CRISPR/Cas9-targeted mutagenesis ofSlCMT4causes changes in plant architecture and reproductive organs in tomato

DNA methylation participates widely in the regulation of gene expression in plants. To date, the regulation and function of DNA methylation is still unknown in tomato plants. Here, we generated the SlCMT4 mutants using CRISPR-Cas9 gene editing system and observed severe developmental defects including small and thick leaves, increased lateral buds, defective stamens and pistils, small fruit size with reduced setting rate, and defective seed development in CRISPR-Cas9 mediated SlCMT4 mutants. The alterations at hormonal levels (IAA, tZR, strigol) were consistent with the multi-branching phenotype in SlCMT4 mutant plants. CRISPR-Cas9-mediated knockout of SlCMT4 induced the expression of two pollen-specific genes (PMEI and PRALF), suppressed the development of pollen wall and pollen tube elongation, which is responsible for irregular and defective pollen. Small sized-fruit phenotype probably is associated with up-regulated expression of IMA gene and reduced seeds in the mutant lines. Furthermore, we performed whole-genome bisulfite sequencing (WGBS) of fruits and found SlCMT4 knockout reduced genome-wide cytosine methylation. A reduction of methylation was also observed in 2-kp region of the IMA and LOXB promoters in the SlCMT4-mutant fruits, indicating that the hypermethylation status of the CHH context is critical for the inhibition of IMA and LOXB promoter activity. Our results depict that SlCMT4 is required for normal development of tomato vegetative and reproductive organs. This study illuminates the function of SlCMT4 and shed light on the molecular regulatory mechanism of tomato plant architecture, fruit development and ripening.