An improved,versatile and efficient modular plasmid assembly system for expression analyses of genes in Xanthomonas oryzae

Xanthomonas oryzae pathovars oryzae (Xoo) and oryzicola (Xoc) infect rice, causing bacterial blight and bacterial leaf streak, respectively, which are two economically important bacterial diseases in paddy fields. The interactions of Xoo and Xoc with rice can be used as models for studying fundamental aspects of bacterial pathogenesis and host tissue specificity. However, an improved vector system for gene expression analysis is desired for Xoo and Xoc because some broad host range vectors that can replicate stably in X. oryzae pathovars are low-copy number plasmids. To overcome this limitation, we developed a modular plasmid assembly system to transfer the functional DNA modules from the entry vectors into the pHM1-derived backbone vectors on a high-copy number basis. We demonstrated the feasibility of our vector system for protein detection, and quantification of virulence gene expression under laboratory conditions and in association with host rice and nonhost tobacco cells. This system also allows execution of a mutant complementation equivalent to the single-copy chromosomal integration system and tracing of pathogens in rice leaf. Based on this assembly system, we constructed a series of protein expression and promoter-probe vectors suitable for classical double restriction enzyme cloning. These vector systems enable cloning of all genes or promoters of interest from Xoo and Xoc strains. Our modular assembly system represents a versatile and highly efficient toolkit for gene expression analysis that will accelerate studies on interactions of X. oryzae with rice.     

Novel broad-spectrum bacteriophages against Xanthomonas oryzae and their biocontrol potential in rice bacterial diseases

Bacterial leaf blight (BLB) and bacterial leaf streak (BLS)—caused by Xanthomonas oryzae pv. oryzae (Xoo) and Xanthomonas oryzae pv. oryzicola (Xoc), respectively—are two major bacterial diseases that threaten the safe production of rice, one of the most important food crops. Bacteriophages are considered potential biocontrol agents against rice bacterial pathogens, due to their host specificity and environmental safety. It is common for BLB and BLS to occur together in fields, which highlights the need for broad-spectrum phages capable of infecting both Xoo and Xoc. In this study, two lytic broad-spectrum phages (pXoo2106 and pXoo2107) that can infect various strains of Xoo and Xoc were assessed. Both phages belong to the class Caudoviricetes and one of them to the family Autographiviridae, while the other belongs to an unclassified family. Two phages alone or combined in a phage cocktail could effectively inhibit Xoo and Xoc growth in vitro. In an in vivo biocontrol experiment, the phage cocktail reduced the total CFU and significantly eased the symptoms caused by Xoo or Xoc. Our results suggest that pXoo2106 and pXoo2107 have a broad-spectrum host range targeting different X. oryzae strains, and have strong biocontrol potential in field applications against both BLB and BLS.     

Elucidation of the hrp Clusters of Xanthomonas oryzae pv. oryzicola That Control the Hypersensitive Response in Nonhost Tobacco and Pathogenicity in Susceptible Host Rice

Xanthomonas oryzae pv. oryzicola, the cause of bacterial leaf streak in rice, possesses clusters of hrp genes that determine its ability to elicit a hypersensitive response (HR) in nonhost tobacco and pathogenicity in host rice. A 27-kb region of the genome of X. oryzae pv. oryzicola (RS105) was identified and sequenced, revealing 10 hrp, 9 hrc (hrp conserved), and 8 hpa (hrp-associated) genes and 7 regulatory plant-inducible promoter boxes. While the region from hpa2 to hpaB and the hrpF operon resembled the corresponding genes of other xanthomonads, the hpaB-hrpF region incorporated an hrpE3 gene that was not present in X. oryzae pv. oryzae. We found that an hrpF mutant had lost the ability to elicit the HR in tobacco and pathogenicity in adult rice plants but still caused water-soaking symptoms in rice seedlings and that Hpa1 is an HR elicitor in nonhost tobacco whose expression is controlled by an hrp regulator, HrpX. Using an Hrp phenotype complementation test, we identified a small hrp cluster containing the hrpG and hrpX regulatory genes, which is separated from the core hrp cluster. In addition, we identified a gene, prhA (plant-regulated hrp), that played a key role in the Hrp phenotype of X. oryzae pv. oryzicola but was neither in the core hrp cluster nor in the hrp regulatory cluster. A prhA mutant failed to reduce the HR in tobacco and pathogenicity in rice but caused water-soaking symptoms in rice. This is the first report that X. oryzae pv. oryzicola possesses three separate DNA regions for HR induction in nonhost tobacco and pathogenicity in host rice, which will provide a fundamental base to understand pathogenicity determinants of X. oryzae pv. oryzicola compared with those of X. oryzae pv. oryzae.     

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.     

