Characterization of a Novel Insect-Induced Sesquiterpene Synthase GbTPS1 Based on the Transcriptome of Gossypium barbadense Feeding by Cotton Bollworm

Abstract

When attacked by insect herbivores, plants initiate sophisticated defenses mediated by complex signaling networks and usually release a blend of functional volatiles such as terpenes against infestation. The extra-long staple cotton Gossypium barbadense cultivated worldwide as natural textile fiber crop is frequently exposed to a variety of herbivores, such as cotton bollworm Helicoverpa armigera. However, little is known about insect-induced transcriptional changes and molecular mechanisms underlying subsequent defense responses in G. barbadense. In the current study, transcriptome changes in G. barbadense infested with chewing H. armigera larvae were investigated, and we identified 5,629 differentially expressed genes (DEGs) in the infested cotton leaves compared with non-infested controls. H. armigera feeding triggered complex signaling networks in which almost all (88 out of 90) DEGs associated with the jasmonic acid (JA) pathway were upregulated, highlighting a central role for JA in the defense responses of G. barbadense against target insects. All DEGs involved in growth-related photosynthesis were downregulated, whereas most DEGs associated with defense-related transcript factors and volatile secondary metabolism were upregulated. It was noteworthy that a terpene synthase gene in the transcriptome data, GbTPS1, was strongly expressed in H. armigera-infested G. barbadense leaves. The upregulation of GbTPS1 in qPCR analysis also suggested an important role for GbTPS1 in herbivore-induced cotton defense. In vitro assays showed that recombinant GbTPS1 catalyzed farnesyl pyrophosphate and neryl diphosphate to produce three sesquiterpenes (selinene, α-gurjunene, and β-elemene) and one monoterpene (limonene), respectively. Moreover, these catalytic products of GbTPS1 were significantly elevated in G. barbadense leaves after H. armigera infestation, and elemene and limonene had repellent effects on H. armigera larvae in a dual-choice bioassay and increased larval mortality in a no-choice bioassay. These findings provide a valuable insight into understanding the transcriptional changes reprogramming herbivore-induced sesquiterpene biosynthesis in G. barbadense infested by H. armigera, which help elucidate the molecular mechanisms underlying plant defense against insect pests.

Keywords: GbTPS1; Helicoverpa armigera; extra-long staple cotton; induced defense; sesquiterpene biosynthesis; transcriptome sequencing; β-elemene.

FIGURE 1

Gene Ontology enrichment analysis of differentially expressed genes (DEGs) in Gossypium barbadense leaves after Helicoverpa armigera infestation. The DEGs were judged as differentially expressed when log₂| Ratio| ≥ 1 with FDR ≤ 0.05.

FIGURE 2

The top 20 pathways of differentially expressed genes (DEGs) identified in Gossypium barbadense leaves after Helicoverpa armigera infestation.

FIGURE 3

Log₂(fold change) of selected genes in RNA-seq (black bar) and qPCR (gray bar) analysis.

FIGURE 4

Phylogenetic tree of amino acid sequences of 13 TPSs identified in the transcriptome from G. barbadense leaves and previously characterized TPSs (GhTPS1-GhTPS15) from Gossypium hirsutum. The tree was constructed with the neighbor-joining method and 1,000 replications for bootstrapping.

FIGURE 5

Gas chromatography-mass spectrometry analysis of the products catalyzed by recombinant GbTPS1. 1, selinene; 2, α-gurjunene; 3, β-elemene; 4, limonene. Other peaks are contamination.

FIGURE 6

Representative chromatograms of volatile terpenes from Gossypium barbadense infested by Helicoverpa armigera (A) and from undamaged control plants (B). Numbered compounds correspond to those in Table 1. IS, internal standard (ethyl nonanoate); #1, Non-anal; #2, 1,2,4,5-tetramethylbenzene.

FIGURE 7

Behavioral preference of Helicoverpa armigera larvae to β-elemene (A) and limonene (B). Data were tested for significant differences using χ² analysis (**P < 0.01; ***P < 0.001).

FIGURE 8

Larval mortality of Helicoverpa armigera caused by β-elemene (A) and limonene (B). Data were tested to evaluate differences using Student’s t-test (*P < 0.05; **P < 0.01; ***P < 0.001).