Kunitz Trypsin Inhibitor Research Articles

A Novel Kunitz Trypsin Inhibitor from Enterolobium gummiferum Seeds Exhibits Antibiofilm Properties against Pathogenic Yeasts.

Plant peptidase inhibitors play crucial roles in plant defence mechanisms and physiological processes. In this study, we isolated and characterised a Kunitz trypsin inhibitor from Enterolobium gummiferum seeds named EgPI (E. gummiferum peptidase inhibitor). The purification process involved two chromatography steps using size exclusion and hydrophobic resins, resulting in high purity and yield. EgPI appeared as a single band of ~20 kDa in SDS-PAGE. Under reducing conditions, the inhibitor exhibited two polypeptide chains, with 15 and 5 kDa. Functional characterisation revealed that EgPI displayed an inhibition stoichiometry of 1:1 against trypsin, with a dissociation constant of 8.4 × 10-9 mol·L-1. The amino-terminal sequencing of EgPI revealed the homology with Kunitz inhibitors. Circular dichroism analysis provided insights into the secondary structure of EgPI, which displayed the signature typical of Kunitz inhibitors. Stability studies demonstrated that EgPI maintained the secondary structure necessary to exhibit its inhibitory activity up to 70 °C and over a pH range from 2 to 8. Microbiological screening revealed that EgPI has antibiofilm properties against pathogenic yeasts at 1.125 μmol·L-1, and EgPI reduced C. albicans biofilm formation by 82.7%. The high affinity of EgPI for trypsin suggests potential applications in various fields. Furthermore, its antibiofilm properties recommended its usefulness in agriculture and antimicrobial therapy research, highlighting the practical implications of our research.

Validation of Molecular Markers for Low Kunitz Trypsin Inhibitor Content in European Soybean (Glycine max L. Merr.) Germplasm.

Trypsin inhibitors (TI) in raw soybean grain, mainly represented by the Kunitz trypsin inhibitor protein (KTI), prevent the normal activity of the digestive enzymes trypsin and chymotrypsin in humans and monogastric livestock. The inactivation of TI is achieved through costly and time-consuming heat treatment. Thermal processing also impairs the solubility and availability of the soybean grain protein. Therefore, the genetic elimination of KTI has been proposed as a suitable alternative to heat treatment. The aim of this study was to screen the collection of European soybean cultivars with six genetic markers (one SSR marker and five SNP markers) previously proposed as tightly linked to the KTI3 gene encoding the major Kunitz trypsin inhibitor seed protein of soybean and validate their usability for marker-assisted selection (MAS). The six markers were validated on a subset of 38 cultivars with wide variability in KTI content and in the F2 and F3:5 progenies of two crosses between the known high- and low-KTI cultivars. Three genetic markers (SSR Satt228 and two SNP markers, Gm08_45317135_T/G and Gm08_45541906_A/C) were significantly associated with KTI content in a subset of 38 cultivars. Low-KTI alleles were detected in both low- and high-KTI genotypes and vice versa, high-KTI alleles were found in both high- and low-KTI genotypes, indicating a tight but not perfect association of these markers with the KTI3 gene. The genetic marker SSR Satt228 showed a significant association with KTI content in the F2 progeny, while the SNP markers Gm08_45317135_T/G and Gm08_45541906_A/C allowed significant discrimination between progeny with high- vs. low-KTI progenies in the F3:5 generation. These three markers could be applied in MAS for low-KTI content but not without the additional phenotyping step to extract the desired low-KTI genotypes.

Overexpression of RpKTI2 from Robinia pseudoacacia Affects the Photosynthetic Physiology and Endogenous Hormones of Tobacco.

Kunitz trypsin inhibitor genes play important roles in stress resistance. In this study, we investigated RpKTI2 cloned from Robinia pseudoacacia and its effect on tobacco. RpKTI2 was introduced into the tobacco cultivar NC89 using Agrobacterium-mediated transformation. Six RpKTI2-overexpressing lines were obtained. Transgenic and wild-type tobacco plants were then compared for photosynthetic characteristics and endogenous hormone levels. Transgenic tobacco showed minor changes in chlorophyll content, fluorescence, and photosynthetic functions. However, the maximum photochemical efficiency (Fv/Fm) increased significantly while intercellular CO2 concentration (Ci) decreased significantly. Stomatal size and hormone content (indole-3-acetic acid, zeatin riboside, gibberellin, and indole-3-propionic acid) were reduced, while brassinosteroid content increased. Random forest regression revealed that RpKTI2 overexpression had the biggest impact on carotenoid content, initial fluorescence, Ci, stomatal area, and indole-3-acetic acid. Overall, RpKTI2 overexpression minimally affected chlorophyll synthesis and photosynthetic system characteristics but influenced stomatal development and likely enhanced the antioxidant capacity of tobacco. These findings provide a basis for future in-depth research on RpKTI2.

