EcpA, an extracellular protease, is a specific virulence factor required by Xanthomonas oryzae pv. oryzicola but not by X. oryzae pv. oryzae in rice.
Zou Hua-Song,Song Xue,Zou Li-Fang,Yuan Liang,Li Yu-Rong,Guo Wei,Che Yi-Zhou,Zhao Wen-Xiang,Duan Yong-Ping,Chen Gong-You
Microbiology (Reading, England)
Previously, 12 protease-deficient mutants of the Xanthomonas oryzae pv. oryzicola (Xoc) RS105 strain were recovered from a Tn5-tagged mutant library. In the current study, the Tn5 insertion site in each mutant was mapped. Mutations in genes encoding components of the type II secretion apparatus, cAMP regulatory protein, integral membrane protease subunit, S-adenosylmethionine decarboxylase proenzyme and extracellular protease (ecpA(Xoc)) either partially or completely abolished extracellular protease activity (ECPA) and reduced virulence in rice. Transcription of ecpA(Xoc) was induced in planta in all the mutants except RΔecpA. Complementation of RΔecpA with ecpA(Xoc) in trans restored ECPA, virulence and bacterial growth in planta. Purified EcpA(Xoc) induced chlorosis- and necrosis-like symptoms similar to those induced by the pathogen when injected into rice leaves. Heterologous expression of ecpA(Xoc) conferred ECPA upon the vascular bacterium X. oryzae pv. oryzae (Xoo) and upon non-pathogenic Escherichia coli. Genetic analysis demonstrated that the C-terminal residues of EcpA in Xoo PXO99(A) and Xoc RS105 are different, and a frame shift in ecpA(Xoo) may explain the absence of EcpA activity in Xoo. Collectively, these results suggest that EcpA(Xoc) is a tissue-specific virulence factor for Xoc but not Xoo, although the two pathovars are closely related bacterial pathogens of rice.
Virulence analysis and gene expression profiling of the pigment-deficient mutant of Xanthomonas oryzae pathovar oryzae.
Park Young-Jin,Song Eun-Sung,Noh Tae-Hwan,Kim Hyungtae,Yang Kap-Seok,Hahn Jang-Ho,Kang Hee-Wan,Lee Byoung-Moo
FEMS microbiology letters
Xanthomonas oryzae pathovar oryzae (Xoo) causes bacterial blight disease in rice (Oryza sativa L.). For a study of function, we constructed a random insertion mutant library of Xoo using a Tn5 transposon and isolated the mutant strain (M11; aroK::Tn5) that had extremely low pigment production. In addition, M11 had decreased virulence against the susceptible rice cultivar IR24. Thermal asymmetric interlaced-PCR and sequence analysis of M11 revealed that the transposon was inserted into the aroK gene (which encodes a shikimate kinase). To investigate the expression patterns of the pigment- and virulence-deficient mutant, DNA microarray analysis was performed. In addition, reverse transcriptase-PCR was performed to confirm the expression levels of several genes, including the aro genes of the aroK mutant. Our findings reveal that several crucial genes for virulence, including cellulase and hypersensitive response and pathogenicity (hrp) genes, were regulated by mutations in the aroK gene.
The genome sequence of Xanthomonas oryzae pathovar oryzae KACC10331, the bacterial blight pathogen of rice.
Lee Byoung-Moo,Park Young-Jin,Park Dong-Suk,Kang Hee-Wan,Kim Jeong-Gu,Song Eun-Sung,Park In-Cheol,Yoon Ung-Han,Hahn Jang-Ho,Koo Bon-Sung,Lee Gil-Bok,Kim Hyungtae,Park Hyun-Seok,Yoon Kyong-Oh,Kim Jeong-Hyun,Jung Chol-hee,Koh Nae-Hyung,Seo Jeong-Sun,Go Seung-Joo
Nucleic acids research
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.
Genetic and pathogenic variability of Indian strains of Xanthomonas campestris pv. campestris causing black rot disease in crucifers.
Singh Dinesh,Dhar Shri,Yadava D K
Xanthomonas campestris pv. campestris (Pammel) Dowson (Xcc) causing black rot of crucifers is a serious disease in India and causes >50% crop losses in favorable environmental conditions. Pathogenic variability of Xcc, X. oryzae pv. oryzae (Xoo), and X. axonopodis pv. citri (Xac) were tested on 19 cultivars of cruciferae including seven Brassica spp. viz., B. campestris, B. carinata, B. juncea, B. napus, B. nigra, B. oleracea and B. rapa, and Raphanus sativus for two consecutive years viz., 2007-2008 and 2008-2009 under field conditions at Indian Agricultural Research Institute, New Delhi. Xcc (22 strains) and other species of Xanthomonas (2 strains), they formed three distinct groups of pathogenic variability i.e., Group 1, 2, and 3 under 50% minimum similarity coefficient. All strains of Xcc clustered under Groupl except Xcc-C20. The strains of Xcc further clustered in 6 subgroups viz., A, B, C, D, E, and F based on diseases reaction on host. Genetic variability of 22 strains of Xcc was studied by using Rep-PCR (REP-, BOX- and ERIC-PCR) and 10 strains for hrp (hypersensitive reaction and pathogenecity) gene sequence analysis. Xcc strains comprised in cluster 1, Xac under cluster 2, while Xoo formed separate cluster 3 based on >50% similarity coefficient. Cluster 1 was further divided into 8 subgroups viz., A, B, C, D, E, F, G, and H at 75% similarity coefficient. The hrpF gene sequence analysis also showed distinctness of Xcc strains from other Xanthomonads. In this study, genetic and pathogenic variability in Indian strains of Xcc were established, which will be of immense use in the development of resistant genotypes against this bacterial pathogen.
Mutations in the N-terminal coding region of the harpin protein Hpa1 from Xanthomonas oryzae cause loss of hypersensitive reaction induction in tobacco.
Wang Xiao-yu,Song Cong-feng,Miao Wei-guo,Ji Zhao-lin,Wang Xiben,Zhang Yan,Zhang Jia-huan,Hu John S,Borth Wayne,Wang Jin-sheng
Applied microbiology and biotechnology
Harpins encoded by many gram-negative phytopathogenic bacterial hrp genes induce hypersensitive response (HR) and associated defense responses on nonhost plants. Hpa1(Xoo) and Hpa1(Xoc), two harpin proteins from Xanthomonas oryzae pathovars, induce HR when infiltrated into tobacco leaves. N- and C-terminal mutations of Hpa1(Xoo) and Hpa1(Xoc), respectively, were tested for their ability to elicit HR on tobacco. Deletion of codons for 12 highly hydrophilic amino acids (H(2)N-QGISEKQLDQLL-COOH) that partially overlap the N-terminal alpha-helical regions of respective proteins was found to be critical for the elicitation of HR in tobacco. Furthermore, two single missense mutants Hpa1(Xoo) (L51P) and Hpa1(Xoc) (L53P) that are predicted to destroy the coiled-coil integrity and inhibit the dimer formation eliminated HR elicitation activity in tobacco. However, both wild-type proteins and derivative mutants retained the ability to induce systemic acquired resistance in tobacco against tobacco mosaic virus. Accumulations of npr1 (nonexpressor of pathogenesis-related protein 1), hsr515 (hypersensitivity-related protein 515), and pr2 (pathogenesis-related protein 2) transcripts were found in tobacco plants infiltrated with wild-type or mutated proteins.
Xanthomonas oryzae pv. oryzae requires H-NS-family protein XrvC to regulate virulence during rice infection.
Liu Yongting,Long Juying,Shen Dan,Song Congfeng
FEMS microbiology letters
Histone-like nucleoid-structuring (H-NS) proteins, which are conserved in Gram-negative bacteria, bind DNA and act as the global transcriptional repressors. In this study, we identified and characterized the xrvC gene encoding a H-NS protein in Xathomonas oryzae pv. oryzae (Xoo) Philippines strain PXO99(A) Compared with the wild type, the xrvC-deficient mutant of PXO99(A) (named PXO99ΔxrvC) showed a reduced growth rate in both nutrient-rich and nutrient-limited media. Interestingly, PXO99ΔxrvC exhibited significantly reduced virulence on rice cultivar IRBB214, but its virulence on 31 other rice cultivars was not affected. Transcriptional analysis revealed that the expression of hrpG, hrpX and hpa1 and of 15 out of 18 tested non-TAL (transcription activator-like) effector genes was decreased significantly in the xrvC mutant compared with that in the wild type. In addition, loss of xrvC also impaired the induction of the rice susceptibility gene Os8N3 in IRBB214 by PXO99(A) Our results suggest that the xrvC gene is involved in bacterial growth, and it plays a vital role in virulence by positively regulating the expression of hrp genes and non-TAL effector genes in PXO99(A) and the susceptibility gene Os8N3 in rice.
Two overlapping two-component systems in Xanthomonas oryzae pv. oryzae contribute to full fitness in rice by regulating virulence factors expression.
Zheng Dehong,Yao Xiaoyan,Duan Meng,Luo Yufeng,Liu Biao,Qi Pengyuan,Sun Ming,Ruan Lifang
Two-component signal transduction systems (TCSs) are widely used by bacteria to adapt to the environment. In the present study, StoS (stress tolerance-related oxygen sensor) and SreKRS (salt response kinase, regulator, and sensor) were found to positively regulate extracellular polysaccharide (EPS) production and swarming in the rice pathogen Xanthomonas oryzae pv. oryzae (Xoo). Surprisingly, the absence of stoS or sreKRS did not attenuate virulence. To better understand the intrinsic functions of StoS and SreKRS, quantitative proteomics isobaric tags for relative and absolute quantitation (iTRAQ) was employed. Consistent with stoS and sreK mutants exhibiting a similar phenotype, the signalling circuits of StoS and SreKRS overlapped. Carbohydrate metabolism proteins and chemotaxis proteins, which could be responsible for EPS and swarming regulation, respectively, were reprogrammed in stoS and sreK mutants. Moreover, StoS and SreKRS demonstrated moderate expression of the major virulence factor, hypersensitive response and pathogenicity (Hrp) proteins through the HrpG-HrpX circuit. Most importantly, Xoo equipped with StoS and SreKRS outcompetes strains without StoS or SreKRS in co-infected rice and grows outside the host. Therefore, we propose that StoS and SreKRS adopt a novel strategy involving the moderation of Hrp protein expression and the promotion of EPS and motility to adapt to the environment.
Comparative transcriptome profiling reveals different expression patterns in Xanthomonas oryzae pv. oryzae strains with putative virulence-relevant genes.
Zhang Fan,Du Zhenglin,Huang Liyu,Vera Cruz Casiana,Zhou Yongli,Li Zhikang
Xanthomonas oryzae pv. oryzae (Xoo) is the causal agent of rice bacterial blight, which is a major rice disease in tropical Asian countries. An attempt has been made to investigate gene expression patterns of three Xoo strains on the minimal medium XOM2, PXO99 (P6) and PXO86 (P2) from the Philippines, and GD1358 (C5) from China, which exhibited different virulence in 30 rice varieties, with putative virulence factors using deep sequencing. In total, 4,781 transcripts were identified in this study, and 1,151 and 3,076 genes were differentially expressed when P6 was compared with P2 and with C5, respectively. Our results indicated that Xoo strains from different regions exhibited distinctly different expression patterns of putative virulence-relevant genes. Interestingly, 40 and 44 genes involved in chemotaxis and motility exhibited higher transcript alterations in C5 compared with P6 and P2, respectively. Most other genes associated with virulence, including exopolysaccharide (EPS) synthesis, Hrp genes and type III effectors, including Xanthomonas outer protein (Xop) effectors and transcription activator-like (TAL) effectors, were down-regulated in C5 compared with P6 and P2. The data were confirmed by real-time quantitative RT-PCR, tests of bacterial motility, and enzyme activity analysis of EPS and xylanase. These results highlight the complexity of Xoo and offer new avenues for improving our understanding of Xoo-rice interactions and the evolution of Xoo virulence.
