<Emphasis Type="Italic">In planta</Emphasis> gene expression analysis of <Emphasis Type="Italic">Xanthomonas oryzae</Emphasis> pathovar <Emphasis Type="Italic">oryzae</Emphasis>, African strain MAI1

Background  

Bacterial leaf blight causes significant yield losses in rice crops throughout Asia and Africa. Although both the Asian and African strains of the pathogen, Xanthomonas oryzae pv. oryzae (Xoo), induce similar symptoms, they are nevertheless genetically different, with the African strains being more closely related to the Asian X. oryzae pv. oryzicola (Xoc).     

Detection of <Emphasis Type="Italic">Xanthomonas oryzae</Emphasis> pv. <Emphasis Type="Italic">oryzae</Emphasis> in seeds using a specific TaqMan probe

Xanthomonas oryzae pv. oryzae is the pathogen that causes bacterial leaf blight in rice. Bacterial leaf blight is the main cause for severe rice underproduction in many countries. However, with conventional methods it is difficult to quickly and reliably distinguish this pathogen from other closely related pathogenic bacteria, especially X. oryzae pv. oryzicola, the causal organism of bacterial leaf streak in rice. We have developed a novel and highly sensitive real-time method for the identification of this specific bacteria based on a TaqMan probe. This probe is designed to recognize the sequence of a putative siderophore receptor gene cds specific to X. oryzae pv. oryzae, and can be identified from either a bacterial culture or naturally infected rice seeds and leaves in only 2 h. The sensitivity of the method is 100 times higher than that of the current polymerase chain reaction (PCR) gel electrophoresis method for diagnosis.     

Fine genetic mapping of <Emphasis Type="Italic">xa24</Emphasis>, a recessive gene for resistance against <Emphasis Type="Italic">Xanthomonas oryzae</Emphasis> pv. <Emphasis Type="Italic">oryzae</Emphasis> in rice

Bacterial blight, caused by Xanthomonas oryzae pv. oryzae (Xoo), is the most devastating plant bacterial disease worldwide. Different bacterial blight resistance (R) genes confer race-specific resistance to different strains of Xoo. We fine mapped a fully recessive gene, xa24, for bacterial blight resistance to a 71-kb DNA fragment in the long arm of rice chromosome 2 using polymerase chain reaction-based molecular markers. The xa24 gene confers disease resistance at the seedling and adult stages. It mediates resistance to at least the Philippine Xoo races 4, 6 and 10 and Chinese Xoo strains Zhe173, JL691 and KS-1-21. Sequence analysis of the DNA fragment harboring the dominant (susceptible) allele of xa24 suggests that this gene should encode a novel protein that is not homologous to any known R proteins. These results will greatly facilitate the isolation and characterization of xa24. The markers will be convenient tools for marker-assisted selection of xa24 in breeding programs. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Effect of modified glucose catabolism on xanthan production in <Emphasis Type="Italic">Xanthomonas oryzae</Emphasis> pv. <Emphasis Type="Italic">oryzae</Emphasis>

In this study, the glucose 6-phosphate dehydrogenase gene (XOO2314) was inactivated in order to modulate the intracellular glucose 6-phosphate, and its effects on xanthan production in a wild-type strain of Xanthomonas oryzae were evaluated. The intracellular glucose 6-phosphate was increased from 17.6 to 99.4 μmol g⁻¹ (dry cell weight) in the gene-disrupted mutant strain. The concomitant increase in the glucose 6-phosphate was accompanied by an increase in xanthan production of up to 2.23 g l⁻¹ (culture medium). However, in defined medium supplemented with 0.4% glucose, the growth rate of the mutant strain was reduced to 52.9% of the wild-type level. Subsequently, when a family B ATP-dependent phosphofructokinase from Escherichia coli was overexpressed in the mutant strain, the growth rate was increased to 142.9%, whereas the yields of xanthan per mole of glucose remained approximately the same.     

