Abstract
Organisms such as microorganisms and plants have cell walls that encircle their cells and execute diverse functions by generating turger pressure inside the cell. For instance, in Colletotrichum orbiculare, which is one of a type of plant-pathogenic fungus, spores differentiate into appressoriums on the plant surface and make a turger pressure to punch a hole in the plant cell wall for mycelium to invade. The cell wall structure of the appressorium that supports such strong turger pressure attracted extensive interest from numerous research fields. Despite this, the nanoscale structure of the cell wall has yet to be elucidated through microscopic techniques, such as optical microscopy and electron microscopy. Thus, a method for visualizing this is strongly required.In this study, we developed a method for the nanoscale analysis of appresorium cell walls using atomic force microscopy (AFM). We used appressoria of Colletotrichum orbicular with both wild-type and melanin mutant (where the melanin gene was knocked out) as our model sample. Melanin mutant cells are known to have lower turger pressure in the appressorium than wild-type cells, and we expected differences in the cell wall structure as well. The appressoria were grown on a plastic dish and immersed in ultrapure water. We performed AFM measurements using a NanoWizard4 (JPK, Bruker) and 240AC (OPUS) cantilever. The cantilever was brought close to the appressorium surface, and the surface topography image was obtained using the QI mode in JPK. The obtained high resolution AFM images of the cell wall surface of both wild-type and melanin mutant appressoria, revealing the fine fibrous structures that make up the cell wall with a resolution of less than 10 nm. Our analysis found that the diameter of the fibrous structures was larger in the wild-type cell wall than in the melanin mutant and that particle-like structures with a size of several tens of nanometers were present in the wild-type cell wall, forming a robust membrane structure that creates strong turger pressure inside the appressorium. This method is expected to provide new insights into the mechanism by which appressoria can form such high turger pressures and contribute to the field of biology and materials science.
