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African Crop Science Journal
African Crop Science Society
ISSN: 1021-9730 EISSN: 2072-6589
Vol. 4, Num. 1, 1996, pp. 79-87
African Crop Science Journal,Vol. 4. No.l, pp. 79-87, 1996

Levels of three antifungal proteins during development, germination and in response to fungal infection in grain sorghum

R. SUNITHA KUMARI. A. CHANDRASHEKAR^1 and R.A. FREDERIKSEN

Department of Plant Pathology and Microbiology, Texas A&M University College Station Texas-77843 USA.
^1 Plant Molecular Biology Unit, Central Food Technological Research Institute, Mysore- 570013, India

(Received 11 September 1995; accepted 8 January 1996)


Code Number: CS96043
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ABSTRACT

An enzyme linked immunosorbent assay (ELISA) for the levels of three antifungal proteins (18, 26 and 30 kDa) in sorghum (Sorghum bicolor (L.) Moench) varieties susceptible and resistant to grain moulds was analysed. The results suggested that hard and mould resistant grains have higher levels of the 18 kDa and 30 kDa antifungal proteins. The levels of these proteins were also analysed at 7, 14, 21, 28, 35 and 42 days alter anthesis during seed development using SDS-PAGE and ELISA in a variety that was hard and resistant to fungal infection (E-35-1) and a soft susceptible variety(M-35-I). A 30 kDa antifungal protein increased in amount in developing seeds from 7 days after anthesis up to 28 days and decreased thereafter. Two other antifungal proteins (18 and 26 kDa) appeared in seed sample 14 days after anthesis. The resistant variety contained more of 30 and 18 kDa antifungal proteins at all developmental stages. Levels of the 26 kDa protein increased in the susceptible variety after inoculation of grains with Fusarium moniliforme, suggesting its inducibility. There was little change in the resistant variety in the levels Of all the three antifungal proteins after inoculation. During germination the levels of the three proteins, especially that of the 18 kDa, increased in extractibility. A complex regulatory mechanism controlling the levels of three proteins during development and germination is shown by the present study.

Key Words: Disease resistance, ELISA, grain moulds, inducibility, kDa protein

RESUME

Un test d'ELISA etait utilise pour determiner les niveaux de trois proteines antichampignons (18. 26 et 30 KDa) sur les varietes du sorgho (Sorghum bicolor (L.) Moench) susceptibles et resistantes au "grain mould". Les resultats ont montre que les grains resistants ont des niveaux eleves (18 KDa et 30 KDa) de proteines antichampignons. Les niveaux de ces proteines etaient aussi analyses a 7 14 21 28 35 et 42 jours apres l'anthese pendant le developpement de la semence moyennant les tests de SDS-PAGE et ELISA sur une variete a grains durs et resistante a l'infection au champignon (E-35-1) et une variete susceptible a grains moux (M-35-1 ). La quantite de proteine antichampignon 30 KDa augmentait dans les semences a partir de 7 jours apres l'anthese jusqu'a 28 jours puis decroissait. Deux autres proteines antichampignons (18 et 26 KDa) apparaissaient 14 jours apres l'anthese dans l'echantillon de semences. La variete resistante contenait plus de proteines antichampignons 10 et 18 KDa a tous les stades de developpement. Des niveaux de proteines de 26 KDa augmentaient sur la variete susceptible apres inoculation de grains avec le Fusarium monoliforme, ce qui prouve son effet par induction. Il y a peu de changement pour une variete resistante aux niveaux de tous les trois proteines antichampignons apres leur inoculation. Durant la germination, les niveaux de trois proteines particulierement le 18 KDa, augmentaient leur extraction. Un mecanisme complexe de regulation controllant les niveaux de trois proteines durant le developpement et la germination est montre dans la presente etude.

Mots Cles: Resistance a la maladie, ELISA, grain mould, effet d'induction, proteine kDa

INTRODUCTION

Pathogenesis-related proteins belonging to different classes are known to be induced in plants during infection (Carr and Klessig, 1989), wounding (Green and Ryan, 1972) and chemical treatment (Nasser et al., 1990). Belonging to this class are the proteinase inhibitors (Pengand Black, 1976), chitinases and beta 1,3-glucanases (Leah et al. 1991) and those that belong to the thaumatin class of protein (Vigers et al., 1992). It has been shown that osmotin, which belongs to this group, is induced by salt stress (King etal., 1982). Most of these proteins have been studied in leaves and stems.

