1.13.6
EXPRESSED GENE PROBES AS MOLECULAR TOOLS FOR DETAILED ANALYSIS OF THE OBLIGATE PARASITE POLYMYXA BETAE AND ITS ASSOCIATION WITH SUGAR BEET

ES MUTASA-GOTTGENS, DM CHWARSZCZYNSKA and MJC ASHER

IACR-Broom's Barn, Higham, Bury St Edmunds, Suffolk 1P28 6NP, UK

Background and objectives
Comparatively little progress has been made in the study of Polymyxa betae at the molecular level even though it is a pathogen of significant economic importance as a virus vector. This is largely due to its obligately parasitic habit and until now, molecular studies have focused on the development of accurate, sensitive detection methods [1]. Evidence showing biological differences between fungal isolates with respect to host range already exists [2]; further studies may indicate variation in pathogenicity and the ability to transmit beet necrotic yellow vein virus (BNYVV). A more detailed knowledge of P. betae at the molecular level may begin to reveal answers to these questions. We describe how probes from highly expressed transcripts are not only starting to provide clues about possible mechanisms underlying the P. betae/sugar beet interaction, but are also useful for examining intra-specific variation amongst fungal isolates.

Materials and methods
Poly(A)+ RNA was isolated from sugar beet roots heavily infected with P. betae using the Promega PolyATract mRNA Isolation System, and used to make a lambda Zap-cDNA library (Stratagene). The phage particles were excised in vivo and the resultant pBluescript plasmids gridded on Hybond-N membranes in a 96 well format. Duplicate filters were then probed in parallel under stringent conditions with total RNA probes generated from infected and uninfected sugar beet roots. Clones that were unique to infected material were recovered and analysed further by Southern and/or Northern blot hybridisation to nucleic acids isolated from infected and uninfected sugar beet roots. This further differentiated fungal and host clones, and indicated the expression levels in infected vs uninfected roots. Selected fungal and host clones were sequenced and database searches used to deduce possible function. Fungal clones were also tested for isolate specific RFLPs in Southern blots or by restriction digestion of PCR products from the same single copy loci.

Results and conclusions
Out of 288 clones screened in the initial round, 150 hybridised to the infected sugar beet root probe. Of these, 123 clones (82%) were specifically expressed in infected roots, and 27 clones (18%) were also expressed in the absence of P. betae. An additional 8 sugar beet clones that appeared to be repressed during infection were also identified. Southern analysis of a random sample of 25 clones showed that 28% were of sugar beet origin, 44% of fungal origin and 8% were mixed, containing both sugar beet and fungal sequences. Results on the remaining 20% were unclear and require further testing. This is therefore an efficient method for isolating expressed genes from an obligate parasitic fungus. All P. betae cDNAs tested generated RFLPs in genomic DNA digested with PstI and were able to differentiate our standard test isolates originally obtained from soils at separate geographic locations. RFLP patterns of PCR products from these single copy loci were also used to identify and track isolates grown and passaged through 9 different weed hosts commonly found on sugar beet fields. Data revealed that isolates grown on Chenopodium quinoa were distinct from all others. All isolates adopted the same pattern whenever they were also transferred to C. quinoa. Northern analysis of 6 sugar beet clones showed at least two that were developmentally regulated in P. betae-free roots, with possible early induction caused by infection. Sequence data suggest that one of these transcripts may be a member of the chitinase multigene family, implicated in host defence. One of the P. betae clones sequenced so far may have a possible role in protecting the fungus against the hypersensitive response by the host.

References
1. Mutasa ES, Chwarszczynska DM, Asher MJC, 1996. Phytopathology 86, 493-97.
2. Barr KJ, Asher MJC, 1992. Plant Pathology 41, 64-68.