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.

     

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.     

Salicylic acid-induced accumulation of biochemical components associated with resistance against Xanthomonas oryzae pv. oryzae in rice

Seed treatment and foliar sprays of salicylic acid (SA) provided protection in rice against bacterial leaf blight (BLB) caused by bacterial Xanthomonas oryzae pv. oryzae (Xoo). Treatment of rice with exogenous SA reduced disease severity by more than 38%. Superoxide anion production and hypersensitive response increased approximately 28% and 110% at 6 and 48?h after Xoo inoculation, respectively, for plants treated with SA. Moreover, the Xoo in treated rice plants grew more slowly, resulting in a population that was half of that observed in the control. Fourier transform infrared spectroscopy analysis revealed that the higher ratios of 1233/1517, 1467/1517, and 1735/1517?cm^?1 observed in treated rice suggested alteration of monomer composition of lignin and pectin in the rice cell wall. Exogenous SA-treated rice had more amide I β-sheet structure and lipids as shown by the peaks at 1629, 2851, and 1735?cm^?1. These biochemical changes of rice treated with SA and inoculated with Xoo were related to primed resistance of the rice plants to BLB disease.     

A proteomic study of Xanthomonas oryzae pv. oryzae in rice xylem sap

Xanthomonas oryzae pv. oryzae (Xoo) is the second most important rice pathogen, causing a disease called bacterial leaf blight. Xoo colonizes and infects the vascular tissue resulting in tissue necrosis and wilting causing significant yield losses worldwide. In this study Xoo infected vascular fluid (xylem sap) was recovered and analyzed for secreted Xoo proteins. Three independent experiments resulted in the identification of 324 different proteins, 64 proteins were found in all three samples which included many of the known virulence-associated factors. In addition, 10 genes encoding for the identified proteins were inactivated and one mutant displayed statistically a significant loss in virulence when compared to the wild type Xoo, suggesting that a new virulence-associated factor has been revealed. The usefulness of this approach in understanding the lifestyle and unraveling the virulence-associated factors of phytopathogenic vascular bacteria is discussed.     

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.     

Evaluation of resistance genes in rice against local isolates of Xanthomonas oryzae pv. oryzae in Punjab Province of Pakistan

Absence of resistance/tolerance against bacterial leaf blight (BLB), incited by Xanthomonas oryzae pv. oryzae, in famous basmati varieties is one of the main reason for BLB epidemic in Punjab in 2007–2008. For developing resistance against BLB, the response of 26 IRBB lines of IRRI including 10 near isogenic lines (NILs) and 16 gene pyramids carrying two to five resistance genes (Xa series) was evaluated against 61 indigenous Xoo isolates under artificial inoculation field conditions. None of the NILs or gene pyramid provides complete protection against all the isolates. However, Xa21 and xa13 were found resistant against the majority of Xoo isolates, followed by Xa14 and Xa7. Of the 16 gene pyramids used in this study, IRBB-54 (Xa5 + Xa21), IRBB-55 (Xa13 + Xa21) followed by IRBB-58 (Xa4 + Xa13 + Xa21) were found effective against the majority of the Xoo isolates. These resistance genes (individually and in combinations) can be incorporated for the improvement of basmati rice cultivars cultivated in Punjab province of Pakistan. Effectiveness of gene combination supports the strategy of pyramiding appropriate resistance genes. Newly identified resistant genes may also be evaluated for achieving broad spectrum resistance against more Xoo isolates of the area.     

A LuxR homologue of Xanthomonas oryzae pv. oryzae is required for optimal rice virulence

In Gram-negative bacteria a typical quorum sensing (QS) system usually involves the production and response to acylated homoserine lactones (AHLs). An AHL QS system is most commonly mediated by a LuxI family AHL synthase and a LuxR family AHL response regulator. This study reports for the first time the presence of a LuxR family-type regulator in Xanthomonas oryzae pv. oryzae ( Xoo ), which has been designated as OryR. The primary structure of OryR contains the typical signature domains of AHL QS LuxR family response regulators: an AHL-binding and a HTH DNA binding motif. The oryR gene is conserved among 26 Xoo strains and is also present in the genomes of close relatives X. campestris pv. campestris and X. axonopodis pv. citri . Disrupting oryR in three Xoo strains resulted in a significant reduction of rice virulence. The wild-type Xoo strains do not seem to produce AHLs and analysis of the Xoo sequenced genomes did not reveal the presence of a LuxI-family AHL synthase. The OryR protein was shown to be induced by macerated rice and affected the production of two secreted proteins: a cell-wall-degrading cellobiosidase and a 20-kDa protein of unknown function. By expressing and purifying OryR it was then observed that it was solubilized when grown in the presence of rice extract indicating that there could be a molecule(s) in rice which binds OryR. The role of OryR as a possible in planta induced LuxR family regulator is discussed.     

