The HERBIVORE ELICITOR-REGULATED1 gene enhances abscisic acid levels and defenses against herbivores in Nicotiana attenuata plants

Abstract

Nicotiana attenuata plants can distinguish the damage caused by herbivore feeding from other types of damage by perceiving herbivore-associated elicitors, such as the fatty acid-amino acid conjugates (FACs) in oral secretions (OS) of Manduca sexta larvae, which are introduced into wounds during feeding. However, the transduction of FAC signals into downstream plant defense responses is still not well established. We identified a novel FAC-regulated protein in N. attenuata (NaHER1; for herbivore elicitor regulated) and show that it is an indispensable part of the OS signal transduction pathway. N. attenuata plants silenced in the expression of NaHER1 by RNA interference (irHER1) were unable to amplify their defenses beyond basal, wound-induced levels in response to OS elicitation. M. sexta larvae performed 2-fold better when reared on irHER1 plants, which released less volatile organic compounds (indirect defense) and had strongly reduced levels of several direct defense metabolites, including trypsin proteinase inhibitors, 17-hydroxygeranyllinallool diterpene glycosides, and caffeoylputrescine, after real and/or simulated herbivore attack. In parallel to impaired jasmonate signaling and metabolism, irHER1 plants were more drought sensitive and showed reduced levels of abscisic acid (ABA) in the leaves, suggesting that silencing of NaHER1 interfered with ABA metabolism. Because treatment of irHER1 plants with ABA results in both the accumulation of significantly more ABA catabolites and the complete restoration of normal wild-type levels of OS-induced defense metabolites, we conclude that NaHER1 acts as a natural suppressor of ABA catabolism after herbivore attack, which, in turn, activates the full defense profile and resistance against herbivores.

Figure 1.

NaHER1 is regulated by OS and FAC independently of JA. A, NaHER1 transcript abundances (±se; n ≥ 3) in wild-type leaves wounded and immediately treated with 20 µL of water (WW) or 1:5 water-diluted OS (WOS) from M. sexta larvae. Samples were collected at the designated time points and analyzed by qPCR. B, NaHER1 transcript abundances in wild-type plants wounded and treated with OS (WOS) or with C18:3-Glu (0.26 mm) suspended in water (WW+FAC) and complemented with 150 µg (0.625 µmol) of MeJA in 20 µL of lanolin paste as described; 20 µL of pure lanolin was used as a control. Samples were collected after 1 h of treatment. C, NaHER1 transcript abundances in JA biosynthesis-deficient (irLOX3) and perception-deficient (irCOI1) plants after WOS and WOS + MeJA treatments; samples were collected after 1 h of treatment. Different letters show significant differences determined by one-way ANOVA followed by Fisher’s Plsd post hoc test (P ≤ 0.05). WT, Wild type.

Figure 2.

WOS-induced volatile emissions are compromised in irHER1 plants. Relative emission rates (means ± se; n ≥ 6) are shown for green leaf volatiles (A), monoterpenes (B), diterpenes (C), and benzenoid/phenylpropanoid derivatives (D) released from the systemic leaves of wild-type (WT) and irHER1 plants treated with WW or WOS. One local rosette leaf in each 44-d-old N. attenuata plant was mechanically wounded and treated with 20 µL of water (WW) or diluted OS (WOS) 18 h before the start of volatile collections. Volatiles were collected for 3 h from the head space of a systemic leaf at position +8 relative to the leaf undergoing its transition from source to sink (position 0). Different letters show significant differences determined by one-way ANOVA followed by Fisher’s Plsd post hoc test (P ≤ 0.05). IS, Internal standard. [See online article for color version of this figure.]

Figure 3.

Silencing of NaHER1 suppressed plant defense metabolite accumulation and increased the performance of the M. sexta specialist herbivore. A, Fresh masses (±se; n ≥ 10) of M. sexta neonates feeding on wild-type (WT) and irHER1 plants recorded at designated time points to estimate the specialist herbivore performance. B, Relative transcript abundances (±se; n = 5) of NaMYB8 and NaMYC2 genes determined by qPCR. C, Accumulations (means ± se; n = 5) of secondary metabolites CP, DCS, and total HGL-DTGs in wild-type and irHER1 leaves fed by M. sexta neonates for 4 d. D, TPI activity (means ± se; n = 5) determined in local WOS-treated leaves 24 h after elicitation. Asterisks represent significant differences between the wild type and irHER1 lines determined by ANOVA followed by Fisher’s Plsd post hoc test (*P ≤ 0.05, ***P ≤ 0.01). CHA eq, CHA equivalents; FM, fresh mass. [See online article for color version of this figure.]

Figure 4.

