Different multicellular trichome types coordinate herbivore mechanosensing and defense in tomato

Abstract

Herbivore-induced wounding can elicit a defense response in plants. However, whether plants possess a surveillance system capable of detecting herbivore threats and initiating preparatory defenses before wounding occurs remains unclear. In this study, we reveal that tomato (Solanum lycopersicum) trichomes can detect and respond to the mechanical stimuli generated by herbivores. Mechanical stimuli are preferentially perceived by long trichomes, and this mechanosensation is transduced via intra-trichome communication. This communication presumably involves calcium waves, and the transduced signals activate the jasmonic acid (JA) signaling pathway in short glandular trichomes, resulting in the upregulation of the Woolly (Wo)-SlMYC1 regulatory module for terpene biosynthesis. This induced defense mechanism provides plants with an early warning system against the threat of herbivore invasion. Our findings represent a perspective on the role of multicellular trichomes in plant defense and the underlying intra-trichome communication.

Figure 1.

Insect crawling induces the biosynthesis of insect-repellent terpenes in glandular trichomes. A) Schematic representation of tomato at different developmental stages (up). Left, juvenile stage (10 days), showing true leaves, cotyledons, and hypocotyl. Right, adult stage (30 days), showing top, middle, bottom leaves, and stem. Schematic representation of dominant trichome types at different developmental stages (down). The juvenile stage (10 days) includes type I, I, VI, VII trichomes, while the adult stage (30 days) includes type II, III, IV, V, VI, VII trichomes. B) Observation of insect crawling on leaf surfaces. 1st-2nd instar bollworm crawled on the true leaf (A, left) in the juvenile stage (10 d), the top leaf and the stem (A, right) in the adult stage (30 d). I, II, and VI represent type I, type II, and type VI trichomes, respectively. Scale bar, blue-500 µm, white-100 µm. Also see Supplementary Movies S1, S4 and S7. C) Schematic representation of the isolation of type VI glandular trichomes after artificial mechanical stimulation or insect crawling. D–E) Heatmap of SlTPS genes induced after insect crawling or artificial mechanical stimulation in WT. 30-day-old tomato plants were subjected to insect crawling or artificial mechanical stimulation (MS) of long trichomes (II + III) 10 times. After 0, or 1 h, type VI glandular trichomes of the stimulated plants were isolated, and the RNA was extracted for RT-qPCR analysis. Crawling-II + III, stimulation of type II and III trichomes by cotton bollworm crawling. MS-II + III, mechanical stimulation of type II and type III trichomes. Color scale bar represents the relative expression level of insect crawling or artificial mechanical stimulation (MS) for 1 h compared to 0 h. F) The RT-qPCR showing the expression change of SlTPS1/5/12 after mechanical stimulation (MS) in h/hl double mutants. 30-day-old tomato plants were subjected to artificial mechanical stimulation of trichomes (IV + V) 10 times. After 0, 1, or 2 h, type VI glandular trichomes of the stimulated plants were isolated, and the RNA was extracted for RT-qPCR analysis. Data are means ± SD (n = 3, biological replicates). Significant differences were determined by one-way ANOVA with Holm-Šídák's test compared to the MS 0 h. ns, no significance. MS-IV + V, mechanical stimulation of type IV and V trichomes. G) The RT-qPCR analysis of the expression of SlTPS1/5/12 after trichome-brushing in cr-w/m#1 mutant. 30-day-old tomato mutants were subjected to brushing 10 times, and the RNA extraction and statistics are as described in (F). MB, mechanical brushing. Data are means ± SD (n = 4, biological replicates). Significant differences were determined by one-way ANOVA with Holm-Šídák's test compared to the MS 0 h. **P < 0.01, ***P < 0.001, ****P < 0.0001. MB-VI + VII, mechanical brushing of type VI and VII trichomes. H) The RT-qPCR analysis of the expression of SlTPS1/5/12 after 10 times of insect crawling in the juvenile plants. 10-day-old tomato plants were subjected to artificial mechanical stimulation of long trichomes (type I trichome) 10 times. RNA extraction and statistics are as described in (F). Data are means ± SD (n = 3, biological replicates). Significant differences were determined by one-way ANOVA with Holm-Šídák's test compared to the MS 0 h. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. MS-I, mechanical stimulation of type I trichomes. MT, Micro-Tom.

