A paralog of the MtN3/saliva family recessively confers race-specific resistance to Xanthomonas oryzae in rice

Approximately one third of the identified 34 rice major disease resistance (R) genes conferring race-specific resistance to different strains of Xanthomonas oryzae pv. oryzae (Xoo), which causes rice bacterial blight disease, are recessive genes. However, only two of the recessive resistance genes have been characterized thus far. Here we report the characterization of another recessive resistance gene, xa25, for Xoo resistance. The xa25, localized in the centromeric region of chromosome 12, mediates race-specific resistance to Xoo strain PXO339 at both seedling and adult stages by inhibiting Xoo growth. It encodes a protein of the MtN3/saliva family, which is prevalent in eukaryotes, including mammals. Transformation of the dominant Xa25 into a resistant rice line carrying the recessive xa25 abolished its resistance to PXO339. The encoding proteins of recessive xa25 and its dominant allele Xa25 have eight amino acid differences. The expression of dominant Xa25 but not recessive xa25 was rapidly induced by PXO339 but not other Xoo strain infections. The nature of xa25-encoding protein and its expression pattern in comparison with its susceptible allele in rice-Xoo interaction indicate that the mechanism of xa25-mediated resistance appears to be different from that conferred by most of the characterized R proteins.     

Inhibition of resistance gene-mediated defense in rice by Xanthomonas oryzae pv. oryzicola

Xanthomonas oryzae pv. oryzae and the closely related X. oryzae pv. oryzicola cause bacterial blight and bacterial leaf streak of rice, respectively. Although many rice resistance (R) genes and some corresponding avirulence (avr) genes have been characterized for bacterial blight, no endogenous avr/R gene interactions have been identified for leaf streak. Genes avrXa7 and avrXa10 from X. oryzae pv. oryzae failed to elicit the plant defense-associated hypersensitive reaction (HR) and failed to prevent development of leaf streak in rice cultivars with the corresponding R genes after introduction into X. oryzae pv. oryzicola despite the ability of this pathovar to deliver an AvrXa10:Cya fusion protein into rice cells. Furthermore, coinoculation of X. oryzae pv. oryzicola inhibited the HR of rice cultivar IRBB10 to X. oryzae pv. oryzae carrying avrXa10. Inhibition was quantitative and dependent on the type III secretion system of X. oryzae pv. oryzicola. The results suggest that one or more X. oryzae pv. oryzicola type III effectors interfere with avr/R gene-mediated recognition or signaling and subsequent defense response in the host. Inhibition of R gene-mediated defense by X. oryzae pv. oryzicola may explain, in part, the apparent lack of major gene resistance to leaf streak.     

Are the dominant and recessive plant disease resistance genes similar? A case study of rice R genes and Xanthomonas oryzae pv. oryzae races.

The resistance of rice to its bacterial blight pathogen Xanthomonas oryzae pv. oryzae (Xoo) has both qualitative and quantitative components that were investigated using three near-isogenic line sets for four resistance (R) genes (Xa4, xa5, xa13, and Xa21) and 12 Xoo races. Our results indicate that these two resistance components of rice plants were associated with the properties of the R genes. The qualitative component of the R genes was reflected by their large effects against corresponding avirulent Xoo races. The quantitative component of the R genes was their residual effects against corresponding virulent races and their epistatic effects, which together could lead to high-level resistance in a race-specific manner. Our results revealed important differences between the different types of R genes. Two R genes, Xa4 and Xa21, showed complete dominance against the avirulent Xoo races and had large residual effects against virulent ones. They acted independently and cumulatively, suggesting they are involved in different pathways of the rice defensive system. The third R gene, xa5, showed partial dominance or additivity to the avirulent Xoo races and had relatively small but significant residual effects against the virulent races. In contrast, xa13 was completely recessive, had no residual effects against the virulent races, and showed more pronounced race specificity. There was a strong interaction leading to increased resistance between xa13 and xa5 and between either of them and Xa4 or Xa21, suggesting their regulatory roles in the rice defensive pathway(s). Our results indicated that high-level and durable resistance to Xoo should be more efficiently achieved by pyramiding different types of R genes.     

