alexa Expressional Regulation of the Virulence Gene eglXoA Encoding Endoglucanase, Dependent on HrpXo and Cyclic AMP Receptor-Like Protein (Clp) in Xanthomonas oryzae pv. oryza
ISSN: 2157-7471
Journal of Plant Pathology & Microbiology

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Expressional Regulation of the Virulence Gene eglXoA Encoding Endoglucanase, Dependent on HrpXo and Cyclic AMP Receptor-Like Protein (Clp) in Xanthomonas oryzae pv. oryza

Temuujin U1 and Kang HW2,3*

1Department of Biotechnology and Breeding, Mongolian University of Life science, Ulaanbaatar 17024, Mongolia

2Department of Horticulture, Hankyong National University, Ansung 17579, Korea

3Institute of Genetic Engineering, Hankyong National University, Ansung 17579, Korea

*Corresponding Author:
Kang HW
PhD, Professor, Department of Horticulture
Hankyong National University, Ansung 17579
Tel: 82-31-670-5420
E-mail: [email protected]

Received date: July 26, 2017; Accepted date: August 12, 2017; Published date: August 15, 2017

Citation: Temuujin U, Kang HW (2017) Expressional Regulation of the Virulence Gene eglXoA Encoding Endoglucanase, Dependent on HrpXo and Cyclic AMP Receptor-Like Protein (Clp) in Xanthomonas oryzae pv. oryza. J Plant Pathol Microbiol 8: 416. doi: 10.4172/2157-7471.1000416

Copyright: © 2017 Temuujin U, et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

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An eglXoA encoding endoglucanase that clustered with eglXoB and eglXoC in Xanthomonas oryzae pv. oryza genome (accession No. AE013598) is a pathogenicity related gene. RT-PCR showed that the in trans eglXoA was transcriptionally regulated by HrpX, a type III secretion regulator, and cyclic AMP receptor-like protein in X. oryzae pv. oryzae (ClpXo), which has been known as a global regulator. Western blot analysis showed that EglXoA is secreted via a type II secretion system and was detected in wild-type strain KACC10859, but not in the mutant strains hrpX::Tn5 and clpXo::Tn5. In an electrophoretic mobility shift assay, the promoter region of eglXoA directly bound to ClpXo. The two consensus eglXoA upstream regions were found to include putative Clp-binding sites with a perfect TCACA-N block in the left arm and a 2/5 matched block, TGT, in the right arm. eglXoA, which encodes endoglucanase, appears to be the first gene of Xoo known to be activated by ClpXo via direct binding to the promoter region. Molecular interaction between HrpX and ClpXo shows that ClpXo acts as transcription regulator of hrpX and binds to promoter region of hrpX.


ClpXo; eglXoA gene; Expression; HrpX; Xanthomonas oryzae pv. oryzae


Xanthomonas oryzae pv. oryzae (Xoo), which causes bacterial leaf blight, is the most economically important bacterial disease in rice. Bacterial blight is prevalent in many rice-growing countries [1]. During pathogenesis, plant cell walls act as the first barrier of defense against bacterial invasion and multiplication. Nevertheless, the enzymatic activities of Cell Wall-Degrading Enzymes (CWDEs) may facilitate pathogen invasion into the cells of host plants by digesting the plant cell walls [2-4]. Research regarding CWDEs in phytopathogenic bacteria has mainly focused on enzymatic activities, the identification of genes encoding them, and their roles in virulence [5,6]. The type II secretion system (T2SS) allows most gram-negative bacteria to secrete extracellular hydrolytic enzymes and toxins [7,8], many of which are responsible for pathogenesis in plants, into their surroundings and hosts. CWDEs such as cellulases, pectinases, xylanases, and proteases are secreted by plant pathogens to degrade the components of host cell walls and may play a crucial role in virulence and bacterial nutrition [9,10]. Currently, genes encoding CWDEs such as cellulase and xylanase are thought to play a role in the virulence of Xoo [11-14]. The T2SS-related gene cluster consists of 11 genes, xpsEFGHIJKLMND, in the Xoo genome. Mutations in the xpsD and xpsF structural genes of the Xoo T2SS reduce virulence and cause xylanase accumulation in the periplasmic space [5,12].

