【作者】 张桂英；

【导师】 王金生；

【作者基本信息】 南京农业大学 ， 植物病理学， 2009， 博士

【摘要】 水稻黄单胞菌的两个致病变种水稻白叶枯病菌(Xanthomonas oryzae pv. oryzae, Xoo)和水稻条斑病菌(Xanthomonas oryzae pv.oryzicola, Xooc)是模式植物水稻(Oryza sativa)上的两种模式病原菌,分别引起水稻白叶枯病(bacterial blight, BB)和水稻细菌性条斑病(bacterial leaf streak, BLS),影响了亚洲大部分、非洲部分地区主要粮食作物的生产。水稻白叶枯菌(Xoo)从被发现至今100多年的历史中,一直是植物病理学研究者关注的焦点。研究水稻抗病基因(R)和Xoo无毒基因(A)的互作是阐明抗病基因和Xoo的致病性分化、鉴定小种、无毒基因、致病基因等的基本依据,也是发展有效的病害防治措施的需要。Xoo的小种分化涉及到对现有抗病品种的潜在利用价值。各国的Xoo代表菌株在科研和生产实践中都具有十分重要的意义。在寄主植物上的过敏反应HR最初被认为是一种“抗病现象”受到广泛关注,而病原细菌在非寄主烟草上的HR症状也是判定一个细菌分离物是否具有植物致病性的重要指标。由于HR现象与植物病原菌的致病性及寄主和非寄主植物的抗病性或内生免疫有着重要的关系,因此,研究Xoo菌株在寄主水稻上产生HR相关的avrBs3/pthA家族无毒基因和使非寄主烟草丧失产生HR的文库克隆pA254的组成和功能为更好地阐释植物细菌致病机理,植物病害抗病机理和植物与微生物学的其他方面的基本原理,同时也为内生免疫等方面带来重要启示。1 Xoo代表菌株致病多样性分析通过苗期注射接种法和成株期剪叶接种法,研究了20个来自中国、日本和菲律宾的Xoo不同致病型的代表菌株与36个水稻品种的互作关系。Xoo注射接种入14d的36个水稻品种的叶片中进行非亲和互作的HR表型测定,并对接种后第2-5d的接种叶片进行DAB溶液染色,检测接种叶片中活性氧爆发的情况,来观察HR发生情况。结果显示OS-198与所测试36个水稻品种苗期叶片中都发生有不同程度的非亲和互作,PX061、JXOI、OS-189菌株等与所测试36个品种中大多数品种之间存在特异性互作关系。Xoo注射接种所产生的症状是一复杂的表型,JXOI与IRBB1、IRBB2等发生特异性的互作,产生肉眼可见的典型的HR症状,表现出质量性状,但是更普遍现象是多数的Xoo在水稻品种上产生一种混合症状,即water-soaking症状和HR反应同时存在,但是病斑长度没有明显的变化,推测Xoo中所含有多数的avrBs3/pthA基因具有的无毒性属于一种数量累加效应,不是质量性状。研究结果明确了白叶枯菌与水稻近等基因系等36个水稻品种的互作关系,报道了在Xoo-水稻互作系统中的非亲和性互作与氧爆发的关系,推测不同代表菌株中可能含有的无毒基因,这为克隆对应R基因的无毒基因以及揭示水稻黄单胞菌致病性变异机制奠定了工作基础和提供了科学线索和依据。成株期剪叶接种结果显示在36个水稻品种中,基因累加品系IRBB54(xa5+Xa21)和IRBB55(xa13+Xa21)抗所有测试菌株,在20个测试菌株中,没有发现对36个水稻品种都致病的菌株。带有显性抗病基因Xa4、Xa7、Xal7、Xa21和隐性抗病基因xa5、xa13、xa24的水稻品种对50%以上的测试代表菌株抗病,而带显性抗病基因Xa1、Xa2、Xa10、Xa11、Xa12、Xa14、Xa18和隐性抗病基因xa8、xa19、xa20的水稻品种对50%以上的测试的代表菌株感病,籼稻IRBB3(Xa3)感病的品种也超过50%。IRBB21(Xa21)拥有最广的单基因抗性谱。IRBB54和IRBB55是抗病表现最好的双抗病基因组合的品种。常规品种TN-1感所有的测试菌株,IR26、BJ1、Asminori、Wase Aikoku 3和DV85对60%以上的测试菌有抗性.IR24、Java14、Tetep和Cas209对60%以上的测试菌感病。Xoo在36个水稻品种上的致病表型把测试菌株分成19个致病型,每个代表菌株的致病性各不相同。根据致病型的相似性,19个致病型可聚成7个类群(Cluster),聚类群3是优势群,分布最广,包含有测试的3国的代表菌株。2 Xoo代表菌株的小种遗传多样性分析有证据表明水稻黄单胞菌都含有15个以上的avrBs3/pthA家族基因,它们具有几乎一样的5’端和3’端,不同的只是中间102 bp重复单元的重复数不同。本研究参照PXO99A全基因组序列中已知的avrBs3/pthA基因的组成、数目和序列,以avrBs3/pthA家族成员avrXa3的中间保守区域大小为2.4kb-BamHI和1.4kb-SphI的中间片段为探针,对20个Xoo的代表菌株和Xooc菌株RS105的分别用BamHI和SphI酶切的基因组DNA进行Southern杂交作RFLP分析。杂交结果显示Xoo中很高的avrBs3/pthA家族成员遗传组成多样性。发现Xoo的不同小种间所含有的avrBs3/pthA基因的拷贝数不同,有些条带在所有的小种中都存在,有些条带是某个小种所特有的。发现探针的同源片段主要集中在1000bp到5000bp这个区间,意味着102bp的重复数大约有9-50个,每个菌株有12-38个同源拷贝。各国水稻白叶枯菌间至少共有4.3kb、4.0kb、3.9kb、3.8kb、3.5kb、3.1kb、2.8kb大小的BamHI片段或3.2kb、2.9kb、2.8kb、2.5kb、2.3kb、2.1kb、1.8kb大小的SphI片段的7个大小相同或相近的avrBs3/pthA家族基因。这些共有的基因在维护和展现水稻白叶枯菌的共性特征方面发挥着重要的作用。20个参试菌中总共鉴定了19种RFLP分子型(haplotype),根据分子型的相似性分为7个系群(Lineage).3.Xoo菌株PXO99A的无毒基因缺失突变分析水稻白叶枯菌中含有数目不等的无毒基因,但对大多数无毒基因的功能所知甚少。通过对同源重组获得的一个PXO99A无毒基因的突变体PXO99Δavr,进行Southern杂交验证、突变序列分析和水稻成株期剪叶接种的致病性测定。结果证实了突变体缺失了5个无毒基因,同源重组发生在PXO99A全基因组中一个有5个无毒基因串连的位点上。水稻成株期致病性测定结果表明突变体PXO99Δavr在IRBB10等15个水稻品种上的病斑长度比PXO99A明显缩短,在IRBB14、IRBB21和IRBB55上病斑变长,缺失的5个无毒基因的的综合表现为毒性因子功能。推断在缺失的基因中含有无毒基因avrXa14、avrXa21、avrxal3以及和抗病基因Xa3、Xa4、xa5、Xa10、Xal7亲和互作有关的毒性因子。为利用自杀式载体pKNG101建立模式菌株PXO99A的无毒基因缺失突变体库,进一步研究PXO99A中各无毒基因的功能做了一些初步的探索。4.无毒基因avrXa3介导的水稻白叶枯病菌在不同水稻品种上的致病适应性分析从水稻白叶枯JXOIII菌株中克隆到一个无毒基因avrXa3,它属于avrBs3/pthA家族成员。