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The antigen reactive to an anti-white pine blister rust fungal monoclonal antibody (Mab 7) is a homologue of 70-kDa heat shock proteins (a BiP protein)

Abstract The production and characterization of monoclonal antibodies (Mab) to the white pine blister rust (WPBR) fungus (Cronartium ribicola) were described by Ekramoddoullah and Taylor (1996). One of the monoclonal antibodies, Mab 7, detected a major mycelial antigen on blister rust This monoclonal antibody did not cross-react with proteins from the white pine host or with proteins from other fungal species that were known to infect western white pine. As part of our ongoing work on the potential use of Mab 7 as a plantibody for engineering WPBR-resistant white pine, we report here on the cloning and characterization of cDNA encoding the antigen reactive to Mab 7. Five rounds of immunoscreening of a Uni-ZAP expression cDNA library from poly (A) + mRNA of C. ribicola mycelia with Mab 7 led to the identification of three positive clones. One of them was completely sequenced. Sequence analysis indicated an open reading frame of 2010 bases encoding a protein (designated as Cro r II) of 669 amino acid residues with a molecular mass of 72.9 kDa and a predicted isoelectric point of 5.0. Two-dimensional silver-stained gels and 2-D Western analyses detected multiple spots with the same molecular mass and slightly different pI around pH 5.0, suggesting isoforms of Cro r II. A BLAST search of the NCBI database with the deduced Cro r II protein sequence indicated homology with a group of 70-kDa heat shock proteins. Southern blot hybridizations indicated that the C. ribicola genome contained at least one copy of the Cro r II gene. Western immunoblot analyses revealed that the Cro r II protein was present in mycelial culture, in the infected white pine tissues, in the alternate infected Ribes stage, and in the five different spore stages, suggesting a constitutive role for Cro r II in the fungus.