Tomato DOR encodes a signal peptide peptidase-like protein required for adventitious organogenesis and rooting

The identification of the tomato DOR gene that is essential for adventitious organogenesis provides a genetic target for improving regeneration efficiency and root system performance in tissue culture and plant breeding. Plant rooting and adventitious organogenesis represent major bottlenecks in the application of plant tissue culture techniques. In this study, we characterize the dor mutant (defective in organogenesis and rooting) identified in our collection of tomato T-DNA lines. This mutant exhibits normal callus proliferation but fails to differentiate adventitious buds. Additionally, it displays underdeveloped embryonic roots, and its adventitious roots derived from various explants are also altered. Grafting experiments revealed compromised in vivo development primarily due to its abnormal root system. Upon identifying an allelic mutant in another tomato line, we observed no co-segregation between a T-DNA insert and the phenotype in either of the identified allelic mutants. Through mapping-by-sequencing, we identified Solyc12g098670 as the gene responsible for this mutation, which is homologous to SIGNAL PEPTIDE PEPTIDASE-LIKE (SPPL) genes from Arabidopsis thaliana, particularly SPPL3 and SPPL5. The expression pattern of DOR in tomato is nearly ubiquitous, similar to SPPL3 in Arabidopsis, yet the regeneration and rooting of sppl3 Arabidopsis mutants resemble the wild-type. In both tomato and Solanum pennellii (Correll) D'Arcy, RNAi lines and plants edited by CRISPR/Cas exhibit a dor mutant phenotype. However, by increasing the expression level of DOR, the plants become indistinguishable from the wild-type in terms of in vitro and in vivo development. Overexpression of this gene in the mutant has enabled the regeneration of plants with a wild-type phenotype. These results demonstrate that DOR is the first member of the tomato SPPL gene family known to be associated with root development and adventitious organogenesis.