白叶枯病菌胁迫下云南普通野生稻SSH文库的构建及抗病相关基因分析

以云南普通野生稻为材料,利用抑制差减杂交技术（SSH）,构建了白叶枯病菌胁迫的云南普通野生稻特异表达基因的差减文库.通过对文库所有阳性单克隆进行测序,聚类分析后共获得494条高质量的表达序列标签（EST）.经过BlastN分析,有417条与已知功能的序列有较高同源性;经BlastX分析,有104条EST与未知功能蛋白或假定蛋白有较高相似性,49条EST未能找到同源匹配,341条EST与已知功能蛋白有较高同源性.初步分析发现,这些基因主要涉及能量代谢、蛋白质代谢、核酸代谢、防御与抗逆应答反应、信号转导、光合作用及膜运输等代谢过程.使用半定量RT-PCR研究了7个可能与白叶枯病抗性相关的EST序列在云南普通野生稻对照和白叶枯病菌处理的叶片中的表达情况,并获得这些基因的表达谱.结果发现,克隆编号为OR7,OR68和OR826的EST受白叶枯病菌胁迫诱导上调表达,其中OR826 EST在蛋白数据库中无同源序列,可能是一类新的白叶枯病抗性基因,而组成型表达的OR143 EST在对照和接菌处理的叶片中均能检测到其mRNA的表达,但其表达量在白叶枯病菌胁迫48 h后逐渐增强,推测这些基因直接参与了云南普通野生稻抗病防御反应.本研究为从云南普通野生稻中发掘和克隆新的白叶枯病抗性基因提供了理论依据,为进一步研究云南普通野生稻抗白叶枯病的分子机制奠定了基础.     

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.     

Molecular determinants of disease and resistance in interactions of Xanthomonas oryzae pv. oryzae and rice

Xanthomonas oryzae pv. oryzae is the causal agent of rice bacterial blight disease. Numerous genes critical for virulence have been identified. This article reviews current knowledge on the molecular mechanisms of X. oryzae pv. oryzae virulence.     

Virulence deficiency caused by a transposon insertion in the purH gene of Xanthomonas oryzae pv. oryzae

Xanthomonas oryzae pv. oryzae causes bacterial leaf blight, a serious disease of rice. We have identified a Tn5-induced virulence-deficient mutant (BXO1704) of X. oryzae pv. oryzae. The BXO1704 mutant exhibited growth deficiency in minimal medium but was proficient in inducing a hypersensitive response in a non-host tomato plant. Sequence analysis of the chromosomal DNA flanking the Tn5 insertion indicated that the Tn5 insertion is in the purH gene, which is highly homologous to purH genes of other closely related plant pathogenic bacteria Xanthomonas axonopodis pv. citri and Xanthomonas campestris pv. campestris. Purine supplementation reversed the growth deficiency of BXO1704 in minimal medium. These results suggest that the virulence deficiency of BXO1704 may be due to the inability to use sufficient purine in the host.     

Functional analysis of the aroC gene encoding chorismate synthase from Xanthomonas oryzae pathovar oryzae

Xanthomonas oryzae pv. oryzae causes bacterial blight in rice, and this bacterial blight has been widely found in the major rice-growing areas. We constructed a transposon mutagenesis library of X. oryzae pv. oryzae and identified a mutant strain (KXOM9) that is deficient for pigment production and virulence. Furthermore, the KXOM9 mutant was unable to grow in minimal medium lacking aromatic amino acids. Thermal asymmetric interlaced-PCR and sequence analysis of KXOM9 revealed that the transposon was inserted into the aroC gene, which encodes a chorismate synthase in various bacterial pathogens. In planta growth assays revealed that bacterial growth of the KXOM9 mutant in rice leaves was severely reduced. Genetic complementation of this mutant with a 7.9-kb fragment containing aroC restored virulence, pigmentation, and prototrophy. These results suggest that the aroC gene plays a crucial role in the growth, attenuation of virulence, and pigment production of X. oryzae pv. oryzae.     