The genome sequence of Xanthomonas oryzae pathovar oryzae KACC10331, the bacterial blight pathogen of rice

The nucleotide sequence was determined for the genome of Xanthomonas oryzae pathovar oryzae (Xoo) KACC10331, a bacterium that causes bacterial blight in rice (Oryza sativa L.). The genome is comprised of a single, 4 941 439 bp, circular chromosome that is G + C rich (63.7%). The genome includes 4637 open reading frames (ORFs) of which 3340 (72.0%) could be assigned putative function. Orthologs for 80% of the predicted Xoo genes were found in the previously reported X.axonopodis pv. citri (Xac) and X.campestris pv. campestris (Xcc) genomes, but 245 genes apparently specific to Xoo were identified. Xoo genes likely to be associated with pathogenesis include eight with similarity to Xanthomonas avirulence (avr) genes, a set of hypersensitive reaction and pathogenicity (hrp) genes, genes for exopolysaccharide production, and genes encoding extracellular plant cell wall-degrading enzymes. The presence of these genes provides insights into the interactions of this pathogen with its gramineous host.     

Novel Candidate Virulence Factors in Rice Pathogen Xanthomonas oryzae pv. oryzicola as Revealed by Mutational Analysis

Bacterial leaf streak, caused by Xanthomonas oryzae pv. oryzicola, is an important disease of rice. Transposon-mediated mutational analysis of the pathogen with a quantitative assay revealed candidate virulence factors including genes involved in the pathogenesis of other phytopathogenic bacteria, virulence factors of animal pathogens, and genes not previously associated with virulence.     

Domain Dissection of AvrRxo1 for Suppressor,Avirulence and Cytotoxicity Functions

AvrRxo1, a type III effector from Xanthomonas oryzae pv. oryzicola (Xoc) which causes bacterial leaf streak (BLS) in rice, can be recognised by non-host resistance protein Rxo1. It triggers a hypersensitive response (HR) in maize. Little is known regarding the virulence function of AvrRxo1. In this study, we determined that AvrRxo1 is able to suppress the HR caused by the non-host resistance recognition of Xanthomonas oryzae pv. oryzae (Xoo) by Nicotiana benthamiana. It is toxic, inducing cell death from transient expression in N. benthamiana, as well as in yeast. Among the four AvrRxo1 alleles from different Xoc strains, we concluded that the toxicity is abolished by a single amino acid substitution at residue 344 in two AvrRxo1 alleles. A series of truncations from the carboxyl terminus (C-terminus) indicate that the complete C-terminus of AvrRxo1 plays an essential role as a suppressor or cytotoxic protein. The C-terminus was also required for the avirulence function, but the last two residues were not necessary. The first 52 amino acids of N-terminus are unessential for toxicity. Point mutagenesis experiments indicate that the ATP/GTP binding site motif A is required for all three functions of AvrRxo1, and NLS is required for both the avirulence and the suppression of non-host resistance. The putative thiol protease site is only required for the cytotoxicity function. These results determine that AvrRxo1 plays a role in the complex interaction with host proteins after delivery into plant cells.     

Multilocus sequence analysis and type III effector repertoire mining provide new insights into the evolutionary history and virulence of Xanthomonas oryzae

Multilocus sequence analysis (MLSA) and type III effector (T3E) repertoire mining were performed to gain new insights into the genetic relatedness of Xanthomonas oryzae pv. oryzae (Xoo) and Xanthomonas oryzae pv. oryzicola (Xoc), two major bacterial pathogens of rice. Based on a collection of 45 African and Asian strains, we first sequenced and analysed three housekeeping genes by MLSA, Bayesian clustering and a median-joining network approach. Second, we investigated the distribution of 32 T3E genes, which are known to be major virulence factors of plant pathogenic bacteria, in all selected strains, by polymerase chain reaction and dot-blot hybridization methods. The diversity observed within housekeeping genes, as well as within T3E repertoires, clearly showed that both pathogens belong to closely related, but distinct, phylogenetic groups. Interestingly, these evolutionary groups are differentiated according to the geographical origin of the strains, suggesting that populations of Xoo and Xoc might be endemic in Africa and Asia, and thus have evolved separately. We further revealed that T3E gene repertoires of both pathogens comprise core and variable gene suites that probably have distinct roles in pathogenicity and different evolutionary histories. In this study, we carried out a functional analysis of xopO, a differential T3E gene between Xoo and Xoc, to determine the involvement of this gene in tissue specificity. Altogether, our data contribute to a better understanding of the evolutionary history of Xoo and Xoc in Africa and Asia, and provide clues for functional studies aiming to understand the virulence, host and tissue specificity of both rice pathogens.     