Figure 2.

Intra-trichome communication relies on calcium waves. (A, C, E, G, I and K) Ca²⁺ imaging of tomato trichomes expressing 35Spro:GCaMP6f (Micro-Tom) marker. Multicellular trichomes of 10 or 30 day old plants were mechanically stimulated (MS). A) Mechanical stimulation of type I trichome transmits calcium wave signals to type I and VI trichomes. The examined tomato plant is 10-day-old. I, VI indicate type I and type VI trichomes, respectively. 1, 5, and 6 represent basal cells; 2, 3, and 4 mark the positions of VI-type trichomes; 7 marks glandular head of type I trichome. NMS, no mechanical stimulation. MS, mechanical stimulation. Scale bars for all images, 100 µm. Also see Supplementary Movie S15. C) Mechanical stimulation of type II trichome transmits calcium wave signals to VI-type trichomes. The examined tomato plant is 30-day-old. II, VI indicate type II and type VI trichomes, respectively. Numbers (1, 2, 3, 4, 5) on the white dashed lines mark the positions for the quantification of [Ca²⁺]_cyt after mechanical stimulation. 1 represent basal cells; 2 and 3 represent leaf surface; 4 represents VI-type trichome. MS, mechanical stimulation. Scale bars for all images, 100 µm. Also see Supplementary Movie S16. E) Mechanical stimulation of type III trichome barely transmits calcium waves. The examined tomato plant is 30-day-old. III indicates type III trichome. Numbers (1, 2) on the white dashed lines mark the positions for the quantification of [Ca²⁺]_cyt after mechanical stimulation. 1 represents the position at the base of type III trichome. 2 represents leaf surface. MS, mechanical stimulation. Scale bars for all images, 100 µm. Also see Supplementary Movie S17. G) Mechanical stimulation of type IV trichome weakly transmits calcium wave signals to the leaf surface. 1 represents leaf surface. The examined tomato plant is 10-day-old. IV indicates type IV trichome. Number (1) and white dashed circles mark the position of the base of the Type IV trichomes for the quantification of [Ca²⁺]_cyt after mechanical stimulation. MS, mechanical stimulation. Scale bars for all images, 100 µm. Also see Supplementary Movie S22. I) Mechanical stimulation of type V trichome weakly transmits calcium wave signals to the leaf surface. The examined tomato plant is 30-day-old. V indicates type V trichome. Numbers (1, 2) on the white dashed lines mark the positions for the quantification of [Ca²⁺]_cyt after mechanical stimulation. 1 represents the position at the base of type V trichome. 2 represents leaf surface. MS, mechanical stimulation. Scale bars for all images, 100 µm. Also see Supplementary Movie S23. K) Mechanical stimulation of type VI trichome transmits calcium wave signals to the leaf surface or type II, VI trichome. The examined tomato plant is 30-day-old. II, VI indicate type II and type VI trichomes, respectively. Numbers (1, 2, 3, 4, 5) on the white dashed lines mark the positions for the quantification of [Ca²⁺]_cyt intensity after mechanical stimulation. 1 represents the position at the base of type VI trichome. 2 and 3 represent leaf surface. 4 represent type VI trichome. 5 and 6 represent type II trichomes. MS, mechanical stimulation. Scale bars for all images, 100 µm. Also see Supplementary Movie S20. (B, D, F, H, J and L) are the quantification of [Ca²⁺]_cyt shown in (A, C, E, G, I and K) respectively.

Figure 3.

