Roles of signaling compounds and WRKY31 in the defense of Pinus massoniana L. against Dendrolimus punctatus

Dendrolimus punctatus is an important pest affecting Masson pine (Pinus massoniana L.) forests and can cause serious economic and ecological losses. WRKY transcription factors play important roles in coping with various environmental stresses. In particular, recent studies have shown that WRKY transcription factors play an important role in plant responses against herbivorous insects. However, the mechanisms underlying the actions of these genes in the defense responses of P. massoniana L. are still unclear. Our previous study provided evidence that WRKY may play an important role in the insect resistance of P. massoniana L. In this study, application of semiochemicals such as exogenous hormones and Ca2+ by spraying increased the concentrations of endogenous hormones, terpenoid synthases, and volatile substances in P. massoniana L. and effectively improved its resistance to D. punctatus. After analyzing the WRKY family of P. massoniana L., the PmWRKY31 gene was selected and studied. Yeast two-hybrid assays showed that the LP8 gene interacted with PmWRKY31. Fluorescence-based quantitative polymerase chain reaction showed that after treatment with exogenous hormones and Ca2+, the expression levels of the PmWRKY31 gene, hormonal signal–related genes, and terpene biosynthetic pathway–related genes were significantly increased, whereas the expression of the LP8 gene was decreased. Therefore, the PmWRKY31 and LP8 genes affected downstream gene expression by positively and negatively regulating the hormone signaling pathways, respectively. This result provides theoretical support for the involvement of WRKY transcription factors in the insect resistance of P. massoniana L. through their regulation of hormone signaling.

Encyclopedia of Genes and Genomes (KEGG) data were used for metabolic pathway 160 analysis.
161 Subcellular localization 162 The constructed pBWA(V)HS-wrky-GLosgfp vector plasmid was transferred into 163 Agrobacterium. After Agrobacterium-coated plates were incubated at 30 °C for 2 days, 164 Agrobacterium was inoculated into 10 mL YEB liquid medium and resuspended in 10 165 mM MgCl 2 suspension (containing 120 µM AS), and the optical density measured at a 166 wavelength of 600 nm (OD600) was adjusted to approximately 0.6. The suspension 167 was injected into the epidermis of a tobacco leaf with a 1-mL syringe (needle removed). 168 After injection, the tobacco plants were cultured under low light intensity for 2 days. 169 Next, the tobacco leaves were collected and imaged directly under a laser confocal  primers specific for resistance genes (Table S1) were used to amplify and detect the 181 presence of PmWRKY31 in tobacco seedlings using the conventional PCR method.

182
Transgenic lines were screened from the F3 generation of tobacco plants transduced 183 with PmWRKY31, morphological indicators were observed, and hormones, volatile 184 substances, and resistance were determined.

185
Yeast two-hybrid assay 186 Construction of the cDNA library 187 After RNA extraction, cDNA was synthesized and purified. The purified cDNA was   Pull-down assay 219 Primers for the PmLP8 and PmWRKY31 genes were designed in CmSuite8 software 220 ( the typical WRKY domains of the WRKY family (Fig.1b, Fig. 1c, Table S2). WRKY2 280 was successfully annotated in the KEGG Orthology (KO), indicating that WRKY2 is 281 involved in plant pathogen defense (Fig. 1d). Therefore, we hypothesized that WRKY2 282 is involved in the defense of P. massoniana L. against herbivorous insects.  (Fig. 2c). Sequencing of the PCR amplification product revealed the 297 calcium-binding protein LP8 (Fig. 2d). This gene contained three EF-hand 298 calcium-binding domains and a secreted protein acidic and rich-in-cysteine

301
To further confirm the interaction between PmWRKY31 and LP8, we performed 302 pull-down experiments (Fig. 3a). We also used BiFC technology to further confirm the 303 protein-protein interactions between LP8 and the three WRKY genes of P. massoniana 304 L. (Fig. S4). The results showed that LP8 interacted with WRKY2, WRKY6, and 305 WRKY 31 in the nuclei of plant cells (Fig. 3b). Subcellular localization of WRKY31 306 also indicated that WRKY31 was located in the nucleus (Fig. 3c, d). β-cubebene, and γ-elemene (Fig. 5c). After treatment, the food intake and weight of D. 344 punctatus were significantly lower than those in the control group (Fig. 5d). This 345 indicated that the treatments prevented D. punctatus from feeding on P. massoniana L.

346
Ca treatment did not significantly increase the GA concentration, while the GA and GA 347 + Ca treatments did (Fig. 5b). At the same time, the expression level of LP8 was Under different treatments, the expression level of PmWRKY31 was significantly 357 higher than it was in the control (Fig. 5c, Fig. 5d). The expression of PmLP8 was 358 downregulated under both JA and SA treatments (Fig. 5c, Fig. 5d). Overall, SA 359 treatment alone and ABA treatment alone significantly increased the expression levels 360 of TPS biosynthesis-related genes in the SA and ABA pathways (Fig. 5c, Fig. 5d).    (Fig. 8)

526
Table S1 Information on the primers in the experiment.

527
Table S2 Bioinformatics analysis of 4 genes and domain prediction.

773
The GST protein, GST-LP8 protein with GST resin were incubated with His-Wrky protein 774 overnight and eluted with reduced glutathione the next day. The following day, the elution was 775 performed with reduced glutathione. Western blot was performed after appropriate amount of the growth condition were selected. 1 mL syringe without gun tip was used to inject from the lower