Homology between the HrpO protein of Pseudomonas solanacearum and bacterial proteins implicated in a signal peptide-independent secretion mechanism

A region of approximately 22 kb of DNA defines the large hrp gene cluster of strain GMI1000 of Pseudomonas solanacearum. The majority of mutants that map to this region have lost the ability to induce disease symptoms on tomato plants and are no longer able to elicit a hypersensitive reaction (HR) on tobacco, a nonhost plant. In this study we present the complementation analysis and nucleotide sequence of a 4772 by region of this hrp gene cluster. Three complete open reading frames (ORFs) are predicted within this region. The corresponding putative proteins, HrpN, HrpO and HpaP, have predicted sizes of 357, 690 and 197 amino acids, respectively, and predicted molecular weights of 38607, 73 990 and 21959 dalton, respectively. HrpN and HrpO are both predicted to be hydrophobic proteins with potential membrane-spanning domains and HpaP is rich in proline residues. A mutation in hpaP (for hrp associated) does not affect the HR on tobacco or the disease on tomato plants. None of the proteins is predicted to have an N-terminal signal sequence, which would have indicated that the proteins are exported. Considerable sequence similarities were found between HrpO and eight known or predicted prokaryotic proteins: LcrD of Yersinia pestis and Y. enterocolitica, FlbF of Caulobacter crescentus, F1hA of Bacillus subtilis, MxiA and VirH of Shigella flexneri, InvA of Salmonella typhimurium and HrpC2 of Xanthomonas campestris pv. vesicatoria. These homologies suggest that certain hrp genes of phytopathogenic bacteria code for components of a secretory system, which is related to the systems for secretion of flagellar proteins, Ipa proteins of Shigella flexneri and the Yersinia Yop proteins. Furthermore, these homologous proteins have the common feature of being implicated in a distinct secretory mechanism, which does not require the cleavage of a signal peptide. The sequence similarity between HrpO and HrpC2 is particularly high (66% identity and 81 % similarity) and the amino acid sequence comparison between these two proteins presented here reveals the first such sequence similarity to be shown between Hrp proteins of P. solanacearum and X. campestris. An efflux of plant electrolytes was found to be associated with the interactions between P. solanacearum and both tomato and tobacco leaves. This phenomenon may be part of the mechanism by which hrp gene products control and determine plant-bacterial interactions, since hrpO mutants induced levels of leakage which were significantly lower than those induced by the wild type on each plant.     

The HD-GYP Domain Protein RpfG of Xanthomonas oryzae pv. oryzicola Regulates Synthesis of Extracellular Polysaccharides that Contribute to Biofilm Formation and Virulence on Rice

Bacterial leaf streak caused by Xanthomonas oryzae pv. oryzicola (Xoc) is one of the most important diseases in rice. However, little is known about the pathogenicity mechanisms of Xoc. Here we have investigated the function of three HD-GYP domain regulatory proteins in biofilm formation, the synthesis of virulence factors and virulence of Xoc. Deletion of rpfG resulted in altered production of extracellular polysaccharides (EPS), abolished virulence on rice and enhanced biofilm formation, but had little effect on the secretion of proteases and motility. In contrast, mutational analysis showed that the other two HD-GYP domain proteins had no effect on virulence factor synthesis and tested phenotypes. Mutation of rpfG led to up-regulation of the type III secretion system and altered expression of three putative glycosyltransferase genes gumD, pgaC and xagB, which are part of operons directing the synthesis of different extracellular polysaccharides. The pgaABCD and xagABCD operons were greatly up-regulated in the Xoc ΔrpfG mutant, whereas the expression of the gum genes was unaltered or slightly enhanced. The elevated biofilm formation of the Xoc ΔrpfG mutant was dramatically reduced upon deletion of gumD, xagA and xagB, but not when pgaA and pgaC were deleted. Interestingly, only the ΔgumD mutant, among these single gene mutants, exhibits multiple phenotype alterations including reduced biofilm and EPS production and attenuated virulence on rice. These data indicate that RpfG is a global regulator that controls biofilm formation, EPS production and bacterial virulence in Xoc and that both gumD- and xagB-dependent EPS contribute to biofilm formation under different conditions.     

Crystal structure of malonyl CoA-Acyl carrier protein transacylase from Xanthomanous oryzae pv. oryzae and its proposed binding with ACP

Xanthomonas oryzae pv. oryzae (Xoo) is a plant bacterial pathogen that causes bacterial blight (BB) disease, resulting in serious production losses of rice. The crystal structure of malonyl CoA-acyl carrier protein transacylase (XoMCAT), encoded by the gene fabD (Xoo0880) from Xoo, was determined at 2.3 Å resolution in complex with N-cyclohexyl-2-aminoethansulfonic acid. Malonyl CoA-acyl carrier protein transacylase transfers malonyl group from malonyl CoA to acyl carrier protein (ACP). The transacylation step is essential in fatty acid synthesis. Based on the rationale, XoMCAT has been considered as a target for antibacterial agents against BB. Protein-protein interaction between XoMCAT and ACP was also extensively investigated using computational docking, and the proposed model revealed that ACP bound to the cleft between two XoMCAT subdomains.