Department of Molecular Biology and Genetic Engineering
G. B. Pant University of Agriculture and Technology
PANTNAGAR 263 145. UP. India.
ABSTRACT
Pathotoxin(s) produced by Alternaria brassicae, the causal agent of Alternaria blight in rape seed and mustard inhibit the growth of Brassica calli and induce chlorosis in leaves of Brassica campestris. Similar symptoms were also observed during nutritional depletion at the decaying stage of the growth. The possible involvement of apoptosis in these conditions was investigated by comparing the levels of p53 in pathotoxin treated and nutritionally depleted leaves and calli with the normal leaves and calli. Studies were also performed to compare the levels of p53 in pathotoxin treated and nutritionally depleted calli with proliferating calli. Antibodies raised against human p53 were used to detect and quantitate p53 in B. campestris. Expression of p53 was continuously increased from proliferating to static growth stage but then remains constant in decaying stage. Both ELISA and dot blot immunoassay binding assay showed that p53 protein was overexpressed in toxin treated and nutritionally depleted calli. Almost similar changes were seen senescent leaves when compared with normal leaves. We concluded that toxin related damage in Brassica species may involve p53 dependent pathways.
KEY WORDS Brassica campestris, Alternaria blight, Pathotoxin, Apoptosis, p53 protein.
INTRODUCTION
Alternaria blight characterizing necrotic-chlorotic lesions, is one of the most recalcitrant diseases caused by four different species. The major pathogen, Alternaria brassicae produces toxin(s) which are responsible for these lesions. Understanding the mechanism of action, particularly, host specific toxins (HSTs) provide a better appreciation of host pathogen interactions and resistance mechanisms. Two approaches have been used to study the mode of action of HSTs. One is the study on the molecular level of host selectivity and the other at the cellular level. It causes amongst other aberrations in chloroplast and mitochondria (Agarwa; et al., 1992; Pandey, 1996). This simple antagonism of toxin action by phytohormone is difficult to explain on the basis of signal recognition by which plant cells transmit the toxin(s) effects to downstream events in the form of chlorotic symptoms (senescence). The cells undergoing senescence often exhibit similar morphological changes as in case of programmed cell death (apoptosis) (Arends et al., 1990; Wyllie, 1987). Thus it is quite pertinent to study the induction of apotopsis in response to pathotoxin that could contribute to explain the molecular changes associated with chlorosis.
The apoptosis is regulated by a stochiometric balance between death and suravival signals (Brown, 1996). Senescence in plant system is also known to be regulated by phytohormones and many known pathogenic triggers (Noodin, 1988; Thomas, 1982). Hence, the question how does apoptotic pathway operate in Brassica? Therefore, considering the role of p53 gene and protein in apopotisis, p53 protein was chosen to study its presence during different stages of growth of Brassica cells for possible changes during aging and nutritional starvation. Moreover, to study the influence of pathotoxin on p53 expression, the level of p53 were compared by ELISA reactivity in pathotoxin treated calli with proliferating and nutritionally depleted calli as well as pathotoxin treated leaves with senescent and normal leaves.
MATERIALS AND METHODS
Partial purification of alternaria toxin
Alternaria toxin (dextruxin B) was partially purified by the procedure developed by Tyagi (1990). Liquid culture of Alternaria brassicae was grown on Frie’s medium (Agarwal, 1992) for 35 days. The toxic component was extracted in ethyl acetate and purified by employing Sephadex G-10 and LH-20 column chromatography. The elution profile of the components was recorded at 235, 254 and 280 nm. Peak fractions were pooled, lyophilized and checked for the toxicity by foliar bioassays.
Callus culture
Hypocotyl segments were cut into very small pieces. These pieces were teased with the help of a scalpel blade prior to their transfer into MS medium supplemented with 2 ppm BAP and 1 ppm NAA. Induction of callus was observed after 10 days. The volume of growth callus were estimated by measuring length, width and height from outside of the tube with the help of a scale.
Growth curve
Growth includes both cell enlargement and cell division. Increase in volume, dry weight/fresh weight can be taken as a measurement of growth of the callus. Length, width and height of growing callus were estimated by taking dimension of callus at three particular directions and volume was calculated. The logarithm of volume of callus at 5 days interval were plotted on Y axis and number of day on X axis to obtain the growth curve of the callus.
Partially purified toxin treatment of callus
Callus pieces of about 30-35 mm2 in size were taken in sterilized petri plates. Ten μl of toxin (chlorotic fraction of the eluent of partial purification) was applied on callus piece as control. These were transferred aseptically into each tube containing MS medium. Protein was extracted from 7 and 14 days old callus under toxin treatment.
Extraction of total protein
Total protein was extracted from callus and leaves of Brassica as described by Fielere and Jacob (1990) with slight modification. The protein was estimated by Bradford method (1976).
Immunodetection of p53
The p53 expression in alternaria treated toxin Brassica calli and leaves were detected by performing dot ELISA and microtitre ELISA.
RESULTS
The cell cycle as well as apoptic proteins have been highly conserved from yeast to mammals in order to study the effect of toxin on the expression of p53 in different calli, attempts have been made to detect the apparent differences of the p53 during different stages of growth of normal Brassica calli and pathotoxin treated calli by dot immunoblot techniques using antibodies for p53 raised in rabbit against p53 proteins of human.
