Bmapaf-1 is Involved in the Response against BmNPV Infection by the Mitochondrial Apoptosis Pathway

Abstract

Discovery of the anti-BmNPV (Bombyx mori nuclearpolyhedrovirus) silkworm strain suggests that some kind of antiviral molecular mechanism does exist but is still unclear. Apoptosis, as an innate part of the immune system, plays an important role in the response against pathogen infections and may be involved in the anti-BmNPV infection. Several candidate genes involved in the mitochondrial apoptosis pathway were identified from our previous study. Bombyx mori apoptosis protease-activating factor-1 (Bmapaf-1) was one of them, but the antiviral mechanism is still unclear. In this study, sequences of BmApaf-1 were characterized. It was found to contain a unique transposase_1 functional domain and share high CARD and NB-ARC domains with other species. Relatively high expression levels of Bmapaf-1 were found at key moments of embryonic development, metamorphosis, and reproductive development. Further, the significant difference in expression of Bmapaf-1 in different tissues following virus infection indicated its close relationship with BmNPV, which was further validated by RNAi and overexpression in BmN cells. Briefly, infection of budded virus with enhanced green fluorescent protein (BV-EGFP) was significantly inhibited at 72 h after overexpression of Bmapaf-1, which was confirmed after knockdown of Bmapaf-1 with siRNA. Moreover, the downstream genes of Bmapaf-1, including Bmnedd2-like caspase (BmNc) and Bmcaspase-1 (Bmcas-1), were upregulated after overexpression of Bmapaf-1 in BmN cells, which was consistent with the RNAi results. Furthermore, the phenomenon of Bmapaf-1 in response to BmNPV infection was determined to be related to apoptosis using the apoptosis inducer NSC348884 and inhibitor Z-DEVD-FMK. Therefore, Bmapaf-1 is involved in the response against BmNPV infection by the mitochondrial apoptosis pathway. This result provides valuable data for clarifying the anti-BmNPV mechanism of silkworms and breeding of resistant silkworm strains.

Keywords: BmNPV; Bombyx mori; apoptotic protease-activating factor 1; mitochondrial apoptosis pathway; response analysis.

Figure 1

The spatiotemporal expression analysis of Bmapaf-1 using RT-qPCR. Relative expression levels of Bmapaf-1 among different egg development times (A); different developmental stages (B); and different tissues (C). 1st day, period of the longest embryo; 2nd day, period of protuberance occurred; 3rd day, prophase of protuberance rapid development; 4th day, period of shortening; 5th day, period of embryonic reversal; 6th day, head pigmentation period. BmGAPDH was used to normalize the data that were shown as the mean ± standard error, and the mean is from three independent repeats. The 2^−△△Ct method was adopted to calculate the relative expression level. Differences among triple repeats were analyzed using the SPSS Statistics 20 software (IBM, USA) with the one-way ANOVA method. Different letters represented the significant difference (a, b, c; p < 0.05).

Figure 2

The analysis of Bmapaf-1 expression level in different tissues of different resistant strains following BmNPV infection using RT-qPCR. YeA was a resistant silkworm strain, and YeB was a susceptible silkworm strain. BC, blank control; NC, negative control. BmGAPDH was used to normalize the data that were shown as the mean ± standard error, and the mean was from three independent repeats. The 2^−△△Ct method was adopted to calculate the relative expression level. Differences among triple repeats were analyzed using the SPSS Statistics 20 software (IBM, USA) with the one-way ANOVA method. Asterisks represented the significant difference, as follows: ** p < 0.01; *** p < 0.001.

Figure 3

Expression analysis of selected downstream genes after knockdown of Bmapaf-1 with siRNA at different time points using RT-qPCR. (A) Expression level analysis of Bmapaf-1 after transfection with siRNA at 24, 48, and 72 h. Analysis of BmNc (B) and Bmcas-1 (C) expression levels after knockdown of Bmapaf-1 at different time points. BmGAPDH was used to normalize the data that were showed as the mean ± standard error, and the mean was from three independent repeats. The 2^−△△Ct method was adopted to calculate the relative expression level. Differences among triple repeats were analyzed using the SPSS Statistics 20 software (IBM, USA) with the one-way ANOVA method. Asterisks represent the significant difference, as follows: * p < 0.05; ** p < 0.01.

Figure 4

Analysis of BmNPV infection following knockdown of Bmapaf-1 at different times in BmN cells: 24 h (A); 48 h (B), and 72 h (C) after BV-EGFP infection. (D) The analysis of vp39 expression after knockdown of Bmapaf-1 at different times. Scale bar = 200 μm. Trans (white), optical transmission. EGFP (green), expressed following the replication of BV. BmGAPDH was used to normalize the data that were showed as the mean ± standard error, the mean was from three independent repeats. The 2^−△△Ct method was adopted to calculate the relative expression level. Differences among triple repeats were analyzed using the SPSS Statistics 20 software (IBM, USA) with the one-way ANOVA method. Asterisks represent the significant difference, * p < 0.05.

Figure 5

Expression analysis of selected downstream genes after overexpression of Bmapaf-1 at different times in BmN cells. (A) Overexpression detection of Bmapaf-1 after transfection with pIZT-mCherry-Bmapaf-1 in BmN cells. Scale bar = 200 μm. Trans (white), optical transmission. mCherry (Red), fused expression with Bmapaf-1 protein. (B) The construction of pIZT-mCherry-Bmapaf-1: (1) validation of the recombinant vector using double enzyme digestion and (2) amplification of the functional domain of Bmapaf-1. (C) The expression level analysis of Bmapaf-1 after transfecting with recombinant vector using RT-qPCR. Expression level analysis of BmNc (D) and Bmcas-1 (E) after overexpression of Bmapaf-1. BmGAPDH was used to normalize the data that were shown as the mean ± standard error, and the mean was from three independent repeats. The 2^−△△Ct method was adopted to calculate the relative expression level. Differences among triple repeats were analyzed using the SPSS Statistics 20 software (IBM, USA) with the one-way ANOVA method. Asterisks represent the significant difference, as follows: *** p < 0.001.

Figure 6

Analysis of BmNPV infection after overexpression of Bmapaf-1 at different times in BmN cells, 24 h (A), 48 h (B), and 72 h (C), after BV-EGFP infection. (D) The analysis of vp39 expression after overexpression of Bmapaf-1 at different times. Scale bar = 200 μm. Trans (white), optical transmission. EGFP (green), expressed following the replication of BV. BmGAPDH was used to normalize the data that were shown as the mean ± standard error, the mean was from three independent repeats. The 2^−△△Ct method was adopted to calculate the relative expression level. Differences among triple repeats were analyzed using the SPSS Statistics 20 software (IBM, USA) with the one-way ANOVA method. Asterisks represent the significant difference, as follows: ** p < 0.01.

Figure 7

The analysis of BmNPV replication in the RNAi and overexpression group following regulation of apoptosis in BmN cells using RT-qPCR. (A) The replication of BmNPV in the RNAi group after treatment with inducer NSC348884. (B) The analysis of BmNPV replication in the transgenic BmN cell line following treatment with apoptosis inhibitor Z-DEVD-FMK. BmGAPDH was used to normalize the data that were shown as the mean ± standard error, the mean was from three independent repeats. The 2^−△△Ct method was used to calculate the relative expression level. Differences among triple repeats were analyzed using the SPSS Statistics 20 software (IBM, USA) with the one-way ANOVA method. Asterisks represented the significant difference, as follows: ** p < 0.01.

Figure 8

The proposed function of BmApaf-1 in response against BmNPV infection by the mitochondrial apoptosis pathway. MMP, mitochondrial membrane potential.