Seeds are perhaps the most important part of plants in the human diet. Seeds contain a variety of antifungal proteins (Roberts and Selitrennikoff, 1986, 19 8 8, 1990) which appear to play a defensive role against pests and pathogens. Many of these proteins belong to the group anti-nutritional proteins and include the a-amylase and protease inhibitors (Pusztai, 1967) and the lectins (Chrispeels and Raikhel, 1991 ).

Three antifungal proteins have been identified in sorghum and antibodies developed against them (Sunitha Kumari and Chandrashekar, 1994a). An ELISA was used for the quantification of the antifungal proteins in flour extracts (Sunitha Kumari and Chandrashekar, 1994b) and it was shown that the relative values for the three antifungal proteins in high prolamin varieties was higher than those with low prolamin content. Earlier studies (Sunitha Kumari et al., 1992) indicated that grains depositing more prolamin appear to be more resistant to grain mould than those depositing less prolamin during development. It was of interest to assess differences in the levels of three antifungal proteins in grains that were hard and resistant to grain moulds.

Furthermore, since fungal infection occurs during kernel development, the levels of these proteins in a resistant and a susceptible variety was estimated. The levels of antifungal proteins were studied at different stages of maturity. Most antifungal protein chitinases and beta 1,3 glucanases that have recently been studied are induced by stress, wounding, or by pathogen invasion. The second objective of this study was to monitor the level of the three antifungal proteins after infection. The third objective was to quantify the level of these antifungal proteins in germinated samples. This would provide information on the possible role of these proteins in resisting fungal growth during germination.

MATERIALS AND METHODS

Materials. Two varieties of sorghum (Sorghum bicolor (L) Moench) hard resistant (E-35-1) and soft susceptible (M-35-1) to grain moulds were selected. The crushing strength/hardness values for the grains of variety E-35-1 were 13.4 kg cm^2 and that of M-35-1 was 5.6 kg cm^2 (Sunitha Kumari et al., 1992). Antibodies raised against three antifungal proteins (Sunitha Kumari and Chandrashekar, 1994a, 1994b) that were specific with no cross reactivity were used for the study.

Fungal culture. The grain mould pathogen Fusarium moniliforme Sheldon used in the study was isolated from moulded sorghum grains and maintained on potato dextrose agar. For inoculation studies, the inoculum was obtained by culturing F. moniliforme in potato dextrose broth. The mycelial mats from seven day old cultures were removed and resuspended in distilled water. The suspension was filtered through muslin cloth to remove mycelial pieces and the filtrate containing the conidial suspension was used. The conidial concentration taken for spraying the panicles was approximately 10^5 conidia ml^-1.

Developmental studies. Grains were planted in the field at the experimental farm of the Department of Studies in Applied Botany, University of Mysore, India. Random plants were tagged at anthesis and the earheads were harvested at seven day intervals starting from 14 days after anthesis (DAA) up to 42 DAA. The grains were lyophilised and powdered in a Udy cyclone mill using a 0.4 mm screen. Pre-pollinated florets (ovaries and stamens) were also collected and lyophilised. The extracts of pre-pollinated florets were run on SDS-PAGE using the procedure of Laemmli (1970).

Flour (100 mg) was extracted with 10 ml of Tris buffered saline (TBS; tris 50 mM and 200 mM NaCl, pH 7.2) for 4 hr at room temperature, and centrifuged at 6,000 g for 10 min. The protein content in the extracts were estimated by the modified Lowry's method (Schacterle and Pollack, 1973). The levels of the antifungal proteins were quantified by ELISA.

