VipR positively regulates cry genes expression during the vegetative phase

Previous studies have reported that VipR positively regulates expression of the vip3Aa located in BtPAI-1 during the vegetative phase [34]. Further bioinformatic analysis also identified putative VipR-binding motifs in the promoter regions of several cry genes located within the same pathogenicity island [34], suggesting that VipR may be involved in regulating their expression. Based on this, we hypothesized that VipR may regulate the expression of cry genes on BtPAI-1. Therefore, we constructed a vipR knockout mutant (HD-1ΔvipR) in Bt subsp. kurstaki strain HD-1 (S1A and S1B Fig). Growth curve analyses showed no significant differences in growth dynamics between HD-1 and HD-1ΔvipR (S1C Fig), indicating that deletion of vipR does not affect bacterial growth.

Parasporal crystal formation was then examined at different growth stages by microscopy observations. At the vegetative phase (9 h), no obvious spores were observed in either strain. However, large fuchsin-stained inclusion bodies were readily detected in HD-1, whereas only small inclusion-like structures were observed in a subset of HD-1ΔvipR cells (Figs 1A and S2). At the early (24 h) and mid (36 h) sporulation phases, both HD-1 and HD-1ΔvipR began to form spores and visible parasporal crystal inclusions. However, spores and crystals remained closely associated and could not be reliably distinguished for quantitative analysis at these stages. At the late sporulation phase (42 h), spores were almost completely formed in both strains. However, the crystal-to-spore ratio remained significantly higher in HD-1 (99.6% ± 6.3%) than in HD-1ΔvipR (71.7% ± 8.0%) (Fig 1A and 1B).

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Fig 1. VipR promotes early Cry proteins accumulation during vegetative growth and enhances insecticidal activity of Bt.

(A) Representative microscopic images of HD-1 and HD-1ΔvipR cells at 9 h, 24 h, 36 h and 42 h of growth. The images shown are 4 × enlarged views of the original micrographs, and the corresponding original images are provided in S2 Fig. Scale bar = 2.5 μm. (B) Crystal-to-spore ratio in HD-1 and HD-1ΔvipR at 42 h. The ratios were determined by counting crystals and spores in six randomly selected microscopic fields. Data represent the means ± SD (n = 6 biological replicates). Statistical procedures are specified in Materials and methods. ***P < 0.001, ****P < 0.0001. (C) Western blot detection of Cry1A protein in HD-1 and HD-1ΔvipR strains at 9 h, 24 h and 36 h. Cry1A was detected using an anti-Cry1A primary antibody and IRDye 680-conjugated goat anti-rabbit secondary antibody. All protein samples were extracted from an identical number of cells (1 × 10⁷ colony-forming units (CFU)/mL), and 20 µL of each sample (containing approximately 2 × 10⁵ CFU equivalents) was loaded per lane. (D) Larval mortality of Spodoptera frugiperda larvae after exposure to fermentation broth samples collected from HD-1, HD-1Δvip3Aa and HD-1ΔvipR cultures at different growth phases. The fermentation broth samples were not lyophilized; fresh cultures were used directly. Prior to the bioassay, the cell concentration was determined using the dilution plate technique, and all samples were adjusted to the same cell density (1 × 10⁷ CFU/mL) with sterile ddH₂O and then further diluted 4-fold (2.5 × 10⁶ CFU/mL). Subsequently, 3 mL of each diluted sample was mixed with 15 g of artificial diet, resulting in a final concentration of 5 × 10⁵ CFU per gram of diet, and the mixture was aliquoted into 24-well plates (one larva per well). Data represent the means ± SD (n = 3 biological replicates). **P < 0.01, ***P < 0.001, ****P < 0.0001. (E) Comparison of LC₅₀ values of spore-crystal mixtures from HD-1, HD-1Δvip3Aa and HD-1ΔvipR strains against S. frugiperda. Prior to LC₅₀ determination, the cell concentration in each fermentation broth sample was determined using the dilution plate technique, and all samples were adjusted to the same cell density (1 × 10⁷ CFU/mL) with sterile ddH₂O. The normalized samples were then lyophilized. The lyophilized spore-crystal powders were resuspended in ddH₂O and then serially diluted from 1000 μg/mL to 100, 10, 5, 2.5, 1.25, and 0.625 μg/mL. 3 mL of each dilution was mixed with 15 g of artificial diet, resulting in final concentrations of 200, 20, 2, 1, 0.5, 0.25, and 0.125 μg per gram of diet. LC₅₀ is the protein concentration causing 50% mortality of target insects, expressed as μg lyophilized spore-crystal mixture per gram of diet. The LC₅₀ values include their corresponding 95% confidence limits (95% CL), intercept, slope, and Chi-square (χ²) statistics are shown.

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To validate these phenotypic differences at the protein level, Cry1Aa production was analyzed by western blot. Cry1Aa was clearly detected in HD-1 during the vegetative phase (9 h) but was undetectable in HD-1ΔvipR (Fig 1C). Upon entry into the sporulation phase (24–36 h), Cry1Aa expression was detected in both strains after normalization to equivalent cell numbers. Analysis of these data showed higher protein levels of Cry1Aa in HD-1 than in HD-1ΔvipR (Fig 1C). To determine whether the reduced Cry accumulation in the vipR mutant affected insecticidal activity, bioassays against Spodoptera frugiperda larvae were performed. Because VipR positively regulates vip3Aa expression [34] and Vip3Aa represents a major vegetative-phase insecticidal protein, a vip3Aa deletion mutant (HD-1Δvip3Aa) was constructed in the HD-1 background as a control strain to distinguish the respective contributions of Vip3Aa and Cry proteins in toxicity against S. frugiperda (S3 Fig). The insecticidal activities of HD-1, HD-1ΔvipR and HD-1Δvip3Aa were evaluated at different growth phases. The results showed that during the vegetative phase (9 h), the fermentation broth of HD-1 caused 80.1% ± 2.9% larval mortality, whereas that of HD-1ΔvipR caused almost 6.4-fold lower values with 12.5% ± 1.4% mortality (Fig 1D). Notably, although the insecticidal activity of HD-1Δvip3Aa at 9 h (26.4% ± 1.4%) remained significantly lower than that of wild-type HD-1, it was substantially higher than that of HD-1ΔvipR. These results indicate that the severe reduction in early insecticidal activity caused by vipR deletion cannot be explained solely by the loss of vip3Aa, but is also associated with the reduced accumulation of Cry proteins during the vegetative phase. During the sporulation phase (24 h and 36 h), HD-1 caused mortality rates of 92.1% ± 0.8% and 94.4% ± 1.4%, respectively, whereas HD-1ΔvipR exhibited substantially reduced mortalities of 31.5% ± 3.5% and 64.4% ± 3.2%, respectively (Fig 1D). However, the insecticidal activity of the HD-1Δvip3Aa fermentation broth at the sporulation phases (24 h and 36 h) remained significantly lower than that of HD-1 but higher than that of HD-1ΔvipR, with mortality rates of 49.1% ± 5.6% and 78.7% ± 2.9%, respectively. These results suggest that the reduced insecticidal activity observed in HD-1ΔvipR during sporulation cannot be explained solely by the loss of Vip3Aa. Rather, it is also attributable to the reduced accumulation of Cry proteins caused by vipR deletion.

