection,the mutant could not induce HR on tobacco,tomto and peper,but done in Chinese cabbage. These indicate that the hpa2 gene possesses a pathogenicity in rice,and may induce HR in some nonhosts plants,but not in the others.Our result suggests that Hpa2 protein is conserved in X.oryzae,and has a lytic activity against the bacterial cell wall.We speculate that the Hpa2 contributes to the assembly of the TTSS by enlarging gaps in the peptidoglycan meshwork of bacterial cell wall,furtherly affect the pathogenicity of the pathogen.As this is the first of such enzyme activity identified in the Hrp protein family,our result offers a new clue to better understand the interaction between bacteria and their hosts.