Enzyme Linked Immunosorbent Assay. Tris buffered saline extracts of flour were used for coating the wells of the microtitre plate (Corning, USA). To each well, different aliquots of flour extracts were added and the final volume was brought to 100 ul with TBS. The antigen was immobilised by incubating the plates at 37 C for 16 hr. The wells were washed with TBS thrice for 5 min and were then blocked with 100 ul of BSA (2%) for 1 hr. Wells were washed with TBS-T (TBS with 0. 05% Tween 20) and 100 ul of the antibody at 1:1000 dilution was added and incubated for 2 hr at 37 C. To the wells, 100 ul of anti-rabbit-goat IgG biotin conjugate (1:10,000 Sigma) was added and incubated for 2 hr at 37 C and washed with TBS-T. Avidin peroxidase (100 ul, at 1:500 dilution, Sigma) was added to the wells and the plates incubated for an hour and the enzyme activity was detected with 100 ul of 0.005% hydrogen peroxide in 0.034% O-phenylene diamine (Sigma). The reaction was stopped after 30 min with 50 ul of 2 M sulphuric acid. The colour was read at 492 nm using an Organon Teknika micro ELISA reader. Both antigen and antibody blanks were used.

All optical density values were normalised to a percentage of that obtained with those of E-35-1 and were averages of four replicates from two separate experiments. Standard deviation was calculated for the ELISA values. The student's test of significance was applied for ELISA data. The procedure followed was according to Hawkes et al. (1982) using Avidin-biotin system.

Artificial inoculation with Fusarium moniliforme. Panicles were sprayed with 10 ml of conidial suspension of Fusarium moniliforme (10^5 conidia ml^-1) and were kept bagged. The ml^-1 inoculation was conducted at the following stages of development; anthesis, 7 DAA, 14 DAA and 21 DAA. The middle portion of the particles were sampled 6 days after inoculation. Along with the inoculated samples, respective controls were also sampled. The controls were particles sprayed with distilled water and were kept bagged. The samples were lyophilised and ground to flour in a Udy cyclone mill using a 0.4 mm screen. The estimation of protein and ELISA was carried out as described before.

Mycoflora analysis. The identification of mycoflora associated with the infected and uninfected samples at all stages of development was carried out as described earlier (Sunitha Kumari et al., 1992).

Germination. Grains were soaked in distilled water containing 1% sodium hypochlorite for 6 hr. The grains were thoroughly washed and kept for germination in an incubator BOD (Biological Oxygen Demand) maintained at 22 C on moist filter paper in petriplates. Samples were removed after 1, 3, 4, 5 and 6 days after germination, lyophilised, the roots and shoots were separated from the kernels. The kernels were ground to flour in a Udy cyclone mill using a 0.4 mm screen. Analysis was carried out as for the samples obtained during development.

Proteolysis. Flour (50 mg) was extracted with one ml of TBS to which 100 ug of trypsin (Sigma) and 100 ug of Subtilisin (Boehringer Mannheim) were added separately. The flour with enzyme was incubated at 37 C for 15, 30 and 60 min. After incubation, 2 mM phenyl methyl sulphonyl fluoride (Sigma) was added to the flour containing the enzyme. The samples were mixed by vortexing before centrifugation. The extracts were centrifuged at 6,000 g for 10 min and the supernatant was then used for the quantification of the antifungal proteins by ELISA. The values from enzyme treated samples were normalised to those obtained from flour of either variety not treated with proteases.

RESULTS AND DISCUSSION

Developmental profile of antifungal proteins. In order to determine changes in the amount of the three antifungal proteins during development, SDS-PAGE of extracts of the flowers, and developing grains from a resistant and a susceptible variety harvested from 14 to 42 DAA were analysed. SDS-PAGE indicated the presence of a 30 kDa protein band in the extracts from florets. ELISA of florets could not be conducted as there was too much background colour, so a dot immunobinding assay was conducted. This assay showed that the anti-30 kDa antibody reacted with the extract from the pre-pollinated florets (Fig. 1), which was also shown by westerm blotting (data not shown). Proteins soluble in tris buffered saline decreased in amount steadily from 14 to 42 DAA in both varieties and were generally slightly higher in the hard variety than in the soft (Table 1 ).

