Abstract
Withania somnifera is a high value medicinal plant which is used against large number of ailments. The medicinal properties of the plant attributes to a wide array of important secondary metabolites. The plant is predominantly infected with leaf spot pathogen Alternaria alternata, which leads to substantial biodeterioration of pharmaceutically important metabolites. To develop an effective strategy to combat this disease, proteomics based approach could be useful. Hence, in the present study, three different protein extraction methods tris-buffer based, phenol based and trichloroacetic acid-acetone (TCA-acetone) based method were comparatively evaluated for two-dimensional electrophoresis (2-DE) analysis of W. somnifera. TCA-acetone method was found to be most effective and was further used to identify differentially expressed proteins in response to fungal infection. Thirty-eight differentially expressed proteins were identified by matrix assisted laser desorption/ionization time of flight-mass spectrometry (MALDI TOF/TOF MS/MS). The known proteins were categorized into eight different groups based on their function and maximum proteins belonged to energy and metabolism, cell structure, stress and defense and RNA/DNA categories. Differential expression of some key proteins were also crosschecked at transcriptomic level by using qRT-PCR and were found to be consistent with the 2-DE data. These outcomes enable us to evaluate modifications that take place at the proteomic level during a compatible host pathogen interaction. The comparative proteome analysis conducted in this paper revealed the involvement of many key proteins in the process of pathogenesis and further investigation of these identified proteins could assist in the discovery of new strategies for the development of pathogen resistance in the plant.
Highlights
MethodsPlant materialW. somnifera seedlings were germinated in earthen pots containing a combination of soil: sand: vermicompost in the ratio of 1: 1: 8. After 30 days, the developed plants were shifted to individual pots
We have reported that leaf spot disease of W. somnifera which is caused by Alternaria alternata leads to biodeterioration of pharmaceutically important secondary metabolites and human health promoting components [15,16]
The quantitative comparison showed that the phenol based method and TCA-acetone method provided highest protein yields as compared to tris buffer based extraction protocols (Table 1)
Summary
Plant materialW. somnifera seedlings were germinated in earthen pots containing a combination of soil: sand: vermicompost in the ratio of 1: 1: 8. After 30 days, the developed plants were shifted to individual pots. Leaf proteome analysis of Withania somnifera tissues were frozen in liquid nitrogen instantly after harvest and stored at -80 ̊C for subsequent experimentation. The causative pathogen was isolated from leaf spot infected plant and was further used for disease induction. The concentration of spore suspension was adjusted to 6×105 spores/ml with the help of a haemocytometer. These spores were sprayed on the healthier plants and pots were held in wet chamber to maintain relative humidity of 75 ± 5% at 25 ̊C. Plants treated in the same way with sterile water and 0.01% Tween-20 (v/v) served as control. Extended leaves demonstrating disease symptoms (brown spots) were immediately solidified in fluid nitrogen and kept at -80 ̊C for further experimentations
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