A protein associated with citrus blight (CB), a disease of unknown cause, was partially characterized. The 12 kDa protein, designated p12, is diagnostic of CB and is present in leaves and xylem fluid from roots and stems of CB-affected trees. The protein, and up to six other CB-specific proteins, are readily detected by SDS-PAGE of xylem fluid from CB-affected trees. The partial N-terminal amino acid sequence of p12 was found to be unique based on database searches. A cDNA library from CB-affected root cambium was screened with a 60 bp fragment, obtained by PCR amplification of cDNA with degenerate primers designed using the amino acid sequence of p12, and two clones were selected. These clones were sequenced revealing a 674 nucleotide cDNA with a 393 nt ORF which included sequence predicted by the N-terminal amino acid sequence of p12. The amino acid sequence based on the p12 ORF was found to be up to 49% similar and 31% identical to expansins. Bacterial expression of the cloned ORF, which encodes an 11.8 kDa protein plus an N-terminal hydrophobic signal peptide, produced an immunoreactive protein of the expected size. By northern blot analysis, it was determined that p12 transcripts are present in root and stem cambium, but not in leaves of CB-affected trees, suggesting transport of the protein to leaves. Southern hybridization analysis of citrus genomic DNA indicated that p12 is a citrus encoded protein.p>
Plant Molecular Biology – Springer Journals
Published: Oct 6, 2004
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