The engineering and application of multiepitope recombinant proteins (MEPs) enhanced resistance to Botrytis cinerea in tomatoes, presenting a new paradigm in the creation of plant immune activators

Xiaixa Man ¹ Huang You ¹ Zhi-Qiang Cheng ¹ Jun-Hao Li ¹ Dun-Chao Yao ¹ Hao-Feng Wang ¹ Zhi-Hong Diao ¹ Xiao-Song Yu ¹ Wei Wu ² Cheng Zhou ² YAN HUANG ¹ Jinbo SHEN ³ Xiaohong Zhuang ⁴ Yi Cai ^1*

• ¹ Sichuan Agricultural University, Ya'an, China
• ² Chengdu Lusyno Biotechnology Co., Ltd, Chengdu, China
• ³ Zhejiang Agriculture and Forestry University, Hangzhou, China
• ⁴ The Chinese University of Hong Kong, Shatin, Hong Kong Region, China

Plant elicitors have emerged as key agents in effectively invoking immune responses across various plant species, gaining attention for their role in sustainable agricultural protection strategies.Nevertheless, the utility of peptide elicitors such as flg22, flgII-28, and systemin is economically limited when considering broader agricultural applications. This study presents a novel recombinant protein, SlRP5, which integrates five active epitopes of flg22, csp22, flgII-28, SIPIP1 and systemin to activate immune responses and significantly enhanced resistance to Botrytis cinerea in tomatoes (Solanum lycopersicum). SlRP5 significantly induced reactive oxygen species (ROS), MAPK phosphorylation, and callose deposition in tomato leaves during in vitro experiments.Transcriptomic analysis revealed that SlRP5 more effectively activated key immune-related pathways compared to traditional peptides, upregulating critical genes involved in calcium-binding proteins and phenylpropanoid biosynthesis. In further in vivo experiments, SlRP5 alleviated B. cinerea-induced membrane damage by reducing MDA and REC levels, while simultaneously enhancing the activities of antioxidant enzymes such as SOD, CAT, and POD to mitigate the excess ROS generated by infection. Additionally, SlRP5 elicited significant immunological effects in tobacco and eggplant characterized by ROS bursts and callose deposition, amplified tobacco's resistance to the TMV, and mitigated B. cinerea-induced damage in eggplant. These findings underscore the substantial potential of SlRP5 as a plant immune activator, integrating multiple immune-eliciting peptides, offering a novel strategy for the cultivation of new biopesticides that can induce immune responses and heighten disease resistance in various crops.