ELISA data for the three antifungal proteins during development are presented in Figure 2. The level of 26 kDa antifungal protein did not show much difference between the two varieties at 21 DAA. The 30 kDa antifungal protein peaks at 21 DAA and the values for E-35-1 were lower when compared with that for M-35-1 at that same stage of development. The values then decreased towards maturity. The level of 18 kDa protein was less in E-35-1 than in M-35-1 at all stages of development, except 7 DAA.

Level of antifungal proteins after artificial inoculation. The amount of protein soluble in tris buffered saline was generally more in M-35-1 than in the E-35-1 at all stages except at anthesis (Table 2). After inoculation at four points of development, the level of proteins increased in the variety M-35-1, whereas there was no increase in the protein level in the variety E-35-1 except at 7 DAA.

There was more 26 kDa protein in response to infection at the first three stages of inoculation (the values relative to its respective control were 120, 136 and 125) in comparison to the control in M-35 - 1. The values for E-35 - I were not different from the control (Fig. 3). For the 30 kDa protein, the values for both varieties were lower than those of control inoculated early during development, but were equal or greater than control in samples inoculated at 14 and 21 DAA (the values were 109 and 130, Fig. 3). The level of the 18 kDa increased from being lower than that of control when infected at anthesis and at 7 DAA to being higher than the control in samples inoculated at 14 and 21 DAA for M-35-1. The estimated value for the 18 kDa in E-35-1 was the same as in the control sample in any of the inoculated material at 7, 14 and 21 DAA (Fig. 3). The SDS-PAGE data corroborated the ELISA estimates after artificial inoculation during development (data not shown). In samples that were inoculated at anthesis and harvested at physiological maturity the values for 26, 30 and 18 kDa protein relative to the uninfected mature sample of E-35-1 was 37, 66 and 74% in E-35-1 and 52,74 and 42% in M-35-1.

TABLE 1. Amount of tris buffered saline soluble protein from a soft (M-35-1 ) and a hard (E-35-1 ) sorghum variety during development

Days after      Protein mg/100g*
anthesis      M-35-1        E-35-1 
--------------------------------------
   14         1476+/-4.4    1630+/-4.7 
   21         1533+/-3.0    1267+/-4.7 
   28         1263+/-3.0    1223+/-4.4 
   35         1009+/-3.1    1242+/-5.3 
   42          940+/-2.8     995+/-3.1

* Data show mean +/-SE

    Figure 1. Dot ELISA of pre-pollinated flowers and probed with anti-30 kDa antibodies using the biotin-avidin peroxidase system (a) Endosperm extract of flour (b) M-35-1 flower extract, (c) E-35-1 flower extract.

TABLE 2. Amount of TBS soluble protein in a hard and soft sorghum variety in control and inoculated samples

Stage of             TBS soluble protein mg/100 g*
development     
--------------------------------------------------------------
                  M-35-1                 E-35-1
          ------------------------   -------------------------
               C           I            C           I
--------------------------------------------------------------
At anthesis    
          2368+/-4.4     2480+/-3.1    3177+/-3.6  2980+/- 377 
  7DAA    2150+/- 3.6    2356+/-3.1    1626+/-2.2  2065+/- 2.6 
 14DAA    1505+/- 3.6    1600+/-5.0    1423+/-1.7  1311+/- 3.0 
21 DAA    1239+/- 3.0    1319+/-3.7   1170 +/-3.0 11.40+/-3.0
--------------------------------------------------------------

*Data show mean +/- SE
C: Control l: Inoculated with Fusarium moniliforme

    Figure 2. ELISA estimates for three antifungal proteins during development in soft (M-35-1) and hard (E-35-1) sorghum grain. All values are expressed as a relative percentage of those obtained with mature E-35-1. Data show mean +/- SE.

    Figure 3, ELISA estimates for sorghum samples inoculated at anthesis 7, 14 and 21 days after anthesis and sampled 6 days thereafter in comparison with the uninoculated otherwise similarly treated control from soft (M-35-1) and a hard (E-35-1) variety. Data show mean +/- SE.

