Department of Plant Sciences, University of Cambridge, Cambridge CB2 3EA, UK

Background and objectives
Pyrenopeziza brassicae is a hemibiotrophic ascomycete which causes light leaf spot disease of brassicas, including many agriculturally important crops such as oilseed rape (Brassica napus ssp. oleifera). After penetration of the leaf surface, the fungus proliferates in the sub-cuticular region, causing minimal tissue damage. During this biotrophic phase, the fungus obtains all its nutritional requirements from living host tissue, although the mechanism by which this is achieved is unknown. The ability of biotrophic and hemibiotrophic pathogens to maintain a close physiological relationship with their host without causing obvious tissue damage has been attributed to the capacity of these fungi to induce translocation of host photosynthates and other nutrients to the site of infection. It has been proposed that fungal-derived cytokinins act as a metabolic sink in several plant-pathogen interactions. Early symptoms of infection of B. napus by P. brassicae include leaf curl and green island formation, both of which are indicative of a cytokinin imbalance. The aim of this work is to ascertain whether cytokinin production by P. brassicae is a contributing factor in early pathogenesis on oilseed rape.

Materials and methods
A number of strategies are being employed to determine the role of P. brassicae-derived cytokinins in light leaf spot disease. Cytokinin production in vitro is being studied using HPLC-ELISA. To analyse the effect of cytokinin over-production, P. brassicae has been co-transformed with pnipt, a vector carrying a cytokinin biosynthesis gene (ipt), and pAN7-1, for hygromycin selection. Cytokinin production by transformants both in vitro and in planta is being analysed. Pathogenicity towards oilseed rape cotyledons and whole plants is being investigated, and compared to pathogenicity of the wild type. A rapid, efficient and reliable UV mutagenesis protocol for this fungus has been devised, which employs the UV crosslinker (Stratalinker). Mutants are currently being screened for cytokinin production by crude ELISA, using a method adapted from Lichter et al. [1].

Results and conclusions
A range of P. brassicae field isolates of both mating types and differing geographical locations were analysed for cytokinin production in vitro, and correlation between pathogenicity and cytokinin production is under way. Three co-transformants carrying the Agrobacterium tumefaciens ipt gene are being investigated for cytokinin production in vitro and in planta, and for altered symptomology in planta. RT-PCR and Northern analyses have demonstrated in vitro transcription of ipt. HPLC-ELISA analysis of cytokinin production in vitro showed an increase in cytokinin production by the transformants compared to wild type. Analysis of in planta cytokinin production is under way. Symptom expression of the transformants greatly differed from that of the wild type in both cotyledon-based and whole plant-based pathogenicity tests, indicative of there being increased cytokinin levels within infected tissue.

The results support the hyptothesis that P. brassicae-derived cytokinins play a role in pathogenesis of oilseed rape. Current progress in this research will be presented.

1. Lichter A, Manulis S, Sagee O et al., 1995. Molecular Plant-Microbe Interactions 8, 114-121.
2. Murphy AM, Pryce-Jones E, Johnstone K, Ashby AM, 1997. Physiological and Molecular Plant Pathology 50, 53-65.