Reciprocal responses in the interaction between Arabidopsis and the cell-content-feeding chelicerate herbivore spider mite

Abstract

Most molecular-genetic studies of plant defense responses to arthropod herbivores have focused on insects. However, plant-feeding mites are also pests of diverse plants, and mites induce different patterns of damage to plant tissues than do well-studied insects (e.g. lepidopteran larvae or aphids). The two-spotted spider mite (Tetranychus urticae) is among the most significant mite pests in agriculture, feeding on a staggering number of plant hosts. To understand the interactions between spider mite and a plant at the molecular level, we examined reciprocal genome-wide responses of mites and its host Arabidopsis (Arabidopsis thaliana). Despite differences in feeding guilds, we found that transcriptional responses of Arabidopsis to mite herbivory resembled those observed for lepidopteran herbivores. Mutant analysis of induced plant defense pathways showed functionally that only a subset of induced programs, including jasmonic acid signaling and biosynthesis of indole glucosinolates, are central to Arabidopsis's defense to mite herbivory. On the herbivore side, indole glucosinolates dramatically increased mite mortality and development times. We identified an indole glucosinolate dose-dependent increase in the number of differentially expressed mite genes belonging to pathways associated with detoxification of xenobiotics. This demonstrates that spider mite is sensitive to Arabidopsis defenses that have also been associated with the deterrence of insect herbivores that are very distantly related to chelicerates. Our findings provide molecular insights into the nature of, and response to, herbivory for a representative of a major class of arthropod herbivores.

Figure 1.

Arabidopsis damage by spider mites. A, Extent of damage, as assessed by the total area of chlorotic spots, of 26 Arabidopsis accessions exposed to 10 mites for 4 d (n = 6 plants per accession). Shown are means ± se. B, Representative damage and trypan blue staining of Bla-2 and Kon leaves after 24 h of spider mite feeding. C, Spider mite accession preference in a choice experiment. Shown are means ± se percentage of recovered mites (n = 22 sets of plants inoculated with 40 mites per set). D, Spider mite performance on Bla-2 and Kon as assessed by mean ± se days required for larvae to become nymphs and mean ± se percentage of larval mortality (n = 5 sets of 50 larvae). Asterisks represent significantly different comparisons (unpaired Student’s t test, *P < 0.05, **P < 0.01, ***P < 0.001). Replicated experiments of the same comparisons produced similar results. Full names and sources of studied accessions are listed in Supplemental Table S1.

Figure 2.

Transcriptional responses to spider mite feeding in Arabidopsis accessions Bla-2 and Kon. A, Hierarchical clustering analysis of log₂ FC (LFC) exhibited by DEGs with absolute FC > 2 and BH-adjusted P < 0.01 detected upon spider mite feeding for 1, 3, 6, 12, and 24 h and feeding of hundreds of mites for 1 h (FS sample). The distance metric was Euclidean, and the clustering method was Ward's. B, Principal component analysis of expression measures data for Bla-2 and Kon. C, Analysis of DEGs from 1 h, 3- to 24-h, and FS treatments. D, GO analysis of DEGs detected in FS and 3- to 24-h samples. Up to 50 biological process, molecular function, and cellular component GO categories with weighted Fisher’s P < 0.05 are compared. Blue samples, Bla-2; red samples, Kon; NT, nontreated samples.

Figure 3.

Analysis of a preexisting transcriptional difference between Bla-2 and Kon and its stability upon spider mite feeding. A, Analysis of DEGs detected between nontreated Bla-2 and Kon. B, GO analysis of DEGs detected between nontreated Bla-2 and Kon. Up to 50 biological process GO categories with weighted Fisher’s P < 0.05 are compared. C, Hierarchical clustering analysis of expression measures of 124 DEGs that were detected as inducible by spider mite feeding and exhibited differences of expression levels in nontreated Bla-2 and Kon. The distance metric was Pearson’s correlation coefficient, and the clustering method was average. NT, Nontreated samples.

Figure 4.

Meta-analysis of Arabidopsis transcriptional responses to a range of biotic, abiotic, and hormonal stimuli. The dendrogram shows the levels of similarity between individual responses. Edge labels are bootstrap P values (bp) in green and approximately unbiased P values (au) in red. The distance metric was Pearson’s correlation coefficient, and the clustering method was average. ACC, 1-Aminocyclopropane-1-carboxylic acid; BR, brassinosteroids; IAA, indole-3-acetic acid; MeJA, methyl jasmonate; ZTN, zeatin.

Figure 5.