Identification of phenolic compounds that suppress the virulence of Xanthomonas oryzae on rice via the type III secretion system.
Fan Susu,Tian Fang,Li Jianyu,Hutchins William,Chen Huamin,Yang Fenghuan,Yuan Xiaochen,Cui Zining,Yang Ching-Hong,He Chenyang
Molecular plant pathology
The targeting of bacterial type III secretion systems (T3SSs), which are critical virulence factors in most Gram-negative pathogens, is regarded as an alternative strategy for the development of novel anti-microbial drugs. Xanthomonas oryzae pv. oryzae (Xoo) and X. oryzae pv. oryzicola (Xoc) are two of the most important bacterial pathogens on rice, which cause leaf blight and leaf streak diseases, respectively. To identify potential anti-virulence drugs against these two pathogens, we screened a library of plant phenolic compounds and derivatives for their effects on the Xoo T3SS. Ten of 56 compounds significantly inhibited the promoter activity of a harpin gene, hpa1. These inhibitors were further tested for their impact on the hypersensitive response (HR) caused by Xoo on non-host tobacco plants. The results showed that pretreatment of Xoo with TS006 (o-coumaric acid, OCA), TS010, TS015 and TS018 resulted in significantly attenuated HR without affecting bacterial growth or survival. In addition, Cya translocation assays demonstrated that the translocation of two T3 effectors was suppressed by the four inhibitors. Quantitative reverse transcription-polymerase chain reaction (qRT-PCR) analysis showed that mRNA levels of representative genes in the hrp (hypersensitive response and pathogenicity) cluster, as well as the regulatory genes hrpG and hrpX, were reduced by treatment with the four inhibitors, suggesting that expression of the Xoo T3SS was suppressed. The expression of other virulence factors was not suppressed, which indicated possible T3SS-specific inhibition. Finally, we demonstrated that these inhibitors reduced the disease symptoms of Xoo and Xoc on the rice cultivar (Oryza sativa) IR24 to varying extents.
Synthesis and bioactivity of 1,3-thiazolidine-2-thione derivatives against type III secretion system of Xanthomonas oryzae.
Tao Hui,Tian Hao,Jiang Shan,Xiang Xuwen,Ahmed Wasim,Tang Riyuan,Cui Zi-Ning
Bioorganic & medicinal chemistry
Targeting virulence factors of bacterial without affecting their growth and survival, has been an initiative strategy for the development of novel anti-microbial agents. The type III secretion system (T3SS), one of essential and highly conserved virulence factors in most Gram-negative pathogenic bacteria, has been regarded as an effective target that developed new anti-microbial drugs. Xanthomonas oryzae pv. oryzae (Xoo) is one of the most important bacterial pathogens on rice, which causes leaf blight disease. To discover potential anti-virulence agents against the pathogens, a new series of 1,3-thiazolidine-2-thione derivatives containing 5-phenyl-2-furan were designed and synthesized. Their structures were characterized by H NMR, C NMR, MS, and elemental analysis. All the title compounds inhibited the promoter activity of a harpin gene hpa1, significantly, that were further checked for the impact on bacterial growth. The results indicated that treatment of Xoo with the title compound III-7 did not affect bacterial growth or survival. Moreover, quantitative reverse transcription-polymerase chain reaction (qRT-PCR) analysis showed that the expression of the Xoo T3SS was suppressed by treatment with the inhibitor. The mRNA levels of representative genes in the hrp (hypersensitive response and pathogenicity) cluster, as well as the regulatory genes hrpG and hrpX, were reduced. Finally, the in vivo test demonstrated that the compounds could reduce the disease symptoms of Xoo on the rice cultivar (Oryza sativa) IR24.
Transcriptome-Based Identification of Differently Expressed Genes from Xanthomonas oryzae pv. oryzae Strains Exhibiting Different Virulence in Rice Varieties.
Noh Tae-Hwan,Song Eun-Sung,Kim Hong-Il,Kang Mi-Hyung,Park Young-Jin
International journal of molecular sciences
Xanthomonas oryzae pv. oryzae (Xoo) causes bacterial blight (BB) in rice (Oryza sativa L.). In this study, we investigated the genome-wide transcription patterns of two Xoo strains (KACC10331 and HB1009), which showed different virulence patterns against eight rice cultivars, including IRBB21 (carrying Xa21). In total, 743 genes showed a significant change (p-value < 0.001 in t-tests) in their mRNA expression levels in the HB1009 (K3a race) strain compared with the Xoo KACC10331 strain (K1 race). Among them, four remarkably enriched GO terms, DNA binding, transposition, cellular nitrogen compound metabolic process, and cellular macromolecule metabolic process, were identified in the upregulated genes. In addition, the expression of 44 genes was considerably higher (log2 fold changes > 2) in the HB1009 (K3a race) strain than in the Xoo KACC10331 (K1 race) strain. Furthermore, 13 and 12 genes involved in hypersensitive response and pathogenicity (hrp) and two-component regulatory systems (TCSs), respectively, were upregulated in the HB1009 (K3a race) strain compared with the Xoo KACC10331 (K1 race) strain, which we determined using either quantitative real-time PCR analysis or next-generation RNA sequencing. These results will be helpful to improve our understanding of Xoo and to gain a better insight into the Xoo-rice interactions.
[Identification of a hrp-associated gene hpaB in the role of pathogenicity of Xanthomonas oryzae pv. oryzae].
Zeng Shen-yan,Hu Jun,Huang Gui-xiu,He Chao-zu
Wei sheng wu xue bao = Acta microbiologica Sinica
Xanthomonas oryzae pv. oryzae (Xoo), a Gram-negative bacterium, is the causal agent of rice bacterial blight disease, which can cause severe yield loss of rice worldwide. To identify genes contributing to virulence and explore the possible mechanism of pathogenicity, transposon mutagenesis was used to isolate nonpathogenic mutants. By screening of a high-quality Tn5-like transposon (EZ: :TN) insertional mutant library of Xoo PXO99 against a host plant (rice cultivar IR24), one virulence-deficient mutant, XOG11, was identified. Genomic fragment flanking the insertion site of the mutant was amplified by thermal asymmetric interlaced polymerase chian reaction ( TAIL-PCR) and sequenced. The result of NCBI blast homologue searching of the fragment shows that the transposon was inserted into a hrp associated gene, hpaB. Xoo hpaB gene is one of the hrp gene cluster members that encode a type [I secretion system (TTSS) and locates at the downstream of hrpE. The product of hpaB in Xoo is a small (Molecular Weight, 17.6kDa), acidic (PI, 4.28) and Leucine-rich (14.4%) protein and shares high homology with corresponding proteins in other Xanthomonas. It suggests that HpaB may play as a TTSS chaperone. Mutant XOGl1 was confirmed both by PCR and Southern blotting: The PCR result by using primers upstream and downstream of hpaB respectively verified Tn5 insertion in hpaB and excluded the rare case of second transfer of the transposon associated with flanking sequence; Southern blot of digested genomic DNA with the probe of Km resistance gene aph proved that XOG11 was inserted by a single-copy transposon, indicating that the loss of pathogenicity in XOG11 was due to the Tn5 insertion in hpaB gene. Genetic complementation by cloning hpaB in the wide host range plasmid pHMI and transferring the recombinant plasmid into XOG11 restored its pathogenicity in IR24. These results suggest that the pathogenicity deficiency of XOG11 is due to the mutation of hpaB gene.
Discovery of Ethyl 2-Nitro-3-Arylacrylates Molecules as T3SS Inhibitor Reducing the Virulence of Plant Pathogenic Bacteria .
Jiang Shan,Li Hui,Ahmed Wasim,Xiang Xuwen,Song Gaopeng,Cui Zi-Ning
Frontiers in microbiology
pv. () is a gram-negative pathogen which causes leaf blight disease. Known traditional bactericides are not much more effective in inhibiting this bacteria than before. Selecting the virulence factor of the bacteria as the target without affecting their growth has been considered as a novel method for developing new anti-microbial drugs. Type III secretion systems (T3SS) are one of the important and highly conserved virulence factors in most gram-negative pathogens, which has been considered as an effective target to develop new anti-microbial drugs. In order to discover potential anti-microbial drugs against pathogens, a series of ethyl 2-nitro-3-arylacrylates compounds were screened. Among them, the compounds I-9, I-12, and I-13 could highly inhibit the promoter activity of a harpin gene , which were used to further check for the influence on bacterial growth and on the hypersensitive response (HR) caused by bacteria on non-host plants. The results showed that above compounds could reduce HR without affecting bacterial growth and survival. Moreover, qRT-PCR analysis indicated that treatment with the three inhibitors (I-9, I-12, and I-13) could suppress the expression of the T3SS in different extent. The mRNA levels of representative genes in the cluster, including the key regulatory genes and , were decreased. Last but not least, test ensured that the above compounds reduced the disease symptoms of on the rice and on the Chinese radish.
Small Molecule Inhibitors Specifically Targeting the Type III Secretion System of on Rice.
Tao Hui,Fan Su-Su,Jiang Shan,Xiang Xuwen,Yan Xiaojing,Zhang Lian-Hui,Cui Zi-Ning
International journal of molecular sciences
The initiative strategy for the development of novel anti-microbial agents usually uses the virulence factors of bacteria as a target, without affecting their growth and survival. The type III secretion system (T3SS), one of the essential virulence factors in most Gram-negative pathogenic bacteria because of its highly conserved construct, has been regarded as an effective target that developed new anti-microbial drugs. pv. () causes leaf blight diseases and is one of the most important pathogens on rice. To find potential anti-virulence agents against this pathogen, a number of natural compounds were screened for their effects on the T3SS of . Three of 34 compounds significantly inhibited the promoter activity of the harpin gene, , and were further checked for their impact on bacterial growth and on the hypersensitive response (HR) caused by on non-host tobacco plants. The results indicated that treatment of with CZ-1, CZ-4 and CZ-9 resulted in an obviously attenuated HR without affecting bacterial growth and survival. Moreover, quantitative reverse transcription-polymerase chain reaction (qRT-PCR) analysis showed that the expression of the T3SS was suppressed by treatment with the three inhibitors. The mRNA levels of representative genes in the hypersensitive response and pathogenicity () cluster, as well as the regulatory genes and , were reduced. Finally, the in vivo test demonstrated that the compounds could reduce the disease symptoms of on the rice cultivar () IR24.
An H-NS-like protein involved in the negative regulation of hrp genes in Xanthomonas oryzae pv. oryzae.
Kametani-Ikawa Yumi,Tsuge Seiji,Furutani Ayako,Ochiai Hirokazu
FEMS microbiology letters
hrp genes encode components of a type III secretion (T3S) system and play crucial roles in the pathogenicity of the rice pathogen Xanthomonas oryzae pv. oryzae (Xoo). A histone-like nucleoid-structuring (H-NS) protein binds DNA and acts as a global transcriptional repressor. Here, we investigated the involvement of an h-ns-like gene, named xrvB, in the expression of hrp genes in Xoo. Under the hrp-inducing culture condition, the expression of a key hrp regulator HrpG increased in the XrvB mutant, followed by activation of the downstream gene expression. Also, in planta, the secretion of a T3S protein (XopR) was activated by the mutation in xrvB. Gel retardation assay indicated that XrvB has DNA-binding activity, but without a preference for the promoter region of hrpG. The results suggest that XrvB negatively regulates hrp gene expression and that an unknown factor(s) mediates the regulation of hrpG expression by XrvB.
