The maize anthracnose stalk rot and leaf blight diseases caused by the fungal pathogen Colletotrichum graminicola is emerging as an important threat to corn production worldwide. In this work, we p...

Maize (Zea mays L.) is a major economic crop highly susceptible to Colletotrichum graminicola, the causal agent of anthracnose leaf blight, which causes substantial annual yield losses. This fungal pathogen employs numerous effectors to manipulate plant immunity, yet the functions of many secreted proteins during biphasic infection remain poorly characterized. In this study, we identified CgMas2, a candidate secreted protein in C. graminicola and a homolog of Magnaporthe oryzae MoMas2. Deletion of CgMAS2 in the wild-type strain CgM2 did not affect fungal vegetative growth or conidial morphology but significantly impaired virulence on maize leaves. Leaf sheath infection assays revealed that CgMas2 is required for biotrophic invasive hyphal growth, as the mutant showed defective spreading of invasive hyphae to adjacent cells. Subcellular localization analysis indicated that CgMas2 localizes to the cytoplasm of conidia and to the primary infection hyphae. Furthermore, DAB staining demonstrated that disrupt of CgMAS2 leads to host reactive oxygen species (ROS) accumulation. Comparative transcriptome analysis of maize infected with ΔCgmas2 versus CgM2 revealed enrichment of GO terms related to peroxisome and defense response, along with up-regulation of benzoxazinoid biosynthesis genes (benzoxazinone biosynthesis 3, 4 and 5) at 60 h post-inoculation (hpi). Conversely, six ethylene-responsive transcription factors (ERF2, ERF3, ERF56, ERF112, ERF115 and ERF118) involved in ethylene signaling pathways were down-regulated at 96 hpi. These expression patterns were validated by RT-qPCR. Collectively, our results demonstrate that CgMas2 not only promotes invasive hyphal growth during the biotrophic stage but may also modulate phytohormone signaling and defense compound biosynthesis during the necrotrophic phase of infection.

Phenol amides (PAs), a class of plant secondary metabolites, play crucial roles in plant defense, yet the genes regulating their biosynthesis and functional mechanisms against pathogens in maize remain poorly characterized. Here, we demonstrated that pathogen infection upregulates ZmPHT3, which encodes a BAHD acyltransferase essential for driving the accumulation of p-coumaroylputrescine (Cou-put), N-caffeoylputrescine (Caf-put), and N-feruloylputrescine N-glucoside (Fer-put-glu). Overexpression of ZmPHT3 boosted the levels of these compounds, consequently suppressing hyphal growth and strengthening resistance. Conversely, disruption of ZmPHT3 diminished PA accumulation, leading to increased hyphal expansion and higher susceptibility. Exogenous application of Cou-put and Caf-put partially restored fungal growth restriction in zmpht3 mutants, confirming the antimicrobial function of PAs. Mechanistically, we found that ZmPHT3 modulates the phloem protein ZmPP2-B8 via 2 distinct pathways: (i) direct protein interaction and (ii) through the action of its enzymatic products. This model is supported by multiple lines of evidence: ZmPHT3 physically interacted with ZmPP2-B8; a catalytically inactive ZmPHT3 variant failed to promote ZmPP2-B8 protein accumulation; and treatment with Caf-put, Cou-put, or Fer-put elevated ZmPP2-B8 protein levels. Silencing ZmPP2-B8 impaired disease resistance, and silencing both ZmPHT3 and ZmPP2-B8 demonstrated that their encoded proteins act synergistically to promote immunity. Furthermore, ZmPHT3 and ZmPP2-B8 cooperatively upregulated lignin biosynthesis and cellulose synthase genes, enhancing cell wall fortification. Our findings unveil a defense mechanism in which pathogen-induced ZmPHT3 promotes accumulation of PAs that function dually as antimicrobial agents and ZmPP2-B8 stabilizers, forming a synergistic module that reinforces the cell wall and broadens the resistance spectrum in maize.