Silencing of NaHER1 affects transpiration rates and plant growth under drought stress. A, Wild-type (WT; left) and irHER1-6/4 (right) plants after 12 d without watering. B, Transpiration rates (±se; n = 4) determined by an LI-6400 Portable Photosynthesis System in the intact leaves of glasshouse-grown wild-type and irHER1 plants. C, Water loss (±se; n = 9) from detached leaves of wild-type and irHER1 plants determined at designated time points after spraying with 0.5% ethanol (control) or with 0.5% ethanol supplied with 300 µm ABA. Transpiration rates were compared by ANOVA followed by Fisher’s Plsd post hoc test; water loss data in C were analyzed by the Mann-Whitney U test (*P ≤ 0.05, **P ≤ 0.01). [See online article for color version of this figure.]

Figure 5.

Silencing of NaHER1 affects ABA, JA, and defense metabolite levels. Rosette-stage N. attenuata leaves were treated with WOS in zone 2, and samples were collected after 30 min and 48 h and analyzed by LC-MS/MS (ABA, JA, JA-Ile, SA [±se; n = 4]) and HPLC (CHA, CP, DCS, HGL-DTGs [±se; n = 4]), respectively. Leaves were divided into four equal parts during sampling, and each part was analyzed separately. Different letters show significant differences determined by one-way ANOVA followed by Fisher’s Plsd post hoc test (P ≤ 0.05). CHA eq, CHA equivalents; FM, fresh mass; WT, wild type. Red dashed lines show treated areas. [See online article for color version of this figure.]

Figure 6.

Local and systemic defense metabolite levels in irHER1 cannot be complemented by exogenous MeJA. Local (A) and systemic (B) accumulations (means ± se; n ≥ 4) are shown for CHA, DCS, CP, and total HGL-DTGs in 44-d-old N. attenuata plants that had their rosette leaves mechanically wounded and treated with 20 µL of diluted OS from M. sexta (WOS) in combination with MeJA in lanolin paste as described. After 72 h, local and systemic leaves (+8 position) from each plant were collected and analyzed by HPLC. Asterisks represent significant differences between wild-type (WT) and irHER1-6/4 plants determined by Student’s t test: *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001. CHA eq, CHA equivalents; FM, fresh mass. [See online article for color version of this figure.]

Figure 7.

ABA is required for the induction of OS-dependent defense metabolite accumulation. NaHER1 or NaPYL4 (ABA receptor) were silenced by VIGS in wild-type N. attenuata plants. A, Phytohormone (ABA, JA, JA-Ile) contents (means ± se; n = 6) in mechanically wounded leaves treated with 20 µL of diluted OS from M. sexta (WOS), collected after 1 h, and analyzed by LC-MS/MS. B, Secondary metabolites (CP, DCS, HGL-DTGs) in the leaves attacked by M. sexta neonates (one per leaf) for 4 d and analyzed by HPLC. Different letters show significant differences between samples determined by one-way ANOVA followed by Fisher’s Plsd post hoc test (P ≤ 0.05). EV, Empty vector. CHA eq, CHA equivalents; FM, fresh mass.

Figure 8.

Exogenous application of ABA restores local and systemic defense metabolite levels in irHER1 leaves. Secondary metabolite contents (means ± se; n = 4) are shown for rosette-stage wild-type (WT) and irHER1 leaves wounded with a pattern wheel in zone 2 and treated with 5 µL of diluted OS from M. sexta. Immediately after treatment, leaves were sprayed with 0.5% (v/v) ethanol in water (control) or 100 µm ABA diluted in 0.5% ethanol to complement ABA deficiency in irHER1 plants. Samples were collected after 48 h and analyzed by HPLC. Asterisks represent significant differences (P ≤ 0.05) between wild-type and irHER1-6/4 plants determined by Student’s t test: *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001. CHA eq, CHA equivalents; FM, fresh mass. [See online article for color version of this figure.]

Figure 9.

NaHER1 silencing alters ABA metabolism in N. attenuata. Accumulations (means ± se; n = 3) are shown for ABA-derived metabolites in detached leaves of wild-type (WT) and irHER1 plants supplied via petiole with 2 mL of 0.5% (v/v) ethanol in water supplemented with 0.5% (w/v) mannitol and 0.02% (v/v) OS from M. sexta larvae (control) or with a control solution supplied with 100 µg of ABA. Samples were collected after 3 h and analyzed by LC-MS/MS for individual ABA metabolites. Different letters show significant differences between samples determined by one-way ANOVA followed by Fisher’s Plsd post hoc test (P ≤ 0.05). 7(9)-OH-ABA, 7(9)-Hydroxy-ABA; FM, fresh mass; neoPA, neophaseic acid; Me, methyl ester. The levels of individual ABA metabolites were determined relative to six atoms of deuterium (D₆)-ABA internal standard. [See online article for color version of this figure.]

Figure 10.

Proposed model of NaHER1 function in N. attenuata. The perception and signal transduction of FACs in OS of herbivores induce the expression of the NaHER1 gene, which, in turn, increases ABA levels by suppressing the catabolism of the hormone (ABA_cat). Higher ABA levels promote the accumulation of defense metabolites and JA, which promotes the defense of N. attenuata plants and increases resistance against feeding herbivores. [See online article for color version of this figure.]