Identification of the flagellar chaperone FlgN in the phytopathogen <Emphasis Type="Italic">Xanthomonas axonopodis</Emphasis> pathovar <Emphasis Type="Italic">citri</Emphasis> by its interaction with hook-associated FlgK

Genome annotation of the plant pathogen Xanthomonas axonopodis pv. citri (Xac), identified flagellar genes in a 15.7 kb gene cluster. However, FlgN, a secretion chaperone for hook-associated proteins FlgK and FlgL, was not identified. We performed extensive screening of the X. axonopodis pv. citri genome with the yeast two-hybrid system to identify a protein with the characteristics of the flagellar chaperone FlgN. We found a candidate (XAC1990) encoded by an operon for components of the flagellum apparatus that interacted with FlgK. In order to further support this finding, Xac FlgK and XAC1990 were cloned, expressed, and purified. The recombinant proteins were characterized by spectroscopic methods and their interaction in vitro confirmed by pull-down assays. We, therefore, conclude that XAC1990 and its homologs in other Xanthomonas species are, in fact, FlgN proteins. These observations extend the sequence diversity covered by this family of proteins.     

Mining <Emphasis Type="Italic">Xanthomonas</Emphasis> and <Emphasis Type="Italic">Streptomyces</Emphasis> genomes for new pectinase-encoding sequences and their heterologous expression in <Emphasis Type="Italic">Escherichia coli</Emphasis>

Microbial genome sequencing has left a legacy of annotated yet uncharacterized genes or open reading frames, activities that may have useful applications in health and/or the environment. We are interested in the discovery and characterization of potentially new pectinolytic activities for the enzymatic retting of natural bast fibers such as hemp and flax. A highlight in this study is the discovery of a cold-active pectate lyase among five pectate-lyase-encoding sequences and two polygalacturonase-encoding sequences that we have cloned from the genomes of Xanthomonas campestris pv. campestris and Streptomyces coelicolor A3(2). Heterologous expression of these sequences as active pectate lyases and polygalacturonases required their subcloning in Escherichia coli Rosetta™ cells. The most active recombinant pectate lyase (XcPL NP_638163), a cold-active pectate lyase (XcPL NP_636037), and a polygalacturonase (XcPG NP_638805) were purified to near homogeneity and their kinetic parameters were determined. A significant amount of pectin degradation products was shown to be released by the two pectate lyases but not the polygalacturonase when hemp fiber pectin was used as substrate. Results of this study showed that genome data mining, besides an economical approach to new gene acquisition, may uncover new findings such as the discovery of a cold-active pectate-lyase-encoding sequence from X. campestris, a mesophilic microorganism.     

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.     

Identification of quantitative trait loci for resistance to <Emphasis Type="Italic">Xanthomonas campestris</Emphasis> pv. <Emphasis Type="Italic">campestris</Emphasis> in <Emphasis Type="Italic">Brassica rapa</Emphasis>

Resistance to six known races of black rot in crucifers caused by Xanthomonas campestris pv. campestris (Pammel) Dowson is absent or very rare in Brassica oleracea (C genome). However, race specific and broad-spectrum resistance (to type strains of all six races) does appear to occur frequently in other brassica genomes including B. rapa (A genome). Here, we report the genetics of broad spectrum resistance in the B. rapa Chinese cabbage accession B162, using QTL analysis of resistance to races 1 and 4 of the pathogen. A B. rapa linkage map comprising ten linkage groups (A01–A10) with a total map distance of 664 cM was produced, based on 223 AFLP bands and 23 microsatellites from a F₂ population of 114 plants derived from a cross between the B. rapa susceptible inbred line R-o-18 and B162. Interaction phenotypes of 125 F₂ plants were assessed using two criteria: the percentage of inoculation sites in which symptoms developed, and the severity of symptoms per plant. Resistance to both races was correlated and a cluster of highly significant QTL that explained 24–64% of the phenotypic variance was located on A06. Two additional QTLs for resistance to race 4 were found on A02 and A09. Markers closely linked to these QTL could assist in the transference of the resistance into different B. rapa cultivars or into B. oleracea.     

<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).     

