# The Candidate Effector Cgmas2 Orchestrates Biphasic Infection of Colletotrichum graminicola in Maize by Coordinating Invasive Growth and Suppressing Host Immunity

## TL;DR

The study identifies a fungal protein, CgMas2, that helps a pathogen infect maize by promoting growth and suppressing plant defenses.

## Contribution

The novel contribution is the functional characterization of CgMas2 in the biphasic infection process of Colletotrichum graminicola in maize.

## Key findings

- CgMas2 is essential for invasive hyphal growth during the biotrophic phase of infection.
- Disruption of CgMas2 leads to increased reactive oxygen species accumulation in host plants.
- CgMas2 modulates defense compound biosynthesis and phytohormone signaling during the necrotrophic phase.

## Abstract

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.