Molecular interactions between the specialist herbivore Manduca sexta (Lepidoptera, Sphingidae) and its natural host Nicotiana attenuata. II. Accumulation of plant mRNAs in response to insect-derived cues

Abstract

The transcriptional changes in Nicotiana attenuata Torr. ex Wats. elicited by attack from Manduca sexta larvae were previously characterized by mRNA differential display (D. Hermsmeier, U. Schittko, I.T. Baldwin [2001] Plant Physiol 125: 683-700). Because herbivore attack causes wounding, we disentangled wound-induced changes from those elicited by M. sexta oral secretions and regurgitant (R) with a northern analysis of a subset of the differentially expressed transcripts encoding threonine deaminase, pathogen-induced oxygenase, a photosystem II light-harvesting protein, a retrotransposon homolog, and three unknown genes. R extensively modified wound-induced responses by suppressing wound-induced transcripts (type I) or amplifying the wound-induced response (type II) further down-regulating wound-suppressed transcripts (type IIa) or up-regulating wound-induced transcripts (type IIb). It is interesting that although all seven genes displayed their R-specific patterns in the treated tissues largely independently of the leaf or plant developmental stage, only the type I genes displayed strong systemic induction. Ethylene was not responsible for any of the specific patterns of expression. R collected from different tobacco feeding insects, M. sexta, Manduca quinquemaculata, and Heliothis virescens, as well as from different instars of M. sexta were equally active. The active components of M. sexta R were heat stable and active in minute amounts, comparable with real transfer rates during larval feeding. Specific expression patterns may indicate that the plant is adjusting its wound response to efficiently fend off M. sexta, but may also be advantageous to the larvae, especially when R suppress wound-induced plant responses.

Figure 1

Transcript accumulation in sink and source leaves. Leaves growing at node 1 and node 4 of separate plants with the youngest fully expanded leaf defining node 3 were analyzed. Leaves of five replicate N. attenuata plants were continuously wounded and supplied with W or R from M. sexta larvae for 105 min, creating one row of puncture wounds every 15 min and harvesting 15 min after the final treatment. Untreated leaves were harvested as controls (C). Probe pDH63.5, coding for a GAL83 homolog, was used as a positive indicator of source-sink differences. Hybridization with an 18S rRNA probe demonstrates equal loading. The type of expression pattern (I, IIa, and IIb) is indicated.

Figure 2

Transcript accumulation in rosette or stem leaves of flowering plants. Leaves (being two positions older than the youngest rosette leaf or two positions younger than the first stem-born leaf) of five replicate N. attenuata plants were continuously wounded and supplied with W or R from M. sexta larvae for 80 min, creating one row of puncture wounds every 20 min and harvesting 20 min after the final treatment. Untreated leaves were harvested as controls (C). Hybridization with an 18S rRNA probe demonstrates equal loading. The type of expression pattern (I, IIa) is indicated.

Figure 3

Local and systemic transcript accumulation in rosette stage plants. The node 4 leaf of four replicate N. attenuata plants was wounded and supplied with W or R from M. sexta larvae every 15 to 20 min, creating one row of puncture wounds at each harvest. The treated leaf (local), all leaves younger (syst. young), and all leaves older (syst. old) than the node 4 leaf were harvested at eight different time intervals (min) after the first treatment when they had received one, two, four, six, or eight (the last three harvests) rows of puncture wounds, respectively. Harvests at 30, 50, 85, 115, and 135 min were during the continuous treatment period, whereas the 190- and 315-min harvests were taken 1 and 3 h after the final treatment event, respectively. Untreated plants were harvested as controls (C). Hybridization with an 18S rRNA probe demonstrates equal loading.

Figure 4

Transcript accumulation in the absence (−) or presence (+) of 1-MCP, the competitive inhibitor of ethylene receptors. The node 4 leaf of five replicate rosette-stage N. attenuata plants was continuously wounded and supplied with W or R from M. sexta larvae for 80 min, creating one row of puncture wounds every 20 min and harvesting 20 min after the final treatment. Alternatively, seven 2nd and 3rd instar M. sexta larvae (L) were allowed to systemically feed on four replicate plants for 4 h. Untreated node 4 leaves or plants were harvested as controls (C). Hybridization with an 18S rRNA probe demonstrates equal loading.

Figure 5

Transcript accumulation in response to different R solutions. The node 4 leaf of four replicate rosette-stage N. attenuata plants was continuously wounded and supplied with W or different R solutions for 80 min, creating one row of puncture wounds every 20 min and harvesting 20 min after the final treatment. R from Manduca quinquemaculata (Q), Spodoptera littoralis (S), Heliothis virescens (H) and 3rd to 5th instar M. sexta (M) larvae were tested, as well as boiled, sterile filtered, and freshly collected M. sexta R. Untreated node 4 leaves were harvested as controls (C). Hybridization with an 18S rRNA probe demonstrates equal loading.

Figure 6

Transcript accumulation in response to real and simulated herbivory. On seven replicate N. attenuata plants, one 3rd instar M. sexta larva (L) was allowed to feed on the node 4 source leaf for 150 to 180 min. Feeding was simultaneously mimicked with larval mandibles (M). The damaged leaf (local), as well as all leaves younger (syst. young) and all leaves older (syst. old) than the node 4 leaf were analyzed. Untreated node 4 leaves were harvested as controls (C). Hybridization with an 18S rRNA probe demonstrates equal loading.