The circular RNA circP5CS1 coordinates plant immunity by sequestering miR167 to modulate ARF6/8 signaling and...

Circular RNAs (circRNAs) are a type of noncoding RNA formed by backsplicing, characterized by covalently closed loop structures with enhanced stability. Although growing evidence highlights their regulatory roles in plants, their precise biological functions remain largely unclear. In this study, we identified Arabidopsis thaliana circP5CS1, a circRNA derived from the delta-1-pyrroline-5-carboxylate synthase (P5CS1) gene through backsplicing of exons 11-13. The junction between exons 11 and 13 generates a de novo miR167 binding site, confirmed by sequence alignment and RNA pull-down assays, indicating that circP5CS1 preferentially binds miR167a/b. circP5CS1 acts as a miR167 sponge to promote the expression of auxin response factor 6 (ARF6) and ARF8, fine-tuning salicylic acid- and jasmonic acid-mediated immunity during infection by Pseudomonas syringae pv. tomato DC3000. In addition, circP5CS1 suppresses its host gene P5CS1, a rate-limiting enzyme for proline biosynthesis, through both splicing competition and post-transcriptional mechanisms, thus linking circRNA biogenesis with the control of gene expression. Our results suggest that circP5CS1 directly modulates plant immunity through both hormone and proline pathways. circP5CS1 expression can be induced by both P. syringae pv. tomato DC3000 and AvrRpt2 infection. Accordingly, resistance to these pathogens is compromised in circP5CS1-overexpression (OE), p5cs1, and miR167 short tandem target mimic plants, whereas circP5CS1-RNAi, P5CS1-1-OE, and miR167-OE plants show enhanced resistance. Proline levels are increased in P5CS1-1-OE and circP5CS1-RNAi plants but reduced in circP5CS1-OE and p5cs1 mutants, supporting a link between circP5CS1 activity and proline metabolism. Our findings suggest that circP5CS1 may be an immune regulatory hub that integrates hormone signaling and proline homeostasis through microRNA sponging and host-gene suppression, revealing its dual regulatory role in coordinating RNA-mediated regulation with plant immune response.