Development of a green soybean line with a green cotyledon and a tetra null genotype for P34, lectin, Kunitz trypsin inhibitor, and lipoxygenase proteins

Development of a green soybean line with a green cotyledon and a tetra null genotype for P34, lectin, Kunitz trypsin inhibitor, and lipoxygenase proteins

Enzymatic and Algebraic Methodology to Determine the Contents of Kunitz and Bowman-Birk Inhibitors and Their Contributions to Total Trypsin or Chymotrypsin Inhibition in Soybeans.

Soybeans are the number one source of plant proteins for food and feed, but the natural presence of protein protease inhibitors (PIs), namely, the Kunitz trypsin inhibitor (KTI) and the Bowman-Birk inhibitor (BBI), exerts antinutritional effects. This communication describes a new methodology for simultaneously quantitating all parameters of PIs in soybeans. It consists of seven steps and featured enzymatically measuring trypsin and chymotrypsin inhibitory activities, respectively, and subsequently determining the contents of reactive KTI and BBI and the contributions of each toward total PI mass and total trypsin or chymotrypsin inhibition by solving a proposed system of linear equations with two variables (C = dB + eK and T = xB + yK). This enzymatic and algebraic (EA) methodology was based on differential inhibitions of KTI and BBI toward trypsin and chymotrypsin and validated by applications to a series of mixtures of purified KTI and BBI, two KTI-null and two conventional soybeans, and by sodium dodecyl sulfate polyacrylamide gel electrophoresis. The EA methodology allowed calculations of PI composition and the contributions of individual inhibitors toward total inhibition with ease. It was first found that although BBI constituted only about 30% of the total PI mass in conventional raw soybeans, it contributed about 80% toward total chymotrypsin inhibitor activity and about 45% toward trypsin inhibitor activity. Therefore, BBI caused more total protease inhibitions than those of KTI. Furthermore, the so-called KTI-null soybean mutants still contained measurable KTI content and thus should be named KTI-low soybeans.

246 The effect of trypsin inhibitor unit concentrations from raw soybeans in grower pig performance and protein digestibility

Abstract Soybeans contain two major protease inhibitors, Kunitz trypsin and Bowman-Birk protease inhibitors, both of which are anti-nutritional components that can be directly linked to a reduction in amino acid bioavailability and growth performance in swine. Therefore, the objective of this study was to determine the level at which trypsin inhibitor units (TIU, expressed as TIU/mg of complete feed), coming from raw soybeans, in pig diets result in reduction in growth performance and digestibility. This study used 45 grower gilts initial body weight (BW) = 39.7 ± 2.1 kg] that were individually penned and fed one of five dietary TIU treatments (n = 9 pigs/trt) in a complete randomized design. Diets were representative of a commercial corn-soybean meal diet and formulated to have ranging TIU/mg of complete feed of 0.89, 1.77, 3.55, 6.99, and 11.54 TIU/mg utilizing raw ground soybeans and soybean meal at incremental levels. Analyzed diets contained 0.99, 2.23, 3.07, 6.49, and 9.38 active TIU/mg. Diets were balanced to the same grams of SID Lys:ME of 2.85 and NDF concentration of 7.4% utilizing soybean oil and soybean hulls, respectively. Individual feed intake was recorded throughout, and pig BW were collected on d 0, 21, and 27 to calculate average daily gain (ADG), average daily feed intake (ADFI), and feed efficiency gain to feed ratio (G:F). All pigs were placed into metabolism crates on d 21 for 6 d to determine apparent total tract digestibility (ATTD) of nitrogen (N), N excretion and N balance. Growth performance and ATTD data were analyzed with pig as the experimental unit and diet TIU level as a fixed effect using ANOVA, linear, and quadratic contrast models. Overall, ADG decreased as diet TIU increased (1.07, 1.03, 1.02, 0.91, and 0.83 kg/d, respectively, linear P < 0.001). However, no differences in ADFI (P > 0.10) were reported and this resulted in a decrease in G:F (0.48, 0.45, 0.43, 0.41, 0.36, respectively, linear P < 0.001) as dietary TIU concentrations increased from 0.99 to 9.38 TIU/mg. Compared with 0.99 TIU/mg, pigs fed 9.38 TIU/mg had a 9.32% reduction in final BW (68.7 vs. 62.3 kg, linear P < 0.001). Apparent total tract digestibility of N decreased when pigs were fed 9.38 TIU/mg compared with 0.99 and 2.23 TIU/mg (83.26% vs. 90.29% and 89.57%, respectively, P < 0.001). In conclusion, as total trypsin inhibitor units increase in complete feed of grower pigs, digestibility of protein, growth rates and feed efficiency are reduced in a linear manner.

