Identification of New Candidate Pathogenicity Factors in the Xylem-Invading Pathogen Xanthomonas albilineans by Transposon Mutagenesis

Abstract

Xanthomonas albilineans is a xylem-invading pathogen that produces the toxin albicidin that blocks chloroplast differentiation, resulting in disease symptoms of sugarcane leaf scald. In contrast to other xanthomonads, X. albilineans does not possess a hypersensitive response and pathogenicity type III secretion system and does not produce xanthan gum. Albicidin is the only previously known pathogenicity factor in X. albilineans, yet albicidin-deficient mutant strains are still able to efficiently colonize sugarcane. To identify additional host adaptation or pathogenicity factors, sugarcane 'CP80-1743' was inoculated with 1,216 independently derived Tn5 insertions in X. albilineans XaFL07-1 from Florida. Sixty-one Tn5 mutants were affected in development of leaf symptoms or in stalk colonization. The Tn5 insertion sites of these mutants were determined and the interrupted genes were identified using the recently available genomic DNA sequence of X. albilineans GPE PC73 from Guadeloupe. Several pathogenicity-related loci that were not previously reported in Xanthomonas spp. were identified, including loci encoding hypothetical proteins, a membrane fusion protein conferring resistance to novobiocin, transport proteins, TonB-dependent outer-membrane transporters, and an OmpA family outer-membrane protein.

Concomitant Loss of the Glyoxalase System and Glycolysis Makes the Uncultured Pathogen “Candidatus Liberibacter asiaticus” an Energy Scavenger

“…Full-length gloA genes from L. crescens strain BT-1 and X. albilineans strain FL07-1 were PCR amplified, cloned in pCR2.1-TOPO (Invitrogen, Carlsbad, CA), verified for sequence fidelity, and finally subcloned into a broad-host-range repW shuttle vector pUFR071 (27). Electrocompetent cells of L. crescens (9) and X. albilineans (47) were prepared and transformed as described previously.…”

The RpfCG two-component system negatively regulates the colonization of sugar cane stalks by Xanthomonas albilineans

“…Primer sequences and PCR conditions for each target gene (rpfF, rpfG, rpfC) or gene region (rpfG+rpfC, rpfG+rpfC+rpfF) are described in Table 2. Each plasmid containing a splice-overlap PCR product that carried both ends of the target ORF but not the target region was electroporated into electrocompetent cells of Xanthomonas albilineans as described by Rott et al (2011). Following electroporation, 1 ml liquid GW medium (MW medium without sucrose but supplemented with 10 g glucose l 21 ) was added to the mix and the culture was grown at 30 uC for 2.5-4 h with shaking at 120 r.p.m.…”

“…A gene cluster from rpfB to prfB comprising rpfF, rpfC and rpfG of Xanthomonas albilineans GPE PC73 was cut from pPYOAAB9CC10 with BamHI and ligated into the same sites of pUFR047 to make pPR017 (Table 1). Plasmid pPR017 were electroporated into electrocompetent cells of Xanthomonas albilineans as described by Rott et al (2011). Following electroporation, 1 ml liquid MW medium was added to the mix and the culture was grown at 30 uC for 2.5 h with shaking at 120 r.p.m.…”

“…Toxindeficient mutant isolates of strain XaFL07-1 from Florida are even able to cause disease symptoms, showing that albicidin is not always necessary for expression of leaf scald symptoms . Other putative pathogenicity factors of Xanthomonas albilineans include hypothetical proteins, cell surface polysaccharides, a membrane fusion protein conferring resistance to novobiocin, transport proteins, TonB-dependent outer-membrane transporters and an OmpA family outer-membrane protein (Pieretti et al, 2012;Rott et al, 2011).…”

“…Sugar cane stalks with at least four internodes were inoculated with 10 8 c.f.u. ml 21 bacterial suspensions in sterile distilled water by a decapitation method, as described by Rott et al (2011). In this method, the spindle leaves on a stalk were cut off just below the third visible dewlap with pruning shears and 0.2-0.5 ml inoculum was then deposited onto the cut surface.…”