Identification of novel pathogenicity-related cellulase genes in Xanthomonas oryzae pv. oryzae

Physiological and Molecular Plant Pathology 76 (2011) 152e157

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Physiological and Molecular Plant Pathology journal homepage: www.elsevier.com/locate/pmpp

Identification of novel pathogenicity-related cellulase genes in Xanthomonas oryzae pv. oryzae Ulambayar Temuujina, Jae-Won Kima, Jong-Kun Kimc, Byoung-Moo Leeb, Hee-Wan Kanga, c, * a

Graduate School of Bio and Information Technology, Hankyong National University, Ansung 456-749, Republic of Korea National Academy of Agricultural Science, Suwon 441-707, Republic of Korea c Institute of Genetic Engineering, Hankyong National University, Ansung 456-749, Republic of Korea b

a r t i c l e i n f o

a b s t r a c t

Article history: Accepted 17 August 2011

Twelve genes encoding cellulases, including endo- and exoglucanases, were identified from the genomic database of Xanthomonas oryzae pv. oryzae KACC10331. The genes were amplified by polymerase chain reaction (PCR) from X. oryzae pv. oryzae KACC10859 and mutated by transposon insertion; further, marker exchange was performed with the target genes of the wild type strain. Homologous recombination events were confirmed by PCR and Southern hybridization analysis. We found that the mutant strains eglXoA::Tn5, eglXoB::Tn5, and celXoB::Tn5 were completely virulence-deficient. In addition, mutants celbXoA::Tn5, bglXoC::Tn5, and bglXoF::Tn5 showed attenuated virulence, while the virulence of other mutants was not affected. Ó 2011 Elsevier Ltd. All rights reserved.

Keywords: Xanthomonas oryzae pv. oryzae Cellulase genes Pathogenicity

1. Introduction Xanthomonas oryzae pv. oryzae (Xoo) is the causal agent of bacterial blight in rice (Oryza sativa L.). Many virulence related genes, including hypersensitive response and pathogenicity (hrp) genes and those involved in the production of extracellular polysaccharides (EPS) and lipopolysaccharides (LPS), have been isolated and characterized [2,13,27]. The different novel virulence genes, including hypothetical genes and predicted functional genes for cell-wall degrading enzymes, fimbrial and flagella assembly regulators, and metabolic regulators, were identified by transposon mutagenesis [31], which suggested that various genes are involved in the virulence of Xoo. The plant cell wall is the first barrier to intracellular invasion by bacteria [30]. The primary cell wall is initially poor in cellulose and rich in pectic compounds, polysaccharides, and hemicelluloses. Cellulose and xylan are the main constituents of the xylem [23]. Extracellular enzymes, including pectinases, cellulases, proteases, and xylanases, have long been considered to play a role in the virulence of phytobacterial pathogens [1,11,14,17,22] and a cellulase gene, eglXoB, have been reported as important virulence factors in Xoo [10]. Interestingly, it was reported Type II secretion

* Corresponding author. Graduate School of Bio and Information Technology, Hankyong National University, Jungang-ro, Ansung 456-749, Republic of Korea. Tel.: þ82 31 670 5420; fax: þ82 31 676 2602. E-mail address: [email protected] (H.-W. Kang). 0885-5765/$ e see front matter Ó 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.pmpp.2011.08.004

