In order to analyze the inducible defense response of hybrid poplar (Populus trichocarpa x P. deltoides), macroarrays were used to profile transcript patterns elicited by wounding and by regurgitant from forest tent caterpillar (FTC; Malacosoma disstria), a poplar defoliator. FTC regurgitant proved to be a potent elicitor of defense gene expression and was shown to contain the insect-derived elicitor volicitin. Comparison of inducible defense responses elicited by FTC regurgitant and wounding with pliers revealed qualitatively similar responses in terms of transcript accumulation. Extensive overlap was also observed in the sets of induced genes from locally- and systemically-induced leaves. Systemic responses were further investigated and shown to also be inducible in roots, which implies shoot-root systemic signaling. Comparative macroarray analysis showed similarities between inducible responses in leaves and roots, including genes that encode previously identified leaf herbivore defense genes. The macroarray analysis also established a suite of marker genes for future studies of herbivore defense in poplar, many of which may play key roles in the defense response and are candidates for further study. Among these genes were several inducible Kunitz trypsin inhibitors (KTIs), which were investigated further with biochemical analyses. The sequenced poplar genome was used to select KTI genes that represent the diversity of this family. Recombinant proteins were generated and showed that the poplar KTI genes encode functional proteinase inhibitors and that they are functionally distinct, i.e. they
have specific proteinase substrate preferences. Moreover, wounding increases accumulation of KTI proteins, as well as protease inhibitor activity in leaves, supporting a defensive role for this protein family. These proteins were therefore tested for their ability to inhibit insect digestive proteases from FTC and bertha armyworm. The poplar KTIs tested all inhibited at least some protease activity from FTC midgut extracts. The strongest inhibitor of FTC proteases, TI3. was further tested in bioassays and shown to reduce larval growth of FTC when incorporated into insect diet. confirming that this KTI functions as an anti-herbivore protein. In addition, analysis of FTC midguts from T13 feeding experiments showed that larvae responded to TI3 by producing more gut proteases. This hyperproduction of proteases may exacerbate the antinutritive effects of TI3 by reducing pools of essential amino acids.
| Identifer | oai:union.ndltd.org:uvic.ca/oai:dspace.library.uvic.ca:1828/2406 |
| Date | 30 March 2010 |
| Creators | Major, Ian |
| Contributors | Constabel, Carsten Peter |
| Source Sets | University of Victoria |
| Language | English, English |
| Detected Language | English |
| Type | Thesis |
| Format | application/pdf |
| Rights | Available to the World Wide Web |
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