根据无毒基因avrXa3的序列,构建了水稻白叶枯PXO99A菌株缺失5个串联的无毒基因的突变体PXO99Δavr。将avrXa3导入PXO99A和PXO99Δavr后,得到衍生菌株PXO99A/avrXa3, PXO99Δavr/avrXa3,与36个含不同抗病基因的水稻品种进行互作分析。结果显示导入avrXa3后的菌株在不同的水稻品种上表现出了明显的寄生适应性变化。进一步分析证明无毒基因avrXa3在Wase Aikoku 3、IRBB2、IRBB3、IRBB203、IRBB204、IRBB205、IRBB211、IRBB53、IR24、TN-1等11个水稻上病斑缩短,表现明显的无毒活性；在IRBB21、IRBB10、IRBB14、IR26、Cas209、Java14等6个品种病斑明显变长,体现无毒基因的毒性功能。实验结果表明,无毒基因avrXa 3对PXO99A中其它无毒基因的表达有明显干扰作用,单个效应分子的变化也会使细菌在不同水稻品种上的寄生适应性发生复杂的改变。5.Xoo菌株与非寄主HR相关的PXO99A基因文库克隆pA254的功能分析来源于水稻白叶枯病菌PXO99A的文库克隆pA254,分别使水稻黄单胞菌PXO99A和RS105的致病力下降及丧失在烟草上激发过敏反应的能力。用SacI、EcoRI、KpnI和BamHI四种内切酶进行了交叉消化,构建了文库克隆pA254的酶切物理图谱。亚克隆导入RS105进行功能互补,在烟草上进行HR测定。结果显示亚克隆获得SacI, KpnI, EcoRl, BamHI完全和不完全酶切的29个亚克隆,导入RS105得到的接合子,都可以使烟草产生HR反应,没有与文库克隆pA254表型一致的亚克隆,即还未获得pA254的最小的功能片段。对所获得的pA254的亚克隆进行测序分析,通过序列同源性分析发现pA254与已经公布的PX099A的全基因组序列100%同源,与另外2个水稻白叶枯病菌具有99%以上同源性。这个克隆带有的外源片段大小为42572bp,位于PX099A基因组序列中相对保守的区域,包含约46个基因,也分别和MAFF11081和KACC10331中的37和42个基因同源。其中发现有毒力调控子基因、毒力蛋白基因、转录抑制子和含HTH基序的转录调控子等毒力相关因子,另外还有12个为未知功能基因,推测为保守的蛋白,4个转座酶基因,4个DNA连接酶,3个细胞色素C氧化还原酶,2个谷胱甘肽S转移酶,2个氨基酸-tRNA(Asp)合成酶,2个饥饿胁迫蛋白,2个抑制子(多羟基链烷酸合成抑制子),有一个乙酰辅酶Co-A还原酶,GumN蛋白和脂蛋白,YjeF家族蛋白,DNA错配修复酶,铁硫簇结合蛋白,乙酰丙氨酸酰胺酶,脱氧核糖核酸酶(亦称DNase),核糖核酸酶,可溶解胞壁转糖基酶等。文库克隆中有一大小约21kb的序列具有基因岛或致病岛的许多典型特征：带有1个或多个毒力相关因子、它们有明显的GC含量的变化,在连接处有整合酶基因,边界有tRNA作为整合位点,还有多个遗传移动性基因转座酶基因以及氨基酸偏爱密码子等。推测文库克隆pA254是hrp致病岛以外另一个和非寄主HR有关新的致病岛或基因岛(genomic island)。更多还原

【Abstract】 Xanthomonas oryzae pv. oryzae (Xoo) and Xanthomonas oryzae pv. oryzicola (Xooc) cause bacterial blight (BB) and bacterial leaf streak (BLS) in rice (Oryza sativa), respectively. BB is one of the most destructive diseases of rice in Asia and Africa and BLS is emerging in importance in rice. Xoo and Xooc are considered as the model pathogens of rice, which is taken as the model cereal crop. The relationship between host resistance genes (R) and bacterial avr genes (A) offers important information about pathogenicity differentiation, race identification, and isolation of avr, pth and R genes. Understanding the complex interactions between the bacterial avr gene and its host rice R gene is imperative for the development of effective disease controls. Race differentiation of Xoo is involved in the potential value of resistant varieties of the existing resistant varieties. Analysis on the interaction between the rice and the representative of Xoo strains are of great significance in the practice of scientific research and production. The hypersensitive response (HR) on the host is recognized as a type of resistance phenomenon with the centenary of the first descriptions of’hypersensitiveness’and HR on the nonhost used as an important standard of phytopathogenesis for a pathogen.For the HR is so crucial with the pathogenesis and resistance or innate immunity, Therefore, the elucidation of the composing and function of the HR-associated genes surely facilitate our understanding molecular mechanisms of bacterial pathogenesis of plants and plant disease resistance, as well as other aspects of plant and microbial biology, with implications also for animal innate