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Fruit softening in tomato (Solanum lycopersicum) is closely associated with cell wall disassembly, which is brought about through the action of a range of cell wall structure-related enzymes and other proteins such as expansins. Xyloglucan endotransglucosylase/hydrolase (XTH) (EC 2.4.1.207 and/or EC 3.2.1.151) has been proposed to be key player involved in xyloglucan metabolism. SlXTH5 showed the highest expression level among all SlXTHs during tomato ripening. In this study, the role of SlXTH5 involved in tomato softening was investigated in CRISPR-based knockout mutants of SlXTH5. Loss-of-function of SlXTH5 in transgenic tomato lines resulted in slightly firmer fruit pericarp, but significantly decreased their color index compared with azygous wild type (WT) control fruits. Increased paste viscosity was detected in CRISPR mutants, indicating that the activity of SlXTH5 is responsible for maintaining cell wall structural integrity. Immunocytochemistry studies were performed using the monoclonal antibody probe LM25 to examine the localization and distribution of xyloglucan in the pericarp cells of the CRISPR mutant fruits. The data indicated more xyloglucan was retained in the pericarp of CRISPR mutant fruit than in WT control fruit. This study revealed the link between SlXTH5 and xyloglucan metabolism and indicated the potential of manipulating SlXTH5 to regulate fruit softening. [ABSTRACT FROM AUTHOR]