Consistent with these results, determination of the median lethal concentration (LC₅₀) using spore-crystal mixtures showed that the LC₅₀ value of HD-1 was 2.23 μg/g (with 95% confident values of 1.65-3.14 μg/g), whereas HD-1ΔvipR displayed a significantly higher LC₅₀ value of 31.74 μg/g (with 95% confidence values of 24.02-38.62 μg/g). The absence of overlapping in the 95% confidence values confirmed a 15-fold reduction in toxicity of HD-1ΔvipR compared with HD-1 (Fig 1E). The HD-1Δvip3Aa strain exhibited an intermediate LC₅₀ value of 22.72 μg/g (with 95% confidence limits of 17.94-28.78 μg/g), representing an approximately 10-fold reduction in toxicity compared with HD-1, consistent with previous reports describing the contribution of Vip3Aa to insecticidal activity [35]. Although HD-1Δvip3Aa exhibited higher toxicity than HD-1ΔvipR, the overlap between their 95% confidence intervals indicated that this difference was not statistically significant. This may be due to partial compensation by the substantial Cry protein production during the late sporulation phase.

Collectively, these results demonstrate that VipR not only regulates Vip3Aa expression but also promotes Cry protein production during vegetative growth. Also, the premature expression of Cry proteins significantly contributes to the insecticidal activity of the Bt HD-1 strain.

VipR directly binds to the promoters of cry genes in BtPAI-1 and activates their expression during vegetative growth

To evaluate whether VipR modulates the transcription of cry genes located within BtPAI-1, we analyzed the expression profiles of several cry genes in HD-1 and HD-1ΔvipR at different growth stages by using quantitative reverse transcription PCR (qRT-PCR). During the vegetative phase (9 h), transcription of cry1Aa, cry2Ab, cry1Ia and cry2Aa in HD-1ΔvipR was markedly reduced compared with HD-1, with relative expression levels of approximately 0.046-fold, 0.040-fold, 0.067-fold and 0.117-fold, respectively (Fig 2A–2D). Upon entry into the early sporulation phase (24 h), the expression of these genes in HD-1ΔvipR remained lower than in HD-1, showing 0.437-fold, 0.470-fold, 0.239-fold, and 0.610-fold of HD-1, respectively (Fig 2A–2D). By the late sporulation (36 h), transcription levels were similar in both strains with differences of less than 0.2-fold (Fig 2A–2D). These results indicate that in HD-1, VipR promotes transcription of multiple cry genes during vegetative growth and early sporulation, but has little or no effect on their expression during late sporulation.

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Fig 2. VipR directly activates transcription of multiple cry genes within BtPAI-1 by binding to their promoter regions.

(A–D) Relative transcriptional levels of cry2Ab (A), cry1Aa (B), cry1Ia (C), and cry2Aa (D) in HD-1 and HD-1ΔvipR at 9 h, 24 h, and 36 h, determined by quantitative reverse transcription PCR (qRT-PCR). The gapdh and 16S rRNA were used as internal reference genes. Data represent the means ± SD (n = 3 biological replicates). Statistical significance was determined as described in Materials and methods. ns, P > 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001. (E-H) β-galactosidase reporter assays showing promoter activities of amidase (E), cry2Ab (F), cry1Ia (G), and cry2Aa (H) in HD-1 and HD-1ΔvipR. Promoter regions were fused to a lacZ reporter gene, and β-galactosidase activity was measured at the indicated time points. Data represent the means ± SD from three independent experiments. Different letters indicate significant differences between groups (P < 0.05). (I–L) Electrophoretic mobility shift assays (EMSA) demonstrating direct binding of VipR to the promoter regions of amidase (I), cry2Ab (J), cry1Ia (K), and cry2Aa (L). FAM-labeled DNA promoter probes (P_amidase-FAM, P_cry2Ab-FAM, P_cry1Ia-FAM and P_cry2Aa-FAM), were incubated with increasing concentrations of purified recombinant VipR protein. To exclude non-specific binding, 100 ng of poly(dI-dC) was added to each reaction mixture. For competition assays, a 10-fold molar excess of unlabeled specific cold probe was added. To further verify binding specificity, promoter probes carrying deletions in the predicted VipR-binding motifs were generated for amidase (I), cry2Ab (J), cry1Ia (K), and cry2Aa (L) promoters. The positions of protein-DNA complexes (shifted bands) and free probe are indicated.

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To further investigate the role of VipR in regulating cry gene expression, we first examined transcriptional organization within the genomic region of BtPAI-1. RT-PCR analysis showed that each one of cry2Ab, cry1Ia and cry2Aa genes is transcribed independently, whereas cry1Aa is co-transcribed with the upstream amidase gene (S4 Fig). Based on these results, the promoter regions of cry2Ab (P_cry2Ab), cry1Ia (P_cry1Ia), and cry2Aa (P_cry2Aa), as well as the amidase promoter (P_amidase) (S5 Fig) were fused to a lacZ reporter gene. Each promoter-lacZ fusion construction was introduced separately into both HD-1 and HD-1ΔvipR, and β-galactosidase activity was determined throughout bacterial growth. Promoter activities driven by P_amidase, P_cry2Ab and P_cry1Ia were significantly higher in HD-1 than in HD-1ΔvipR across all tested growth phases (Fig 2E–2H). Specifically, at 9 h (vegetative phase), P_amidase-driven β-galactosidase activity in HD-1 reached approximately 100 units, whereas it was nearly undetectable (0 units) in HD-1ΔvipR (Fig 2E). At 12 h (vegetative phase), the P_cry2Ab-driven β-galactosidase activity in HD-1 was 153 units, about seven-fold higher than that in HD-1ΔvipR (Fig 2F). Similarly, at 14 h (late vegetative phase), the P_cry1Ia-driven activity in HD-1 reached 56 units, but remained nearly undetectable in HD-1ΔvipR (Fig 2G). In each case, peak promoter activity in HD-1 was 5.3-fold (P_amidase), 3.8-fold (P_cry2Ab) and 1.3-fold (P_cry1Ia) higher than that in HD-1ΔvipR, respectively (Fig 2E–2G). In contrast, activity of the P_cry2Aa promoter showed a different temporal pattern. Before 28 h, the β-galactosidase activity driven by P_cry2Aa was significantly higher in HD-1 than in HD-1ΔvipR, with a peak activity of approximately 4.9-fold higher in the wild type strain. After 28 h, however, promoter activity became higher in HD-1ΔvipR than in HD-1 (Fig 2H). Taken together, these results indicate that in HD-1, VipR activates cry gene transcription during vegetative growth by modulating promoter activity.

