Identification of a family of avirulence genes from Xanthomonas oryzae pv. oryzae.

Races of Xanthomonas oryzae pv. oryzae, the causal agent of bacterial blight of rice, interact with cultivars of rice in a gene-for-gene specific manner. Multiple DNA fragments of various sizes from all strains of X. o. pv. oryzae hybridized with avrBs3, an avirulence gene from Xanthomonas campestris pv. vesicatoria, in Southern blots; this suggests the presence of several homologs and possibly a gene family. A genomic library of a race 2 strain of X. o. pv. oryzae, which is avirulent on rice cultivars carrying resistance genes xa-5, Xa-7, and Xa-10, was constructed. Six library clones, which hybridized to avrBs3, altered the interaction phenotype with rice cultivars carrying either xa-5, Xa-7, or Xa-10 when present in a virulent race 6 strain. Two avirulence genes, avrXa7 and avrXa10, which correspond to resistance genes Xa-7 and Xa-10, respectively, were identified and partially characterized from the hybridizing clones. On the basis of transposon insertion mutagenesis, sequence homology, restriction mapping, and the presence of a repeated sequence, both genes are homologs of avirulence genes from dicot xanthomonad pathogens. Two BamHI fragments that are homologous to avrBs3 and correspond to avrXa7 and avrXa10 contain a different number of copies of a 102-bp direct repeat. The DNA sequence of avrXa10 is nearly identical to avrBs3. We suggest that avrXa7 and avrXa10 are members of an avirulence gene family from xanthomonads that control the elicitation of resistance in mono- and dicotyledonous plants.     

水稻白叶枯病菌和水稻细菌性条斑病菌的实时荧光PCR快速检测鉴定

成功建立了水稻白叶枯菌与水稻细菌性条斑病菌快速检测鉴定的实时荧光PCR方法。根据含铁细胞接受子基因设计两菌的通用引物PSRGF/PSRGR（扩增一个152bpDNA片段）和特异性探针（Baiprobe和Tiaoprobe）,并对13种细菌和1种植原体进行实时荧光PCR。结果表明,两个特异性探针能分别特异性检测到目标病原菌产生荧光信号而其它参考菌不产生荧光信号。检测的绝对灵敏度是30.6fg/μL质粒DNA和103CFU/mL的菌悬浮液,相当于1个细菌细胞的基因,比常规PCR电泳检测高约100倍,相对灵敏度为105CFU/mL。整个检测过程只需2h,完全闭管,降低了污染的机会,无需PCR后处理。 用这两个特异性探针分别对自然感染白叶枯菌和条斑菌的叶片DNA提取液和种子浸泡液进行实时荧光PCR,结果均可特异性检测到目标菌的存在并完全可将两种病原细菌区分开来,且只需03g叶片和10g种子。     

中国、日本和菲律宾水稻白叶枯病菌遗传多样性比较分析

用IS-PCR和Rep-PCR对19个来自中国、日本和菲律宾的水稻白叶枯病菌(Xanthomonas oryzae pv.oryzae)群体进行遗传多样性分析.4个专化引物中的IS1113和ERIC能较好的区分三国水稻白叶枯病菌.UPGMA聚类结果表明,三国菌株主要呈现第2簇和第3簇遗传型;中国和菲律宾菌株在第2簇和笫3簇遗传型基础上有各自的特异性分化.病原菌的遗传分簇与致病群之间没有相关性.     

Recombinant expression and purification of functional XorII, a restriction endonuclease from Xanthomonas oryzae pv. oryzae

An endonuclease from Xanthomonas oryzae pathovar oryzae KACC 10331, XorII, was recombinantly produced in Escherichia coli using a T7 system. XorII was purified using a combination of ion exchange and immobilized metal affinity chromatography (IMAC). An optimized washing protocol was carried out on an IMAC in order to obtain a high purity product. The final amount of purified XorII was approximately 2.5 mg/L of LB medium. The purified recombinant XorII was functional and showed the same cleavage pattern as PvuI. The enzyme activity tested the highest at 25 degrees in 50 mM NaCl, 10 mM Tris-HCl, 10 mM MgCl2, and 1 mM dithiothreitol at a pH of 7.9.     