Identification and characterization of two <Emphasis Type="Italic">uvrA</Emphasis> genes of <Emphasis Type="Italic">Xanthomonas axonopodis</Emphasis> pathovar citri

Two uvrA-like genes, designated uvrA1 and uvrA2, that may be involved in nucleotide excision repair in Xanthomonas axonopodis pv. citri (X. a. pv. citri) strain XW47 were characterized. The uvrA1 gene was found to be 2,964 bp in length capable of encoding a protein of 987 amino acids. The uvrA2 gene was determined to be 2,529 bp with a coding potential of 842 amino acids. These two proteins share 71 and 39% identity, respectively, in amino acid sequence with the UvrA protein of Escherichia coli. Analyses of the deduced amino acid sequence revealed that UvrA1 and UvrA2 have structures characteristic of UvrA proteins, including the Walker A and Walker B motifs, zinc finger DNA binding domains, and helix-turn-helix motif with a polyglycine hinge region. The uvrA1 or uvrA2 mutant, constructed by gene replacement, was more sensitive to DNA-damaging agents methylmethane sulfonate (MMS), mitomycin C (MMC), or ultraviolet (UV) than the wild type. The uvrA1 mutant was four orders of magnitude more sensitive to UV irradiation and two orders of magnitude more sensitive to MMS than the uvrA2 mutant. The uvrA1uvrA2 double mutant was one order of magnitude more sensitive to MMS, MMC, or UV than the uvrA1 single mutant. These results suggest that UvrA1 plays a more important role than UvrA2 in DNA repair in X. a. pv. citri. Both uvrA1 and uvrA2 genes were found to be constitutively expressed in the wild type and lexA1 or lexA2 mutant of X. a. pv. citri, and treatment of these cells with sublethal dose of MMC did not alter the expression of these two genes. Results of electrophoresis mobility shift assays revealed that LexA1 or LexA2 does not bind to either the uvrA1 or the uvrA2 promoter. These results suggest that uvrA expression in X. a. pv. citri is not regulated by the SOS response system.     

Two coiled-coil regions of <Emphasis Type="Italic">Xanthomonas oryzae</Emphasis> pv. <Emphasis Type="Italic">oryzae</Emphasis> harpin differ in oligomerization and hypersensitive response induction

Hpa1_Xoo (harpin) is a type III secreted protein of the rice blight bacterial pathogen Xanthomonas oryzae pv. oryzae that elicits a hypersensitive response (HR) in nonhost tobacco. Hpa1_Xoo is predicted to contain two potential coiled-coil (CC) regions, one at the N-terminus with a high probability of formation, and one at the C-terminus with a lower probability of formation. We constructed several CC-equivalent peptides by a chemosynthetic method, and investigated the structure–function of the predicted Hpa1_Xoo CC regions, using biophysical and biochemical approaches. Both peptides elicited an HR in tobacco. Mutant versions of the N- and C-terminal peptides that were predicted to disrupt or favor CC formation were generated. The resulting altered HR activity and oligomerization indicated that the N-terminal CC region is essential for eliciting HR, but the C-terminus is not. The results also indicate that a 14-residue fragment (LDQLLCQLISALLQ) within the N-terminal CC region is a minimal and independent functional element for HR-induction in tobacco leaves. We propose that HR-induction requires a specific oligomerization of the CC regions of Hpa1_Xoo.     

Zinc Uptake Regulator (<Emphasis Type="Italic">zur</Emphasis>) Gene Involved in Zinc Homeostasis and Virulence of <Emphasis Type="Italic">Xanthomonas oryzae</Emphasis> pv. <Emphasis Type="Italic">oryzae</Emphasis> in Rice

