Pathogenesis-Related Proteins in Trifolium subterraneum: A General Survey and Subsequent Characterisation of a Protein Inducible by Ethephon and Redlegged Earth Mite Attack

Abstract

One of the chief predators of subterranean clover (Trifolium subterraneum) pastures is redlegged earth mite (Halotydeus destructor; RLEM). Subterranean clover pathogenesis-related (PR) proteins induced by RLEM attack and ethephon treatment were surveyed, and PR proteins with peroxidase, β-1,3-glucanase and chitinase activities were detected. A protein co-migrating with a chitinase activity, induced by RLEM predation and treatment with ethephon, was isolated. It was purified and the N-terminal amino acid sequence determined. Using a degenerate oligonucleotide primer designed from this sequence, a corresponding cDNA fragment was amplified by reverse transcriptase-PCR, then cloned, and used as a probe to screen a subterranean clover cv. Karridale genomic library. The cDNA and a 97% homologous genomic clone were sequenced and the deduced amino acid sequence revealed an open reading frame of 157 amino acids capable of encoding a peptide of 16 478 Da. Significant homology (80%) was found between this protein and an abscisic acid (ABA)-responsive protein from Pisum sativum of unknown function which is an intracellular pathogenesis-related (IPR) protein. The gene encoding this protein also has homology to pea ‘disease response resistance genes’ and to proteins from other plant species in the PR-10 family. The induced protein was designated TsPR-10a due to its homology to other PR-10 proteins. Genomic Southern analysis indicates that the gene encoding this protein, designated Ypr10a, is a member of a multigene family with at least three members. Northern blot analysis indicates that the subterranean clover Ypr10a mRNA, or homologous transcript, level is strongly induced by ethephon treatment in both root and aerial tissues of 3 week old plants. The rapid induction kinetics of Ypr10a mRNA under ethephon treatment, its correlation with a putative chitinase activity, and homology to other PR-protein genes, suggests a pathogenesis-related role for TsPR-10a protein in subterranean clover.