Phase-contrast microtomography unveils mechanisms of root colonization by a vascular fungal pathogen

: Soil-borne vascular pathogens pose serious threats to agriculture with complex invasion strategies that remain poorly characterized compared to foliar pathogens. While foliar pathogens like Magnaporthe oryzae employ specialized appressoria to penetrate plant surfaces through a combination of mechanical force and enzymatic degradation, the invasion mechanisms of vascular pathogens that lack classical appressoria have remained largely theoretical. The nanoscale processes governing root penetration and colonization by these pathogens are particularly challenging to visualize due to technical limitations of conventional microscopy. Here we show, using phase-contrast X-ray computed microtomography and advanced microscopy, that Fusarium oxysporum (Fo) employs distinct mitogen-activated protein kinase (MAPK) cascades to orchestrate root invasion through unprecedented morphological plasticity. We identify previously undocumented appressoria-like structures that facilitate physical penetration, while demonstrating that Fo exhibits remarkable cellular adaptability, reducing hyphal diameter by more than 20-fold (from 5 μm to 220 nm) to navigate confined plant spaces, a dramatic morphological transition previously thought impossible. By using cellulase-deficient mutants, we demonstrate that cellulolytic activity is dispensable for surface breach and submicrometric hyphal colonization, establishing that mechanical force generation rather than enzymatic degradation is the primary determinant of successful host penetration. Three-dimensional reconstruction reveals a quantitative correlation between fungal proliferation and progressive embolism formation, with distinct MAPK pathways differentially regulating penetration force generation (Fmk1), osmotic adaptation during apoplastic colonization (Hog1), and directional growth toward vascular tissues (Mpk1). These findings provide a mechanistic framework for vascular wilt pathogenesis and reveal potential targets for controlling these economically devastating plant diseases.