Avoidance of host recognition by alterations in the repetitive and C-terminal regions of AvrXa7, a type III effector of Xanthomonas oryzae pv. oryzae

avrXa7 is a member of the avrBs3/pthA gene family. The gene is a critical type III effector in several strains of Xanthomonas oryzae pv. oryzae (virulence activity), and in the presence of the Xa7 host gene for resistance, controls the elicitation of resistance in rice (avirulence activity). The ability of strains containing avrXa7 to adapt to the presence of Xa7 in the host population is dependent, in part, on the genetic plasticity of avrXa7. The potential for the conversion of avrXa7 to a virulence effector without Xa7-dependent elicitor activity was examined. Internal reorganization of avrXa7 by artificially deleting a portion of the central repetitive region resulted in gene pthXo4, which retained virulence activity and lost Xa7-dependent avirulence activity. Similarly, spontaneous rearrangements between repetitive regions of avrXa7 during bacterial culture gave rise to gene pthXo5, which also had virulence activity without Xa7-dependent avirulence activity. pthXo5 appeared to be the result of recombination between avrXa7 and a related gene in the genome. Loss of avirulence activity and retention of virulence activity also resulted from replacement of a portion of the C-terminal coding region of avrXa7 with the corresponding sequence from avrBs3. The results demonstrated the potential for a critical virulence effector to lose avirulence activity while retaining effector function. The results also demonstrated that features of both repetitive and nonrepetitive C-terminal regions of AvrXa7 are involved in avirulence specificity.     

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.     

TAL effector-dependent Bax gene expression in transgenic rice confers disease resistance to Xanthomonas oryzae pv. oryzae

The hypersensitive response (HR) is a form of programmed cell death of plant cells occurring in the local region surrounding pathogen infection site to prevent the spread of infection by pathogens. Bax, a mammalian pro-apoptotic member of Bcl-2 family, triggers HR-like cell death when expressed in plants. However, constitutive expression of the Bax gene negatively affects plant growth and development. The Xa10 gene in rice (Oryza sativa) is an executor resistance (R) gene that confers race-specific disease resistance to Xanthomonas oryzae pv. oryzae strains harboring TAL effector gene AvrXa10. In this study, the Xa10 promoter was used to regulate heterologous expression of the Bax gene from mouse (Mus musculus) in Nicotiana benthamiana and rice. Cell death was induced in N. benthamiana after co-infiltration with the P_Xa10:Bax:T_Xa10 gene and the P_PR1:AvrXa10:T_Nos gene. Transgenic rice plants carrying the P_Xa10:Bax:T_Xa10 gene conferred specific disease resistance to Xa10-incompatible X. oryzae pv. oryzae strain PXO99^A(pHM1AvrXa10), but not to the Xa10-compatible strain PXO99^A(pHM1). The resistance specificity was confirmed by the AvrXa10-dependent induction of the P_Xa10:Bax:T_Xa10 gene in transgenic rice. Our results demonstrated that the inducible expression of the Bax gene in transgenic rice was achieved through the control of the executor R gene promoter and the heterologous expression of the pro-apoptosis regulator gene in rice conferred disease resistance to X. oryzae pv. oryzae.

     

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.     

Xa26, a gene conferring resistance to Xanthomonas oryzae pv. oryzae in rice, encodes an LRR receptor kinase-like protein

Rice bacterial blight, caused by Xanthomonas oryzae pv. oryzae (Xoo), is one of the most serious rice diseases worldwide. A rice gene, Xa26, conferring resistance against Xoo at both seedling and adult stages was isolated by map-based cloning strategies from the rice cultivar Minghui 63. Xa26 belongs to a multigene family consisting of four members. It encodes a leucine-rich repeat (LRR) receptor kinase-like protein and is constitutively expressed. Sequence analysis revealed that IRBB3 and Zhachanglong lines that are resistant to a broad range of Xoo strains, also carry Xa26. However, significant difference in lesion length was observed among these lines after inoculation with a set of Xoo strains. Moreover, transgenic plants carrying Xa26 showed enhanced resistance compared with the donor line of the gene in both seedling and adult stages. These results suggest that the resistance conferred by Xa26 is influenced by the genetic background.     