Xanthomonas oryzae pv. oryzae causes bacterial leaf blight, one of the most widespread and destructive bacterial diseases in rice. In order to understand the gene of zinc uptake regulator (zur) involved in virulence of the pathogen in rice, we generated a mutant OSZRM by homologous suicide plasmid integration. The mutant failed to grow in NYGB medium supplemented with Zn²⁺ or Fe³⁺ at a concentration of 500 μM or 6 mM, whereas the wild-type strain grew well at the same conditions. The zur mutant was hypersensitive to hydrogen peroxide and exhibited reduction catalase activity and the production of extracellular polysaccharide (EPS). Interestingly, the mutant showed a reduction in virulence on rice but still kept triggering hypersensitive response (HR) in tobacco. When the mutant was complemented with the zur gene, the response was recovered to wild-type. These results suggested that zur gene is a functional member of the Zur regulator family that controls zinc and iron homeostasis, oxidative stress, and EPS production, which is necessary for virulence in X. oryzae pv. oryzae. Wanfeng Yang and Yan Liu contributed equally to this work     

Mutational analysis of the <Emphasis Type="Italic">gum</Emphasis> gene cluster required for xanthan biosynthesis in <Emphasis Type="Italic">Xanthomonas</Emphasis><Emphasis Type="Italic">oryzae</Emphasis> pv <Emphasis Type="Italic">oryzae</Emphasis>

Genome sequence analysis of Xanthomonas oryzae pv. oryzae has revealed a cluster of 12 ORFs that are closely related to the gum gene cluster of Xanthomonas campestris pv. campestris. The gum gene cluster of X. oryzae encodes proteins involved in xanthan production; however, there is little experimental evidence supporting this. In this study, biochemical analyses of xanthan produced by a defined set of X. oryzae gum mutant strains allowed us to preliminarily assign functions to most of the gum gene products: biosynthesis of the pentasaccharide repeating unit for GumD, GumM, GumH, GumK, and GumI, xanthan polymerization and transport for GumB, GumC, GumE, and GumJ, and modification of the pentasaccharide repeating unit for GumF, GumG, and GumL. In addition, we found that the exopolysaccharides are essential but not specific for the virulence of X. oryzae. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users. Sang-Yoon Kim and Jeong-Gu Kim contributed equally to this work.     

Characteristic expression of rice pathogenesis-related proteins in rice leaves during interactions with <Emphasis Type="Italic">Xanthomonas oryzae</Emphasis> pv. <Emphasis Type="Italic">oryzae</Emphasis>

Pathogenesis-related (PR) proteins play an important role in the disease resistance response. To better understand the function of rice PR proteins, we examined the expressions of ten PR proteins in rice leaves at different developmental stages with or without the interaction between rice and Xanthomonas oryzae pv. oryzae (Xoo). The results showed that most of the PR proteins were expressed in rice leaves in normal growth conditions, suggesting that they play a role in rice growth. Six out of ten PR proteins (PR1, PR2, PR3, PR4b, PR8, and PR-pha) showed enhanced expression in Xa21-mediated resistance responses at late stages after inoculation with Xoo. The remaining four PR proteins (PR5, PR6, PR15, and PR16) did not show changes in expression in the resistance response. The expressions of PR proteins in the resistance reaction were further compared with those in the susceptible reaction and a mock treatment. Interestingly, several of the PR proteins were expressed at the highest levels in the susceptible reaction and at the lowest levels in the mock treatment. Among the other four PR proteins, PR5 and PR16 showed changes in the abundance only in the susceptible response, while PR6 and PR15 showed no detectable difference in expression. These data provide fundamental knowledge about the expression of PR proteins in the interaction between rice and Xoo.     

Compositional Difference of the Exopolysaccharides Produced by the Virulent and Virulence-Deficient Strains of <Emphasis Type="Italic">Xanthomonas oryzae</Emphasis> pv. <Emphasis Type="Italic">oryzae</Emphasis>

Xanthomonas oryzae pv. oryzae, the bacterial blight pathogen of rice, is known to produce phytotoxic polysaccharides. The extracellular polysaccharide (EPS) was isolated from virulent (BXO1) and virulence-deficient gum G mutant (BXO1002) strains of X. oryzae pv. oryzae and characterized using fourier transform infrared (FT-IR) and nuclear magnetic resonance (NMR). Data from the FT-IR suggested that the aldehyde (R-CHO) group and C=O of acid anhydride are present in BXO1 but absent in BXO1002. The ¹H-NMR spectra showed the presence of an acetyl amine of hexose or pentose, free amines of glucose, an β-anomeric carbon of hexose and pentose, hydrogen next to hydroxyl group, an acetyl amine of hexose and pentose in the polysaccharides of both BXO1 and BXO1002, and the absence of α-anomeric carbon of hexose or pentose and the glucuronic acid in the polysaccharides produced by BXO1002. The test for glucuronic acid also confirmed the absence of glucuronic acid in the polysaccharides of BXO1002 and the presence glucuronic acid (32 μg/mg) in the polysaccharides produced by BXO1. Received: 14 May 2002 / Accepted: 21 June 2002     