To determine whether VipR directly regulates these promoters, we purified recombinant VipR protein and performed electrophoretic mobility shift assays (EMSA). DNA probes corresponding to P_amidase, P_cry2Ab, P_cry1Ia and P_cry2Aa all showed clear binding to VipR protein (Fig 2I–2L). These interactions were specific, as the shifted bands were efficiently competed away by addition of excess unlabeled competitor DNA probes (Fig 2I–2L). Deletion of the predicted VipR-binding motifs in the promoter regions of amidase (Fig 2I), cry2Ab (Fig 2J), cry1Ia (Fig 2K), and cry2Aa (Fig 2L) completely abolished VipR binding to these mutant probes, further supporting the sequence-specific interaction between VipR and these promoters. Collectively, these data demonstrate that VipR directly binds to the promoter regions of multiple cry genes within BtPAI-1 of HD-1 strain and activates their transcription during vegetative growth.

Overexpression of vipR increases transcription of BtPAI-1 toxin genes during the vegetative phase and enhances insecticidal activity

Given that transcription factor-mediated regulation is often dose-dependent [36], we hypothesized that increasing the transcriptional level of vipR could result in enhanced transcription of cry genes, thereby improving the insecticidal activity of the modified strains. To test this hypothesis, vipR was placed under the control of the constitutive promoter P_kan and introduced into two Bt strains (HD-1 and CT-43), which harbor BtPAI-1. The qRT-PCR analysis confirmed that vipR transcript levels were significantly increased in all strains carrying the P_kan-vipR construct, with increases of 43-fold in HD-1, and 74-fold in CT-43 relative to their respective wild-type controls (S6 Fig).

We next assessed the transcriptional response of insecticidal genes encoded within BtPAI-1 during the vegetative phase (9 h). In HD-1, overexpression of vipR resulted in significant upregulation of vip3Aa, cry2Ab, cry1Aa, cry1Ia and cry2Aa, with significant fold changes of approximately 19.3-fold, 8.8-fold, 6.6-fold, 7.0-fold, and 8.8-fold, respectively (Fig 3A). In CT-43, the same genes were upregulated by approximately 5.8-fold, 2.6-fold, 4.7-fold, 4.8-fold and 3.2-fold, respectively (Fig 3B). These results indicate that increased vipR expression promotes the transcription of multiple toxin genes in the HD-1 and CT-43 strains.

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Fig 3. Overexpression of vipR significantly upregulates the transcriptional levels of multiple insecticidal protein genes in BtPAI-1 during the vegetative phase and enhances insecticidal activity.

(A, B) The qRT-PCR analysis of vip3Aa, cry2Ab, amidase, cry1Aa, cry1Ia and cry2Aa transcriptional levels in HD-1, CT-43, and their respective vipR overexpression strains (HD-1 (P_kan-vipR) and CT-43 (P_kan-vipR)) at 9 h. The gapdh and 16S rRNA genes were used as internal reference genes. Data represent the means ± SD of three biological replicates (n = 3). Statistical significance was determined using appropriate tests as explained in Materials and methods. Significance thresholds: ns, P > 0.05, *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. (C, D) Larval mortality of Spodoptera frugiperda following exposure to spore-crystal mixture samples derived from vipR-overexpressing strains and their corresponding wild-type strains. Spore-crystal mixtures were prepared as described in Fig 1E (60 h cultures, complete sporulation, adjusted to 1 × 10⁷ CFU/mL, lyophilized) and serially diluted to final concentrations of 1000, 100, 10, 5, 2.5, 1.25 and 0.625 μg/mL. The serially diluted spore-crystal suspensions were mixed with artificial diet for bioactivity assays. Data represent the means ± SD (n = 3 biological replicates). ns, P > 0.05, *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. (E) Toxicity assays of spore-crystal mixture from HD-1, CT-43, and their respective vipR overexpression strains (HD-1 (P_kan-vipR) and CT-43 (P_kan-vipR)) against S. frugiperda larvae. LC₅₀ refers to the concentration causing 50% mortality of target insects, expressed as μg lyophilized spore-crystal mixture per gram of diet. LC₅₀ values, along with their 95% confidence limits (95% CL), intercept, slope, and Chi-square (χ²) values with corresponding degrees of freedom (df), are presented in the figure.

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We then assessed whether these transcriptional changes affected insecticidal activity. Spore-crystal mixtures from vipR-overexpressing and wild-type strains were tested against S. frugiperda larvae across a range of concentrations. At high concentrations, both HD-1 and its vipR-overexpressing derivative exhibited potent insecticidal activity, with no significant differences in larval mortality (Fig 3C). In contrast, at lower concentrations, the vipR-overexpressing strain of HD-1 consistently showed higher larval mortality than its corresponding wild-type strains (Fig 3C). For strain CT-43, vipR overexpression resulted in increased larval mortality relative to the wild type across all tested concentrations (Fig 3D). Consistent with these phenotypic trends, LC₅₀ estimates indicated an approximately two-fold increase in insecticidal activity for the HD-1 vipR-overexpressing strain and a three-fold increase for the CT-43 counterpart compared with their respective wild-type controls (Fig 3E). However, it is important to mention that the overlap in the 95% confidence limits for HD-1 indicates that this difference was not statistically significant (Fig 3E). This discrepancy is likely attributable to the compensation in crystal production that is activated at the late stages of sporulation.