Susceptibility of Arabidopsis JA-related mutants aos and myc2 myc3 myc4 to spider mite herbivory. A, Levels of OPDA, JA, and JA-Ile (ng g⁻¹ fresh weight) in 3-week-old Bla-2, Col-0, and Kon plants after spider mite herbivory for 1, 6, and 24 h. Values are means ± se (n = 3). Crosses indicate significant differences for planned comparisons relative to corresponding control plants (P < 0.05, pairwise Student’s t tests with pooled sd and the Holm-Bonferroni adjustment of P values). B and C, Susceptibility of Arabidopsis JA-related mutants aos (Columbia-6 background) and myc2 myc3 myc4 (myc2,3,4; Col-0 background) to spider mite herbivory. Mean ± se chlorotic area is expressed relative to the mean chlorotic area of corresponding wild-type plants (n = 12). D, Spider mite performance as assessed by mean ± se days required for larvae to become nymphs and mean ± se percentage of larval mortality upon feeding on Arabidopsis JA-related mutants (n = 5 sets of 50 larvae). Asterisks indicate significant differences from corresponding wild-type plants (unpaired Student’s t test, *P < 0.05, ***P < 0.001). Individual subgroups of comparisons in C and D are not directly comparable, as they were performed as separate experiments. Replicated experiments of the same comparisons produced similar results.

Figure 6.

Role of IGs in the Arabidopsis response to spider mite herbivory. A, Relative levels of the IGs indol-3-ylmethyl glucosinolate (I3M), 1-methoxy-indol-3-ylmethyl glucosinolates (neoglucobrassicins; 1-MeO-I3M), 4-hydroxyindol-3-ylmethyl glucosinolate (4-OH-I3M), and 4-methoxyl-3-ylmethyl glucosinolate (4-MeO-I3M; shown as normalized peak areas) in 3-week-old Bla-2, Col-0, and Kon plants after spider mite herbivory for 1, 6, and 24 h. Values are means ± se (n = 3). Crosses indicate significant differences for planned comparisons relative to corresponding control plants (P < 0.05, pairwise Student’s t tests with pooled sd and the Holm-Bonferroni adjustment of P values). B, Induction of CYP79B2, CYP79B3, MYB28, and MYB29 genes in response to spider mite feeding. Shown are means ± se for FCs of expression levels detected by RT-qPCR in Columbia-6 and aos plants (n = 4). Letters indicate significant comparisons within a genotype (lowercase, aos; uppercase, Columbia-6; Tukey’s HSD test, P < 0.001). Asterisks indicate significant differences in expression levels within treatment type and between genotypes (Tukey’s HSD test, **P < 0.01, ***P < 0.001). C and D, Susceptibility of Arabidopsis mutants with altered levels of glucosinolates to spider mite herbivory. cyp79b2 cyp79b3 (cyp79b2,b3) and cyp81f2 plants lack all or a subset of IGs, qKO plants lack both indole and aliphatic glucosinolates, and atr1D plants have elevated levels of IGs. Mean ± se chlorotic area is expressed relative to the mean chlorotic area of corresponding wild-type plants (n = 12). E, Spider mite performance as assessed by mean ± se days required for larvae to become nymphs and mean ± se percentage of larval mortality upon feeding on Arabidopsis mutants with altered levels of glucosinolates (n = 5 sets of 50 larvae). Asterisks indicate significant differences from corresponding wild-type plants (unpaired Student’s t test: *P < 0.05, **P < 0.01, ***P < 0.001). Individual subgroups of comparisons in D and E are not directly comparable, as they were performed as separate experiments. Replicated experiments of the same comparisons produced similar results. n.a., Developmental time data was not collected due to 100% larval mortality.

Figure 7.

Transcriptional responses of spider mites to feeding on Arabidopsis plants containing different levels of IGs. A, Relative mortality of adult female spider mites 24 h post transfer from bean plants to Arabidopsis lines with varying levels of IGs. Values represent means ± se percentage of adult female mites that died after 24 h of feeding (n = 4 sets of 100 mites; critical P = 0.05). B, Number of detected DEGs upon spider mite feeding on qKO, Col-0, and atr1D Arabidopsis plants that contain none, normal, and increased levels of IGs, respectively. Overlap categories from the Venn diagram are shown for up- and down-regulated DEGs at right. C, Hierarchical clustering analysis of DEGs with consistent increase or decrease of gene expression levels by at least 25% (FC > 1.25, BH-adjusted P < 0.05) within the qKO-Col-0-atr1D continuum. Genes with descriptions in red demonstrated FC > 1.5 per step. The distance metric was Euclidean, and the clustering method was average. D, Comparison between DEGs identified in host transfer from bean plants to tomato (Dermauw et al., 2013) and from bean plants to Arabidopsis lines with different IG contents.