1.8.53
ASPECTS OF CLADOSPORIUM FULVUM METABOLISM IN RELATION TO PATHOGENICITY AND OXIDATIVE STRESS

KJ BLISSEU1, H-J BUSSINK1, AJ CLARK1, M PIKE2, G SEGERS1 and RP OLIVER1

1Department of Physiology, Carlsberg Laboratory, Gamle Carlsberg Vej 10, DK-2500 Copenhagen Valby, Denmark; 2School of Biological Sciences, University of East Anglia, Norwich NR4 7UJ, UK

Background and objectives
Cladosporium fulvum is a well characterized biotrophic pathogen of tomato (Lycopersicon esculentum). During a compatible interaction, this fungus is capable of invasion and growth within the tomato leaf without causing significant tissue damage or eliciting active plant defence mechanisms. The fungus obtains its nutrients exclusively from the apoplastic fluid. We are interested in determining the molecular events involved in maintaining this state of biotrophy. As many fungal genes induced during growth in planta, including pathogenicity determinants, are also activated with the onset of starvation during axenic growth, it has been reasoned that the nutritional states of the invading organism and its host must be important to the outcome of any pathogenic interaction [1].

Results and conclusions
One of the most abundant compounds in growing C. fulvum mycelium is mannitol. Experiments have been undertaken to investigate the role of mannitol metabolism on the ability of C. fulvum to be pathogenic. In order to determine the levels and flux of intracellular fungal metabolites, we have employed HPLC with pulsed amperometric detection (PAD) and in vivo NMR. These have demonstrated that the accumulation pattern of mannitol in C. fulvum mycelium, grown under hyper-osmotic stress, was not consistent with mannitol acting as a compatible solute. Instead, arabinitol and to a lesser extent glycerol levels were more likely to be involved in mediating salt stress. We have also demonstrated that the fungus is under constant hyper-osmotic stress whilst growing in planta, regardless of the plant water status.

Mannitol has been demonstrated to mediate protection against active oxygen species (AOS) in a number of different systems [2]. We have recently shown that a C. fulvum plant-expressed gene [1] encodes a peroxisomal alcohol oxidase. This may suggest that fungal metabolism during growth in planta generates AOS. Additionally, it has been shown that AOS can accumulate in tomato leaves to appreciable levels [3]. Therefore, we are currently studying the role of mannitol in protection against AOS during a compatible interaction. We will also present the latest data on cloning and characterization of genes involved in direct and indirect mediation of oxidative stress.

This work indicates a role for fungal polyol metabolism with respect to successful colonization during pathogenesis. A much more complex picture of compatibility is now developing and the further elucidation of fungal metabolism may help to better understand biotrophic plant pathogens.

References
1. Coleman M, Henricot B, Arnau J, Oliver RP, 1997. Molecular Plant-Microbe Interactions 10, 1106-1109.
2. Chaturvedi V, Bartiss A, Wong B, 1997. Journal of Bacteriology 179, 157-162.
3. May MJ, Hammond-Kosack KE, Jones JDG, 1996. Journal of Plant Physiology 110, 1367-1379.