Characterisation of Pathogenesis related proteins for anthracnose resistance in vegetable cowpea, Vigna spp.

Abstract

Cowpea (Family: Fabaceae) is an important pulse cum vegetable crop of suitable for the tropical and sub-tropical regions of the world. The grain type cowpeas better tolerates the biotic and abiotic stresses against the vegetable types. Under humid conditions, vegetable types, especially the pole types are susceptible to many diseases and among them, anthracnose caused by Colletotrichum lindemuthianum (Sacc. & Magn.) Br. and Cav. is very severe. In Kerala, complete yield loss in vegetable cowpea is reported due to anthracnose during monsoons. The study entitled “Characterization of pathogenesis related proteins for anthracnose resistance in vegetable cowpea, Vigna spp.” was carried out with objective to develop the protein profiles of resistant and susceptible bush and pole genotypes through SDS-PAGE analysis at different time intervals of infection and to characterize the differentially expressed proteins by MALDI-TOF followed by in-silico analyses. Two bush type varieties Pusa Komal and Kanakamony, the former reported to be highly susceptible and the latter immune to anthracnose and two pole type varieties Lola and Arimbra Local, of which the former susceptible and the latter resistant were used in the study. Pure culture of the pathogenic fungus was developed and maintained on selective medium (Neopeptone-Glucose-Agar) at the Dept. of Plant Pathology. The identity of Colletotrichum lindemuthianum has been established from the spore characteristics observed under phase contrast microscope and the pathogenicity was confirmed through artificial inoculation under controlled conditions. The pot culture experiment was conducted with 50 pots per variety. Artificial inoculation of pathogenic fungus was done and the leaf samples were collected at 0, 6, 12, 18, 24, 48, 72, 96, 120, 144,168 and 192 hours after artificial inoculation. The total protein was extracted using Tris-HCl buffer (pH-7.5), quantified using spectrophotometer and analyzed by SDS-PAGE method. The defense enzymes like peroxidase (PO), polyphenol oxidase (PPO) and phenylalanine ammonia-lyase (PAL) were assayed. By artificial inoculation, disease responses for anthracnose were confirmed to be highly susceptible in Pusa Komal and Lola; highly resistant in Arimbra Local and immune in Kanakamony. Protein expression was found to be higher from the initial hours in resistant varieties whereas in susceptible varieties, the expression was reduced immediately after infection then peaked at 18hr and gradually decreased later on. Two prominent and differentially expressed protein bands at 56 kD and 14 kD were sequenced in MALDI-TOF to obtain the peptide mass fingerprint. Through in-silico analyses using Mascot server software, they were identified to be the large and small subunits of the chloroplastic enzyme RuBisCo. Thus the capability of a variety to maintain high levels of RuBisCo was found to be the deciding factor for anthracnose disease resistance. Further, protein profiles developed after purification of proteins by dialysis have clearly identified the differentially expressed band at 29 kD in the resistant varieties which is in the size range of already reported PR proteins. PO and PAL activities were proportionate to the resistance behavior, with the peak values at 18 and 24 hr after inoculation. With the results of this study, these defense enzymes are recommended as biochemical markers for identifying the resistance in the accessions. Capability to maintain higher levels of RuBisCo, PO and PAL enzymes is the characteristic of anthracnose resistant vegetable cowpeas and the future breeding programmes could be oriented in this direction