Revised phylogeny and novel horizontally acquired virulence determinants of the model soft rot phytopathogen Pectobacterium wasabiae SCC3193

Abstract

Soft rot disease is economically one of the most devastating bacterial diseases affecting plants worldwide. In this study, we present novel insights into the phylogeny and virulence of the soft rot model Pectobacterium sp. SCC3193, which was isolated from a diseased potato stem in Finland in the early 1980s. Genomic approaches, including proteome and genome comparisons of all sequenced soft rot bacteria, revealed that SCC3193, previously included in the species Pectobacterium carotovorum, can now be more accurately classified as Pectobacterium wasabiae. Together with the recently revised phylogeny of a few P. carotovorum strains and an increasing number of studies on P. wasabiae, our work indicates that P. wasabiae has been unnoticed but present in potato fields worldwide. A combination of genomic approaches and in planta experiments identified features that separate SCC3193 and other P. wasabiae strains from the rest of soft rot bacteria, such as the absence of a type III secretion system that contributes to virulence of other soft rot species. Experimentally established virulence determinants include the putative transcriptional regulator SirB, two partially redundant type VI secretion systems and two horizontally acquired clusters (Vic1 and Vic2), which contain predicted virulence genes. Genome comparison also revealed other interesting traits that may be related to life in planta or other specific environmental conditions. These traits include a predicted benzoic acid/salicylic acid carboxyl methyltransferase of eukaryotic origin. The novelties found in this work indicate that soft rot bacteria have a reservoir of unknown traits that may be utilized in the poorly understood latent stage in planta. The genomic approaches and the comparison of the model strain SCC3193 to other sequenced Pectobacterium strains, including the type strain of P. wasabiae, provides a solid basis for further investigation of the virulence, distribution and phylogeny of soft rot bacteria and, potentially, other bacteria as well.

Figure 1. Phylogenetic tree constructed of 51 orthologous protein groups from 53 bacterial strains and yeast as an outgroup.

For the selection of the orthologous groups, reciprocal best hits were determined based on similarities detected using the fast protein sequence database search tool SANS. The ortholog groups were aligned using Muscle and bootstrapped trees were created with RAxML. Consensus tree was built using Consense and visualized with iTOL. The values presented in the figure indicate bootstrap values calculated based on 51000 bootstrapped trees. The Pectobacterium and Dickeya groups are highlighted.

Figure 2. Comparison of proteomes of Pectobacterium and Dickeya strains.

The strain numbers correspond to the following species: Yersinia pestis CO92 (outgroup), Dickeya dadantii Ech703, Dickeya dadantii Ech586, Dickeya dadantii 3937, Dickeya zeae Ech1591, Pectobacterium carotovorum subsp. brasiliensis PBR1692, Pectobacterium carotovorum WPP14, Pectobacterium carotovorum subsp. carotovorum PC1, Pectobacterium atrosepticum SCRI1043, Pectobacterium wasabiae WPP163, Pectobacterium wasabiae SCC3193 and Pectobacterium wasabiae CFBP 3304^T. (A) OrthoMCL clusters were converted into an orthologs vs. species matrix and visualized as a heat map. The core genome is visualized in the middle of the figure, and species and strain-specific protein clusters can be found above and below the core. (B) The correlations between proteomes were calculated and visualized to indicate their phylogenetic relationships.

Figure 3. Synteny of Pectobacterium wasabiae and Pectobacterium atrosepticum genomes.

Pairwise alignments of genomes were generated using Mauve. P. wasabiae CFBP 3304^T contigs were aligned according to P. wasabiae SCC3193. The sequence similarity in the pairwise alignment of P. wasabiae SCC3193 and CFBP 3304^T was 80.6%. The similarity between SCC3193 and WPP163 was 92.6% and between WPP163 and CFBP 3304^T was 80.9%. The sequence similarity compared to P. atrosepticum was 71.3% in the case of SCC3193, 69.6% for CFBP 3304^T and 72.2% for WPP163.

Figure 4. Circular representation of the chromosome of Pectobacterium wasabiae SCC3193.

The circles from outer to inner represent open reading frames on both strands, tRNAs (green) and rRNAs (orange), manually curated genomic islands (red), SIGI-HMM predicted islands (orange), IslandPath-DIMOB predicted islands (blue), IslandPick predicted islands (green) and GC percentage (gray).

Figure 5. sirB⁻, Vic1, Vic2 and T6SS-double mutants showed decreased virulence in planta.

The virulence of mutants was experimentally determined on tobacco and potato. Experiments were repeated a minimum of three times. The data from one experiment is shown for tobacco, and the combined data from five experiments are shown for potato. The strains shown are Pectobacterium wasabiae SCC3193, sirB⁻, sirB⁻(pSirB), sirB⁻(pSirB1), sirB⁻(pSirB2), sirB⁻(pMW119), Vic1, Vic2 and T6SS-double mutant. (A) Axenic tobacco seedlings (cv. Samsun) were inoculated locally with 5×10⁴ cfu, and symptoms were documented after 48 h as follows: 0 = no maceration, 1 = maceration in less than half of the inoculated leaf, 2 = maceration in more than half of the inoculated leaf, 3 = inoculated leaf was completely macerated, 4 = maceration spread to other leaves, but less than half of the plant was macerated, 5 = maceration spread to other leaves, and more than half of the plant was macerated and 6 = the whole plant was macerated. (B) To determine the bacterial growth on tobacco, samples were taken at 24 and 44 h post-inoculation, and the amount of living bacteria was measured by plate counting. For one sample, two inoculated plants were combined, and the result shows the average of 8 samples. Bars represent standard deviations. (C) Potato tuber slices (cv. Van Gogh) were inoculated with 1×10⁴ cfu, and after three days the macerated area (%) on the tuber slice was documented as follows: 0 = 0%, 1 = ∼5%, 2 = ∼25%, 3 = ∼50%, 4 = ∼75%, 5 = ∼90% and 6 = 100%.

Figure 6. Virulence cluster 2 locus comparison.

Visualization and a comparison of the Vic2 locus was completed in Pectobacterium wasabiae SCC3193, which contains a putative lipoprotein transporting system and a HopL1-like protein. A comparison of the gene cluster was conducted using blastn and blastp against a nucleotide collection and against non-redundant protein sequences to obtain strains with similar loci. ORFs are indicated using colored and scaled arrows, except in case of P. wasabiae CFPB 3304T for which operons were not predicted.