Induced plant defense responses against chewing insects. Ethylene signaling reduces resistance of Arabidopsis against Egyptian cotton worm but not diamondback moth

Abstract

The induction of plant defenses by insect feeding is regulated via multiple signaling cascades. One of them, ethylene signaling, increases susceptibility of Arabidopsis to the generalist herbivore Egyptian cotton worm (Spodoptera littoralis; Lepidoptera: Noctuidae). The hookless1 mutation, which affects a downstream component of ethylene signaling, conferred resistance to Egyptian cotton worm as compared with wild-type plants. Likewise, ein2, a mutant in a central component of the ethylene signaling pathway, caused enhanced resistance to Egyptian cotton worm that was similar in magnitude to hookless1. Moreover, pretreatment of plants with ethephon (2-chloroethanephosphonic acid), a chemical that releases ethylene, elevated plant susceptibility to Egyptian cotton worm. By contrast, these mutations in the ethylene-signaling pathway had no detectable effects on diamondback moth (Plutella xylostella) feeding. It is surprising that this is not due to nonactivation of defense signaling, because diamondback moth does induce genes that relate to wound-response pathways. Of these wound-related genes, jasmonic acid regulates a novel beta-glucosidase 1 (BGL1), whereas ethylene controls a putative calcium-binding elongation factor hand protein. These results suggest that a specialist insect herbivore triggers general wound-response pathways in Arabidopsis but, unlike a generalist herbivore, does not react to ethylene-mediated physiological changes.

Figure 1

Regulation of Arabidopsis genes by insect feeding or wounding. Total RNA (10 μg) was extracted from rosette tissue and RNA gel blots were hybridized with probes indicated on the left. In contrast to loading controls, abbreviations of genes related to insect feeding are in bold. A, Plants were untreated, exposed to one diamondback moth (DBM) larvae per plant for 10 or 30 h, or mechanically wounded (Wnd) 10 or 30 h prior to harvest. Blots were stripped and re-probed with ACT2, a loading control that is constitutively expressed. Size estimates for the different mRNAs are indicated on the right. B, Plants were untreated, mechanically wounded, or diamondback moths (four larvae per plant) were applied prior to harvest at the indicated time points in minutes. Size estimates are listed on the right. A probe for 25S rRNA served as a loading control. Additional controls (not shown) found no trace of circadian or light-dependent changes in expression of these genes.

Figure 2

Regulation of stress-response genes by MeJA. Plants were untreated or sprayed with 150 μm MeJA 10 h or 30 h prior to harvest. Total RNA (10 μg) was extracted from rosette tissue and RNA gel blots were hybridized with probes indicated on the left. ACT2 or rRNA was used as loading controls.

Figure 3

Regulation of stress-response genes by ethephon. Plants were untreated or sprayed with 50 μm ethephon 1, 3, 6, 9, or 27 h prior to harvest. Total RNA (10 μg) was extracted from rosette tissue and RNA gel blots were hybridized with probes indicated on the left.

Figure 4

Regulation of stress-response genes by SA. Plants were untreated or sprayed with 5 mm SA 10 or 30 h prior to harvest. A, Total RNA (10 μg) was extracted from rosette tissue and RNA gel blots were hybridized with probes indicated on the left. B, SA-regulation of specific genes was confirmed by semiquantitative PCR. We observed more GST2 and PR-1 product upon SA treatment than in controls after 25, 27, or 29 PCR cycles, suggesting a real difference.

Figure 5

Measure of leaf damage caused by insect feeding on Arabidopsis. Representative examples of plants are shown that were grouped into categories (0–6) based on the amount of leaf area removed by herbivores (0%–100%). Arrows indicate leaves that were attacked.

Figure 6

Ethylene perception compromises resistance of Arabidopsis to Egyptian cotton worm, but not to diamondback moth. Resistance against Egyptian cotton worm is enhanced in hls1-1 compared with wild-type (Col-0) Arabidopsis and reduced by ethephon application. Resistance against diamondback moth is neither significantly affected by genotype nor by ethylene treatment. Damage is a measure of the amount of leaf area consumed by larvae, scored on a scale from 0 (resistant) to 6 (susceptible). Ethe, Ethephon; Ethy, ethylene. Error bars indicate se. Statistical analysis of the Egyptian cotton worm data set is provided in Table I.

Figure 7

The ein2-1 mutation enhances resistance against Egyptian cotton worm, but not diamondback moth relative to wild type (Col-0). Damage is a measure of the amount of leaf area consumed by larvae, scored on a scale from 0 (resistant) to 6 (susceptible). Error bars indicate se.

Figure 8

Consensus phylogenetic tree from genes belonging to the glucosyl hydrolase family 1 (Henrissat and Bairoch, 1993) based on coding sequence data. The tree is a majority rule consensus of 1,000 trees, each inferred from parametric distances (Lake, 1994) by the neighbor joining method (Felsenstein, 1993). Branch lengths were fitted using the Fitch-Margoliash algorithm, as implemented in PHYLIP. The numbers are percentages based on how many trees out of 1,000 supported the clades. Bar = genetic distance. BGL1 falls into a clade of β-glucosidases from Arabidopsis and Brassica that is separate from myrosinases, cyanogenic β-glucosidases, and other more distantly related genes. Cyanogenesis has not been demonstrated experimentally for all of the enzymes in the middle group, and some may have alternative functions. BG, β-Glucosidases; DH, dhurrinase; FG-BG, furostanol glycoside BG; PH, prunasin hydrolase; AH, amygdlin hydrolase; N-CBG, non-cyanogenic BG; LIN, linamarase; MYR, myrosinase; TGG, thioglucosidase; LPH, lactase-phlorizin hydrolase; PBG, phospho-BG. Note that BG7 and BG8 of Arabidopsis have been mistakenly annotated as myrosinases in the databases. In contrast to myrosinases, these two genes contain the active site catalyst Glu found in β-glucosidases instead of Gln found in myrosinases. Accession numbers are available at http://vanilla. ice.mpg.de/departments/Gen/publications/stotz_tree.html.

Figure 9

BGL1 encodes a predicted protein of 60.5 kD. The arrow indicates a potential cleavage site of the signal peptide. Putative N-glycosylation sites are underlined, a putative O-glycosylation site is double underlined. Residue Glu-207 is the acid catalyst that is conserved in all β-glucosidases, but not found in myrosinases. The predicted endoplasmic reticulum retention signal REEL is shown in bold.