The hrp genes encode proteins involved in the type III secretion system (T3SS), which is involved in the secretion of effector proteins from bacteria to plants [15]. The hrp gene cluster in Xoo is composed of 27 genes, from hrpA2 to hrpF [16], and the expression of these genes is regulated by two regulators, hrpG and hrpX, which are separate from the hrp gene cluster [17]. HrpG belongs to the OmpR family and activates the expression of hrpX, an AraC-like transcription activator that controls hrp genes along with some effector proteins [17,18]. Moreover, HrpXo regulates the transcriptional expression of genes associated with T2SS proteins such as cysteine proteases [19]. Recently, it was reported that polygalacturonase and extracellular proteases in X. campestris pv. campestris (Xcc) are regulated by HrpX [20,21]. These reports suggest that HrpX can potentially regulate expression of other genes in addition to the hrp genes. It has been reported that purified cellulase and lipase induce defense responses in rice that are suppressible by Xoo in a T3SSdependent manner [22]. Therefore, it is plausible that genes encoding CWDEs can participate in diverse virulence functions associated directly or indirectly with the expression of key pathogenicity-related genes, such as hrp genes.

Catabolite-Activator Protein (CAP), also called the cAMP receptor protein (CRP)-like protein (Clp), belongs to the CRP/FNR superfamily of transcriptional factors, which is one of the largest groups of bacterial environmental sensors [23-25]. Also, it has been known to be a global transcriptional regulator for the expression of virulence factors in Xcc. Clp transcriptionally activates more than 150 genes, including those that encode for extracellular enzymes and the production of exopolysaccharides (EPS), and other macromolecules such as flagellin and Hrp proteins [24]. Clp contains nucleotide- and DNA-binding domains and binds to the promoters of an endoglucanase (engA) from Xcc [26].

Recently, 12 genes that encode cellulases, including endoglucanases and exoglucanases, were isolated from the Xoo genome and mutated, and novel pathogenicity-related cellulase genes were identified and characterized [14]. Interestingly, the eglXoABC genes arranged as a cluster in genome of X. oryzae pv. oryzae. Of them, it was revealed that transposon insertion mutant of eglXoA and eglXoA displayed virulence-deficient phenotype, but not in the eglXoB. However, little is known about expressional regulation of eglXo genes in the pathogenesis of Xoo. The goal of this study was to elucidate expressional regulation of eglXoA. We demonstrate that HrpX and ClpXo act as regulators of expression of eglXoA. Furthermore, the electrophoretic mobility shift assay (EMSA) showed that ClpXo binds to the promoter region of eglXoA.

Materials and Methods

Bacterial strains, plasmids, and culture conditions

The bacterial strains and plasmids used in this study are listed in Table 1. Wild-type strain Xoo KACC10859 was obtained from the Korean Agricultural Culture Collection (KACC) at the National Institute of Agricultural Biotechnology, Suwon, Korea. Xoo strains were cultured at 28°C on peptone sucrose agar (PSA: peptone, 10 g/L; sucrose, 10 g/l; and agar, 15 g/L) or XOM2 medium [27]. E. coli was grown in Luria-Bertani (LB) broth (Difco, Detroit, MI, USA) at 37°C for 18 h. Antibiotics were added at the following final concentrations for E. coli and Xoo, respectively: ampicillin, 80 μg/mL and 50 μg/mL; gentamycin, 50 μg/mL and 20 μg/mL; and kanamycin, 50 μg/mL and 20 μg/mL.

