Comparative genomic and transcriptome analyses of pathotypes of Xanthomonas citri subsp. citri provide insights into mechanisms of bacterial virulence and host range

Abstract

Background: Citrus bacterial canker is a disease that has severe economic impact on citrus industries worldwide and is caused by a few species and pathotypes of Xanthomonas. X. citri subsp. citri strain 306 (XccA306) is a type A (Asiatic) strain with a wide host range, whereas its variant X. citri subsp. citri strain A(w)12879 (Xcaw12879, Wellington strain) is restricted to Mexican lime.

Results: To characterize the mechanism for the differences in host range of XccA and Xcaw, the genome of Xcaw12879 that was completed recently was compared with XccA306 genome. Effectors xopAF and avrGf1 are present in Xcaw12879, but were absent in XccA306. AvrGf1 was shown previously for Xcaw to cause hypersensitive response in Duncan grapefruit. Mutation analysis of xopAF indicates that the gene contributes to Xcaw growth in Mexican lime but does not contribute to the limited host range of Xcaw. RNA-Seq analysis was conducted to compare the expression profiles of Xcaw12879 and XccA306 in Nutrient Broth (NB) medium and XVM2 medium, which induces hrp gene expression. Two hundred ninety two and 281 genes showed differential expression in XVM2 compared to in NB for XccA306 and Xcaw12879, respectively. Twenty-five type 3 secretion system genes were up-regulated in XVM2 for both XccA and Xcaw. Among the 4,370 common genes of Xcaw12879 compared to XccA306, 603 genes in NB and 450 genes in XVM2 conditions were differentially regulated. Xcaw12879 showed higher protease activity than XccA306 whereas Xcaw12879 showed lower pectate lyase activity in comparison to XccA306.

Conclusions: Comparative genomic analysis of XccA306 and Xcaw12879 identified strain specific genes. Our study indicated that AvrGf1 contributes to the host range limitation of Xcaw12879 whereas XopAF contributes to virulence. Transcriptome analyses of XccA306 and Xcaw12879 presented insights into the expression of the two closely related strains of X. citri subsp. citri. Virulence genes including genes encoding T3SS components and effectors are induced in XVM2 medium. Numerous genes with differential expression in Xcaw12879 and XccA306 were identified. This study provided the foundation to further characterize the mechanisms for virulence and host range of pathotypes of X. citri subsp. citri.

Figure 1

Maximum likelihood phylogenetic tree of the genome of Xanthomonas citri subsp. citri A^w 12879 showing the relationship to other Xanthomonads and related species. The tree was constructed using concatenated protein sequences of nine housekeeping genes (UvrD, SecA, CarA, RecA, GroEL, DnaK, AtpD, GyrB and InfB) aligned using Clustal W. Phylogenic tree from concatenated sequences was constructed in CLC Genomics workbench v6.0 using the Maximum likelihood method. The percentage of replicate trees in which the associated taxa clustered together in the bootstrap test (1000 replicates) are shown next to the branches. Horizontal scale bar (0.11) at the bottom represents number of amino-acid substitutions per site.

Figure 2

MAUVE alignment of the genome sequences of X. citri subsp. citri str. 306 and X. citri subsp. citri A^w 12879. Conserved and highly related regions are colored and low identity unique region are in white (colorless). The colored lines indicate translocations of the genome sections. Same colored blocks on opposite sides of the line indicate inversion.

Figure 3

Comparison of the LPS gene clusters of X. citri subsp. citri str. 306, X. citri subsp. citri A^w 12879 and X. oryzae pv. oryzicola str. BLS256. Conserved and highly related genes (over 80% identity) are colored and syntenic regions between the bacteria are shaded in grey (over 50% identity).

Figure 4

Pathogenicity assay in planta. Inoculation by pressure infiltration of X. citri subsp. citri str. 306, X. citri subsp. citri str. A^w12879 and X. citri subsp. citri str. A^wΔavrGf1 mutant on young Duncan grapefruit, Valencia and Hamlin leaves. The culture concentration of 10⁸ cfu/ml was used for inoculation and leaves were photographed after 10 days of incubation. XccA306 infects all three citrus varieties; Xcaw12879 shows hypersensitive reaction only on grapefruit. XcawΔavrGf1 mutant shows reduced symptoms as compared to XccA306 on grapefruit and no symptoms on Valencia and Hamlin.

Figure 5

XopAF contributes to the growth of Xcaw12879 strain in planta. XccA306 (A), Xcaw12879 (Aw), Xcaw12879ΔxopAF (AwΔxopAF), Xcaw12879ΔxopAF-53:xopAF (AwΔAF:53:xopAF, complement strain), Xcaw12879ΔavrGf1 (AwΔavrGf1), Xcaw12879ΔavrGf1-34:avrGf1 (AwΔGf1:34:avrGf1, complement strain), Xcaw12879ΔxopAFΔavrGf1 (AwΔxopAFΔavrGf1) and Xacw12879 ΔxopAFΔavrGf1-34:avrGf1-53:xopAF (AwΔGf1ΔAF:34:avrGf1:53:xopAF, complement strain) were inoculated at approximately 10⁶ cfu/ml concentration into A. Duncan grapefruit, B. Mexican lime and C. Valencia sweet orange leaves using a needleless syringe. Bacterial cells from the inoculated leaves were recovered at different time-points, diluted and counted to plot the growth curve. The values at each time point represent means of three replicates. Means ± SD are plotted.

Figure 6

Number of differentially expressed genes when comparing expression of common genes in X. citri subsp. citri str. A^w 12879 to X. citri subsp. citri str. 306 in NB and XVM2 growth conditions. Gene expression of orthologous genes between Xcaw (W) and XccA (A) was compared when grown in Nutrient broth (NB, nutrient rich medium) and XVM2 (XVM, hrp inducing medium).

Figure 7

Protease and Pectate lyase activity of X. citri subsp. citri str. 306, and X. citri subsp. citri str. A^w 12879. (A) Protease activity was tested by inoculating 1 μl culture on 10% milk agar plates at 28°C for 6 days. Zone of clearance was used as the measure of protease activity. (B) Pectate lyase activity was tested by inoculating 1 μl culture on Hildebrand’s agar medium at 28°C for 6 days. More pitting can be seen on medium at pH 8.5 for XccA strain compared to Xcaw.