A single gene that promotes interaction of a phytopathogenic bacterium with its insect vector, Drosophila melanogaster

Abstract

Insects are major vectors of plant and animal disease, and bacterial phytopathogens are often disseminated by flies. We have previously reported that some isolates of the phytopathogenic bacterial species Erwinia carotovora infect Drosophila and activate an immune response. Using a genetic screen, we have now identified two genes that are required by E. carotovora to infect Drosophila. One of these genes has a regulatory role whereas the other, evf, confers an infectious phenotype: its transfer to non-infectious Erwinia strains or to several enterobacteria improves survival in the gut and triggers the immune response. Overexpression of Erwinia virulence factor (evf) allowed bacteria to colonize the apical side of the gut epithelium and in some cases to spread to the body cavity. Our results demonstrate a specific interaction between plant pathogens and flies that promote their dissemination.

Figure 1

Identification of two genes of Erwinia required to infect Drosophila larvae. (A) GFP expression in larvae carrying a Diptericin–GFP reporter gene after infection by wild-type (wt) Ecc15, evf and hor mutants. Top, Local expression of a Diptericin–lacZ reporter gene by the same strains. Middle, LacZ staining (Tzou et al., 2000) was performed on gut collected 1 d after infection. Bottom, Infection of potatoes by the same bacterial strains. Potatoes were infected as described previously (Jones et al., 1993). (B) Northern blot analysis of Diptericin gene expression after natural infection of wild-type Drosophila with wild-type Ecc15, evf and hor mutants. RNA samples were extracted from Drosophila larvae collected at different time points after ingestion (24, 48, 72 and 96 h, and 'ad' for adults) and processed as described previously (Basset et al., 2000). rp49 hybridization was performed to normalize RNA samples. (C) Schematic representation of NKBOR insertion in regions containing evf and hor genes. Insertions of transposon NKBOR are represented by triangles with arrows. Genes located around evf are indicated. (D) Southern blot hybridizations of ³²P-labelled evf and hor genes with EcoRI- and PvuII-cleaved total DNA from Ecc15 (lane 1), Ecc1488 (lane 2), Ecc1401 (lane 3), Ecc2140 (lane 4), Ecc2145 (lane 5) and Ecc2046 (lane 6).

Figure 2

Epistatic relationship between evf and hor. (A) Quantitative measurements of β-galactosidase activity were performed with Drosophila larvae carrying the Diptericin–lacZ reporter gene collected 24 h after natural infection (Basset et al., 2000) by Ecc15 derivatives. Each mutant was transformed by pOM1, pOM1-evf and pOM1-hor. Bars represent mean results of three samples of five Drosophila larvae. wt, wild type. (B) RNA expression was monitored by RT–PCR with RNA isolated from the gut of 30 Drosophila larvae 6 h after infection by wild-type (wt) Ecc15, evf mutant and hor mutant. evf mRNA, hor mRNA and 16S rRNA were analysed as indicated; 16S rRNA was used as a positive control. (C) Genetic networks required for Drosophila and plants infection by Ecc15.

Figure 3

Immune response and persistence of bacteria expressing evf. (A) β-Galactosidase assay revealing the immune response in Ecc15, Ecc2046, E. coli MG1655, Salmonella typhimurium LT2 and Serratia marcescens expressing evf constitutively. Wild-type Ecc15 and Ecc15 carrying pOM1 were used as positive controls; Ecc2046, E. coli MG1655, Salmonella typhimurium LT2 and Serratia marcescens carrying pOM1 plasmid were used as negative controls. Lane C, banana alone. (B) Bacterial persistence was measured in wild-type CantonS Drosophila larvae (Basset et al., 2000). The number of colony-forming units per larva obtained at each time point after infection is the mean of 120 larvae infected. (C) Visualization of Ecc15 derivatives carrying a GFP marker gene in Drosophila larvae. Drosophila larvae were infected with Ecc15 carrying pOM1–GFP (a, d), with evf mutant carrying pOM1–GFP (b, e), and with evf mutant carrying pOM1-evf–GFP (c, f). Pictures were taken 2 h (a–c) and 8 h (d–f) after infection. Exposure times were shorter in (a–c) than in (d–f). Magnification × 10.

Figure 4

Representative immunostained sections of Drosophila larvae infected with evf mutant carrying pOM1–GFP (A) and with evf mutant carrying pOM1-evf–GFP (B, C). Thirty larvae infected with evf mutant carrying pOM1–GFP or with evf mutant carrying pOM1-evf–GFP were sectioned and bacteria were detected by anti-GFP antibodies. The evf mutant did not persist and rare bacteria can be detected with food in the lumen of the anterior midgut (A). Similar results were obtained with wild-type Ecc15 (data not shown). The evf mutant overexpressing evf colonized the epithelium of the entire midgut (B). In 30% of the larvae, bacteria carrying pOM1-evf were detected in the haemolymph (C, a case of major systemic infection). FB, fat body; G, gut; Cu, external cuticle. Magnification × 40.