Bacterial strains, plasmids and PCR primers Characteristics References
Bacterial strains
E. coliBL21 (DE3) fhuA2 [lon] ompT gal (λ DE3) [dcm] ∆hsdSλ DE3=λ sBamHIo ∆EcoRI-B int::(lacI::PlacUV5::T7 gene1) i21 ∆nin5 Lab collection
Xanthomonas oryzae pv.oryzaestrains
KACC10859 Wild type strain, Korean race 1 KACC
eglxoA::Tn5 Transposon insertion in eglXoA of KACC10859, Kmr This study
clpXo::Tn5 Transposon insertion in clpXo of KACC10859, Kmr This study
hrp X::Tn5 Transposon insertion in hrpXof KACC10859, Kmr This study
xps F::Tn5 Transposon insertion in xpsF of KACC10859, Kmr This study
CeglXoA eglXoA::Tn5 harboring  pMLeglXoA, Gm rKm r  
pGEM-TEasy vector T- cloning vector, Ampr Promega
pQE-80L Overexpression vector, Ampr Lab collection
pQE-clpXo pQE-80L harboring clpXo, Ampr This study
pET15b Overexpression vector, Ampr Novagen
pET-eglXoA pET15b harboring eglxoA,Ampr This study
pML122 Broad-host-range vector, Gmr Lab collection
pMLeglxoA pML122 harboring eglxoA This study
Primers for RT-PCR
This study
This study
This study
This study
This study
Primer for probes used in EMSA
hrpX Probe 1: F: 5’-CTT ACA TAA CGG GCA TGT GGG-3’
This study
eglXoA Probe 1: F: 5’- GTG CTC ATC TGA AAA CTC CGG -3’
Probe3: F: 5’- ACG CAG CAG CCG ATC ACC CTG -3’
This study

Table 1: Bacterial strains, plasmids and PCR primers used in the study.

Pathogenicity assays

Inoculums (approximately 1 × 106 cells/ml) prepared from wild-type and mutant strains of Xoo were grown on PSA for 3 days. Pathogenicity assays were performed on 60-days-old leaves of a susceptible rice cultivar (Milyang 23) by the leaf-punching method by Temujin et al. [14]. Pathogenicity was observed at 14 days post inoculation.

Northern blot analysis

Xoo strains were first incubated in NB to OD600=1.0 and then collected by centrifugation. The pelleted cells were washed twice with XOM2 media and resuspended in XOM2 to OD600=0.5 and cultured in XOM2 to OD600=1.0. The total RNAs were extracted with TRIzol Reagent (Invitrogen, Carlsbad, CA, USA) and electrophoresed. Probes were labeled with the DIG Northern Starter Kit (Roche) according to the manufacturer’s protocol. Hybridization signals were detected on exposure of the samples to X-ray film (Fujifilm, Tokyo, Japan).

Reverse transcription (RT)-PCR analysis

The transposon mutants and wild-type Xoo KACC10859 were cultured in nutrient broth (NB) until OD600=0.5, pelleted by centrifugation at 3,000 g for 10 min, and washed with distilled water. The bacterial cells were suspended in 5 mL of XOM2 medium and additionally cultured in a shaking incubator (180 rpm) at 28°C for 36 h. Total RNA was extracted by Trizol Reagent according to manufacturer’s instructions and treated with RNase-free water. Then, DNase I (Promega, Madison, WI, USA) was used to remove potential traces of DNA according to the manufacturer’s instructions. The cDNA synthesis and PCR were conducted using a SuperScript First-Strand RT-PCR kit (Invitrogen) with the RT-PCR primers listed in Table 1 under the following conditions: 1 cycle of 1 min at 94°C; 30 cycles of 30 sec at 94°C, 30 secs at 60°C, 1 min at 72°C; and a final extension cycle of 10 min at 72°C. PCR products were visualized in agarose gels by staining with ethidium bromide.