Dot immunoblot analysis was done for detection of p53 during different stages of Brassica calli i.e., proliferating (15 days), stationary (30 days) and decaying (45 days) stages. Varying amounts (200-1000 μg) of protein extract of proliferating and decaying callus were spotted on the membrane (9x7) in duplicates for 1:500 and 1:1000 dilution of antibody. The first antibody was applied on the protein spot with the help of grid. All the spots with 1:1000 dilution gave very faint purple colour on development with BCIP/NBT. Antibody dilution 1:500 has revealed a linear relationship between colour developed and concentration of antigen. The p53 expression was found to increase from 15 days to 30 days and afterward remained constant at 45 days. The dots having protein extracts of 45 days were expected to have higher concentration of p53 but similar dot intensity both at static and decaying stage was interpreted due to saturated amount of anti-p53 antibodies (1:500 dilution) used.
Dot immunoblot binding assays for p53 of proliferating, decaying and alternaria toxin treated cells further confirmed increased level of p53 in decaying and pathotoxin treated callus. Levels of p53 was quite comparable in decaying and pathotoxin treated callus. Similar experiments were performed with healthy, senescent and pathotoxin treated leaf. Results were similar to that of callus proteins.
In order to confirm the qualitative differences the microtitre ELISA was performed. The microtitre ELISA was developed by using optimum antigen concentration of 0.6 μg from calli and 0.2 μg from leaf protein and 1:500 dilution of anti-p53 antibody was extrapolated from dot immunoblot (Table 1). It was seen that relative concentration as indicated by OD 405 showed that p53 antigen were present almost 2-3 times more in decaying callus compared to proliferating callus. The ELISA reactivity pattern also showed that the senescent leaf and pathotoxin treated leaves had similar concentration of p53 protein which was more than twice observed in healthy leaves.
Table 1 ELISA reading using rabbit anti-human p53 antibodies for calluses and leaves of B. campestris.
Antigen |
OD405 nm |
Mean OD |
ELISA reactivity | |
I. |
Callus proteins |
|||
1. |
Proliferating |
0.340 |
0.326 |
43 |
2 |
Decaying callus |
0.752 |
0.759 |
100 |
3 |
Pathotoxin treated callus |
0.656 |
0.645 |
85 |
II. |
Leaf protein |
|||
1. |
Healthy leaf |
0.257 |
0.241 |
46 |
2 |
Senescent leaf |
0.545 |
0.522 |
100 |
3 |
Pathotoxin treated leaf |
0.558 |
0.504 |
46 |
DISCUSSION
The cell death in the plants occurs due to senescent effect aging as well as due to some biotic and abiotic triggers. The rapid cell death that follows pathogen recognition is a genetically determined event, since mutants have been isolated and spontaneously form HR-like necrotic lesions in the absence of pathogen infection (Greenberg et al., 1994; Dietrich et al., 1994).
Alternaria blight is characterized by dark brown to blackish necrotic lesions surrounded by chlorotic areas on leaf stem and pod. It has been observed that Alternaria brassica, the destructive plant pathogen produce multiple host selective toxins that mimick the characteristic chlorotic and necrotic symptoms of the disease (Tyagi, 1990, Bains and Tewari, 1987; Buchawald and Green, 1992). The plants do not exhibit natural resistance to this disease. The disease also caused HR-response in some cultivars of Brassica. The question of cell death and tissue necrosis at the site of pathogen ingress by elaboration of toxic metabolites. Whether the toxin is responsible for it or the hypersensitive response of the host itself. However, no reports are available to suggest that chlorotic and necrotic lesions exhibited in Alternaria blight disease are the consequences of any resistance mechanisms or due to phytotoxic effect of Alternaria toxin(s).
In the present study, we developed a model to investigate the effects of alternaria toxin on death in Brassica system. These toxins on foliar application induced chlorotic lesions similar to those seen in the disease. Though effects on mitochondria and chloroplast were suspected they could not be unequivocally shown under in vitro condition. Hence, in our laboratory attempts have been made to study the effect of alternaria toxin at molecular level using Brassica calli which underwent slow growth and browning in response to alternaria toxin. In order to understand the cell death caused by toxin, it was postulated that pathotoxin could be inducing the apoptosis in Brassica in association with morphological changes. In our laboratory, the ultrastructure of chlorotic lesion seen in naturally infected leaves and toxin treated leaves was compared (Agarwal et al., 1994) and showed swollen mitochondria with reduced number of crystae and vesiculation of the envelope. Thus, our findings suggests that the alternaria toxin induces apoptosis rather than necrosis. Changes in p53 protein both in decaying and pathotoxin treated cells can form the ground to further investigate the antagonism between pathotoxin and phytohormone at cell cycle level.
The nutritionally decaying calli and detached leaves also exhibited similar symptoms of browning and chlorosis after kept for longer duration. The browning and chlorosis induced by pathotoxin treatment and nutrient depletion lend to cell death. Further, to ascertain the chlorotic cell death of Brassica cells are due to apoptosis, the crucial protein involved in apoptotic cell death was monitored using heterologous anti-p53 polyclonal antibody probes. ELISA reactivity by dot immunoblot binding assay showed that p53 protein was overexpressed in toxin treated calli and leaves. The levels of p53 was quantitatively measured by micotitre ELISA and found to be double in toxin treated calli and leaves as compared to control. We conclude that toxin treatment kills brassica cells via p53 dependent apoptotic cell death pathway and extent of damage may be related with p53 expression.
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