Proteomic Analysis and Immunoprofiling of Persian Horned Viper Venom, Pseudocerastes Persicus, from Central Part of Iran.

Numerous species of venomous snakes of medical importance exist in Iran. Pseudocerastes persicus (P. persicus), one of the medically important snakes, also called the Persian horned viper, has a geographical spread that extends to the east, southwest, and central areas of Iran and is endemic across the wider region. As a result, this species is responsible for many snakebite occurrences. Venom from P. persicus found in the central province of Semnan contains phospholipase A2 and L-amino acid oxidase activities, and high toxic potency. The venom was fractionated by reverse-phase high-performance liquid chromatography (HPLC) and analyzed by Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), Western blotting and two-dimensional electrophoresis. Using liquid chromatography with tandem mass spectrometry (LC-MS/MS), a range of components were identified, consistent with the biochemical and toxicological properties of the venom. Proteins identified from 2D electrophoresis and shotgun methods included metallo- and serine proteases, phospholipases, oxidases, and Kunitz trypsin inhibitors, along with many other components at lower qualitative abundance. This study provides a more detailed understanding of the protein profile of Iranian P. persicus venom, which can be effective in the production of an effective antidote against it. The analysis of the resulting data shows that there is a wide range of proteins in the venom of the Persian horned viper. This information can provide a better understanding of how venom is neutralized by polyclonal antivenom. Considering the wide presence of this snake and its related species in Iran and surrounding countries, knowing the venom protein profile of this family can be of great support to antivenom producers such as Razi Vaccine & Serum Research Institute in the preparation of regional antivenoms.

Exploring peptidomes of by-products generated during chhurpi production using Lactobacillus delbrueckii WS4 for identification of novel bioactive peptides.

The considerable value of whey is evident from its significant potential applications and contributions to the functional food and nutraceutical market. The by-products were individually obtained during functional chhurpi and novel soy chhurpi cheese production using defined lactic acid bacterial strains of Sikkim Himalaya's traditional chhurpi. Hydrolysis of substrate proteins by starter proteinases resulted in a comparable peptide content in whey and soy whey which was associated with antioxidant and ACE inhibition potential. Peptidome analysis of Lactobacillus delbrueckii WS4 whey and soy whey revealed the presence of several bioactive peptides including the multifunctional peptides PVVVPPFLQPE and YQEPVLGPVRGPFPIIV. In silico analyses predicted the antihypertensive potential of whey and soy whey peptides with strong binding affinity for ACE active sites. QSAR models predicted the highest ACE inhibition potential (IC50) for the β-casein-derived decapeptide PVRGPFPIIV (0.95 μM) and the Kunitz trypsin inhibitor protein-derived nonapeptide KNKPLVVQF (16.64 μM). Chhurpi whey and soy whey can be explored as a valuable source of diverse and novel bioactive peptides for applications in designer functional foods development.

Pulsed electric field treatment of soymilk: Impact on Kunitz trypsin inhibitor allergenicity, antinutritional factor, and aroma characteristics.

Allergens, antinutritional factors, and lipoxygenase (LOX) enzyme present in soymilk limit its consumption as vegan milk. Therefore, the present study focuses on reducing these limiting factors using pulsed electric field (PEF) treatment. In this regard, 20-40kV/cm electric field was applied to soymilk for the effective treatment periods of 450, 1350, and 2250ms. After the treatment, a reduction in pH (6.60±0.10 to 6.47±0.12) and an increase in the conductivity (173.03±0.40 to 177.33±0.72 µS) were observed. Furthermore, FTIR (Fourier Transform Infrared Spectroscopy), UV (Ultra Violet)intrinsic spectra, and CD (Circular Dichroism)spectra (α-helix reduction and β-sheet increase) data indicated mild structural changes in the proteins of soymilk. As a result, PEF treatment reduced the soymilk allergenicity (67.33±20.48%), LOX activity (69.45±9.38%), and trypsin inhibitor activity (75.61±4.04%). Apart from that, the color, viscosity, and volatiles of soymilk also had significant changes due to PEF treatment. The aroma changes in PEF-treated soymilk were highly influenced by two major principal component (PC1 & PC2) groups and they accounted for about 70% of the aroma variations. However, these changes were mild and did not induce any off-flavors and the treatment remained effective against the quality hazards like allergens, antinutritional factors, and LOX enzyme. PRACTICAL APPLICATION: PEF treatment of soymilk reduces the possible allergic reactions in human body at least by 30%. Further, it reduces the antinutritional factor and off-odor inducing compounds. Therefore, the PEF treatment can be used in industries as a pre-treatment to produce allergen and antinutritional compounds free protein isolates from soybeans.

