Abstract
Citrus bacterial canker caused by Xanthomonas axonopodis pv. citri is a serious disease that impacts citrus production worldwide, and X. axonopodis pv. citri is listed as a quarantine pest in certain countries. Biofilm formation is important for the successful development of a pathogenic relationship between various bacteria and their host(s). To understand the mechanisms of biofilm formation by X. axonopodis pv. citri strain XW19, the strain was subjected to transposon mutagenesis. One mutant with a mutation in a two-component response regulator gene that was deficient in biofilm formation on a polystyrene microplate was selected for further study. The protein was designated as BfdR for biofilm formation defective regulator. BfdR from strain XW19 shares 100% amino acid sequence identity with XAC1284 of X. axonopodis pv. citri strain 306 and 30–100% identity with two-component response regulators in various pathogens and environmental microorganisms. The bfdR mutant strain exhibited significantly decreased biofilm formation on the leaf surfaces of Mexican lime compared with the wild type strain. The bfdR mutant was also compromised in its ability to cause canker lesions. The wild-type phenotype was restored by providing pbfdR in trans in the bfdR mutant. Our data indicated that BfdR did not regulate the production of virulence-related extracellular enzymes including amylase, lipase, protease, and lecithinase or the expression of hrpG, rfbC, and katE; however, BfdR controlled the expression of rpfF in XVM2 medium, which mimics cytoplasmic fluids in planta. In conclusion, biofilm formation on leaf surfaces of citrus is important for canker development in X. axonopodis pv. citri XW19. The process is controlled by the two-component response regulator BfdR via regulation of rpfF, which is required for the biosynthesis of a diffusible signal factor.
Highlights
Our previous study indicated that Bacillus subtilis TKS1-1 and Bacillus amyloliquefaciens WG6-14 can interfere with phyllosphere biofilm formation by X. axonopodis pv. citri, which may contribute to the attenuation of citrus bacterial canker symptom development [6]
The twocomponent system (TCS) consists of a histidine kinase (HK) and a response regulator (RR) and plays a major role in a prokaryote’s ability to sense and respond to environmental stimuli [39]
In X. axonopodis pv. citri XW19, we identified a histidine kinase (BfdS) flanking BfdR, and we found that this kinase possesses a conserved asparagine at position four downstream from histidine; BfdS is classified as a group II HK
Summary
The bacteria are presumably considered as epiphytes on the plant surface before infection [2]. They infect leaves, stems, thorns and fruits and enter the citrus plant mainly through wounds and stomata [3]. Rigano et al (2007) showed that biofilm formation is necessary for epiphytic fitness and canker development in X. axonopodis pv. Citri, which may contribute to the attenuation of citrus bacterial canker symptom development [6]. These results and findings from other plant-associated bacteria suggest that biofilm formation plays important roles in pathogenesis [5,6,7,8]. Experiments were conducted to uncover genes and gene clusters that are involved in biofilm formation and virulence as well as to reveal potential interactions between these two processes in X. axonopodis pv. citri, which may aid in the development of a strategy for disease management
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