XopR, a type III effector secreted by Xanthomonas oryzae pv. oryzae, suppresses microbe-associated molecular pattern-triggered immunity in Arabidopsis thaliana

Xanthomonas oryzae pv. oryzae is the causal agent of bacterial blight of rice. The XopR protein, secreted into plant cells through the type III secretion apparatus, is widely conserved in xanthomonads and is predicted to play important roles in bacterial pathogenicity. Here, we examined the function of XopR by constructing transgenic Arabidopsis thaliana plants expressing it under control of the dexamethasone (DEX)-inducible promoter. In the transgenic plants treated with DEX, slightly delayed growth and variegation on leaves were observed. Induction of four microbe-associated molecular pattern (MAMP)-specific early-defense genes by a nonpathogenic X. campestris pv. campestris hrcC deletion mutant were strongly suppressed in the XopR-expressing plants. XopR expression also reduced the deposition of callose, an immune response induced by flg22. When transiently expressed in Nicotiana benthamiana, a XopR::Citrine fusion gene product localized to the plasma membrane. The deletion of XopR in X. oryzae pv. oryzae resulted in reduced pathogenicity on host rice plants. Collectively, these results suggest that XopR inhibits basal defense responses in plants rapidly after MAMP recognition.     

Constitutive heterologous expression of avrXa27 in rice containing the R gene Xa27 confers enhanced resistance to compatible Xanthomonas oryzae strains

The vascular pathogen Xanthomonas oryzae pv. oryzae ( Xoo ) and nonvascular pathogen Xanthomonas oryzae pv. oryzicola ( Xoc ) cause bacterial blight (BB) and bacterial leaf streak (BLS) diseases of rice, respectively. We have previously identified the avirulence gene avrXa27 from Xoo PXO99^A, which specifically induces the expression of the rice resistance gene Xa27 , ultimately leading to resistance against BB disease in rice. In this study, we have generated a transgenic rice line (L24) that expresses avrXa27 constitutively under the control of the PR1 promoter, and have examined its role in the host–pathogen interaction. L24 is not more susceptible to BB, indicating that avrXa27 does not contribute to virulence. AvrXa27 retains avirulence activity in L24 and, after crossing with a line containing Xa27 , progeny display phenotypic changes including inhibition of tillering, delay in flowering, stiff leaves, early leaf senescence and activation of pathogenesis-related ( PR ) genes. On challenge with a variety of compatible strains of Xoo and Xoc strain L8, lines with both avrXa27 and Xa27 also show enhanced resistance to bacterial infection. The induction of Xa27 and subsequent inhibition of Xoc growth in Xa27 plants are observed on inoculation with Xoc L8 harbouring avrXa27 . Our results indicate that the heterologous expression of avrXa27 in rice containing Xa27 triggers R gene-specific resistance and, at the same time, confers enhanced resistance to compatible strains of Xoo and Xoc . The expression of AvrXa27 and related proteins in plants has the potential to generate broad resistance in plants.     

Dissection of the genetic architecture of rice resistance to Xanthomonas oryzae pv. oryzae using a genomewide association study

Bacterial blight, caused by Xanthomonas oryzae pv. oryzae (Xoo), usually causes serious rice yield loss in many countries. Rice breeders have used resistance (R) genes to control the disease but many of the resistant cultivars become susceptible few years after releasing. Identification of new R genes to Xoo is one of the main objectives in rice breeding programs. In this study, we used a genomewide association study (GWAS) to analyse the resistance against the Xoo race C1 using the Rice Diversity Panel 1 (RDP1). Disease evaluation of the RDP1 population to C1 indicated that the AUS subgroup conferred a higher level of resistance to C1 than other subgroups. Genomewide association mapping identified 15 QTLs that are distributed on chromosomes 1, 2, 3, 4, 5, 6, 8, 9, 10 and 12. Some of them are located in the regions without known resistance loci or QTLs. This study demonstrated the effectiveness of GWAS on the genetic dissection of rice resistance to Xoo and provided many Xoo resistance‐associated SNP markers for rice breeding.     

