Proteome wide identification of iron binding proteins of <Emphasis Type="Italic">Xanthomonas translucens</Emphasis> pv. <Emphasis Type="Italic">undulosa</Emphasis>: focus on secretory virulent proteins

Xanthomonas translucens pv. undulosa (Xtu) causes Bacterial Leaf Streak disease in the staple food crops such as wheat and barley. The survival strategies of pathogen and host are determined by the complex interactions occurring between the host plants and the pathogenic microbes. Iron binding proteins are important in the plant–microbe interactions as they are indulged in enzyme catalysis, virulence, metabolic and transport activities. In the presented study, we have identified that ~9.8% of Xtu proteome possess iron binding sequence motifs. Further, the analysis of Xtu proteome for secretory iron binding virulent proteins (IBVPs) revealed the fact that iron co-regulate the function of secretory proteins in virulence. We have found 26 secretory IBVPs and observed that these proteins are diverse in their biological functions ranging from transport to antimicrobial resistance, Reactive oxygen species detoxification and carbohydrate catabolism. The inferences may instigate to design the new strategies to combat and control the microbial diseases of staple food crops.     

High-resolution mapping and gene prediction of <Emphasis Type="Italic">Xanthomonas Oryzae</Emphasis> pv. <Emphasis Type="Italic">Oryzae</Emphasis> resistance gene <Emphasis Type="Italic">Xa7</Emphasis>

Bacterial blight (BB) caused by Xanthomonas oryzae pv. oryzae (Xoo) is a devastating disease in rice worldwide. The resistance gene Xa7, which provides dominant resistance against the pathogen with avirulence (Avr) gene AvrXa7, has proved to be durably resistant to BB. A set of SSR markers were selected from the “gramene” database based on the Xa7 gene initial mapping region on chromosome 6. These markers were used to construct a high-resolution genetic map of the chromosomal region surrounding the Xa7 gene. An F₂ mapping population with 721 highly susceptible individuals derived from a cross between the near isogenic lines (NILs) IRBB7 and IR24 were constructed to localize the Xa7 gene. In a primary analysis with eleven polymorphic SSR markers, Xa7 was located in approximately the 0.28-cM region. To walk closer to the target gene, recombinant F₂ individuals were tested using newly developed STMS (sequence tagged microsatellite) markers. Finally, the Xa7 gene was mapped to a 0.21-cM interval between the markers GDSSR02 and RM20593. The Xa7-linked markers were landed on the reference sequence of cv. Nipponbare through bioinformatics analysis. A contig map corresponding to the Xa7 gene was constructed. The target gene was assumed to span an interval of approximately 118.5-kb which contained a total of fourteen genes released by the TIGR Genome Annotation Version 5.0. Candidate-gene analysis of Xa7 revealed that the fourteen genes encode novel domains that have no amino acid sequence similar to other cloned Xa(xa) genes. Shen Chen and Zhanghui Huang are contributed equally to this work.     

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.     

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.     

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.     

<Emphasis Type="Italic">STAYGREEN</Emphasis> (<Emphasis Type="Italic">CsSGR</Emphasis>) is a candidate for the anthracnose (<Emphasis Type="Italic">Colletotrichum orbiculare</Emphasis>) resistance locus <Emphasis Type="Italic">cla</Emphasis> in Gy14 cucumber

Key message

Map-based cloning identified a candidate gene for resistance to the anthracnose fungal pathogen Colletotrichum orbiculare in cucumber, which reveals a novel function for the highly conserved STAYGREEN family genes for host disease resistance in plants.

Abstract

Colletotrichum orbiculare is a hemibiotrophic fungal pathogen that causes anthracnose disease in cucumber and other cucurbit crops. No host resistance genes against the anthracnose pathogens have been cloned in crop plants. Here, we reported fine mapping and cloning of a resistance gene to the race 1 anthracnose pathogen in cucumber inbred lines Gy14 and WI 2757. Phenotypic and QTL analysis in multiple populations revealed that a single recessive gene, cla, was underlying anthracnose resistance in both lines, but WI2757 carried an additional minor-effect QTL. Fine mapping using 150 Gy14?×?9930 recombinant inbred lines and 1043 F₂ individuals delimited the cla locus into a 32 kb region in cucumber Chromosome 5 with three predicted genes. Multiple lines of evidence suggested that the cucumber STAYGREEN (CsSGR) gene is a candidate for the anthracnose resistance locus. A single nucleotide mutation in the third exon of CsSGR resulted in the substitution of Glutamine in 9930 to Arginine in Gy14 in CsSGR protein which seems responsible for the differential anthracnose inoculation responses between Gy14 and 9930. Quantitative real-time PCR analysis indicated that CsSGR was significantly upregulated upon anthracnose pathogen inoculation in the susceptible 9930, while its expression was much lower in the resistant Gy14. Investigation of allelic diversities in natural cucumber populations revealed that the resistance allele in almost all improved cultivars or breeding lines of the U.S. origin was derived from PI 197087. This work reveals an unknown function for the highly conserved STAYGREEN (SGR) family genes for host disease resistance in plants.

     

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.     