EVALUATION OF SOYBEAN FLOURS OBTAINED FROM WHOLE AND SHELLED GRAINS SUBJECTED TO HEAT TREATMENT

This study aimed to obtain and characterize soy flours from whole (WG) and shelled grains (SG) of the conventional cultivar Vmax and special cultivars used for human consumption, BRS 267 and BRS 257. For lipoxygenase enzyme inactivation, the WGs and SGs of BRS 267 and Vmax were bleached at 98 °C for 5 min, roasted at 150 °C for 30 min, milled and finally sieved through a size 40 mesh to obtain the flours. These flours were analyzed for proteins, lipids, isoflavones, Kunitz trypsin inhibitor, their nitrogen solubility index and their protein dispersibility index. The grain heat treatments caused a reduction in protein solubility, an increase in aglycone isoflavones and an 80% reduction in levels of the Kunitz trypsin inhibitor. Flours from bleached WGs were brown in color. Flours from BRS 267 had higher protein levels, while those from the cultivar Vmax had the highest lipid content. Flours from BRS 257 had greater protein solubility. Thus, varieties of special soybeans, genetically enhanced for human consumption, can be processed for better quality soy foods, promoting their acceptance.

Identification of a Novel KTi-1 Allele Associated with Reduced Trypsin Inhibitor Activity in Soybean Accessions

Trypsin inhibitors (TIs) in soybean seeds reduce the availability of processed soybean foods and animal feed. This study aimed to evaluate the trypsin inhibitor activity (TIA) in 999 Korean soybean accessions and conduct molecular characterization of soybean accessions with low TIA. TIA was evaluated using colorimetric analysis through a substrate–enzyme reaction. The average TIA of the 999 soybean accessions was 90.31%, ranging from 43.70% to 99.51%. Kunitz trypsin inhibitor 3 (KTi-3) gene target sequencing analysis was performed on seven soybean germplasm accessions (IT105782, IT170889, IT273590, IT274513, IT274515, IT276197, and IT022891) showing less than 60% TIA. Four soybean accessions (IT274513, IT274515, IT276197, and IT022891) exhibited the same mutations (a G-to-T transversion and an AG deletion) in the KTi-3 gene (Glyma.08g341500) in PI542044. In addition, whole-genome re-sequencing was performed on three soybean accessions with no mutations in the KTi-3 gene. Compared with the reference soybean genome, an in-frame insertion and five missense mutations were identified in the coding sequencing of the KTi-1 gene (Glyma.01g09500) in IT105782. A RT-qPCR analysis showed that the mRNA expression level of KTi-1 was reduced by approximately 17% in IT105782 during seed development. In this study, we identified a previously unreported mutation in the KTi-1 gene and developed a KASP marker using this new KTi-1 variant.

Protein Concentration Affects the Food Allergen γ-Conglutin Uptake and Bacteria-Induced Cytokine Production in Dendritic Cells.

γ-Conglutin (γ-C) from lupin seeds has been identified as a potent allergen with cross reactivity to peanuts. Here, we investigated how γ-C affected the response in bone marrow-derived dendritic cells (DCs) to bacterial stimuli. γ-C enhanced L. acidophilus NCFM (LaNCFM)-induced IL-12, IL-10, and IL-23 dose-dependently. In contrast, together with E. coli Nissle or LPS, γ-C reduced the production of IL-12 but not of IL-23 and IL-10. Enzyme-hydrolyzed γ-C also enhanced LaNCFM-induced IL-12 and IL-23 production. All preparations induced ROS production in the DCs. The mannose receptor ligands mannan and dextran and the clathrin inhibitor monodansylcadaverine partly inhibited the endocytosis of γ-C. Kunitz trypsin inhibitor and the scavenger receptor ligand polyG also enhanced LaNCFM-induced IL-12, indicating the involvement of receptors other than C-type lectin receptors. The endocytosis of labeled γ-C increased dose-dependently by addition of unlabeled γ-C, which coincided with γ-C's tendency to aggregate. Taken together, γ-C aggregation affects endocytosis and affects the cytokine production induced by gram-positive and gram-negative bacteria differently. We suggest that γ-C is taken up by the same mechanism as other food proteins but due to aggregation is present in higher concentration in the DCs. This could influence the resulting T-cell response in a microbial stimuli-dependent way.