Collectively, these findings show that vipR overexpression significantly enhances the transcription of BtPAI-1-encoded cry genes in HD-1 and CT43, and this transcriptional upregulation contributes to a moderate improvement of insecticidal activity.

The vipR is widely distributed among Bt strains and associated with BtPAI-1 and its variants

To assess the distribution of vipR and BtPAI-1 among Bt populations, we performed a systematic analysis in 889 publicly available Bt genomes from the NCBI database. BtPAI-1 or its homologous sequences (BtPAI-1 variants) were identified in 327 strains (37%). Among these, 196 strains contained a complete BtPAI-1, whereas 131 strains carried partial regions, hereafter referred to as BtPAI-1 variants (Figs 4 and S7).

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Fig 4. vipR is widely distributed in Bt strains and is associated with BtPAI-1 and its variants.

The maximum likelihood (ML) phylogenetic tree of Bt strains was inferred using PhyML with the best-fit substitution model (JTT + I + G), based on alignments of concatenated protein sequences of single-copy core genes. Bootstrap support values were calculated from 1000 replicates. The outermost ring indicates the presence of vipR (light coral, vipR-positive; and light teal green, vipR-negative strains). The inner ring denotes the distribution of BtPAI-1 and its variants. The bar plot surrounding the tree represents the number of insecticidal protein genes identified in each Bt strain.

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We further analyzed the distribution of vipR across these Bt genomes. A total of 296 strains (33%) harbored vipR (Fig 4). The presence of vipR was strongly associated with BtPAI-1 or its variants (Fig 4). Among these vipR-positive strains, 196 strains (66%) contained vipR within complete BtPAI-1 elements, while the remaining 100 strains carried vipR located adjacent to BtPAI-1 variants (Fig 4). Conversely, only 24% of the strains harboring BtPAI-1 variants lacked vipR (Fig 4). Furthermore, all strains harboring vipR encoded more than five major insecticidal protein genes (Fig 4). These findings indicate that vipR is widely distributed in Bt, and is closely associated with BtPAI-1 and variants, suggesting that vipR as a conserved regulator of toxin gene expression.

Conserved role of VipR among different Bt backgrounds

As mentioned above, 24% of analyzed Bt strains that harbor BtPAI-1 or its variants, lack a vipR gene. Bioinformatic analysis of these strains further revealed that the cry genes and their corresponding promoter regions within BtPAI-1 and its variants exhibit a high nucleotide identity (>80%), with putative VipR-binding sites within their promoter regions (S8 Fig). Based on these observations, we hypothesized that introducing vipR into these strains would trigger premature activation of insecticidal gene expression.

To test this hypothesis, vipR was heterologously expressed in strains HD73 [37] and BGSC 4J5 [19], both of which lack an endogenous vipR gene. We then examined the transcriptional levels of cry genes within their respective BtPAI-1 variants and evaluated the insecticidal activity of the engineered strains. The qRT-PCR analysis revealed that cry1Ac transcription in HD73 (transformed with vipR) was significantly increased during the vegetative phase (9 h), exhibiting an approximately 6.0-fold increase relative to the parental HD73 strain (Fig 5A). Similarly, BGSC 4J5 (transformed with vipR) displayed increased transcript levels of cry2Ab, cry1Aa and cry1Ia transcripts at 9 h, with approximately 2.3-fold, 6.4-fold and 4.3-fold increases, respectively (Fig 5B–5D). Notably, the elevated transcription of these genes persisted in the vipR-expressing strains after entry into the stationary phase (24 h-36 h) compared to the wild-type strains (Fig 5A–5D). Consistent with the transcriptional data, western blot analysis demonstrated early accumulation of Cry proteins in the vipR-expressing strains. Cry1Ac was detected as early as 9 h in both HD73 (vipR) and BGSC 4J5 (vipR), whereas prominent Cry1A signals in the parental strains were only observed after the stationary phase (24 h and 36 h) (Fig 5E). Then, we performed bioassays using spore-crystal mixtures against the susceptible insect larvae. At low concentrations, strains expressing heterologous vipR gene exhibited higher larval mortality than the wild-type strain (Fig 5F and 5G). In contrast, at high concentrations, all strains displayed potent insecticidal activity, with no significant differences in larval mortality among groups (Fig 5F and 5G). Consistent with this trend, LC₅₀ estimates indicated that the insecticidal activity of the vipR-expressing strains, HD73 (vipR) and BGSC 4J5 (vipR), increased by approximately two-thirds relative to the wild type. However, again the 95% confidence limits overlapped, indicating that these differences were not statistically significant (Fig 5H). Heterologous expression of vipR markedly enhanced the transcription of cry genes during the vegetative phase in Bt strains HD73 and BGSC 4J5 carrying BtPAI-1 variants. However, this elevated transcription did not translate into a statistically significant increase in the insecticidal activity of sporulated cultures. A plausible explanation, consistent with the overexpression scenario, is that compensatory crystal production during the late stages of sporulation offsets the potential advantages conferred by increased gene expression.

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Fig 5. VipR promotes premature Cry proteins expression during the vegetative phase and enhances insecticidal activity in vipR-deficient Bt.

(A) qRT-PCR analysis of cry1Ac transcript levels in wild-type HD73 and HD73 transformed with vipR gene, at 9 h, 24 h, and 36 h of growth. The gapdh and 16S rRNA genes were used as internal reference genes. Data represent the means ± SD from three independent biological replicates (n = 3). Statistical significance was described in Materials and methods. Significance thresholds: ns, P > 0.05, *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. (B–D) qRT-PCR analysis of cry2Ab, cry1Aa and cry1Ia transcriptional levels in wild-type BGSC 4J5 and BGSC 4J5(vipR) transformed with vipR gene, at 9 h, 24 h, and 36 h of growth. The internal reference genes, data representation and statistical analyses are as described in Fig 5A. (E) Western blot analysis of Cry1A protein accumulation in HD73, BGSC 4J5, HD73 (vipR) and BGSC 4J5 (vipR) at 9 h, 24 h and 36 h. Sample preparation and loading were performed as described in Fig 1C (cells adjusted to 1 × 10⁷ CFU/mL; 20 µL per lane, equivalent to 2 × 10⁵ CFU per lane). Cry1A protein was detected using anti-Cry1A primary antibody and a goat anti-rabbit IRDye 680 secondary antibody. Data are representative of three independent experiments. (F, G) Larval mortality of Spodoptera frugiperda larvae after exposure to spore-crystal mixture samples collected from HD73, BGSC 4J5, HD73(vipR) and BGSC 4J5(vipR) cultures. Sample preparation was performed as described in Fig 1E (60 h cultures, complete sporulation, adjusted to 1 × 10⁷ CFU/mL, lyophilized) and serially diluted to final concentrations of 1000, 100, 10, 5, 2.5, 1.25, and 0.625 μg/mL. The serially diluted spore-crystal suspensions were mixed with artificial diet for bioactivity assays. Data represent the means ± SD (n = 3 biological replicates). ns, P > 0.05, *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. (H) Insecticidal activity of spore-crystal mixture from HD73, BGSC 4J5, HD73(vipR) and BGSC 4J5(vipR) against S. frugiperda larvae. LC₅₀ values refer to the sample concentration causing 50% mortality of target insects, expressed as μg of lyophilized spore-crystal mixture per gram of diet. LC₅₀ values, along with their 95% confidence limits (95% CL), intercept, slope, and Chi-square (χ²) values with corresponding degrees of freedom (df), are indicated in the figure.

