Characterization of Mutants in Xanthomonas axonopodis pv. citri Lipopolysaccharide Biosynthesis

PETROCELLI, S.; OTTADO, J.; ORELLANO, E.G.

Rosario, Argentina

Congreso; V Congreso Argentino de Microbiología General; 2008

Sociedad Argentina de Microbiología General (SAMIGE)

Plants rely on innate immunity to defend against most potential pathogens. A broad range of pathogens is recognized by plants through so-called pathogen-associated molecular patterns (PAMPs), which are highly conserved fragments of pathogenic molecules. Lipopolysaccharide (LPS) is an essential outer membrane component of gram-negative bacteria. In plant pathogenic bacteria, LPS is an important virulence factor. LPS is also being increasingly recognized as a major PAMP for plants. The genes wzt and rfb303, present in Xac genome, code for an ATP-binding component of the O antigen ABC transporter and a core glycosiltransferase, respectively. Xac mutant strains defective in wzt and rfb303 genes were constructed. The aim of this study is to determine the role of Xathomonas axonopodis pv. citri (Xac) LPS in citrus canker. The motilities of Xac wild-type, Xac-wzt and Xac-rfb303 have been analyzed. Samples of over night cultures were stabbed in swimmer agar (0,3 % w/v) and swarmer agar (0,7 % w/v) plates.  Xac-wzt showed loss of motility in these conditions compared with Xac wild-type and Xac-rfb303. In this context a western blot assay with antibodies anti-flagellin of Serratia marcescens was made. Xac-wzt expresses lower levels of flagellin than Xac wild-type and Xac-rfb303. No differences were observed in the adhesion capacity of the three bacteria strains to orange leaves and to a plastic surface. Protease secretion was determined on milk agar plates and no differences were observed between Xac wild-type and the mutants. The degree of host plants (Citrus sinensis) cell membrane injury induced by biotic stress was estimated through measurements of ion leakage from the cells into an aqueous medium. Xac-wzt exhibited the lowest values of injury index compared with Xac wild-type and Xac-rfb303. This was consistent with the phenotype of infected orange leaves. On the other side, infection of non-host plants of cotton with wild-type and mutant bacteria produced the typical hypersensitive response (HR). Trypan blue was used to selectively stain dead cells. Cotton leaves were stained with DAB to detect H2O2 accumulation throughout the inoculation site. The Xac-wzt mutant was less virulent in orange plants and produced lower values of injury index than Xac wild-type and Xac-rfb303, suggesting that Xac LPS is involved in pathogenicity during citrus canker disease. On the other hand, Xac wild-type, Xac-wzt and Xac-rfb303 produced the HR in non-host plants of cotton.