Trichomes serve as the calcium reservoir in plants. A and E) Tomato and Arabidopsis calcium indicator (35Spro:GCaMP6f marker, in tomato Micro-Tom and Arabidopsis Col-0 backgrounds) noninvasive confocal imaging. The calcium indicator lines were grown in 1/2 MS medium for 3 weeks prior to imaging. For noninvasive confocal imaging, the plants were inverted with the imaging site mounted in water in the cavity of a concavity slide. The red line indicates type I trichomes, with red arrows pointing to the basal cells and the leaf surface in (A). The white line marks the trichome, with white arrows pointing to the skirt cells and the leaf surface in (E). Scale bars for all images, 50 µm. B and F) Quantification of calcium indicator fluorescence shown in (A and E). Fluorescence was quantified using Image J. Data are means ± SD ((B): stalk, basal cell, leaf surface, n = 12, biological replicates. F): trichome, skirt cell, leaf surface, n = 11, biological replicates). Significant differences were determined by one-way ANOVA with Holm-Šídák's test: a, b, c, d, P < 0.05. C and G) Images of calcium probes (Calbryte 520 AM) showing the highly enriched signals in trichomes of tomato and Arabidopsis. White dashed lines outline the trichomes in (C). +indicates Calbryte 520 AM addition, -indicates without Calbryte 520 AM addition. D and H) Quantification of Calbryte 520 AM shown in (C and G). Fluorescence was quantified using Image J. Data are means ± SD (D): II, basal cell, leaf surface, n > 46, biological replicates; (H): trichome, skirt cell, leaf surface, n > 78, biological replicates). Significant differences were determined by two-way ANOVA with Holm-Šídák's test: a, b, c, d, P < 0.05. A, B, C, D, P < 0.01. Box plots show the 2nd and 3rd quartiles along with the median; whiskers represent 1.5 times the interquartile range, and each point indicates an individual value. I–J) μXRF analysis of the Ca distribution in tomato (MT) and Arabidopsis (Col-0) leaves. The leaves were placed both horizontally and vertically to observe the distribution of calcium ions on the leaf surface and trichomes. The white dashed circles outline the basal cells in (I). Color scale bar represents calcium ion intensity. K) The pattern diagram of calcium ion distribution. I and II represent type I and type II trichomes, respectively. The green scale bar represents calcium ion concentration.

Figure 4.

Wounding induces calcium waves in type I and VI trichomes. A–C and F) Ca²⁺ is imaged using 35Spro:GCaMP6f (Micro-Tom). The leaf surface of a 10-day-old tomato plant was mechanically cut. A) The tomato leaf was divided by dashed lines, and different parts are labeled as 1, 2, 3, and 4. Calcium wave in different parts were quantified and shown in (D and E). The green scale bars in (B) and (C) represent cytosolic calcium ion concentration. Scale bars, (A), (B), and (C), 1 mm; (F), 100 μm. Also see Supplementary Movie S26. D, E, and G) Quantification of [Ca²⁺]_cyt changes under different levels of mechanical cut. Fluorescence was quantified using Image J.

Figure 5.

Basal (skirt) cells of trichomes are critical for calcium mediated signal transmission. A–D) Imaging Ca²⁺ using 35Spro:GCaMP6f (slbrc2a). The calcium waves are barely transmitted in the type I (A) or II (C) trichome after the mechanical stimulation. [Ca²⁺]_cyt changes in (A) and (C) are quantified in (B) and (D), respectively. I and the white arrow point to type I trichome in (A). II and the white arrow point to type II trichome in (B). Numbers (1, 2, 3) on the white dashed lines mark the positions for the quantification of [Ca²⁺]_cyt after mechanical stimulation. 1 represents the position at the base of the trichome. 2 and 3 represent the positions on the leaf surface. 1st MS, first mechanical stimulation; 2nd MS, Second mechanical stimulation. MS-I, mechanical stimulation of type I trichomes; MS-II, mechanical stimulation of type II trichomes. The green scale bar represents cytosolic calcium ion concentration. Fluorescence was quantified using Image J. Scale bars for all images, 100 µm. Also see Supplementary Movies S27 and S28. E–G) Mechanical stimulation triggers calcium wave in the trichomes on the different developmental stages of Arabidopsis leaves. Calcium wave can be transmitted downward at the 3rd-6th stages (G), but cannot at the 1st-2nd stages (E). The quantification of [Ca²⁺]_cyt changes is shown in (F) and (H). White dashed lines outline of trichomes in (E and G). White arrows indicate skirt cells. Numbers (1, 2, 3) on the white dashed lines mark the positions for the quantification of [Ca²⁺]_cyt after mechanical stimulation. The green scale bar represents cytosolic calcium ion concentration. Fluorescence was quantified using Image J. Scale bars for all images, 100 µm. Also see Supplementary Movies S29 and S30. I–J) Model diagrams showing the loss of calcium waves in the trichomes lacking basal cells (tomato) or skirt cells (Arabidopsis).

