Use of chitin:DNA ratio to assess growth form in fungal cells

Abstract

Background Dimorphism, the ability to switch between a ‘yeast-like’ and a hyphal growth form, is an important feature of certain fungi, including important plant and human pathogens. The switch to hyphal growth is often associated with virulence, pathogenicity, biofilm formation and stress resistance. Thus, the ability to accurately and efficiently measure fungal growth form is key to research into these fungi, especially for discovery of potential drug targets. To date, fungal growth form has been assessed microscopically, a process that is both labour intensive and costly. Results Here, we unite quantification of the chitin in fungal cell walls and the DNA in nuclei to produce a methodology that allows fungal cell shape to be estimated by calculation of the ratio between cell wall quantity and number of nuclei present in a sample of fungus or infected host tissue. Using the wheat pathogen Zymoseptoria tritici as a test case, with confirmation in the distantly related Fusarium oxysporum, we demonstrate a close, linear relationship between the chitin:DNA ratio and the average polarity index (length/width) of fungal cells. We show the utility of the method for estimating growth form in infected wheat leaves, differentiating between the timing of germination in two different Z. tritici isolates using this ratio. We also show that the method is robust to the occurrence of thick-walled chlamydospores, which show a chitin:DNA ratio that is distinct from either ‘yeast-like’ blastospores or hyphae. Conclusions The chitin:DNA ratio provides a simple methodology for determining fungal growth form in bulk tissue samples, reducing the need for labour-intensive microscopic studies requiring specific staining or GFP-tags to visualise the fungus within host tissues. It is applicable to a range of dimorphic fungi under various experimental conditions.

Myo5B plays a significant role in the hyphal growth and virulence of the human pathogenic fungus Mucor lusitanicus

“…The polarity index is the quotient of cell length divided by cell width [ 35 ] and is an indicator of germination and hyphae formation. The polarity index can be used as an indicator of fungal growth and virulence [ 26 36 ]. In this study, the polarity index of the myo5BΔ mutant cells was considerably significantly reduced compared to the wild-type strain R7B, especially after 3 h of growth in liquid culture ( Fig.…”