中国、日本和菲律宾水稻白叶枯病菌遗传多样性比较分析

用IS-PCR和Rep-PCR对19个来自中国、日本和菲律宾的水稻白叶枯病菌(Xanthomonas oryzae pv. oryzae)群体进行遗传多样性分析。4个专化引物中的IS1113和ERIC能较好的区分三国水稻白叶枯病菌。UPGMA聚类结果表明, 三国菌株主要呈现第2簇和第3簇遗传型;中国和菲律宾菌株在第2簇和第3簇遗传型基础上有各自的特异性分化。病原菌的遗传分簇与致病群之间没有相关性。     

Genetic Diversity of Xanthomonas oryzae pv. oryzae in Asia

Restriction fragment length polymorphism and virulence analyses were used to evaluate the population structure of Xanthomonas oryzae pv. oryzae, the rice bacterial blight pathogen, from several rice-growing countries in Asia. Two DNA sequences from X. oryzae pv. oryzae, IS1112, an insertion sequence, and avrXa10, a member of a family of avirulence genes, were used as probes to analyze the genomes of 308 strains of X. oryzae pv. oryzae collected from China, India, Indonesia, Korea, Malaysia, Nepal, and the Philippines. On the basis of the consensus of three clustering statistics, the collection formed five clusters. Genetic distances within the five clusters ranged from 0.16 to 0.51, and distances between clusters ranged from 0.48 to 0.64. Three of the five clusters consisted of strains from a single country. Strains within two clusters, however, were found in more than one country, suggesting patterns of movement of the pathogen. The pathotype of X. oryzae pv. oryzae was determined for 226 strains by inoculating five rice differential cultivars. More than one pathotype was associated with each cluster; however, some pathotypes were associated with only one cluster. Most strains from South Asia (Nepal and India) were virulent to cultivars containing the bacterial blight resistance gene xa-5, while most strains from other countries were avirulent to xa-5. The regional differentiation of clusters of X. oryzae pv. oryzae in Asia and the association of some pathotypes of X. oryzae pv. oryzae with single clusters suggested that strategies that target regional resistance breeding and gene deployment are feasible.     

Effect of phenol on ultra structure and plasmid DNA of Xanthomonas oryzae pv. oryzae

Most phenolic substances of plant origin are toxic to microorganisms and they confer some degree of protection to plants against phytopathogens. Xanthomonas oryzae pv. oryzae, bacterial blight pathogen of rice (Oryza sativa) was treated with phenol (monohydroxy benzene) and its effects on the morphology and cytological changes of the bacterium were studied. Total lysis of cells occurred with 5 mM conc of phenol while at 2 mM conc, the cell walls became rough and cell contents started shrinking. Plasmids isolated from both treated (2 mM) and control cells did not show any marked difference under electron microscope except that they differed in their quantity and might influence pathogenicity.     

Pathotypic variation of Xanthomonas oryzae pv. oryzae in Bangladesh

Bacterial Blight (BB) caused by Xanthomonas oryzae pv. oryzae (Xoo), a destructive disease of rice. Altogether, 96 isolates of Xoo were collected from 19 rice growing districts of Bangladesh in irrigated and rainfed seasons during 2014 to assess pathotypic variation. Pathotypic analyses on a set of 12 Near Isogenic Lines (NILs) of rice containing resistance genes viz. Xa1, Xa2, Xa3, Xa4, Xa5, Xa7, Xa8, Xa10, Xa11, Xa13, Xa14 and Xa21 and two check varieties IR24 and TN1 by leaf clip-inoculation technique. A total of 24 pathotypes were identified based on their virulence patterns on NILs tested. Among these, pathotypes VII, XII, and XIV considered as major, containing maximum number of isolates, (9.38% each) frequently distributed in North to Mid-Eastern districts of Bangladesh. Most virulent pathotype I recorded from Habiganj and Brahmanbaria. This pathotypic variation explained the pathogenic relatedness of X. oryzae pv. oryzae populations from diverse geographic areas in Bangladesh.     