https://doi.org/10.1371/journal.ppat.1014476.g005

Collectively, these results demonstrate that heterologous expression of vipR is sufficient to induce vegetative-phase transcription of cry genes in the Bt strains HD73 and BGSC 4J5 carrying BtPAI-1 variants, leading to earlier accumulation of Cry proteins. Moreover, these findings also indicate that the regulatory function of VipR may be conserved across diverse Bt strains harboring distinct BtPAI-1 variants.

VipR activates transcription of non-PAI insecticidal genes during the vegetative phase

We have demonstrated that VipR directly binds to the promoter regions of different BtPAI-1-associated insecticidal genes and activates their expression in multiple Bt strains. Our EMSA assays confirmed that deletion of the predicted VipR-binding motif abolishes DNA binding, identifying this sequence as a key determinant site for VipR-promoter interaction (Fig 2I–2L). Furthermore, similar motifs have been identified in the promoter region of additional insecticidal genes, including cry9Ca-orf1 and cry9Aa genes [38], suggesting that VipR may regulate a broader set of targets (Fig 6A and 6B). Consistent with this possibility, our systematic genome-wide bioinformatic analysis performed on 889 available Bt genomes, where insecticidal protein genes were identified using BtToxin_Digger [39], and their promoter regions were scanned for the potential VipR-binding motif using the FIMO tool (MEME Suite v5.5.4, http://meme-suite.org/tools/fimo) [40], revealed that potential VipR-binding motifs are widely distributed among different classes of Bt insecticidal genes: detected in 49.32% of cry1-type genes (831/1685), 77.57% of cry2-type genes (377/486), 70.75% of cry9-type genes (104/147), and 90.3% of vip3-type genes (307/341) (Fig 6C–6F). We also found that 59.9% of cry1-type, 76.1% of cry2-type, and 93.5% of vip3-type genes are located on BtPAI-1 or its variants (Fig 6C–6F). In contrast, 40.1% of cry1-type genes, 23.9% of cry2-type genes, 6.5% of vip3-type genes and 100% of cry9-type genes are located on non-BtPAI-1 plasmids (Fig 6C–6F). Furthermore, when we introduced vipR into the cry9Aa-harboring strain BGSC 4AE1, we observed a significant increase in the transcription level of the cry9Aa gene (Fig 6G), suggesting that VipR may regulate both BtPAI-1-encoded and non-BtPAI-1-encoded insecticidal genes across diverse Bt genetic backgrounds such as HD-1, CT-43, HD73, BGSC 4J5 and BGSC 4AE1 strains.

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Fig 6. VipR activates the transcription of multiple potential target genes distributed across the Bt genome during the vegetative phase.

(A) Identification of potential regulatory targets of VipR based on its conserved binding motifs. Proportion of genes harboring conserved VipR-binding motifs among homologous insecticidal protein gene families. (B) Predicted conserved VipR-binding motifs in the promoter regions of individual target genes. (C–F) Pie chart showing the percentage of cry1-type genes (C), cry2-type genes (D), cry9-type genes (E) and vip3-type genes (F) carrying VipR-binding motifs located on BtPAI-1 (and its variants) or in independent genomic regions. (G) Detection of cry9Aa transcriptional levels in BGSC 4AE1 and BGSC 4AE1(vipR). The qRT-PCR analysis of cry9Aa in BGSC 4AE1 and BGSC 4AE1 (vipR) was performed at 9 h, 24 h and 36 h. The gapdh and 16S rRNA genes were used as internal reference genes. Data represent the means ± SD (n = 3 biological replicates). Statistical significance was determined using appropriate tests (see Materials and methods). Significance thresholds: *P < 0.05, **P < 0.01, ***P < 0.001.

https://doi.org/10.1371/journal.ppat.1014476.g006

Bacterial strains, plasmids, and cultural conditions

Escherichia coli DH5α was used as the host strain for plasmid construction and E. coli BL21(DE3) [45] was used for VipR protein production. All bacterial strains and plasmids used in this study are listed in S1 and S2 Tables, respectively. E. coli strains were cultured in Luria-Bertani (LB) medium (0.5% yeast extract, 1% NaCl, 1% peptone) [46] 37 °C with shaking at 220 rpm. Bacillus thuringiensis (Bt) was cultured in insecticidal crystal protein medium (ICPM: 0.6% peptone, 0.5% glucose, 0.05% MgSO₄·7H₂O, 0.1% CaCO₃, 0.05% KH₂PO₄; pH 7.0) at 28 °C with shaking at 220 rpm. Antibiotics were used at the final concentrations: erythromycin (10 μg/mL), kanamycin (100 μg/mL), ampicillin (100 μg/mL), and spectinomycin (100 μg/mL).

DNA manipulation

Standard molecular biology procedures were performed according to Sambrook and Russell, 1983 [47]. DNA fragments from gels or PCR products were purified using the E.Z.N.A. Gel Extraction Kit (Omega). Primers used in this study are listed in S3 Table. All recombinant plasmids were verified by Sanger sequencing (Augct, Wuhan, China). E. coli was transformed using the standard chemical method [47], while Bt was transformed by electroporation as previously described [48].