(T2S)-dependent induction of basal plant defense responses is suppressed by Xoo wild type strains that contain a functional type III secretion (T3S) system [13]. This suggests that type III effector proteins counteract basal plant defense responses elicited by T2S substrates. Therefore, it was reasonably assumed that extracellular cellulases participate in diverse virulence functions associated directly or indirectly with the expression of main virulence genes, e.g., hrp genes. Generally, cell-wall degrading enzymes, such polygalacturonases, cellulases, xylanases, and proteases are secreted into the extracellular space through the T2SS [19]. In Xanthomonas species, Xanthomonas protein secretion (xps) genes are secreted through the T2SS [28]. Xanthomonas genomes, including those of Xoo, Xanthomonas axonopodis pv. citri, and Xanthomonas campestris pv. campestris, have been completely sequenced [5,8,16]; the sequence information provides important insights regarding the general and specific features of virulence genes in the Xanthomonas genomes. On the basis of the sequence information, the genes for extracellular enzymes, including 12 cellulases, 6 proteases, a polygalacturonase, pectin-degrading enzymes (1 pectinesterase and 2 pectate lyases), 4 xylanases, and 6 xylosidases, were identified in Xoo KACC1033 [16]. However, no direct evidences for the associations of most cellulase genes with pathogenicity in Xoo were found. This study aimed to identify the virulent cellulase genes in Xoo KACC10859. Twelve cellulase genes were cloned from the wild type strain Xoo KACC10859 and mutated by transposon insertion; then, marker exchange was performed with the target genes of the wild type strain. Finally, the virulence genes were identified by pathogenicity assays.

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Wild type strain, Xoo KACC10859, was obtained from the Korean Agricultural Culture Collection (KACC) at the National Institute of Agricultural Biotechnology (NIAB), Suwon, Korea. Xoo strains were cultured at 28  C on peptone sucrose agar (PSA; peptone, 10 g; sucrose, 10 g; and agar, 15 g/L) or nutrient agar (NA; Difco). Escherichia coli was grown in LuriaeBertani medium (Duchefa) at 37  C for 18 h. Antibiotics were added at the following final concentrations: for E. coli: ampicillin, 80 mg/L; gentamycin, 50 mg/L; and kanamycin, 50 mg/L and for Xoo: ampicillin, 50 mg/L; gentamycin, 20 mg/L; and kanamycin, 20 mg/L.

following conditions: one cycle of 4 min at 94  C; 30 cycles of 30 s at 94  C, 30 s at 60  C, and 2 min at 72  C; and one final extension cycle of 7 min at 72  C. The PCR products were separated on a 1.5% agarose gel in TAE buffer and visualized by staining with ethidium bromide (1 mg/L). For Southern blot hybridization, about 5 mg of genomic DNA was digested with the restriction enzyme EcoRI, fractionated on a 0.9% agarose gel in TAE buffer (10 mM Triseacetate, 1 mM EDTA, pH 8.0), and transferred to a HybondÔ Nþ nylon membrane (Amersham Biosciences, UK). The DNA fragments on the membrane were hybridized with DNA probes labeled with AlkPhos Direct Labeling Reagents (GE Healthcare, UK). Hybridization signals were detected on radiographs by using detection systems, according to the protocol provided by the manufacturer (Amersham Biosciences).

2.2. Transposon mutagenesis and marker exchange

2.5. Complementation test

Twelve cellulase genes, as mentioned in Table 1, were amplified from Xoo KACC10859 by polymerase chain reaction (PCR) using primers designed on the basis of the sequence information of the open reading frames (ORFs); the amplification products were cloned in pGEM-T easy vector (Promega) and transformed into E. coli DH5a cells by using the MicroPulserÔ electroporation apparatus (Bio-Rad). The target genes on plasmids were disrupted by transposon insertion using the EZ::TN insertion kit (Epicentre Technologies), according to the manufacturer’s instructions. Each plasmid containing a transposon-disrupted target gene was transformed into Xoo KACC10859 by electroporation, and the cells were plated on NA containing kanamycin (20 mg/L). The kanamycin-resistant colonies were selected as the markerexchange clones.