The rsmA-like gene rsmA(Xoo) of Xanthomonas oryzae pv. oryzae regulates bacterial virulence and production of diffusible signal factor.
Zhu Pei-Liang,Zhao Shuai,Tang Ji-Liang,Feng Jia-Xun
Molecular plant pathology
The plant-pathogenic prokaryote Xanthomonas oryzae pv. oryzae (Xoo) causes bacterial leaf blight, one of the most destructive diseases of rice. A nonpolar mutant of the rsmA-like gene rsmA(Xoo) of the Xoo Chinese strain 13751 was constructed by homologous integration with a suicide plasmid. Virulence tests on a host plant, namely the hybrid rice cultivar Teyou 63, showed that the mutant had lost its virulence almost completely, whereas tests on a nonhost, namely castor-oil plant (Ricinus communis), showed that the mutant had also lost the ability to induce a hypersensitive response in the nonhost. In addition, the rsmA(Xoo) mutant produced significantly smaller amounts of the diffusible signal factor, extracellular endoglucanase, amylase and extracellular polysaccharide, but showed significantly higher glycogen accumulation, bacterial aggregation and cell adhesion. The expression of most hrp genes, genes encoding AvrBs3/PthA family members, rpfB, xrvA, glgA, eglXoB and XOO0175 (encoding an α-amylase) was down-regulated in the rsmA(Xoo) mutant. All phenotypes and expression levels of the tested genes in the rsmA(Xoo) mutant were restored to their levels in the wild-type by the presence of rsmA(Xoo) in trans. These results indicate that rsmA(Xoo) is essential for the virulence of Xoo.
GamR, the LysR-Type Galactose Metabolism Regulator, Regulates hrp Gene Expression via Transcriptional Activation of Two Key hrp Regulators, HrpG and HrpX, in Xanthomonas oryzae pv. oryzae.
Rashid M Mamunur,Ikawa Yumi,Tsuge Seiji
Applied and environmental microbiology
UNLABELLED:Xanthomonas oryzae pv. oryzae is the causal agent of bacterial leaf blight of rice. For the virulence of the bacterium, the hrp genes, encoding components of the type III secretion system, are indispensable. The expression of hrp genes is regulated by two key hrp regulators, HrpG and HrpX: HrpG regulates hrpX, and HrpX regulates other hrp genes. Several other regulators have been shown to be involved in the regulation of hrp genes. Here, we found that a LysR-type transcriptional regulator that we named GamR, encoded by XOO_2767 of X. oryzae pv. oryzae strain MAFF311018, positively regulated the transcription of both hrpG and hrpX, which are adjacent to each other but have opposite orientations, with an intergenic upstream region in common. In a gel electrophoresis mobility shift assay, GamR bound directly to the middle of the upstream region common to hrpG and hrpX The loss of either GamR or its binding sites decreased hrpG and hrpX expression. Also, GamR bound to the upstream region of either a galactose metabolism-related gene (XOO_2768) or a galactose metabolism-related operon (XOO_2768 to XOO_2771) located next to gamR itself and positively regulated the genes. The deletion of the regulator gene resulted in less bacterial growth in a synthetic medium with galactose as a sole sugar source. Interestingly, induction of the galactose metabolism-related gene was dependent on galactose, while that of the hrp regulator genes was galactose independent. Our results indicate that the LysR-type transcriptional regulator that regulates the galactose metabolism-related gene(s) also acts in positive regulation of two key hrp regulators and the following hrp genes in X. oryzae pv. oryzae. IMPORTANCE:The expression of hrp genes encoding components of the type III secretion system is essential for the virulence of many plant-pathogenic bacteria, including Xanthomonas oryzae pv. oryzae. It is specifically induced during infection. Research has revealed that in this bacterium, hrp gene expression is controlled by two key hrp regulators, HrpG and HrpX, along with several other regulators in the complex regulatory network, but the details remain unclear. Here, we found that a novel LysR-type transcriptional activator, named GamR, functions as an hrp regulator by directly activating the transcription of both hrpG and hrpX Interestingly, GamR also regulates a galactose metabolism-related gene (or operon) in a galactose-dependent manner, while the regulation of hrpG and hrpX is independent of the sugar. Our finding of a novel hrp regulator that directly and simultaneously regulates two key hrp regulators provides new insights into an important and complex regulation system of X. oryzae pv. oryzae hrp genes.
Mechanism of HRP-catalyzed nitrite oxidation by HO revisited: Effect of nitroxides on enzyme inactivation and its catalytic activity.
Samuni Amram,Maimon Eric,Goldstein Sara
Free radical biology & medicine
The peroxidative activity of horseradish peroxidase (HRP) undergoes progressive inactivation while catalyzing the oxidation of nitrite by HO. The extent of inactivation increases as the pH increases, [nitrite] decreases or [HO] increases, and is accompanied by a loss of the Soret peak of HRP along with yellow-greenish coloration of the solution. HRP-catalyzed nitrite oxidation by HO involves not only the formation of compounds I and II as transient heme species, but also compound III, all of which in turn, oxidize nitrite yielding NO. The rate constant of nitrite oxidation by compound III is at least 10-fold higher than that by compound II, which is also reducible by NO where its reduction by nitrite is the rate-determining step of the catalytic cycle. The extent of the loss of the Soret peak of HRP is lower than the loss of its peroxidative activity implying that deterioration of the heme moiety leading to iron release only partially contributes toward heme inactivation. Cyclic stable nitroxide radicals, such as 2,2,6,6-tetramethyl-piperidine-N-oxyl (TPO), 4-OH-TPO and 4-NH-TPO at µM concentrations detoxify NO thus protecting HRP against inactivation mediated by this radical. Hence, HRP inactivation proceeds via nitration of the porphyrin ring most probably through compound I reaction with NO, which partially leads to deterioration of the heme moiety. The nitroxide acts catalytically since its oxidation by NO yields the respective oxoammonium cation, which is readily reduced back to the nitroxide by HO, superoxide ion radical, and nitrite. In addition, the nitroxide catalytically inhibits tyrosine nitration mediated by HRP/HO/nitrite reactions system as it efficiently competes with tyrosyl radical for NO. The inhibition by nitroxides of tyrosine nitration is demonstrated also in the case of microperoxidase (MP-11) and cytochrome c revealing an additional role played by nitroxide antioxidants.
A two-genome microarray for the rice pathogens Xanthomonas oryzae pv. oryzae and X. oryzae pv. oryzicola and its use in the discovery of a difference in their regulation of hrp genes.
Seo Young-Su,Sriariyanun Malinee,Wang Li,Pfeiff Janice,Phetsom Jirapa,Lin Ye,Jung Ki-Hong,Chou Hui Hsien,Bogdanove Adam,Ronald Pamela
BACKGROUND:Xanthomonas oryzae pv. oryzae (Xoo) and X. oryzae pv. oryzicola (Xoc) are bacterial pathogens of the worldwide staple and grass model, rice. Xoo and Xoc are closely related but Xoo invades rice vascular tissue to cause bacterial leaf blight, a serious disease of rice in many parts of the world, and Xoc colonizes the mesophyll parenchyma to cause bacterial leaf streak, a disease of emerging importance. Both pathogens depend on hrp genes for type III secretion to infect their host. We constructed a 50-70 mer oligonucleotide microarray based on available genome data for Xoo and Xoc and compared gene expression in Xoo strains PXO99A and Xoc strain BLS256 grown in the rich medium PSB vs. XOM2, a minimal medium previously reported to induce hrp genes in Xoo strain T7174. RESULTS:Three biological replicates of the microarray experiment to compare global gene expression in representative strains of Xoo and Xoc grown in PSB vs. XOM2 were carried out. The non-specific error rate and the correlation coefficients across biological replicates and among duplicate spots revealed that the microarray data were robust. 247 genes of Xoo and 39 genes of Xoc were differentially expressed in the two media with a false discovery rate of 5% and with a minimum fold-change of 1.75. Semi-quantitative-RT-PCR assays confirmed differential expression of each of 16 genes each for Xoo and Xoc selected for validation. The differentially expressed genes represent 17 functional categories. CONCLUSION:We describe here the construction and validation of a two-genome microarray for the two pathovars of X. oryzae. Microarray analysis revealed that using representative strains, a greater number of Xoo genes than Xoc genes are differentially expressed in XOM2 relative to PSB, and that these include hrp genes and other genes important in interactions with rice. An exception was the rax genes, which are required for production of the host resistance elicitor AvrXa21, and which were expressed constitutively in both pathovars.
Identification of seven Xanthomonas oryzae pv. oryzicola genes potentially involved in pathogenesis in rice.
Guo Wei,Cui Yi-Ping,Li Yu-Rong,Che Yi-Zhou,Yuan Liang,Zou Li-Fang,Zou Hua-Song,Chen Gong-You
Microbiology (Reading, England)
Xanthomonas oryzae pv. oryzicola (Xoc) causes bacterial leaf streak (BLS) in rice, an emerging and destructive disease worldwide. Identification of key virulence factors is a prerequisite for understanding the pathogenesis of Xoc. In this study, a Tn5-tagged mutant library of Xoc strain RS105 was screened on rice, and 27 Tn5 mutants were identified that were either non-pathogenic or showed reduced virulence in rice. Fourteen of the non-pathogenic mutants were also unable to elicit the hypersensitive response (HR) in tobacco and were designated Pth(-)/HR(-) mutants; 13 mutants showed attenuated virulence and were able to induce an HR (Vir(-)/HR(+)). Sequence analysis of the Tn5-tagged genes indicated that the 14 Pth(-)/HR(-) mutants included mutations in hrcC, hrcT, hrcV, hpaP, hrcQ, hrpF, hrpG and hrpX. The 13 Vir(-)/HR(+) mutants included tal-C10c-like (a transcriptional activator-like TAL effector), rpfC (regulator of pathogenicity factors), oxyR (oxidative stress transcriptional regulator), dsbC (disulfide isomerase), opgH (glucan biosynthesis glucosyltransferase H), rfbA (glucose-1-phosphate thymidylyltransferase), amtR (aminotransferase), purF (amidophosphoribosyltransferase), thrC (threonine synthase), trpA (tryptophan synthase alpha subunit) and three genes encoding hypothetical proteins (Xoryp_02235, Xoryp_00885 and Xoryp_22910). Collectively, the 27 Tn5 insertions are located in 21 different open reading frames. Bacterial growth and in planta virulence assays demonstrated that opgH, purF, thrC, trpA, Xoryp_02235, Xoryp_00885 and Xoryp_22910 are candidate virulence genes involved in Xoc pathogenesis. Reduced virulence in 13 mutants was restored to wild-type levels when the cognate gene was introduced in trans. Expression profiles demonstrated that the seven candidate virulence genes were significantly induced in planta, although their roles in Xoc pathogenesis remain unclear.
Allelic variation for broad-spectrum resistance and susceptibility to bacterial pathogens identified in a rice MAGIC population.
