1.1.13
IN VITROAND IN VIVOMUTAGENESIS OF THE BACTERIAL AVIRULENCE GENE, AVRPTO

JH CHANG1, CM TOBIAS2, BJ STASKAWICZ1,3 and RW MICHELMORE1,4

1NSF Center for Engineering Plants for Resistance Against Pathogens (CEPRAP), University of California, Davis, CA 95616, USA; 2DNA Plant Technologies (DNAP), Oakland, CA 94608, USA; 3Department of Plant and Microbial Biology, University of California, Berkeley, CA 94720, USA; 4Department of Vegetable Crops, University of California, Davis, CA 95616, USA

Background and objectives
AvrPtoof Pseudomonas syringaepv. tomato (Pst) is recognized by Pto of tomato in a gene-for-gene interaction. avrPto is 165 amino acids in length and is a hydrophilic protein. avrPto lacks homology to any known protein. We and others have shown a direct interaction between avrPto and Pto in the yeast two-hybrid system [1, 2]. DEB-mutants of Pto that could no longer mount a complete resistance response against Pst failed to interact with avrPto. Likewise, avrPto mutants which did not elicit an HR in N. benthamiana expressing Pto from a CaMV35S promoter did not interact with Pto. These data suggest the interaction between avrPto and Pto is vital in eliciting resistance to Pst. We conducted both systematic in vitro and random in vivo mutagenesis of avrPto to further define the regions of avrPto necessary for Pto resistance, and to investigate the molecular events that occur during recognition and subsequent signal transduction. These mutants will also provide information on the mechanisms by which bacterial pathogens secrete avirulence proteins. AvrPto mutants may aid in future studies on possible pathogenicity or virulence functions of avrPto.

Results and conclusions
Systematic deletions of avrPto were created and tested against Pto in the two-hybrid system. A fragment lacking 30 amino acids from the amino-terminus and 40 amino acids from the carboxy terminus still interacted with Pto. AvrPto with deletions of an additional 10 or more amino acids from either end was not recognized by Pto. Therefore, we have localized the binding determinant of avrPto to a region of no larger than 95 amino acids.

We took advantage of the low fidelity of RNA polymerases to create random mutations of avrPto. We cloned avrPto into potato virus X (PVX) and inoculated Pto-carrying and Pto-lacking genotypes. PVX normally causes a systemic chlorosis on plants. Pto-carrying plants had no phenotype when challenged with PVX::avrPto. Pto-lacking plants showed systemic necrosis. To identify avrPto mutations, we performed RT-PCR on RNA isolated from plants that exhibited wild-type PVX symptoms. Of the avrPto point mutations that we have identified to date, all lie within the smallest fragment of avrPto recognized by Pto.

We are currently in the process of testing all avrPto mutants in vivo using Pst and transient Agrobacterium assays to determine if the in vivo results correlate with the two-hybrid data. Results from expression of avrPto mutants in Pst may be influenced by the interplay between avrPto and the pathogen's secretion machinery.

References
1. Scofield SR, Tobias CM, Rathjen JP et al., 1997. Science 274, 2063-2065.
2. Tang X, Frederick RD, Zhou J et al., 1997. Science 274, 2060-2063.