Quantitative determination of trypsin inhibitor as a breeding marker in maize varieties with different resistance to fungal diseases

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Abstract


Determination of plant resistance to fungal pathogens is an important breeding component. Thus, the study of protein profiles from corn kernels (13 genotypes) revealed a constitutively pronounced 14 kDa protein, trypsin inhibitor (TI), which is present at a relatively high level of concentration in seven Aspergíllus flavus - resistant maize lines, but at low concentrations or is absent in six sensitive lines. The 14 kDa trypsin inhibitor (TI) also showed antifungal activity against other mycotoxicogenic species. In this regard, the task was to determine the content of TI in varieties of maize with known properties, resistance, or sensitivity to such fungal pathogens of maize as head smut, common smut, and Fusarium stalk rot. According to the data obtained, the content of TI varies in different varieties and can vary by 4 times. However, in disease-resistant varieties its content is increased, which may be the primary marker of resistance of the variety to fungal pathogens.


Introduction Plants are exposed to a large number of infectious pathogenic fungi. For maize, common smut, head smut, stem rot, fusarium disease are the most common fungal pathogens in the Russian Federation. While in countries with a hot and arid climate, pathogens of the genus Aspergillus are most common, the effects of which are most fully studied. Thus, a study of protein profiles from maize grains (13 genotypes) revealed a constitutively expressed 14 kDa protein, a trypsin inhibitor (TI), which was present in relatively high concentrations in seven maize A. flavus resistant lines, but in low concentrations or was absent altogether in six sensitive lines [1]. 14 kDa TI also showed antifungal activity against other mycotoxicogenic species [2]. Although plants do not have an immune system, they use protective mechanisms, including the synthesis of low molecular weight compounds, proteins and peptides with antifungal activity. To date, 13 classes of antifungal proteins are known, among them PR-1 proteins, 1,3-β-glucanases, chitinases, chitin-binding proteins, thaumatin-like proteins, defensins, cyclophilin-like proteins, proteinase inhibitors and other proteins [3]. Proteinase inhibitors are an integral part of protection system against pests and diseases [4]. It was shown that pathogenic fungi Fusarium and Helminthosporium secrete enzymes into the environment that break down protein and synthetic substrates. Different types of fungi are characterized by unequal activity of extracellular proteinases. The high content of extracellular proteinase inhibitors of the pathogenic fungi Fusarium and Helminthosporium in the seeds of pea, buckwheat, and corn can be one of the important factors ensuring the resistance of these crops to root rot [5]. Similarly, TI content of 14 kDa in maize seeds was shown to correlate with resistance to A. flavus disease [3] and, therefore, aflatoxin accumulation. In addition to trypsin-inhibiting activity, TI from 14 kDa maize was shown to have alpha-amylase activity, and, when added to the nutrient medium, it suppressed growth of conidia and growth of phytopathogen hyphae Aspergillus flavus, Asp. paprasiticus, F. moniliforme, and also showed antifungal activity against other mycotoxicogenic species [2, 6]. We held experiments to study TI content of 14 kDa in maize samples with known properties, resistance or sensitivity to fungal pathogens, such as head smut (pathogen - Sphacelotheca reiliana), common smut (pathogen - Ustilago maydis, U. zeae) and stalk rot of maize (pathogen - Fusarium moniliforme fungus). Materials and methods Object of study. Maize varieties with known properties of resistance (sensitivity) to fungal pathogens (head smut, common smut and stalk rot) were provided from Russian Research Institute of Maize in Pyatigorsk. Six varieties resistant to fungal infections and 12 varieties sensitive to fungal pathogens were investigated. Sample preparation. The seeds were ground, each maize sample (10 g) was extracted with 20 ml of a 0.2 M NaCl solution, stirred for 2.5 hours, then filtered, and approximately 15 ml of extract was obtained. To each sample, 6 g of ammonium sulfate (60% concentration) was added, left in the refrigerator for 18 hours overnight. Then it was centrifuged for 10 minutes, the precipitate was dissolved in 5 ml of buffer (20 mM Tris-HCl, 0.15 M NaCl, pH 6 ,8) and used for application on trypsin agarose or on HPLC. Trypsin agarose affinity chromatography. An affinity sorbent, trypsin agarose, was chosen as the isolation method [7]. Trypsin agarose was obtained by immobilizing TPCK trypsin on BrCN agarose. To quantify the method, a standard sample of 14 kDI TI, 200 μg in 1 ml of equilibration buffer was used. Samples of 1.2 ml of each extract were applied to a trypsin-agarose column (4 ml) at a rate of 0.5 ml/min. After washing with equilibration buffer (10 mM Tris-HCl, 0.3 M NaCl, pH 7.0), an elution with 0.15 M acetic acid was performed. HPLC (hydrophobic chromatography). Sample analysis was performed on an RP-304 (250´4.6) Bio Rad column. The following conditions were used: 3' - 0% CH3CN; 40' - 100% CH3CN; 50'- 100% CH3CN, 65' - 0% CH3CN. 200 μg of 14 kDI TI was previously used as a standard. Electrophoresis in page. SDS-PAGE 15% gel electrophoresis was used to analyze samples. Results 6 smut-resistant maize varieties and 12 varieties susceptible to smut and stalk rot were studied. The samples of maize grains with known properties of resistance (sensitivity) to fungal pathogens were provided from Russian Research Institute of Maize in Pyatigorsk. Peak areas obtained by trypsin-agarose chromatography were calculated by mathematical methods. The amount of 14 kDa TI was determined on the basis of calibration; for this, a sample containing 200 μg TI was used (Fig. 1). A typical chromatography picture of extracts of maize kernels on trypsin agarose is shown in Fig. 2. Fig. 1. Standard chromatography curve on trypsin-agarose. In this case, 200 μg of 14 kDa TI is applied for calibration Fig. 2. Typical chromatography curve of maize grains extract on trypsin-agarose: second peak - TI elution Fig. 3. Typical elution profile of maize grain extract on HPLC Fig. 4. 14 kDa trypsin inhibitor as a standard in HPLC (200 μg) According to HPLC, 4 extracts of different maize varieties were studied (Fig. 3, 4). The following results were obtained: 1st sample - 4.14% 14 kDa TI (resistant to Sphacelotheca, R); 2nd sample - 0.09% 14 kDa TI (susceptible to Sphacelotheca, S); 3rd sample - 3.40% 14 kDa TI (resistant to Ustilago, R); 4th sample - 3.60% 14 kDa TI (susceptible to Ustilago, S). Subsequently, an analysis of extracts of different varieties of maize grains was carried out (R - resistant varieties, S - susceptible) using affinity chromatography on trypsin agarose (Table 1). An additional 18 samples were examined. A standard sample containing 200 μg of 14 kDI TI was used for calibration. Results of screening maize varieties by quantity of 14 kDa trypsin inhibitor Table 1 Elution peak area Calculated TI per 1 g of corn grains Resistance to fungal diseases 11.3 290 mcg R Ustilago (580/14) 6.85 174 mcg VR Sphacelotheca (4716/11) 8.94 228 mcg R Ustilago (3867/11) 13.59 347 mcg R Ustilago (3941/11) 6.3 162 mcg VR Sphacelotheca (1358/12) 10.9 278 mcg VR Sphacelotheca (4683/11) 3.29 84 mcg S Ustilago, Fusarium (2617/09) 4.53 115 mcg S Fusarium 2597/09 3.59 86 mcg S Sphacelotheca П2/27 2013 6.35 162 mcg S Sphacelotheca (3398/14) 5.83 149 mcg S Fusarium (2613/09) 7.73 197 mcg S Ustilago (2599/09) 7.6 190 mcg S Ustilago (2075/12) 8.09 200 mcg