Bossa-Castro Ana M,Tekete Cheick,Raghavan Chitra,Delorean Emily E,Dereeper Alexis,Dagno Karim,Koita Ousmane,Mosquera Gloria,Leung Hei,Verdier Valérie,Leach Jan E
Plant biotechnology journal
Quantitative trait loci (QTL) that confer broad-spectrum resistance (BSR), or resistance that is effective against multiple and diverse plant pathogens, have been elusive targets of crop breeding programmes. Multiparent advanced generation intercross (MAGIC) populations, with their diverse genetic composition and high levels of recombination, are potential resources for the identification of QTL for BSR. In this study, a rice MAGIC population was used to map QTL conferring BSR to two major rice diseases, bacterial leaf streak (BLS) and bacterial blight (BB), caused by Xanthomonas oryzae pathovars (pv.) oryzicola (Xoc) and oryzae (Xoo), respectively. Controlling these diseases is particularly important in sub-Saharan Africa, where no sources of BSR are currently available in deployed varieties. The MAGIC founders and lines were genotyped by sequencing and phenotyped in the greenhouse and field by inoculation with multiple strains of Xoc and Xoo. A combination of genomewide association studies (GWAS) and interval mapping analyses revealed 11 BSR QTL, effective against both diseases, and three pathovar-specific QTL. The most promising BSR QTL (qXO-2-1, qXO-4-1 and qXO-11-2) conferred resistance to more than nine Xoc and Xoo strains. GWAS detected 369 significant SNP markers with distinguishable phenotypic effects, allowing the identification of alleles conferring disease resistance and susceptibility. The BSR and susceptibility QTL will improve our understanding of the mechanisms of both resistance and susceptibility in the long term and will be immediately useful resources for rice breeding programmes.
Genetic diversity of transcriptional activator-like effector genes in Chinese isolates of Xanthomonas oryzae pv. oryzicola.
Ji Zhi-Yuan,Zakria Muhammad,Zou Li-Fang,Xiong Li,Li Zheng,Ji Guang-Hai,Chen Gong-You
Xanthomonas oryzae pv. oryzicola causes bacterial leaf streak (BLS), a devastating disease of rice in Asia countries. X. oryzae pv. oryzicola utilizes repertoires of transcriptional activator-like effectors (TALEs) to manipulate host resistance or susceptibility; thus, TALEs can determine the outcome of BLS. In this report, we studied genetic diversity in putative tale genes of 65 X. oryzae pv. oryzicola strains that originated from nine provinces of southern China. Genomic DNAs from the 65 strains were digested with BamHI and hybridized with an internal fragment of avrXa3, a tale gene originating from the related pathogen, X. oryzae pv. oryzae, which causes bacterial leaf blight (BLB). Southern blot analysis indicated that the strains contained a variable number (9 to 22) of avrXa3-hybridizing fragments (e.g., putative tale genes). Based on the number and size of hybridizing bands, strains were classified into 14 genotypes (designated 1 to 14), and genotypes 3 and 10 represented 29.23 and 24.64% of the total, respectively. A high molecular weight BamHI fragment (HMWB; ≈6.0 kb) was present in 12 of the 14 genotypes, and sequence analysis of the HMWB revealed the presence of a C-terminally truncated tale, an insertion element related to IS1403, and genes encoding phosphoglycerate mutase and endonuclease V. Primers were developed from the 6.0-kb HMWB fragment and showed potential in genotyping X. oryzae pv. oryzicola strains by polymerase chain reaction. Virulence of X. oryzae pv. oryzicola strains was assessed on 23 rice cultivars containing different resistance genes for BLB. The X. oryzae pv. oryzicola strains could be grouped into 14 pathotypes (I to XIV), and the grouping of strains was almost identical to the categories determined by genotypic analysis. In general, strains containing higher numbers of putative tale genes were more virulent on rice than strains containing fewer tales. The results also indicate that there are no gene-for-gene relationships between the tested rice lines and X. oryzae pv. oryzicola strains. To our knowledge, this is the first description of genetic diversity of X. oryzae pv. oryzicola strains based on tale gene analysis.
Productivity and biochemical properties of green tea in response to full-length and functional fragments of HpaG Xooc, a harpin protein from the bacterial rice leaf streak pathogen Xanthomonas oryzae pv. oryzicola.
Wu Xiaojing,Wu Tingquan,Long Juying,Yin Qian,Zhang Yong,Chen Lei,Liu Ruoxue,Gao Tongchun,Dong Hansong
Journal of biosciences
Harpin proteins from plant pathogenic bacteria can stimulate hypersensitive cell death (HCD), drought tolerance, defence responses against pathogens and insects in plants, as well as enhance plant growth. Recently, we identified nine functional fragments of HpaG;Xooc, a harpin protein from Xanthomonas oryzae pv.oryzicola, the pathogen that causes bacterial leaf streak in rice. Fragments HpaG;1-94'HpaG;10-42, and HpaG;62-138, which contain the HpaG;Xooc regions of the amino acid sequence as indicated by the number spans, exceed the parent protein in promoting growth, pathogen defence and HCD in plants. Here we report improved productivity and biochemical properties of green tea (Camellia sinensis) in response to the fragments tested in comparison with HpaG;Xooc and an inactive protein control. Field tests suggested that the four proteins markedly increased the growth and yield of green tea, and increased the leaf content of tea catechols, a group of compounds that have relevance in the prevention and treatment of human diseases. In particular, HpaG;1-94 was more active than HpaG;Xooc in expediting the growth of juvenile buds and leaves used as green tea material and increased the catechol content of processed teas. When tea shrubs were treated with HpaH;Xooc and HpaG;1-94 compared with a control, green tea yields were over 55% and 39% greater, and leaf catechols were increased by more than 64% and 72%, respectively. The expression of three homologues of the expansin genes, which regulate plant cell growth, and the CsCHS gene encoding a tea chalcone synthase, which critically regulates the biosynthesis of catechols, were induced in germinal leaves of tea plants following treatment with HpaG;1-94 or HpaG;Xooc. Higher levels of gene expression were induced by the application of HpaG;1-94 than HpaG;Xooc. Our results suggest that the harpin protein, especially the functional fragment HpaG;1-94, can be used to effectively increase the yield and improve the biochemical properties of green tea, a drink with medicinal properties.
Difficidin and bacilysin from Bacillus amyloliquefaciens FZB42 have antibacterial activity against Xanthomonas oryzae rice pathogens.
Wu Liming,Wu Huijun,Chen Lina,Yu Xinfang,Borriss Rainer,Gao Xuewen
Bacterial blight and bacterial leaf streak are serious, economically damaging, diseases of rice caused by the bacteria Xanthomonas oryzae pv. oryzae and X. oryzae pv. oryzicola. Bacillus amyloliquefaciens FZB42 was shown to possess biocontrol activity against these Xanthomonas strains by producing the antibiotic compounds difficidin and bacilysin. Analyses using fluorescence, scanning electron and transmission electron microscopy revealed difficidin and bacilysin caused changes in the cell wall and structure of Xanthomonas. Biological control experiments on rice plants demonstrated the ability of difficidin and bacilysin to suppress disease. Difficidin and bacilysin caused downregulated expression of genes involved in Xanthomonas virulence, cell division, and protein and cell wall synthesis. Taken together, our results highlight the potential of B. amyloliquefaciens FZB42 as a biocontrol agent against bacterial diseases of rice, and the utility of difficidin and bacilysin as antimicrobial compounds.
Dual RNA-seq of Xanthomonas oryzae pv. oryzicola infecting rice reveals novel insights into bacterial-plant interaction.
Liao Zhou-Xiang,Ni Zhe,Wei Xin-Li,Chen Long,Li Jian-Yuan,Yu Yan-Hua,Jiang Wei,Jiang Bo-Le,He Yong-Qiang,Huang Sheng
The Gram-negative bacterium Xanthomonas oryzae pv. oryzicola (Xoc) is the causal agent of rice bacterial leaf streak (BLS), one of the most destructive diseases of rice (Oryza sativa L.) that is the important staple crop. Xoc can invade host leaves via stomata and wounds and its type three secretion system (T3SS) is pivotal to its pathogenic lifestyle. In this study, using a novel dual RNA-seq approach, we examined transcriptomes of rice and Xoc in samples inoculated with wild type Xoc GX01 and its T3SS defective strain (T3SD), to investigate the global transcriptional changes in both organisms. Compared with T3SD strain, rice inoculated with wild type Xoc GX01 resulted in significant expression changes of a series of plant defence related genes, including ones altered in plant signalling pathway, and downregulated in phenylalanine metabolism, flavonoid and momilactone biosynthesis, suggesting repression of plant defence response and reduction in both callose deposition and phytoalexin accumulation. Also, some known transcription activator-like effector (TALE) targets were induced by Xoc GX01, e.g. OsSultr3;6 which contributes to rice susceptibility. Some cell elongation related genes, including several expansin genes, were induced by GX01 too, suggesting that Xoc may exploit this pathway to weaken cell wall strength, beneficial for bacterial infection. On the other hand, compared with wild type, the T3SD strain transcriptome in planta was characterized by downregulation of ATP, protein and polysaccharide synthesis, and upregulation of antioxidation and detoxification related genes, revealing that T3SD strain faced serious starvation and oxidation stresses in planta without a functional T3SS. In addition, comparative global transcript profiles of Xoc in planta and in medium revealed an upregulation of virulence factor synthesis and secretion in planta in favour of bacterial infection. Collectively, this study provides a comprehensive representation of cross talk between the host and bacterial pathogen, revealing insights into the Xoc-rice pathogenic dynamic and reveals novel strategies exploited by this important pathogen to cause disease.
Molecular characterization of bacterial leaf streak resistance in hard winter wheat.
Ramakrishnan Sai Mukund,Sidhu Jagdeep Singh,Ali Shaukat,Kaur Navjot,Wu Jixiang,Sehgal Sunish K
Bacterial leaf streak (BLS) caused by is one of the major bacterial diseases threatening wheat production in the United States Northern Great Plains (NGP) region. It is a sporadic but widespread wheat disease that can cause significant loss in grain yield and quality. Identification and characterization of genomic regions in wheat that confer resistance to BLS will help track resistance genes/QTLs in future wheat breeding. In this study, we evaluated a hard winter wheat association mapping panel (HWWAMP) containing 299 hard winter wheat lines from the US hard winter wheat growing region for their reactions to BLS. We observed a range of BLS responses among the lines, importantly, we identified ten genotypes that showed a resistant reaction both in greenhouse and field evaluation. -Genome-wide association analysis with 15,990 SNPs was conducted using an exponentially compressed mixed linear model. Five genomic regions ( < 0.001) that regulate the resistance to BLS were identified on chromosomes 1AL, 1BS, 3AL, 4AL, and 7AS. The QTLs , , , and explain a total of 42% of the variation. In silico analysis of sequences in the candidate regions on chromosomes 1AL, 1BS, 3AL, 4AL, and 7AS identified 10, 25, 22, eight, and nine genes, respectively with known plant defense-related functions. Comparative analysis with rice showed two syntenic regions in rice that harbor genes for bacterial leaf streak resistance. The ten BLS resistant genotypes and SNP markers linked to the QTLs identified in our study could facilitate breeding for BLS resistance in winter wheat.
The Type III Effector AvrBs2 in Xanthomonas oryzae pv. oryzicola Suppresses Rice Immunity and Promotes Disease Development.
Li Shuai,Wang Yanping,Wang Shanzhi,Fang Anfei,Wang Jiyang,Liu Lijuan,Zhang Kang,Mao Yuling,Sun Wenxian
Molecular plant-microbe interactions : MPMI
Xanthomonas oryzae pv. oryzicola, the causal agent of bacterial leaf streak, is one of the most important bacterial pathogens in rice. However, little is known about the functions of individual type III effectors in virulence and pathogenicity of X. oryzae pv. oryzicola. Here, we examined the effect of the mutations of 23 putative nontranscription activator-like effector genes on X. oryzae pv. oryzicola virulence. The avrBs2 knock-out mutant was significantly attenuated in virulence to rice. In contrast, the xopAA deletion caused enhanced virulence to a certain rice cultivar. It was also demonstrated that six putative effectors, including XopN, XopX, XopA, XopY, XopF1, and AvrBs2, caused the hypersensitive response on nonhost Nicotiana benthamiana leaves. Virulence function of AvrBs2 was further confirmed by transgenic technology. Pathogen-associated molecular pattern-triggered immune responses including the generation of reactive oxygen species and expression of pathogenesis-related genes were strongly suppressed in the AvrBs2-expressing transgenic rice lines. Although not inhibiting flg22-induced activation of mitogen-activated protein kinases, heterologous expression of AvrBs2 greatly promotes disease progression in rice caused by two important bacterial pathogens X. oryzae pvs. oryzae and oryzicola. Collectively, these results indicate that AvrBs2 is an essential virulence factor that contributes to X. oryzae pv. oryzicola virulence through inhibiting defense responses and promoting bacterial multiplication in monocot rice.