Phylogenetic analysis

Protein sequences from Bt genomes were subjected to BlastP analysis (E-value < 10⁻⁵), followed by clustering analysis using the Markov cluster algorithm (MCL) with an inflation parameter of 2 [49]. Single-copy core proteins from each cluster were aligned using MUSCLE [50], and poorly aligned regions were trimmed with TrimaAl [51]. A maximum likelihood (ML) phylogenetic tree was constructed using PhyML with the best-fit substitution model (JTT + I + G).

Genome assemblies were retrieved from GenBank, and those with scaffold N50 < 50 kb were excluded. Core genome single nucleotide polymorphisms (SNPs) were identified using the Northern Arizona SNP Pipeline (NASP) (https://github.com/TGenNorth/NASP).

To determine the distribution of BtPAI-1 and its variants, BLASTP searches (E-value <1 x 10^-5) were performed for VipR, Vip3Aa, Cry2Ab, amidase, Cry1Aa, and Cry1Ia across all Bt genomes. The positional relationships of these genes were analyzed, and the phylogenetic tree was annotated using iTOL [52].

CRISPR-Cas9 mediated gene deletion

The vipR and vip3Aa gene in Bt was knocked out using the CRISPR-Cas9 system based on plasmid pJOE8999, as previously described [53]. Single guide RNA (sgRNA) targeting vipR and vip3Aa were designed using CRISPy-web (https://crispy.secondarymetabolites.org/#/input) [54] (see S3 Table for details). The corresponding sgRNA cassettes were generated using primer pairs delvipR-sgRNA-F/R and delvip3Aa-sgRNA-F/R and cloned into Eco31I-digested pJOE8999, respectively. For vipR, the upstream (507 bp) and downstream (422 bp) homologous arms of vipR gene were amplified using the delvipR-sfiI-up-F/R and delvipR-sfiI-down-F/R primers, respectively. For vip3Aa, the upstream (497 bp) and downstream (500 bp) homologous arms of vip3Aa gene were amplified using the delvip3Aa-sfiI-up-F/R and delvip3Aa-sfiI-down-F/R primers, respectively. These fragments were amplified and fused by using splicing by overlap extension (SOE) PCR technology. For each target gene, the homologous arms were fused by splicing by overlap extension (SOE)-PCR and inserted into SfiI-digested pJOE8999, generating plasmids vipR-pJOE8999 and vip3Aa-pJOE8999, respectively. Each construct contained both the sgRNA cassette and the corresponding repair template. These construct were transformed into the HD-1 strain by electroporation to obtain the vipR-pJOE8999/HD-1 and vip3Aa-pJOE8999/HD-1 strains, respectively.

Transformants were cultured in LB medium at 28 °C with shaking at 220 rpm for 24 h. Next, 1 mL of the bacterial culture was diluted 10⁴–10⁶ times in LB medium, and 100 μL of this diluted culture was spread on LB solid medium. After overnight incubation at 28 °C, single colonies were picked from the plate and cultured overnight at 28 °C for further verification. Individual colonies were screened by colony PCR. Deletion of vipR was verified using primer pairs delvipR-upup-F/R and delvipR-dodo-F/R, whereas deletion of vip3Aa was verified using primer pairs delvip3Aa-upup-F/R and delvip3Aa-dodo-F/R. The resulting mutants were further confirmed by Sanger sequencing (S1 and S3 Figs).

To cure the temperature-sensitive pJOE8999 plasmid [55], the deletion mutant strain ΔvipR-pJOE8999/HD-1 and Δvip3Aa-pJOE8999/HD-1 were inoculated into LB medium and cultured at 42 °C with shaking at 220 rpm for 24–48 h. Elimination of the pJOE8999 plasmid was confirmed by antibiotic sensitivity screening and PCR using primers pJOE8999-F/R.and

Deletion of genes and absence of off-target mutations were confirmed by Sanger sequencing of the mutated region. Sequencing reads were aligned to the reference genome of the wild-type HD-1 strain using SnapGene software with default parameters, and visualization was performed using Jalview to confirm the precise deletion. No off-target mutations were detected, confirming the accuracy of the knockout process.

Bacterial growth curves

The wild-type HD-1 and the mutant deletion HD-1ΔvipR strains were cultured overnight at 28 °C in LB medium and subsequently diluted 1:100 into fresh LB medium. Cultures were grown to OD₆₀₀ value of 0.4-0.5 at 28 °C with shaking at 220 rpm. Next, 200 μL aliquots were transferred to a 100-well honeycomb plate. Growth was monitored using a Bioscreen C Pro automated growth analyzer at 28°C with shaking at 480 rpm, recording OD₆₀₀ every 30-min. All experiments were performed in triplicate.

Insect bioassay

Strains HD-1 [56], CT-43 [57], HD-1ΔvipR, HD-1Δvip3Aa, HD-1(P_kan-vipR), CT-43(P_kan-vipR), HD73, BGSC 4J5, HD73(vipR) and BGSC 4J5(vipR) were cultured in LB medium. Samples of fermentation broths of these strains were collected at the vegetative (9 h), the early sporulation (24 h) and late the sporulation (36 h) phases. Cell density of each sample was determined using the dilution plate counting method. Briefly, serial dilutions were prepared and plated onto LB agar plates, and colony-forming units (CFU) were calculated after incubation. Based on CFU determination, all samples were adjusted to a uniform density of 1 × 10⁷ CFU/mL using sterile double-distilled water (ddH₂O). For bioassays, the standardized fermentation broth was further diluted four-fold (2.5 × 10⁶ CFU/mL). Subsequently, 3 mL of each diluted sample was mixed with 15 g of artificial diet, resulting in a final concentration of approximately 5 × 10⁵ CFU per gram of diet. The mixture was then aliquoted into 24-well plates for insecticidal activity assays (one larva per well).

In addition, for crystal-spore preparations, strains were cultured in ICPM medium at 28 °C with shaking at 220 rpm for 60 h (until complete sporulation, confirmed by microscopy). Cells were harvested by centrifugation (10,000 rpm, 4°C), washed 3–5 times with EDTA-NaCl solution (10 mM Ethylenediaminetetraacetic acid, 1M NaCl, pH 7.0), followed by three-times wash with ddH₂O. The cell density of the washed sample was determined using the dilution plate counting method, and all samples were adjusted to a uniform density (1 × 10⁷ CFU/mL) with sterile ddH₂O. The normalized samples were then lyophilized using a freeze dryer to obtain spore-crystal powders (Life Technologies). The lyophilized preparations were resuspended at 1000 μg/mL and serially diluted to final concentrations of 100, 10, 5, 2.5, 1.25 and 0.625 μg/mL. These suspensions were mixed with artificial diet for insect bioassays and LC₅₀ values determinations.

