Abstract
Hydroxyproline-rich glycopeptides (HypSys peptides) are recently discovered 16-20-amino acid defense signals in tobacco and tomato leaves that are derived from cell wall-associated precursors. The peptides are powerful wound signals that activate the expression of defensive genes in tobacco and tomato leaves in response to herbivore attacks. We have isolated a cDNA from petunia (Petunia hybrida) leaves encoding a putative protein of 214 amino acids that is a homolog of tobacco and tomato HypSys peptide precursors and is inducible by wounding and MeJA. The deduced protein contains a leader sequence and four predicted proline-rich peptides of 18-21 amino acids. Three of the four peptides were isolated from leaves, and each peptide contained hydroxylated prolines and glycosyl residues. Each of the peptides has a -GR- motif at its N terminus, indicating that it may be the substrate site for a processing enzyme. The peptides were active in a petunia suspension culture bioassay at nanomolar concentrations, but they did not induce the expression of defense genes that are directed against herbivores, as found in tobacco and tomato leaves. They did, however, activate expression of defensin 1, a gene associated with inducible defense responses against pathogens.
Highlights
Peptide signals that activate receptor-mediated defense signaling pathways in plants are derived from either pathogens [1,2,3] or from host plants (4 –7) in response to wounding or infection
Three PhHypSys peptides were isolated from petunia leaf extracts that were encoded by PhpreproHypSys I, but no peptides encoded by PhpreproHypSys II were found during the isolations
Isolation of PhpreproHypSys I and II cDNAs—A consensus sequence derived from a highly conserved region of 30 nucleotides within the coding regions of NtpreproHypSys [5] and LepreproHypSys [6] (Fig. 1) was used to synthesize a primer for RT-PCR to search for an ortholog in petunia (Fig. 1B)
Summary
Peptide Assays—Petunia suspension cells utilized for the alkalinization assay were maintained as previously described [8], and the medium was adjusted to pH 5.6 with KOH. CDNA Isolation—The cDNAs that were previously isolated from tobacco and tomato each contain a 30-nucleotide region overlapping the processing site of the leader peptide From these sequences, a consensus, degenerative oligonucleotide primer, 5Ј-GGAGCTNAAGCAAGAACTTTRCTAGNAAAT-3Ј (where N represents G/C/T/A, and R is G/A), was synthesized for 3Ј RT-PCR (Ambion, Austin, TX) to seek orthologs of the two genes in petunia. The 3Ј-end of the sequence was obtained by synthesizing a primer, 5Ј-GGAGCTGAAGCAAGATCTTTGC-3Ј, from within the region contained in the degenerative 30-mer (Fig. 1B) with all of the correct bases using 3Ј-rapid amplification of cDNA ends RT-PCR This resulted in the complete sequence of a cDNA that encoded the sequence of the peptides isolated from Peaks 1, 2, and 3 (Fig. 3A). The leaf samples were collected for assays at 0, 1, 2, 4, 6, 8, 12, and 24 h after spraying and immediately frozen in liquid nitrogen and stored at Ϫ80 °C
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