Two genes, namely, eglXoA, celXoB, celbXoA, bglXoC, and bglXoF containing promoters upstream of the initial codons, were amplified by using Pyrobest DNA polymerase (Takara) and ligated in the SmaI site of pML122 (resistant to gentamycin), thus yielding pMLeglA, pMLcelB, pMLcelbA, pMLbglC and pMLbglF. The plasmids were introduced into the transposon insertion mutants eglXoA::Tn5,celXoB::Tn5,celbXoA::Tn5, bglXoC::Tn5, bglXoF::Tn5 by electroporation and plated on NA containing gentamycin and kanamycin. After 5 days, the colonies formed on the medium were selected as complementation clones, namely, CeglXoA CcelXoB, CcelbXoA, CbglXoC, and CbglXoF, respectively.

2. Materials & methods 2.1. Bacterial strain and culture media

2.3. Pathogenicity assays Inoculums (about 1  106 cells) prepared from wild type and mutant strains of Xoo were grown on PSA for 3 days. Pathogenicity assays were performed on 60-day-old leaves of a susceptible rice cultivar (rice variety, Milyang 23) by the leaf-punch method. Pathogenicity was observed 14 d after inoculation. 2.4. PCR and Southern hybridization analysis Genomic DNA was extracted from Xoo cells grown in 5 mL peptone sucrose broth for 3 d at 28  C by using a previously described method [26]. The PCR reaction was performed in a 50 mL mixture containing 10 mM TriseHCl, 50 mM KCl, 1.5 mM MgCl2, 0.01% (w/v) gelatin, 200 mM each dNTP, 100 ng primer, and 2.5 units Taq polymerase (JK Biotech, Korea). PCR amplification was carried out in the PTC-200Ô (MJ Research, Inc.) thermocycler under the

Table 1 Cellulase encoding genes in X. oryzae pv. oryzae KACC10331 genome. Gene ID

Start

End

Length Gene names Functions (Families)

XOO1076 XOO1077 XOO2352 XOO2356 XOO0281 XOO0282 XOO0283 XOO4019 XOO4035 XOO4036 XOO4123 XOO4423

1099419 1100446 2491994 2499565 289805 291788 293570 4291073 4313615 4316042 4398993 4712121

1100177 1101396 2494315 2502231 291106 292921 294700 4292674 4315633 4317814 4401164 4714835

759 951 2322 2667 1302 1134 1131 1602 2019 1773 2172 2715

bglXoA bglXoB bglXoC bglXoD eglXoA eglXoB eglXoC bglXoE celbXoA celXoB bglXoF bglXoG

Celluase S (12) Celluase S (12) Beta-glucosidase (12) 1,4-Beta-glucosidase (12) Endoglucanase (5) Endoglucanase (5) Endoglucanase (5) Cellulase (12) 1,4-Beta-cellobiosidase (6) Cellulase (5) Beta-glucosidase (12) 1,4-Beta-glucosidase (12)

2.6. Sequence analysis The DNA sequence data analysis and similarity searches were with the BLAST program at the National Center for Biotechnology Institute and amino acid alignments were constructed using clustal W method of MEGALIGN software (DNASTAR). Potential signal peptide at an N-terminus was predicted by SIG-Pred program. 3. Results 3.1. Molecular characterization of cellulase genes in Xoo Table 1 shows 12 cellulase genes that are present in Xoo. Cellulases have been classified on the basis of their structural characteristics and active mode. “Cellulase” is a general term for a synergistic system of 3 major categories of enzymes, including endoglucanase (endo-b-1,4-glucanase), exoglucanase (b-1,4cellobiohydrolase), and b-glucosidase [3,9], that hydrolyze the b-1,4-glucosidic bonds. These enzymes have been classified according to their mode of action and substrate specificity. Endoglucanases cleave b-glucosidic bonds in the amorphous regions of cellulose, thus creating cleavage sites for exoglucanases, which cleave cellobiose from the nonreducing ends of cellulose [4]. Subsequently, b-glucosidase hydrolyzes the resultant cellobiose to glucose, thus preventing cellobiose accumulation and exoglucanase inhibition. In this study, the whole-genome sequencing data of Xoo KACC10331 was used to identify the cellulase genes in this organism [16]. These cellulases were classified into 3 families. EglXoA, EglXoB, EglXoC, and CelXoB, which possess endo-b-1,4glucanase activity, belonged to family 5. CelbXoA, with the activity of cellobiohydrolases, was classified into family 6. Other cellulases, with the activity of xyloglucan hydrolase, were grouped into family 12. Further, eglXoA/,eglXoBeglXoC, celbXoA/celXoB, and bglXoA/bglXoB formed clusters with multiple genes in the Xoo genome (Table 1). It would be interesting to determine the biological features of the cellulase gene clusters, since it may provide

