1.6.3S
THE RALSTONIA SOLANACEARUM HRP MACHINERY

M ARLAT, B BRITO, F VAN GIJSEGEM, J VASSE, M MARENDA, JC CAMUS, M GUENERON, P BARBERIS, S GENIN and C BOUCHER

Laboratoire de Biologie Moleculaire des Relations Plantes Microorganismes, INRA-CNRS, BP27, 31326 Castanet tolosan cedex, France

Background and objectives
Ralstonia solanacearum, causal agent of bacterial wilt, is responsible for severe losses to many important crops in tropical, sub-tropical and warm temperate regions of the world. An hrp gene cluster that is essential for the development of disease symptoms on host plants and the elicitation of the hypersensitive response (HR) on tobacco has been isolated from strain GM1000. This gene cluster spans more than 23 kb and is organized in five transcriptional units which altogether code for 20 genes. Nine of these genes are conserved in hrp gene clusters isolated from other phytopathogenic bacteria (Pseudomonas syringae, Xanthomonas campestris pv. vesicatoria and Erwinia), and share sequence similarities with virulence genes characterized in several animal pathogenic bacteria (Salmonella, Shigella, Yersinia and Pseudomonas aeruginosa). These conserved genes, renamed hrc genes, are components of the so-called type III secretion/injection machinery [1]. In R. solanacearum we have established that the Hrp machinery allows the secretion of PopA, a protein inducing a hypersensitive-like response [2]. The aim of our work is to answer to the following questions: (i) which are the proteins which transit through the Hrp system and what are their functions? (ii) does the Hrp machinery require the formation of exo-structures at the interface with plant cells, and (iii) how is hrp gene expression regulated and what are the inducing signals in planta?

Results and conclusions
To further analyse the hrp gene cluster, we constructed non-polar mutants in each of the hrp genes. These mutants were tested for their ability to wilt host plants, to elicit the HR on tobacco and to secrete PopA. The results of these experiments suggest that two of the hrp genes are not involved in the secretion process, but are nevertheless required for disease development. The corresponding proteins might therefore be transported by the Hrp machinery. In hrp gene expression-inducing conditions, R. solanacearum produces an exostructure made of rigid filaments of 5-10 nm in diameter at one pole of the bacterial cell. The production of these filaments is dependent on the integrity of the Hrp secretion machinery and is required for the secretion of PopA. The role of these filaments during the interaction with plant cells is under investigation. The expression of hrp genes is induced in minimal medium and depends on the hrp regulatory gene, hrpB. Recently, we have established that upon co-culture with plant cell suspensions, the expression of hrp genes (including hrpB) is induced up to 10-20-fold more than in minimal medium. This induction is not triggered by diffusible signals but requires the presence of plant cells. Moreover this plant cell-specific induction is controlled by prhA (plant regulator of hrp genes), a gene located next to popA. This gene codes for a putative outer membrane protein which could act as a receptor for plant-specific signals [3]. Four new regulatory genes located to the right of the hrp gene cluster have been identified. These four genes are involved in the transduction of the plant-specific signals and act upstream of hrpB in the hrp regulatory pathway. A model integrating our latest results will be discussed.

References
1. Van Gijsegem F, Gough C, Zischek C et al., 1995. Molecular Microbiology 15, 1095-1114.
2. Arlat M, Van Gijsegem F, Huet JC et al., 1994. EMBO Journal 13, 543-553.
3. Marenda M, Brito B, Callard D et al., 1998. Molecular Microbiology 27, 437-453.