Functional analysis of <Emphasis Type="Italic">pilQ</Emphasis> gene in <Emphasis Type="Italic">Xanthomanas oryzae</Emphasis> pv. <Emphasis Type="Italic">oryzae</Emphasis>, bacterial blight pathogen of rice

Bacterial blight (BB) of rice, caused by Xanthomonas oryzae pv. oryzae (Xoo), is the most devastating bacterial disease in rice. A virulence-attenuated mutant strain HNU89K9 of X. oryzae pv. oryzae (KACC10331), with a transposon insertion in the pilQ gene was used for this study. The pilQ was involved in the gene cluster pilMNOPQ of the Xoo genome. Growth rate of the pilQ mutant was similar to that of wild-type. At level of amino acids, PilQ of Xoo showed that a high sequence identities more than 94% and 70% to Xanthomonas species and to Xyllela fastidiosa, respectively but a low sequence homology less than 30% to other bacterial species. The twitching motility forming a marginal fringe on PSA media was observed on colony of the wild-type strain KACC10331, but not in mutant HNU89K9. Wild-type Xoo cells formed a biofilm on the surface of the PVC plastic test tube, while the mutant strain HNU89K9 did not form a biofilm. The results suggest that the pilQ gene of X. oryzae pv. oryzae plays a critical role in pathogenicity, twitching motility, and biofilm formation.     

Functional analysis of <Emphasis Type="Italic">Xa3/Xa26</Emphasis> family members in rice resistance to <Emphasis Type="Italic">Xanthomonas</Emphasis><Emphasis Type="Italic">oryzae</Emphasis> pv. <Emphasis Type="Italic">oryzae</Emphasis>

Plant disease resistant (R) genes are frequently clustered in the genome. The diversity of members in a complex R-gene family may provide variation in resistance specificity. Rice Xa3/Xa26, conferring resistance to Xanthomonas oryzae pv. oryzae (Xoo) encodes a leucine-rich repeat (LRR) receptor kinase-type protein and belongs to a multigene family, consisting of Xa3/Xa26, MRKa, MRKc and MRKd in rice cultivar Minghui 63. MRKa and MRKc are intact genes, while MRKd is a pseudogene. Complementary analyses showed that MRKa and MRKc could not mediate resistance to Xoo when regulated by their native promoters, but MRKa not MRKc conferred partial resistance to Xoo when regulated by a strong constitutive promoter. Plants carrying truncated XA3/XA26, which lacked the kinase domain, were compromised in their resistance to Xoo. However, the kinase domain of MRKa could partially restore the function of the truncated XA3/XA26 in resistance. MRKa and MRKc showed similar expression pattern as Xa3/Xa26, which expressed only in the vascular systems of different tissues. The expressional characteristic of MRKa and MRKc perfectly fits the function of genes conferring resistance to Xoo, a vascular pathogen. These results suggest that although MRKa and MRKc cannot mediate bacterial blight resistance nowadays, they may be once effective genes for Xoo resistance. Their expressional characteristic and sequence similarity to Xa3/Xa26 will provide templates for generating novel recognition specificity to face the evolution of Xoo. In addition, both LRR and kinase domains encoded by Xa3/Xa26 and MRKa are the functional determinants and MRKa-mediated resistance is dosage-dependent. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Rice bacterial blight pathogen <Emphasis Type="Italic">Xanthomonas oryzae</Emphasis> pv. <Emphasis Type="Italic">oryzae</Emphasis> produces multiple DSF-family signals in regulation of virulence factor production

Background  

Xanthomonas oryzae pv. oryzae (Xoo) is the causal agent of rice bacterial blight disease. Xoo produces a range of virulence factors, including EPS, extracellular enzyme, iron-chelating siderophores, and type III-secretion dependent effectors, which are collectively essential for virulence. Genetic and genomics evidence suggest that Xoo might use the diffusible signal factor (DSF) type quorum sensing (QS) system to regulate the virulence factor production. However, little is known about the chemical structure of the DSF-like signal(s) produced by Xoo and the factors influencing the signal production.     