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Table 2 Amino acid similarity among cellulase genes of different bacterial species and X. oryzae pv. oryzae.

information concerning the transcriptional regulation, coordinate expression, and evolutionary relationship of the genes. Many bacterial cellulase genes are distributed as clusters on genomes. Large cellulase gene clusters have been cloned and sequenced from Clostridium spp. [25]. The amino acid sequences of cellulases in Xoo were compared to those of members of glycosyl hydrolase family GH5 (endoglucanases) in a wide range of microorganisms, including X. campestris pv. campestris, Erwinia chrysanthemi, Ralstonia solanacearum, and Streptomyces lividans; the sequences were obtained from GenBank (Table 2). The sequences of EglXoA, EglXoB, and EglXoC showed 79.8%, 61.4%, and 59.6% identity, respectively, to endoglucanase 3 from X. campestris pv. campestris; further, they showed high identity (>80%) to endoglucanases 1, 2, and 3 from X. campestris pv. vesicatoria and X. axonopodis pv. citri (data not shown) but low identity (<26%) to endoglucanases from other Xanthomonas species. 3.2. Pathogenicity of cellulase genes The 12 cellulase genes shown in Table 1 were PCR-amplified, cloned, and disrupted by transposon insertion; further, marker exchange was performed with target genes of wild type strain KACC10859. Finally, marker-exchange mutants, namely, eglXoA::Tn5, eglXoB::Tn5, eglXoC::Tn5, celbXoA::Tn5, celXoB::Tn5, bglXoA::Tn5, bglXoB::Tn5, bglXoC::Tn5, bglXoD::Tn5, bglXoE::Tn5, bglXoF::Tn5, and bglXoG::Tn5 were selected for this study.

A

Verification of the transposon insertions in the target genes and single transposon insertion in each gene was further confirmed by PCR and Southern blot analysis. Fig. 1A represents PCR analysis of eglXo genes. Primers targeting eglXoA eglXoB and eglXoC amplified PCR fragments of the expected sizes with 1.3 kb, 1.1 kb and 1.1 kb in wild type strain. The transposon insertional mutants in eglXo genes were greater than those from wild type strain KACC10859 (1221 bp), which indicated that the transposon inserted eglXo genes were exchanged with the target genes in wild type strain by homologous recombination. In the case of the complementation clone CeglXoA, two PCR amplicons of sizes 1.3 kb and 2.6 kb, which were derived from pMLeglA and eglXoA::Tn5, were obtained from the genomic DNA sample of CeglXoA. To further investigate the number of Tn5 insertions in the target genes, genomic DNA was extracted from both wild type and mutant strains, and DNA from the mutant strains was digested with EcoRI, which does not cleave the transposon. These samples were subjected to Southern hybridization analysis by using labeled eglXoA and Tn5 genes as the probes. Fig. 1B shows the Southern blot analysis of mutant eglXoA::Tn5. The probe eglXoA gene detected the hybridized bands around 4.3 kb and 6.5 kb in the genomes of KACC10859 and the mutant eglXoA::Tn5 (Fig. 1B(a)). On the other hand, a single hybridized band of 6.5 kb was detected in genomic DNA of the mutant by probe transposon, but was not in the wild type Xoo KACC10331 (Fig. 1B (b)). The result indicated that a single copy of the transposon had been inserted in each target gene. The growth rate of the mutants on PSA was largely similar to that of the wild

