5.3.15
ANALYSIS OF THE POSSIBLE INVOLVEMENT OF PEA DEFENSINS IN DISEASE RESISTANCE

F-M LAI , K MEI, C DELONG and P FOBERT

Background and objectives
Mycosphaerella pinodes is a serious fungal pathogen of peas in the Canadian prairies, for which there is currently no effective source of natural resistance. As a possible means of enhancing resistance to this fungus, we are producing transgenic plants expressing high levels of antifungal molecules. One class of molecules being evaluated is the defensins - small, cationic, antimicrobial peptides present in a variety of organisms. In peas, two cDNA clones isolated following fungal challenge, PI39 and PI230 [1], encode peptides that share sequence homology to known defensins. The objective of this study was to determine the possible role of these genes in disease resistance and to evaluate their potential effectiveness in enhancing tolerance to fungal pathogens in transgenic plants.

Results and conclusions
To test whether the proteins encoded by PI39 and PI230 possess antifungal activity, their coding regions were fused separately to the strong, constitutive CaMV 35S promoter and used to produce transgenic tobacco plants. Compared to peptide preparations from untransformed plants, those from plants expressing high levels of pea defensin transcripts were considerably more effective at reducing growth of the test fungus Trichoderma reesei in a microtitre plate bioassay. These preparations appeared to be less effective at inhibiting the growth of M. pinodes. In contrast, peptide preparations from transgenic tobacco plants expressing the radish defensin RsAFP2 appeared to be equally effective at inhibiting growth of T. reesei and M. pinodes. We are currently testing a range of fungi to determine if M. pinodes may be more tolerant to the pea defensins.

We also examined the expression pattern of the pea defensins in detail. Transcripts of both genes were detected at high levels in numerous tissues from unchallenged plants, including mature leaflets, tendrils, stems, flowers and radicles from axenically germinated seedlings. Immature seed pods, leaflets from seedlings and mature roots expressed PI39 and PI230 at either lower or non-detectable levels. Following challenge of mature leaflets with compatible, incompatible and non-host strains of Pseudomonas syringae, moderate increases in transcript levels were observed for both genes. In comparison, levels of a PR10 homologue were barely detectable prior to challenge, and increased dramatically thereafter. Overall, the expression pattern of PI39 and PI230 suggests that they may represent preformed resistance factors, or possibly be involved in non-defence-related processes.

References
1. Chiang CC, Hadwiger LA, 1991. Molecular Plant-Microbe Interactions 4, 324-331.