Variation suggestive of horizontal gene transfer at a lipopolysaccharide (<Emphasis Type="Italic">lps</Emphasis>) biosynthetic locus in <Emphasis Type="Italic">Xanthomonas oryzae</Emphasis> pv. <Emphasis Type="Italic">oryzae</Emphasis>, the bacterial leaf blight pathogen of rice

Background  

In animal pathogenic bacteria, horizontal gene transfer events (HGT) have been frequently observed in genomic regions that encode functions involved in biosynthesis of the outer membrane located lipopolysaccharide (LPS). As a result, different strains of the same pathogen can have substantially different lps biosynthetic gene clusters. Since LPS is highly antigenic, the variation at lps loci is attributed to be of advantage in evading the host immune system. Although LPS has been suggested as a potentiator of plant defense responses, interstrain variation at lps biosynthetic gene clusters has not been reported for any plant pathogenic bacterium.     

<Emphasis Type="Italic">Trichoderma harzianum</Emphasis>: a biocontrol agent against <Emphasis Type="Italic">Bipolaris oryzae</Emphasis>

Rice brown spot, caused by Bipolaris oryzae, can be a serious disease causing a considerable yield loss. Trichoderma harzianum is an effective biocontrol agent for a number of plant fungal diseases. Thus, this research was carried out to investigate the mechanisms of action by which T. harzianum antagonizes Bipolaris oryzae in vitro, and the efficacy of spray application of a spore suspension of T. harzianum for control of rice brown spot disease under field conditions. In vitro, the antagonistic behavior of T. harzianum resulted in the overgrowth of B. oryzae by T. harzianum, while the␣antifungal metabolites of T.␣harzianum completely prevented the linear growth of B. oryzae. Light and scanning electron microscope (SEM) observations showed no evidence that mycoparasitism contributed to the aggressive nature of the tested isolate of T. harzianum against B. oryzae. Under field conditions, spraying of a spore suspension of T. harzianum at 10⁸ spore ml⁻¹ significantly reduced the disease severity (DS) and disease incidence (DI) on the plant leaves, and also significantly increased the grain yield, total grain carbohydrate, and protein, and led to a significant increase in the total photosynthetic pigments (chlorophyll a and b and carotenoids) in rice leaves.     

A novel thermostable and organic solvent-tolerant lipase from <Emphasis Type="Italic">Xanthomonas oryzae</Emphasis> pv. <Emphasis Type="Italic">oryzae</Emphasis> YB103: screening,purification and characterization

Thermostable lipases offer major biotechnological advantages over mesophilic lipases. In this study, an intracellular thermostable and organic solvent-tolerant lipase-producing strain YB103 was isolated from soil samples and identified taxonomically as Xanthomonas oryzae pv. oryzae. The lipase from X. oryzae pv. oryzae YB103 (LipXO) was purified 101.1-fold to homogeneity with a specific activity of 373.9 U/mg. The purified lipase showed excellent thermostability, exhibiting 51.1 % of its residual activity after incubation for 3 days at 70 °C. The enzyme showed optimal activity at 70 °C, suggesting it is a thermostable lipase. LipXO retained 75.1–154.1 % of its original activity after incubation in 20 % (v/v) hydrophobic organic solvents at 70 °C for 24 h. Furthermore, LipXO displayed excellent stereoselectivity (e.e._p >99 %) toward (S)-1-phenethyl alcohol in n-hexane. These unique properties of LipXO make it promising as a biocatalyst for industrial processes.