This is the report from a BSPP Undergraduate ‘Vacation’ Bursary.
Click here to read more/apply for one yourself.
Zymoseptoria tritici is responsible for Septoria Tritici Blotch (STB), a disease of wheat which, during severe epidemics, can result in up to 50% yield losses. The economic burden of STB, in terms of crop losses and fungicide applications, costs roughly €790 million a year in the UK alone. My project, funded by the BSPP, investigated whether Z. tritici had the capacity to form biofilms. The discovery of biofilms production would further our knowledge regarding Z. tritici’s survival mechanisms and proliferation, which could provide a future target for epidemic control.
Firstly, I sought to identify the media conditions which resulted in the largest proportion of adhered growth (deemed biofilm-like growth). I used three strains of Z. tritici; IPO323 (reference strain) as well as SG13 and SG14, both of which show predominantly yeast-like growth on agar and on the outer surface of the leaf. This was achieved by using 96-well plates containing various media conditions and an optimised crystal violet assay that stained and quantified adhered growth and extracellular matrix using a plate reader. Across three strains, it was found that the minimal media with no carbon resulted in the most biofilm-like growth. Minimal media with no carbon was determined as the biofilm stimulating media condition to be used going forward. Furthermore, yeast extract peptone dextrose (YPD) was found to suppress biofilm growth since it produced the least adhered growth. These findings were supported by optical microscopy quantification of growth in larger, 6-well plates. After conducting the CV assay, the YPD condition was almost absent of cells whilst even after washing high growth was observed in the no carbon condition. In addition, thresholding analysis of crystal violet staining suggested the no carbon minimal media resulted in the most stained material around the cells, which could indicate higher presence of an extracellular matrix.
Due to the vast morphological differences in growth under our experimental conditions, IPO323 and SG14 were selected going forward for our next phase of investigation. Furthermore, minimal media with no carbon, YPD and minimal media with a high concentration of xylose (a plant sugar that might act as a host signal) were selected as our media of interest since each represented high, intermediate and low biofilm growth conditions. The next phase of investigation focused on imaging the biofilm and exploring its features.
Microscopy slides coated in different plastic materials were used to grow Z. tritici in the different media conditions. I carried out scanning electron microscopy, to resolve the presence of an extracellular matrix. From the images taken, it was noted that SG14 cells appeared to be embedded on the surface material with less clear cell boundaries. In comparison, IPO323 cells were distinct from the surface of the slide with defined cell edges. This morphological feature could indicate SG14 cells are secreting an extracellular matrix, which as a result may provide adhesion to surface substrates.
Along with morphological distinctness, biofilms are often associated with changes in gene expression. Using bioinformatic tool BLAST, I identified Z. tritici orthologues of genes expressed in biofilms in other fungi. I designed primers for genes scoring the highest sequence similarity involved in adhesion and the production of an extracellular matrix. I conducted qRT-PCR amplification using these primers which amplified an adhesin gene and an extracellular matrix gene to assess if the orthologues are expressed, and if so, was expression higher in our biofilm-forming condition/strain. I used a reference rRNA gene for normalisation of gene expression. Since our no carbon condition had extremely low growth that was mostly adhered to the surface substrate, obtaining enough RNA was difficult and therefore the reliability of our results from this condition were questionable. However, in the other conditions, not only were there strain-dependent differences in expression of our genes of interest across media conditions, but also the genes were up-regulated in the high xylose condition compared to the YPD in both SG14 and IPO323. Therefore, SG14, which appeared to produce an extracellular matrix when visualised by SEM, also has higher expression of adhesins and extracellular matrix genes compared to IPO323, a strain not showing this growth form. These genes also show increased expression in response to the host-derived sugar, xylose, in both strains.
This BSPP studentship has confirmed my passion for research and heightened my ambition to secure a PhD project after I graduate. I would like to thank Helen Fones, for not only supervising the project, but for the continual encouragement and inspiration, giving me the confidence to pursue a career in research.
Tegan Tyzack
University of Exeter
Scanning electron microscopy of SG14 (left) and IPO323 (right) grown in minimal media with a high concentration of xylose on plastic mounted microscopy slides for 7 days. Compared to the IPO323 cells, which have clean cell boundaries, the SG14 cells appear to be associated with the surface substrate and have less distinct cell boundaries. Circled in red are cells thought to be embedded in an extracellular matrix.
This is the report from a BSPP Undergraduate ‘Vacation’ Bursary.
Click here to read more/apply for one yourself.