Marker assisted selection of bacterial blight resistance genes in rice

Bacterial leaf blight caused by Xanthomonas oryzae pv. oryzae is one of the most important diseases affecting rice production in Asia. We were interested in surveying rice genotypes that are popularly used in the Indian breeding program for conferring resistance to bacterial blight, using 11 STMS and 6 STS markers. The basis of selection of these DNA markers was their close linkage to xa5, xa13, and Xa21 genes and their positions on the rice genetic map relative to bacterial blight resistance genes. Eight lines were found to contain the xa5 gene while two lines contained Xa21 gene and none of the lines contained the xa13 gene with the exception of its near-isogenic line. Using the polymorphic markers obtained in the initial survey, marker-assisted selection was performed in the F3 population of a cross between IR-64 and IET-14444 to detect lines containing multiple resistance genes. Of the 59 progeny lines analyzed, eight lines contained both the resistance genes, xa5 and Xa4.     

rpfF mutants of Xanthomonas oryzae pv. oryzae are deficient for virulence and growth under low iron conditions

Xanthomonas oryzae pv. oryzae causes bacterial leaf blight, a serious disease of rice. In the related bacterium Xanthomonas campestris pv. campestris, the rpfF gene is involved in production of a diffusible extracellular factor (DSF) that positively regulates synthesis of virulence-associated functions like extracellular polysaccharide (EPS) and extracellular enzymes. Transposon insertions in the rpfF homolog of X. oryzae pv. oryzae are deficient for virulence and production of a DSF but are proficient for EPS and extracellular enzyme production. The rpfF X. oryzae pv. oryzae mutants exhibit an unusual tetracycline susceptibility phenotype in which exogenous iron supplementation is required for phenotypic expression of a tetracycline resistance determinant that is encoded on an introduced plasmid. The rpfF X. oryzae pv. oryzae mutants also overproduce one or more siderophores and exhibit a growth deficiency under low iron conditions as well as in the presence of reducing agents that are expected to promote the conversion of Fe+3 to Fe+2. Exogenous iron supplementation promotes migration of rpfF X. oryzae pv. oryzae mutants in rice leaves. The results suggest that rpfF may be involved in controlling an iron-uptake system of X. oryzae pv. oryzae and that an inability to cope with the conditions of low iron availability in the host may be the reason for the virulence deficiency of the rpfF X. oryzae pv. oryzae mutants.     

Fine genetic mapping of <Emphasis Type="Italic">xa24</Emphasis>, a recessive gene for resistance against <Emphasis Type="Italic">Xanthomonas oryzae</Emphasis> pv. <Emphasis Type="Italic">oryzae</Emphasis> in rice

Bacterial blight, caused by Xanthomonas oryzae pv. oryzae (Xoo), is the most devastating plant bacterial disease worldwide. Different bacterial blight resistance (R) genes confer race-specific resistance to different strains of Xoo. We fine mapped a fully recessive gene, xa24, for bacterial blight resistance to a 71-kb DNA fragment in the long arm of rice chromosome 2 using polymerase chain reaction-based molecular markers. The xa24 gene confers disease resistance at the seedling and adult stages. It mediates resistance to at least the Philippine Xoo races 4, 6 and 10 and Chinese Xoo strains Zhe173, JL691 and KS-1-21. Sequence analysis of the DNA fragment harboring the dominant (susceptible) allele of xa24 suggests that this gene should encode a novel protein that is not homologous to any known R proteins. These results will greatly facilitate the isolation and characterization of xa24. The markers will be convenient tools for marker-assisted selection of xa24 in breeding programs. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

RaxH/RaxR: a two-component regulatory system in Xanthomonas oryzae pv. oryzae required for AvrXa21 activity

