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DIFFERENTIAL INDUCTION OF b-1,3-GLUCANASES IN RESPONSE TO SCLEROSPORA GRAMINICOLA INFECTION IN PEARL MILLET
DIFFERENTIAL INDUCTION OF b-1,3-GLUCANASES IN RESPONSE TO SCLEROSPORA GRAMINICOLA INFECTION IN PEARL MILLET
RAMACHANDRA KINI K., VASANTHI U. KUMAR and SHETTY H. S.
Downy Mildew Research Laboratory, Department of Studies in Applied Botany, University of Mysore, Mysore- 570 006, INDIA
Background
Sclerospora graminicola an obligate, oomycetous fungus causing downy mildew disease of pearl millet (Pennisetum glaucum which is the major constraint in the production of pearl millet. Even though many cultivars are resistant to the disease, the resistance is not durable and often there is breakdown of resistance in the cultivars. The exact reasons for the breakdown of resistance are not clearly known as there is a lack of complete understanding of the biochemical basis of resistance of pearl millet to downy mildew. b-1,3-glucanases, found in most of the plant species have been shown to have antifungal properties and are implicated in the defense reactions of a number of plants. In the present study, involvement of b-1,3-glucanase in pearl millet resistance against downy mildew was investigated.
Materials and methods
The enzyme was assayed in the crude extracts of resistant and susceptible cultivars of pearl millet seedlings inoculated with the pathogen. b-1,3-glucanase was purified and characterized from pearl millet seedlings. lsozyme pattern of b-1,3-glucanase was studied using isoelectric focusing. Antibodies were produced against the purified enzyme and were used in western blotting analysis of crude extracts of pearl millet seedlings.
Results and conclusions
Inoculation of seedlings with the downy mildew pathogen S. graminicola resulted in accumulation of b-1,3-glucanase enzyme in resistant cultivars of pearl millet seedlings. lsoelectric focusing of total proteins from resistant and susceptible cultivars of pearl millet revealed four basic isoforms with pl 9.6, 9.0, 8.9 and 8.2 and two acidic isoforms with pl 4.9 and 6.2 of b-1,3-glucanase. Not all the isoforms of b-1,3-glucanases identified in the study were associated with resistance of pearl millet to S. graminicola infection. The basic isoform of pl 9.6 appears to have a developmental role as it was present in both resistant and susceptible cultivars of pearl millet before and after inoculation. The pl 8.2 isoform is probably involved in the expression of resistance as its activity was very high in incompatible reaction. The acidic isoform of pl 4.9 seemed to be selectively induced only in the compatible interaction during pathogenesis at the expense of the two basic isoforms of pl 8.9 and 9.0 which disappeared in the inoculated seedlings. Another acidic isoform of b-1,3-glucanase (pl 6.2) which was unique to resistant cultivar was highly induced after inoculation. The immunoblot analysis using antiserum raised against the purified b-1,3-glucanase has identified this isoform of the enzyme in the total proteins of crude extracts of resistant and susceptible cultivars of pearl millet seedlings. Even though the antiserum was raised against this 20.5 kDa isoform, it reacted more strongly with another isoform of 30 kDa only in the resistant samples. It appears that this is related to the isoform of pl 6.2 which was detected prominently in the resistant samples after inoculation and shown to be absent in both control and inoculated seedlings of susceptible cultivars of pearl millet. Thus, probably during infection of the host there is a programmed rearrangement of b-1,3-glucanase synthesis that induces an isoform which effects plant resistance in incompatible interaction. However, further work is necessary to substantiate this observation. In conclusion, our data suggest that b-1,3-glucanases are involved in the resistance of pearl millet against the downy mildew pathogen. In addition to the constitutive expression, different isoforms of the enzyme are also induced during infection.