Identification of a protein secreted by the blister rust fungus Cronartium ribicola in infected white pines and its cDNA cloning and characterization

Previously we showed that a white pine protein Pin m III (a member of PR10 family of pathogenesis-related proteins) is up-regulated by infection in the white pine blister rust pathosystem. In this study, a blister rust fungal protein, Cro r I, which is similar in size to Pin m III (19 kDa), was detected in the infected white pine tissues. The N-terminal amino acid sequence of Cro r I isolated from infected pine foliage and from fungal mycelia was identical. Rabbit antibody was prepared to a synthetic N-terminal peptide and was purified by immunoaffinity. The purified antibody was used in a Western immunoblot to quantify the amount of Cro r I in various tissues. In western white pine seedlings the amount of Cro r I was significantly (p < 0.0001) higher in infected tissues of cankered seedlings than the infected tissues of resistant seedlings. In sugar pine seedlings, the amount of Cro r I was also significantly (p < 0.01) higher in infected tissues of susceptible seedlings than in resistant seedlings. Furthermore, Cro r I is secreted by the blister rust fungus and was found to be translocated to the healthy tissues of cankered white pines. Cro r I is a major protein that could be extracted from infected foliage by vacuum infiltration. The level of Cro r I detected in the mycelium of different isolates varied. The cDNA of Cro r I was isolated by reverse transcription - polymerase chain reaction. Comparison of the DNA sequence and the deduced protein sequence with data bases revealed that it is a previously undescribed protein. The calculated molecular weight from the deduced protein sequence of Cro r I was 16.7 kDa and the calculated isoelectric point was 9.55. Protein sequence analysis showed that Cro r I has two potential N-linked glycosylation sites in its sequence.