<Emphasis Type="Italic">Ty</Emphasis>1<Emphasis Type="Italic">/copia</Emphasis>- and <Emphasis Type="Italic">Ty</Emphasis>3<Emphasis Type="Italic">/gypsy</Emphasis>-like DNA sequences in <Emphasis Type="Italic">Helianthus </Emphasis>species

Two repeated DNA sequences isolated from a partial genomic DNA library of Helianthus annuus, p HaS13 and p HaS211, were shown to represent portions of the int gene of a Ty3 /gypsy retroelement and of the RNase-Hgene of a Ty1 /copia retroelement, respectively. Southern blotting patterns obtained by hybridizing the two probes to BglII- or DraI-digested genomic DNA from different Helianthus species showed p HaS13 and p HaS211 were parts of dispersed repeats at least 8 and 7 kb in length, respectively, that were conserved in all species studied. Comparable hybridization patterns were obtained in all species with p HaS13. By contrast, the patterns obtained by hybridizing p HaS211 clearly differentiated annual species from perennials. The frequencies of p HaS13- and p HaS211-related sequences in different species were 4.3x10(4)-1.3x10(5) copies and 9.9x10(2)-8.1x10(3) copies per picogram of DNA, respectively. The frequency of p HaS13-related sequences varied widely within annual species, while no significant difference was observed among perennial species. Conversely, the frequency variation of p HaS211-related sequences was as large within annual species as within perennials. Sequences of both families were found to be dispersed along the length of all chromosomes in all species studied. However, Ty3 /gypsy-like sequences were localized preferentially at the centromeric regions, whereas Ty1/ copia-like sequences were less represented or absent around the centromeres and plentiful at the chromosome ends. These findings suggest that the two sequence families played a role in Helianthusgenome evolution and species divergence, evolved independently in the same genomic backgrounds and in annual or perennial species, and acquired different possible functions in the host genomes.     

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.     

Niches and routes of transmission of <Emphasis Type="Italic">Xanthomonas citri</Emphasis> pv. <Emphasis Type="Italic">fuscans</Emphasis> to bean seeds

Aims

Seeds are vectors of a diversified microbiota including plant pathogens. To better understand transmission of common bacterial blight (CBB) agents to bean seeds, we analyzed the role of non-pathogenic xanthomonads on seed transmission efficiency and investigated the location of Xanthomonas citri pv. fuscans (Xcf) into seeds and plantlets.

Methods

Competition between CBB and NP strains was initially assessed in vitro and then extended in planta to monitor the impact of co-inoculation on Xcf seed transmission. Moreover, location of Xcf strains in seeds and seedlings was visualized using a combination of gfp-tagged strain and DOPE-FISH/CSLM.

Results

Whereas CBB agent growth was inhibited in vitro by some seed-borne non-pathogenic xanthomonads strains, these strains did not transmit efficiently to seed through floral pathway and did not affect Xcf seed transmission. Xcf cells were observed entering seed through vascular elements and parenchyma of funiculus, but also micropyle and testa. Xcf cells were observed, moreover, among other bacteria on radicle surfaces, especially tip, in cotyledons, and plumules.

Conclusions

CBB agents are more efficient than non-pathogenic xanthomonads in using the floral route to colonize seeds. CBB agents are located within different niches in the seed tissues up to the embryonic axis.

     

Populations of <Emphasis Type="Italic">Xanthomonas citri</Emphasis> pv. <Emphasis Type="Italic">mangiferaeindicae</Emphasis> from Asymptomatic Mango Leaves Are Primarily Endophytic

Terrestrial organic carbon is exported to freshwater systems where it serves as substrate for bacterial growth. Temporal variations in the terrigenous organic carbon support for aquatic bacteria are not well understood. In this paper, we demonstrate how the combined influence of landscape characteristics and hydrology can shape such variations. Using a 13-day bioassay approach, the production and respiration of bacteria were measured in water samples from six small Swedish streams (64° N, 19° E), draining coniferous forests, peat mires, and mixed catchments with typical boreal proportions between forest and mire coverage. Forest drainage supported higher bacterial production and higher bacterial growth efficiency than drainage from mires. The areal export of organic carbon was several times higher from mire than from forest at low runoff, while there was no difference at high flow. As a consequence, mixed streams (catchments including both mire and forest) were dominated by mire organic carbon with low support of bacterial production at low discharge situations but dominated by forest carbon supporting higher bacterial production at high flow. The stimulation of bacterial growth during high-flow episodes was a result of higher relative export of organic carbon via forest drainage rather than increased drainage of specific “high-quality” carbon pools in mire or forest soils.     

A Simple Method for In Vivo Expression Studies of <Emphasis Type="Italic">Xanthomonas axonopodis</Emphasis> pv. <Emphasis Type="Italic">citri</Emphasis>

A major problem in studying bacterial plant pathogens is obtaining the microorganism directly from the plant tissue to perform in vivo expression (protein or mRNA) analyses. Here we report an easy and fast protocol to isolate Xanthomonas axonopodis pv. citri directly from the host plant, in sufficient amounts to perform protein fingerprinting by 2-D gel electrophoresis as well as RNA expression assays. The protein profile obtained was very similar to that of X. axonopodis pv. citri grown in the presence of a leaf extract of Citrus sinensis; however, some differential proteins expressed in vivo were observed. Total RNA extraction revealed typical 16S and 23S bands in the agarose gel, and RT-PCR reactions using primers specific for genes of the bacterium confirmed the quality of the RNA preparation. Also, RT-PCR reactions using plant ribosomal primers were employed, and no amplification product was obtained, indicating that plant RNA is not present in the bacterium RNA sample.     

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.