Figure 6.

The threshold of [Ca²⁺]_cyt response in multicellular trichomes. A–D) [Ca²⁺]_cyt changes with different magnitudes of mechanical stimuli. A) Morphology of type II, VI trichomes on the leaf surface. II and VI represent type II and type VI trichomes. White arrows indicate type II and type VI trichomes, and 1 and purple arrows point to the basal cell of type II trichomes. The white dashed line indicates the path of calcium wave in (B). Scale bar, 100 µm. B) Visualization of 35Spro:GCaMP6f (Micro-Tom) marker under different magnitudes of mechanical stimuli. The examined tomato plant is 30-day-old. VI and white arrows indicate type VI trichomes. The green scale bar represents cytosolic calcium ion concentration. Scale bars for all images, 100 µm. C) shows the quantified [Ca²⁺]_cyt changes shown in (B). D) Schematic diagram of different magnitudes of mechanical stimuli. NMS, no mechanical stimulation. Light MS, light mechanical stimulation. Force MS, force mechanical stimulation. Also see Supplementary Movie S32. E–H) [Ca²⁺]_cyt threshold with different frequencies of mechanical stimuli. E) Morphology of type II trichomes on the leaf surface. II represent type II trichomes. Scale bar, 100 µm. F) Visualization of 35Spro:GCaMP6f (Micro-Tom) marker under different frequencies of mechanical stimuli. The examined tomato plant is 30-day-old. Numbers (1, 2, 3) on the white dashed lines (E and F) mark the positions for the quantification of calcium wave in (F) and (H). The green scale bar represents cytosolic calcium ion concentration. Scale bars for all images, 100 µm. G) shows the quantified [Ca²⁺]_cyt changes shown in (F). H) Schematic diagram of different frequencies of mechanical stimuli. F–H) NMS, no mechanical stimulation. 1st MS-1 time, mechanical stimulation once for the first time. 2nd MS-5 times, mechanical stimulation five times for the second time. 3rd MS-10 times, mechanical stimulation ten times for the third time. 4th MS-5 times, mechanical stimulation five times for the fourth times. Also see Supplementary Movie S33. I–L) Repeated mechanical stimuli promotes the transmission of calcium waves to type VI trichomes. I) Morphology of type I, VI trichomes on the leaf surface. I, VI and the white arrows indicate type I and type VI trichomes, respectively. Numbers (1, 2, 3, 4, and 5) on the white dashed lines and magenta dashed lines mark the positions for the quantification of calcium wave in (J) and (K). The dashed circle outlines the range of calcium waves shown in (J). Scale bar, 100 µm. J) Visualization of 35Spro:GCaMP6f (Micro-Tom) marker with repeated mechanical stimuli. The examined tomato plant is 10-day-old. Numbers (1, 2, 3, 4, and 5) mark the positions for the quantification of calcium waves. The green scale bar represents cytosolic calcium ion concentration. Scale bars for all images, 100 µm. K) shows the quantification of [Ca²⁺]_cyt shown in (J). L) Schematic diagram of repeated mechanical stimuli. J–L) NMS, no mechanical stimulation. 1st-MS-1time, mechanical stimulation once for the first time. 2nd-MS-4 times, mechanical stimulation four times for the second time. 1st MS-1time, mechanical stimulation once for the first time. 2nd MS-5 times, mechanical stimulation five times for the second time. 3rd MS-10 times, mechanical stimulation ten times for the third time. 4th MS-5 times, mechanical stimulation five times for the fourth time. Also see Supplementary Movie S34. Fluorescence in B, F and J was quantified using Image J.

