This is the report from a BSPP Undergraduate ‘Vacation’ Bursary.
Click here to read more/apply for one yourself.
Fusarium Head Blight (FHB) is a disease of wheat that causes considerable yield losses worldwide. The causal agent, Fusarium graminearum, is an ascomycete fungal pathogen, and produces harmful mycotoxins which contaminate the grain and make it unsafe for consumption. Fusarium graminearum lacks specialised cell wall penetration structures such as appressoria and so relies heavily on wounds and natural openings such as stomata to gain entry to its host. The aim of my summer studentship project in Professor Kim Hammond-Kosak’s lab at Rothamsted Research was to gain insight into the role of plant cell wall components during Fusarium graminearum infection using the model organism Arabidopsis thaliana.
Plant cell walls consist of 3 main layers, i) the Primary cell wall, which is formed mostly of Celluloses, ii) the middle lamella which is formed mostly of pectin, iii) and the secondary cell wall which has a similar composition to the primary cell wall, but with an added lignin component. Cell walls are important in plant-pathogen interactions as cell wall material is deposited at the site of pathogen entry to provide a physical and spatial restriction to further colonisation. The various components of cell walls are also targets for Cell Wall Degrading Enzymes (CWDEs) such as pectinases, produced by phytopathogens to digest host cells.
During this project I screened a range of Lignin, Pectin and Cellulose mutants, in addition to peroxidase mutants and a Wild type (WT) control line. The disease phenotype of each mutant was assessed via detached leaf and floral assays, in which detached leaves were pressed into water agar and inoculated with a WT spore solution of F.graminearum. The agar plates were incubated for 5 days, then imaged using a digital camera and sampled. gDNA from infected leaves was extracted for analysis via qPCR to measure the ratio of fungal to plant DNA. The images taken of the leaves were inputted into the Pliman™ package in R, which measures the percentage of total diseased and healthy area, using RGB pixel values. The flowers were given a disease score from 0-3, where 0 is No disease, 1 – ariel mycelium, 2- Necrosis, and 3- stem constriction within the flower head. Alongside the floral and leaf analysis, all the plants were genotyped to verify they were carrying the mutation in question.
The results of this study suggest that the most important cell wall components in the context of fusarium infection are lignin and pectin, as the lignin and pectin mutants had the most significant levels of disease when compared to the WT. Pectin is crucial for holding cell walls together, and with lower pectin levels cell walls become brittle and weak. Lignin provides a dual function as it is deposited at the site of entry, and without this, infection progresses further. Lignin also provides a hydrophobic barrier, and without this water soluble CWDE can digest cell walls with ease. These functions may explain why lignin, and pectin knockout mutants, show increased susceptibility to Fusarium graminearum.
Unfortunately, during my time at Rothamsted Research, the growth room was experiencing a mystery soil fungus outbreak, which unfortunately managed to infect the mock plants used as negative controls. To investigate whether this would affect the qPCR results, I cultured a sample of the fungus and carried out a PCR using the qPCR primers. This confirmed that unfortunately there was some cross reactivity between the soil fungus and the qPCR primers, sadly undermining all of the qPCR fungal burden results.
Completing this BSPP studentship has been an extremely valuable experience, and the skills and techniques I have learnt during my time at Rothamsted Research have given me a lot more confidence working independently in the lab which will in no doubt prove helpful in my final year lab projects. Working alongside professionals in the field has inspired me to consider undertaking a plant pathology related PhD programme, and possibly a future career in plant pathology. I am grateful for the BSPP for providing me this opportunity and I would like to especially thank Victoria Armer and Professor Kim Hammond-Kosak at Rothamsted Research for their help and guidance.
Max Fontaine
This is the report from a BSPP Undergraduate ‘Vacation’ Bursary.
Click here to read more/apply for one yourself.
