1.6.1S
PROTEIN TRAFFICKING IN XANTHOMONAS-PLANT INTERACTIONS

U BONAS1,2, A BALLVORA2, K HAHN2, E HUGUET2, M PIERRE2, O ROSSIER2, B SZUREK1 and G VAN DEN ACKERVEKEN1

1Institut für Genetik, Martin-Luther-Universität Halle-Wittenberg, D-06099 Halle, Germany; 2Institut des Sciences Vegetales, CNRS, F-91198 Gif-sur-Yvette, France

Background
Basic pathogenicity of bacterial plant pathogens is determined by hrp (hypersensitive reaction and pathogenicity) genes which are conserved among all major Gram-negative plant pathogenic bacteria except Agrobacterium. We are studying the interaction between Xanthomonas campestris pv. vesicatoria (Xcv), the causal agent of bacterial spot, and its host plants pepper and tomato. hrp mutants no longer cause disease symptoms and fail to grow in the susceptible plant, and they do not induce the hypersensitive reaction (HR) in resistant host or non-host plants. Most hrp genes of Xcv are clustered in a 23-kb chromosomal region, which has been sequenced and contains six operons, hrpA to hrpF. Nine out of more than 20 Xcv Hrp proteins are conserved among both plant and mammalian pathogenic bacteria (e.g. Shigella, Yersinia) and encode components of a type III protein-secretion pathway. Another research topic concerns recognition of Xcv by resistant host plants, which is governed by an avirulence gene in the bacterium and a corresponding resistance gene in the plant. For example, Xcv strains expressing the avirulence gene avrBs3 are recognized by pepper lines carrying the Bs3 resistance gene. Upon recognition, the plant tissue undergoes an HR. The AvrBs3 protein contains 17.5 nearly identical direct-repeat units of 34 amino acids which determine the specificity of recognition.

Results and conclusions
To assess the role of each individual gene in hrp operons consisting of more than one gene, i.e. hrpB, hrpC and hrpD, non-polar mutants were generated. Plant infection tests revealed that most of the genes are indeed true hrp genes and that at least three genes play a more quantitative role in plant interaction. Whether the latter genes encode secreted proteins is not yet known. We are currently establishing an in vitro system to study Hrp type III secretion. One of the candidate proteins to be secreted is the avirulence protein AvrBs3. We recently showed that the AvrBs3 protein itself is the signal molecule and that recognition occurs inside the plant cell [1, 2]. Since hrp genes are essential for recognition of Xcv by the plant, we believe that the AvrBs3 protein is 'injected' via the Hrp secretion pathway into the plant cell. We propose that AvrBs3 has its activity in the plant cell nucleus, as its C-terminal domain carries two functional nuclear localization signals (NLSS) and an acidic transcription activation domain. Mutation analysis showed that the NLSs and the activation domain are both required for HR induction. We are using the interaction-trap yeast two-hybrid system to isolate pepper proteins that interact with AvrBs3. A first interactor has been characterized and encodes importin-a. Results on AvrBs3 suggest that the product of the Bs3 resistance gene might represent a novel type of R-gene protein.

References
1. Van den Ackerveken G, Marois E, Bonas U, 1996. Cell 87, 1307-1316.
2. Bonas U, Van den Ackerveken G, 1997. Plant Journal 12, 1-7.