Figure 7.

The mechanical sensing by trichomes initiates both the biosynthesis and signaling of jasmonic acid (JA). A) The RT-qPCR showing the changed expression of key genes involved in JA biosynthesis pathway after artificial mechanical stimulation. Type I (10 days old) or II (30 days old plants) trichomes were mechanically stimulated (MS). After 0, 1, and 2 h, type VI glandular trichomes were isolated, and RNA was extracted for RT-qPCR analysis. Data are means ± SD (left image, n = 4, biological replicates; right image, n = 3, biological replicates). Significant differences were determined by one-way ANOVA with Dunnett's test compared to the MS 0 h. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. MS-I, mechanical stimulation of type I trichome. MS-II + III, mechanical stimulation of type II and III trichomes. B) VENUS and mCherry fluorescence confocal imaging in type VI glandular trichomes after artificial mechanical stimulation (type II trichomes) and wounding. Schematic representation of the JA marker construct (top). Confocal imaging of JA marker transgenic materials in tomato (bottom). MS, mechanical stimulation. MD, mechanical damage. Scale bars for all images, 10 µm. C) Quantification of JA marker fluorescence normalized to mCherry signals in type VI glandular trichomes after artificial mechanical stimulation (type II trichomes) and wounding. Data are means ± SD (MS/MD 0 h, n = 44, biological replicates; MS 2 h, n = 46, biological replicates; MD 2 h, n = 45, biological replicates). Significant differences were determined by one-way ANOVA with Dunnett's test compared to the MS/MD 0 h. ****P < 0.0001. Box plots show the 2nd and 3rd quartiles along with the median; whiskers represent 1.5 times the interquartile range, and each point indicates an individual value. D) GUS staining of pSlMYC1::GUS#1 and pWoolly::GUS transgenic plants (30-day-old) after artificial mechanical stimulation (type II trichomes) and wounding. GUS staining was observed for 10 h. VI represents type VI trichomes. MS, mechanical stimulation. MD, mechanical damage. Scale bars for all images, red-50 µm, white-100 µm. E) The visual imaging of calcium wave transmission in type II trichomes. Visual imaging was performed after 0.05% (v/v) Silwet-77 (control) and LaCl₃ (a blocker for plasma-membrane Ca²⁺-channel) treatment for 20 min. White arrows indicate the location near the basal cells to show the subsequent calcium wave propagation. The green scale bar represents cytosolic calcium ion concentration. Ca²⁺ imaging marker, 35Spro:GCaMP6f (Micro-Tom). II represents type II trichomes. MS, mechanical stimulation. Scale bars for all images, 100 µm. F) shows the quantified [Ca²⁺]_cyt changes shown in (E). G and H) The RT-qPCR analysis of key genes involved in JA synthesis (G) and SlTPS1/5/12 (H) after artificial mechanical stimulation (MS-II + III). 