Overexpression and purification of EglXoA and ClpXo

eglXoA was amplified from genomic DNA of Xoo using a forward primer (eglXoA-F: 5′-CAGAATCTCATATGTCCAACCGCACCAC-3′) containing an NdeI restriction site (underlined) at the start codon of the ORF and a reverse primer (eglXoA-R: 5′-CTGCTCGAGTCAATTTTGATTCACCAAC-3′) containing an XhoI restriction site after the stop codon. The PCR amplicons were double digested with NdeI and XhoI, ligated into the pET-15b expression vector (Novagen) containing a 6× His tag upstream of a thrombin cleavage site and the multiple cloning site, and transformed into E. coli BL21 (DE3) pLysS, yielding the recombinant clone pET-eglA. To construct the clpXo overexpression vector, clpXo was amplified with the forward primer containing a BamHI restriction site (underlined) at the start codon of the ORF clpXo-F: (5′-GGATCCATGAGCTCAGCAAAC-3′) and the reverse primer clpXo-R: (5′-AAGCTTTTAGCGCGTGCCGTA-3′) containing a Hind III restriction site spanning the stop codon. The PCR product was digested with BamHI and Hind III, ligated in the pQE-80L vector, and then transformed into E. coli BL21 (DE3) pLysS, yielding recombinant clone pQE-ClpXo. For protein overexpression of EglXoA and ClpXo in E. coli, clones pET-eglXoA and pQE-ClpXo were grown in 1 L of LB liquid medium containing ampicillin at 37°C until OD600=0.5 and overexpression was induced by adding 0.5 mM IPTG for 3 h. To purify EglXoA, the bacterial cells were pelleted and suspended in 200 mL buffer (20 mM Tris, 5 mM imidazole, pH 8.0), sonicated for 2 min at 20 kHz and the acoustic power ranged between 35 W and 95 W, and centrifuged at 6,500 g for 15 min. The supernatant was loaded on a column packed with nickel-nitrilotriacetic acid (Ni-NTA) equilibrated with buffer solution (20 mM Tris-HCl, 0.5 M NaCl, 5 mM imidazole, 8.0 M urea, pH 8.0). The column was first washed with the same buffer containing 50 mM imidazole and the fusion protein was then eluted using a 250-mM imidazole in the same buffer. To purify ClpXo, the bacterial cells were pelleted and suspended in 200 mL buffer (300 mM NaCl, 50 mM NaH2PO4, 10 mM imidazole, pH 8.0), sonicated 6 times, and centrifuged at 6,500 g for 15 min. The pelleted cells were washed with 0.5% Triton X-100 in 1 × PBS and solubilized by the buffer. The mixture was centrifuged at 6,500 g for 15 min, and the supernatant purified using an Ni-NTA column as for EglXoA.

Western blot analysis

The 30 N-terminal amino acids (NH2- LILYQKNAKAAELSKKILGLQAQDLPGNLA) corresponding to residues 98 to 127 in EglXoA were deduced from nucleotide sequence data and synthesized, and antiserum against the peptide was commercially produced (Peptron, Daejeon, Korea). To isolate, extraction of extracellular secreted proteins from bacterial cells, Xoo strains were precultured in 5 mL NB media for 3 days and pelleted and then suspended in 30 mL XOM2 liquid media and cultured additionally to OD600=0.8. The bacterial cells were harvested by centrifugation at 15,000 rpm for 20 min and further filtrated by membrane filter (0.45 μm pore size) to remove remnant cells. The supernatant was precipitated by 30% ammonium sulfate addition on ice for 30 min. After centrifugation at 15,000 rpm for 30 min, protein precipitates were washed with ester and suspended in 1/30 original volume of 50 mM Tris-HCl (pH 8.0) and resuspended in 2x Laemmli buffer. Protein samples were boiled for 5 min and separated by 10% of sodium dodecyl sulfate SDS-PAGE by CBB and Silver staining methods. For western blot analysis, protein samples from different Xoo strains were electrophoresed in 12% SDS-PAGE gels and transferred onto a polyvinylidene fluoride (PVDF) membrane (GE Healthcare, Little Chalfont, UK). The blotted membrane was incubated at room temperature for 2 h in a blocking solution and then hybridized with anti-EglXo antibody. After 3 washes with the washing solution, the PVDF membrane was incubated with anti-rabbit secondary antibody HRF (GE Healthcare) for 1 h according to the manufacturer’s protocol. The signals on the membrane were detected by exposure to X-ray film (Fujifilm).

