Complete Genome Sequence Analysis of Ralstonia solanacearum Strain PeaFJ1 Provides Insights Into Its Strong Virulence in Peanut Plants

Abstract

The bacterial wilt of peanut (Arachis hypogaea L.) caused by Ralstonia solanacearum is a devastating soil-borne disease that seriously restricted the world peanut production. However, the molecular mechanism of R. solanacearum-peanut interaction remains largely unknown. We found that R. solanacearum HA4-1 and PeaFJ1 isolated from peanut plants showed different pathogenicity by inoculating more than 110 cultivated peanuts. Phylogenetic tree analysis demonstrated that HA4-1 and PeaFJ1 both belonged to phylotype I and sequevar 14M, which indicates a high degree of genomic homology between them. Genomic sequencing and comparative genomic analysis of PeaFJ1 revealed 153 strain-specific genes compared with HA4-1. The PeaFJ1 strain-specific genes consisted of diverse virulence-related genes including LysR-type transcriptional regulators, two-component system-related genes, and genes contributing to motility and adhesion. In addition, the repertoire of the type III effectors of PeaFJ1 was bioinformatically compared with that of HA4-1 to find the candidate effectors responsible for their different virulences. There are 79 effectors in the PeaFJ1 genome, only 4 of which are different effectors compared with HA4-1, including RipS4, RipBB, RipBS, and RS_T3E_Hyp6. Based on the virulence profiles of the two strains against peanuts, we speculated that RipS4 and RipBB are candidate virulence effectors in PeaFJ1 while RipBS and RS_T3E_Hyp6 are avirulence effectors in HA4-1. In general, our research greatly reduced the scope of virulence-related genes and made it easier to find out the candidates that caused the difference in pathogenicity between the two strains. These results will help to reveal the molecular mechanism of peanut-R. solanacearum interaction and develop targeted control strategies in the future.

Keywords: Ralstonia solanacearum; comparative genomic analysis; genome sequencing; pathogenicity; peanut.

FIGURE 1

R. solanacearum PeaFJ1 shows different virulent profiles compared with HA4-1 in peanut varieties. Bacterial wilt symptoms (A) and survival ratio (B) of the peanut varieties A184 and A281 infected with R. solanacearum PeaFJ1 and HA4-1, respectively. (A) Photographs from representative plants were taken at 10 days post- inoculation. White scale bars = 5 cm, red scale bars = 10 cm. (B) The percentage of surviving peanuts was recorded for 15 days. The data used for the survival ratio were collected from three independent experiments. Gehan–Breslow–Wilcoxon test p-values are < 0.0001 and 0.6471 in A184 and A281, respectively. ****indicates p < 0.0001, (ns) means no significantly different.

FIGURE 2

Circos plot of the genome of R. solanacearum strain PeaFJ1. The genome of strain PeaFJ1 consists of a chromosome contig with a full length of 3,800,378 bp (A) and a megaplasmid contig with a full length of 2,008,534 bp (B). From the outer circle to the inner circle, it represents the length of chromosomes, CDS, tRNAs, GC content, and GC skew curve, respectively. (C,D) The distribution of genes with COG functional categories in the chromosome (C) and the megaplasmid (D) of PeaFJ1. Different colors represent different COG functional classifications that were explained in the right side.

FIGURE 3

Comparison of R. solanacearum PeaFJ1 with other representative sequenced R. solanacearum strains. (A) Synteny blocks identified in PeaFJ1 across HA4-1, Rs-P.362200, HZAU091, GMI1000, and CQPS-1. (B) Illustration of structural variation types between PeaFJ1 and HA4-1. Reference is HA4-1, and query is PeaFJ1.

FIGURE 4

Circular plots of genomic islands identified in R. solanacearum PeaFJ1 genome. The orange- and blue-colored shapes determine the predicted genomic islands as identified by SIGI-HMM (orange) and IslandPath-DIMOB (blue), and red shows the integrated genomic island search results.

FIGURE 5

Comparison of orthologs of strain PeaFJ1 with other R. solanacearum strains. (A) A big Venn diagram of orthologs for PeaFJ1, HA4-1, Rs-P.362200, HZAU091, GMI1000, and CQPS-1. (B) Statistical analysis of common/specific orthologs between PeaFJ1 and the other five strains. The x-axis indicates the number of gene families; the y-axis means the different R. solanacearum strains. Green bar, blue bar, and red bar mean common gene families, specific gene families, and PeaFJ1- specific gene families when PeaFJ1 is compared with the other five strains, respectively. (C) A Venn diagram of the common/specific genes between PeaFJ1 and HA4-1. (D) Top 20 enriched KEGG pathways of strain-specific genes in PeaFJ1 whole genome. The q-value results from the p-value via multi-test correction. The ranges of q-value are from 0 to 1, and a higher enrichment is achieved when the q-value reaches to 0.