Comprehensive genome sequence analysis of the devastating tobacco bacterial phytopathogen Ralstonia solanacearum strain FJ1003

Abstract

Due to its high genetic diversity and broad host range, Ralstonia solanacearum, the causative phytopathogen of the bacterial wilt (BW) disease, is considered a "species complex". The R. solanacearum strain FJ1003 belonged to phylotype I, and was isolated from the Fuzhou City in Fujian Province of China. The pathogen show host specificity and infects tobacco, especially in the tropical and subtropical regions. To elucidate the pathogenic mechanisms of FJ1003 infecting tobacco, a complete genome sequencing of FJ1003 using single-molecule real-time (SMRT) sequencing technology was performed. The full genome size of FJ1003 was 5.90 Mb (GC%, 67%), containing the chromosome (3.7 Mb), megaplasmid (2.0 Mb), and small plasmid (0.2 Mb). A total of 5133 coding genes (3446 and 1687 genes for chromosome and megaplasmid, respectively) were predicted. A comparative genomic analysis with other strains having the same and different hosts showed that the FJ1003 strain had 90 specific genes, possibly related to the host range of R. solanacearum. Horizontal gene transfer (HGT) was widespread in the genome. A type Ⅲ effector protein (Rs_T3E_Hyp14) was present on both the prophage and genetic island (GI), suggesting that this gene might have been acquired from other bacteria via HGT. The Rs_T3E_Hyp14 was proved to be a virulence factor in the pathogenic process of R. solanacearum through gene knockout strategy, which affects the pathogenicity and colonization ability of R. solanacearum in the host. Therefore, this study will improve our understanding of the virulence of R. solanacearum and provide a theoretical basis for tobacco disease resistance breeding.

Keywords: Ralstonia solanacearum; Rs_T3E_Hyp14; bacterial wilt; disease resistance; effector proteins; genome sequencing; tobacco.

FIGURE 1

Circular map showing the genome of the R. solanacearum strain FJ1003. The outermost circle indicates the marker of genome size, with each scale of 0.1 Mb; the second and third circles indicate the genes on the positive and negative strands of the genome, respectively, and different colors represent the different functional categories of COG; the fourth circle represents repetitive sequences, and the fifth circle is tRNAs. The innermost layer indicates the GC content.

FIGURE 2

COG and GO functional categories of protein-coding genes in the R. solanacearum FJ1003 genome. (A) Statistical results of COG classification; the abscissa indicates the content of COG classification; the ordinate is the number of genes. (B) Clustering results of GO annotation; the abscissa indicates the content of each GO classification; the left side of the ordinate indicates the percentage of the number of genes, and the right side indicates the number of genes.

FIGURE 3

Enrichment analysis results based on the NR and PHI databases. (A) Statistical results of species distribution by the NR database. Different colors represent different homologous species. (B) The gene enrichment annotation to the PHI database. Different colors represent different types of disease-related genes.

FIGURE 4

Molecular phylogenetic analysis between FJ1003 and other strains. This figure is mainly based on the sequence similarity of endoglucanase in different genomes. The percentage of tree in which the associated taxa were clustered together, is shown next to the branches. The tree is drawn to scale, with the corresponding branch lengths indicating the number of substitutions per site. The pink font on the far right is the name of the different species of R. solanacearum.

FIGURE 5

Venn diagrams showing the number of specific and shared genes among the R. solanacearum strain FJ1003 and other bacterial species. (A) Venn diagrams of the sequenced strains and reference strains from different phylotypes. (B) The hosts of the three strains are tobacco. Note: the overlapping regions in the figure indicate the number of gene families shared among the different species, while the regions that do not overlap with other species indicate the number of gene families specific to that species.

FIGURE 6

The collinear relationship between the FJ1003 strain and other strains. The genome location coordinates of the reference near-source species are measured on the left, while the assembled genome location coordinates are on the right. The lines in the figure represent the collinear regions between the two species. Lines of different colors represent different collinear areas between different chromosomes. The color of the lines is automatically assigned by the software.

FIGURE 7

Functional identification of RS-T3E-Hyp14 (A) Tertiary structures of Rs_T3E_Hyp14 in FJ1003. (B) Molecular phylogenetic analysis of predicted RS-T3E-Hyp14 effectors. The phylogenetic tree was constructed by the maximum likelihood method. (C) Symptoms of tobacco after 8 days of inoculation with Ralstonia solanacearum. (D) Disease index of Ralstonia solanacearum at different time after inoculation (E) Colonization of Ralstonia solanacearum in tobacco stems after inoculation for 3 days. Values are means ± SE for three replicates. (WT: wild type strains FJ1003, M: mutant strains ΔRs_T3E_Hyp14, C: complementary strains CRs_T3E_Hyp 14ΔRs_T3E_Hyp14).