Recombinant expression, purification, and characterization of XorKII: a restriction endonuclease from Xanthomonas oryzae pv. oryzae

An endonuclease from Xanthomonas oryzae pathovar oryzae (Xoo) KACC10331, XorKII, was recombinantly produced in Escherichia coli by applying the stationary state induction method, which was necessary to prevent the unwanted lysis of E. coli cells. XorKII was purified by immobilized metal affinity chromatography on an FPLC system. The yield was 3.5 mg of XorKII per liter of LB medium. The purified recombinant XorKII showed that it recognized and cleaved to the same site as PstI. It behaved as a dimer as evidenced by the size exclusion chromatography. The specific activity of the purified XorKII was determined to be 31,300 U/mg. The enzyme activity was monitored by cleaving lambda DNA or YEp24 plasmid as substrates. The enzyme was the most active at 10 mM Tris–HCl pH 7.0, 10 mM MgCl₂, 1 mM dithiothreitol at 37 °C. XorKII was easily inactivated by heating at 65 °C for 5 min, but retained most of the original activity after incubation at 37 °C for 24 h.     

Identification of the XorII methyltransferase gene and a vsr homolog from Xanthomonas oryzae pv. oryzae

We have changed the translation initiation codon of the COX2 mRNA of Saccharomyces cerevisiae from AUG to AUA, generating a mutation termed cox2-10. This mutation reduced translation of the COX2 mRNA at least five-fold without affecting the steady-state level of the mRNA, and produced a leaky nonrespiratory growth phenotype. To address the question of whether residual translation of the cox2-10 mRNA was initiating at the altered initiation codon or at the next AUG codon downstream (at position 14), we took advantage of the fact that the mature coxll protein is generated from the electrophoretically distinguishable coxII precursor by removal of the amino-terminal 15 residues, and that this processing can be blocked by a mutation in the nuclear gene PET2858. We constructed a pet2858, cox2-10 double mutant strain using a pet2858 allele from our mutant collection. The double mutant accumulated low levels of a polypeptide which comigrated with the coxII precursor protein, not the mature species, providing strong evidence that residual initiation was occurring at the mutant AUA codon. Residual translation of the mutant mRNA required the COX2 mRNA-specific activator PET111. Furthermore, growth of cox2-10 mutant strains was sensitive to alterations in PET111 gene dosage: the respiratory-defective growth phenotype was partially suppressed in haploid strains containing PET111 on a high-copy-number vector, but became more severe in diploid strains containing only one functional copy of PET111.     

Status of Streptomycin Resistance Development in Xanthomonas oryzae pv. oryzae and Xanthomonas oryzae pv. oryzicola in China and their Resistance Characters

Rice leaves with bacterial blight or bacterial leaf streak symptoms were collected in southern China in 2007 and 2008. Five hundred and thirty‐four single‐colony isolates of Xanthomonas oryzae pv. oryzae and 827 single‐colony isolates of Xanthomonas oryzae pv. oryzicola were obtained and tested on plates for sensitivity to streptomycin. Four strains (0.75%) of X. oryzae pv. oryzae isolated from the same county of Province Yunnan were resistant to streptomycin, and the resistance factor (the ratio of the mean median effective concentration inhibiting growth of resistant isolates to that of sensitive isolates) was approximately 226. The resistant isolate also showed streptomycin resistance in vivo. In addition to resistant isolates, isolates of less sensitivity were also present in the population of X. oryzae pv. oryzae from Province Yunnan. However, no isolates with decreased streptomycin‐sensitivity were obtained from the population of X. oryzae pv. oryzicola. Mutations in the rpsL (encoding S12 protein) and rrs genes (encoding 16S rRNA) and the presence of the strA gene accounting for streptomycin resistance in other phytopathogens or animal and human pathogenic bacteria were examined on sensitive and resistant strains of X. oryzae pv. oryzae by polymerase chain reaction amplification and sequencing. Neither the presence of the strA gene nor mutations in the rpsL or rrs were found, suggesting that different resistance mechanisms are involved in the resistant isolates of X. oryzae pv. oryzae.     

Assessment of genetic diversity and population structure of Xanthomonas oryzae pv. oryzae with a repetitive DNA element.

