Tomato MADS-BOX PROTEIN 22 interacts with AGAMOUS-like-6 to suppress fertilization-independent fruit set

Fruit set and development are key determinants of yield and thus of significant agronomic importance in all edible fruit crops. Fruit set is established during and soon after fertilization of the ovules inside the quiescent ovary, yet the molecular mechanisms underlying it remains obscure. The tomato CRISPR loss-of-function mutant of the MIKCC group MADS-box transcription factor gene AGAMOUS-like6 (SlAGL6) (slagl6CR-sg1) is capable of fertilization-independent setting of normal, yet seedless (parthenocarpic), fruit. To identify SlAGL6 interactors that function in ovule development and parthenocarpy, a yeast two-hybrid library of tomato anthesis ovary was constructed and screened with full-length SlAGL6, resulting in the identification of eight putative MADS-box partnering proteins. Four of them (SlMBP22, SlMBP3, SlAGL11, SlFYFL) were found to be expressed in anthesis ovary ovules and therefore further validated by pull-down assays that validated SlMBP22, SlMBP3, SlAGL11 interaction with SlAGL6. These putative interactors were then functionally analyzed to confirm their role in ovule development and parthenocarpy. CRISPR KO mutants of their encoding genes (slfyflCR-20, slmbp3CR-20 and slmbp22CR-20) were isolated and their phenotypes were compared to that of slagl6CR-sg1. The slfyflCR-20 and slmbp3CR-20 mature ovules were similar to wild-type and they set seeded fruits, suggesting that SlFYFL and SlMBP3 do not function with in ovule development and parthenocarpy. In contrast, the slmbp22CR-20 mature ovules exhibited enlarged integument and failed to differentiate an endothelium, a phenotype that is reminiscent that of slagl6CR-sg1 ovules. Moreover, around 30% of the slmbp22CR-20 fruits were seedless, by their parthenocarpy status is yet to be confirmed. Taken together, our results indicate that SlMBP22 and SlAGL6 function in the same protein complex to regulate ovule integument development.