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
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.