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. 2023 Feb 17:14:1129454.
doi: 10.3389/fpls.2023.1129454. eCollection 2023.

Overexpression of soybean trypsin inhibitor genes decreases defoliation by corn earworm (Helicoverpa zea) in soybean (Glycine max) and Arabidopsis thaliana

Mst Shamira Sultana  1   2 , Mitra Mazarei  1   2 , Juan Luis Jurat-Fuentes  3 , Tarek Hewezi  1 , Reginald J Millwood  1 , C Neal Stewart Jr  1   2
Affiliations

Overexpression of soybean trypsin inhibitor genes decreases defoliation by corn earworm (Helicoverpa zea) in soybean (Glycine max) and Arabidopsis thaliana

Mst Shamira Sultana et al. Front Plant Sci. .

Abstract

Trypsin inhibitors (TIs) are widely distributed in plants and are known to play a protective role against herbivores. TIs reduce the biological activity of trypsin, an enzyme involved in the breakdown of many different proteins, by inhibiting the activation and catalytic reactions of proteins. Soybean (Glycine max) contains two major TI classes: Kunitz trypsin inhibitor (KTI) and Bowman-Birk inhibitor (BBI). Both genes encoding TI inactivate trypsin and chymotrypsin enzymes, which are the main digestive enzymes in the gut fluids of Lepidopteran larvae feeding on soybean. In this study, the possible role of soybean TIs in plant defense against insects and nematodes was investigated. A total of six TIs were tested, including three known soybean trypsin inhibitors (KTI1, KTI2 and KTI3) and three genes encoding novel inhibitors identified in soybean (KTI5, KTI7, and BBI5). Their functional role was further examined by overexpression of the individual TI genes in soybean and Arabidopsis. The endogenous expression patterns of these TI genes varied among soybean tissues, including leaf, stem, seed, and root. In vitro enzyme inhibitory assays showed significant increase in trypsin and chymotrypsin inhibitory activities in both transgenic soybean and Arabidopsis. Detached leaf-punch feeding bioassays detected significant reduction in corn earworm (Helicoverpa zea) larval weight when larvae fed on transgenic soybean and Arabidopsis lines, with the greatest reduction observed in KTI7 and BBI5 overexpressing lines. Whole soybean plant greenhouse feeding bioassays with H. zea on KTI7 and BBI5 overexpressing lines resulted in significantly reduced leaf defoliation compared to non-transgenic plants. However, bioassays of KTI7 and BBI5 overexpressing lines with soybean cyst nematode (SCN, Heterodera glycines) showed no differences in SCN female index between transgenic and non-transgenic control plants. There were no significant differences in growth and productivity between transgenic and non-transgenic plants grown in the absence of herbivores to full maturity under greenhouse conditions. The present study provides further insight into the potential applications of TI genes for insect resistance improvement in plants.

Keywords: corn earworm; overexpression; soybean cyst nematode; tissue-specific promoter; transgenic soybean and Arabidopsis; trypsin and chymotrypsin enzymes inhibition; trypsin inhibitors.

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Conflict of interest statement

