| The interaction between plants and destructive insects is complicated. Ecologists have long understood that plants exhibit multimechanistic resistance towards herbivores, but the molecular mechanisms underlying these complicated responses remain elusive. Soybean (Glycine max L. Merrill) is a main oil-seed crop worldwide. The demand for soybean has grown rapidly because of its use in a wide range of applications in food, feed and industrial products. However, insect pests greatly increase the cost of soybean production. The study of induced resistance is of great significance for improving plant resistance to pests and reducing pesticide use.In this study, two different local soybean lines, Wanxianbaidongdou (WX) and Nannong 99-10 (NN), constitutively resistant and susceptible to the common cutworm, were respectively chosen to test the progression of induced resistance levels at six time points after insect feeding. The results showed that the induced resistance peaked at 5 and 1 dai in the WX and NN lines, respectively. To facilitate a better understanding of the common and special induced resistance mechanisms at the molecular level, two sets of transcriptome profiles from the WX and NN lines at the peak of their induced resistance were compared using microarray combined with RNA-Seq technology. In total,827 and 349 transcripts were detected by microarray analysis to be differentially expressed in the WX and NN lines, respectively, with 80 probes common regulated and seven regulated in the opposite direction. All common- and unique-regulated genes were grouped into 10 functional categories based on sequence similarity searches, showing that most of the genes were related to stress and defense responses in common gene list and NN upregulated gene list, most genes in WX upregulated gene list were related to transcriptional regulation and most genes in NN downregulated gene list were related to primary metabolism. On the other hand,1004 and 1580 unigenes were detected to be differently expressed in response to insects in resistant and susceptible soybean line respectively from RNA-Seq data. Functional analysis of these unigenes based on sequence similarity searches identified the same significant function categories as in the microarray results, most of which were related to stress and defense responses.Based on the combination of microarray and RNA-Seq analyses, a list of commonly regulated genes in the WX and NN lines as well as gene lists uniquely regulated in WX or NN line were generated as candidate genes involved in general induced resistant mechanisms and/or special induced resistant mechanisms in soybean. qRT-PCR for a subset of 21 genes selected from those gene lists confirmed that the results of the microarray data and RNA-Seq data were reliable and rigorous. Furthermore, the spatial and temporal expression analysis of six genes, including SVL, CysPl3, IFS1, N.IFR, VSPβ and pHSP, revealed that genes expressed in different parts of the plant responding differently to insect stress across various time points. The six genes investigated in this study were all quickly regulated after herbivory treatment in both the WX and NN local leaves, which demonstrated a larger magnitude of expression changes than the systemic leaves. Because of the quick response and reduction of local expression, the peak time of the local response often occurred before 1 dai, whereas the peak time of the systemic expression of most genes occurred later. These results were in consistent with the timing of the resistance response observed in previous bioassay experiments, either by exhibiting the same peak time or occurring earlier than in the previous experiments, suggesting that the time of the defence induction was important for the common cutworm resistance in the WX and NN lines.The integration of microarray and RNA-Seq data provided a list of genes which were significantly regulated in both resistant and susceptible soybean lines. Among them, two genes, vegetative storage protein encoding gene (GmVSPβ) (involved in response to water deficit, wounding, and JA, but not contribute to plant productivity as normal storage proteins) and NADPH isoflavone reductase encoding gene (GmN:IFR) (involved in soybean phytoalexin biosynthesis pathway), are considered to be good candidates for common cutworm resistance and were stably introduced into tobacco plants to evaluate their potential function in insect resistance. Two insect bioassays were performed to test the insecticidal activity of transgenic tobaccos. In the free-feeding experiment, leaves of GmVSPP and GmN:IFR transgenic tobacco plants were significantly less damaged compared with control plants. Meanwhile, the RGR of common cutworms fed on the transgenic tobacco were significantly decreased. While in dual-choice feeding test, most of common cutworms chosen to eat control leaves, and transgenic tobacco leaves were significantly less damaged compared to the control. After 24 hours, consumed leaves in transgenic tobacco were significantly less than in control, and the size and RGR of insects feeding on transgenic tobacco leaves were significantly less than those fed on control leaves. Comparative analysis of GmVSPβ and GmN:IFR transgenic tobaccos proved their function to confer resistance against insects, which may also be associated with the constitutive upregulation of two genes encoding key enzymes in JA biosynthesis pathway and two genes related to nicotine biosynthesis pathway. These results suggest the better potential of the GmVSPβ gene for engineering transgenic plants with enhanced insect resistance.A high portion of transcriptional regulators were specifically identified from transcriptome analysis in the resistant line in response to insects feeding. To further explore the regulatory roles of these transcriptional factors in plant defense net and their effects on the promoters of GmVSPa、GmVSPβ and GmN:IFR genes, we successfully cloned the GmVSPa、GmVSPβ and GmN:IFR promoter sequences and encoding sequences of 12 transcriptional factors including MYBs, WRKYs, NACs, bZIP and DREB. Cis-element prediction analysis of the three promoters using PLACE and PlantCARE online databases revealed a lot of cis-acting elements related to stress and defense. The number of cis-acting elements in GmVSPβ and GmN:IFR promoters were more than the number of cis-acting elements in GmVSPa promoter. Co-transfection assays were performed in Arabidopsis leaf protoplasts with 12 effectors vectors constructed with the 12 transcription factors and 3 reporter vectors constructed with the three promoters. Among them, GmbZIP110 and GmWRKY39 significantly have positive effects on the activity of GmVSPa promoter; more transcription factors mainly including GmbZIP110 and GmWRKY39 have positive effects on the activity of GmVSPβ promoter; GmMYB75, GmWRKY39 and GmWRKY20 notably have positive effects on the activity of GmN:IFR promoter. These transcriptional regulators may cooperatively or independently constitute gene networks in soybean, with the regulatory elements coordinating the complex metabolic reconfiguration changes. Detailed research on these transcription factors will provide additional information about their expression patterns and the regulation functions in plant responses to herbivores. |
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