miR398-SlCSD1 module participates in the SA-H2O2 amplifying feedback loop in Solanum lycopersicum

Abstract

Introduction

Salicylic acid (SA) is essential for immune response signal transduction in higher plants, with its signaling thought to be enhanced through interactions with reactive oxygen species (ROS). However, the exact mechanisms behind this SA self-amplifying signaling are still not well understood.

Objectives

In this study, we report the involvement of the miR398b-SlCSD1 module in the SA-H₂O₂ amplifying feedback loop in tomato (Solanum lycopersicum).

Methods

Experiments were conducted using various concentrations of SA to assess its impact on ROS metabolism and the expression of SlCSD1 and sly-miR398. CRISPR/Cas9 was employed to knock out sly-miR398 and SlCSD1. Bioinformatics analyses, dual-luciferase reporter assays (Dual-Luc), and electrophoretic mobility shift assays (EMSA) were used to identify SA-responsive transcription factors and validate their regulation of sly-miR398b. The role of miR398 in endogenous SA synthesis was examined using overexpression and knockout tomato lines.

Results

Low SA concentrations stimulated H₂O₂ accumulation, increased superoxide dismutase (SOD) activity, and suppressed sly-miR398 expression, effects absent in NahG plants with reduced SA levels. Knockout of SlCSD1 via CRISPR/Cas9 partially inhibited SA-induced H₂O₂ accumulation, confirming SlCSD1′s role in SA-dependent H₂O₂ signaling. Furthermore, Dual-Luc and EMSA results revealed that TGACG-sequence-specific binding protein 2 (TGA2) mediated the regulation of miR398-SlCSD1 module by SA in tomato. Additionally, overexpression and mutation of sly-miR398b promoted SA synthesis via the phenylalanine ammonia-lyase (PAL) and isochorismate synthase (ICS) pathways, highlighting its regulatory role in SA biosynthesis.

Conclusion

Taken together, our results shed light on the involvement of the miR398-SlCSD1 module in the SA-H₂O₂ amplifying feedback loop, providing new insights into SA signaling in tomato. These findings contribute to understanding SA-ROS interactions and offer a potential strategy for enhancing stress tolerance in crops by targeting microRNA-regulated pathways.