Association of Rho4 Gene Deletion in Penicillium expansum with Activation of Phenylpropanoid Metabolism at the Disease-Health Interface of Apple Fruits

To investigate the role of Rho4, a small GTPase, in host resistance during fruit-pathogen interactions, we inoculated apple fruits separately with a wild-type (WT) strain, a Rho4-deleted mutant (ΔPeRho4) and a complemented strain (ΔPeRho4-C) of Penicillium expansum. The lesion size was assessed, and transcriptomic analysis of tissues at the disease-health interface was performed to identify differentially expressed genes (DEGs). The expression and activity of key enzymes involved in phenylpropanoid metabolism were measured and changes in related metabolites were examined. Our results showed that fruits inoculated with ΔPeRho4 exhibited significantly smaller lesions compared with those inoculated with the WT strain, and the lesion size of ΔPeRho4-C was similar to the WT level. Transcriptomic profiling identified 216 DEGs, among which four were associated with the phenylpropanoid pathway and up-regulated in ΔPeRho4 inoculated tissues. Moreover, ΔPeRho4 inoculation significantly increased the expression and activities of phenylalanine ammonia lyase (PAL), cinnamate-4-hydroxylase (C4H), 4-coumaroyl-coenzyme A ligase (4CL), and cinnamyl alcohol dehydrogenase (CAD). It also promoted the accumulation of phenolic acids, lignin monomers, total phenolics, flavonoids, and lignin. These results suggest that the deletion of Rho4 attenuated the pathogenicity of P. expansum, which may indirectly trigger the activation of phenylpropanoid metabolism at the disease-health interface and enhance defense responses in apple fruits. This study provides new insights into the molecular mechanisms by which fungal small GTPases influence host resistance and offers potential strategies for postharvest disease control.