30-day-old tomato plants were treated with 10 mm LaCl₃ (A control group was treated with 0.05% (v/v) Silwet-77). After 0 and 2 h, type VI glandular trichomes were isolated, and RNA was extracted for RT-qPCR analysis. Data are means ± SD (n = 3, biological replicates). Significant differences were determined by one-way ANOVA with Tukey's test. a, b, c, P < 0.05. MS-II + III, mechanical stimulation of type II and III trichomes. I) The ratio of injured leaves was evaluated in WT, wo (wo^W106R and cr-wo) and slmyc1(slmyc1-1 and slmyc1-2) mutants. 30-day-old tomato plants were subjected to artificial mechanical stimulation (MS) of long trichomes (WT and slmyc1 mutants, II + III) or manual brush of trichomes (wo mutants) for 5 min every 5 h, and the bollworm larvae of 1st-2nd instar was inoculated onto tomato plants. After 7 days of treatment (mechanical stimulation or manual brush), a fixed number of leaves were selected from the top to the middle of the plant, and the ratio of damaged leaves to total leaves was quantified. Data are means ± SD (WT and wo mutants, n = 20, biological replicates; WT and slmyc1 mutants, n = 18, biological replicates). Significant differences were determined by two-way ANOVA with Tukey's test. a, b, c, P < 0.05. Box plots show the 2nd and 3rd quartiles along with the median; whiskers represent 1.5 times the interquartile range, and each point indicates an individual value. NMB, no manual brush. NMS, no mechanical stimulation. MS, mechanical stimulation. MS-II + III, mechanical stimulation of type II and III trichomes. WT: wild type (Micro-Tom). J) Quantification of the weight of cotton bollworms. The weight of cotton bollworm was counted after 7 days of feeding. Mechanical stimulation or manual brush of 30-day-old plants was described in (I). Data are means ± SD (WT and wo mutants, n = 10, biological replicates; WT and slmyc1 mutants, n = 11, biological replicates). Significant differences were determined by two-way ANOVA with Tukey's test. a, b, c, P < 0.05. Box plots show the 2nd and 3rd quartiles along with the median; whiskers represent 1.5 times the interquartile range, and each point indicates an individual value. NMB, no manual brush. NMS, no mechanical stimulation. MS, mechanical stimulation. MS-II + III, mechanical stimulation of type II + III. WT: wild type (Micro-Tom). K) The RT-qPCR showing the expression change of SlGAME11/25 after mechanical stimulation in WT, slmyc1(slmyc1-1 and slmyc1-2) and slmyc2(slmyc2-1 and slmyc2-2) mutants. 30-day-old tomato plants were subjected to artificial mechanical stimulation of long trichomes (II + III) 10 times. After 0, 1 h, leaf RNA was extracted for RT-qPCR analysis. Data are means ± SD (n = 3, biological replicates). Significant differences were determined by two-way ANOVA with Holm-Šídák's test. a, b, c, P < 0.05. MS-II + III, mechanical stimulation of type II and III trichomes. WT: wild type (Micro-Tom).

