Biomolecular condensation of two RNA binding proteins modulates plant NLR immunity

Biomolecular phase separation is emerging as a critical mechanism for regulating diverse biochemical processes, yet its role in maintaining immune homeostasis remains poorly understood. In Arabidopsis, disruption of the MEKK1–MKK1/2–MPK4 MAPK cascade triggers NLR SUMM2-mediated autoimmunity by activating the transcription of the MAPK kinase kinase MEKK2. To understand the mechanisms underlying this MAPK cascade-mediated immune homeostasis, we performed an RNA interference-based genetic screen for suppressors of mekk1 cell death and identified several lethality suppressors of mekk1 (let). Among them, LET8, an RNA-recognition motif (RRM)-type RNA-binding protein, modulates SUMM2-mediated autoimmunity by regulating MEKK2 pre-mRNA processing. Interestingly, LET8 contains two intrinsically disordered regions (IDRs): one promotes nuclear biomolecular condensate formation upon immune activation, while the other inhibits condensate formation under normal growth conditions. This dual functionality ensures that the phase separation process is tightly regulated to maintain immune homeostasis.  LET8 functions in a complex with Cleavage and Polyadenylation Specificity Factor 30 (CPSF30), a zinc-finger protein involved in pre-mRNA processing. Together, LET8 and CPSF30 undergo co-phase separation in vivo and in vitro, facilitating the binding and processing of a specific intron in MEKK2 pre-mRNA. In the absence of either LET8 or CPSF30, MEKK2 mRNA containing a cryptic intron is rapidly degraded. Notably, LET8 exhibits strong RNase activity, and mutations in the RNA cleavage active sites of LET8 or CPSF30 severely impair their functions. Our findings uncover a novel regulatory mechanism involving immune-induced biomolecular condensation that modulates LET8 and CPSF30 RNase activity in MEKK2 mRNA processing, thereby regulating NLR immunity.