水稻黄单胞菌致病相关基因hpa2的研究
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摘要
植物病原细菌水稻黄单胞引起水稻病害,包括水稻白叶枯病和水稻细条斑病。水稻白叶枯病由水稻黄单胞白叶枯致病变种引起,是水稻上最严重的病害之一;水稻细条斑病由水稻黄单胞细条斑致病变种引起,是在水稻主产区逐渐流行的一个重要病害。作为重要的植物病原,水稻黄单胞菌的侵入机制还没有被很好地阐明。病菌与寄主互作的过程与经过分泌系统泌出的效应蛋白相联系。在植物病原细菌中,Ⅲ型泌出通道是一个重要的分泌系统。这个系统涉及病原致病性,过敏性反应,以及效应蛋白的分泌。
在植物病原黄单胞菌中,引起过敏反应和致病性的蛋白由其hrp基因簇上的基因编码,这些蛋白包括Hrc蛋白、Hrp蛋白和Hpa蛋白等。hrc基因共有11个,即hrcC、T、N、L、J、U、V、Q、R、S和D,这些基因对应的Hrc蛋白是Ⅲ型泌出系统的组成成分。hrp基因共有9个,即hrpB7、B5、B4、B2、B1、D5、D6、E和F。hpa基因共有8个,即hpa2、1、P、A、B、4、3和F。已经确定,在hrp基因簇一些操纵子的启动子区域含有植物诱导的PIP盒,即一个保守的序列TTCGC-N_(15)-TTCGC。这个PIP包括完整的PIP盒和不完整的PIP盒。
为了研究hpa2基因的功能,我们选定了5个水稻黄单胞菌菌株,即Xanthomonasoryzae pv.oryzae PXO99(Xoo PXO99)、Xanthomonas oryzae pv.oryzae ZHE173(XooZHE173)、Xanthomonas oryzae pv.oryzae JXOⅢ(Xoo JXOⅢ)、Xanthomonas oryzaepv.oryzicola HAN1(Xooc HAN1)和Xanthomonas oryzae pv.oryzicola RS105(XoocRS105),克隆得到相应的hpa2基因。对这些基因及其推测的氨基酸序列进行分析,结果表明:具有712 bp的hpa2基因由564 bp的ORF区域和148 bp的启动子区域构成,在水稻黄单胞菌中高度保守。在启动子区域的上游都存在一个相同的不完整的植物诱导的PIP盒,即TTCGC-N_(15)-TTCGT。在核苷酸序列中,只有个别碱基发生变化。在启动子区域,菌株Xooc HAN1只有一个碱基被替换,即G_(80)→A_(80)。在ORF区域,菌株Xooc RS105有三个碱基被替换,即C_(430)→T_(430),T_(592)→C_(592),T_(673)→C_(673)。菌株XooPXO99有两个碱基被替换,即A_(601)→T_(601),A_(618)→G_(618)。由于密码子的简并性,这五个hpa2基因编码一致的氨基酸序列。
蛋白质数据库搜索结果显示,Hpa2蛋白的氨基酸序列中含有可溶性的裂解转糖基酶(Soluble Lytic Transglycosylase,SLT)的功能域。在酶分子中,具有总酸碱催化能力的氨基酸残基有七个,即谷氨酸、天冬氨酸、赖氨酸、精氨酸、酪氨酸、半胱氨酸和组氨酸等。它们在Hpa2蛋白中的含量为30.5%,接近于在SLT70中28.6%的含量和在T4噬菌体溶菌酶(Enterobacteria phage T4 Lysozyme,T4L)中32.3%的含量。
使用pET-30a(+)载体,我们成功地表达了来自Xoo PXO99菌株hpa2基因564 bp的ORF,得到了一个Hpa2蛋白。该蛋白含有187个氨基酸,分子量为20.8 kDa,等电点为9.62。与推算的结果相一致,Hpa2蛋白呈现强碱性,在pH10.1的TE缓冲液中,能够提取到足量的Hpa2蛋白。为了研究Hpa2蛋白的分泌特性,我们将Hpa2蛋白的过量表达菌株564-6与菌株Micrococcus luteus进行了共培养,观察OD_(600)值的变化情况。与菌株M.luteus的单独培养相比较,共培养菌液的OD_(600)值没有发生明显的变化;而与含有Hpa2蛋白的两个正对照相比却发生了明显的变化。这表明,Hpa2蛋白没有从它的过量表达菌株E.coli的细胞内分泌至细胞外。
我们对M.luteus、Clavibacter michiganense subsp.sepedonicum、Bacillus subtilis、Escherichia coli、Xoo PXO99和Xooc RS105等六个菌株分别进行了含有Hpa2蛋白上清液、含有pET-30a(+)载体的上清液(阴性对照)、溶菌酶(阳性对照)和pH值为6.8的TE缓冲溶液(阴性对照)的处理。对所有处理液的6个时间段,即2、4、6、8、10和12小时,分别进行了OD_(600)值的测定。结果显示,随着处理时间的延长,所有处理液的OD_(600)值都有下降的趋势。与两个阴性对照相比,经溶菌酶和Hpa2上清液处理的都发生了明显的变化。但是,两种处理变化的情况不同。除M.luteus外,溶菌酶与Hpa2上清液处理的OD_(600)变化趋势存在差异。在2小时后,对于C.m.subsp.sepedonicum、E.coli和Xoo PXO99菌株,Hpa2上清液低于溶菌酶的OD_(600)值。在8小时以后,而对于C.m.subsp.sepedonicum和E.coli菌株,Hpa2上清液却高于溶菌酶的OD_(600)值。这一结果表明的Hpa2的酶动力性高于溶菌酶,但酶活性要低于溶菌酶。与另外五个菌株的变化趋势相比,M.luteus菌株4个处理溶液的OD_(600)值一直呈稳定变化的趋势。在所有时间段里,溶菌酶处理的OD_(600)值都明显低于Hpa2上清液。在8小时之前,溶菌酶和Hpa2上清液与两个阴性对照[TE缓冲溶液和pET-30a(+)上清液]相比,都明显降低。但在10小时之后,Hpa2上清液与两个阴性对照相比,却无差异。
