Genetic Loci of Plant Pathogenic Dickeya solani IPO 2222 Expressed in Contact with Weed-Host Bittersweet Nightshade (Solanum dulcamara L.) Plants

Abstract

Dickeya solani, belonging to the Soft Rot Pectobacteriaceae, are aggressive necrotrophs, exhibiting both a wide geographic distribution and a wide host range that includes many angiosperm orders, both dicot and monocot plants, cultivated under all climatic conditions. Little is known about the infection strategies D. solani employs to infect hosts other than potato (Solanum tuberosum L.). Our earlier study identified D. solani Tn5 mutants induced exclusively by the presence of the weed host S. dulcamara. The current study assessed the identity and virulence contribution of the selected genes mutated by the Tn5 insertions and induced by the presence of S. dulcamara. These genes encode proteins with functions linked to polyketide antibiotics and polysaccharide synthesis, membrane transport, stress response, and sugar and amino acid metabolism. Eight of these genes, encoding UvrY (GacA), tRNA guanosine transglycosylase Tgt, LPS-related WbeA, capsular biosynthesis protein VpsM, DltB alanine export protein, glycosyltransferase, putative transcription regulator YheO/PAS domain-containing protein, and a hypothetical protein, were required for virulence on S. dulcamara plants. The implications of D. solani interaction with a weed host, S. dulcamara, are discussed.

Keywords: Erwinia chrysanthemi; Tn5; alternative plant host; colonization; qRT-PCR; random transposon mutagenesis.

Figure 1

Protein networking for the 16 proteins of D. solani strain IPO 2222, whose expression was induced exclusively by the presence of S. dulcamara tissues. The analysis was performed using STRING with the proteome of D. solani strain IPO 2222 as a reference, and the image was manually curated. Each node represents a protein of IPO 2222 with a protein accession number. Nodes with dark color variants represent proteins whose expression was up-regulated, as evidenced by the GUS reporter assay [22], nodes with light color variants belong to the enriched group identified by STRING. Network-based on a full STRING network (the edges indicate both functional and physical protein associations). Network edges indicate confidence, line thickness indicates the strength of data support, and dotted lines represent clustering according to k-means. Interaction based on all available sources for STRING version 12.0, minimal interaction score = 0.4 (medium). Disconnected nodes were disabled. The M value near the node refers to the particular D. solani Tn mutant.

Figure 2

Disease symptoms observed after inoculation of the stem bases of Solanum dulcamara plants with eight Dickeya solani Tn5 mutants (M241, M253, M264, M271, M277, M278, M281 and M596), shown as the percentage of affected plants. Symptoms were evaluated at 14 days post inoculation. Percentages do not sum up to 100 as several symptoms per plant were observed. As a control, D. solani strain IPO 2222 (WT) was used.

Figure 3

Population size of D. solani IPO 2222 (WT) and eight D. solani Tn5 mutants within stems of S. dulcamara plants after inoculation into stem-base. Results were considered significant at p = 0.05, and the pair-wise differences were obtained using the t-test. The means that do not share the same letters above each bar differ. Vertical lines represent standard deviation (SD).

Figure 4

qRT-PCR analysis of the eight D. solani IPO 2222 loci found to be differentially regulated by S. dulcamara in a former transposon-based study. For each gene, we compared the expression levels in cells grown in a culture medium alone (control) to those in cells grown in a medium supplemented with in vitro-derived S. dulcamara plantlets. Genes lpxC and yhb were used as reference genes for data normalization. Per locus, the fold change under inductive to non-inductive conditions normalized to the expression of the control genes is shown [22]. Per locus, the fold change (log₂FC) in gene expression of the target genes in the presence of S. dulcamara was calculated in relation to the control. Five biological replicates were analyzed per locus, and the results were averaged [22].