The Two Tomato Ubiquitin E1 Enzymes Play Unequal Roles in Host Immunity

Plants typically encode multiple ubiquitin-activating enzymes (E1s or UBAs), but their functional equivalence or divergence remains unclear. Here, we demonstrate that the two tomato (Solanum lycopersicum) E1s, SlUBA1 and SlUBA2, differentially regulate development and immunity. Knockdown of SlUBA1 or SlUBA2 caused distinct growth and developmental defects in tomato, while silencing both genes resulted in severe abnormalities, rapid etiolation, and plant death within 5-7 weeks. Notably, silencing SlUBA2, but not SlUBA1, compromised plant immunity against the bacterial pathogen Pseudomonas syringae pv. tomato (Pst). SlUBA1 and SlUBA2 exhibited distinct charging efficiencies for E2s from groups IV (SlUBC32/33/34), V (SlUBC7/14/35/36), VI (SlUBC4/5/6/15) and XII (SlUBC22), with SlUBA2 showing significantly higher efficiency. Swapping the C-terminal ubiquitin-folding domains (UFDs) between SlUBA1 and SlUBA2 largely reversed their E2-charging efficiency for these groups. Furthermore, mutating a key residue (SlUBA2^Q1009) in the UFD or deleting a conserved 13-amino-acid sequence unique to group V E2s altered the E2-charging profiles of both E1s. These findings suggest dual ubiquitin-activating systems (DUAS) operate in tomato. Given the established role of group IV E2s in plant immunity against Pst, the SlUBA2-group IV E2 module likely plays a central role in modulating host defence. Similarly, the Arabidopsis E1s, AtUBA1 and AtUBA2, differentially charge homologues of tomato group IV E2s, suggesting a conserved mechanism by which plant E1s fulfil distinct physiological roles.