immunity.1.Virulence diversity of Xoo interaction with 36 rice cultivarsHere we test 20 representative Xoo strains, comprising 4 Japanese strains (JXOI, JXOIII, JXOIV, JXOV),8 Philippine strains (PXO124, PXO112, PXO99, PXO99A, PXO86, PXO79, PXO71, PXO61) and 8 Chinese strains (OS-40, OS-86, OS-189, OS-198, OS-225, KS-6-6,GD-3, GD-5) interaction with 36 rice cultivars at the nursery and adult stage. In the seedling through the water-soaking method to detect the imcompatible interaction, the 20 Xoo strains were inoculated into 14-day rice seedlings of 36 cultivars by an needleless injection method, respectively. Photography and analysis the third day water soking lesions in rice. The HR phenomenon of oxidative burst was detected by DAB staining after needleless syringe-inoculation from the second day to the fifth day. The results of water-soaking lesions and DAB stain showed that no strain was virulence to all these R genes tested, while IRBB21, IRBB54, IRBB55 could overcome all these representative Xoo strains, strain OS-198 have imcompatible interaction with all 36 cultivars in seedlings, and PXO61, JXOI and OS-189, has speciality HR with a majority of detecting rice lines. This test analyses the avirulence and virulence of the 20 representative strains of Xoo from China, Japan and Philippines on 36 rice varieties. These results can be speculated that Xoo strains may contain avirulence genes and provide useful information for subsequent cloning experiments of the avr genes of the representative Xoo strains.In adult stage, the clip-inoculation results show that IRBB55 (Xa21+xal3) and IRBB54 (xa5+Xa21) have the best combination of R gene against BB. No strain was virulence to all these R genes tested, while IRBB54, IRBB55 could overcome all these representative Xoo strains. The lines with the gene Xa4, Xa7, Xa17, Xa21 or the recessive genes xa5. xa13. xa24, were resistant against over eighty percent of tested Xoo strains. While with the gene Xa1. Xa2. Xa10, Xall. Xal2,Xa14, Xa18 or xa8, xa19, xa20, were susceptible wih over fifty percent of tested strains. The gene, xa5, Xa7, and Xa21, exhibited the best broad resistance against about eighty percent of all tested strains. So the resistant gene xa5, Xa7, Xa21 is the best candidate R gene for breeding against Xoo. Among traditional varieties, IR26、BJ1、Asminori、Wase Aikoku 3,DV85 are resistance to over fifty percent of tested strains. Among the 20 strains tested,19 pathotypes were identified based on the 36 rice lines. Each strain has its pathotype. Further analysis of virulence data using the consensus of three clustering statistics and UPGMA revealed 7 clusters. Cluster 3 was the most heterogeneous and contained 1 from Philippines,2 from Japan and 6 from China, respectively.2.Genotypic diversity of Xoo with 2.4kb-BamHI and 1.4kb-Sphl fragment of avrXa3Evidence indicates that each race of Xanthomonas oryzae contain more than 15 members of avrBs3/pthA family genes. They have almost identical the 5’and 3’terminals. The difference is the repeat number