Electrophoretic mobility shift assay

The DNA probes used for the EMSA were prepared by PCR amplification of the desired regions of the eglXoA promoter under the following conditions: 1 cycle of 4 min at 94°C; 30 cycles of 30 secs at 94°C, 30 secs at 58°C, 1 min at 72°C; and a final extension cycle of 10 min at 72°C, using 5′-end biotin-labeled synthetic oligonucleotides as the primers in Table 1. The amplicons were purified from agarose gels and used for gel-shift experiments. The EMSA reaction mixture (10 μL) contained ca. 1.0 pmol of biotin-labeled probe and various amounts of ClpXo in a 5 × binding buffer containing 1 μg of nonspecific competitor DNA poly d(I-C) (Panomics, Inc). Following incubation at room temperature for 5 min, the DNA-protein complexes were resolved by electrophoresis in a 6% non-denaturing polyacrylamide gel in 0.5 × TBE buffer (40 mM Tris-Cl, pH 8.3, 45 mM boric acid, 1 mM EDTA). The gel was transferred onto a positively charged Hybond N+ nylon membrane (GE Healthcare) for 30 min at 300 mA. After UV cross-linking, biotinylated probes in the membrane were detected corresponding to protocol provided by the EMSA Gel Shift Kit (Panomics, Inc).


eglXoA is essential for the virulence and its expression is nonpolar

The virulence of the eglXoA mutant strain assayed on leaves of a susceptible rice variety, Milyang 23. The degree of pathogenicity was checked in 14 days after inoculation. Figure 1, shows the disease symptom of brown stripes was observed on rice leaf inoculated with the wild-type strain. In contrast, the mutant strains eglXoA::Tn5 was virulence deficient. Mutation was confirmed by complementation with the entire sole eglXoA gene. The complemented X. oryzae pv. oryzae strain CeglXoA::Tn5 was constructed by introducing the recombinant plasmid pMLeglxoA containing the entire wild-type eglXoA gene into the mutant eglXoA::Tn5 (Table 1). Consequently, CeglXoA::Tn5 recovered virulence and produced disease lesions similar to those of the wild-type strain KACC10859 (Figure 1), suggesting that eglXoA is essential for the virulence.


Figure 1: Pathogenicity assay of eglXoA in a gene cluster encoding endoglucanases of Xanthomonas oryzae pv. oryzae. The mutant strain eglXoA::Tn5, wild type strain KACC10859 and complement strain pMLeglA were inoculated on leaves of rice variety, Milyang 23 using leaf-punching method and pathogenicity was checked after 14 days of inoculation. Distilled water (DW) was used as negative control.

The eglXoA was located upstream eglXoB and it was observed long intergenic regions of 682 bp between eglXoA and eglXoB and 649 bp between eglXoB and eglXoC (Figure 1). The transcriptional linkage in the gene cluster was analyzed by RT-PCR. Two cDNA products synthesized from total RNA samples of eglXoA::Tn5 and wild-type strain KACC10859 were used as template. The primer pair targeting eglXoABC genes amplified RT-PCR products of the predicted length in the wild-type RNA sample, while mutation in eglXoA did not affect transcriptional expression of eglXoB and eglXoB, as they continued to generate RT-PCR products (Figure 2). These results reasonably assumed that each eglXoA, eglXoB, and eglXoC is monocistronically transcribed without polar expressional fashion dependent on a promoter.


Figure 2: Reverse transcription (RT)-PCR analysis of eglXo genes of Xanthomonas oryzae pv. oryzae on transcriptome of eglXoA::Tn5 and wild type strains.

eglXoA expression is regulated by HrpX and ClpXo

In this experiment, the in trans transcriptional regulation of eglXoA was investigated with regard to HrpX and ClpXo. The total RNA samples were extracted from the mutants and wild-type strain KACC10859 after cultivation in hrp-inducing XOM2 medium. RT-PCR analysis was performed by using primer pairs targeting eglXoA. The primer set targeting 16S rRNA, which was used as a positive control, amplified relatively intense RT-PCR bands from the wild-type strain as well as the hrpX::Tn5 and clpXo::Tn5 mutants. On the other hand, the primer pair targeting eglXoA amplified the RT-PCR band only in the wild-type strain KACC10859 RNA sample and not in those of the hrpX::Tn5 and clpXo::Tn5 mutants (Figure 3A). These results indicate that transcriptional expression of eglXoA is regulated by hrpX and clpXo.