Mycoflora analysis. The level of mycoflora was greater in inoculated samples than in the control samples and increased as development proceeded. Only F. moniliforme was seen in the inoculated samples. Cladosporium (15%) and Aspergillus (5%) were seen in the control samples along with F. moniliforme. The percent incidence of F. moniliforme in inoculated samples in the variety M-35-1 at anthesis, 7 DAA, 14 DAA and 21 DAA were 60, 80, 80 and 100% whereas the control samples showed O, 21, 37 and 43% at corresponding stages. On the other hand the variety E-35-1 showed 45, 50, 65 and 80% incidence when inoculated samples were analysed whereas the control samples showed O, 4, 6, and 8% infestation.

TABLE 3. Amount of tris buffered saline soluble protein from a soft (M-35-1) and a hard (E-35-1) sorghum variety during germination

Days after        Protein g/100 mg*
germination  ---------------------------
                 M-35-1       E-35-1
    
    0        837 +/- 3.3     997 +/- 3.3 
    1       1766 +/- 3.4    1432 +/- 3.4 
    3       1624 +/- 5.1    1827 +/- 2.4 
    4       1972 +/- 2.4    2032 +/- 2.4 
    5       2083 +/- 5.0    2308 +/- 3.4 
    6       2311 +/- 3.6    2364 +/- 3.16

* Data show mean + SE

Germination and antifungal proteins. The amount of protein extracted with tris buffered saline from the two varieties during germination increased steadily and was slightly higher for extracts from E-35-1 (Table 3). ELISA estimates showed an increase for the 26 kDa protein between 0 and 2 days of germination (Fig. 4). The variety E-35-1 contained more of this protein at 1,3,4 and 5 days after germination than did M-35-1. The level of 26 kDa antifungal protein in germinated M-35-1 sample (1, 3, 4 and 5 days after the germination) was lower than that of ungerminated samples. Estimates for the amount of the 30 kDa protein was generally higher for M-35 - 1 than for E-35 -1 and decreased slightly as germination proceeded (Fig. 4). The values for the 18 kDa protein increased in both the varieties during germination, more so with the resistant cultivar (Fig. 4). The ELISA values for the two varieties were found to be significant at 1% level. In E-351 the extractable level of the 18 kDa protein increased by 60% during five days of germination. In M-35-1, the amount of the protein measured increased by 20% during the same period.

Proteolysis. In order to explain the apparent increase in the amount of the proteins detected after germination and to check the stability of protein against protease attack, flour was treated with trypsin or subtilisin prior to quantification by ELISA.

    Figure 4. ELISA estimates for three antifungal proteins at different days after germination in a soft (M-35-1) and a hard (E-35-1) sorghum variety. All values are expressed as a relative percentage to those obtained with mature E-35-1. Data shows mean +/- standard deviation
The 30 and 26 kDa proteins seem to be more resistant to trypsin attack, while there was generally greater digestion with subtilisin with time (Table 4). Moreover, for the variety M-351, the level of extractable protein for 26 and 30 kDa protein actually increased during incubation with trypsin compared to the control not treated with the enzyme. The 18 kDa was more susceptible to protease attack than the other two proteins in either variety.

Prolamins were shown (Sunitha Kumari et al., 1992) to be deposited in hard grains continuously up to maturity, while soft grains deposited prolamins mostly during earlier stage of development. It has also been shown (Sunitha Kumari et al., 1992) that infection occurs during early stages of development. Bass et al. (1992) have shown a relationship in maize between prolamin level and that of a ribosomal inactivating protein in maize which is antifungal. We sought here to relate fungal infection with the levels of three antifungal proteins. The fungal infection that occurs during development often manifests itself during germination. We therefore investigated the levels of three antifungal proteins during germination.

The 30 kDa antifungal protein was present in flower. The extractable level of the 30 kDa protein increased steadily up to 28 DAA in both varieties, failing in extractability thereafter. The fall in extractability during later stages of development may be due to the complexing or protein with other grain constituents. There is little change, however, inextractibility of protein with increased germination indicating stability of the complex. The level of the 30 kDa protein did not change with infection in the hard variety, whereas it was lower than the control in M-35-1, in samples inoculated at O, 7 and 14 DAA. This indicated synthesis of the 30 kDa may be inducible by infection at an early stage of development.