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.     

N-terminal HrpE from Xanthomonas oryzae pv. oryzae mediates the regulation of growth and photosynthesis in rice

Plant Growth Regulation - Harpin proteins, produced by gram-negative bacteria, can enhance the plant growth. The growth enhancing potential of the harpins are beneficial and may be associated with...     

Role of an in planta-expressed xylanase of Xanthomonas oryzae pv. oryzae in promoting virulence on rice

Xanthomonas oryzae pv. oryzae is the causal agent of bacterial leaf blight, a serious disease of rice. We demonstrated earlier that the type II secretion system (T2S) is important for virulence of X. oryzae pv. oryzae and that several proteins, including a xylanase, are secreted through this system. In this study, the xynB gene encoding for the secreted xylanase was cloned as a 6.9-kb EcoRI fragment (pRR7) that also included a paralog called xynA. As in X. oryzae pv. oryzae, xynA and xynB are adjacent to each other in X. axonopodis pv. citri, whereas only the xynA homolog is present in X. campestris pv. campestris. Mutations in xynB but not xynA affect secreted xylanase activity. Western blot analysis using anti-XynB antibodies on exudates from infected rice leaves indicated that this xylanase is expressed during in planta growth. Another T2S-secreted protein was identified to be a lipase/esterase (LipA) based on the sequence tags obtained by tandem mass spectrometry analysis and biochemical assays. Mutations in either xynB or lipA partially affected virulence. However, a lipA-xynB double mutant was significantly reduced for virulence, and the pRR7 clone containing an intact xynB gene could complement the virulence-deficient phenotype of the lipA-xynB mutant. Our results suggest that there is functional redundancy among the T2S secreted proteins of X. oryzae pv. oryzae in promoting virulence on rice.     

Rice cationic peroxidase accumulates in xylem vessels during incompatible interactions with Xanthomonas oryzae pv oryzae.

A cationic peroxidase, PO-C1 (molecular mass 42 kD, isoelectric point 8.6), which is induced in incompatible interactions between the vascular pathogen Xanthomonas oryzae pv oryzae and rice (Oryza sativa L.), was purified. Amino acid sequences from chemically cleaved fragments of PO-C1 exhibited a high percentage of identity with deduced sequences of peroxidases from rice, barley, and wheat. Polyclonal antibodies were raised to an 11-amino acid oligopeptide (POC1a) that was derived from a domain where the sequence of the cationic peroxidase diverged from other known peroxidases. The anti-POC1a antibodies reacted only with a protein of the same mobility as PO-C1 in extracellular and guttation fluids from plants undergoing incompatible responses collected at 24 h after infection. In the compatible responses, the antibodies did not detect PO-C1 until 48 h after infection. Immunoelectron microscopy was used to demonstrate that PO-C1 accumulated within the apoplast of mesophyll cells and within the cell walls and vessel lumen of xylem elements of plants undergoing incompatible interactions.     

A “defeated” rice resistance gene acts as a QTL against a virulent strain of Xanthomonas oryzae pv. oryzae

The genetic components responsible for qualitative and quantitative resistance of rice plants to three strains (CR4, CXO8, and CR6) of Xanthomonas oryzae pv. oryzae (Xoo) were investigated using a set of 315 recombinant inbred lines (RILs) from the cross Lemont (japonica) × Teqing (indica) and a complete linkage map with 182 well distributed RFLP markers. We mapped a major gene (Xa4) and ten quantitative trait loci (QTLs) which were largely responsible for segregation of the resistance phenotype in the RILs. The Teqing allele at the Xa4 locus, Xa4 ^T , acted as a dominant resistance gene against CR4 and CXO8. The breakdown of Xa4 ^T -associated resistance mediated by the mutant allele at the avrXa4 locus in the virulent strain CR6 results from significant changes in both gene action (lose of dominance) and the magnitude of gene effect (≈50% reduction). Nevertheless, Xa4 ^T still acted as a recessive QTL with a significant residual effect against CR6. The mutant alleles at the avrXa4 locus in CXO8 and CR6 that lead to a reduction in effect, or “breakdown”, of Xa4 ^T were apparently accompanied by corresponding penalties for their fitness. The quantitative component of resistance to Xoo in the RILs was largely due to a number of resistance QTLs. Most resistance QTLs mapped to genomic locations where major resistance genes and/or QTLs for resistance to Xoo, blast and sheath blight were identified in the same cross. Most QTLs showed consistent levels of resistance against all three Xoo strains. Our results suggest that a high level of durable resistance to Xoo may be achieved by the cumulative effects of multiple QTLs, including the residual effects of “defeated” major resistance genes.     