High pressure processing of whole soymilk: Effect on allergenicity, anti-nutritional factor, lipoxygenase activity and E-nose-aroma characteristics

Soymilk was subjected to high pressure processing (HPP) at different pressure levels (300–600 MPa) and time (5, 15 and 25 min) intervals. After the HPP treatment, a mild reduction was observed in soymilk pH and conductivity values; however, a sudden surge in the protein content (17.34±0.10 mg/ml) was recorded at 600 MPa (5 min) treatment. When soymilk proteins were evaluated for their structural changes, FTIR (amide regions), CD spectra, and UV inflorescence showed the positive responses and as the result, soy Kunitz trypsin inhibitor (SKTI) antigen binding ability reduced from 100% to 44.39±8.50% at 300 MPa (5 min). However, prolong exposure at 300 MPa had a negative effect on allergenicity. Nevertheless, HPP treatment reduced the lipoxygenase (100.00% to 68.13±12.80%) and trypsin inhibitor (100.00% to 91.30±1.45%) activities. In addition, the treatment also slightly altered the viscosity, whiteness, and volatile aroma components of soymilk after HPP treatment. Principal component analysis revealed that among 22 selected volatiles, only those belong to PC1 and PC2 caused 68% variance in aroma changes. Overall, HPP treatment reduced allergenicity, trypsin inhibitor activity and lipoxygenase activity without modifying the soymilk nature. Further, it also reduced the beany flavor inducing hexanal volatile content after the treatment.

StMLP1, as a Kunitz trypsin inhibitor, enhances potato resistance and specifically expresses in vascular bundles during Ralstonia solanacearum infection.

Miraculin-like proteins (MLPs), members of the Kunitz trypsin inhibitor (KTI) family that are present in various plants, have been discovered to have a role in defending plants against pathogens. In this study, we identified a gene StMLP1 in potato that belongs to the KTI family. We found that the expression of StMLP1 gradually increases during Ralstonia solanacearum (R. solanacearum) infection. We characterized the promoter of StMLP1 as an inducible promoter that can be triggered by R. solanacearum and as a tissue-specific promoter with specificity for vascular bundle expression. Our findings demonstrate that StMLP1 exhibits trypsin inhibitor activity, and that its signal peptide is essential for proper localization and function. Overexpression of StMLP1 in potato can enhance the resistance to R. solanacearum. Inhibiting the expression of StMLP1 during infection accelerated the infection by R. solanacearum to a certain extent. In addition, the RNA-seq results of the overexpression-StMLP1 lines indicated that StMLP1 was involved in potato immunity. All these findings in our study reveal that StMLP1 functions as a positive regulator that is induced and specifically expressed in vascular bundles in response to R. solanacearum infection.

Purification of soybean Kunitz trypsin inhibitor and the mechanism of its passivation by lysine and disulfide bond modifications

Soybean trypsin inhibitor（STI）was selectively recovered from soybean whey, a by-product of soybean protein production, by ammonium sulfate salting and the STI was purified up to 5.46-fold (Compared to STI in soybean whey). After purification with a Superdex 75 increase gel filtration preloader column, a 91.5% electrophoretically pure soybean Kunitz trypsin inhibitor（SKTI）was obtained by optical density analysis with a purification ploidy of 33.32-fold and a specific activity of 1072.9 U/mg, and a single band protein with a molecular weight of 20 kDa on SDS-PAGE. SKTI's 85.65% inhibitory activity was maintained after heating at 80 °C for 30 min, and there was no significant change in inhibitory activity in the range of pH 3–10. The secondary structure of SKTI did not change with temperature and pH showing good stability. By chemical bond modification of SKTI, the activity of SKTI was found to have an important connection with lysine and disulfide bond. This study set up a process for the rapid preparation of STI by salinization and the related properties of SKTI was explored to provide more potential for the treatment of soy protein production waste and the application of STI in the food field in the future.