综合上述OD_(600)值测定结果,我们得出结论:Hpa2蛋白对供试的6个细菌菌株都具有溶菌作用,但对不同菌株的溶菌活性有差异。M.luteus、C.m.subsp.sepedonicum和B.subtilis为革兰氏阳性菌;E.coli、Xoo PXO99和Xooc RS105为革兰氏阴性菌。这说明,Hpa2蛋白对不同类型的细菌都具有溶解活性。
为了进一步确定Hpa2蛋白溶解细菌细胞的位点,根据上述实验结果,我们选定了M.luteus作为供试菌株。经过Hpa2蛋白处理后,在透射电子显微镜下观察,细菌的细胞壁明显地被裂解。细胞壁由光滑变为凹凸形状,部分被毁坏,甚至呈现崩溃的、细胞内物质溢出的状态。这说明,Hpa2蛋白能够裂解细菌的细胞壁。
为了更好地探明Ⅲ型泌出系统与hpa2基因的关系,我们利用自杀性的敲除载体pKNOCK-Cm和具有功能互补作用的粘粒载体pUFR034,构建了hpa2基因的突变载体pKNO464-16和互补载体pUFRcom712-22,并筛选得到hpa2基因的突变体464-16及其功能互补子712-22。对突变体和互补转化子进行水稻接种,观察植株反应情况。接种水稻的观察结果:突变菌株接种叶片的病斑明显小于野生菌株Xoo PXO99的病斑,互补菌株的病斑虽然比野生菌株的小,但比突变菌株的大。细菌繁殖的增长量测定结果:在水稻叶片中,接种两天后的野生菌株细胞的增长量大约为1000倍,突变体的大约为10倍,互补的大约为100倍。接种非寄主的结果:突变体不能诱导烟草、番茄和辣椒产生HR,但可以诱导大白菜产生HR。上述结果表明,hpa2基因能够在水稻上引起致病性,可以在某些非寄主植物上引产生敏性反应,也有个别非寄主植物不能产生过敏性反应。
综合全部实验结果,我们得出结论:Hpa2蛋白在水稻黄单胞菌中是保守的,对细菌细胞壁具有裂解活性。我们推测Hpa2蛋白通过拉大细菌细胞壁肽聚糖网络孔洞的方式辅助Ⅲ型泌出系统的装配。这表明hpa2基因可以通过TTSS起作用,进而影响病菌的致病性。Hpa2是Hrp蛋白家族中第一个被鉴定的具有裂解活性的蛋白,所以,我们的实验结果有助于更好地理解细菌与寄主的互作关系。
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,typeⅢsecretion 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-N_(15)-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 JXOⅢ、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-N_(15)-TTCGT) was identified in upstream of the promoter region.Several bases change in the nucleotide sequences.In promoter region,the base A_(80) substitutes for G_(80) in strain Xooc HAN1.In ORF region,the base T_(430) substitutes for C_(430),C_(592) for T_(592) and C_(673) for T_(673) in strain Xooc RS105;the base T_(601) substitutes for A_(601) and the G_(618) for A_(618) 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 OD_(600) value.Compared with M.luteus alone,the OD_(600) 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 OD_(600) values of treatments were measured in 2,4,6,8,10 and 12 hours.The result indicates that the OD_(600) 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 OD_(600) 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 OD_(600) value change of the strain M.luteus did not fluctuate.The OD_(600) value of lysozyme was obviously lower than that of the Hpa2 in all treatments.Before 8 hours,the OD_(600) values of lysozyme and Hpa2 were lower than the pET-30a(+) and TE buffer. However,after 10 hours the OD_(600) 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 OD_(600) 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.
