Detecting herbivore-specific transcriptional responses in plants with multiple DDRT-PCR and subtractive library procedures

Differential display-reverse transcriptase PCR (DDRT-PCR) and subtractive hybridization with magnetic beads (SHMB) procedures were modified to compare the transcriptional responses of the postfire desert annual, Nicotiana attenuata when it is attacked by its two most abundant herbivores: the voracious Lepidopteran caterpillars of Manduca sexta and the piercing/sucking nymphs and adults of the mirid bug, Tupiocoris notatus. The relative merits of the two procedures were compared. DDRT-PCR requires less starting material, allows for comparisons of multiple herbivores, and identifies both down- and up-regulated responses, but is more laborious than SHMB. SHMB produced a greater proportion of known sequences (43.8 versus 35.6%), but the sequences were not significantly longer than those obtained by DDRT-PCR, suggesting that the SMART (switching mechanism at 5' end of RNA transcript) modification of the SHMB procedure did not produce the desired result. Both procedures produced apparent false positives and microarray-based verification of differential expression would be a powerful approach to identify novel genes involved in ecological interactions. During the N. attenuata-T. notatus interaction, the expression of several photosynthesis-related genes (cytochrome f, psaE, rubisco, rubisco activase, two aldolases, and phosphoglycerate kinase) was altered, which suggests that reprogramming of photosynthesis contributes to the mechanisms mediating tolerance to mirid damage. In addition, T. notatus-induced changes in the expression of transcripts coding for a rhamnosyl transferase, a thionin, and an S-adenosylmethionine-decarboxylase are particularly interesting in the context of what is known about defence against herbivores in N. attenuata.