S Ustilago (2613/09) 5.8 140 mcg S Ustilago (1200/13) 5.5 132 mcg S Ustilago (3358/14) 5.7 138 mcg S Ustilago (1506/14) 7.7 190 mcg S Ustilago (605/12) 7.83 200 mcg TI, standard sample Results and discussion Earlier [1], in the study of maize grain extracts (13 genotypes), the presence of constitutive 14 kDa TI at a relatively high concentration level was shown in seven A. flavus resistant varieties of maize, but in six susceptible varieties TI was present in low concentration or was absent altogether. The mechanism of action of TI against fungal growth may be partially due to the inhibition of fungal amylase, which restricts access of A. flavus to simple sugars [5], which is necessary not only for the growth of pathogenic fungi, but also for their production of toxins. TI also demonstrated antifungal activity against other mycotoxigenic species [2]. In [6], TI content was studied in different varieties of Indian maize, resistant and susceptible to A. Parasiticus, the data are shown in Table 2. Proteomic analysis of wheat germ proteins and endosperm proteins of maize varieties resistant and susceptible to Aspergillus flavus, separated by two-dimensional electrophoresis (2 D PAGE), showed that there were 5 more constitutive marker proteins, namely - storage proteins (globulin 1 and globulin 2), proteins of late embryogenesis (LEA), proteins associated with drought (LEA3 and LEA14) or osmotic stress (WSI18 and aldose reductase) and proteins associated with heat stress (HSP16.9). Aldose reductase activity, measured in resistant and susceptible genotypes before and after infection, indicates the importance of constitutive levels of this enzyme for resistance [7]. Trypsin inhibitor concentration in Indian maize varieties [6] Table 2 Variety Trypsin inhibitor concentration in Indian maize varieties Response to A. parasiticus infections LM-6 242.2 Resistant P(Y)S-8-185-6-B8B 258 GY-37-1-328 420 CML-142 421-2 CML-176 90 Susceptible Pob-24-FSRS-C-1 90 Hyd-9745 132 Maduri 134 CML-185 158 CML-430 188 CML-291 266 Moderately susceptible CML-161 36 African tail 130 CML-150 130 MPQ-13 131 Pob31(ALM) HHH-XB 132 Local 190.8 Shaktiman-1 211.6 CM-119 302 Panchaganga 356 Most proteomic studies are related to resistance to genus Aspergillus (flavus and parasiticus) [8]; our studies of the content of TI in different varieties of maize resistant (susceptible) to smut and stalk rot showed that the number of TI was increased in resistant varieties and reduced in susceptible varieties, however, there were intermediate variants, called in the literature as “moderately susceptible” [6]. Conclusions Based on the importance of presence of proteinase inhibitors in plants, various maize varieties were screened for trypsin inhibitor content. Isolation of TI was performed using an affinity sorbent, trypsin agarose. Estimating the amount of TI in maize grains can serve as a primary marker of resistance (susceptibility) to fungal pathogens such as smut and stalk rot. The number of TIs is increased in resistant varieties and reduced in susceptible varieties, but there are also intermediate variants - varieties, which are called moderately susceptible in the literature.

Galina Vladimirovna Shekhvatova

Scientific Research Production Company Gamma

Author for correspondence.
Email: shgal06@gmail.com
Pushchino, Russian Federation

Senior Researcher

Viktor Vasilievich Ashin

Skryabin Institute of Biochemistry and Physiology of Microorganisms RAS

Email: shgal06@gmail.com
Pushchino, Russian Federation

Candidate of Biological sciences, Junior Researcher, Laboratory of Adaptation of Microorganisms

Elena Fedorovna Sotchenko

Russian Research Institute of Maize

Email: shgal06@gmail.com
Pyatigorsk, Russian Federation

Candidate of Biological sciences, leading researcher, Department of Selection for immunity

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