Isolation and structural characterization of the active molecule from Sirex noctilio woodwasp venom inducing primary physiological symptoms in attacked pine species
Bordeaux, John Michael
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Sirex noctilio F., a non-native, invasive woodwasp recently introduced into North America, attacks and kills living pines, including economically important species. S. noctilio introduces phytotoxic venom and a pathogenic basidiomycete, Amylostereum areolatum, into trees during oviposition. S. noctilio venom alone induces pine needle chlorosis and abscission, phloem collapse, altered respiration, loss of carbohydrate translocation, and reduced growth rate. A 26,496-feature loblolly pine cDNA microarray was used to identify two genes useful as biomarkers in qRT-PCR bioassays for pine response to venom exposure, PR4 and TLP. Expression of both was strongly induced in response to venom, while expression of an apparent actin gene (ACT1) was stable in response to the venom. The S. noctilio venom gland transcriptome was assembled from >260 million Illumina HiSeq reads using a minimum length cutoff for assemblies of 100 nucleotides. This dataset is the first transcriptome-scale resource available for a basal-order species of Hymenoptera. Of the 68,887 transcripts generated from this dataset, 24,471 (35%) returned annotations with E- value < 1 x 10 10. No annotations were returned for the seven most abundant transcripts. The single most abundant transcript present encodes the molecule responsible for initial wilt and gene responses in pines. The eighth most highly-expressed sequence recovered was a transcript encoding a putative laccase-like multicopper oxidase. The bioassay above was utilized to isolate from S. noctilio venom an 11-amino acid peptide (SEGPROGTKRP) of 1850 Da. This peptide, noctilisin, induced the wilt phenotype in pine seedling explants. Primary sequence and structure were established using Edman degradation, NMR, and MS-MS. Post-translational modifications include O-glycosylation of serine and threonine, and hydroxylation of the 6th residue (proline). Two identical O-glycans present were characterized as N-acetylgalactose, modified by an O-linked phosphoethanolamine on the glycan C6. Noctilisin activity is glycan-dependent. Wide-scale screening for resistance to woodwasp attack across pine populations now is feasible. A further RNA-Seq experiment was performed to compare the effects of whole venom, noctilisin alone, and a biologically-inactive, non-glycosylated peptide on sensitive P. radiata explants. These results should elucidate suites of genes responding specifically to noctilisin and distinguish them from genes responding to other active factors in whole venom.