Gene expression analysis identifies hypothetical genes that may be critical during the infection process of Xanthomonas citri subsp. citri

Background: Gene expression analysis via microarray is widely used in phytobacteria to validate differential gene expression associated with virulence or to compare biological profiles of wild type and mutant strains. Here, we employed DNA microarrays to study the early stages of the infection process (24, 72 and 120 h post-inoculation) of Xanthomonas citri subsp. citri (Xac) infecting Citrus sinensis to interrogate the expression profiles of hypothetical genes. Results: Under infective conditions, 446 genes were up- and 306 downregulated. Outstanding among genes upregulated during infection were those involved in synthesizing the Type 3 Secretion System and effectors, xanthan gum and quorum-sensing induction, and flagellum synthesis and regulation. Additionally, 161 hypothetical genes were up- and 100 were downregulated, 49 of which are known to have a significant biological role. To understand hypothetical gene co-regulation or -expression, nine expression profiles including 158 genes were identified during the three infection phases. Of these, 47 hypothetical genes were identified as having expression profiles associated with at least one connected to a gene associated with adaptation and virulence. Conclusions: Expression patterns of six differentially expressed genes were validated by quantitative reverse transcription polymerase chain reaction, thus demonstrating the effectiveness of this tool in global gene expression analysis in Xac.

Molecular methods for diversity assessment among xanthomonads of Bulgarian and Macedonian pepper

ABSTRACT Bacterial spot is an important disease of pepper in Bulgaria and Macedonia. For characterization of Xanthomonas species associated with bacterial spot, 161 strains were collected from various field pepper-growing regions. Among them, 131 strains were identified as Xanthomonas euvesicatoria and 30 as Xanthomonas vesicatoria using species-specific primers and polymerase chain reaction followed by restriction fragment length polymorphism analysis. To assess the genetic diversity of the strains, two methods (Random Amplified Polymorphic DNA and Repetitive Element Palindromic-Polymerase Chain Reaction) were applied. Discriminatory index was calculated and analysis of molecular variance was carried out.Combined random amplified polymorphic DNA analysis of the X. euvesicatoria strains with primers CUGEA-4 and CUGEA-6 had greater discriminative power (0.60) than repetitive element palindromic-polymerase chain reaction with ERIC and BOX A1R primers, which makes this method applicable for strain diversity evaluation. Discrimination among the X. vesicatoria strains was achieved by the use of ERIC primers and only for the Bulgarian strains. The results demonstrated that X. euvesicatoria was more diverse than X. vesicatoria and heterogeneity was observed mainly in the Bulgarian populations. According to the analysis of molecular variance, genetic variations in X. euvesicatoria were observed among and within populations from different regions, while the differences between the two countries were minor. Following the principal coordinates analysis, a relation between the climatic conditions of the regions and a genetic distance of the populations may be suggested.

A protein expression system for tandem affinity purification in Xanthomonas citri subsp. citri

Abstract Citrus canker, caused by the Gram-negative bacterium Xanthomonas citri subsp. citri (Xac), is one of the most devastating diseases to affect citrus crops. There is no treatment for citrus canker; effective control against the spread of Xac is usually achieved by the elimination of affected plants along with that of asymptomatic neighbors. An in depth understanding of the pathogen is the keystone for understanding of the disease; to this effect we are committed to the development of strategies to ease the study of Xac. Genome sequencing and annotation of Xac revealed that ∼37% of the genome is composed of hypothetical ORFs. To start a systematic characterization of novel factors encoded by Xac, we constructed integrative-vectors for protein expression specific to this bacterium. The vectors allow for the production of TAP-tagged proteins in Xac under the regulation of the xylose promoter. In this study, we show that a TAP-expression vector, integrated into the amy locus of Xac, does not compromise its virulence. Furthermore, our results also demonstrate that the polypeptide TAP can be overproduced in Xac and purified from the soluble phase of cell extracts. Our results substantiate the use of our vectors for protein expression in Xac thus contributing a novel tool for the characterization of proteins and protein complexes generated by this bacterium in vivo.

AFLP analysis of Xanthomonas axonopodis and X arboricola strains used in xanthan production studies reveal high levels of polymorphism

Amplified fragment length polymorphism (AFLP) was used to analyze the genetic diversity of 14 strains of Xanthomonas arboricola pv. pruni and seven strains of X. axonopodis pv. phaseoli, which are used in xanthan production studies. Relationships identified by the AFLP profiles were assessed for xanthan production capacity, geographical location and host plant. Strains were isolated from 10 different geographic regions in South and Southeast States in Brazil. Data were analyzed for genetic similarity using the Dice coefficient and subjected to UPGMA cluster analysis. A total of 128 AFLP fragments were generated from four primer combinations: EcoRI+C/MseI+0, EcoRI+A/MseI+0, EcoRI+G/MseI+T and EcoRI+G/MseI+A. Of these, 96.1 percent were polymorphic. X. axonopodis pv. phaseoli (S D = 0.27) was shown to be more polymorphic than X. arboricola pv. pruni (S D = 0.58). All 14 pathovar pruni strains were included in a single main group (S D = 0.58), while the pathovar phaseoli strains were divided into three separate groups, with one group containing five strains (S D = 0.38) and two isolated groups (S D = 0.31 and 0.27) composed of only one strain each. Species were distinguished by three and eight specific AFLP markers present in the pathovar phaseoli and the pathovar pruni, respectively. For the unique strain without xanthan production capacity (X. axonopodis pv. phaseoli str. 48), nine specific AFLP bands were found. There was no evidence that geographic area or host plant influenced genetic heterogeneity. Correlations between AFLP patterns and xanthan production capacity were found in some strains, but were not consistent enough to establish a relationship.

Genetic diversity of Xanthomonas axonopodis pv. citri based on plasmid profile and pulsed field gel electrophoresis

Xanthomonas axonopodis pv. citri strains that cause disease in citrus were investigated by pulsed field and plasmid profile analysis. For the first method, genomic DNA was digested by the rare-cutting enzymes Xba I and Vsp I. The strains evaluated were collected in seven different States of Brazil and in Argentina, Bolivia, Paraguay and Uruguay. Genetic variability was found among strains of X. axonopodis pv. citri from different geographical areas Argentina, Bolivia and Uruguay, with similarities varying from 0.62 to 0.83. However, the strains collected in Brazil, despite being from different States, have shown a genetic similarity ranging from 0.83 to 1.00. Cluster analysis showed a relationship between genomic similarity and geographical origin of the strains. Plasmids were observed in all strains, with a total of five different plasmids, with sizes between 57.7 and 83.0 kilobases. The 72.6 kb plasmid was the most frequent, present in 15 out of 22 strains, while the 68.1 kb plasmid was observed in two strains only. Although the plasmid diversity detected in the present study was not very great, the X. axonopodis pv. citri strains evaluated showed a considerable degree of diversity with regard to this extrachromosomal genetic element.

Effect of phenol on ultra structure and plasmid DNA of Xanthomonas oryzae pv. oryzae.

Most phenolic substances of plant origin are toxic to microorganisms and they confer some degree of protection to plants against phytopathogens. Xanthomonas oryzae pv. oryzae, bacterial blight pathogen of rice (Oryza sativa) was treated with phenol (monohydroxy benzene) and its effects on the morphology and cytological changes of the bacterium were studied. Total lysis of cells occurred with 5 mM conc of phenol while at 2 mM conc, the cell walls became rough and cell contents started shrinking. Plasmids isolated from both treated (2 mM) and control cells did not show any marked difference under electron microscope except that they differed in their quantity and might influence pathogenicity.