Xanthomonas campestris pv. campestris Requires a Functional pigB for Epiphytic Survival and Host Infection

Abstract

When cauliflower plants (Brassica oleraceae) were misted with bacterial suspensions of Xanthomonas campestris pv. campestris (causal agent of black rot of cruciferous plants), two separate populations of the pathogen were associated with the leaves. Initially, bacteria removable by sonication and sensitive to sodium hypochlorite treatment predominated (easily removable epiphytic bacteria, EREB). However, after 2 weeks, bacteria not removable by sonication and insensitive to sodium hypochlorite treatment were dominant. Although the exact location of this second population of the pathogen was not determined, evidence is presented to support its location in protected sites on the leaf surface, pigB of this pathogen is required for production of extracellular polysaccharide (EPS), xanthomonadin pigments, and the diffusible signal molecule, DF (diffusible factor). DF can extracellularly restore EPS and xanthomonadin production to pigB mutant strains. Parent strain B-24 and pigB mutant strain B24-B2 were identical for in planta growth and symptomatology after artificial infection by injection in leaf mid-veins. Subsequently, X. campestris pv. campestris parent strain B-24, Tn3HoHo1 pigB insertion mutation strain B24-B2, chromosomally restored pigB mutation strain B24-B2R, and strain B24-79 with a Tn3HoHo1 insertion in an unrelated part of the genome were compared for epiphytic survival on, and natural infection of, cauliflower. After application, strains B-24, B24-B2R, and B24-79 all maintained leaf EREB populations of between approximately 3 and 6 (log [1 + CFU per g of fresh weight]) over a 3-week period, whereas B24-B2 populations fell to nearly undetectable levels. Plants sprayed with strains B-24, B24-B2R, and B24-79 averaged between 1.0 and 1.2 lesions, whereas those sprayed with B24-B2 averaged only 0.03 lesions per plant after 3 weeks. Differences in EREB population levels did not explain the observed differences in host infection frequencies, and the results indicated that strain B24-B2 was reduced in its ability to infect the host via the hydathodes, but unaffected in infection via wounds. When strains B-24 and B24-B2 were mixed in equal numbers and sprayed on plants together, B24-B2 epiphytic populations were intermediate between those of B-24 applied alone and B24-B2 applied alone. These results indicate that a functional pigB is required for epiphytic survival and natural host infection under the experimental conditions tested, and suggest that DF, xanthomonadins, and EPS could all be important for survival of this pathogen on the leaf surface, and/or for host infection.

Biological Role of Xanthomonadin Pigments in Xanthomonas campestris pv. Campestris

“…Chromosomally restored derivatives of mutant strains B24-C4 and B24-G14 were constructed by homologous recombination with pIG102 ( Fig. 1) as previously described (16).…”

“…Mutations in the other pig transcriptional units did not appear to have pleiotropic affects. When tested on the host plant, pigB mutants were significantly reduced in epiphytic survival and natural host infection via hydathodes (16). DF extracellularly restored all of these traits to a pigB mutant strain (4,15,16), indicating that DF is needed for xanthomonadin and EPS production, as well as for epiphytic survival and host infection.…”

“…When tested on the host plant, pigB mutants were significantly reduced in epiphytic survival and natural host infection via hydathodes (16). DF extracellularly restored all of these traits to a pigB mutant strain (4,15,16), indicating that DF is needed for xanthomonadin and EPS production, as well as for epiphytic survival and host infection. These results suggest that DF acts as a signal for the initiation of xanthomonadin and EPS production and that xanthomonadins and EPS may play a role in host infection and/or epiphytic survival.…”

Insights into Genome Plasticity and Pathogenicity of the Plant Pathogenic BacteriumXanthomonas campestrispv. vesicatoria Revealed by the Complete Genome Sequence

“…VESICATORIA GENOME SEQUENCE 7259 sensing systems participate in the regulation of exopolysaccharide, an important virulence factor in X. campestris pv. campestris (75,78). Based on the conserved gene content, these systems are probably functional in X. campestris pv.…”

Systems and synthetic biology perspective of the versatile plant-pathogenic and polysaccharide-producing bacterium Xanthomonas campestris

“…Upon its perception by the Rpf two-component system, the level of the Xanthomonas second messenger cyclic di-GMP is modulated, which has an influence on the activity of the high-ranking transcriptional regulator Clp [144,145]. Besides the DSF, Xcc utilizes a second quorum sensing signal termed the diffusible factor (DF) which is less well characterized, but which also has an effect on xanthan generation [146,147]. It is very likely that more metabolic pathways are influenced by these regulatory and signalling pathways, but so far this has not been systematically analysed.…”