Hsp70 and Hsp90 Elaborately Regulate RNAi Efficiency in Plutella xylostella

Abstract

Heat-shock proteins (HSPs) serve as molecular chaperones in the RNA interference (RNAi) pathway of eukaryotic organisms. In model organisms, Hsp70 and Hsp90 facilitate the folding and remodeling of the client protein Argonaute (Ago). However, the specific function of HSPs in the RNAi pathway of Plutella xylostella (L.) (Lepidoptera: Plutellidae) remains unknown. In this study, we identified and analyzed the coding sequences of PxHsc70-4 and PxHsp83 (also known as PxHsp90). Both PxHsc70-4 and PxHsp83 exhibited three conserved domains that covered a massive portion of their respective regions. The knockdown or inhibition of PxHsc70-4 and PxHsp83 in vitro resulted in a significant increase in the gene expression of the dsRNA-silenced reporter gene PxmRPS18, leading to a decrease in its RNAi efficiency. Interestingly, the overexpression of PxHsc70-4 and PxHsp83 in DBM, Sf9, and S2 cells resulted in an increase in the bioluminescent activity of dsRNA-silenced luciferase, indicating a decrease in its RNAi efficiency via the overexpression of Hsp70/Hsp90. Furthermore, the inhibition of PxHsc70-4 and PxHsp83 in vivo resulted in a significant increase in the gene expression of PxmRPS18. These findings demonstrated the essential involvement of a specific quantity of Hsc70-4 and Hsp83 in the siRNA pathway in P. xylostella. Our study offers novel insights into the roles played by HSPs in the siRNA pathway in lepidopteran insects.

Keywords: Hsp70; Hsp90; Plutella xylostella; RNA interference.

Figure 1

Schematic diagram of the domains of PxHsc70-4 and PxHsp83. Gray, intron; cyan, ATPase nucleotide-binding domain (ATPase_NBD), N-terminal domain (NTD); orange: peptide-binding domain (peptide-BD), ribosomal protein S5 domain 2-type fold (Ribosomal_S5_D2); green: C-terminal domain (CTD). Numbers represent the amino acid sites.

Figure 2

Phylogenetic analysis of Hsc70-4 and Hsp83 across 21 insect species. (A) Hsc70-4 genes were from four Coleoptera (purple), two Orthoptera (blue), three Hymenoptera (green), three Diptera (orange) and ten Lepidoptera (red). (B) Hsp83 genes were from three Coleoptera, two Orthoptera, three Hymenoptera, three Diptera and ten Lepidoptera. The maximum likelihood method was used to construct the phylogenetic tree with 1000 bootstrap replications, and the tree scale was provided.

Figure 3

Expression profiles of PxHsc70-4 and PxHsp83 in different tissues (A,B) and developmental stages (C,D). HD: head, MG: midgut, MT: Malpighian tubule, SG: silk gland, EP: epidermis, TE: testis, HL: hemolymph, FB: fat body, OV: ovary, L: larvae that contain four stages. RPL32 was used to normalize the mRNA expression levels. Each experiment was repeated three times (mean ± SE, n = 3). Statistical significance was determined via one-way ANOVA with Tukey’s test (ns, no significance; **, p < 0.01; ***, p < 0.001).

Figure 4

Effect of HSP knockdown on the expression of RNAi genes in vitro. (A) Relative mRNA expression of PxHsc70-4 after dsHsc70-4 transfection in 24–48 h. (B) Relative mRNA expression of PxHsp83 after dsHsp83 transfection in 24–48 h. (C,D) Relative mRNA expression of PxAgo2 and PxDicer2 after dsHSP transfection in 24–48 h. DsEGFP and H₂O were used as a negative control and a blank control, respectively. Each experiment was repeated three times (mean ± SE, n = 3). Statistical significance was determined via one-way ANOVA with Fisher’s LSD (*, p < 0.05; **, p < 0.01; ***, p < 0.001) and Tukey’s test (*, p < 0.05; **, p < 0.01; ***, p < 0.001), respectively.

Figure 5

Effect of HSP knockdown on the RNAi efficiency of PxmRPS18 in vitro. Relative mRNA expression of PxHsc70-4 (A), PxHsp83 (B) and PxmRPS18 (C) after dsHSP and dsmRPS18 transfection in turn. DsEGFP was used as a control. Each experiment was repeated three times (mean ± SE, n = 3). Statistical significance was determined via one-way ANOVA with Tukey’s test (ns, no significance; *, p < 0.05; **, p < 0.01; ***, p < 0.001).

Figure 6

Effect of HSP inhibition on RNAi efficiency in vitro. Effect of HSP inhibitor transfection on the mRNA expression of PxmRPS18. DMSO and dsEGFP were, respectively, used as controls of the inhibitor and dsRNA. Each experiment was repeated three times (mean ± SE, n = 3). Statistical significance was determined via one-way ANOVA with Tukey’s test (***, p < 0.001).

Figure 7

Effect of PxHSP overexpression on the RNAi efficiency of Luc in DBM, Sf9 and S2 cells. (A) Schematic diagram of RNAi-of-overexpression, with 1st co-transfection of pIZT constructs expressing Hsp70/Hsp90 and Luc, and 2nd transfection of dsmRPS18. (B) Effect of pIZT-HSP and pIZT-Luc co-transfection followed by Luc knockdown on the luciferase activity. The pIZT vector and pIZT-Ago2 were used as a negative control and a positive control, respectively. Each experiment was repeated three times (mean ± SE, n = 3). Statistical significance was determined via one-way ANOVA with Tukey’s test (ns, no significance; *, p < 0.05; **, p < 0.01; ***, p < 0.001).

Figure 8

Effect of HSP inhibition on the RNAi efficiency of PxmRPS18 in vivo. Effect of PES/17-AAG and dsmRPS18 co-injection on the mRNA expression of PxmRPS18 in vivo. DsEGFP and DMSO were used as negative controls. Each experiment was repeated three times (mean ± SE, n = 3). Statistical significance was determined via one-way ANOVA with Tukey’s test (*, p < 0.05).