在植物病原黄单胞菌中,引起过敏反应和致病性的蛋白由其hrp基因簇上的基因编码,这些蛋白包括Hrc蛋白、Hrp蛋白和Hpa蛋白等。hrc基因共有11个,即hrcC、T、N、L、J、U、V、Q、R、S和D,这些基因对应的Hrc蛋白是Ⅲ型泌出系统的组成成分。hrp基因共有9个,即hrpB7、B5、B4、B2、B1、D5、D6、E和F。hpa基因共有8个,即hpa2、1、P、A、B、4、3和F。已经确定,在hrp基因簇一些操纵子的启动子区域含有植物诱导的PIP盒,即一个保守的序列TTCGC-N_(15)-TTCGC。这个PIP包括完整的PIP盒和不完整的PIP盒。
为了研究hpa2基因的功能,我们选定了5个水稻黄单胞菌菌株,即Xanthomonasoryzae pv.oryzae PXO99(Xoo PXO99)、Xanthomonas oryzae pv.oryzae ZHE173(XooZHE173)、Xanthomonas oryzae pv.oryzae JXOⅢ(Xoo JXOⅢ)、Xanthomonas oryzaepv.oryzicola HAN1(Xooc HAN1)和Xanthomonas oryzae pv.oryzicola RS105(XoocRS105),克隆得到相应的hpa2基因。对这些基因及其推测的氨基酸序列进行分析,结果表明:具有712 bp的hpa2基因由564 bp的ORF区域和148 bp的启动子区域构成,在水稻黄单胞菌中高度保守。在启动子区域的上游都存在一个相同的不完整的植物诱导的PIP盒,即TTCGC-N_(15)-TTCGT。在核苷酸序列中,只有个别碱基发生变化。在启动子区域,菌株Xooc HAN1只有一个碱基被替换,即G_(80)→A_(80)。在ORF区域,菌株Xooc RS105有三个碱基被替换,即C_(430)→T_(430),T_(592)→C_(592),T_(673)→C_(673)。菌株XooPXO99有两个碱基被替换,即A_(601)→T_(601),A_(618)→G_(618)。由于密码子的简并性,这五个hpa2基因编码一致的氨基酸序列。
蛋白质数据库搜索结果显示,Hpa2蛋白的氨基酸序列中含有可溶性的裂解转糖基酶(Soluble Lytic Transglycosylase,SLT)的功能域。在酶分子中,具有总酸碱催化能力的氨基酸残基有七个,即谷氨酸、天冬氨酸、赖氨酸、精氨酸、酪氨酸、半胱氨酸和组氨酸等。它们在Hpa2蛋白中的含量为30.5%,接近于在SLT70中28.6%的含量和在T4噬菌体溶菌酶(Enterobacteria phage T4 Lysozyme,T4L)中32.3%的含量。
使用pET-30a(+)载体,我们成功地表达了来自Xoo PXO99菌株hpa2基因564 bp的ORF,得到了一个Hpa2蛋白。该蛋白含有187个氨基酸,分子量为20.8 kDa,等电点为9.62。与推算的结果相一致,Hpa2蛋白呈现强碱性,在pH10.1的TE缓冲液中,能够提取到足量的Hpa2蛋白。为了研究Hpa2蛋白的分泌特性,我们将Hpa2蛋白的过量表达菌株564-6与菌株Micrococcus luteus进行了共培养,观察OD_(600)值的变化情况。与菌株M.luteus的单独培养相比较,共培养菌液的OD_(600)值没有发生明显的变化;而与含有Hpa2蛋白的两个正对照相比却发生了明显的变化。这表明,Hpa2蛋白没有从它的过量表达菌株E.coli的细胞内分泌至细胞外。
我们对M.luteus、Clavibacter michiganense subsp.sepedonicum、Bacillus subtilis、Escherichia coli、Xoo PXO99和Xooc RS105等六个菌株分别进行了含有Hpa2蛋白上清液、含有pET-30a(+)载体的上清液(阴性对照)、溶菌酶(阳性对照)和pH值为6.8的TE缓冲溶液(阴性对照)的处理。对所有处理液的6个时间段,即2、4、6、8、10和12小时,分别进行了OD_(600)值的测定。结果显示,随着处理时间的延长,所有处理液的OD_(600)值都有下降的趋势。与两个阴性对照相比,经溶菌酶和Hpa2上清液处理的都发生了明显的变化。但是,两种处理变化的情况不同。除M.luteus外,溶菌酶与Hpa2上清液处理的OD_(600)变化趋势存在差异。在2小时后,对于C.m.subsp.sepedonicum、E.coli和Xoo PXO99菌株,Hpa2上清液低于溶菌酶的OD_(600)值。在8小时以后,而对于C.m.subsp.sepedonicum和E.coli菌株,Hpa2上清液却高于溶菌酶的OD_(600)值。这一结果表明的Hpa2的酶动力性高于溶菌酶,但酶活性要低于溶菌酶。与另外五个菌株的变化趋势相比,M.luteus菌株4个处理溶液的OD_(600)值一直呈稳定变化的趋势。在所有时间段里,溶菌酶处理的OD_(600)值都明显低于Hpa2上清液。在8小时之前,溶菌酶和Hpa2上清液与两个阴性对照[TE缓冲溶液和pET-30a(+)上清液]相比,都明显降低。但在10小时之后,Hpa2上清液与两个阴性对照相比,却无差异。
综合上述OD_(600)值测定结果,我们得出结论:Hpa2蛋白对供试的6个细菌菌株都具有溶菌作用,但对不同菌株的溶菌活性有差异。M.luteus、C.m.subsp.sepedonicum和B.subtilis为革兰氏阳性菌;E.coli、Xoo PXO99和Xooc RS105为革兰氏阴性菌。这说明,Hpa2蛋白对不同类型的细菌都具有溶解活性。
