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

Abstract

Xanthomonas citri subsp. citri is the causal agent of citrus canker, which has a significant impact on citrus production. In this study, we characterized the galU gene of X. citri subsp. citri. Two galU mutants (F6 and D12) were identified in an X. citri subsp. citri EZ-Tn5 <R6K␥ori/KAN-2> Tnp transposon library. Rescue cloning, sequence analysis, and Southern blot analysis indicated that both of these mutants had a single copy of the EZ-Tn5 transposon inserted in galU in the chromosome. Further study showed that galU was required for biosynthesis of extracellular polysaccharides (EPS; xanthan gum) and capsular polysaccharide (CPS) and biofilm formation. Mutation of galU resulted in a loss of pathogenicity for grapefruit. The loss of pathogenicity of a galU mutant resulted from its inability to grow in planta rather than from the effect on virulence genes. Quantitative reverse transcription-PCR assays indicated that mutation of galU did not impair the expression of key virulence genes, such as pthA of X. citri subsp. citri. Although D12 had a growth rate similar to that of the wild-type strain in nutrient broth, no D12 population became established in the intercellular spaces of citrus leaves. Coinoculation of a galU mutant with the wild-type strain did not promote growth of the galU mutant in planta. Defects in EPS and CPS production, pathogenicity, and growth in planta of the galU mutant were complemented to the wild-type level using plasmid pCGU2.1 containing an intact galU gene. These data indicate that the galU gene contributes to X. citri subsp. citri growth in intercellular spaces and is involved in EPS and CPS synthesis and biofilm formation.

The ColR/ColS Two-Component System Plays Multiple Roles in the Pathogenicity of the Citrus Canker Pathogen Xanthomonas citri subsp. citri

“…citri strain 306. To isolate virulence-deficient mutants of strain 306, an EZ-Tn5 transposon mutagenesis library was screened as described in our previous study (16). Four mutants, 256A10, 421E7, 386C6, and 417E10, which were virulence deficient following inoculation onto citrus leaves were isolated (Fig.…”

“…The bacterial cells were resuspended in sterile tap water at a concentration of 10 6 CFU ml Ϫ1 and used to infiltrate cv. Duncan grapefruit leaves with a needleless syringe (16). To evaluate the growth of strain 306 and its derivatives in the intercellular spaces of plants, leaf disks (leaf area, 1 cm 2 in diameter) from inoculated leaves (four leaves for each sample) were excised with a cork borer and then ground in 1 ml sterile tap water.…”

“…Assays of biofilm formation in glass tubes were performed as described previously (16). Briefly, X. citri subsp.…”

“…Pathogenicity assays were conducted as described previously (16). Briefly, fully expanded, immature grapefruit (Citrus paradise Macf.…”

Identification and characterization of biofilm formation-defective mutants of Xanthomonas citri subsp. citri

“…Some features of biofilm, known to be required in other micro-organisms, were also found necessary for the biofilm establishment of Xcc. Xanthan production (Rigano et al, 2007), quorum sensing (Siciliano et al, 2006), a filamentous haemagglutinin-like adhesin (Gottig et al, 2009), flagellum synthesis (Malamud et al, 2011), a UTPglucose-1-phosphate uridylyltransferase (Guo et al, 2010), LPS biosynthesis (Li & Wang, 2011a; and IP: 13.66.222.141…”