B

Fig. 1. PCR (A) and Southern blot (B) analysis of eglXo mutants. Genomic DNA was extracted from wild type strain KACC10859 and eglXo marker-exchange mutants and PCR-amplified by using primer sets targeting eglXo genes. For Southern blot hybridization, the genomic DNA of the eglXoA::Tn5 mutant was digested with EcoRI and electrophoresed on agarose gel, blotted onto nylon membranes, and hybridized with probes eglXoA gene (a) and transposon (b). Lanes M are 1-kb ladder.

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Fig. 2. Pathogenicity assay of cellulase genes in Xanthomonas oryzae pv. oryzae KACC10859. Pathogenicity values are represented as the mean  standard deviation from 3 independent experiments.

type strain, which indicated that the cellulase genes did not affect the growth of Xoo. Furthermore, the pathogenicity of the mutant strains was assayed on leaves of susceptible rice variety, Milyang 23, by using the leaf-punch method. The results, as shown in Fig. 2, suggested that the mutant strains eglXoA::Tn5, eglXoB::Tn5, and celXoB::Tn5 were completely virulence-deficient. In addition, mutants celbXoA::Tn5, bglXoC::Tn5, and bglXoF::Tn5 showed attenuated pvirulece, while the virulence of other mutants such as eglXoC::Tn5 was not affected. Moreover, mutants with phenotypes EglXoA, EglXoB, and CelXoB completely lost their pathogenicity, which suggested that these genes were absolutely required for pathogenicity. The association of the eglXoB gene with virulence has already been reported [11], but that of the eglXoA, celXoB, celbXoA, bglXoC, and bglXoF genes still remains to be determined. To confirm the pathogenicity the novel virulence genes, pML122 harboring each gene were introduced into mutant strains. Pathogenecity of the complementation clones, CeglXoA CcelXoB, CcelbXoA, CbglXoC, and CbglXoF was assayed using rice leaves. They recovered virulence producing disease lesion comparing to that of the wild type strain KACC10859, which confirmed that the genes are required for pathogenicity. Fig. 3 shows disease symptoms caused by Xoo strains with mutations in eglXoA/eglXoB/eglXoC. Nonpathogenic disease symptoms were noted on rice leaves infected with eglXoA::Tn5 and eglXoB::Tn5, while the wild type strain and eglXoC::Tn5 was associated with typical disease lesions with brown strip.In conclusion, in the present study, it was shown that eglXoA celXoB, celbXoA, bglXoC, and bglXoF were novel cellulase genes associated with pathogenicity in Xoo.

components and their variously modified forms. Therefore, the high number of cellulase genes in the Xoo genome may be required for pathogenicity. In this study, we induced mutations in 12 cellulase genes by transposon mutagenesis for identifying the cellulase genes involved in the pathogenicity of Xoo. We found that eglXoA, celXoB, celbXoA, bglXoC, and bglXoF are novel cellulase genes associated with pathogenicity. Mutations in the eglXoA and celXoB genes resulted in complete virulence deficiency. This was logical, since bacterial blight is a vascular disease and Xoo multiplies and spreads in the xylem vessel, where xylan and cellulose are abundant [6]. These enzymes enhance the ability of pathogens to penetrate the plant tissue and also enhance the degradation of the plant cell wall, which can further be utilized for the nutrition of phytopathogens. Among the phytopathogenic bacteria, R. solanacearum [17,24] and Clavibacter michiganensis subsp. michiganensis [12] produce endoglucanases that are required for pathogenicity. In addition, cellulase-deficient mutants of C. michiganensis subsp. sepedonicus showed markedly reduced pathogenicity to the eggplant [15]. The celV1 gene of Erwinia carotovora subsp. carotovora SCC3193, encoding a secreted cellulase (CelV1), was cloned, and its

