Expression Profiling of Virulence and Pathogenicity Genes ofXanthomonas axonopodispv. citri

Abstract

DNA macroarrays of 279 genes of Xanthomonas axonopodis pv. citri potentially associated with pathogenicity and virulence were used to compare the transcriptional alterations of this bacterium in response to two synthetic media. Data analysis indicated that 31 genes were up-regulated by synthetic medium XVM2, while only 7 genes were repressed. The results suggest that XVM2 could be used as an in vitro system to identify candidate genes involved in pathogenesis of X. axonopodis pv. citri. Within the genus Xanthomonas, several genes have been found associated with pathogenicity and virulence. Of these genes, the avr (avirulence), rpf (named for regulation of pathogenicity factors), and hrp (named for hypersensitive response and pathogenicity) genes are perhaps the most widely studied elements. The avr genes encode a known group of effector proteins responsible for controlling the ability of bacteria to elicit the hypersensitive reaction in resistant hosts (26) and may also participate in pathogenicity or virulence in compatible interactions (38,47). The rpf operon is thought to control the production of important pathogenicity factors, such as proteases, endoglucanases, polygalacturonate ligases, and extracellular polysaccharides (5, 17). Finally, the hrp genes are thought to encode proteins involved in the type III secretion system, responsible for delivering effector proteins inside the host plant cells (8,9,20,21).Even though pathogenicity and virulence of Xanthomonas axonopodis pv. citri have been traditionally associated with the activity of a single avirulence-like gene known as pthA (14,22,45,46), little is known about other gene products involved in these processes. In this respect, transcription profiling under natural conditions may be a good alternative to identify all the elements involved in pathogenicity and virulence of this microbial pathogen. However, leaf spot pathogens, such as X. axonopodis pv. citri, do not reach high population levels in infected tissues and do not yield enough material (either bacterial cells or RNA) to conduct gene expression studies. Therefore, in an attempt to develop an alternative system for gene expression studies, an in vitro system was evaluated in order to determine whether it could be used to model pathogen responses to host tissues under controlled conditions. The two media selected were NB (nutrient broth), commonly used for growing this bacterium, and XVM2, suspected to mimic the environment of the plant intercellular spaces (1, 41, 51, 55).Construction of DNA macroarrays. To study the expression profile of X. axonopodis pv. citri, 279 candidate genes, associated with pathogenicity or virulence by sequence similarity, were selected from the genome of this bacterium (13). Specific primers were designed to amplify either the entire open reading frame or a fragment of it. PCR amplifications were performed in two rounds using 96-well plates and a Mastercycler Eppendorf (Eppendorf). The quality of amplimers was analyzed by agarose gel electrophoresis.Prior to the cons...

hpaR, a PutativemarRFamily Transcriptional Regulator, Is Positively Controlled by HrpG and HrpX and Involved in the Pathogenesis, Hypersensitive Response, and Extracellular Protease Production ofXanthomonas campestrisPathovar campestris

“…In xanthomonads, the hrp gene cluster comprises six operons (hrpA to hrpF) and is under the positive control of HrpG and HrpX (8,61,62). It has long been considered that HrpX is the regulator of genes with a TTSS-associated function, such as other hrp genes and the TTSS effector genes (6,7,39,61). However, in this study, we have demonstrated that X. campestris pv.…”

Requirement of the galU Gene for Polysaccharide Production by and Pathogenicity and Growth In Planta of Xanthomonas citri subsp. citri

“…XVM2 medium has been reported to mimic the environment of plant intercellular spaces, and most virulence factors of Xanthomonas spp. can be induced in this medium (2,52). Therefore, we grew D12 and the wild-type strain in XVM2 medium, removed samples at the exponential phase, and compared the levels of transcription of 15 genes using QRT-PCR.…”

Differential expression of pectolytic enzyme genes in Xanthomonas citri subsp. citri and demonstration that pectate lyase Pel3 is required for the formation of citrus canker

“…campestris strain 8004, the expressions of two polygalacturonases genes, pghAxc and pghBxc, were regulated by HrpG and HrpX (Wang et al, 2008). Similar involvement of the T3SS regulators in controlling the expression of pectolytic enzyme-coding genes was found in two strains of Xcc: the expression of one pel gene in Xcc strain 306 can be induced in XVM2 medium (Astua-Monge et al, 2005); the other example was found in Xcc strain NA-1 that two polygalacturonase-coding genes, pglA and peh-1, were regulated by HrpG (Yamazaki et al, 2008). Thus, we hypothesized that the regulation of the pectolytic enzyme genes in Xcc XW19 might also be dependent on HrpG and HrpX.…”

“…in vitro (Astua- Monge et al, 2005;Koebnik et al, 2006;Yamazaki et al, 2008). The Xanthomonas t3ss genes are tightly regulated by HrpG and HrpX.…”