Fungal phytopathogens cause devastating losses in global agriculture. All plant-infecting fungi use conserved MAPK signaling pathways to successfully locate, enter, and colonize their hosts.

Hyphae are filamentous structures that are crucial for the lifestyle of many fungi, allowing rapid colonization of diverse substrates such as dead organic material or living host tissue. One of the key features of fungal hyphae is their remarkable morphogenetic plasticity. During host colonization, phytopathogenic fungi must traverse extremely narrow spaces between plant cells such as the apoplastic space or the plasmodesmata. The mechanisms underlying hyphal plasticity during growth into microspaces are poorly understood. Here, we tested a set of isogenic mutants of Fusarium oxysporum , an important plant pathogen that causes devastating vascular wilt diseases on more than a hundred crops, for their ability to cross extremely narrow 1 µm channels, approximately one-quarter the width of hyphae. We found that the conserved Mpk1 cell wall integrity mitogen-activated protein kinase (MAPK) cascade is required for efficient growth of hyphae through the microchannels, whereas the two other MAPK pathways, the Fmk1 invasive growth and the Hog1 hyperosmolarity response cascades, are dispensable for this process. Staining with specific probes showed a reduction in both chitin and glucan content in the cell wall of hyphae growing within the 1 µm channel. We further establish that channel passage in cell wall remodeling mutants is impaired due to their inability to constrict hyphal diameter, and this defect can be rescued by increasing extracellular osmolarity. Both the cell wall integrity mutants impaired in microchannel passage, as well as the invasive growth mutants unable to penetrate cellophane membranes, caused significantly reduced mortality in tomato plants. Our study reveals that two morphogenetic processes essential for plant infection, which are governed by distinct cellular pathways, contribute independently to fungal phytopathogenicity. IMPORTANCE: This study highlights the critical role of hyphal plasticity and cell wall remodeling in the pathogenicity of Fusarium oxysporum , a major plant pathogen affecting over a hundred crops. The ability of fungal hyphae to traverse narrow plant tissue spaces, such as apoplastic gaps and plasmodesmata, is essential for successful host colonization. The research demonstrates that the Mpk1 mitogen-activated protein kinase (MAPK) pathway, responsible for cell wall integrity, is crucial for hyphal growth through microspaces, while other MAPK pathways are not. Staining with specific probes showed a reduction in both chitin and glucan content in the cell wall of hyphae growing within 1 µm width channels. Both the cell wall integrity mutants impaired in microchannel passage, as well as the invasive growth mutants unable to penetrate cellophane membranes, caused significantly reduced mortality in tomato plants. Our study reveals that two morphogenetic processes essential for plant infection, which are governed by distinct cellular pathways, contribute independently to fungal phytopathogenicity.

Like many hemibiotrophic plant pathogens, the root-infecting vascular wilt fungus Fusarium oxysporum induces an increase in the pH of the surrounding host tissue. How alkalinization promotes fungal infection is not fully understood, but recent studies point towards the role of cytosolic pH (pH_c) and mitogen-activated protein kinase (MAPK) signaling. In fungi, pH_c is mainly controlled by the essential plasma membrane H⁺-ATPase Pma1. Here we created mutants of F. oxysporum lacking casein kinase 1 (Ck1), a known negative regulator of Pma1. We found that the ck1Δ mutants have constitutively high Pma1 activity and exhibit reduced alkalinization of the surrounding medium as well as decreased hyphal growth and conidiation. Importantly, the ck1Δ mutants exhibit defects in hyphal chemotropism towards plant roots and in pathogenicity on tomato plants. Thus, Ck1 is a key regulator of the development and virulence of F. oxysporum.

Fungi are essential members across ecosystems, yet phytopathogenic fungi pose an increasing risk to crop yields. Despite their ecologic importance, cell–cell communication in fungi is underexplored, partly due to the lack of high-throughput techniques. Here, we developed a Yeast Mating Platform (YeMaP) to investigate the interaction between fungal G protein–coupled receptors (GPCRs) and pheromone peptides. We used YeMaP for high-throughput screening of 8,000 pheromone sequences and identified peptides with improved agonism or antagonism action. We found that these peptides can be applied in a native fungal system such as the plant pathogen Fusarium oxysporum , to control hyphal chemotropism and reduce plant root penetration. Additionally, we utilized YeMaP in a one-pot assay to investigate how abiotic factors influence the communication of multiple pheromone–GPCR combinations and found that the cell–cell communication mediated by the GPCR Ste2 from F. oxysporum signaled robustly across different abiotic factors, while other fungal GPCR–pheromone interactions were more sensitive to changes. Taken together, YeMaP accelerates the identification of fungal GPCR–peptide interactions by enabling one-pot assays, and serves as a model system for studying fungal cell–cell communication.