# Circular RNA Vv‐circCOR27 modulates thermotolerance through attenuating VvHSP90.2b‐VvHsfA7a interaction in grapevine

## TL;DR

A circular RNA called Vv-circCOR27 in grapevines reduces heat tolerance by blocking a key protein interaction.

## Contribution

This study reveals a novel regulatory mechanism of thermotolerance in grapevines via a circRNA that inhibits a protein interaction.

## Key findings

- Vv-circCOR27 expression is suppressed under heat stress and is lower in thermotolerant grapevine cultivars.
- Overexpression of Vv-circCOR27 worsens thermotolerance by inhibiting the interaction between VvHsfA7a and VvHSP90.2b.
- Vv-circCOR27 binds directly to VvHSP90.2b, leading to downregulation of small heat shock protein genes under heat stress.

## Abstract

Circular RNAs (circRNAs) are single‐stranded, covalently closed RNA molecules that arise from exon back‐splicing. The identification and function investigation of circRNAs have been comprehensively explored in plants, however, the regulatory mechanisms are largely unknown.This study employed genetic transformation, circRNA pull‐down assay, ribonucleoprotein immunoprecipitation (RIP), and the circRNA trimolecular fluorescence complementation (cTriFC) system to investigate the function and regulatory mechanisms of a circRNA Vv‐circCOR27 in grapevine.
Vv‐circCOR27 expression was suppressed under heat stress and was markedly lower in thermotolerant cultivars. In addition, overexpression of Vv‐circCOR27, using an expression cassette with endogenous introns inserted into circRNA‐producing exons to minimize the background level of cognate linear RNA, was shown to exacerbate thermotolerance in grapevine embryogenic calli and seedlings. Biochemical assays demonstrate that Vv‐circCOR27 binds directly to heat shock protein 90.2b (VvHSP90.2b). Further evidence indicated that Vv‐circCOR27 inhibits the interaction between VvHsfA7a and VvHSP90.2b in vivo, which resulted in the downregulation of small heat shock protein (sHSP) genes under heat stress in Vv‐circCOR27 overexpression calli.Collectively, this finding suggests a model in which thermotolerance is fine‐tuned by Vv‐circCOR27 via competitively binding to VvHSP90.2b, thereby attenuating its interaction with VvHsfA7a and subsequent transactivation. This work advances understanding of circRNA regulatory mechanisms in plants.

Circular RNAs (circRNAs) are single‐stranded, covalently closed RNA molecules that arise from exon back‐splicing. The identification and function investigation of circRNAs have been comprehensively explored in plants, however, the regulatory mechanisms are largely unknown.

This study employed genetic transformation, circRNA pull‐down assay, ribonucleoprotein immunoprecipitation (RIP), and the circRNA trimolecular fluorescence complementation (cTriFC) system to investigate the function and regulatory mechanisms of a circRNA Vv‐circCOR27 in grapevine.

Vv‐circCOR27 expression was suppressed under heat stress and was markedly lower in thermotolerant cultivars. In addition, overexpression of Vv‐circCOR27, using an expression cassette with endogenous introns inserted into circRNA‐producing exons to minimize the background level of cognate linear RNA, was shown to exacerbate thermotolerance in grapevine embryogenic calli and seedlings. Biochemical assays demonstrate that Vv‐circCOR27 binds directly to heat shock protein 90.2b (VvHSP90.2b). Further evidence indicated that Vv‐circCOR27 inhibits the interaction between VvHsfA7a and VvHSP90.2b in vivo, which resulted in the downregulation of small heat shock protein (sHSP) genes under heat stress in Vv‐circCOR27 overexpression calli.

Collectively, this finding suggests a model in which thermotolerance is fine‐tuned by Vv‐circCOR27 via competitively binding to VvHSP90.2b, thereby attenuating its interaction with VvHsfA7a and subsequent transactivation. This work advances understanding of circRNA regulatory mechanisms in plants.