Comprehensive analysis of VQ motif-containing gene expression in rice defense responses to three pathogens.
Li Na,Li Xianghua,Xiao Jinghua,Wang Shiping
Plant cell reports
KEY MESSAGE:Expression levels of rice VQ motif-containing genes in response to pathogen infection vary among pathogens, and some of the genes are co-expressed with defense-response WRKY genes. Recent studies have revealed that some VQ (FxxxVQxLTG) motif-containing proteins in plants partner with WRKY transcription factors to participate in their functions. Accumulating information suggests that WRKY proteins play important roles in the response of rice plants to pathogen infection. However, the functions of rice VQ motif-containing proteins are unknown. To explore whether VQ motif-containing proteins are involved in defense against pathogens in rice, we performed a comprehensive expression analysis of the genes for these proteins. The rice VQ motif-containing family consists of 40 genes, all of which encode proteins harboring a 21-amino acid VQ-containing motif, which in turn contains the known VQ motif. On the basis of their phylogenetic relationships and tissue-specific and developmental stage-specific expression characteristics, we transcriptionally analyzed 13 representative genes in rice responses to three pathogens: Xanthomonas oryzae pv. oryzae, which causes bacterial blight disease; X. oryzae pv. oryzicola, which causes bacterial streak disease; and Magnaporthe oryzae, which causes fungal blast disease. The expression of some of the genes changed markedly in response to infection by at least one of the pathogen species, and some of the genes also showed markedly different expression in resistant and susceptible reactions. In addition, some defense-responsive VQ motif-containing genes were co-expressed with defense-response WRKY genes. These results provide a new perspective on the putative roles of rice VQ motif-containing proteins and their putative WRKY partners in rice-pathogen interactions.
Addition of transcription activator-like effector binding sites to a pathogen strain-specific rice bacterial blight resistance gene makes it effective against additional strains and against bacterial leaf streak.
Hummel Aaron W,Doyle Erin L,Bogdanove Adam J
The New phytologist
Xanthomonas transcription activator-like (TAL) effectors promote disease in plants by binding to and activating host susceptibility genes. Plants counter with TAL effector-activated executor resistance genes, which cause host cell death and block disease progression. We asked whether the functional specificity of an executor gene could be broadened by adding different TAL effector binding elements (EBEs) to it. We added six EBEs to the rice Xa27 gene, which confers resistance to strains of the bacterial blight pathogen Xanthomonas oryzae pv. oryzae (Xoo) that deliver the TAL effector AvrXa27. The EBEs correspond to three other effectors from Xoo strain PXO99(A) and three from strain BLS256 of the bacterial leaf streak pathogen Xanthomonas oryzae pv. oryzicola (Xoc). Stable integration into rice produced healthy lines exhibiting gene activation by each TAL effector, and resistance to PXO99(A) , a PXO99(A) derivative lacking AvrXa27, and BLS256, as well as two other Xoo and 10 Xoc strains virulent toward wildtype Xa27 plants. Transcripts initiated primarily at a common site. Sequences in the EBEs were found to occur nonrandomly in rice promoters, suggesting an overlap with endogenous regulatory sequences. Thus, executor gene specificity can be broadened by adding EBEs, but caution is warranted because of the possible coincident introduction of endogenous regulatory elements.
Overexpression of OsHSP18.0-CI Enhances Resistance to Bacterial Leaf Streak in Rice.
Ju Yanhu,Tian Hongjuan,Zhang Ruihua,Zuo Liping,Jin Guixiu,Xu Qian,Ding Xinhua,Li Xiangkui,Chu Zhaohui
Rice (New York, N.Y.)
BACKGROUND:The small heat shock proteins represent a large family of proteins that respond to a wide range of abiotic and biotic stresses. OsHsp18.0-CI confers tolerance to salt and cadmium and interacts with viral RNA-dependent RNA polymerase (RdRp). However, the direct function of OsHsp18.0-CI in resistance against biotic stresses remains unclear in rice. RESULTS:Here, we report that the expression of OsHsp18.0-CI was up-regulated upon inoculation with RS105, a strain of Xanthomonas oryzae pv. oryzicola (Xoc) that causes bacterial leaf streak in rice. In comparison with wild-type, OsHsp18.0-CI overexpression (OE) lines exhibited enhanced resistance to RS105, whereas repression lines exhibited compromised resistance to RS105. In addition, the transcriptional profiles of wild type and OE lines were compared with and without inoculation with RS105. After inoculation with RS105, most of the genes with up-regulated expression were commonly stimulated in the wild type and OE lines, with stronger induction in the OE lines than in wild type. CONCLUSION:Our study reveals that OsHsp18.0-CI positively regulates resistance to Xoc by mediating an enhanced version of the basal defense response in rice.
New multilocus variable-number tandem-repeat analysis tool for surveillance and local epidemiology of bacterial leaf blight and bacterial leaf streak of rice caused by Xanthomonas oryzae.
Poulin L,Grygiel P,Magne M,Gagnevin L,Rodriguez-R L M,Forero Serna N,Zhao S,El Rafii M,Dao S,Tekete C,Wonni I,Koita O,Pruvost O,Verdier V,Vernière C,Koebnik R
Applied and environmental microbiology
Multilocus variable-number tandem-repeat analysis (MLVA) is efficient for routine typing and for investigating the genetic structures of natural microbial populations. Two distinct pathovars of Xanthomonas oryzae can cause significant crop losses in tropical and temperate rice-growing countries. Bacterial leaf streak is caused by X. oryzae pv. oryzicola, and bacterial leaf blight is caused by X. oryzae pv. oryzae. For the latter, two genetic lineages have been described in the literature. We developed a universal MLVA typing tool both for the identification of the three X. oryzae genetic lineages and for epidemiological analyses. Sixteen candidate variable-number tandem-repeat (VNTR) loci were selected according to their presence and polymorphism in 10 draft or complete genome sequences of the three X. oryzae lineages and by VNTR sequencing of a subset of loci of interest in 20 strains per lineage. The MLVA-16 scheme was then applied to 338 strains of X. oryzae representing different pathovars and geographical locations. Linkage disequilibrium between MLVA loci was calculated by index association on different scales, and the 16 loci showed linear Mantel correlation with MLSA data on 56 X. oryzae strains, suggesting that they provide a good phylogenetic signal. Furthermore, analyses of sets of strains for different lineages indicated the possibility of using the scheme for deeper epidemiological investigation on small spatial scales.
AvrXa7-Xa7 mediated defense in rice can be suppressed by transcriptional activator-like effectors TAL6 and TAL11a from Xanthomonas oryzae pv. oryzicola.
Ji Zhi-Yuan,Xiong Li,Zou Li-Fang,Li Yu-Rong,Ma Wen-Xiu,Liu Liang,Zakria Muhammad,Ji Guang-Hai,Chen Gong-You
Molecular plant-microbe interactions : MPMI
The closely related plant pathogens Xanthomonas oryzae pv. oryzicola and X. oryzae pv. oryzae cause bacterial leaf streak (BLS) and bacterial leaf blight (BLB), respectively, in rice. Unlike X. oryzae pv. oryzae, endogenous avirulence-resistance (avr-R) gene interactions have not been identified in the X. oryzae pv. oryzicola-rice pathosystem, though both X. oryzae pv. oryzicola and X. oryzae pv. oryzae possess transcriptional activator-like effectors (TALE), which are known to modulate R or S genes in rice. In this report, avrXa7, avrXa10, and avrXa27 from X. oryzae pv. oryzae were transferred into YNB0-17 and RS105, hypovirulent and hypervirulent strains, respectively, of X. oryzae pv. oryzicola. When YNB0-17 containing avrXa7, avrXa10, or avrXa27 was inoculated to rice, hypersensitive responses (HR) were elicited in rice cultivars containing the R genes Xa7, Xa10, and Xa27, respectively. By contrast, RS105 expressing avrXa27 elicited an HR in a rice cultivar containing Xa27 but the expression of avrXa7 and avrXa10 in RS105 did not result in HR in rice cultivars containing Xa7 and Xa10, correspondingly. Southern blot analysis demonstrated that YNB0-17 possesses only approximately nine putative tale genes, whereas the hypervirulent RS105 contains at least 20. Although YNB0-17 contains an intact type III secretion system (T3SS), its genome is lacking the T3SS effector genes avrRxo1 and xopO, which are present in RS105. The introduction of avrRxo1 and xopO into YNB0-17 did not suppress avrXa7- or avrXa10-triggered immunity in rice. However, the transference of individual tale genes from RS105 into YNB0-17 led to the identification of tal6 and tal11a that suppressed avrXa7-Xa7-mediated defense. Thus, YNB0-17 may be a useful recipient for discovering such suppressors. This is the first report that co-evolutionally generated tale genes in X. oryzae pv. oryzicola suppress gene-for-gene defense against BLB, which may explain the lack of BLS-resistant cultivars.
Antimicrobial activity of UV-induced phenylamides from rice leaves.
Park Hye Lin,Yoo Youngchul,Hahn Tae-Ryong,Bhoo Seong Hee,Lee Sang-Won,Cho Man-Ho
Molecules (Basel, Switzerland)
Rice produces a wide array of phytoalexins in response to pathogen attacks and UV-irradiation. Except for the flavonoid sakuranetin, most phytoalexins identified in rice are diterpenoid compounds. Analysis of phenolic-enriched fractions from UV-treated rice leaves showed that several phenolic compounds in addition to sakuranetin accumulated remarkably in rice leaves. We isolated two compounds from UV-treated rice leaves using silica gel column chromatography and preparative HPLC. The isolated phenolic compounds were identified as phenylamide compounds: N-trans-cinnamoyltryptamine and N-p-coumaroylserotonin. Expression analysis of biosynthetic genes demonstrated that genes for arylamine biosynthesis were upregulated by UV irradiation. This result suggested that phenylamide biosynthetic pathways are activated in rice leaves by UV treatment. To unravel the role of UV-induced phenylamides as phytoalexins, we examined their antimicrobial activity against rice fungal and bacterial pathogens. N-trans-Cinnamoyltryptamine inhibited the growth of rice brown spot fungus (Bipolaris oryzae). In addition to the known antifungal activity to the blast fungus, sakuranetin had antimicrobial activity toward B. oryzae and Rhizoctonia solani (rice sheath blight fungus). UV-induced phenylamides and sakuranetin also had antimicrobial activity against rice bacterial pathogens for grain rot (Burkholderia glumae), blight (Xanthomonas oryzae pv. oryzae) and leaf streak (X. oryzae pv. oryzicola) diseases. These findings suggested that the UV-induced phenylamides in rice are phytoalexins against a diverse array of pathogens.
Genomics-Informed Molecular Detection of pv. Strains Causing Severe Bacterial Leaf Streak of Corn.
