Comparative and functional genomic analyses of the pathogenicity of phytopathogen Xanthomonas campestris pv. campestris

Abstract

Xanthomonas campestris pathovar campestris (Xcc) is the causative agent of crucifer black rot disease, which causes severe losses in agricultural yield world-wide. This bacterium is a model organism for studying plant-bacteria interactions. We sequenced the complete genome of Xcc 8004 (5,148,708 bp), which is highly conserved relative to that of Xcc ATCC 33913. Comparative genomics analysis indicated that, in addition to a significant genomic-scale rearrangement cross the replication axis between two IS1478 elements, loss and acquisition of blocks of genes, rather than point mutations, constitute the main genetic variation between the two Xcc strains. Screening of a high-density transposon insertional mutant library (16,512 clones) of Xcc 8004 against a host plant (Brassica oleraceae) identified 75 nonredundant, single-copy insertions in protein-coding sequences (CDSs) and intergenic regions. In addition to known virulence factors, full virulence was found to require several additional metabolic pathways and regulatory systems, such as fatty acid degradation, type IV secretion system, cell signaling, and amino acids and nucleotide metabolism. Among the identified pathogenicity-related genes, three of unknown function were found in Xcc 8004-specific chromosomal segments, revealing a direct correlation between genomic dynamics and Xcc virulence. The present combination of comparative and functional genomic analyses provides valuable information about the genetic basis of Xcc pathogenicity, which may offer novel insight toward the development of efficient methods for prevention of this important plant disease.

Figure 1.

Circular representation of the Xcc 8004 genome and EZ::TN transposon insertion sites of virulence-reduced mutants. Circles range from 1 (outer circle) to 7 (inner circle). (Circle 1) EZ::TN insertional locations, gene names, or codes are according to Supplemental Table 3; (IG) intergenic region; ori, the origin of DNA replication; Red arrowheads indicate Xcc 8004 strain-specific chromosomal segments. (Circle 2) CDSs on forward strand. (Circle 3) CDSs on reverse strand. (Circle 4) tRNA genes. (Circle 5) rRNA genes. (Circle 6) GC skew [(G-C)/(G+C)], red indicates values >0 and green <0. (Circle 7) G+C contents. Colors in circle 2 and 3 represent functional categories; orange for intermediary metabolism; green for biosynthesis of small molecules; blue for macromolecule metabolism; magenta for cell structure; khaki for cellular processes; green for biosynthesis of small molecules; light-blue for mobile genetic elements; pink for pathogenicity, virulence, and adaptation; yellow for hypothetical and conserved hypothetical proteins; dark-green for ORFs with an undefined category.

Figure 2.

Linear genomic comparison of Xanthomonas campestris pv. campestris (Xcc) 8004, Xcc ATCC 33913, and X. axonopodis pv. citri (Xac 306). The red lines represent similar DNA sequences (BLASTN search, e-value < 10^-5) between genomes. A-N designate the chromosomal segments, with strain-specific segments (D and G in Xcc ATCC 33913 and M and N in Xcc 8004) colored red. Black triangles indicate IS1478, the preferred recombination sites for genomic scale rearrangement. (ori) The origin of DNA replication. Genomic sequences of Xcc ATCC 33913 and Xac 306 are according to da Silva et al. (2002).

Figure 3.

A schematic illustration of the experimentally determined interaction between Xcc and host cell. The principle pathways are shown in bold. Putative metabolic pathways were predicted by KEGG. Bacterial genes and gene products associated with pathogenicity are arranged within colored frames. Genes identified in this study are framed in red, while genes that were reported previously in xanthomonads are framed in green. (EPS) Exopolysaccharides; (FAAH) fatty acid α hydroxylase; (Hrp) hypersensitive response and pathogenicity genes; (LPS) lipopolysaccharides; (ROS) reactive oxygen species; (R-protein) resistance gene protein; (Xps) Xanthomonas protein secretion.