为了进一步确定Hpa2蛋白溶解细菌细胞的位点,根据上述实验结果,我们选定了M.luteus作为供试菌株。经过Hpa2蛋白处理后,在透射电子显微镜下观察,细菌的细胞壁明显地被裂解。细胞壁由光滑变为凹凸形状,部分被毁坏,甚至呈现崩溃的、细胞内物质溢出的状态。这说明,Hpa2蛋白能够裂解细菌的细胞壁。
为了更好地探明Ⅲ型泌出系统与hpa2基因的关系,我们利用自杀性的敲除载体pKNOCK-Cm和具有功能互补作用的粘粒载体pUFR034,构建了hpa2基因的突变载体pKNO464-16和互补载体pUFRcom712-22,并筛选得到hpa2基因的突变体464-16及其功能互补子712-22。对突变体和互补转化子进行水稻接种,观察植株反应情况。接种水稻的观察结果:突变菌株接种叶片的病斑明显小于野生菌株Xoo PXO99的病斑,互补菌株的病斑虽然比野生菌株的小,但比突变菌株的大。细菌繁殖的增长量测定结果:在水稻叶片中,接种两天后的野生菌株细胞的增长量大约为1000倍,突变体的大约为10倍,互补的大约为100倍。接种非寄主的结果:突变体不能诱导烟草、番茄和辣椒产生HR,但可以诱导大白菜产生HR。上述结果表明,hpa2基因能够在水稻上引起致病性,可以在某些非寄主植物上引产生敏性反应,也有个别非寄主植物不能产生过敏性反应。
综合全部实验结果,我们得出结论:Hpa2蛋白在水稻黄单胞菌中是保守的,对细菌细胞壁具有裂解活性。我们推测Hpa2蛋白通过拉大细菌细胞壁肽聚糖网络孔洞的方式辅助Ⅲ型泌出系统的装配。这表明hpa2基因可以通过TTSS起作用,进而影响病菌的致病性。Hpa2是Hrp蛋白家族中第一个被鉴定的具有裂解活性的蛋白,所以,我们的实验结果有助于更好地理解细菌与寄主的互作关系。
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,typeⅢsecretion 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-N_(15)-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 JXOⅢ、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-N_(15)-TTCGT) was identified in upstream of the promoter region.Several bases change in the nucleotide sequences.In promoter region,the base A_(80) substitutes for G_(80) in strain Xooc HAN1.In ORF region,the base T_(430) substitutes for C_(430),C_(592) for T_(592) and C_(673) for T_(673) in strain Xooc RS105;the base T_(601) substitutes for A_(601) and the G_(618) for A_(618) 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 OD_(600) value.Compared with M.luteus alone,the OD_(600) 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 OD_(600) values of treatments were measured in 2,4,6,8,10 and 12 hours.The result indicates that the OD_(600) 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 OD_(600) 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 OD_(600) value change of the strain M.luteus did not fluctuate.The OD_(600) value of lysozyme was obviously lower than that of the Hpa2 in all treatments.Before 8 hours,the OD_(600) values of lysozyme and Hpa2 were lower than the pET-30a(+) and TE buffer. However,after 10 hours the OD_(600) 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 OD_(600) 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.
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