Complete Genome Sequence of Sequevar 14M Ralstonia solanacearum Strain HA4-1 Reveals Novel Type III Effectors Acquired Through Horizontal Gene Transfer

Abstract

Ralstonia solanacearum, which causes bacterial wilt in a broad range of plants, is considered a "species complex" due to its significant genetic diversity. Recently, we have isolated a new R. solanacearum strain HA4-1 from Hong'an county in Hubei province of China and identified it being phylotype I, sequevar 14M (phylotype I-14M). Interestingly, we found that it can cause various disease symptoms among different potato genotypes and display different pathogenic behavior compared to a phylogenetically related strain, GMI1000. To dissect the pathogenic mechanisms of HA4-1, we sequenced its whole genome by combined sequencing technologies including Illumina HiSeq2000, PacBio RS II, and BAC-end sequencing. Genome assembly results revealed the presence of a conventional chromosome, a megaplasmid as well as a 143 kb plasmid in HA4-1. Comparative genome analysis between HA4-1 and GMI1000 shows high conservation of the general virulence factors such as secretion systems, motility, exopolysaccharides (EPS), and key regulatory factors, but significant variation in the repertoire and structure of type III effectors, which could be the determinants of their differential pathogenesis in certain potato species or genotypes. We have identified two novel type III effectors that were probably acquired through horizontal gene transfer (HGT). These novel R. solanacearum effectors display homology to several YopJ and XopAC family members. We named them as RipBR and RipBS. Notably, the copy of RipBR on the plasmid is a pseudogene, while the other on the megaplasmid is normal. For RipBS, there are three copies located in the megaplasmid and plasmid, respectively. Our results have not only enriched the genome information on R. solanacearum species complex by sequencing the first sequevar 14M strain and the largest plasmid reported in R. solanacearum to date but also revealed the variation in the repertoire of type III effectors. This will greatly contribute to the future studies on the pathogenic evolution, host adaptation, and interaction between R. solanacearum and potato.

Keywords: Ralstonia solanacearum; genome sequencing; plasmid; potato; type III effectors.

FIGURE 1

Pathogenicity of three Ralstonia solanacearum strains in potato. (A) Pathogenicity characterization of R. solanacearum HA4-1, GMI1000, and UW551 to six different potato genotypes (S. tuberosum cv. E3, S. chacoense acc. C9701, S. albicans acc. ALB28-1 and ALB28-3, S. stoloniferum acc. STO80-5 and STO80-6). The disease index was calculated using the data collected at 14 days post inoculation (dpi) (mean ± SD, n = 3, ^∗∗P < 0.01, ^*P < 0.05, Dunnett-test). Asterisks indicate significant differences compared to S. tuberosum cv. E3. (B) A representative showing of different symptoms caused by different pathogen strains on the same genotype (ALB28-1). Pictures were taken at 14 dpi.

FIGURE 2

The illustration of the gap closure of the megaplasmid with BAC sequences. The black line and the purple line represent the megaplasmid sequence and the BAC sequence, respectively. The region between the two short red vertical lines belongs to the small plasmid. “1” and “2” indicate the positions of the two gap borders. The dotted portions of the purple line indicate absence of the region in BAC1 and BAC2.

FIGURE 3

The circular maps of the HA4-1 genome chromosome (A), megaplasmid (B), and small plasmid (C). The size of chromosome is 3,890,347 bp, megaplasmid is 1,947,245 bp and small plasmid is 143,755 bp. The circles from outer to inner represent the genome size, forward CDS and reverse CDS (different colors represent different functional classifications), rRNA and tRNA (blue, rRNA; red, tRNA), GC ratio (black, outward means GC ratio of the region is higher than average GC ratio; blue, inward means GC ratio of the region is lower than average GC ratio), GC skew (green represents a region with G content greater than C, orange represents a region with C content greater than G).

FIGURE 4

The distribution of genes with COG functional categories in the chromosome and the megaplasmid of HA4-1.

FIGURE 5

Distribution of genes with COG functional categories of plasmid pRSHA. The gene numbers of each category are as follows (from most to less): 23 genes in L category, 12 in U, 8 in S, 7 in O, 7 in T, 6 in K, 1 in each category of D, E, N, and V. The Vir-type T4SS gene cluster is in the U category. RipBS is in S category. RipBR is not classified into any categories.

FIGURE 6

Analysis of RipBR effectors. (A) Phylogenetic analysis of known and putative YopJ family effectors. (B) Sequence alignment of the RipBR effector of R. solanacearum RSCM and HA4-1 with the known and putative YopJ family effectors (Amino acids from 175 to 261 are shown). The red arrows point out the conserved catalytic triad consisting of His/Glu/Cys key residues required for auto-/trans-acetylation. The GeneBank accession numbers of effectors used for this analysis are as follows: AKN09807 (YopJ), AAN37537 (YopP), AAL21745 (AvrA), AAT08443 (VopA), NP_710166 (AopP), AAG39033 (AvrXv4), AAA27595 (AvrRxv), AAD39255 (AvrBsT), ABM32744 (Aave2166), ABM33278 (Aave2708), WP_011347382 (XopJ), CAD13849 for AAR02168 (HopZ1a), WP_004661226 (HopZ1b), AAL84243 (HopZ1c), CAC16700 (HopZ2), AAF71492 (HopZ3), EKG29639 (HopZ4), AAF63400 (ORFB), and NP_443964 (NopJ). The effectors of R. solanacearum are from the Ralsto T3E database. RipP2 is from GMI1000, RipP3 is from YC45, and RipBC is from Po82. Sequence of RipBR of R. solanacearum RSCM is from NCBI. The effectors R. solanacearum effectors of HA4-1 are from this study. Phylogenetic analysis and sequence alignment were performed by MEGA X.

FIGURE 7

Analysis of RipBS effector. (A) Conserved domains of the RipBS effector predicted in NCBI. The LRR domain spans from 142 to 265 and the Fic domain spans from position 373 to 483. (B) Sequence alignment of the RipBS effector of R. solanacearum HA4-1 with its homologs. Sequences in the red frame is the LRR domain and in the green frame is Fic domain. The GeneBank accession numbers of effectors used for this analysis are as follows: WP_112188676 for the hypothetical protein of Ralstonia sp. GX3-BWBA, AFP74845 for the XopAC of Xanthomonas campestris pv. campestris str. 8004. Sequence alignment was performed by JavaView.