Cloning and characterization of a cDNA of cro rI from the white pine blister rust fungus Cronartium ribicola

White pine blister rust (WPBR) is caused by the fungus Cronartium ribicola which has five spore stages on two unrelated hosts, the five-needle pines and Ribes spp. Recently, during the molecular analysis of the proteins and genes involved in host-pathogen interaction, the WPBR fungal protein Cro rI was identified in infected white pine tissues. To further characterize Cro rI, an expression cDNA library from poly(A)(+) mRNA of C. ribicola axenic mycelial culture was constructed and immunoscreened and the cDNA was cloned. Sequence analysis indicated an open reading frame of 462 bases, which encodes a protein of 153 amino acid residues with a molecular mass of 16.7 kDa and a predicted isoelectric point (pI) of 8.93. Based on the N-terminal amino acid sequences of Cro rI, the secreted portion of Cro rI protein should be 136 amino acids long with several putative posttranslational modification sites and a molecular mass of 14.8 kDa. The predicted pl for the secreted portion was 9.34. The predicted N-terminal signal peptide was 17 amino acids long. The N-terminal 42-amino acid sequence of the predicted mature protein (secreted portion) was identical to the amino terminal sequence of Cro rI that was previously determined. Southern blot hybridizations indicated that the C. ribicola genome contained at least two copies of the cro rI gene. Isolation of the genomic PCR fragment, which was approximately 400 bp longer than the cDNA, and subsequent cloning and sequencing analyses confirmed that there were three introns within the coding regions. Western immunoblot analyses revealed that Cro rI protein accumulated in large amounts only in the infected white pine tissues while no trace was detectable in the alternate Ribes stage or the five different spores, suggesting a critical role of Cro rI in the haploid stage of the fungus (in pine). The translocation of Cro rI was only found to occur in cankered trees, and not in the young infected seedlings. The implications of Cro rI in pathogenesis are discussed. (C) 2002 Elsevier Science (USA).