The level of the 26 kDa protein was higher in the resistant than in the susceptible cultivar from 21 DAA and thereafter. It increased after fungal infection or protease treatment indicating release of bound protein. However, no change in the extractable levels of the protein occurred during germination. The amount of the protein measured may reflect a balance between protein that was released from the sequestered or bound form and that which was degraded by protease. A possible explanation is that both induction and simultaneous sequestration of the protein occurs with the sequestered protein being released after infection or enzyme treatment.

The 18 kDa protein works in the opposite direction. It was synthesised early and decreased steadily after 7 DAA. The 18 kDa protein increased in extractibility during germination and was susceptible to enzyme attack in vitro.

TABLE 4. Effect of externally added enzymes on the ELISA values * for the three antifungal proteins in flour

Sorghum variety
--------------------------------------------------------------
          Trypsin                     Subtilisin 
  min after incubation            min after incubation
----------------------------  --------------------------------
15          30         60        15        30        60
--------------------------------------------------------------
                      18 kDa antifungal protein 
M-35-1         
66+/-1.7  80+/-2.6  83+/-2.0  66+/-1.7  67+/-1.4  69+/-2.0 
E-35-1        
62+/-2.0  65+/-1.7  67+/-1.7  45+/-1.7  48+/-1.7  51+/-1.4

                      26 kDa antifungal protein

M-35-1       
130+/-1.7 143+/-1.4 146+/-1.7 119+/-1.7 121+/-1.7 109+/-2.6 
E-35-1        
 97+/-2.0  96+/-2.0 104+/-2.0  79+/-2.4  81+/-2.0  82+/-1.4

                      30 kDa antifungal protein

M-35-1        
86+/-1.7  115+/-3.0 118+/-1.7  73+/-1.4  64+/-1.7  64+/-1.7

E-35-1        
87+/-2.4   80+/-2.2  75+/-2.0  60+/-2.8  60+/-1.7  53+/-2.0

Data shows means +/- SE
*Values are calculated as percentage of values obtained from extracts of mature M-35-1 or E-35-1 flour, respectively

The susceptible cultivar responds to infection by synthesising increased levels of the antifungal proteins, while no change in the levels of the protein was seen in infected samples of the hard variety that had been inoculated by the fungus. In peas (Pisum sativum L.), a rise in the activity of beta 1,3-glucanase and chitinases, two antifungal proteins, during development was reported (Mauch et al., 1988). The ribosomal inactivating protein, b-32, also an antifungal protein increased in amount during germination (Bass et al., 1992). In contrast, a differential synthesis into bound forms during development with subsequent solubilisation during germination was found in this study. Moreover, the levels of these proteins increased on proteolysis, fungal infection and germination. The antifungal proteins become insolubilised during development and are released on limited proteolysis during germ nation or fungal infection. This may play an important role in protecting the plant during development and germination. Further work at molecular level is being carried out and the genes encoding these and other anti fungal protiens are being cloned and their expression in response to infection is being studied.

ACKNOWLEDGEMENTS

The authors thank Prof H.S. Sherry of Department of Studies in Applied Botany, Mysore University, India, for his interest. The authors also thank Dr. Clint Mogul, of Plant Pathology Department, Texas A&M University for correcting the manuscript. One of the authors (SKR) is grateful to CSIR for a fellowship grant.

REFERENCES

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Carr, J.P. and Klessig, D.F. 1989. The pathogenesis related proteins of plants. In: Genetic Engineering. Principles and Methods. Vol. I 1. Setlow, R.A and Hofleander, A. (Eds.), pp. 65-109. Plenum Press, New York.

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Sunitha Kumari, R. and Chandrashekar, A. 1994a. Isolation and Purification of three antifungal proteins from sorghum endosperm. Journal of the Science of Food and Agriculture 64:357-364.

Sunitha Kumari, R. and Chandrashekar, A. 1994b. Relationships between grain hardness, and contents of prolamin and three antifungal proteins in sorghum. Journal of Cereal Science 20:93-99.

Sunitha Kumari, R., Chandrashekar, A. and Shelly, H.S.1992. Proteins in the sorghum endosperm that may be involved in resistance to grain moulds. Science of Food and Agriculture 60: 275-282.

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Copyright 1996 The African Crop Science Society


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