of the 102 bp repeat unit among them. In this study, we use the 2.4kb-BamHI and 1.4kb-SphI DNA fragment of the avrXa3 gene, a member of avrBs3/pthA family genes, as the probes for genomic DNA Southern blot analysis of the representative Xoo and Xooc strains for RFLP research. The DNA fingerprint pattern generated by the probe revealed the high genetic diversity in Xoo strains.The copies of avrBs3/pthA gene are different in races of these two pathogens and found some bands in all races and some of the bands are specific in certain races.The size of DNA fragments that hybridized mainly ranged from 1 to 5 kbs, therefore, there are about 9 to 50 repeat units inferred with 102 bps in central region between the conservative SphI site among avrBs3/PathA family. The results indicated the presence of multiple homologous copies of the avrBs3/PthA gene family among the pathogens, ranging from 12-38 signal bands. Alignment of detected signal bands indicated that seven bands with the size of 4.3kb,4.0kb,3.9kb,3.8kb,3.5kb,3.1kb,2.8kb-BamHI fragment or 3.2kb, 2.9kb,2.8kb,2.5kb,2.3kb,2.1kb,1.8kb-Sph fragment respectively, are shared by all of tested strains and another one with 1.2 kbs is common for all Xoo strains. The DNA fingerprint pattern was referred to as molecular haplotype. At least 38 BamHI and 43 SphI bands positions were scored for the collections. On the basis of consensus of three clustering statistics, seven lineages were found among these strains. Genetic diversity was high in all lineages and No significant (P< 0.05) differences were revealed by t test.3. Studies on the mutant avr genes of Xoo strain PXO99A by knock-out mutagenesisPXO99△avr, an avr gene deletion mutant of Xoo strain PXO99A was obtained by homologous recombination. The mutant was identified by PCR, Southern blot, flanking sequence analysis and rice inoculation assay. The results showed that five avr genes were deleted in PXO99△avr and the site directed mutagenesis occurred on the one loci of the five series-wound avr genes in PXO99A genome. The inoculation assays of 36 rice varieties indicated that PXO99△avr induced shorter lesion on 15 varieties such as IRBB10 and longer lesion on IRBB14, IRBB21, and IRBB55 than PXO99A. Based on the compositive result, five knock-outed avr genes in PXO99A could coded virulence factors to induce rice leaf lesion. It was concluded that avr genes avrXa14, avrXa21, avrxal3 could be contained in five series-wound avr genes and some pathogenic fitness factors in PXO99A could be associated with the interaction with rice R genes such as Xa3, Xa4, Xa5, Xa10, Xa17. It should facilitate the study to generate avr gene mutant libraries of PXO99A and know more about their function. 