Identification of Key Residues Essential for the Activation of Plant Immunity by Subtilisin From Bacillus velezensis LJ02

Abstract

Subtilisin, a serine protease, can trigger defense responses in a wide variety of plants, both locally and systemically, to protect against pathogens. However, key residues of subtilisin to improve resistance to plant diseases remain unknown. In this study, Nicotiana benthamiana (N. benthamiana) leaves expressing subtilisin from Bacillus velezensis LJ02 were shown to improve protection against Botrytis cinerea (B. cinerea). Furthermore, the underlying mechanism that LJ02 subtilisin improved the protective effect was explored, and the direct inhibitory effect of subtilisin on B. cinerea was excluded in vitro. Subsequently, reactive oxygen species (ROS) burst and upregulation of resistance-related genes in systemic leaves of N. benthamiana further verified that subtilisin could induce systemic protection against B. cinerea. G307A/T308A and S213A/L214A/G215A subtilisin significantly reduced the ability to resist B. cinerea infection in N. benthamiana. Furthermore, the ROS content and expression levels of resistance-related genes of both mutants were significantly decreased compared with that of wild-type subtilisin. This work identified key residues essential for the activation function of subtilisin plant immunity and was crucial in inducing plant defense responses against B. cinerea.

A novel elicitor protein phosphopentomutase from Bacillus velezensis LJ02 enhances tomato resistance to Botrytis cinerea

“…The systemic leaves of tomato were collected and inoculated with B. cinerea mycelium discs at 2 days after infiltration. The diameter of lesions on tomato leaves was measured by the criss-cross method ( Hu et al., 2022 ) and photographed at 3 days after inoculation (DAI).…”

“…The concentration was determined by Bradford method and recorded. The protein expression, purification and detection method was described previously ( Hu et al., 2022 ).…”

“…The tomato leaves were inoculated with B. cinerea mycelium disc, the inoculation method was described previously ( Wang et al., 2016 ). The leaves were placed in a plate containing wet filter paper to maintain high-humidity, then the plate was placed in a phytotron at 25°C for 3 d. The lesion diameter was measured ( Hu et al., 2022 ) and photographed at 3 DAI.…”

“…The ROS detection method was described previously ( Hu et al., 2022 ). The ROS content in the fruits was determined by adding 0.1 g of tomato fruit powder to phosphate buffered saline (pH 7.4) followed by a commercial plant ROS enzyme-linked immunosorbent assay (ELISA) kit (Chundubio).…”

“…Bacillus velezensis ( B.velezensis ) LJ02, as a beneficial microorganism, can enhance plant resistance to B. cinerea and other pathogens, and can also secrete elicitor proteins to induce plant immune responses ( Li et al., 2015 ; Hu et al., 2022 ). In this study, the new elicitor protein BvEP, which can improve the control effect of tomatoes on B. cinerea , was selected from B. velezensis LJ02.…”