Characterization of PR Proteins in selected calliclones of Black Pepper in relation to phytophthora foot rot disease

Debashis Sahoo; KAU

Characterization of PR Proteins in selected calliclones of Black Pepper in relation to phytophthora foot rot disease

Files

174234.pdf (48.61 MB)

Date

2017

Authors

Debashis Sahoo

KAU

Publisher

Centre for Plant Biotechnology and Molecular Biology, College of Horticulture, Vellanikkara

Abstract

Black pepper (Piper nigrum L.), the king of spices is severely affected by Phytophthora foot rot disease caused by Phytophthora capsici. The disease results in severe crop loss, and valuable genotypes are lost every year due to this dreadful disease. The local cultivars and released varieties of black pepper are susceptible to the pathogen, but variation exists in genotypes in the degree of tolerance and mechanism of defense to the disease. The plants resist the pathogen infection by accumulating a number of defense related proteins in the intercellular spaces which are collectively known as pathogenesis related (PR) proteins. The study was aimed to characterize PR proteins in selected eleven calliclones of black pepper along with susceptible variety Panniyur-1 after challenge inoculation with Phytophthora capsici so as to get more insight on the defense mechanism of Phytophthora foot rot. Investigations on disease reaction of the selected calliclones and variety Panniyur-1 after challenge inoculation with Phytophthora capsici, β-1,3-glucanase activity and protein analysis by SDS-PAGE was carried out at 0, 24, 48 and 72 hours after inoculation. Protein profiling by 2D-gel electrophoresis and protein identification by MALDI-TOF MS / MS in the most tolerant and susceptible calliclone and in silico analysis for characterization of proteins were also attempted in the present study. In leaf symptom bioassay, variety Panniyur-1 showed susceptible reaction as compared to calliclones of Cheriakanyakkadan and Kalluvally. Based on β-1,3-glucanase activity and expression of 16.5 kDa band in SDS-PAGE, clone KLCC 89 was selected as the tolerant calliclone. 2D-gel electrophoresis was attempted in the selected tolerant calliclone, KLCC 89 and susceptible variety Panniyur-1. Proteome analysis by 2D-gel electrophoresis could locate 167 differentially expressed protein spots in KLCC 89, 24 hours after inoculation. Analysis by PDQUEST software could select four protein spots (Spot 1, Spot 2, Spot 3 and Spot 4) from 167 spots based on higher degree of differential expression. The selected spots were sent to Rajiv Gandhi Centre for Biotechnology, Thiruvananthapuram for protein identification by MALDI-TOF. Analysis by MALDI-TOF and MASCOT search could identify 15 hit peptides from the selected four protein spots. Analysis and functional characterization of the 15 hit peptides by BLAST2GO revealed the defense response of tolerant calliclone KLCC 89 against Phytophthora foot rot disease. The enhanced expression of plastocyanin protein, TRAF-like family proteins, RUBISCO dependent glycolate and glyoxylate metabolism, light dependent ROS production during photorespiration, F-box proteins, synthesis of antimicrobial metabolites and retrotransposition activity were observed in the tolerant calliclone KLCC 89 as defense related responses. Plastocyanin is involved in regulation of photosynthesis to meet the requirements of nutrient competition by the P. capsici whereas the TRAF-like family proteins is involved in regulation of programmed cell death. The increment in RUBISCO, regulates the glycolate and glyoxylate metabolism for H2O2 production. F-box proteins are involved in regulation of jasmonate regulated defense-related pathways. The study could characterize PR proteins in the selected calliclones and investigate in depth the Phytophthora capsici interaction in black pepper at proteome level. The future research should focus on validation of identified proteins in defense mechanism, characterization of novel protein in KLCC 89, in-depth investigations on retrotransposons in defense mechanism of Phytophthora foot rot tolerance in black pepper, metabolic engineering of the pathways triggering transcription of PR-genes and transgenic / cisgenic research for Phytophthora foot rot resistance.