A repetitive DNA element cloned from Xanthomonas oryzae pv. oryzae was used to assess the population structure and genetic diversity of 98 strains of X. oryzae pv. oryzae collected between 1972 and 1988 from the Philippine Islands. Genomic DNA from X. oryzae pv. oryzae was digested with EcoRI and analyzed for restriction fragment length polymorphisms (RFLPs) with repetitive DNA element as a probe. Twenty-seven RFLP types were identified; there was no overlap of RFLP types among the six races from the Philippines. Most variability (20 RFLP types) was found in strains of races 1, 2, and 3, which were isolated from tropical lowland areas. Four RFLP types (all race 5) were found among strains isolated from cultivars grown in the temperate highlands. The genetic diversity of the total population of X. oryzae pv. oryzae was 0.93, of which 42% was due to genetic differentiation between races. The genetic diversities of strains collected in 1972 to 1976, 1977 to 1981, and 1982 to 1986, were 0.89, 0.90, and 0.92, respectively, suggesting a consistently high level of variability in the pathogen population over the past 15 years. Cluster analysis based on RFLP banding patterns showed five groupings at 85% similarity. The majority of strains from a given race were contained within one cluster, except for race 3 strains, which were distributed in three of the five clusters.     

Culture medium for Xanthomonas campestris pv. oryzae

Y uan , W. 1990. Culture medium for Xanthomonas campestris pv. oryzae. Journal of Applied Bacteriology 69 , 798–805.
Studies on nutrient requirements of four Chinese strains of Xanthomonas campestris pv. oryzae in a modified Watanabe's medium led to the development of a new synthetic medium containing sucrose, sodium glutamate, methionine, KH₂PO₄, NH₄C1 and iron chelated with EDTA. The concentration of each ingredient was optimized based on the number of colonies and time required for their appearance. Various concentrations of some nutrients were compared based upon their effects on growth of the pathogen strains and 34 contaminants from rice materials. Tryp-tone enhanced the growth of X. c. oryzae more than that of many contaminants, including Erwinia herbicola . Peptone stimulated growth of X. c. oryzae without promoting excessive contamination. When compared with other media used for X. c. oryzae , the new culture medium enriched with tryptone and peptone gave the highest recovery and earliest appearance of colonies of Chinese strains of this bacterium.     

Transgenic elite Indica rice varieties,resistant to Xanthomonas oryzae pv. oryzae

The agronomically important Indica (group 1) rice varieties IR64, IR72, hybrid restorer line Minghui 63, and BG90-2 were co-transformed by microbombardment of embryogenic suspensions with plasmids that contain the Xa21 gene which confers resistance to Xanthomonas oryzae pv. oryzae and the hph gene for resistance to hygromycin B. Six of the 55 transgenic R0 plant lines containing the Xa21 gene displayed high levels of resistance to the pathogen, and no partial resistance was observed. The trait was stably inherited in subsequent generations, and transgenic plants are currently in field tests. The ability to transfer agronomically important genes into elite Indica rice varieties demonstrates the applicability of genetic engineering for the agronomic improvement of rice.     

Assessment of genetic diversity and population structure of Xanthomonas oryzae pv. oryzae with a repetitive DNA element.

A repetitive DNA element cloned from Xanthomonas oryzae pv. oryzae was used to assess the population structure and genetic diversity of 98 strains of X. oryzae pv. oryzae collected between 1972 and 1988 from the Philippine Islands. Genomic DNA from X. oryzae pv. oryzae was digested with EcoRI and analyzed for restriction fragment length polymorphisms (RFLPs) with repetitive DNA element as a probe. Twenty-seven RFLP types were identified; there was no overlap of RFLP types among the six races from the Philippines. Most variability (20 RFLP types) was found in strains of races 1, 2, and 3, which were isolated from tropical lowland areas. Four RFLP types (all race 5) were found among strains isolated from cultivars grown in the temperate highlands. The genetic diversity of the total population of X. oryzae pv. oryzae was 0.93, of which 42% was due to genetic differentiation between races. The genetic diversities of strains collected in 1972 to 1976, 1977 to 1981, and 1982 to 1986, were 0.89, 0.90, and 0.92, respectively, suggesting a consistently high level of variability in the pathogen population over the past 15 years. Cluster analysis based on RFLP banding patterns showed five groupings at 85% similarity. The majority of strains from a given race were contained within one cluster, except for race 3 strains, which were distributed in three of the five clusters.     

Construction and physiological analysis of a Xanthomonas oryzae pv. oryzae recA mutant

A Xoo recA insertion inactivation mutant was constructed. The mutant, lacking RecA, showed increased sensitivity towards mutagen killing. This phenotype could be complemented by a cloned, functional recA. Unlike other bacteria, both the recA mutant and the parental strain had similar level of resistance to H₂O₂ killing and peroxide-induced mutagenesis.