Stulberg Michael J,Santillana Gem,Studholme David J,Kasiborski Beth,Ortiz-Castro Mary,Broders Kirk,Arias Silvina,Block Charles,Munkvold Gary,Rascoe John
pv. (syn. pv. ) was initially identified as the causal agent of bacterial leaf streak of corn in South Africa. The pathovar causes disease on sugarcane and corn, but a subset of these strains was noted for its increased disease severity in corn. This subset was reclassified as pv. in the early 1990s and was found to have slightly different biochemical and genetic properties than isolates from sugarcane. There has been an emergence of pv. -like strains of pv. in both the United States and Argentina since 2010. We performed whole genome sequencing on U.S. isolates to confirm their identity. Informed by comparative genomics, we then developed specific TaqMan qPCR and loop-mediated isothermal amplification (LAMP) assays for the detection of this specific subset of pv. strains. The qPCR 4909 assay was tested against 27 xanthomonads (diverse representation), 32 DNA extractions from corn leaves confirmed as positive or negative for the bacterium, 41 pv. isolates from corn in the United States and Argentina, and 31 additional bacteria associated with corn, sugarcane, or sorghum. In all cases the assay was shown to be specific for the pv. isolates that cause more severe disease on corn. We then tested the LAMP 166 assay against the 27 xanthomonads and 32 corn leaf DNA samples, and we found this assay was also specific for this subset of pv. isolates. We also developed a live/dead cells distinction protocol using propidium monoazide prior to DNA extraction for analyzing seed washes using these assays. These two detection assays can be useful for both diagnosticians and researchers to specifically identify the pv. isolates that cause more severe symptoms on corn.
Tal1 in pv. Contributes to Virulence in Bacterial Leaf Streak of Wheat.
Shah Syed Mashab Ali,Haq Fazal,Ma Wenxiu,Xu Xiameng,Wang Sai,Xu Zhengyin,Zou Lifang,Zhu Bo,Chen Gongyou
Frontiers in microbiology
pv. () causes bacterial leaf streak (BLS) of important cereal crops, including wheat () and barley (). Transcription activator-like effectors (TALEs) play vital roles in many plant diseases caused by spp., however, TALEs have not been previously characterized in . In this study, the whole genome of NXtc01, a virulent strain of from Xinjiang, China, was sequenced and compared with genomes of other spp. NXtc01 consists of a single 4,622,298 bp chromosome that encodes 4,004 genes. Alignment of the NXtc01 sequence with the draft genome of strain CFBP 2541 (United States) revealed a single giant inversion and differences in the location of two genes, which were designated and . In NXtc01, both genes are located on the chromosome, whereas is plasmid-encoded in CFBP 2541. The repeat variable diresidues (RVDs) at the 12th and 13th sites within Tal2 repeat units were identical in both strains, whereas Tal1 showed differences in the third RVD. NXtc01 and CFBP 2541 encoded 35 and 33 non-TALE type III effectors (T3Es), respectively. , , and -free deletion mutants of NXtc01 were constructed and evaluated for virulence. The and -free deletion mutants were impaired with respect to symptom development and growth in wheat, suggesting that is a virulence factor in NXtc01. This was confirmed in gain-of-function experiments that showed the introduction of , but not , restored virulence to the -free mutant. Furthermore, we generated a deletion mutant of NXtc01; the mutant was non-pathogenic on wheat and unable to elicit a hypersensitive response in the non-host . Our data provide a platform for exploring the roles of both TALEs and non-TALEs in promoting BLS on wheat.
Detection and Characterization of Xanthomonas vasicola pv. vasculorum (Cobb 1894) comb. nov. Causing Bacterial Leaf Streak of Corn in the United States.
Lang J M,DuCharme E,Ibarra Caballero J,Luna E,Hartman T,Ortiz-Castro M,Korus K,Rascoe J,Jackson-Ziems T A,Broders K,Leach J E
Bacterial leaf streak of corn (Zea mays) recently reached epidemic levels in three corn-growing states, and has been detected in another six states in the central United States. Xanthomonas vasicola was identified as the causal agent of this disease. A multilocus sequence alignment of six housekeeping genes and comparison of average nucleotide identity from draft genome sequence were used to confirm phylogenetic relationships and classification of this bacteria relative to other X. vasicola strains. X. vasicola isolates from Nebraska and South Africa were highly virulent on corn and sugarcane and less virulent on sorghum but caused water-soaking symptoms that are typical of X. vasicola infection on the leaves of all three hosts. Based on host range and phylogenetic comparison, we propose the taxonomic designation of this organism to X. vasicola pv. vasculorum ( Cobb 1894 ) comb. nov. Polymerase chain reaction-based diagnostic assays were developed that distinguish X. vasicola pv. vasculorum and X. vasicola pv. holcicola from each other and from other Xanthomonas spp.
First Report of Xanthomonas oryzae pv. oryzicola Causing Bacterial Leaf Streak of Rice in Uganda.
Afolabi O,Milan B,Poulin L,Ongom J,Szurek B,Koebnik R,Silue D
In June 2013, symptoms reminiscent of bacterial leaf streak (BLS) caused by Xanthomonas oryzae pv. oryzicola were observed on rice plants at the booting stage in the Doho rice irrigation scheme, Butaleja district, and at the tillering stage in Nambale, Iganga district and Magada, Namutumba district of Uganda. In areas surveyed, disease incidence was about 80, 40, and 30% in Doho, Nambale, and Magada, respectively. Outside the irrigation schemes, it was lower but widespread. Affected leaves showed typical BLS symptoms, such as water-soaked lesions, translucent stripes, and yellow-brown to black streaks, sometimes with visible exudates at the leaf surfaces. To check for the presence of the bacteria, symptomatic leaves were ground in sterile water and the suspension obtained was subjected to a multiplex PCR assay for X. oryzae pathovars, leading to the three diagnostic DNA fragments for X. oryzae pv. oryzicola (3). In parallel, bacterial strains were isolated from surface-sterilized symptomatic leaves. To this end, rice leaves were ground in sterile distilled water and serial dilutions of the cell suspensions were plated on semi-selective PSA medium (4). Each of the three samples yielded yellow, mucoid Xanthomonas-like colonies that resembled the positive control strain MAI10 (1). These isolates were named Ug_1, Ug_10, and Ug_14, which originated from Doho, Magada, and Nambale, respectively. Multiplex PCR on the pure cultures strongly supported that these isolates corresponded to X. oryzae pv. oryzicola. Two isolates, Ug_1 and Ug_14, were further subjected to partial DNA sequence analysis of the gyrB gene upon PCR amplification using the primers XgyrB1F and XgyrB1R (5). The 467-bp DNA sequence was identical to the gyrB sequences from the X. oryzae pv. oryzicola strains BLS256 (Philippines), ICMP 12013 (China), and MAI3 (Mali) (2). The partial nucleotide sequence of the gyrB gene of strain Ug_1 was submitted to GenBank (KJ921786). Pathogenicity tests were performed on greenhouse-grown 4-week-old rice plants of the cultivars Nipponbare, Azucena, IRBB 1, IRBB 2, IRBB 3, FKR 14, PNA64F4-56, TCS 10, Gigante, and Adny 11. For this purpose, bacterial cultures were grown overnight in PSA medium and re-suspended in sterile water at a concentration of 1 × 10 CFU/ml. Bacterial suspensions were sprayed on leaves of rice seedlings. Four seedlings per accession and isolate were inoculated. Fifteen days after incubation in a BSL-3 containment facility (27 ± 1°C with a 12-h photoperiod), inoculated leaves exhibited typical water-soaked lesions with yellow exudates that were similar to the symptoms seen in the fields. Re-isolation of the bacteria from the diseased leaves yielded colonies with the typical morphology of Xanthomonas. Multiplex PCR and sequence analysis of portions of the gyrB gene confirmed that these isolates are X. oryzae pv. oryzicola, thus fulfilling Koch's postulates. One of the three isolates, Ug_1, has been deposited in the Collection Française de Bactéries Phytopathogènes (CFBP) as strain CFBP 8171 ( http://www.angers-nantes.inra.fr/cfbp/ ). Further surveys and strain collections in East and Central Africa will help assess the geographic distribution and importance of BLS. References: (1) C. Gonzalez et al. Mol. Plant Microbe Interact. 20:534, 2007. (2) A. Hajri et al. Mol. Plant Pathol. 13:288, 2012. (3) J. M. Lang et al. Plant Dis. 94:311, 2010. (4) L. Poulin et al. Plant Dis. 98:1423, 2014. (5) J. M. Young et al. Syst. Appl. Microbiol. 31:366, 2008.
Confirmation of Bacterial Leaf Streak Caused by Xanthomonas oryzae pv. oryzicola on Rice in Madagascar.
Poulin L,Raveloson H,Sester M,Raboin L-M,Silué D,Koebnik R,Szurek B
Bacterial leaf streak (BLS) caused by Xanthomonas oryzae pv. oryzicola is an important disease of rice. BLS is prevalent in Asia and West Africa, where it was first reported in Nigeria and Senegal in the early 1980s (4). Recently, molecular analysis of strains from Mali (2) and Burkina Faso (5) further confirmed the presence of BLS in West Africa. In Madagascar, BLS symptoms were first reported in the 1980s by Buddenhagen but the causal agent was not unequivocally determined (1). To confirm Buddenhagen's observations using modern molecular typing tools, we surveyed several rice fields in the Antananarivo and Antsirabe districts in March 2013. BLS symptoms were observed on cultivated Oryza sativa grown under both upland and lowland conditions, with a proportion of diseased individuals varying from 30% up to 80%. Symptomatic leaves presenting water-soaked lesions that developed into translucent, yellow streaks with visible exudates at the surface were sampled. One to four centimeter long pieces of diseased leaves were ground using the Qiagen TissueLyser system at 30 rps for 30 s (Qiagen, Courtaboeuf, France). The ground tissue was then macerated in 1 ml of sterile water for 1 h at 4°C. Non-diluted and 10-fold diluted tissue macerates were plated on semi-selective PSA medium (peptone 10 g/liter, sucrose 10 g/liter, glutamic acid 1 g/liter, bacto agar 16 g/liter, actidione 50 mg/liter, cephalexin 40 mg/liter, and kasugamycin 20 mg/liter) and incubated for 3 to 7 days at 28°C. Single, yellow, Xanthomonas-like colonies were isolated on non-selective PSA medium. Diagnostic multiplex PCR was performed on single colonies for pathovar identification (3). Five strains that produced three diagnostic bands corresponding to the X. oryzae pv. oryzicola pattern were further analyzed for pathogenicity on 3-week-old O. sativa cv. Nipponbare plants. Bacteria grown on PSA plates and adjusted to 1 × 10 CFU/ml were infiltrated into rice leaves with a needleless 1-ml syringe (2 × 3 infiltrations per plant and strain). Seven days after incubation in the greenhouse (27 ± 1°C with a 12-h photoperiod), inoculated leaves showed water-soaked lesions that produced yellow exudates corresponding to those initially observed in rice fields and observed for leaves challenged with the X. oryzae pv. oryzicola reference strain BLS256. Symptomatic leaf tissues were ground and plated on non-selective PSA medium, resulting in colonies with typical Xanthomonas morphology that were confirmed as X. oryzae pv. oryzicola by multiplex PCR typing (3), thus fulfilling Koch's postulates. Finally, the five strains were subjected to gyrB sequencing upon PCR amplification using the universal primers XgyrB1F (5'-ACGAGTACAACCCGGACAA-3') and XgyrB1R (5'-CCCATCARGGTGCTGAAGAT-3'). The 743-bp partial gyrB sequences were 100% identical to the gyrB sequence of strain BLS256. As expected, the gyrB sequence of strains KACC10331, MAFF311018, and PXO99 of the X. oryzae pv. oryzae pathovar respectively showed nine, 16, and 10 mismatches in comparison to the Malagasy strains, thus further supporting that they belong to the pathovar oryzicola. References: (1) I. W. Buddenhagen. Int. Rice Comm. Newsl. 34:74, 1985. (2) C. Gonzalez et al. Mol. Plant Microbe Interact. 20:534, 2007. (3) J. M. Lang et al. Plant Dis. 94:311, 2010. (4) D. O. Niño-Liu et al. Mol. Plant Pathol. 7:303, 2006. (5) I. Wonni et al. Plant Dis. 95:72, 2011.
