Cyt toxin expression reveals an inverse regulation of insect and plant virulence factors of Dickeya dadantii Denis Costechareyre, 1–4 Bedis Dridi, 1–4† Yvan Rahbé 5 and Guy Condemine 1–4 * 1 Université de Lyon, •• F-69003, France. 2 Université Lyon 1, •• F-69622, France. 3 INSA-Lyon, Villeurbanne F-69621, France. 4 CNRS, UMR5240, Microbiologie Adaptation et Pathogénie, •• F-69622, France. 5 Université de Lyon, Biologie Fonctionnelle Insectes et Interactions, UMR203 BF2I INRA INSA-Lyon, Villeurbanne F-69621, France. Summary The plant pathogenic bacteria Dickeya dadantii is also a pathogen of the pea aphid Acyrthosiphon pisum. The genome of the bacteria contains four cyt genes, encoding homologues of Bacillus thuringiensis Cyt toxins, which are involved in its pathogenicity to insects. We show here that these genes are tran- scribed as an operon, and we determined the conditions necessary for their expression. Their expression is induced at high temperature and at an osmolarity equivalent to that found in the plant phloem sap. The regulators of cyt genes have also been identified: their expression is repressed by H-NS and VfmE and activated by PecS. These genes are already known to regulate plant virulence factors, but in an opposite way. When tested in a virulence assay by ingestion, the pecS mutant was almost non- pathogenic while hns and vfmE mutants behaved in the same way as the wild-type strain. Mutants of other regulators of plant virulence, GacA, OmpR and PhoP, that do not control Cyt toxin production, also showed reduced pathogenicity. In an assay by injection of bacteria, the gacA strain was less pathogenic but, surprisingly, the pecS mutant was slightly more viru- lent. These results show that Cyt toxins are not the only virulence factors required to kill aphids, and that these factors act at different stages of the infection. Moreover, their production is controlled by general virulence regulators known for their role in plant virulence. This integration could indicate that viru- lence towards insects is a normal mode of life for D. dadantii. Introduction The phytopathogenicity of Dickeya dadantii (formerly Erwinia chrysanthemi) is a complex phenomenon involv- ing many factors and signalling pathways. The bacterium, which has a broad host range, is able to enter the plant, remain latent until the conditions become favourable and then synthesize enzymes that will disorganize the paren- chymatous tissue, producing the soft rot symptoms. The relative importance of several factors during this process has been analysed. During the first steps of the interac- tion, the presence of surface polysaccharides (EPS and LPS) (Schoonejans et al., 1987; Condemine et al., 1999), adhesins (Rojas et al., 2002) and flagella is required for colonization of the host. Two iron uptake systems, anti- bacterial detoxification and cellular repair systems enable the bacteria to survive in the plant (Lopez-Solanilla et al., 1998; El Hassouni et al., 1999; Franza et al., 2005). During the symptomatic phase of the disease, D. dadantii produces and secretes, by the Out type II secretion system, a battery of plant cell wall degrading enzymes: eight pectate lyases, three polygalacturonases, a rham- nogalacturonate lyase and a cellulase (Condemine et al., 1992; Nasser et al., 1999; Laatu and Condemine, 2003). The Hrp type III secretion system is also required for complete virulence (Yang et al., 2002), involving a set of effectors not yet fully characterized. Synthesis of all these virulence factors is tightly con- trolled by a set of regulators with partially overlapping specificity and responding to various signals: KdgR, PecS, PecT, H-NS, Fur, GacA, VfmE, RsmA-RsmB, PhoP and HrpY (Condemine et al., 1987; Reverchon et al., 1994; Surgey et al., 1996; Nasser et al., 2001; Franza et al., 2002; Llama-Palacios et al., 2005; Lebeau et al., 2008; Yang et al., 2008; Yap et al., 2008). Moreover, while most of them control directly the synthesis of the virulence factors, some of these regulators control other regulators within a highly intricate network: for example, H-NS represses pecT and PecS regulates vfmE (Nasser and Reverchon, 2002; Hommais et al., 2008). All these con- trols result in an adequate temporal and spatial expres- sion of the virulence factors (Sepulchre et al., 2007). Received •• ••, ••; revised •• ••, ••; accepted •• ••, ••. *For correspon- dence. E-mail guy.condemine@insa-lyon.fr; Tel. (+33) 472 44 58 27; Fax (+33) 472 43 15 84. † Present address: Laboratoire de Microbi- ologie Clinique, CHU de la Timone, 13385 Marseille Cedex 5. 3 3 emi_2305 Environmental Microbiology (2010) doi:10.1111/j.1462-2920.2010.02305.x © 2010 Society for Applied Microbiology and Blackwell Publishing Ltd 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 66 67 68 69 70 71 72 73 74 75 76 77 78 79 80 81 82 83 84 85 86 87 88 89 90 91 92 93 94 95 96 2