Xanthomonas oryzae pv. oryzae is the causal agent of bacterial leaf blight, one of the most serious diseases in rice. X. oryzae pv. oryzae Philippine race 6 (PR6) strains are unable to establish infection in rice lines expressing the resistance gene Xa21. Although the pathogen-associated molecule that triggers the Xa21-mediated defense response (AvrXa21) is unknown, six rax (required for AvrXa21 activity) genes encoding proteins involved in sulfur metabolism and Type I secretion were recently identified. Here, we report on the identification of two additional rax genes, raxR and raxH, which encode a response regulator and a histidine protein kinase of two-component regulatory systems, respectively. Null mutants of PR6 strain PXO99 that are impaired in either raxR, raxH, or both cause lesions significantly longer and grow to significantly higher levels than does the wild-type strain in Xa21-rice leaves. Both raxR and raxH mutants are complemented to wild-type levels of AvrXa21 activity by introduction of expression vectors carrying raxR and raxH, respectively. These null mutants do not affect AvrXa7 and AvrXa10 activities, as observed in inoculation experiments with Xa7- and Xa10-rice lines. Western blot and raxR/gfp promoter-reporter analyses confirmed RaxR expression in X. oryzae pv. oryzae. The results of promoter-reporter studies also suggest that the previously identified raxSTAB operon is a target for RaxH/RaxR regulation. Characterization of the RaxH/RaxR system provides new opportunities for understanding the specificity of the X. oryzae pv. oryzae-Xa21 interaction and may contribute to the identification of AvrXa21.     

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.     

XA27 depends on an amino-terminal signal-anchor-like sequence to localize to the apoplast for resistance to Xanthomonas oryzae pv oryzae

The rice (Oryza sativa) gene Xa27 confers resistance to Xanthomonas oryzae pv oryzae, the causal agent of bacterial blight disease in rice. Sequence analysis of the deduced XA27 protein provides little or no clue to its mode of action, except that a signal-anchor-like sequence is predicted at the amino (N)-terminal region of XA27. As part of an effort to characterize the biochemical function of XA27, we decided to determine its subcellular localization. Initial studies showed that a functional XA27-green fluorescent protein fusion protein accumulated in vascular elements, the host sites where the bacterial blight pathogens multiply. The localization of XA27-green fluorescent protein to the apoplast was verified by detection of the protein on cell walls of leaf sheath and root cells after plasmolysis. Similarly, XA27-FLAG localizes to xylem vessels and cell walls of xylem parenchyma cells, revealed by immunogold electron microscopy. XA27-FLAG could be secreted from electron-dense vesicles in cytoplasm to the apoplast via exocytosis. The signal-anchor-like sequence has an N-terminal positively charged region including a triple arginine motif followed by a hydrophobic region. Deletion of the hydrophobic region or substitution of the triple arginine motif with glycine or lysine residues abolished the localization of the mutated proteins to the cell wall and impaired the plant's resistance to X. oryzae pv oryzae. These results indicate that XA27 depends on the N-terminal signal-anchor-like sequence to localize to the apoplast and that this localization is important for resistance to X. oryzae pv oryzae.     

水稻抗白叶枯病基因的鉴定、定位和克隆进展

由水稻黄单胞菌水稻变种Xoo引起的水稻白叶枯病是全球性的重要病害之一。已有31个水稻白叶枯抗性基因被鉴定并报道,其中18个被定位到染色体上,5个被克隆。简要综述了水稻白叶枯抗性基因的鉴定、定位和克隆的进展,并讨论了合理利用抗性基因防治白叶枯病的前景。     

Targeting xa13, a recessive gene for bacterial blight resistance in rice

Bacterial blight, caused by Xanthomonas oryzae pv. oryzae (Xoo), is one of the most serious diseases of rice worldwide. Thirty bacterial blight resistance (R) genes (21 dominant genes and 9 recessive genes) in rice have been identified. They are the main sources for the genetic improvement of rice for resistance to Xoo. However, little is known about the recessive R genes. To clone and characterize the recessive R genes, we fine-mapped xa13, a fully recessive gene for Xoo resistance, to a DNA fragment of 14.8 kb using the map-based cloning strategy and a series of sequence-based molecular markers. Sequence analysis of this fragment indicated that this region contains only two apparently intact candidate genes (an extensin-like gene and a homologue of nodulin MtN3) and the 5′ end of a predicted hypothetical gene. These results will greatly facilitate the isolation and characterization of xa13. Four PCR-based markers, E6a, SR6, ST9 and SR11 that were tightly linked to the xa13 locus, were also developed. These markers will be useful tools for the marker-assisted selection of xa13 in breeding programs.