Genomics of hybrid poplar (Populus trichocarpa× deltoides) interacting with forest tent caterpillars (Malacosoma disstria): normalized and full‐length cDNA libraries, expressed sequence tags, and a cDNA microarray for the study of insect‐induced defences in poplar

Abstract

As part of a genomics strategy to characterize inducible defences against insect herbivory in poplar, we developed a comprehensive suite of functional genomics resources including cDNA libraries, expressed sequence tags (ESTs) and a cDNA microarray platform. These resources are designed to complement the existing poplar genome sequence and poplar (Populus spp.) ESTs by focusing on herbivore- and elicitor-treated tissues and incorporating normalization methods to capture rare transcripts. From a set of 15 standard, normalized or full-length cDNA libraries, we generated 139,007 3'- or 5'-end sequenced ESTs, representing more than one-third of the c. 385,000 publicly available Populus ESTs. Clustering and assembly of 107,519 3'-end ESTs resulted in 14,451 contigs and 20,560 singletons, altogether representing 35,011 putative unique transcripts, or potentially more than three-quarters of the predicted c. 45,000 genes in the poplar genome. Using this EST resource, we developed a cDNA microarray containing 15,496 unique genes, which was utilized to monitor gene expression in poplar leaves in response to herbivory by forest tent caterpillars (Malacosoma disstria). After 24 h of feeding, 1191 genes were classified as up-regulated, compared to only 537 down-regulated. Functional classification of this induced gene set revealed genes with roles in plant defence (e.g. endochitinases, Kunitz protease inhibitors), octadecanoid and ethylene signalling (e.g. lipoxygenase, allene oxide synthase, 1-aminocyclopropane-1-carboxylate oxidase), transport (e.g. ABC proteins, calreticulin), secondary metabolism [e.g. polyphenol oxidase, isoflavone reductase, (-)-germacrene D synthase] and transcriptional regulation [e.g. leucine-rich repeat transmembrane kinase, several transcription factor classes (zinc finger C3H type, AP2/EREBP, WRKY, bHLH)]. This study provides the first genome-scale approach to characterize insect-induced defences in a woody perennial providing a solid platform for functional investigation of plant-insect interactions in poplar.

The MYB182 Protein Down-Regulates Proanthocyanidin and Anthocyanin Biosynthesis in Poplar by Repressing Both Structural and Regulatory Flavonoid Genes

“…However, the recombinant UGT78M1 protein was not active with any anthocyanin, flavonol, or flavan-3-ol substrate that we tested, so its biochemical function remains undefined (Veljanovski and Constabel, 2013). The functions of the other UGT are also unknown; however, it is expressed in herbivore-stressed poplars (Ralph et al, 2006). A conspicuous group of down-regulated genes is annotated as encoding flavonoid 39-monooxygenase-like enzymes (i.e.…”

Two Herbivore-Induced Cytochrome P450 Enzymes CYP79D6 and CYP79D7 Catalyze the Formation of Volatile Aldoximes Involved in Poplar Defense

“…The third and most likely mode of volatile nitrile formation in planta is the oxidation of aldoximes by P450 enzymes of the CYP71 or CYP736 family as described for the biosynthesis of cyanogenic glycosides and camalexin (Bak et al, 1998b;Nafisi et al, 2007;Takos et al, 2011). The P. trichocarpa genome contains 25 CYP71 genes and six CYP736 genes (Nelson et al, 2008), and microarray analysis revealed that several of these genes were upregulated after herbivore feeding (Ralph et al, 2006). Therefore, they might play a role in herbivore-induced volatile nitrile formation in poplar.…”

Evaluating Ascorbate Oxidase as a Plant Defense Against Leaf-Chewing Insects Using Transgenic Poplar

“…Similarly, third instars that fed in mesh bags on intact saplings also showed no effect of the high Table 4). It was assumed that AO activities were unaffected by larval feeding, since no evidence of AO induction by caterpillars has been observed in hybrid poplar (Major and Constabel 2006;Ralph et al 2006). Finally, the consumption and growth rates and nutritional indices (AD, ECD, and ECI) of fourth instars did not differ when contrasted between the control and AO-overexpressing genotypes (contrast P's≥ 0.690; Table 5).…”