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Figures

Figure 1
Figure 1
Endogenous expression patterns of the individual trypsin inhibitor genes (KTI1, KTI2, KTI3, KTI5, KTI7, BBI5) in different plant tissues of leaves, stems, and roots (six-week-old and seeds (30 days after flowering) wild-type soybean plants. The relative levels of transcripts were normalized to soybean ubiquitin gene (GmUBI3). Bars represent mean values of six biological replicates (plants) ± standard error. Bars with different letters are significantly different at p < 0.05 as tested by one-way analysis of variance followed by a Fisher’s least significant difference.
Figure 2
Figure 2
Expression analysis of the individual trypsin inhibitor gene construct in leaves of six-week-old T3 transgenic and non-transgenic wild-type (WT) soybean plants. The relative expression of transgene (A) and total gene (B) corresponding to KTI1, KTI2, KTI3, KTI5, KTI7, and BBI5 under the control of 35S CaMV promoter. The relative expression of transgene (C) and total gene (D) corresponding to KTI1, KTI2, KTI3, KTI5, KTI7, and BBI5 under the control of rbcS-SRS4 promoter. The relative levels of transcripts were normalized to soybean ubiquitin gene (GmUBI3). Bars represent mean values of six biological replicates (plants) per each independent line (L1, L2, L3) ± standard error. Each TI gene was statistically analyzed separately. Bars with different letters are significantly different at p < 0.05 as tested by one-way analysis of variance followed by a Fisher’s least significant difference.
Figure 3
Figure 3
Enzyme inhibitory activity of the individual trypsin inhibitor gene (KTI1, KTI2, KTI3, KTI5, KTI7, BBI5) construct in leaves of six-week-old T3 transgenic (A) Arabidopsis plants under the control of 35S CaMV promoter, (B) Soybean plants under the control of 35S CaMV promoter, and (C) Soybean plants under the control of rbcS-SRS4 promoter. Percentage of trypsin and chymotrypsin inhibition activities in leaf total protein extract from transgenic plants with corresponding each type of gene relative to that of non-transgenic wild-type plants. Bars represent mean values of six biological replicates (plants) per each independent line (L1, L2, L3) ± standard error. Bars with different letters are significantly different at p < 0.05 as tested by one-way analysis of variance followed by a Fisher’s least significant difference. Bars with lowercase letters represent trypsin group and uppercase letters represent chymotrypsin group.
Figure 4
Figure 4
Detached leaf-punch bioassay in six-week-old T3 transgenic soybean (under the control of CaMV 35S promoter) using corn earworm (Helicoverpa zea) neonate larvae. (A) Wild-type and transgenic soybean plants detached-leaf punches before corn earworm larval inoculation. (B) Leaf punches were inoculated with corn earworm neonate larvae and eight days after feeding. (C) Larval size after eight days of feeding. (D) Average larval weight after eight days of feeding in WT and transgenic lines (L1, L2, and L3). Bars represent mean values of six biological replicates ± standard error. Bars with different letters are significantly different at p < 0.05 as tested by one-way analysis of variance followed by a Fisher’s least significant difference.
Figure 5
Figure 5
Detached leaf-punch bioassay in six-week-old T3 transgenic soybean (under the control of rbcS-SRS4 promoter) using corn earworm (Helicoverpa zea) neonate larvae. (A) Detached leaf-punches from wild-type and transgenic plants. (B) Neonate corn earworm larvae were inoculated in each well and showing after eight days feeding. (C) Representative corn earworm larval size after eight days of feeding. (D) Average larval weight after feeding in WT and transgenic lines (L1, L2, and L3). Bars represent mean values of six biological replicates ± standard error. Bars with different letters are significantly different at p < 0.05 as tested by one-way analysis of variance followed by a Fisher’s least significant difference.
Figure 6
Figure 6
Whole plant feeding bioassay using six-week-old T3 transgenic soybean plants that contain the 35S cauliflower mosaic virus promoter driving the expression of individual trypsin inhibitor genes (KTI7 and BBI5) grown under greenhouse conditions with corn earworm (Helicoverpa zea) second instar larvae. (A) Representative of transgenic and non-transgenic wild-type soybean plants in a polyester-mesh cage. (B) Ten larvae were added to each plant. (C) Ten days after larvae feeding. (D) Representative defoliated wild-type and transgenic plants. (E) Leaf defoliation rate after 10 days of larvae feeding of wild-type (WT) and transgenic lines. Bars represent mean values of six biological replicates (plants) per each independent line (L1, L2, L3) ± standard error. Bars with different letters are significantly different at p < 0.05 as tested by one-way analysis of variance followed by a Fisher’s least significant difference.
Figure 7
Figure 7
Whole plant feeding bioassay using six-week-old T3 transgenic soybean plants that contain the rbcS-SRS4 promoter driving the expression of trypsin inhibitor genes (KTI7 and BBI5) grown under greenhouse conditions with corn earworm (Helicoverpa zea) second instar larvae. (A) Representative of transgenic and non-transgenic wild-type soybean plants in a polyester-mesh cage. (B) Ten larvae were added to each plant. (C) Ten days after larvae feeding. (D) Representative defoliated wild-type and transgenic plants. (E) Leaf defoliation rate after 10 days of larvae feeding of wild-type (WT) and transgenic lines. Bars represent mean values of six biological replicates (plants) per each independent line (L1, L2, L3) ± standard error. Bars with different letters are significantly different at p < 0.05 as tested by one-way analysis of variance followed by a Fisher’s least significant difference.
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This research was funded by the Tennessee Soybean Promotion Board.

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