4.Analysis on the interaction between avrXa3 mediated Xoo strains and various rice varietiesavrXa3 comes from Xoo(Xanthomonas oryzae pv.oryzae[Ishiyama] Dye)JXOⅢstrain, a member of avrBs3/pthA family. PXO99△avr, an avr gene deletion mutant of Xoo, derivate from PXO99A by homologous recombination. The avrXa3 was introduced into PXO99△avr and PXO99A to produce derivatives,PXO99A/avrXa3, PXO99△avr/avrXa3. The inoculation assays of 36 rice varieties indicated that pathogenic fitness of PXO99A/avrXa3, PXO99△avrlavrXa3on rice varieties was significant modulated as compared with PXO99A, PXO99△avr, respectively. Based on lesion length, avrXa3 shows avirulence on adult plants of rice varieties of Wase Aikoku 3, IRBB2, IRBB3, IRBB203, IRBB204, IRBB205, IRBB211, IRBB53, IR24, TN-1 and virulence on rice varieties of IRBB21, IRBB10, IRBB14, IR26, Cas209, Java14. The avrXa3 showed significant interference for expression of the avr genes in PXO99A. Individual effector change may make subtle alterations for bacterial pathogenicity on relevant rice varieties.5.Function analysis of a Xoo strain PXO99A genomic library clone pA254 associated with nonhost HRBy biparental mating,1200 clones of Xoo strain PXO99A genomic library were transferred into Xooc strain RS105.Clone pA254 was found to significantly reduce the pathogenicity of recipient strain RS105 and its ability to induce HR on nonhost plant tobacco. Same results were obtained when pA254 was transferred into PXO99A. These indicated that pA254 may be carry genes which involved in negative regulation of hrp genes of Xanthomonas oryzae or genes which encoded protein enzymes that can degredate harpins protein produced in Xanthomonas oryzae. Based on the subclone study by SacI digestion, physical map of pA254 was constructed with restriction endonuclease EcoRI, BamHI, KpnI and SacI by cross-digestion method.None of the 29 subclones from 42.6-kb genomic library clone pA254 shows the same phenotype with pA254. We failed to gain the smallest functional fragment. Sequencing 42.6-kb fragment in pA254 demonstrates that pA254 is a 42572bp fragment locates in a relatively conservative region of PXO99A genome, contained 46 annotated genes. There are one virulence regulator xrvA, one virulence protein gene, one repressor etc genes associated with pathogenesis. There are twelve hypothetical protein, four ATP-dependent DNA ligases and transposases, respectively; 3 ubiquinol cytochrome C oxidoreductase,2 glutathione S-transferase,2 stringent starvation protein,2 repressor, and glutamyl-Q tRNA(Asp) synthetase, tRNA-Ala, acetoacetyl-CoA reductase, GumN protein, lipoprotein, DNA mismatch repair protein MutL, N-acetylmuramoyl-L-alanine amidase, YjeF family protein, iron-sulfur cluster binding protein, exodeoxyribonuclease, ribonuclease D, soluble lytic murein transglycosylase et al.. A 21kb fragment of pA254 has many conserved features of a pathogenicity island or genomic island. Include the presence of flanking repeats, mobility genes (e.g. integrases, transposases), proximal transfer RNAs (tRNAs, e.g., an Asp-tRNA gene, a Ala--tRNA gene), and atypical guanine and cytosine content, has a large percentage of phage protein, suggesting a phage-related recombination is involved. It may be speculated as a new pathogenicity island associated with nonhost HR other than the hrp pathogenicity island.更多还原