A Rapid Method for Screening Pathogen-Associated Molecular Pattern-Triggered Immunity-Intensifying Microbes

Abstract

PAMP-triggered immunity (PTI) is the first layer of plant defense response that occurs on the plant plasma membrane. Recently, the application of a rhizobacterium, Bacillus amyloliquefaciens strain PMB05, has been demonstrated to enhance flg22Pst- or harpin-triggered PTI response such as callose deposition. This PTI intensification by PMB05 further contributes to plant disease resistance to different bacterial diseases. Under the demand for rapid and large-scale screening, it has become critical to establish a non-staining technology to identify microbial strains that can enhance PTI responses. Firstly, we confirmed that the expression of the GSL5 gene, which is required for callose synthesis, can be enhanced by PMB05 during PTI activation triggered by flg22 or PopW (a harpin from Ralstonia solanacearum). The promoter region of the GSL5 gene was further cloned and fused to the coding sequence of gfp. The constructed fragments were used to generate transgenic Arabidopsis plants through a plant transformation vector. The transgenic lines of AtGSL5-GFP were obtained. The analysis was performed by infiltrating flg22Pst or PopW in one homozygous line, and the results exhibited that the green fluorescent signals were observed until after 8 h. In addition, the PopW-induced fluorescent signal was significantly enhanced in the co-treatment with PMB05 at 4 h after inoculation. Furthermore, by using AtGSL5-GFP to analyze 13 Bacillus spp. strains, the regulation of PopW-induced fluorescent signal was observed. And, the regulation of these fluorescent signals was similar to that performed by callose staining. More importantly, the Bacillus strains that enhance PopW-induced fluorescent signals would be more effective in reducing the occurrence of bacterial wilt. Taken together, the technique by using AtGSL5-GFP would be a promising platform to screen plant immunity-intensifying microbes to control bacterial wilt.