An N-terminal peptide consisting of 30 amino acids corresponding to residues 98 to 127 of the protein (357 amino acids) was inferred from the nucleotide sequence of eglXoA (1,074 bp), which was artificially synthesized, and polyclonal antibodies were raised against the peptide. The wild-type strain KACC10859 as well as xpsF::Tn5, clpXo::Tn5, and hrpX::Tn5 mutant strains were cultured in XOM2 medium. Their extracellular proteins were isolated, resolved using SDS-PAGE, and then transferred to PVDF membranes. In western blot analysis using anti-EglXoA antibodies, the predicted 37-kDa band was detected in the positive controls, the extracellular protein of the wildtype strain, and the purified protein, but not in extracellular proteins of the xpsF::Tn5, hrpX::Tn5, clpXo::Tn5, and eglXoA::Tn5 mutants (Figure 3B). The putative signal peptide consisting of 38 amino acids was observed on deduced sequence of eglXoA (Figure 3). Consequently, these findings suggest that EglXoA is a secreted protein dependent on T2SS and regulated by HrpX and ClpXo.


Figure 3: Expression of eglXoA dependent on HrpXo and ClpXo. Total RNA samples were extracted from wild-type strain KACC10859, hrpX::Tn5, and clpXo::Tn5. RT-PCR was conducted using a primer pair targeting eglXoA. (A) For western blot analysis, (B) the extracellular proteins were obtained from different Xanthomonas oryzae pv. oryzae strains cultured in XOM2 medium that were blotted onto PVDF membranes and then probed with an anti-EglXoA antibody.

ClpXo binds to the promoter region of eglXoA

RT-PCR and western blot analyses showed that ClpXo regulates transcriptional expression of eglXoA and correspondingly that the gene encoding the protein was not expressed. It has been reported that the Clp protein directly binds to the promoter regions of the endoglucanase (engA) and polygalacturonase (pheA) genes of X. campestris, presumably enhancing transcriptional expression [26,28]. Therefore, it is reasonable to assume that ClpXo can also bind to the promoter region of eglXoA. EMSA was employed to evaluate the binding between soluble ClpXo protein and the promoter region of eglXoA. To obtain this protein, clpXo (693 bp) was subcloned into plasmid pQE-80L, yielding the recombinant clone pQE-Clp, which was then overexpressed in E. coli. The soluble ClpXo protein (27.7 kDa) was obtained by purification with a His-binding affinity column.

Promoter regions (probe A, −316/+127; probe B, −263/+127; and probe C, −85/+127) upstream of eglXoA were generated by nested PCR (Figure 4A). The EMSA was performed with 50 ng of ClpXo protein per reaction. The protein-DNA complex was electrophoresed in a 6% non-denaturing polyacrylamide gel, blotted onto a nylon membrane, and hybridized with biotin-labeled promoter regions. A probe B-ClpXo protein complex was formed, which migrated more slowly than the unbound probe B (Figure 4B). ClpXo do not bind to reside in the region −85/+127 and thus the region −256/−85 appears to possess the complete sequence for the binding of ClpXo. The E. coli CRP-binding site (5′-AAATGTGA-N6-TCACATTT-3′) is 22-bp long, exhibiting perfect 2-fold sequence symmetry, with the bold-faced bases representing the left and right arms for the binding of one subunit of the active CRP dimer. Two putative Clp-binding sites, including a perfect TCACA-N block in the right arm and a 2/5 matched block, TGT, in the left arm, were found from −133 to−153 sequences (Figure 4C).


Figure 4: Electrophoretic mobility shift assay (EMSA) evaluating the binding of the eglXoA promoter region to the ClpXo protein. Different promoter regions upstream of eglXoA, probes A, B, and C, (A) bound to ClpXo. The probe B-ClpXo complex was retarded in SDS-PAGE, (B) The promoter sequence upstream of eglXoA, (C) includes putative ClpXo-binding sites (Boxed sequence), predicted consensus sequence, −10/−35, putative Ribosome-Binding Site (RBS). Amino acids of N-teminal; indicate putative signal peptide.