Identification of genes differentially expressed in cauliflower associated with resistance to <Emphasis Type="Italic">Xanthomonas campestris</Emphasis> pv. <Emphasis Type="Italic">campestris</Emphasis>

Black rot, caused by Xanthomonas campestris pv. campestris (Pammel) Dowson (Xcc), is one of the most damaging diseases of cauliflower and other crucifers. In order to investigate the molecular resistance mechanisms and to find the genes related to black rot resistance in cauliflower, a suppression subtractive hybridization (SSH) cDNA library was constructed using resistant line C712 and its susceptible near-isogenic line C731 as tester and driver, respectively. A total of 280 clones were obtained from the library by reverse northern blotting. Sequencing analysis and homology searching showed that these clones represent 202 unique sequences. The library included many defense/disease-resistant related genes, such as plant defensin gene PDF1.2, lipid transfer protein, thioredoxin h. Gene expression profiles of 12 genes corresponding to different functional categories were monitored by real-time RT-PCR. The results showed that the expression induction of these genes in the susceptible line C712 in response to Xcc was quicker and more intense, while in C731 the reaction was delayed and limited. Our results imply that these up-regulated genes might be involved in cauliflower responses against Xcc infection. Information obtained from this study could be used to understand the molecular mechanisms of disease response in cauliflower under Xcc stress.     

Enhanced fructooligosaccharides and inulinase production by a <Emphasis Type="Italic">Xanthomonas campestris</Emphasis> pv. <Emphasis Type="Italic">phaseoli</Emphasis> KM 24 mutant

Xanthomonas campestris pv phaseoli produced an extracellular endoinulinase (9.24 ± 0.03 U mL⁻¹) in an optimized medium comprising of 3% sucrose and 2.5% tryptone. X. campestris pv. phaseoli was further subjected to ethylmethanesulfonate mutagenesis and the resulting mutant, X. campestris pv. phaseoli KM 24 demonstrated inulinase production of 22.09 ± 0.03 U mL⁻¹ after 18 h, which was 2.4-fold higher than that of the wild type. Inulinase production by this mutant was scaled up using sucrose as a carbon source in a 5-L fermenter yielding maximum volumetric (21,865 U L⁻¹ h⁻¹) and specific (119,025 U g⁻¹ h⁻¹) productivities of inulinase after 18 h with an inulinase/invertase ratio of 2.6. A maximum FOS production of 11.9 g L⁻¹ h⁻¹ and specific productivity of 72 g g⁻¹ h⁻¹ FOS from inulin were observed in a fermenter, when the mutant was grown on medium containing 3% inulin and 2.5% tryptone. The detection of mono- and oligosaccharides in inulin hydrolysates by TLC analysis indicated the presence of an endoinulinase. This mutant has potential for large-scale production of inulinase and fructooligosaccharides.     

Quantitative Measurements of <Emphasis Type="Italic">Xanthomonas Oryzae</Emphasis> pv. <Emphasis Type="Italic">Oryzae</Emphasis> Distribution in Rice Using Fluorescent-Labeling

The rice host sensor, XA21, confers robust resistance to most strains of Xanthomonas oryzae pv. oryzae (Xoo), the casual agent of bacterial blight disease. Using in planta fluorescence imaging of Xoo strain PXO99Az expressing a green fluorescent protein (Xoo-gfp) we show that XA21 restricts Xoo spread at the point of infection. This noninvasive and quantitative method to measure spatial distribution of Xoo populations in planta facilitates detailed assessment of plant disease resistance.     

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.     

Isolation and partial characterization of antibacterial lipopeptide produced by <Emphasis Type="Italic">Paenibacillus polymyxa</Emphasis> HKA-15 against phytopathogen <Emphasis Type="Italic">Xanthomonas campestris</Emphasis> pv. phaseoli M-5

An antibacterial metabolite was isolated from Paenibacillus polymyxa HKA-15, a soybean bacterial endophyte. The purification of the crude metabolite from Paenibacillus polymyxa HKA-15 was done by column chromatography. In TLC, a spot with an R _f value of 0.86 (±0.02) from the purified fraction showed bioactivity against Xanthomonas campestris pv. phaseoli M-5. In SDS-PAGE, the purified antibiotic was separated in the molecular weight range of 3.5 kDa. The exact molecular weight of the active compound was identified as 1,347.7 Da using MS-MS analysis. Infra red spectrum and ¹H NMR analysis showed the presence of amino acids and fatty acids in the active compound. The characterization of the antibacterial compound revealed its lipopeptide nature. In an agar diffusion assay, the crude metabolite showed a broad spectrum of activity, being able to inhibit the growth of the fungal pathogen, Rhizoctonia bataticola, Macrophomina phaseolina and Fusarium udum. A stronger inhibition was observed against bacterial pathogens viz., X. campestris pv.phaseoli M-5, X. campestris pv. phaseoli CP-1-1, Xanthomonas oryzae, Ralstonia solanacearum and Micrococcus luteus.     

