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 <R6Kgammaori/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.

FIG. 1.

Sequence analysis of EZ-Tn5 insertion in the galU mutants. (A) Genomic location of galU on the X. citri subsp. citri chromosome and transposon insertion sites in the galU mutants. kefB encodes a transport protein, galU encodes a UTP-glucose-1-phosphate uridylyltransferase, and XCC2293 encodes a dehydratase protein. Bg, BglII restriction site. (B) PCR analysis confirming insertion of EZ-Tn5 into the galU gene: agarose gel electrophoresis of DNA amplified using primers galU-F1 and galU-R1 targeting the interior region of the galU gene from the X. citri subsp. citri wild-type 306, D12, and F6 strains. Lane 1, Invitrogen 1 Kb Plus DNA size marker; lane 2, D12, lane 3, F6, lane 4, X. citri subsp. citri 306. (C) Southern blot of DNA of X. citri subsp. citri wild-type strain 306 and galU mutants F6 and D12 digested with BglII. The membrane was probed with a 675-bp kan-2 gene fragment that was amplified using PCR with primers Kan-F1 and Kan-R1. Wt, wild type.

FIG. 2.

Effects of galU on the capsular polysaccharide and biofilm formation. (A) Capsule-stained X. citri subsp. citri strains observed with a light microscope (magnification, ×1,000). (B) Biofilm formation by X. citri subsp. citri strains as determined using crystal violet staining. Wt, X. citri subsp. citri wild-type strain 306; D12, galU mutant; CD, strain D12 complemented with pCGU2.1; D12/pUFR053, D12 complemented with empty vector pUFR053 without the galU gene.

FIG. 3.

Pathogenicity assays and growth of X. citri subsp. citri strains in planta. All strains were infiltrated into leaves with needleless syringes. (A) Host responses of cv. Duncan grapefruit inoculated with X. citri subsp. citri wild-type strain 306 (a), galU mutant D12 (b), CD (complemented mutant D12) (c), galU mutant F6 (d), CF (complemented mutant F6) (e), D12 with empty vector pUFR053 without the galU gene (f), F6 with pUFR053 without the galU gene (g), and water control (h). (B) Growth of X. citri subsp. citri wild-type strain 306, galU mutant D12, CD (complemented mutant D12), and D12 with empty vector pUFR053 in grapefruit leaves. (C) Growth of coinoculated X. citri subsp. citri wild-type strain 306 and galU mutant D12 in grapefruit leaves. □, wild-type strain 306; ▪, D12; ○, CD (complemented mutant D12); •, D12 with pUFR053. The in planta growth assays were repeated three times independently with four replicates each time, but the results of only one experiment are shown. The error bars indicate the standard errors of the means.