ClpXo regulates transcriptional expression of hrpX

In our results, it was revealed genes, hrpX and ClpXo are closely related to eglXoA expression. However, it was wonder how hrpX and ClpXo is interracially associated on each gene expression. To investigate expressional control on both genes, Northern blot hybridization was done by using probes hrpX and ClpXo against total RNA samples that were extracted from the hrpX::Tn5 and ClpXo::Tn5 mutants and wildtype strain. Probe ClpXo hybridized to RNA samples of wild-type and hrpX::Tn5 strains, but not to ClpXo::Tn5 (Figure 5). However, probe hrpX detected hybridized signal on wild-type strain KACC10858, but not on the ClpXo::Tn5 and hrpX::Tn5 mutant strains, suggesting ClpXo regulates transcriptional expression of hrpX. Furthermore, the EMSA was conducted to determine if the promoter region of the hrpX gene binds to ClpXo, three types promoter regions (-539/+9, -429/+9 and -238/+9) generating probes A, B and C reacted with ClpXo protein of 50 ng. The protein-DNA complex showing retarded gel migration observed on probe A and B, but not on probe C (Figures 6A and 6B), suggesting ClpXo directly binds to promoter region of hrpXo. The putative ClpXo binding sequence sites with TGTCG-N-TCACA, including a perfect TCACA block in the right arm and a 4/5 matched block, TGTCG, in the left arm, were found in the −388/−409 sequences (Figure 6C).


Figure 5: Northern blot profile of X. oryzae pv. oryzae strains using probes hrpX and clpXo. Total RNA was extracted from wild-type KACC10859 and mutants hrpX::Tn5 and clpXo::Tn5. For northern blot analysis, total RNA was loaded in each lane of 1.5% formaldehyde agarose gels, blotted onto nylon membranes, and hybridized with hrpX and clpXo genes.


Figure 6: Electrophoretic Mobility Shift Assay (EMSA) for evaluating the binding of hrpX promoter region using probe ClpXo. The promoter regions (−539/+19) upstream hrpX, (A) was reacted with ClpXo protein (50 ng), the ClpXo protein-DNA complex was electrophoresed in a polyacrylamide gel, and the DNA-protein complex formation was observed as a retardation of migration in the gel, (B) The sequence region upstream hrpX, (C) includes ClpXo-binding sites (boxed sequence), predicted consensus sequence, −10/−35 and putative ribosome-binding site (RBS); capitalized sequence: initial codon (ATG).


Novel pathogenicity-related genes including eglXoA, which encodes endoglucanase, have been isolated and characterized [14]. Whole genome sequence information of Xoo KACC10331 is available on NCBI’s GenBank. eglXoA (1074 bp), eglXoB (1133 bp), and eglXoB (1131 bp) are organized as a cluster in the same region of the Xoo KACC10331 genome [16]. eglXoA was classified into cellulase family 5, which exhibits endo-1,4-glucanase activities, and had identity over 88.8% to endoglucanase genes, egl1 from X. campestris pv. vesicatoria [14].

Currently, genes encoding CWDEs such as cellulase and xylanase are thought to play a role in the virulence of Xoo [12,14,29]. It has been reported that purified cellulase and lipase proteins induce defense responses in rice that are suppressible by Xoo in a T3SS-dependent manner [22]. Therefore, cellulase genes may play a role in diverse virulence traits that are directly or indirectly associated with the expression of key pathogenicity-related genes, such as hrp genes.

HrpX is the key regulator in the T3SS that controls the expression of hrp and some effector genes. Previous studies have revealed that expression of the virulence genes pghAxc and pghBxc, which encode extracellular polygalacturonase in Xcc, are regulated by the T3SS regulator HrpX [20]. It was also reported that hrpX negatively regulates the α-amylase isozymes in X. axonopodis pv. citri [29] and extracellular proteases in Xcc [21]. In Xoo, only a gene encoding the extracellular T2SS enzyme Cysp2, which is related to pathogenicity, has so far been demonstrated to be regulated by HrpX [19]. These reports support that HrpX is also involved in regulating the expression of some T2SSrelated extracellular enzymes. Using RT-PCR, we discovered that transcriptional products of eglXoA from Xoo did not amplify in the hrpX mutant, showing HrpX-dependent expression. This finding strongly indicates that HrpX is the key regulator of eglXoA expression. The HrpX regulons of Xanthomonas species include a consensus sequence motif called the PIP box (TTCGC-N15-TTCGC) around the promoter regions [30,31]. However, the PIP box was not observed in eglXoA. The PIP box can be an effective marker for screening HrpX regulons from the entire genomic sequence database, and several of these regulons are predicted to be involved in the pathogenicity of xanthomonads and R. solanacearum [31]. Twelve and 20 candidate genes for HrpX regulons, which did not include the genes in hrp clusters, were found in Xanthomonas campestris pv. campestris and Xanthomonas axonopodis pv. citri [32], respectively. However, genes with an imperfect PIP box and genes without a PIP box have been found to be expressed in an HrpX-dependent manner [33]. This regulation by HrpX indicates that the signal transduction networks of pathogens are cross-linked and that the T3SS and T2SS may cooperate via various regulators to promote virulence of the pathogen in the host. Xanthomonas protein secretion (xps) genes encode structural proteins that form the T2SS, which is essential for the secretion of T2SS extracellular enzymes [11,12]. Immunoblot analysis using anti-eglXoA antibodies in this study provided direct evidence of T2SS/Xps-dependent secretion in the culture media of wild-type Xoo and xps mutants.