Some tryptophan pathways in the phytopathogen Xanthomonas oryzae pv. oryzae

Xanthomonas oryzae pv.oryzae, the causal or-ganism of bacterial blight of rice which produces leaf blight as well as kresek (wilt) symptoms in plants were tested for indole, auxin production in culture supplemented withl-tryptophan. On the basis of indoleacetic acid (IAA) production the isolates were grouped into IAA-positive and IAA-negative. Out of 17 isolates, 11 were IAA-positive while 6 were IAA-negative. The isolates metabolized tryptophan through two different routes and the isolates vary in the pathway of tryptophan utilization. The IAA-positive isolates converted tryptophan to IAA as the end product, whereas the IAA-negative isolates formed anthranilate as an intermediate metabolite and finally produced pyrocatecholvia the kynurenine pathway. Quantification of tryptophan metabolism revealed that the maximum production of IAA and pyrocatechol in culture occurred during 2-d incubation at 30±2°C.     

Phosphorylation of proteins in Xanthomonas campestris pv. oryzae

Protein phosphorylation was studied in Xanthomonas campestris pv. oryzae in vivo and in vitro. In vitro labelling showed that the protein kinases in this bacterium used both ATP and GTP as nucleotide substrates at nearly the same efficiency. At least 6 proteins were phosphorylated in vitro, including abundant species of p81, p44, and p32 with M _r of 81000, 44000, and 32000, respectively. Three types of phosphate-protein linkage were found in this bacterium: O-phosphate, N-phosphate and probably acyl phosphate. The p81 and p32 were phosphorylated at histidine. The p44 had mainly phosphoserine and a small part of phosphohistidine. The phosphorylation profile was variable depending on the growth conditions. Furthermore, by a virulent phage Xp10 infection the quantity of phosphorylation increased: for phosphohistinine more than 10-fold, and for phosphoserine about 3-fold. Thus, in this bacterium phosphorylation may be linked with a physiological regulation system and with Xp10 phage development.     

DNA fingerprinting of Indian isolates of Xanthomonas oryzae pv. oryzae

 A high level of genetic polymorphism was detected among Indian isolates of Xanthomonas oryzae pv. oryzae using hypervariable probes such as a microsatellite oligonucleotide, probe (TG)₁₀, a human minisatellite probe, pV47, an avirulence gene probe, avrXa10 and a repeat clone, pBS101. These DNA probes detected multiple loci in the bacterial genome generating complex DNA fingerprints and differentiated all of the bacterial isolates. Analysis of fingerprints indicated that pV47, (TG)₁₀ and pBS101 have a lower probability of identical match than avrXa10 and therefore are potential probes for DNA fingerprinting and variability analysis of Xanthomonas oryzae pv. oryzae pathogen populations. Cluster analysis based on hybridization patterns using all of the above probes showed five groups at 56% similarity. Studies on the methylation patterns of isolates representing the three important races of X. oryzae pv. oryzae indicated more methylation in the most virulent isolate, suggesting a possible role of methylation in pathogenicity. Received: 8 December 1996 / Accepted: 20 December 1996     

Distribution of Xanthomonas oryzae pv. oryzae DNA Modification Systems in Asia

The presence or absence of two DNA modification systems, XorI and XorII, in 195 strains of Xanthomonas oryzae pv. oryzae collected from different major rice-growing countries of Asia was assessed. All four possible phenotypes (XorI⁺ XorII⁺, XorI⁺ XorII⁻, XorI⁻ XorII⁺ and XorI⁻ XorII⁻) were detected in the population at a ratio of approximately 1:2:2:2. The XorI⁺ XorII⁺ and XorI⁻ XorII⁺ phenotypes were observed predominantly in strains from southeast Asia (Philippines, Malaysia, and Indonesia), whereas strains with the phenotypes XorI⁻ XorII⁻ and XorI⁺ XorII⁻ were distributed in south Asia (India and Nepal) and northeast Asia (China, Korea, and Japan), respectively. Based on the prevalence and geographic distribution of the XorI and XorII systems, we suggest that the XorI modification system originated in northeast Asia and was later introduced to southeast Asia, while the XorII system originated in southeast Asia and moved to northeast Asia and south Asia. Genomic DNA from all tested strains of X. oryzae pv. oryzae that were resistant to digestion by endonuclease XorII or its isoschizomer PvuI also hybridized with a 7.0-kb clone that contained the XorII modification system, whereas strains that were digested by XorII or PvuI lacked DNA that hybridized with the clone. Size polymorphisms were observed in fragments that hybridized with the 7.0-kb clone. However, a single hybridization pattern generally was found in XorII⁺ strains within a country, indicating clonal maintenance of the XorII methyltransferase gene locus. The locus was monomorphic for X. oryzae pv. oryzae strains from the Philippines and all strains from Indonesia and Korea.     

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.