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Maize (Zea mays L.) stalk rot is a devastating disease worldwide, causing severe yield losses. Although previous studies have focused on the genetic dissection of maize resistance to stalk rot, the mechanisms of resistance remain largely unknown. We used a comparative proteomics approach to identify candidate proteins associated with stalk rot resistance. Statistical analyses revealed 763 proteins differentially accumulated between Fusarium graminearum and mock-inoculated plants. Among them, the antioxidant protein ZmPrx5, which was up-accumulated in diseased plants, was selected for further study. ZmPrx5 transcripts were present in root, stalk, leaf, ear, and reproductive tissues. The expression of ZmPrx5 in three inbred lines increased significantly upon F. graminearum infection. ZmPrx5 was localized in the cytoplasm. Compared to control plants, maize plants overexpressing ZmPrx5 showed increased resistance to F. graminearum infection, and ZmPrx5 mutant plants were more susceptible than wild-type plants. Defense-associated pathways including plant-pathogen interactions, phenylalanine metabolism, and benzoxazinoid and flavonoid biosynthesis were suppressed in ZmPrx5 homozygous mutant plants compared with wild-type plants. We suggest that ZmPrx5 positively regulates resistance against stalk rot in maize, likely through defense-oriented transcriptome reprogramming. These results lay a foundation for further research on the roles of Prx5 subfamily proteins in resistance to plant fungal diseases, and provide a potential genetic resource for breeding disease-resistance maize lines. [ABSTRACT FROM AUTHOR]