1.9.3
ENZYMES AND PROTEINS INVOLVED IN THE RESISTANCE TO SCLEROTINIA SCLEROTIORUM IN OILSEED RAPE

SY LIU1, BW ZHOU1, QF XIONG2 and HS LI2

1Oil Crops Research Institute, Chinese Academy of Agricultural Science, Wuhan 430062 China; 2Huazhong Agricultural University, Wuhan 430070, China

Background and objectives
Sclerotinia sclerotiorum, a pathogen which can infect more than 400 plant species and cause serious losses of crop yield, produces a toxin, oxalic acid (OA), in pathogenesis and in pure culture. There is a wide variation of resistance in oilseed rape (Brassica sp.) to the fungus and its toxin, and the resistance to the toxin is higher in upper than in basal leaves. A significant correlation was found between resistance of rape to the fungus and to OA. However, the mechanism of such resistance has not been elucidated. The objectives of the studies were to investigate uptake and metabolism of OA by rape seedlings, and the enzymes and proteins involved in the resistance.

Results and conclusions
Close relationships were found between resistance of rape to the pathogen and its toxin, and the activity of peroxidase (PO), polyphenol oxidase (PPO), superoxide dismutase (SOD), phenylalanine ammonia-lyase (PAL) and oxalic acid oxidase (OAO). Inoculations with mycelia or OA induced activity increases in PO, SOD, OAO and PAL, but inhibited the activities of PPO when both leaf extracts and purified enzymes were tested. The activity increase was more in resistant than in susceptible varieties. A significant correlation was found between resistance levels and increases in PO activity. Although the number of the bands of isozymes of both PO and PPO varied irregularly after inoculation, there were more bands in resistant varieties than in susceptible ones among the 30 varieties tested. Resistant and susceptible varieties were clustered into different groups on the basis of distance coefficient computed from the number and intensity of the bands or from integral value of a zymogram scan.

Experiments with rape seedlings fed with 14C-oxalate through roots for 4 h showed significant uptake of the toxin. The autoradiographic profile of seedlings showed that in resistant varieties radioactivity was confined to major veins of leaves and stems, and that the activity in the interveinal tissue was low. In contrast, in susceptible varieties radioactivity was distributed rather uniformly throughout the seedlings. The metabolism of OA was examined.

New protein bands were found by SDS-PAGE 72 h after seedlings were inoculated with the fungus and OA. A 27.5-kDa protein was consistently induced by the fungus, and a 54.8-kDa protein was induced by OA. Two to five bands disappeared in susceptible varieties after inoculation. The 54.8-kDa protein was prepared to immunize rabbits and the antiserum was collected. Double-agar diffusion testing proved that the 54.8-kDa protein was not peroxidase from horseradish or rape leaves. Whether these new proteins are OAO or other functioning enzymes needs to be ascertained.

In conclusion, the studies showed that the following factors contributed toward higher resistance in resistant varieties than susceptible ones: the limited uptake and transport of OA, high rate of metabolism of OA by leaves and more increase of PO, OAO and SOD activity induced by OA or the fungus. The oxidation of H2C2O4 promoted by oxidation of polyphenols in which the first reaction product, H2O2, served as a substrate of the second reaction might play a role in the process of resistance.