Abstract
Stalk rot, primarily caused by Fusarium graminearum (Fg) and Pythium inflatum (Pi), is a major maize disease responsible for significant yield losses. The molecular mechanisms governing defence against these pathogens remain poorly understood. To uncover key miRNAs and their regulatory genes, small RNA, degradome, and transcriptome sequencing data were integrated to explore maize's response to stalk rot. A total of 363 miRNAs, including 113 novel ones, were identified from 12 sRNA libraries, with 305 differentially expressed miRNAs (DEMs) significantly responding to Fg and Pi infection. Degradome analysis detected 120 DEMs. Through transcriptome sequencing and weighted gene co-expression network analysis (WGCNA), 6 Fg/Pi-responsive regulatory modules, centered on hub genes, were identified from 8308 differentially expressed genes activated or repressed by the two pathogens. A machine-learning approach revealed complex regulatory networks within these significant pathogen-responsive modules. WGCNA highlighted ZmMYB74, targeted by zma-miR319, as a key hub gene regulator in these networks. Transgenic plants overexpressing ZmMYB74 showed compromised resistance to stalk rot pathogens, with reduced lignin deposition, whereas knockout or suppression of ZmMYB74 resulted in significantly enhanced resistance. Two In/Dels in the promoter region associated with ZmMYB74 transcription were linked to changes in maize resistance to stalk rot. ZmMYB74 functions as a transcriptional repressor, negatively regulating the expression of ZmCAD, a positive regulator of plant disease resistance involved in lignin biosynthesis. Isolating the resistance gene ZmMYB74 will not only aid in developing durable disease-resistant maize varieties but also enhance understanding of the molecular mechanisms underlying stalk rot resistance.