PtWRKY20 negatively regulates salt tolerance in citrus rootstock <i>Poncirus trifoliata</i>

Abstract Salt stress severely limits global crop productivity. Poncirus trifoliata, a widely used citrus rootstock valued for its disease resistance, exhibits sensitivity to salt stress, presenting a major constraint for sustainable citriculture. A stepwise screening strategy identified PtWRKY20 as a key negative regulator of salt tolerance in Poncirus trifoliata. Transcript analysis revealed that PtWRKY20 expression is rapidly induced within 1 h of exposure to salt stress, peaks at 3 h, and then gradually declines from 6 to 24 h, before dropping below the control level by 48 h. Subcellular localization confirmed that PtWRKY20 is a nuclear-localized transcription factor. Functional validation via CRISPR/Cas9-mediated knockout in hairy roots demonstrated enhanced salt tolerance in Ptwrky20-KO plants, evidenced by reduced chlorosis, higher chlorophyll content, lower electrolyte leakage, and decreased malondialdehyde accumulation under salt stress. Conversely, PtWRKY20 overexpression increased sensitivity, showing opposite physiological trends. Integrated DNA affinity purification sequencing and RNA-seq analyses, validated by yeast one-hybrid and dual-luciferase assays, identified 10 direct target genes bound and transcriptionally regulated by PtWRKY20, including six transcription factor genes (e.g., PtMYB78), a sugar metabolism gene (PtHXK3), an ion homeostasis mediator (PtAMT2) and 2 unannotated genes. Our findings establish PtWRKY20 as a negative regulator of the salt stress response and suggest that its targeted knockout via rootstock-adapted CRISPR is a promising strategy for developing salt-tolerant citrus rootstocks.