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Clone the corresponding genes encoding resistance-associated proteins (RAPs). Study biochemical and physiological role(s) of RAPS in kernel resistance. Examine the expression of RAP genes during kernel development. Study the polymorphism of these genes to see whether there is a particular allele associated with host resistance. Perform promoter analysis of RAP genes and identify possible regulatory genes using the yeast one-hybrid system.
Resistance-associated genes will be cloned using PCR and RACE methods with degenerate primers corresponding to the peptide sequences. The expression of these genes in resistant and susceptible genotypes during kernel development under normal and stress conditions will be examined using northern or real time PCR. The corresponding genes will be overexpressed in E. coli for antibody production, in vitro biochemical and antifungal studies, and also will be expressed in tobacco for testing their biological functions relating to antifungal and stress resistance. The promoters of these genes will be analyzed to identify a common motif, which will then be used in the yeast one-hybrid system to identify the maste regulatory protein/gene(s).