4. Discussion Plant cell walls consist of complex components, which shape the physical barrier for pathogen defense. The majority of these components are proteins, lignin, and exopolysaccharides such as cellulose, hemicelluloses and pectin. The bacterium enters its host through hydathodes and wounds around the leaf edges and then multiplies and spreads in xylem vessels, which results in disease [6]. Genes encoding cell-degrading enzymes, including cellulases and xylanases, have been considered to play a role in the pathogenicity of Xoo [13,21,22,31]. Xoo probably needs a large number of hydrolytic enzymes to degrade the different plant cell wall

Fig. 3. Disease symptoms of mutant and wild type strains. Inoculation was done by using the leaf-punch method. Pathogenicity was checked 14 days after inoculation. CeglXoA is the complementation strain of the eglXoA mutant. Water was used as the negative control.

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nucleotide sequence was determined [18]. The celV1 mutants exhibited reduced pathogenicity, which suggested that CelV1 enhances the ability of strain SCC3193 to macerate the plant tissue, although it is not absolutely required for pathogenicity. Mutants of X. campestris B1459 that were defective in the secretion of both cellulase and amylase were isolated, and they showed reduced pathogenicity for turnip seedlings [29]. X. campestris pv. campestris, the cause of black rot of crucifers, has genes for 4 pectate lyases, 5 xylanases, and 9 cellulases [5]. However, the role of cellulase genes in the pathogenicity of X. campestris pv. campestris has not yet been elucidated. In this study, we found that EglXoA and CelXoB contained a putative signal peptide at the N-terminus, which suggested that they are extracellular enzymes that degrade the plant cell wall. Digestion of plant cell walls by pathogens may have multiple consequences. Generally, extracellular enzymes such as cellulases, pectinases, proteases, and xylanases produced by phytopathogenic bacteria are secreted into the extracellular environment by the general secretory pathway (GSP) [19,20], which is also called the T2SS. Generally, protein secretion in Xanthomonas is performed through the T2SS associated with the xps gene cluster, and the secretion of the aforementioned extracellular enzymes usually involves the GSP encoded by the xps gene cluster [10]. Homologs of the Xps system (xpsEFGHIJKLMN and xpsD) were identified in the Xoo genome and showed >79% amino acid identity to their counterparts in other Xanthomonas strains [16]. In the GSP, proteins are secreted from the cytosol to the extracellular environment in 2 steps. Null mutations in the genes encoding these proteins block the secretion of degradative enzymes from bacterial cells, which causes a substantial loss of pathogenicity [22]. Similarly, a Xoo GSP mutant, which was not able to secrete xylanase, showed reduced pathogenicity to the rice plant [22]. Purified cellulases and lipases induced defense responses in rice, and the induction of these responses was suppressible by Xoo in a T3S-dependent manner [13]. Cell-wall degrading enzymes have been reported to be effective at eliciting defense responses, and thus, several other hydrolytic enzymes of Xoo also might induce host defense responses. Furthermore, hrpXo regulates the transcriptional expressions of genes associated with T3S proteins such as cysteine proteases [7]. In the case of other Xanthomonas species, 2 virulence genes (pghAxc and pghBxc) encoding functional polygalacturonase from X. campestris pv. campestris 8004 are regulated by HrpX [32]. Moreover, hrpX negatively regulates the a-amylase isozymes in X. axonopodis pv. citri [33]. Therefore, it was considered that cellulase genes participate in diverse virulence functions that are directly or indirectly associated with the expressions of main virulence genes such as the hrp genes. Thus, further genetic and biochemical studies need to be performed to investigate the expression of novel virulence genes and their role in the pathogenesis of bacterial blight in rice. Acknowledgments This research was supported by a grant from the Agenda research program (Code #200901FHT020710285) in the Rural Development Administration of Korea. References [1] Barras F. Exracellular enzymes and pathogenesis of soft-rot Erwinia. Annu Rev Phytopathol 1994;32:201e34. [2] Chan JW, Goodwin PH. The molecular genetics of virulence of Xanthomonas campestris. Biotech Adv 1999;17:489e580. [3] Claeyssens M, Henrissat B. Specificity mapping of cellulolytic enzymes: classification into families of structurally related proteins confirmed by biochemical analysis. Protein Sci 1992;1:1293e7.

