**Background:** Jasmonic acid (JA) is a plant hormone that regulates defence against necrotrophic pathogens such as Botrytis cinerea, as well as growth and development. The core JA signalling pathway involves the transcription factor MYC2, which is inhibited by JAZ proteins in the resting state and activated upon JA-Ile perception. MED25, a subunit of the Mediator complex, recruits RNA polymerase II and co-activators to MYC2 target genes. Negative feedback mechanisms, including MTB proteins and alternative splicing of JAZ transcripts, are known to attenuate JA signalling. B-box (BBX) zinc-finger transcription factors regulate photomorphogenesis, stress responses, and pigment accumulation, but their role in JA signalling is largely unexplored. This study investigates the function of SlBBX20 in JA signalling and resistance to B. cinerea in tomato.
**Methods:** The authors generated SlBBX20 overexpression (OE) and CRISPR/Cas9 knockout (KO) lines in tomato (cv. Alisa Craig). SlMED25 OE and KO lines were also generated. Plants were treated with 100 μM MeJA or subjected to mechanical wounding, and expression of JA-responsive genes (JA2L, PI-II, TD) was quantified by RT-qPCR. B. cinerea (strain B05.10) inoculation was performed on whole plants and detached leaves; lesion areas were measured after 3 days using LA-S software. RNA-seq was conducted on SlBBX20-OE, SlBBX20-KO, and WT plants at 0, 1, and 12 h after MeJA treatment (fold change >2, q<0.05). Protein-protein interactions were tested using yeast two-hybrid (Y2H), co-immunoprecipitation (Co-IP) in tobacco protoplasts, and firefly luciferase complementation imaging (LCI) in N. benthamiana. SlMED25 protein levels were quantified by immunoblotting using custom polyclonal antibodies. Dual-luciferase reporter assays were performed with the JA2L promoter. Fruit yield and quality traits (soluble solids, firmness, ascorbic acid) were measured in red ripe fruit.
**Key Results:** SlBBX20 expression was induced within 0.5 h of MeJA treatment and B. cinerea inoculation. SlBBX20-OE lines showed suppressed expression of JA2L, PI-II, and TD under normal conditions, after MeJA treatment, and after wounding, while SlBBX20-KO lines showed elevated expression. Lesion areas after B. cinerea inoculation were 1.7- to 3.9-fold larger in SlBBX20-OE lines than WT, and reduced to 61–69% of WT in SlBBX20-KO lines. MeJA pre-treatment reduced lesion area in WT but not in SlBBX20-OE plants. RNA-seq identified 1778 common DEGs between OE vs WT and KO vs WT before treatment, and 2097 and 4428 DEGs at 1 and 12 h after MeJA, respectively. JA-responsive genes (PI-I, PI-II, TD) were down-regulated in OE and up-regulated in KO lines. Y2H, Co-IP, and LCI assays confirmed that SlBBX20 interacts with SlMED25 via the vWF-A domain (amino acids 1–227). Co-expression of SlBBX20 and SlMED25 in protoplasts and N. benthamiana reduced SlMED25 protein levels. In SlBBX20-OE plants, SlMED25 protein was 53% lower than WT; in SlBBX20-KO plants, it was 2.8-fold higher. SlBBX20 did not affect SlMED25 transcript levels. Dual-luciferase assays showed that SlBBX20 suppressed SlMYC2/SlMED25-mediated activation of the JA2L promoter. In F1 crosses, overexpressing SlMED25 enhanced resistance, but co-overexpression of SlBBX20 and SlMED25 reduced resistance; knocking out SlBBX20 in SlMED25-OE restored resistance. SlBBX20 also interacted with 8 of 11 JAZ proteins and with SlMTB proteins. SlBBX20-KO plants produced significantly higher fruit weight and ascorbic acid content than WT, with no change in fruit number, firmness, or soluble solids.
**Clinical Implications:** This study identifies SlBBX20 as a novel negative regulator of JA signalling that acts by inhibiting SlMED25 protein accumulation. The finding that SlBBX20-KO plants exhibit enhanced resistance to B. cinerea, higher fruit yield, and improved fruit quality (elevated ascorbic acid) positions SlBBX20 as a promising target for CRISPR-Cas9-based crop improvement. The work also provides mechanistic insight into the negative regulation of JA signalling, suggesting that SlBBX20 may form a co-repressor complex with JAZ and MTB proteins. The observation that JA signalling appears more critical than anthocyanin accumulation for B. cinerea resistance is an important conceptual finding. Limitations include the inability to obtain homozygous SlMED25-KO lines, and the lack of field trial data for yield and resistance phenotypes.