Regulatory Mechanism of Transcription Factor AhHsf Modulates AhHsp70 Transcriptional Expression Enhancing Heat Tolerance in Agasicles hygrophila (Coleoptera: Chrysomelidae)

Abstract

Agasicles hygrophila is a classical biological agent used to control alligator weed (Alternanthera philoxeroides). Previous research has indicated that the heat shock factor (HSF) is involved in regulating the transcriptional expression of Hsp70 in response to heat resistance in A. hygrophila. However, the regulatory mechanism by which AhHsf regulates the expression of AhHsp70 remains largely unknown. Here, we identified and cloned a 944 bp AhHsp70 promoter (AhHsp70p) region from A. hygrophila. Subsequent bioinformatics analysis revealed that the AhHsp70p sequence contains multiple functional elements and has a common TATA box approximately 30 bp upstream of the transcription start site, with transcription commencing at a purine base approximately 137 bp upstream of ATG. Promoter deletion analyses revealed that the sequence from -944 to -744 bp was the core regulatory region. A dual-luciferase reporter assay indicated that overexpressed AhHsf significantly enhanced the activity of AhHsp70p. Furthermore, qPCR showed that AhHsp70 expression increased with time in Spodoptera frugiperda (Sf9) cells, and AhHsf overexpression significantly upregulated AhHsp70 expression in vitro. Characterization of the upstream regulatory mechanisms demonstrated that AhHsf binds to upstream cis-acting elements in the promoter region of AhHsp70 from -944 to -744 bp to activate the AhHSF-AhHSP pathway at the transcriptional level to protect A. hygrophila from high temperature damage. Furthermore, we proposed a molecular model of AhHsf modulation of AhHsp70 transcription following heat shock in A. hygrophila. The findings of this study suggest that enhancing the heat tolerance of A. hygrophila by modulating the upstream pathways of the Hsp family can improve the biocontrol of A. philoxeroides.

Keywords: Agasicles hygrophila; cell transfection; heat shock protein 70 promoter (Hsp70p); inverse PCR (I-PCR); real-time quantitative PCR (RT-qPCR); transcription factor AhHsf.

Figure 1

Schematic representation of the promoter sequence of the AhHsp70 gene (AhHsp70p) from Agasicles hygrophila based on bioinformatics analysis. The AhHsp70p sequence contains multiple functional elements and a common TATA box at approximately 30 bp upstream of the transcription start site. Transcription is believed to commence at a purine base approximately 137 bp upstream of the coding region of ATG. The blue circle on the left represents other unknown sequences upstream of the AhHsp70p. −944, 0, 1941, and 2049 refer to the cloned promoter sequence location, the start codon location, the end of the coding region sequence location, and the 3′-UTR sequence location, respectively.

Figure 2

Analysis of the luciferase activity of truncated sequences of AhHsp70p. In this experiment, Spodoptera frugiperda (Sf9) cells were used for the stable expression of AhHsp70 in vitro. Sf9 cells were transfected with recombinant plasmids containing AhHsp70p sequences of differing deletion length or pGL3-Basic and pRL-TK as controls for 48–96 h and the cells were harvested for the luciferase activity assay. qdz represents the AhHsp70 promoter. All values are shown as the mean ± SD. The data were analyzed using the Student’s t-test. ** p < 0.01, extremely significant; *** p < 0.001, extremely extremely significant; ns, not significant.

Figure 3

Overexpressed AhHsf enhanced the activity of AhHsp70p in vitro. In this experiment, Spodoptera frugiperda (Sf9) cells were used for the stable expression of AhHsp70 in vitro. (A) Characterization of the interaction between transcription factor AhHsf and AhHsp70p. Analysis of the luciferase activity of AhHsp70p in response to the overexpression of AhHsf based on a dual-luciferase reporter assay system. pGL3-Basic was used as a control. (B) Schematic diagram of Sf9 cells co-transfected with AhHsf and AhHsp70p luciferase reporter plasmids. (C) Agarose gel electrophoresis of the AhHsf sequence and double-enzyme digestion of the recombinant plasmid. M denotes a Trans DNA marker and lanes 1 and 2 show samples from duplicate analyses. All values are shown as the mean ± SD. The data were analyzed using the Student’s t-test. ** p < 0.01, extremely significant; ns, not significant.

Figure 4

Expression analysis of AhHsp70 gene at 48, 72, and 96 h after transfection in Spodoptera frugiperda (Sf9) cells. Relative mRNA levels were determined using the 2^−ΔΔCt method and normalized to those of the β-actin. The figure shows data on the relative AhHsp70 gene expression levels analyzed using one-way ANOVA followed by the least significant difference (LSD) test and bars with different letters indicate significant differences (p < 0.05). All values are shown as the mean ± SD of three replicates and pGL3-Basic was used as a control.

Figure 5

Effect of AhHsf overexpression on the level of AhHsp70 expression. Relative mRNA levels were determined using the 2^−ΔΔCt method and normalized to those of the β-actin gene. The data were analyzed using the Student’s t-test. * p < 0.05, significant; ** p < 0.01, extremely significant; ns, not significant. All values are shown as the mean ± SD of three replicates and pGL3-Basic was used as a control.

Figure 6

Proposed hypothetical model of the heat shock-induced transcriptional regulation of AhHsp70 gene expression. (HSB-1: Heat shock factor binding protein 1; DDL: WAS protein family homolog).