A luminescent reporter evidences active expression of Ralstonia solanacearum type III secretion system genes throughout plant infection

Abstract

Although much is known about the signals that trigger transcription of virulence genes in plant pathogens, their prevalence and timing during infection are still unknown. In this work, we address these questions by analysing expression of the main pathogenicity determinants in the bacterial pathogen Ralstonia solanacearum. We set up a quantitative, non-invasive luminescent reporter to monitor in planta transcription from single promoters in the bacterial chromosome. We show that the new reporter provides a real-time measure of promoter output in vivo -either after re-isolation of pathogens from infected plants or directly in situ -and confirm that the promoter controlling exopolysaccharide (EPS) synthesis is active in bacteria growing in the xylem. We also provide evidence that hrpB, the master regulator of type III secretion system (T3SS) genes, is transcribed in symptomatic plants. Quantitative RT-PCR assays demonstrate that hrpB and type III effector transcripts are abundant at late stages of plant infection, suggesting that their function is required throughout disease. Our results challenge the widespread view in R. solanacearum pathogenicity that the T3SS, and thus injection of effector proteins, is only active to manipulate plant defences at the first stages of infection, and that its expression is turned down when bacteria reach high cell densities and EPS synthesis starts. INTRODUCTIONDuring infection, pathogens deploy a tightly regulated genetic program to overcome the host natural defences and mobilize metabolic resources to their benefit (Grant et al., 2006;Mudgett, 2005). This program, leading to the appearance of disease symptoms, is still unknown for most pathosystems, although many genes involved in infection have been described and their expression measured in culture.Ralstonia solanacearum is an excellent model to study gene regulation, as the pathways controlling its pathogenicity genes have been characterized in detail (Schell, 2000). This soil-borne b-proteobacterium is the causative agent of bacterial wilt on a wide range of plant hosts, including economically important species such as tomato, potato, peanut and eggplant (Hayward, 2000). R. solanacearum invades plants through root wounds and rapidly colonises the xylem vessels, where it multiplies extensively and produces large amounts of exopolysaccharide (EPS) (Kao et al., 1992;Vasse et al., 2000). EPS accumulation in the vascular system and the ensuing collapse of the water flow causes the wilting symptoms and eventually plant death.Coevolution with its various hosts has led to the emergence of a large number of virulence-promoting genes in R. solanacearum (Poueymiro & Genin, 2009;Schell, 2000). The main pathogenicity determinant is the type III secretion system (T3SS), encoded by the hrp cluster and conserved in most Gram-negative pathogens (van Gijsegem et al., 1995). The T3SS translocates some 70 bacterial effector proteins directly into the host cells (Occhialini et al., 2005;Poueymiro & Genin, 2009) to suppress host defence respons...

Functional analysis of Ralstonia solanacearum PrhG regulating the hrp regulon in host plants

“…g 21 stem (Jacobs et al, 2012). A luminescent reporter fused to hrpB was used to confirm that hrpB was transcribed throughout plant infection, even at late stages, where cells proliferate and reach a high cell density (Monteiro et al, 2012). In this study, we demonstrated that hrpG expression was dramatically decreased in the leaves and in the xylem at high cell density, i.e.…”

A Novel, Sensitive Method to Evaluate Potato Germplasm for Bacterial Wilt Resistance Using a LuminescentRalstonia solanacearumReporter Strain

“…R. solanacearum was routinely grown at 30°C in rich B medium or Boucher's minimal medium (MM) supplemented with 20 mM L-glutamate (Sigma-Aldrich, St. Louis) as a carbon source (Boucher et al 1985;Monteiro et al 2012a). Tetracycline (at 10 and 5 g/ml in liquid cultures) and gentamicin (75 and 5 g/ml in liquid cultures) were used for selection in R. solanacearum.…”

“…To prepare inocula, bacterial strains were grown overnight in liquid rich B medium (Boucher et al 1985;Monteiro et al 2012a) at 30°C with shaking at 200 rpm. Cells were pelleted by centrifugation, suspended in water, and spectrophotometrically adjusted to 10 7 CFU/ml.…”

“…In a previous work, R. solanacearum YN5 was transformed with a plasmid carrying the luxCDABE operon to qualitatively study pathogen colonization in susceptible and resistant tomato (Hikichi et al 1999). Recently, we showed that the R. solanacearum GMI1000 bearing an entire synthetic lux operon could be visualized inside infected plants (Monteiro et al 2012a). An advantage of this system is that the reporter is stably inserted in monocopy in the bacterial chromosome, avoiding reporter loss in competitive conditions (e.g., during plant infection) and variations in reporter copy number.…”

The Virulence Factors of the Bacterial Wilt Pathogen Ralstonia solanacearum

“…The T3SS of R. solanacearum contributes greatly to pathogenesis, but hrp mutants retain the ability to invade tomato roots and systemically colonize the vascular system, although the population size of T3SS mutants in infected tissues was reduced by 10 to 1000 fold compared to wild-type strains [16,17]. Recently, in planta transcriptome study and qRT-PCR tests by Jacobs et al [18] and in planta expression study using green fluorescent protein reporter fusions by Monterio et al [19] found that the T3SS is still active even after R. solanacearum has taken over the xylem, suggesting that the T3SS is functional throughout disease. These results changed the wide spread view from in vitro studies that T3SS is only active at the first stage of infection and is not needed when bacteria reach high cell densities [20,21].…”