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Cloning and Characterization of a Putative Antifungal Peptide Gene (Pm-AMP1) in Pinus monticola

We have been working on proteins that are involved in the defense response of western white pine (WWP) (Pinus monitcola) to the blister rust fungus Cronartium ribicola. Our objective was to identify candidate genes that could be used for improving resistance of WWP to this rust pathogen. During proteomic analysis of bark proteins extracted from WWP trees exhibiting slow-canker-growth (SCG) resistance, a 10.6-kDa peptide, termed Pm-AMP1, was found to be enriched at the receding canker margin. The cDNA encoding this peptide was cloned and characterized. A BLASTX search revealed that the Pm-AMP1 encoded by its cDNA has a 50% homology with MiAMP1, a broad-spectrum antifungal protein isolated from Macadamia integrifolia. Based on the deduced amino acid sequence, an antibody was produced against the Pm-AMP1. Immunochemical quantification of the Pm-AMP1 in bark samples of susceptible WWP trees revealed this protein to be barely detectable in the cankered tissues, but occurring in higher concentrations in healthy tissues away from canker margins. Foliage of SCG-resistant trees contained higher concentrations of the Pm-AMP1 than foliage from susceptible cankered trees. Both wounding and methyl jasmonate treatment of WWP needles induced the expression of this protein, further supporting its putative role as a defense response protein.