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.     

Genetic and Pathogenic Variability of Indian Strains of <Emphasis Type="Italic">Xanthomonas campestris</Emphasis> pv. <Emphasis Type="Italic">campestris</Emphasis> Causing Black Rot Disease in Crucifers

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.     

Patterns of DNA-Sequence Divergence Between <Emphasis Type="Italic">Drosophila miranda</Emphasis> and <Emphasis Type="Italic">D. pseudoobscura</Emphasis>

Contrary to the classical view, a large amount of non-coding DNA seems to be selectively constrained in Drosophila and other species. Here, using Drosophila miranda BAC sequences and the Drosophila pseudoobscura genome sequence, we aligned coding and non-coding sequences between D. pseudoobscura and D. miranda, and investigated their patterns of evolution. We found two patterns that have previously been observed in comparisons between Drosophila melanogaster and its relatives. First, there is a negative correlation between intron divergence and intron length, suggesting that longer non-coding sequences may contain more regulatory elements than shorter sequences. Our other main finding is a negative correlation between the rate of non-synonymous substitutions (d _N) and codon usage bias (F _op), showing that fast-evolving genes have a lower codon usage bias, consistent with strong positive selection interfering with weak selection for codon usage.     

<Emphasis Type="Italic">SulP</Emphasis>, a nuclear gene encoding a putative chloroplast-targeted sulfate permease in<Emphasis Type="Italic"> Chlamydomonas reinhardtii</Emphasis>

Genomic, proteomic, phylogenetic and evolutionary aspects of a novel gene encoding a putative chloroplast-targeted sulfate permease of prokaryotic origin in the green alga Chlamydomonas reinhardtii are described. This nuclear-encoded sulfate permease gene (SulP) contains four introns, whereas all other known chloroplast sulfate permease genes lack introns and are encoded by the chloroplast genome. The deduced amino acid sequence of the protein showed an extended N-terminus, which includes a putative chloroplast transit peptide. The mature protein contains seven transmembrane domains and two large hydrophilic loops. This novel prokaryotic-origin gene probably migrated from the chloroplast to the nuclear genome during evolution of C. reinhardtii. The SulP gene, or any of its homologues, has not been retained in vascular plants, e.g. Arabidopsis thaliana, although it is encountered in the chloroplast genome of a liverwort (Marchantia polymorpha). A comparative structural analysis and phylogenetic origin of chloroplast sulfate permeases in a variety of species is presented.     

<Emphasis Type="Italic">Xanthomonas gardneri</Emphasis> exoenzymatic activity towards plant tissue

Bacterial spot caused by Xanthomonas spp. is an important tomato and pepper disease worldwide. Recent outbreaks of bacterial spot disease in Central Brazil and Canada have been attributed to Xanthomonas gardneri, which is also recognized as group D of Xanthomonas campestris pv. vesicatoria. Carotenoid-like pigments called xanthomonadins, which are diagnostic for yellow Xanthomonas spp., were extracted from X. gardneri. It was shown that the model plant Arabidopsis thaliana, member of the Brassicaceae family, can develop disease symptoms in response to different isolates of X. gardneri. Secretion of enzymes has been shown to play an important role in pathogenicity for different pathogens, and to begin to understand the interaction of X. gardneri and A. thaliana, a biochemical analysis of secreted proteins in the presence of A. thaliana leaves was performed. Different enzymatic activities such as for cellulase, α-arabinofuranosidase, pectinase, invertase and xylanase were assayed. In the presence of leaves, cellulase activity was highest after 60 and 72 h of growth and α-arabinofuranosidase activity was detected between 12 and 72 h of growth. Pectinase, invertase and xylanase activities were not detected. Cellulase and α-arabinofuranosidase activities may be important for X. gardneri acquisition of plant nutrients through degradation of cellulose fibers and hemicellulose of the cell wall, respectively, to the invasion of the host tissue and/or may generate signal molecules that are recognized by the plant. This is the first study to address how X. gardneri responds to host plant tissue.     

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.     

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.