[4] Coughlan MP, Mayer F. The cellulose-decomposing bacteria and their enzyme systems. In: Balows A, Trüper HG, Dworkin M, Harder W, Schleifer KH, editors. The prokaryotes: a handbook on the biology of bacteria. 2nd ed. New York: Springer-Verlag; 1992. p. 460e516. [5] da Silva AC, Ferro JA, Reinach FC, Farah CS, Furlan LR, Quaggio RB, et al. Comparison of genomes of two Xanthomonas pathogens with differing host specificities. Nature 2002;417:459e63. [6] Ezuka A, Kaku H. A historical review of bacterial blight of rice. Bull Natl Inst Agrobiol Resour Jpn 2000;15:1e207. [7] Furutani A, Tsuge S, Ohnishi K, Hikichi Y, Oku T, Tsuno K, et al. Evidence for HrpXo-dependent expression of type II secretory proteins in Xanthomonas oryzae pv. oryzae. J Bacteriol 2004;186:1374e80. [8] He YQ, Zhang L, Jiang BL, Zhang ZC, Xu RQ, Tang DJ, et al. Comparative and functional genomics reveals genetic diversity and determinants of host specificity among reference strains and a large collection of Chinese isolates of the phytopathogen Xanthomonas campestris pv. campestris. Genome Biol 2007;8:R218. [9] Henrissat B, Bairoch A. Updating the sequence-based classification of glycosyl hydrolases. J Biochem 1996;316:695e6. [10] Hu J, Qian W, He C. The Xanthomonas oryzae pv. oryzae eglXoB endoglucanase gene is required for virulence to rice. FEMS Microbiol Lett 2007;269:273e9. [11] Hu NT, Hung MN, Chiou SJ, Tang F, Chiang DC, Huang HY, et al. Cloning and characterization of a gene required for the secretion of extracellular enzymes across the outer membrane by Xanthomonas campestris pv. campestris. J Bacteriol 1992;174:2679e87. [12] Jahr H, Dreier J, Meletzus D, Bahro R, Eichenlaub R. The endo-beta-1,4glucanase CelA of Clavibacter michiganensis subsp. michiganensis is a pathogenicity determinant required for induction of bacterial wilt of tomato. Mol Plant Microbe Interact 2000;13:703e14. [13] Jha G, Rajeshwari R, Sonti RV. Functional interplay between two Xanthomonas oryzae pv. oryazae secretion systems in modulating virulence on rice. Mol Plant Microbe Interact 2007;20:31e40. [14] Kang HW, Park YJ, Lee BM. Functional description and identification of virulence related genes from whole genome sequence of Xanthomonas oyzae pv. oryzae KACC10331. Kor Jour Microbiol 2009;44:1e9. [15] Laine MJ, Haapalainen M, Wahlroos T, Kankare K, Nissinen R, Kassuwi S, et al. The cellulase encoded by the native plasmid of Clavibacter michiganensis ssp. sepedonicus plays a role in virulence and contains an expansin-like domain. Physiol Mol Plant Pathol 2000;57:221e33. [16] Lee BM, Park YJ, Park DS, Kang HW, Kim JG, Song ES, et al. The genome sequence of Xanthomonas oryzae pathovar oryzae KACC10331, the bacterial blight pathogen of rice. Nuci Acids Res 2005;33:577e86. [17] Liu H, Zhang S, Schell MA, Denny TP. Pyramiding unmarked deletions in Ralstonia solanacearum shows that secreted proteins in addition to plant cellwall-degrading enzymes contribute to virulence. Mol Plant Microbe Interact 2005;18:1296e305. [18] Mäe A, Heikinheimo R, Palva ET. Structure and regulation of the Erwinia carotovora subspecies carotovora SCC3193 