Induced Resistance Combined with RNA Interference Attenuates the Counteradaptation of the Western Flower Thrips

Abstract

The western flower thrips, Frankliniella occidentalis Pergande, is an invasive pest that damages agricultural and horticultural crops. The induction of plant defenses and RNA interference (RNAi) technology are potent pest control strategies. This study investigated whether the anti-adaptive ability of F. occidentalis to jasmonic acid (JA)- and methyl jasmonate (MeJA)-induced defenses in kidney bean plants was attenuated after glutathione S-transferase (GST) gene knockdown. The expression of four GSTs in thrips fed JA- and MeJA-induced leaves was analyzed, and FoGSTd1 and FoGSTs1 were upregulated. Exogenous JA- and MeJA-induced defenses led to increases in defensive secondary metabolites (tannins, alkaloids, total phenols, flavonoids, and lignin) in leaves. Metabolome analysis indicated that the JA-induced treatment of leaves led to significant upregulation of defensive metabolites. The activity of GSTs increased in second-instar thrips larvae fed JA- and MeJA-induced leaves. Co-silencing with RNAi simultaneously knocked down FoGSTd1 and FoGSTs1 transcripts and GST activity, and the area damaged by second-instar larvae feeding on JA- and MeJA-induced leaves decreased by 62.22% and 55.24%, respectively. The pupation rate of second-instar larvae also decreased by 39.68% and 39.89%, respectively. Thus, RNAi downregulation of FoGSTd1 and FoGSTs1 reduced the anti-adaptive ability of F. occidentalis to JA- or MeJA-induced defenses in kidney bean plants.

Keywords: Frankliniella occidentalis; RNA interference; counteradaptation; glutathione S-transferase; induced defense; jasmonic acid; metabolites; methyl jasmonate.

Figure 1

Relative expression levels of FoGSTd1, FoGSTs1, FoGSTe1, and FoGSTt1 in second-instar larvae of Frankliniella occidentalis after feeding on exogenous jasmonic acid (JA)-induced kidney bean leaves. (a) Relative expression levels of FoGSTd1; (b) Relative expression levels of FoGSTs1; (c) Relative expression levels of FoGSTe1; (d) Relative expression levels of FoGSTt1. All data are the mean ± standard error (SE). Different uppercase letters above bars indicate significant differences in expression levels among different periods of the same treatment, whereas different lowercase letters indicate significant differences among different treatments at the same time (p < 0.05; one-way ANOVA, followed by Tukey’s test).

Figure 2

Relative expression levels of FoGSTd1, FoGSTs1, FoGSTe1, and FoGSTt1 in second-instar larvae of Frankliniella occidentalis after feeding on exogenous methyl jasmonate (MeJA)-induced kidney bean leaves. (a) Relative expression levels of FoGSTd1; (b) Relative expression levels of FoGSTs1; (c) Relative expression levels of FoGSTe1; (d) Relative expression levels of FoGSTt1. All data are the mean ± SE. Different uppercase letters above bars indicate significant differences in expression levels among different periods of the same treatment, whereas different lowercase letters indicate significant differences among different treatments at the same time (p < 0.05; one-way ANOVA, followed by Tukey’s test).

Figure 3

Jasmonic acid (JA) and methyl jasmonate (MeJA) induce secondary metabolite accumulation in kidney bean leaves and activate glutathione S-transferase (GST) activity in second-instar larvae of Frankliniella occidentalis. (a–e) are contents of tannins, alkaloids, total phenols, flavonoids, and lignin, respectively, in kidney bean leaves. (f) GST activity in second-instar larvae. Values are the mean ± SE. Different lowercase letters indicate significant deference among all treatments (p < 0.05; one-way ANOVA, followed by Tukey’s test).

Figure 4

Orthogonal partial least squares discriminant analysis (OPLS-DA) score plot of jasmonic acid-treated and control samples. (a) Positive ion mode (Pos); (b) negative ion mode (Neg). The abscissa t[1] represents principal component 1, the ordinate t[1] represents principal component 2, and the ellipse represents the 95% confidence interval. Dots of the same color represent each biological replicate within a group, and dot distribution reflects the degree of difference between and within groups.

Figure 5

Heat map of differential metabolites in kidney bean leaves induced by jasmonic acid in positive (a) and negative (b) ion modes. Metabolite names of the same color are in the same superclass. Orthogonal partial least squares discriminant analysis (OPLS-DA) variable importance in the projection (VIP) > 1 and p < 0.05 were used as criteria to screen significantly different metabolites.

Figure 6

Differential metabolite correlation network in positive mode (a) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment (b). The vertical axis in the bar graph represents each KEGG metabolic pathway, and the horizontal axis represents the number of differentially expressed metabolites contained in each KEGG metabolic pathway. The color represents the p-value of the enrichment analysis, and the darker the color is, the smaller the p-value and the more significant the degree of enrichment. The number on the column represents the enrichment factor, and the enrichment factor represents the ratio of the number of differential metabolites in the pathway to the number of annotated metabolites in the pathway.

Figure 7

Downregulation of FoGSTd1 and FoGSTs1 expression levels in second-instar larvae of Frankliniella occidentalis when fed dsRNA solutions. (a) Relative expression level of FoGSTd1; (b) Relative expression level of FoGSTs1. Values are the mean ± SE. Different lowercase letters indicate significant silencing among all treatments (p < 0.05; one-way ANOVA, followed by Tukey’s test).

Figure 8

Effects of downregulation of FoGSTs1 and FoGSTd1 expression levels by RNA interference on glutathione S-transferase activity in second-instar larvae of Frankliniella occidentalis. Values are the mean ± SE. Different lowercase letters indicate significant silencing among all treatments (p < 0.05; one-way ANOVA, followed by Tukey’s test).

Figure 9

Co-silencing of FoGSTd1 and FoGSTs1 expression levels by RNA interference reduced the area of feeding damage on jasmonic acid (JA)- and methyl jasmonate (MeJA)-induced bean leaves by second-instar larvae of Frankliniella occidentalis. Values are the mean ± SE. Different lowercase letters indicate significant differences in feeding damage area on kidney bean leaves with different induction treatments by F. occidentalis fed the same RNAi solution. Different uppercase letters indicate significant differences in feeding damage area of kidney bean leaves with the same induction treatment, but F. occidentalis was fed honey solution (HS) and dseGFP and dsFoGSTs1/d1 solutions (p < 0.05; one-way ANOVA, followed by Tukey’s test).

Figure 10

Co-silencing of FoGSTd1 and FoGSTs1 by RNA interference reduced the pupation rate of second-instar larvae of Frankliniella occidentalis feeding on jasmonic acid (JA)- and methyl jasmonate (MeJA)-induced kidney bean leaves. Values are the mean ± SE. Different lowercase letters indicate that the pupation rate of the second-instar larvae fed with the same solution was significantly different after feeding on the leaves of kidney bean with different induction treatments. Different uppercase letters indicate that the pupation rate of the second-instar larvae fed with different solutions was significantly different after feeding on the kidney bean leaves induced with the same treatment (p < 0.05; one-way ANOVA, followed Tukey’s test).