Quantitative determination of trypsin inhibitor

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

Galina V. Shekhvatova¹*, Viktor V. Ashin²,

Elena F. Sotchenko³

'Scientific Research Production Company Gamma,

Pushchino, Russian Federation

² Skryabin Institute of Biochemistry and Physiology of Microorganisms RAS, Pushchino, Russian Federation

³Russian Research Institute of Maize, Pyatigorsk, Russian Federation

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 Aspergillus 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.

Key words: maize, trypsin inhibitor, trypsin-agarose, fungal pathogens, head smut, common smut, Fusarium stalk rot

Определение количества ингибитора трипсина как маркера селекции сортов кукурузы с различной устойчивостью к грибковым заболеваниям

plant resistance fungal pathogen

Г.В. Шехватова¹*, В.В. Ашин², Е.Ф. Сотченко³

'Научно-производственная фирма «Гамма»,

Пущино, Российская Федерация

²Институт биохимии и физиологии микроорганизмов им. Г.К. Скрябина РАН, Пущино, Российская Федерация

³Всероссийский научно-исследовательский институт кукурузы,

Пятигорск, Российская Федерация

Аннотация

Устойчивость растений к патогенным грибам является важной селекционной составляющей. Механизмы устойчивости очень разнообразны и могут включать в себя взаимодействия ферментов и их ингибиторов. В связи с этим проведено сравнение содержания ингибитора трипсина (ИТ), как возможного маркера селекции, в зрелых зернах сортов кукурузы с известной устойчивостью к пыльной головне, пузырчатой головне и стеблевой гнили. В качестве изучаемых объектов взяты 6 устойчивых и 12 чувствительных сортов. Для гарантированного извлечения из экстрактов молотых зерен кукурузы только ИТ применялась колоночная аффинная хроматография на трипсин-агарозе в аналитическом варианте. В результате выяснилось, что содержание ИТ варьирует внутри обеих групп сортов кукурузы: в устойчивой группе -- максимум 347 и минимум 162 мкг/г, в чувствительной группе -- максимум 200 и минимум 84 мкг/г. Соотношения максимумов к минимумам (2,3) одинаковы в обеих группах, но максимальное и минимальное содержание почти в два раза выше в группе устойчивых сортов кукурузы. В среднем в устойчивых сортах кукурузы содержание ИТ выше в 1,7 раза. Полученные данные подтверждают возможность использования содержащегося в кукурузных зернах ИТ в качестве маркера при выведении новых сортов кукурузы, устойчивых к пыльной головне, пузырчатой головне и стеблевой гнили.

Ключевые слова: кукуруза, ингибитор трипсина, трипсин-агароза, грибковые патогены, пыльная головня, пузырчатая головня, стеблевые гнили.

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-P-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 pg 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 (250x4.6) Bio Rad column. The following conditions were used: 3' -- 0% CH₃CN; 40' -- 100% CH₃CN; 50'-- 100% CH₃CN, 65' -- 0% CH₃CN.

200 pg 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 pg 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 pg of 14 kDa TI is applied for calibration

Fig. 2. Typical chromatography curve of maize grains extract on trypsin-agarose: second peak -- TI elution

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 pg of 14 kDI TI was used for calibration.

Table 1