Identification of the phytosulfokine receptor 1 (OsPSKR1) confers resistance to bacterial leaf streak in rice.
Yang Wei,Zhang Baogang,Qi Guanghui,Shang Luyue,Liu Haifeng,Ding Xinhua,Chu Zhaohui
MAIN CONCLUSION:A rice allele of PSKR1 functioning in resistance to bacterial leaf streak was identified. Phytosulfokine (PSK), a disulfated pentapeptide encoded by precursor genes that are ubiquitously present in higher plants, belongs to the group of plant peptide growth factors. The PSK receptor PSKR1 in Arabidopsis thaliana is an active kinase and has guanylate cyclase activity resulting in dual-signaling outputs. Here, the LOC_Os02g41890 out of three candidates completely rescued root growth and susceptible to Pseudomonas syringae pv. DC3000 in the Arabidopsis pskr1-3 mutant and was identified as OsPSKR1. This protein was localized to plasma membrane similar to AtPSKR1. The expression of OsPSKR1 was upregulated upon inoculation with RS105, a strain of Xanthomonas oryzae pv. oryzicola (Xoc) that cause bacterial leaf streak in rice. OsPSKR1 overexpression (OE) lines had greater resistance to RS105 than the wild type. Consistently, the expression of pathogenesis-related genes involved in the salicylic acid (SA) pathway was upregulated in the transgenic lines. Overall, OsPSKR1 functions as a candidate PSK receptor and regulates resistance to Xoc by activating the expression of pathogenesis-related genes involved in the SA pathway in rice.
First Report of Xanthomonas oryzae pv. oryzicola Causing Bacterial Leaf Streak of Rice in Burundi.
Afolabi O,Milan B,Amoussa R,Koebnik R,Poulin L,Szurek B,Habarugira G,Bigirimana J,Silue D
On May 9, 2013, symptoms reminiscent of bacterial leaf streak (BLS) caused by Xanthomonas oryzae pv. oryzicola were observed on rice plants at the panicle emergence stage at Musenyi, Gihanga, and Rugombo fields in Burundi. Affected leaves showed water-soaked translucent lesions and yellow-brown to black streaks, sometimes with visible exudates on leaf surfaces. Symptomatic leaves were ground in sterile water and the suspensions obtained were subjected to a multiplex PCR assay diagnostic for X. oryzae pathovars (3). Three DNA fragments (331, 691, and 945 bp) corresponding to X. oryzae pv. oryzicola were observed after agarose gel electrophoresis. Single bacterial colonies were then isolated from surface-sterilized, infected leaves after grinding in sterile water and plating of 10-fold dilutions of the cell suspension on semi-selective PSA medium (4). After incubation at 28°C for 5 days, each of four independent cultures yielded single yellow, mucoid Xanthomonas-like colonies (named Bur_1, Bur_2, Bur_6, and Bur_7) that resembled the positive control strain MAI10 (1). These strains originated from Musenyi (Bur_1), Gihanga (Bur_2), and Rugumbo (Bur_6 and Bur_7). Multiplex PCR assays on the four putative X. oryzae pv. oryzicola strains yielded the three diagnostic DNA fragments mentioned above. All strains were further analyzed by sequence analysis of portions of the gyrB gene using the universal primers gyrB1-F and gyrB1-R for PCR amplification (5). The 762-bp DNA fragment was identical to gyrB sequences from the Asian X. oryzae pv. oryzicola strains BLS256 (Philippines), ICMP 12013 (China), LMG 797 and NCPPB 2921 (both Malaysia), and from the African strain MAI3 (Mali) (2). The partial nucleotide sequence of the gyrB gene of Bur_1 was submitted to GenBank (Accession No. KJ801400). Pathogenicity tests were performed on greenhouse-grown 4-week-old rice plants of the cvs. Nipponbare, Azucena, IRBB 1, IRBB 2, IRBB 3, IRBB 7, FKR 14, PNA64F4-56, TCS 10, Gigante, and Adny 11. Bacterial cultures were grown overnight in PSA medium and re-suspended in sterile water (1 × 10 CFU/ml). Plants were inoculated with bacterial suspensions either by spraying or by leaf infiltration (1). For spray inoculation, four plants per accession and strain were used while three leaves per plant and four plants per accession and strain were inoculated by tissue infiltration. After 15 days of incubation in a BSL-3 containment facility (27 ± 1°C with a 12-h photoperiod), the spray-inoculated plants showed water-soaked lesions with yellow exudates identical to those seen in the field. For syringe-infiltrated leaves, the same symptoms were observed at the infiltrated leaf area. Re-isolation of bacteria from symptomatic leaves yielded colonies with the typical Xanthomonas morphology that were confirmed by multiplex PCR to be X. oryzae pv. oryzicola, thus fulfilling Koch's postulates. Bur_1 has been deposited in the Collection Française de Bactéries Phytopathogènes as strain CFBP 8170 ( http://www.angers-nantes.inra.fr/cfbp/ ). To our knowledge, this is the first report of X. oryzae pv. oryzicola causing bacterial leaf streak on rice in Burundi. Further surveys will help to assess its importance in the country. References: (1) C. Gonzalez et al., Mol. Plant Microbe Interact. 20:534, 2007. (2) A. Hajri et al. Mol. Plant Pathol. 13:288, 2012. (3) J. M. Lang et al. Plant Dis. 94:311, 2010. (4) L. Poulin et al. Plant Dis. 98:1423, 2014. (5) J. M. Young et al. Syst. Appl. Microbiol. 31:366, 2008.
Ectopic activation of the rice NLR heteropair RGA4/RGA5 confers resistance to bacterial blight and bacterial leaf streak diseases.
Hutin Mathilde,Césari Stella,Chalvon Véronique,Michel Corinne,Tran Tuan Tu,Boch Jens,Koebnik Ralf,Szurek Boris,Kroj Thomas
The Plant journal : for cell and molecular biology
Bacterial blight (BB) and bacterial leaf streak (BLS) are important diseases in Oryza sativa caused by Xanthomonas oryzae pv. oryzae (Xoo) and Xanthomonas oryzae pv. oryzicola (Xoc), respectively. In both bacteria, transcription activator-like (TAL) effectors are major virulence determinants that act by transactivating host genes downstream of effector-binding elements (EBEs) bound in a sequence-specific manner. Resistance to Xoo is mostly related to the action of TAL effectors, either by polymorphisms that prevent the induction of susceptibility (S) genes or by executor (R) genes with EBEs embedded in their promoter, and that induce cell death and resistance. For Xoc, no resistance sources are known in rice. Here, we investigated whether the recognition of effectors by nucleotide binding and leucine-rich repeat domain immune receptors (NLRs), the most widespread resistance mechanism in plants, is also able to stop BB and BLS. In one instance, transgenic rice lines harboring the AVR1-CO39 effector gene from the rice blast fungus Magnaporthe oryzae, under the control of an inducible promoter, were challenged with transgenic Xoo and Xoc strains carrying a TAL effector designed to transactivate the inducible promoter. This induced AVR1-CO39 expression and triggered BB and BLS resistance when the corresponding Pi-CO39 resistance locus was present. In a second example, the transactivation of an auto-active NLR by Xoo-delivered designer TAL effectors resulted in BB resistance, demonstrating that NLR-triggered immune responses efficiently control Xoo. This forms the foundation for future BB and BLS disease control strategies, whereupon endogenous TAL effectors will target synthetic promoter regions of Avr or NLR executor genes.
Novel 1,3,4-Oxadiazole Derivatives Containing a Cinnamic Acid Moiety as Potential Bactericide for Rice Bacterial Diseases.
Wang Shaobo,Gan Xiuhai,Wang Yanju,Li Shaoyuan,Yi Chongfen,Chen Jixiang,He Fangcheng,Yang Yuyuan,Hu Deyu,Song Baoan
International journal of molecular sciences
Rice bacterial leaf blight and leaf streak are two important bacterial diseases of rice, which can result in yield loss. Currently, effective antimicrobials for rice bacterial diseases are still lacking. Thus, to develop highly effective and low-risk bactericides, 31 novel 1,3,4-oxadiazole derivatives containing a cinnamic acid moiety were designed and synthesized. Bioassay results demonstrated that all compounds exhibited good antibacterial activities in vitro. Significantly, compounds and showed excellent antibacterial activities against pv. () and pv. (), with the 50% effective concentration (EC) values of 0.58 and 0.34, and 0.44 and 0.20 μg/mL, respectively. These compounds were much better than thiodiazole copper (123.10 and 161.52 μg/mL) and bismerthiazol (85.66 and 110.96 μg/mL). Moreover, compound had better protective and curative activities against rice bacterial leaf blight and leaf streak than thiodiazole copper and bismerthiazol in vivo. Simultaneously, the in vivo efficacy of the compounds was demonstrated by real-time quantitative PCR to quantify bacterial titers. In addition, a three-dimensional quantitative structure⁻activity relationship model was created and presented good predictive ability. This work provides support for 1,3,4-oxadiazole derivatives containing a cinnamic acid moiety as a potential new bactericide for rice bacterial diseases.
Inoculation and virulence assay for bacterial blight and bacterial leaf streak of rice.
Yang Bing,Bogdanove Adam
Methods in molecular biology (Clifton, N.J.)
Xanthomonas oryzae pv. oryzae (Xoo) and Xanthomonas oryzae pv. oryzicola (Xoc) cause bacterial blight and bacterial leaf streak in rice, respectively. Despite being very closely related, the pathogens colonize different tissues and cause distinct diseases. The diseases are economically important and also serve as model systems for studying plant-bacterial interactions. Here we describe protocols for Xoo and Xoc inoculation and disease scoring methods that are appropriate to their different modes of infection. These methods are routinely used to evaluate pathogen virulence or host responses under controlled environmental conditions.
Over-expression in the nucleotide-binding site-leucine rich repeat gene DEPG1 increases susceptibility to bacterial leaf streak disease in transgenic rice plants.
Guo Lijia,Li Min,Wang Wujing,Wang Lijuan,Hao Guojing,Guo Chiming,Chen Liang
Molecular biology reports
Bacterial leaf streak of rice (BLS) caused by Xanthomonas oryzae pv. oryzicola (Xoc) is a widely-spread disease in the main rice-producing areas of the world. Investigating the genes that play roles in rice-Xoc interactions helps us to understand the defense signaling pathway in rice. Here we report a differentially expressed protein gene (DEPG1), which regulates susceptibility to BLS. DEPG1 is a nucleotide-binding site (NBS)-leucine rich repeat (LRR) gene, and the deduced protein sequence of DEPG1 has approximately 64% identity with that of the disease resistance gene Pi37. Phylogenetic analysis of DEPG1 and the 18 characterized NBS-LRR genes revealed that DEPG1 is more closely related to Pi37. DEPG1 protein is located to the cytoplasm, which was confirmed by transient expression of DEPG1-GFP (green fluorescent protein) fusion construct in onion epidermal cells. Semi-quantitative PCR assays showed that DEPG1 is widely expressed in rice, and is preferentially expressed in internodes, leaf blades, leaf sheaths and flag leaves. Observation of cross sections of leaves from the transgenic plants with a DEPG1-promoter::glucuronidase (GUS) fusion gene revealed that DEPG1 is also highly expressed in mesophyll tissues where Xoc mainly colonizes. Additionally, Xoc negatively regulates expression of DEPG1 at the early stage of the pathogen infection, and so do the three defense-signal compounds including salicylic acid (SA), methyl jasmonate (MeJA) and 1-aminocyclopropane-1-carboxylic-acid (ACC). Transgenic rice plants overexpressing DEPG1 exhibit enhanced susceptibility to Xoc compared to the wild-type controls. Moreover, enhanced susceptibility to Xoc may be mediated by inhibition of the expression of some SA biosynthesis-related genes and pathogenesis-related genes that may contribute to the disease resistance. Taken together, DEPG1 plays roles in the interactions between rice and BLS pathogen Xoc.
