Fvchli deficiency impairs ABA-mediated stomatal closure and enhances susceptibility to Xanthomonas fragariae in strawberry.

The magnesium chelatase subunit CHLI is well-characterized in chlorophyll biosynthesis, but its role in plant immunity remains unclear. In this study, we generated chimeric Fvchli mutant strawberries via CRISPR/Cas9 and obtained T1-T3 progeny with a wide range of editing efficiencies through self-pollination. Mutants with higher editing efficiency exhibited more leaf spots. Following inoculation with Xanthomonas fragariae, Fvchli mutants showed increased susceptibility, with both lesion area and disease incidence positively correlating with editing efficiency. We further demonstrated that stomata in Fvchli mutants closed significantly more slowly in response to ABA compared to the WT. After 1.5 h of ABA treatment, the stomatal aperture (width-to-length ratio) of the wild type (WT) decreased from 32 % to 2.3 %, whereas in the mutant line with 70 % editing efficiency, it only reduced from 32 % to 16 %. In the edited plants, the expression levels of genes related to ABA biosynthesis and signal transduction were significantly lower than those in the wild type. After pathogen infection, ABA levels increased in both WT and mutant plants, but remained significantly lower in Fvchli mutant. Scanning electron microscopy revealed that Fvchli mutants had wider stomatal apertures at 5 and 12 hpi, facilitating extensive bacterial colonization, whereas WT plants effectively restricted invasion through stomatal closure. Our results reveal a previously unrecognized role for Fvchli in stomatal immunity, where its deficiency impairs ABA accumulation and responsiveness, compromising stomatal defense and promoting bacterial infection.