Figure 8.

Quantitative analysis of insect-resistant phenotypes among different types of trichomes. A and B) Leaf damage (A) and the damaged area quantification (B) in WT and wo (wo^W106R and cr-wo) mutants. Top, middle, and bottom represent leaf positions in different parts of wild-type tomato, respectively. Data are means ± SD (WT, top + middle, n = 77, biological replicates; wo^W106R, top + middle, n = 62, biological replicates; cr-wo, top + middle, n = 66, biological replicates; WT, bottom, n = 79, biological replicates; wo^W106R, bottom, n = 60, biological replicates; cr-wo, bottom, n = 48, biological replicates). Significant differences were determined by two-way ANOVA with Tukey's test compared to the WT: a, b, c, d, P < 0.05. WT, wild type (Micro-Tom). Scale bars for all images, 1 cm. C) Cotton bollworms after 7 days of feeding in wild-type (WT) and wo mutants. 10 d and 30 d represent 10-day-old and 30-day-old tomato plants, respectively. WT, wild type (Micro-Tom). All image scale bars, 2 cm. D) Quantification of the weight of cotton bollworms shown in (C). 10 d and 30 d represent 10-day-old and 30-day-old tomato plants, respectively. Data are means ± SD (10 d, n = 14, biological replicates; 30 d, n = 15, biological replicates). Significant differences were determined by one-way ANOVA with Dunnett's test compared to the WT: a, b, c, d, P < 0.05. WT, wild type (Micro-Tom). E, G, I, and M) Leaf damage under mechanical pretreatment. 30-day-old tomato plants were subjected to mechanical pretreatment (WT and Wo^P635R, slbrc2a, cr-mtr1/2, MS-II + III; h/hl, MS-IV + V; cr-w/m#1, MB-VI + VII) for 5 min every 5 h, and the bollworm larvae of 1st-2nd instar was inoculated onto tomato plants. After 7 days of treatment (mechanical stimulation or manual brush), Leaf damage phenotype was observed. NMS, no mechanical stimulation. MS, mechanical stimulation. NMB, no manual brush. MB-L, manual brush leaves. MB-VI + VII, manual brush type VI + VII trichomes. WT, wild type (Micro-Tom). Scale bars for all images, 1 cm. F, H, J, and O) The ratio of injured leaves was evaluated in (E, G, I, and M). Mechanical stimulation or manual brush of 30-day-old plants and the inoculation of the 1st-2nd instar bollworm larvae were described in (E, G, L, and M). After 7 days of treatment (mechanical stimulation or manual brush), a fixed number of leaves were selected from the top to the middle of the plant, and the ratio of damaged leaves to total leaves was quantified. NMB, no manual brush. MB-L, manual brush leaves. MB-VI + VII, manual brush type VI + VII trichomes. total, total damaged leaf. Data are means ± SD (F) WT, NMS, n = 21, biological replicates; WT, MS, n = 22, biological replicates; Wo^P635R, NMS, n = 20, biological replicates; Wo^P635R, MS, n = 18, biological replicates; slbrc2a-1, NMS, n = 21, biological replicates; slbrc2a-1, MS, n = 26, biological replicates; slbrc2a-2, NMS, n = 20, biological replicates; slbrc2a-2, MS, n = 21, biological replicates; (H) WT and h/hl mutants. WT, NMS, MS, n = 19, biological replicates; h/hl-1, NMS, n = 25, biological replicates; h/hl-1, MS, h/hl-2, NMS, MS, n = 19, biological replicates. J) WT and cr-mtr1/2 mutant. WT, cr-mtr1/2, NMS, n = 21, biological replicates; WT, MS, n = 22, biological replicates; cr-mtr1/2, MS, n = 18, biological replicates; (O) WT and cr-w/m#1 mutants. WT, cr-w/m#1, NMS, MS, n = 21, biological replicates). Significant differences were determined by two-way ANOVA with Tukey's test: a, b, c, d, P < 0.05. Box plots show the 2nd and 3rd quartiles along with the median; whiskers represent 1.5 times the interquartile range, and each point indicates an individual value. WT, wild type (Micro-Tom). K) Cotton bollworms after 7 days of feeding in WT and Wo^P635R, slbrc2a (slbrc2a-1 and slbrc2a-2), h/hl (h/hl-1 and h/hl-2), cr-mtr1/2, cr-w/m#1 mutants. Mechanical stimulation or manual brush of 30-day-old plants and the inoculation of the 1st-2nd instar bollworm larvae were described in (E, G, L, and M). NMS, no mechanical stimulation. MS, mechanical stimulation. NMB, no manual brush. MB-L, manual brush leaves. MB-VI + VII, manual brush type VI + VII trichomes. Scale bars for all images, 2 cm. L and N) Quantification of the weight of cotton bollworms shown in (K). The weight of cotton bollworm was counted after 7 days of feeding. Mechanical stimulation or manual brush of 30-day-old plants and the inoculation of the 1st-2nd instar bollworm larvae were described in (E, G, L, and M). NMS, no mechanical stimulation. MS, mechanical stimulation. NMB, no manual brush. MB-L, manual brush leaves. MB-VI + VII, manual brush type VI + VII trichomes. Data are means ± SD ((L) WT and Wo^P635R, slbrc2a (slbrc2a-1 and slbrc2a-2) mutants, n = 13, biological replicates; WT and h/hl (h/hl-1 and h/hl-2), n = 14, biological replicates; WT and cr-mtr1/2 mutants, n = 12, biological replicates; (N) WT and cr-w/m#1 mutants, n = 27, biological replicates). Significant differences were determined by two-way ANOVA with Tukey's test: a, b, c, d, P < 0.05. Box plots show the 2nd and 3rd quartiles along with the median; whiskers represent 1.5 times the interquartile range, and each point indicates an individual value. WT, wild type (Micro-Tom).