水稻品种次生物质与稻白叶枯病(Xanthomonas oryzae pv. oryzae)抗性的关系

应用高效液相色谱分析了对白叶枯病具有不同抗性水平的12个水稻品种中的19个(组)次生物质色谱峰(面积)的差异及其与白叶枯病抗性水平间关系.结果表明,水稻品种抗性水平与谱峰面积值之间相关极显著(R=0.992,p<0.01),被测的19个组分中,峰1、峰2、峰8、峰10、峰12、峰14、峰16和峰18是影响水稻对稻白叶枯病抗性水平的主要抗原次生物质.建立了水稻品种对白叶枯病抗性级别与以上次生物质含量谱峰面积之间的回归模型:Y=10.7603 0.1823X1-0.2287X2 0.2163X8-2.1975X10 0.0728X12 -0.7438X14 1.1484X16-0.7795X18.研究结果表明水稻品种中起抗病作用的抗原次生物质不止一种,而是几种的组合,而且它们对水稻抗病性的贡献作用是不完全相同的,这与它们的性质与含量密切相关.提出了以抗原次生物质为标记的快速分析、鉴定、预测水稻品种对稻白叶枯病抗性水平的新途径、新方法.     

The Xanthomonas oryzae pv. lozengeoryzae raxP and raxQ genes encode an ATP sulphurylase and adenosine-5'-phosphosulphate kinase that are required for AvrXa21 avirulence activity

Xanthomonas oryzae pv. oryzae (Xoo) Philippine race 6 (PR6) is unable to cause bacterial blight disease on rice lines containing the rice resistance gene Xa21 but is virulent on non-Xa21 rice lines, indicating that PR6 carries avirulence (avrXa21) determinants required for recognition by XA21. Here we show that two Xoo genes, raxP and raxQ, are required for AvrXa21 activity. raxP and raxQ, which reside in a genomic cluster of sulphur assimilation genes, encode an ATP sulphurylase and APS (adenosine-5'-phosphosulphate) kinase. These enzymes function together to produce activated forms of sulphate, APS and PAPS (3'-phosphoadenosine-5'-phosphosulphate). Xoo PR6 strains carrying disruptions in either gene, PR6DeltaraxP or PR6DeltaraxQ, are unable to produce APS and PAPS and are virulent on Xa21-containing rice lines. RaxP and RaxQ are similar to the bacterial symbiont Sinorhizobium meliloti host specificity proteins, NodP and NodQ and the Escherichia coli cysteine synthesis proteins CysD, CysN and CysC. The APS and PAPS produced by RaxP and RaxQ are used for both cysteine synthesis and sulphation of other molecules. Mutation in Xoo xcysI, a homologue of Escherichia coli cysI that is required for cysteine synthesis, blocked APS- or PAPS-dependent cysteine synthesis but did not affect AvrXa21 activity, suggesting that AvrXa21 activity is related to sulphation rather than cysteine synthesis. Taken together, these results demonstrate that APS and PAPS production plays a critical role in determining avirulence of a phytopathogen and reveal a commonality between symbiotic and phytopathogenic bacteria.     

Virulence and type III effector diversities of Xanthomonas citri pv. fuscans and X. phaseoli pv. phaseoli in Brazil

Common bacterial blight (CBB) is caused by four genetic lineages belonging to two species of Xanthomonas, namely Xanthomonas citri pv. fuscans (includes fuscans, NF2 and NF3 lineages) and X. phaseoli pv. phaseoli (lineage NF1). A collection of 117 strains of Xanthomonas isolated from common bean plants grown in several producing regions of Brazil, between 2007 and 2016 was established. For species and lineage identification, the following tests were performed: multiplex PCR with a set of four specific primer pairs, pathogenicity tests on susceptible cultivar BRS Artico and phylogenetic analysis based on housekeeping gene sequences. The presence of the two species were confirmed among the 117 strains, being 62 non-fuscans strains (NF1, NF2 and NF3) and 55 fuscans strains of X. citri pv. fuscans. To select a set of representative strains for the virulence assay, a PCR-based analysis of effector diversity was performed with 42 strains belonging to the two species. PCR with primers for xopL, avrBsT, xopE2 and xopE1 genes were positive for all strains, while for the other six effectors there was variation. Six distinct effector profiles were detected, and one strain representing each type was inoculated in 15 common bean cultivars with varying levels of resistance to CBB. The fuscans strains showed uniformity in their effector profiles and were the most virulent. The phylogenetic analyses of our strain collection revealed that all genetic variants of CBB pathogens (NF1, NF2, NF3 and fuscans) are present in Brazil, with significant variability in virulence to common bean cultivars.