| 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. |
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