Toward the positional cloning of qBlsr5a, a QTL underlying resistance to bacterial leaf streak, using overlapping sub-CSSLs in rice.
Xie Xiaofang,Chen Zhiwei,Cao Jinliang,Guan Huazhong,Lin Degong,Li Chunlan,Lan Tao,Duan Yuanlin,Mao Damei,Wu Weiren
Bacterial leaf steak (BLS) is one of the most destructive diseases in rice. Studies have shown that BLS resistance in rice is quantitatively inherited, controlled by multiple quantitative trait loci (QTLs). A QTL with relatively large effect, qBlsr5a, was previously mapped in a region of ∼ 380 kb on chromosome 5. To fine map qBlsr5a further, a set of overlapping sub-chromosome segment substitution lines (sub-CSSLs) were developed from a large secondary F2 population (containing more than 7000 plants), in which only the chromosomal region harboring qBlsr5a was segregated. By genotyping the sub-CSSLs with molecular markers covering the target region and phenotyping the sub-CSSLs with artificial inoculation, qBlsr5a was delimited to a 30.0-kb interval, in which only three genes were predicted. qRT-PCR analysis indicated that the three putative genes did not show significant response to the infection of BLS pathogen in both resistant and susceptible parental lines. However, two nucleotide substitutions were found in the coding sequence of gene LOC_Os05g01710, which encodes the gamma chain of transcription initiation factor IIA (TFIIAγ). The nucleotide substitutions resulted in a change of the 39th amino acid from valine (in the susceptible parent) to glutamic acid (in the resistant parent). Interestingly, the resistant parent allele of LOC_Os05g01710 is identical to xa5, a major gene resistant to bacterial leaf blight (another bacterial disease of rice). These results suggest that LOC_Os05g01710 is very possibly the candidate gene of qBlsr5a.
Code-assisted discovery of TAL effector targets in bacterial leaf streak of rice reveals contrast with bacterial blight and a novel susceptibility gene.
Cernadas Raul A,Doyle Erin L,Niño-Liu David O,Wilkins Katherine E,Bancroft Timothy,Wang Li,Schmidt Clarice L,Caldo Rico,Yang Bing,White Frank F,Nettleton Dan,Wise Roger P,Bogdanove Adam J
Bacterial leaf streak of rice, caused by Xanthomonas oryzae pv. oryzicola (Xoc) is an increasingly important yield constraint in this staple crop. A mesophyll colonizer, Xoc differs from X. oryzae pv. oryzae (Xoo), which invades xylem to cause bacterial blight of rice. Both produce multiple distinct TAL effectors, type III-delivered proteins that transactivate effector-specific host genes. A TAL effector finds its target(s) via a partially degenerate code whereby the modular effector amino acid sequence identifies nucleotide sequences to which the protein binds. Virulence contributions of some Xoo TAL effectors have been shown, and their relevant targets, susceptibility (S) genes, identified, but the role of TAL effectors in leaf streak is uncharacterized. We used host transcript profiling to compare leaf streak to blight and to probe functions of Xoc TAL effectors. We found that Xoc and Xoo induce almost completely different host transcriptional changes. Roughly one in three genes upregulated by the pathogens is preceded by a candidate TAL effector binding element. Experimental analysis of the 44 such genes predicted to be Xoc TAL effector targets verified nearly half, and identified most others as false predictions. None of the Xoc targets is a known bacterial blight S gene. Mutational analysis revealed that Tal2g, which activates two genes, contributes to lesion expansion and bacterial exudation. Use of designer TAL effectors discriminated a sulfate transporter gene as the S gene. Across all targets, basal expression tended to be higher than genome-average, and induction moderate. Finally, machine learning applied to real vs. falsely predicted targets yielded a classifier that recalled 92% of the real targets with 88% precision, providing a tool for better target prediction in the future. Our study expands the number of known TAL effector targets, identifies a new class of S gene, and improves our ability to predict functional targeting.
Antibacterial activity of two chitosan solutions and their effect on rice bacterial leaf blight and leaf streak.
Li Bin,Liu Baoping,Shan Changlin,Ibrahim Muhammad,Lou Yihan,Wang Yangli,Xie Guanlin,Li Hong-ye,Sun Guochang
Pest management science
BACKGROUND:Bacterial leaf blight and leaf streak are the two most damaging bacterial diseases of rice. However, few bactericidal chemicals are available for controlling both diseases. The antibacterial properties of two kinds of chitosan with different molecular weights and degrees of N-deacetylation and their effect on rice bacterial leaf blight and leaf streak were evaluated. RESULTS:Results showed that the two kinds of chitosan solution possess a strong antibacterial activity against both rice bacterial pathogens and significantly reduced disease incidence and severity by comparison with the control under greenhouse conditions. However, the interaction between chitosan and rice pathogens was affected by the type and concentration of chitosan, the bacterial species and the contact time between chitosan and bacteria. The direct antibacterial activity of chitosan may be attributed to both membrane lysis and the destruction of biofilm. In addition, both chitosan solutions significantly increased the activities of phenylalanine ammonia lyase, peroxidase and polyphenol oxidase in rice seedlings following inoculation of two rice pathogens by comparison with the control. CONCLUSION:The role of chitosan in protection of rice against bacterial pathogens has been shown to involve direct antibacterial activity and indirect induced resistance.
Suppression of expression of the putative receptor-like kinase gene NRRB enhances resistance to bacterial leaf streak in rice.
Guo Lijia,Guo Chiming,Li Min,Wang Wujing,Luo Chengke,Zhang Yuxia,Chen Liang
Molecular biology reports
Bacterial leaf streak (BLS) caused by Xanthomonas oryzae pv. oryzicola (Xoc) is an important disease of rice, which is responsible for the economic losses worldwide. Functional investigation of differentially expressed protein genes (DEPGs) from rice (Oryza sativa L.) upon Xoc infection provides insight into the molecular mechanism of rice-Xoc interactions. Here, we show that one of DEPGs designated NRRB plays a role in rice-Xoc interactions. NRRB, a receptor-like cytoplasmic kinase gene was preferentially expressed in leaf blades and leaf sheaths where the pathogen colonized. Its transcription was depressed by two defense-signal compounds salicylic acid and 1-aminocyclopropane-1-carboxylic-acid, but was activated by wounding and abscisic acid. Additionally, a plenty of cis-elements associated with stress responses were discovered in the promoter region of NRRB. These data suggest that NRRB is involved in stress responses. More importantly, the NRRB-suppressing rice plants exhibited enhanced resistance against BLS, with the markedly shorter average lesion length than that of the wild type. Furthermore, transcription of some salicylic acid synthesis-related and pathogenesis-related genes including PAD4, PR1a and WRKY13 in transgenic plants was activated, implying that enhanced resistance to BLS might be mediated by the activation of the SA signaling pathway. In conclusion, NRRB gene is involved in various stress responses and regulating resistance to BLS, therefore it might be one of useful genes for rice improvement in future.
An operon for production of bioactive gibberellin A phytohormone with wide distribution in the bacterial rice leaf streak pathogen Xanthomonas oryzae pv. oryzicola.
Nagel Raimund,Turrini Paula C G,Nett Ryan S,Leach Jan E,Verdier Valérie,Van Sluys Marie-Anne,Peters Reuben J
The New phytologist
Phytopathogens have developed elaborate mechanisms to attenuate the defense response of their host plants, including convergent evolution of complex pathways for production of the GA phytohormones, which were actually first isolated from the rice fungal pathogen Gibberella fujikuroi. The rice bacterial pathogen Xanthomonas oryzae pv. oryzicola (Xoc) has been demonstrated to contain a biosynthetic operon with cyclases capable of producing the universal GA precursor ent-kaurene. Genetic (knock-out) studies indicate that the derived diterpenoid serves as a virulence factor for this rice leaf streak pathogen, serving to reduce the jasmonic acid-mediated defense response. Here the functions of the remaining genes in the Xoc operon are elucidated and the distribution of the operon in X. oryzae is investigated in over 100 isolates. The Xoc operon leads to production of the bioactive GA , an additional step beyond production of the penultimate precursor GA mediated by the homologous operons recently characterized from rhizobia. Moreover, this GA biosynthetic operon was found to be widespread in Xoc (> 90%), but absent in the other major X. oryzae pathovar. These results indicate selective pressure for production of GA in the distinct lifestyle of Xoc, and the importance of GA to both fungal and bacterial pathogens of rice.
Design, synthesis, and antibacterial activity against rice bacterial leaf blight and leaf streak of 2,5-substituted-1,3,4-oxadiazole/thiadiazole sulfone derivative.
Li Pei,Shi Li,Yang Xia,Yang Lei,Chen Xue-Wen,Wu Fang,Shi Qing-Cai,Xu Wei-Ming,He Ming,Hu De-Yu,Song Bao-An
Bioorganic & medicinal chemistry letters
A series of 2,5-substituted-1,3,4-oxadiazole/thiadiazole sulfone derivatives were synthesized and evaluated for their antibacterial activities against rice bacterial leaf blight and leaf streak caused by Xanthomonas oryzae pv. oryzae and Xanthomonas oryzae pv. oryzicolaby via the turbidimeter test in vitro. Antibacterial bioassay results indicated that most compounds demonstrated good inhibitory effect antibacterial bioactivities against rice bacterial leaf blight and leaf streak. Among the title compounds, compound 6c demonstrated the best inhibitory effect against rice bacterial leaf blight and leaf streak with half-maximal effective concentration (EC50) values of 1.07 and 7.14 μg/mL, respectively, which were even better than those of commercial agents such as Bismerthiazol and Thiediazole Copper. In vivo antibacterial activities tests at greenhouse conditions demonstrated that the controlling effect of compounds 6c (43.5%) and 6g (42.4%) against rice bacterial leaf blight were better than those of Bismerthiazol (25.5%) and Thiediazole Copper (37.5%).
The polygalacturonase-inhibiting protein 4 (OsPGIP4), a potential component of the qBlsr5a locus, confers resistance to bacterial leaf streak in rice.
Feng Chuanshun,Zhang Xia,Wu Tao,Yuan Bin,Ding Xinhua,Yao Fangying,Chu Zhaohui
MAIN CONCLUSION:OsPGIP4 overexpression enhances resistance to bacterial leaf streak in rice. Polygalacturonase-inhibiting proteins are thought to play important roles in the innate immunity of rice against fungi. Here, we show that the chromosomal location of OsPGIP4 coincides with the major bacterial leaf streak resistance quantitative trait locus qBlsr5a on the short arm of chromosome 5. OsPGIP4 expression was up-regulated upon inoculation with the pathogen Xanthomonas oryzae pv. oryzicola strain RS105. OsPGIP4 overexpression enhanced the resistance of the susceptible rice variety Zhonghua 11 to RS105. In contrast, repressing OsPGIP4 expression resulted in an increase in disease lesions caused by RS105 in Zhonghua 11 and in Acc8558, a qBlsr5a resistance donor. More interestingly, upon inoculation, the activated expression of pathogenesis-related genes was attenuated for those genes involved in the salicylic acid pathway, while the activated expression of jasmonic acid pathway markers was increased in the overexpression lines. Our results not only provide the first report that rice PGIP could enhance resistant against a bacterial pathogen but also indicate that OsPGIP4 is a potential component of the qBlsr5a locus for bacterial leaf streak in rice.