Structural studies of Ralstonia solanacearum type3 effectors and RipG4.

Abstract

Most of organisms in earth cannot live without plants. However, there are hundreds of pathogens all around plants. To defend from these pathogens, plants have evolved their own innate immune system, because they cannot move by themselves actively. Especially, many pathogenic organisms can invade plants and cause wilting or other disease symptoms. Plants can recognize microorganisms’ invading through their resistance (R) genes. As responding to invading, they can trigger their own innate immune system, called ‘Zig-zag’ model. ‘Zig-zag’ model is mainly composed of two major steps. First step is a PAMP-Triggered Immunity (PTI) after recognizing Pathogen Associated Molecular Patterns (PAMPs). Second step is an Effector-Triggered Immunity (ETI), responding to effector proteins, which are secreted from the bacteria and injected through a Type3 Secretion System (T3SS) to suppress PTI. Effectors can be modified as time passes to evade ETI. Ralstonia solanaearum is the major pathogenic bacteria for plant wilting and exerts its pathogenicity through more than a hundred secreted effector proteins. Invading process is mostly achieved through T3SS. These gram-negative bacteria are considered as one of the most destructive plant pathogenic bacterium worldwide. Effector proteins, which are secreted from Ralstonia solanacearum, are called the Rip (Ralstonia injected proteins) effectors. Basically, determining three-dimensional protein structure aims to understanding protein activity, function and interactions. Even if overall plant innate immune system is well described, specific interaction mechanisms between Rip effectors and resistance proteins in plants are not well known. To understand this mechanism, structural study of Rip effectors are essential. Therefore, I purified several Rip effectors with high purity and tried crystallization. RipG4 belongs to GALA family, type3 effectors from Ralstonia solanacearum and is one of the purified Rip effectors. GALA family have been shown to be collectively required for disease on Arabidopsis thaliana and tomato. GALA family are conserved well within Ralstonia solanacearum species, and RipG4 exist in all bacterial strains. RipG4 function is reported to interfere with callose deposition. To understand how RipG4 protein can interfere with callose deposition through structural study, I purified and made initial crystals of RipG4 protein. Furthermore, gel-filtration analysis was performed to understand RipG4 protein characteristics.