The transcription factor Clp is a member of a conserved globalregulator family that regulates the expression of approximately 300 genes involved in pathogenesis of Xanthomonas spp. [23]. Clp is a homologue (45% amino acid sequence identity) of the model transcription factor CRP of E. coli. Clp in Xcc also influences the expression of a number of genes, especially the genes in the T2SS [23,24]. The clp gene in Xoo was isolated and characterized [34], and a mutation in ClpXo resulted in a significant decrease in the production of cellulase, xylanase, and EPS. Moreover, a previous study demonstrated the direct binding of Clp to promoter regions [26]. The data from the present study indicate that eglXoA is regulated by ClpXo, which is consistent with previous Xcc studies that show that Clp is involved in the expression of extracellular enzymes of the T2SS [24,26]. Experimental evidence was provided by EMSA, wherein the ClpXo-eglXoA promoter region complex showed gel retardation, indicating that ClpXo directly binds to the eglXoA promoter region. In sequence analysis, a potential Clp-binding site (GTGTT-N9-TCACA) was identified in the eglXoA promoter region. The Clp in X. campestris is homologous to the CRP of E. coli. It was reported that Clp upregulates the transcription of endoglucanaseencoding engA in X. campestris by direct binding to the upstream region of Clp. Two consensus Clp-binding sites were determined on the engA promoter region by site-directed mutagenesis. In this study, two putative Clp-binding sites, including a perfect TCACA-N block in the left arm and a 2/5 matched block in the right arm, TGT, were found in sequence upstream of eglXoA. ClpXo bound to the promoter region that possesses the Clp-binding sites, whereas the promoter region that does not contain Clp-binding sites does not result in a DNA-protein complex, assuming that these sites are responsible for ClpXo binding to DNA. The transcriptional regulator Clp contains nucleotide- and DNAbinding domains that bind to promoters of target genes; DNA binding of Clp from X. axonopodis pv. citri is inhibited in vitro by cyclic di-GMP [35]. In X. campestris pv. campestris, Clp induces the expression of genes belonging to the diffusible signal factor (DSF) regulon, which encodes extracellular enzymes, components of T2SS and T3SS, and genes involved in EPS synthesis. Previous findings regarding DSF-dependent quorum sensing, including the transcriptional self-regulation of Clp, depict a detailed DSF signaling model of the regulation of bacterial virulence. Consequently, the cellular level of free Clp increases, then the regulator acts as a positive transcription factor to induce its own gene transcription and virulence gene expression.


In present study, we concluded that two regulatory genes, hrpX and ClpXo, are interracially associated with expression of the endoglucanase gene eglXoA of X. oryzae pv. oryzae. Furthermore, we investigated the interplay between hrpX and ClpXo. In northern blot analysis, ClpXo mutant inhibited transcriptional expression of hrpX, suggesting ClpXo acts as transcription regulator of hrpX expression. In EMSA, we found that the hrpX promoter region directly binds to the ClpXo protein (Figure 6B). In conclusion, our results suggest that ClpXo leads to dual regulation by binding to the promoter regions of hrpX and eglXoA. In the future, it would be interesting to evaluate how the binding target sequences of ClpXo play a role in regulating expression of eglXoA, either as an activator or an enhancer.


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