Ecology, Environment and Conservation Paper

Vol 27, May Suppl. Issue, 2021; Page No.(390-397)

CLONING, EXPRESSION AND SPECIFICITY EVALUATION OF TYPE III EFFECTOR, RIP4, FROM RALSTONIA SOLANACEARUM

Shalini Bhatt, Neha Faridi, S. Merwyn P. Raj, Dinesh Pathak, Ankur Agarwal, P. Thiru Vardhana Rao and Madhu Bala

Abstract

Ralstonia solanacearum is a Gram-negative bacterial phytopathogen that belongs to the class of beta proteobacterium. It causes one of the most commercially devastating diseases in plants, known as bacterial wilt disease. The bacterium infects a variety of crops and to date the host range has reached to more than 450 species of plants globally. Various Gram-negative phytopathogens interact with their hosts using Type three secretion systems (TTSS) through which it can inject an array of protein known as effectors. These proteins play an important role in the pathogenicity as well as virulence of the bacterium and can be useful in early diagnosis of the pathogen. Although, several detection methods based on biochemical, molecular and immunological techniques are available for the pathogen identification, but most of them are expensive and time consuming. Hence, keeping above in mind this study was aimed with the cloning and expression of gene RSc0321 encoding an important Type III effector (TTE) protein, Ralstonia injected protein 4 (Rip4) of R. solanacearum to produce recombinant Rip4 protein (r-Rip4). The recombinant protein was purified by affinity chromatography using nickel-nitrilotriacetic acid (Ni-NTA) column and further polyclonal antibodies were raised against the r-Rip4. Moreover, the raised antibodies were evaluated for its specificity using Indirect ELISA and Western Blotting. The results generated in the present study, would be helpful for the development of quick and easy immunological detection system for R. solanacearum.