Pathogen-induced protein secretion in plants.

Abstract

The sugar alcohol mannitol is an important carbohydrate in many plants and fungi that has well-documented roles in both metabolism and osmoprotection. In addition, mannitol is an antioxidant, and as such might play a role in host-pathogen interactions. Research suggests that pathogenic fungi secrete mannitol into the plant cell wall to suppress reactive oxygen-mediated host defenses. Previous work suggests that plants counter this by making the enzyme mannitol dehydrogenase (MTD) to catabolize fungal mannitol. Here we show that the normally cytoplasmic enzyme MTD is exported into the extracellular space in response to the endogenous inducer of plant defense responses salicylic acid (SA). This SA-induced secretion is resistant to brefeldin A, an inhibitor of Golgi-mediated protein transport. Together with the absence of MTD in Golgi stacks and the lack of a documented extracellular targeting sequence in MTD, this suggests the secretion of MTD is by a non-Golgi, pathogen-activated protein secretion mechanism in plants. To further characterize pathogen-activated protein secretion in plants, a comprehensive analysis was performed using Arabidopsis suspension culture to study temporal changes in the cell wall proteome in response to different levels of SA. An LC/MSE -based proteomic approach was used as a label-free approach for simultaneous protein identification and absolute quantification. A total of 76 secreted proteins were identified, 66 of which showed differential secretion patterns in response to SA. A majority of induced protein secretion was observed within the first two hours after treatment, suggesting many proteins are involved in the early stage of plant defense response. A number of non-classically secreted proteins were observed, indicating that as in many non-plant systems, alternative Golgi/ER-independent secretion mechanisms might exist in plants. Overall, our results provide new and useful insight into plant apoplastic defense mechanisms, and demonstrate that LC/MSE is a suitable strategy for absolute quantitative proteomic analysis that can be applied for complex experimental designs.