Proteases represent one of the most abundant classes of enzymes in eukaryotes and are known to play key roles in many biological processes in plants. However, little is known about their functions in fruit ripening and disease resistance, which are unique to flowering plants and required for seed maturation and dispersal. Elucidating the genetic mechanisms of fruit ripening and disease resistance is an important goal given the biological and dietary significance of fruit.Through expression profile analyses of genes encoding tomato (Solanum lycopersicum) cysteine proteases, we identify a number of genes whose expression increases during fruit ripening. RNA interference (RNAi)-mediated repression of SlVPE3, a vacuolar protease gene, results in alterations in fruit pigmentation, lycopene biosynthesis, and ethylene production, suggesting that SlVPE3 is necessary for normal fruit ripening. Surprisingly, the SlVPE3 RNAi fruit are more susceptible to the necrotrophic pathogen Botrytis cinerea. Quantitative proteomic analysis identified 314 proteins that differentially accumulate upon SlVPE3 silencing, including proteins associated with fruit ripening and disease resistance. To identify the direct SlVPE3 targets and mechanisms contributing to fungal pathogen resistance, we perform a screening of SlVPE3-interacting proteins using co-immunoprecipitation coupled with mass spectrometry. We show that SlVPE3 is required for the cleavage of the serine protease inhibitor KTI4, which contributes to resistance against the fungal pathogen B. cinerea.Our findings contribute to elucidating gene regulatory networks and mechanisms that control fruit ripening and disease resistance responses.

Reactive oxygen species (ROS) play a vital role in reducing the viability of invading pathogens during plant-pathogen interactions. To understand how oxidative stress caused by ROS reduces cell viability, it is important to identify the proteins affected by ROS. In the present study, we investigated the changes in the expression of proteins from the outer and inner membrane fractions in Pseudomonas syringae pv tomato DC3000 under oxidative stress through membrane subproteomics. A total number of 17 differentially expressed proteins from the outer and inner membrane fractions were identified, among which 11 proteins belong to transporters, such as porins and ABC transporters. Their abundance was all decreased under oxidative stress, indicating that transporters are likely to be affected by oxidative stress. The function of two identified transporters was further characterized by constructing their gene mutant and overexpression strains. We found that mutation of one transporter gene PSPTO_1720 rendered Pseudomonas more sensitive to oxidative stress, whereas overexpression of this gene made the strain more resistant. By comparison, the mutant and overexpression strains of another transporter gene PSPTO_2152 exhibited the same sensitivity to oxidative stress compared with the wild-type. Our data suggest that oxidative stress reduces the viability of bacterial cells by acting on special transporters.