Comparative Binding Affinity Analysis of Soybean Meal Bowman-Birk and Kunitz Trypsin Inhibitors in Interactions with Animal Serine Proteases

Comparative Binding Affinity Analysis of Soybean Meal Bowman-Birk and Kunitz Trypsin Inhibitors in Interactions with Animal Serine Proteases

Preventing Enzymatic Damage to Hides by Timely Inhibition of Trypsin Activity with Soybean Flour during Bating Process

Enzymatic bating is important for producing clean, soft, and high-yield leather. However, it is prone to cause damage to hide collagen fibers and result in loose grain or damaged grain surface due to the longer enzymatic hydrolysis time of the grain layer than the middle layer caused by the slow mass transfer and the rapid hydrolysis reaction of the bating agent in hide. Considering trypsin is the most common bating agent, soybean flour (SF) that contains environmental friendly Kunitz trypsin inhibitor (KTI) was used after delimed hides were treated with trypsin for a certain time to opportunely inhibit its activity in the grain and avoid bating defects. The fluorescence micrographs of hides bated with fluorescently labelled trypsin and KTI showed that KTI could cover only the surface layers when it was added after bating for a certain time, whereas trypsin could penetrate the hide completely. When sufficient SF was added to the bating float at 2 h, the damage to hide collagen caused by trypsin was effectively reduced. In particular, the grain surface remained intact after bating for 8 h. The decrease in the strength of leather caused by bating was also reduced. In addition, the usage of SF scarcely affected the fiber dispersion of the middle layer, thereby ensuring the softness of leather. In summary, opportunely inhibiting the activity of trypsin in the grain could prevent hide damage and improve the quality of leather.

Label-Free Quantification by Liquid Chromatography-Tandem Mass Spectrometry of the Kunitz Inhibitor of Trypsin KTI3 in Soy Products.

The greater awareness of consumers regarding the sustainability of food chains has shifted part of the consumption from animal protein sources to vegetable sources. Among these, of relevance both for human food use and for animal feed, is soy. However, its high protein content is unfortunately accompanied by the presence of antinutritional factors, including Kunitz's trypsin inhibitor (KTI). Now there are few analytical methods available for its direct quantification, as the inhibitory activity against trypsin is generically measured, which however can be given by many other molecules and undergo numerous interferences. Therefore, in this work, a direct label-free liquid chromatography-mass spectrometry (LC-MS) method for the identification and quantification of trypsin Kunitz inhibitor KTI3 in soybean and derivative products has been developed. The method is based on the identification and quantification of a marker peptide, specific for the protein of interest. Quantification is achieved with an external calibration curve in the matrix, and the limit of detection and the limit of quantification of the method are 0.75 and 2.51 μg/g, respectively. The results of the LC-MS method were also compared with trypsin inhibition measured spectrophotometrically, highlighting the complementarity of these two different pieces of information.

Development of new mutant alleles and markers for KTI1 and KTI3 via CRISPR/Cas9-mediated mutagenesis to reduce trypsin inhibitor content and activity in soybean seeds.

The digestibility of soybean meal can be severely impacted by trypsin inhibitor (TI), one of the most abundant anti-nutritional factors present in soybean seeds. TI can restrain the function of trypsin, a critical enzyme that breaks down proteins in the digestive tract. Soybean accessions with low TI content have been identified. However, it is challenging to breed the low TI trait into elite cultivars due to a lack of molecular markers associated with low TI traits. We identified Kunitz trypsin inhibitor 1 (KTI1, Gm01g095000) and KTI3 (Gm08g341500) as two seed-specific TI genes. Mutant kti1 and kti3 alleles carrying small deletions or insertions within the gene open reading frames were created in the soybean cultivar Glycine max cv. Williams 82 (WM82) using the CRISPR/Cas9-mediated genome editing approach. The KTI content and TI activity both remarkably reduced in kti1/3 mutants compared to the WM82 seeds. There was no significant difference in terms of plant growth or maturity days of kti1/3 transgenic and WM82 plants in greenhouse condition. We further identified a T1 line, #5-26, that carried double homozygous kti1/3 mutant alleles, but not the Cas9 transgene. Based on the sequences of kti1/3 mutant alleles in #5-26, we developed markers to co-select for these mutant alleles by using a gel-electrophoresis-free method. The kti1/3 mutant soybean line and associated selection markers will assist in accelerating the introduction of low TI trait into elite soybean cultivars in the future.

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

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.