cellulase gene celV1 and the role of cellulase in phytopathogenicity. Mol Gen Genet 1995;247:17e26. [19] Pugsley AP. The complete general secretory pathway in gram-negative bacteria. Microbiol Rev 1993;57:50e108. [20] Pugsley AP, Francetic O, Possot OM, Sauvonnet N, Hardie KR. Recent progress and future directions in studies of the main terminal branch of the general secretory pathway in gram-negative bacteria e a review. Gene 1997;192: 13e9. [21] Rajeshwari R, Jha G, Sonti RV. Role of an in planta-expressed xylanase of Xanthomonas oryzae pv. oryzae in promoting virulence on rice. Mol Plant Microbe Interact 2005;18:830e7. [22] Ray SK, Rajeshwari R, Sonti RV. Mutants of Xanthomonas oryzae deficient in general secretory pathway are virulent deficient and unable to secrete xylase. Mol Plant Microbe Interact 2000;13:394e401. [23] Roberts K. The plant extracellular matrix:In a net expansive mood. Curr Opin Cell Biol 1994;6:688e94. [24] Saile E, McGarvey JA, Schell MA, Denny TP. Role of extracellular polysaccharide and endoglucanase in Root invasion and Colonization of tomato plants by Ralstonia solanacearum. Phytopathology 1997;87: 1264e71. [25] Sakka M, Goto M, Fujino T, Fujino E, Karita S, Kimura T, et al. Analysis of a Clostridium josui cellulase gene cluster containing the man5A gene and characterization of recombinant Man5A. Biosci Biotech Biochem 2010;74:2077e82. [26] Sambrook J, Russell DW. Molecular cloning: a laboratory manual. 3rd ed. Cold Spring Harbor, NY, USA: Cold Spring Harbor Press; 2001. [27] Shen Y, Ronald P. Molecular determinants of disease and resistance in interactions of Xanthomonas oryzae pv. oryzae and rice. Microbes Infect 2002;4: 1361e7. [28] Sun QH, Hu J, Huang GX, Ge C, Fang RX, He CZ. Type-II secretion pathway structural gene xpsE, xylanase- and celllulase secretion and virulence in Xanthomonas oryzae pv. oryzae. Plant Pathol 2005;54:15e21. [29] Thorne L, Gosink KK, Pollock TJ. Mutants of Xanthomonas campestris defective in secretion of extracellular enzymes. J Ind Microbiol 1989;4:135e44. [30] Vian B, Reis D, Gea L, Grimault V. The cell Wall, first barrier or interface for microorganism: in site approaches to understanding interactions. In: Nicole M, Gianinazzi-Pearson V, editors. Histology, ultrastructure and molecular cytology of plantemicroorgantsms interactions. Dordrecht: Kluwer Academic Publisher; 1996. p. 99e115.

U. Temuujin et al. / Physiological and Molecular Plant Pathology 76 (2011) 152e157 [31] Wang JC, So BH, Kim JH, Park YJ, Lee BM, Kang HW. Genome-Wide Identification of pathogenicity genes in Xanthomonas oryzae pathovar oryzae by Transposon Mutagenesis. Plant Pathol 2008;57:1136e45. [32] Wang L, Wei R, He C. Two Xanthomonas extracellular polygalacturonases, PghAxc and PghBxc, are regulated by type III secretion regulators HrpX and

157

HrpG and are required for virulence. Mol Plant Microbe Interact 2008;21: 555e63. [33] Yamazaki A, Hirata H, Tsuyumu S. Type III regulators HrpG and HrpXct control synthesis of a-amylase, which is involved in planta multiplication of Xanthomonas axonopodis pv.citri. J Gen Plant Pathol 2008;74:254e7.