Exocarp-specific expression of a fungal cutinase in tomato fruits alters cuticle ultrastructure, chemistry and nanomechanics

Fruit cuticles control water and gas diffusion and protect against biotic and environmental stresses. The cuticle is built from the cutin polymer-a composite of C16 and C18 ω-hydroxy fatty acids that are linked via ester bonds, embedded polysaccharides, and phenolics-as well as waxes made primarily from very-long-chain fatty acids that are deposited on the cuticle and incorporated within the cutin matrix. Considerable progress toward understanding fruit cuticle function has been achieved in recent years, but knowledge gaps remain regarding the biosynthesis and assembly of the cuticular constituents and how these processes are linked to the cuticle's macromolecular architecture and nanomechanical properties. Herein, we generated transgenic tomatoes (Solanum lycopersicum) that express, in an exocarp-specific manner, the Fusarium oxysporum f.sp. lycopersici fungal cutinase gene that degrades the host plant cuticle during infection via breakdown of ester bonds between cutin monomers. To track the consequences of this genetic manipulation on fruit cuticle ultrastructure, chemistry and surface behavior, we coordinated information from microscopy, chemical profiling, nanomechanics, biophysical measurements, and transcriptomics. The transgenic fruits deposited unexpectedly thicker cuticles with elevated cutin and wax content as well as distinctive surface cracks and suberized wound periderms; they had enhanced roughness, elastic modulus, stiffness, and adhesion of the cuticle surface; and they showed wide-ranging alterations in gene expression, phase state, and permeability. Taken together, our findings provide valuable knowledge on how the shifted balance among wax, cutin, and suberin that is triggered by exocarp-specific genetic modifications can remodel the fruit cuticle ultrastructure, chemistry and nanomechanics.