Spray-induced gene silencing targeting ZmHSF20 enhances maize thermotolerance

Spray-induced gene silencing (SIGS) is an emerging, time-efficient, and eco-friendly strategy for crop trait manipulation. Heat stress severely threatens global maize production; however, whether SIGS can be effectively applied to improve maize heat tolerance remains largely unexplored. The transcription factor ZmHSF20, a class B2a heat shock factor, has been identified as a negative regulator of maize thermotolerance that represses the ZmHSF4-ZmCESA2 module (Li et al. 2024), yet whether its silencing via a non-transgenic RNA-based approach can confer heat protection is unknown. Here, we designed a specific artificial small RNA atsRNA-ZmHSF20 targeting ZmHSF20 and evaluated its potential in improving maize thermotolerance via the SIGS approach. Compared with untreated plants, foliar application of atsRNA-ZmHSF20 significantly enhanced maize heat tolerance, leading to a remarkable increase in survival rate from 7.94% to 41.37% (~ 5.2-fold improvement). Physiological analyses revealed that exogenous application of atsRNA-ZmHSF20 mitigated heat-induced oxidative damage, as evidenced by reduced ROS accumulation, ion leakage (IL), and malondialdehyde (MDA) content. At the molecular level, downregulating ZmHSF20 prior to heat stress pro-conditioned the plants for thermal tolerance by activating the previously characterized ZmHSF4-ZmCESA2 module, transcriptionally upregulating cell wall organization and cellulose biosynthesis pathways. This study provides an initial proof-of-concept for harnessing SIGS to enhance maize abiotic stress tolerance and offers a promising non-transgenic strategy for future applications in enhancing crop thermotolerance.

The online version contains supplementary material available at 10.1007/s11032-026-01698-x.