3 mL of each diluted sample was thoroughly mixed with 15 g of artificial diet [58]. The mixed diet was transferred to 24-well culture plates, with one newly hatched neonate S. frugiperda larva per well. Each sample was performed in triplicate, and mortality was recorded after seven days. The LC₅₀ values were calculated using probit analysis [59].

Microscopy observation

For general observation, sporulated cultures were stained with fuchsin and observed under an optical microscope (Olympus, USA) using 100 x oil immersion objective.

Western blot analysis

The Bt strains (HD-1, HD-1ΔvipR, HD73, HD73(vipR), BGSC 4J5, and BGSC 4J5(vipR)) were inoculated into 100 mL of LB medium and cultured at 28 °C with shaking at 220 rpm. Samples were collected at different growth phases (9 h, 24 h, and 36 h). For each sample, 2 mL of fermentation broth was centrifuged at 12,000 rpm for 2 min to collect the cell pellets. The pellets were washed twice with sterile ddH₂O. The cell density of each sample was determined using the dilution plate counting method. Based on the results, all samples were adjusted to an equal cell density of 1 × 10⁷ CFU/mL with sterile ddH₂O. Twenty microliters of each normalized sample was mixed with 5 µL of protein loading buffer, and the mixture was loaded per lane (equivalent to 2 × 10⁵ CFU per lane) according to the designated lane order.

Protein samples were separated by 10% SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to a nitrocellulose (NC) membrane (Abclonal Technology, Wuhan, China) at 100 V for 90 min. Membranes were washed three times (5 min each) with PBST (137 mM NaCl, 2.7 mM KCl, 10 mM Na₂HPO₄, 1.8 mM KH₂PO₄, 0.05%(v/v) Tween-20, pH7.5), and blocked 2 h at room temperature with 5% bovine serum albumin, fraction Ⅴ dissolved in PBST (Colaber, Beijing, China). After washing three times with PBST (10 min each), the membranes were incubated 90 min with 1/5,000 dilution in PBST of anti-Cry1A antibody (provided by the Institute of Plant Protection, Chinese Academy of Agricultural Sciences). Following another three washes with PBST (10 min each), the membranes were incubated 45 min in the dark with IRDye 680-conjugated goat anti-rabbit secondary antibody (1/5,000 dilution in PBST) at room temperature. The membranes were washed three times with PBST (15 min each), and signals were finally acquired using a chemiluminescence imaging system (Tanon).

RNA extraction and transcriptional analysis

The wild-type HD-1, the deletion mutant HD-1ΔvipR and additional strains including CT-43, HD-1(P_kan-vipR), CT-43(P_kan-vipR), HD73, BGSC 4J5, HD73(vipR) and BGSC 4J5(vipR) were inoculated into LB medium. Bacterial cultures were collected at the vegetative (9 h), late stationary (24 h), and sporulation (36 h) growth phases, by centrifugation (12,000 rpm, 4 °C). Total RNA was extracted by using the TransZol Up Plus RNA Kit (TransGen) and quantified with a NanoDrop ND-1000 spectrophotometer (Labtech, Wilmington, MA, USA). The cDNA synthesis was performed using PrimeScript RT reagent Kit with gDNA Eraser (Takara, Dalian, China). RT-PCR were conducted to detect the distribution of transcription units on the BtPAI-1, using RT-PCR primers listed in S3 Table. Quantitative reverse transcription PCR (qRT-PCR) assays were conducted using SYBR Green PCR Master Mix (Life Technologies, CA, USA) in an Applied Biosystems 7300 System (Applied Biosystems, Foster City, CA, USA). The gene expression levels were normalized using the gapdh and 16S rRNA genes as an internal reference. All qRT-PCR primers are listed in S3 Table.

Expression and purification of VipR

The codon optimized synthetic vipR gene was cloned into pET15D [60] (previously digested with NdeI and XhoI) via homologous recombination using the ClonExpress II One Step Cloning Kit (Vazyme). Positive clones were selected after verification by Sanger sequencing. The recombinant vector and the empty pET15D vector extracted from E. coli DH5α were introduced into E. coli BL21 (DE3) by chemical transformation. Colonies were screened by their ampicillin resistance and recombinant strains were identified by colony PCR.

The expression strain pET15D-vipR/BL21 was inoculated into LB medium supplemented with ampicillin and cultured at 37°C until the exponential phase (OD₆₀₀ = 0.6-0.8). Subsequently, it was induced with 1 mM isopropyl-β-D-thiogalactopyranoside (IPTG) at 16 °C for 15 h. Cells were collected and homogenized in 40 mL lysis buffer (137 mM NaCl, 2.7 mM KCl, 10 mM Na₂HPO₄, 1.8 mM KH₂PO₄, 10 mM imidazole; pH7.5) on ice using a low-temperature ultra-high pressure continuous cell disruptor (JN-MiniPro). The cell lysate was clarified by centrifugation (60 min, 10,000 × g at 4 °C). The supernatant containing soluble proteins was applied to a Ni²⁺-nitrilotriacetate (Ni-NTA) affinity column (Qiagen) pre-equilibrated with lysis buffer. Nonspecifically bound proteins were removed with wash buffer (187 mM NaCl, 2.7 mM KCl, 10 mM Na₂HPO₄, 1.8 mM KH₂PO₄, 80 mM imidazole; pH7.5), followed by elution of the target protein with elution buffer (187 mM NaCl, 2.7 mM KCl, 10 mM Na₂HPO₄, 1.8 mM KH₂PO₄, 300 mM imidazole; pH7.5). Protein purity was verified by SDS-PAGE and buffer-exchange into 10 mM PBS buffer (137 mM NaCl, 2.7 mM KCl, 10 mM Na₂HPO₄, 1.8 mM KH₂PO₄) was performed by using a HiTrap Desalting Column and stored at -80 °C until used.

