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...

We describe here the construction of a delivery system for stable and directed insertion of gene constructs in a permissive chromosomal site of the bacterial wilt pathogen Ralstonia solanacearum. The system consists of a collection of suicide vectors-the Ralstonia chromosome (pRC) series-that carry an integration element flanked by transcription terminators and two sequences of homology to the chromosome of strain GMI1000, where the integration element is inserted through a double recombination event. Unique restriction enzyme sites and a GATEWAY cassette enable cloning of any promoter::gene combination in the integration element. Variants endowed with different selectable antibiotic resistance genes and promoter::gene combinations are described. We show that the system can be readily used in GMI1000 and adapted to other R. solanacearum strains using an accessory plasmid. We prove that the pRC system can be employed to complement a deletion mutation with a single copy of the native gene, and to measure transcription of selected promoters in monocopy both in vitro and in planta. Finally, the system has been used to purify and study secretion type III effectors. These novel genetic tools will be particularly useful for the construction of recombinant bacteria that maintain inserted genes or reporter fusions in competitive situations (i.e., during plant infection).