1.1.12
CHARACTERIZATION OF AN AVIRULENCE GENE FROM VENTURIA INAEQUALIS

J WIN1,2, KM PLUMMER1,2, JK BOWEN1 and MD TEMPLETON1

1HortResearch, Mt Albert Research Centre, Private Bag 92169, Auckland, NZ; 2University of Auckland, Private Bag 92019, Auckland, NZ

Background and objectives
Venturia inaequalis causes scab disease in apple (Malus). This system offers an excellent opportunity to investigate avirulence. The gene-for-gene nature of the interaction between V. inaequalis and Malus has been demonstrated by genetic analysis of both the host and pathogen. We are investigating the interaction between the resistance gene Vm originally from Malus micromalus and the avirulence gene from V. inaequalis, arbitrarily referred to as AvrVm. Host differential reactions are readily distinguished by a hypersensitive response (HR) in host differential (h5) and susceptibility is characterized by large sporulating lesions in host differential (h1). It has been suggested that the resistance gene product recognizes the avirulence gene product and initiates a signal transduction cascade which leads to resistance. Our main objective is to isolate and characterize the AvrVm gene product using a reverse genetics approach, similar to that used to isolate avr4, avr9 and nip1. V. inaequalis proteins are being screened for their ability to induce HR on the Vm host. HR-evoking peptide(s) will be sequenced and the sequence information will be used to isolate the cDNA encoding the peptide. The putative avirulence gene product will be confirmed by transforming a virulent isolate with the specific cDNA from the avirulent isolate.

Materials and methods
V. inaequalis isolates designated as race 1 and race 5 [1] are characterized by their pathogenicity on differential hosts (h1 and h5). h5 (9AR2T196) [2] carries the Vm resistance gene and h1 (cv. Royal Gala) lacks the Vm resistance gene. Race 1 isolates lack the AvrVm gene. Inheritance of avirulence is being analysed in progeny from sexual crosses. Proteins from in vitro and in planta grown isolates (30 days' growth in potato dextrose broth at 20C) with different avirulence genotypes (races 1 and 5) have been compared for different profiles in SDS PAGE and in a bioassay on h1 and h5.

Results and conclusions
Differential host responses have been observed on h1 and h5 to culture filtrates from in vitro grown race 1 and race 5 isolates. Race 1 and race 5 isolates also have different protein profiles on SDS PAGE, race 1 has proteins in the 4-10 kDa range, whereas race 5 lacks any small proteins; however there is also variation in the larger proteins. Specific protein bands separated by SDS PAGE have been collected using a gel eluter. Individual protein bands are being tested in the bioassay for a differential host response (induction of HR in h5). Viable single ascospore progeny have been collected and are being assessed to ascertain the segregation of proteins, bioassay and avirulence/virulence phenotypes. These results indicate significant progress towards the isolation of the AvrVm gene. We plan to investigate variation in avirulence genes within the V. inaequalis population. This will lead to an understanding of the role of fungal avirulence genes, which in turn will allow the impact of changes in avirulence genes on the durability of a resistance genes to be evaluated. It may also be possible to utilize the avirulence gene product to isolate a specific host resistance gene. An understanding of this interaction could also aid in the design of a durable resistance gene.

References
1. Williams EB, Brown AG, 1968. Plant Disease Reporter 52, 799-801.
2. Kellerhals M, Furrer B, 1994. In Schmidt H, Kellerhals M, eds, Progress in Temperate Fruit Breeding. Kluwer Academic, The Netherlands, pp. 93-97.