The impact of insect egg deposition on Pinus sylvestris transcriptomic and phytohormonal responses to larval herbivory

Abstract

Plants can improve their resistance to feeding damage by insects if they have perceived insect egg deposition prior to larval feeding. Molecular analyses of these egg-mediated defence mechanisms have until now focused on angiosperm species. It is unknown how the transcriptome of a gymnosperm species responds to insect eggs and subsequent larval feeding. Scots pine (Pinus sylvestris L.) is known to improve its defences against larvae of the herbivorous sawfly Diprion pini L. if it has previously received sawfly eggs. Here, we analysed the transcriptomic and phytohormonal responses of Scots pine needles to D. pini eggs (E-pine), larval feeding (F-pine) and to both eggs and larval feeding (EF-pine). Pine showed strong transcriptomic responses to sawfly eggs and-as expected-to larval feeding. Many egg-responsive genes were also differentially expressed in response to feeding damage, and these genes play an important role in biological processes related to cell wall modification, cell death and jasmonic acid signalling. EF-pine showed fewer transcriptomic changes than F-pine, whereas EF-treated angiosperm species studied so far showed more transcriptional changes to the initial phase of larval feeding than only feeding-damaged F-angiosperms. However, as with responses of EF-angiosperms, EF-pine showed higher salicylic acid concentrations than F-pine. Based on the considerable overlap of the transcriptomes of E- and F-pine, we suggest that the weaker transcriptomic response of EF-pine than F-pine to larval feeding damage is compensated by the strong, egg-induced response, which might result in maintained pine defences against larval feeding.

Keywords: biotic interactions; defence; gymnosperm; phytohormone; pine; transcriptome.

Figure 1

Scheme of P. sylvestris treatments and sampling time points. Needles of 3-year-old P. sylvestris trees were treated with natural egg deposition by D. pini (E-pine), larval feeding (F-pine) or natural egg deposition with subsequent feeding (EF-pine). Untreated control (C-pine) trees (grey arrow, no treatment) were included into the experiments. Needles were harvested from E-, F-, EF- and C-pine at different time points after treatments. A new set of trees was used for each sampling time point, thereby avoiding the possibility that sampling at an early time point affects the tree’s response at a later time point. Needles were harvested at 1 h, 24 h and 10 days after egg deposition (yellow arrow). Eleven days after egg deposition, which is an early possible hatching time point after development of D. pini eggs under the abiotic conditions used, 10 D. pini larvae were placed each on egg-free and previously egg-laden pine trees (green arrow and brown arrow, respectively). Needles were harvested after a 1- and 24-h larval feeding period. At equivalent time points, we also harvested needles from egg-laden E-pine trees that had not received any larvae. Needles from control pine trees were harvested at all above-mentioned sampling time points. For the RNA sequencing and phytohormone analysis, n = 4–5 trees were used for each treatment and time point. For the qPCR, we used n = 3–5 trees per treatment and time point.

Figure 2

Number of DEGs. Needles of P. sylvestris were treated with D. pini egg deposition (E; yellow bars), larval feeding on previously egg-free pine (F; green bars) or natural egg deposition with subsequent feeding (EF; brown bars). Needles were sampled 1 h, 24 h and 10 days after egg deposition, as well as 1 and 24 h after the onset of larval feeding. The DEGs were differentially expressed to a significant degree when compared with untreated controls (C; Wald test; corrected P-value ≤ 0.05). Bars above (below) the zero x-axis show the number of upregulated (downregulated) DEGs. Number of replicates: N = 4–5 for each treatment and time point.

Figure 3

Gene Ontology term enrichment. Shown are significantly DEGs in needles of P. sylvestris 1 h, 24 h and 10 days after D. pini egg deposition, and 1 and 24 h after the onset of larval feeding. Top figure: enrichment with upregulated genes; bottom figure: enrichment with downregulated genes. Differently coloured horizontal bars below the figure show groups of GO terms related to similar biological processes, i.e., GO terms related to ‘photosynthesis’, ‘cell wall modification’ (cell wall mod.) (including lignin), ‘cell death’ (CD) (including ‘hypersensitive response’ (HR)), ‘secondary metabolites’ (sec. Metabolites) (including ‘phenylpropanoids’ (PP), ‘flavonoids’ (FL), ‘terpenes’ (TP) and ‘others’ (OT)), ‘response to chitin’ (RC) and those related to ‘phytohormones’ (including ‘jasmonic acid’ (JA), ‘salicylic acid’ (SA), ‘abscisic acid’ (ABA), ‘auxin’ (AUX), ‘ethylene’ (ET) and ‘others’ (OT)) are grouped here. The GO term identities included in these groups are listed in Table S4 available as Supplementary data at Tree Physiology Online (compare GO term ID numbers given above the figure with numbers in Table S4 available as available as Supplementary data at Tree Physiology Online). The enrichment of each GO term is shown by different circles for each treatment and sampling time point. The fold enrichment is illustrated by the size of each circle (highest enrichment = 25 in top figure; highest enrichment = 10 in bottom figure). The P-value (modified Fisher’s exact test; P < 0.05) is visualized by the colour of each circle. Numbers in the yellow (egg deposition)/green/brown (feeding) arrows on the left side of the figure indicate the different sampling time points. The enrichments of GO terms for the treatments of egg deposition (E), larval feeding (F) and natural egg deposition with subsequent feeding (EF) were all compared to the respective, untreated control (C). Additionally, EF was compared with F. Horizontal, dashed lines separate data from E samples from those of EF and F samples, and data from EF and F samples at the 1 and 24 h sampling time points. Vertical, dashed lines separate the different GO term groups. A list of all significantly enriched GO terms is provided in Table S6 available as Supplementary data at Tree Physiology Online.

Figure 4

Phytohormone concentrations in needles of P. sylvestris. Shown are the results 1 h, 24 h and 10 days after D. pini egg deposition, as well as 1 and 24 h after the onset of larval feeding. The non-normally distributed data are visualized as boxplots with the median as centre and all data points as dots. (a) Salicylic acid (SA), (b) jasmonic acid (JA), (c) jasmonic acid-isoleucine (JA-Ile) and (d) abscisic acid (ABA). Treatments were: natural egg deposition (E; yellow), larval feeding (F; green), egg deposition with subsequent feeding (EF; brown) and an untreated control (C; grey). Significant differences between concentrations in C-pine and E-pine 1 h, 24 h and 10 days after egg deposition are indicated by asterisks (Mann–Whitney U test; ^*P < 0.05, ^***P < 0.001). Significant differences between control, E-, F- and EF-pine at the 1 and 24 h time points of larval feeding (and equivalent time points in C- and E-pine) are indicated by different letters (P < 0.05; Kruskal–Wallis test with Tukey post hoc test). For each treatment and time point: N = 4–5 replicates. In some cases, fewer dots than four are visible per treatment; these dots (data) are overlapping.

Figure 5

Overlapping DEGs in differently treated P. sylvestris trees. Venn diagrams are showing the number of pine genes uniquely and commonly (overlapping) differentially expressed in trees that were treated with natural egg deposition (E; yellow), larval feeding (F; green) or natural egg deposition with subsequent feeding (EF; brown). (a) Differentially expressed genes of E-trees and F-trees at all sampling time points; (b) DEGs in F- and EF-pine 1 and 24 h after the onset of larval feeding and in E-trees at equivalent time points; (c) DEGs in F- and EF-pine 1 h and 24 h after the onset of larval feeding and in E-trees 1 h, 24 h and 10 days after egg deposition. Black numbers show upregulated, and blue numbers downregulated, genes, all normalized to untreated controls (C).