Quantification of protein by SDS-PAGE and densitometry

The concentration of VipR was determined using SDS-PAGE combined with grayscale-based quantification using a BSA standard curve. Briefly, 40 µL of the protein sample was mixed with 10 µL of 5 × loading buffer and denatured by boiling for 10 min. For the standard curve, bovine serum albumin (BSA) was serially diluted to prepare protein standards at concentrations of 1000, 500, 250, 100, 50, 25, 12.5, 6.25, and 3.125 ng per lane. Both the VipR sample and BSA standards were resolved on a 10% SDS-polyacrylamide gel. After electrophoresis, the gel was stained with Coomassie Brilliant Blue R-250 and destained until clear background. The band intensities of both the BSA standards and the VipR protein were quantified using ImageJ software. A standard curve was generated by plotting the integrated density of BSA bands against their respective loading amounts, and the protein concentration of the VipR sample was calculated by interpolation.

Electrophoretic mobility shift assays (EMSA)

The expression and purification of vipR-His protein were done as described above. The -10 and -35 promoter boxes of each candidate promoter was predicted using the Berkeley Drosophila Genome Project (BDGP) neural network promoter prediction tool [61]. The promoter DNA fragments were PCR amplified from Bt genomic DNA using specific primers labeled with a 5′-end FAM modification and confirmed by gel electrophoresis. EMSA assays were done as follows: In a 20 μL reaction system, the fluorescent labeled P_vip3Aa, P_cry2Ab, P_amidase, and P_cry1Ia promoter fragments probes were incubated with purified VipR protein at different concentrations in binding buffer (10 mM Tris-HCl, 0.5 mM dithiothreitol (DTT), 50 mM NaCl, pH7.5, 4% (v/v) glycerol) at 37°C for 30 min. In addition, to exclude non-specific binding, 100 ng of poly(dI-dC) was added to each reaction mixture. For competition assays, a 10-fold molar excess of unlabeled specific cold probe was added. To further verify binding specificity, promoter probes carrying deletions in the predicted VipR-binding motifs were generated for amidase, cry2Ab, cry1Ia, and cry2Aa promoters and used as labeled probes in parallel reactions. The DNA-protein mixtures were applied to non-denaturing 5% (w/v) polyacrylamide gel in TBE buffer (90 mM Tris base, 90 mM boric acid, 2 mM EDTA, pH8.0) and electrophoresed at 120 V for 90 min. The labeled fragments were visualized using a Typhoon Scanner System (GE Healthcare).

β-galactosidase activity assay

The promoter regions of vip3Aa (P_vip3Aa, 413 bp), cry2Ab (P_cry2Ab, 455 bp), amidase (P_amidase, 347 bp), and cry1Ia (P_cry1Ia, 457 bp) genes were amplified from HD-1 genomic DNA using Lacz-vip3Aa-F/R, Lacz-cry2Ab-F/R, Lacz-ami-F/R, and Lacz-cry1Ia-F/R primers, respectively (S3 Table). The P_vip3Aa, P_cry2Ab, P_amidase, and P_cry1Ia fragments were digested with PstI and BamHI, and ligated into the linearized pHT304-18z [62,63] plasmid which contains a promoter-less lacZ gene [58], to obtain the recombinant plasmids pHT304-P_vip3Aa-lacZ, pHT304-P_cry2Ab-lacZ, pHT304-P_amidase-lacZ, and pHT304-P_cry1Ia-lacZ. These recombinant plasmids were introduced into HD-1 and HD-1ΔvipR, respectively, to obtain recombinant strains HD-1(P_cry2Ab-lacZ), HD-1(P_vip3Aa-lacZ), HD-1(P_amidase-lacZ), HD-1(P_cry1Ia-lacZ), HD-1ΔvipR(P_cry2Ab-lacZ), HD-1ΔvipR(P_vip3Aa-lacZ), HD-1ΔvipR(P_amidase-lacZ), and HD-1ΔvipR(P_cry1Ia-lacZ). The successful construction of each strain was verified by PCR.

The β-galactosidase activity was measured in 2 ml samples collected by centrifugation (12,000 × g, 1min) from 100 ml cultures that were grown from 0 h to 36 h in LB medium. Pellets were stored at -20 °C util used. For the assay, the samples were resuspended in 0.5 mL of Z-buffer (0.06 M Na₂HPO₄, 0.04 M NaH₂PO₄, 0.01 M KCL, 1 mM MgSO₄, 1 mM dithiothreitol). Cells were disrupted with glass beads (0.1 mm; BioSpec, USA) on ice using a JXFSTPRP-24 automatic rapid sample grinder (Jingxin, Shanghai, China). After centrifugation, the cell extracts were collected. Finally, 0.7 mL of Z-buffer supplemented with 0.2 mL of 4 mg mL-1 2-nitrophenyl β-D-galactopyranoside (sigma) were added to 100 µL of cell extract (ONPG; Sigma). The mixture was incubated at 37 °C and stopped by adding 0.5 mL of 1 M Na₂CO₃. Subsequently, the optical density of the reaction mixture was measured at 420 nm for the β-galactosidase assay. The protein concentration in the samples was determined using a Bio-Rad Protein Assay Kit with bovine serum albumin (BSA) as the standard. The specific activity of β-galactosidase was expressed as enzymatic activity units per milligram of protein (Miller units). These β-galactosidase activities were calculated using the formula: Unit value = (OD₄₂₀ × 1500)/ (T × V × Protein concentration). Where T, is the reaction time (min) and V, is the volume of the sample added (μL). All data represent the mean ± standard error (SE) of at least three independent experiments.

Construction of a strain overexpressing the vipR gene from Bt HD-1

The vipR overexpression vector was constructed using pHT315-gfp [62] plasmid, which contains the constitutive promoter P_kan and green fluorescent protein gene (gfp) under its control. The gfp gene was excised with the restriction endonucleases XbaI and HindIII, and ligated it with the vipR gene product, digested with the same restriction enzymes, to construct the recombinant plasmid pHT315-P_kan-vipR. Subsequently, pHT315-P_kan-vipR and pHT315-gfp were introduced into Bt HD-1 via electroporation to construct the vipR overexpression strain and the control Bt strains.

Statistical analysis

Statistical analyses were performed using GraphPad Prism version 8.0. Data were compared between two groups, with detailed sample sizes. For normally distributed data, Levene’s test was first used to assess the homogeneity of variances. On the basis of outcome, either the independent samples t test (for equal variances) or Welch’s t test (for unequal variances) was applied. For non-parametric distributed data, the Mann-Whitney U test was used.

Statistical significance thresholds were set as follows: ns, P > 0.05; *P < 0.05; **P < 0.01; ***P < 0.001.