Written by Juliet Smith, University of Sheffield. This is the report from a BSPP Undergraduate Summer Vacation Bursary. Click here to read more/apply for one yourself.
Yellow (stripe) rust, caused by the fungal pathogen Puccinia striiformis f.sp. tritici (Pst), is the most significant biotic threat to wheat production globally. Pst currently causes annual losses of 5.74 million tonnes, equating to US$979 million . To attenuate production losses, a greater understanding of the Pst-induced wheat defence response is required. Molecular interactions between wheat and Pst are poorly understood compared to other plant-pathogen systems, as rust fungi cannot be cultured artificially, and common transformation methods are ineffective for wheat and rust.
Being involved in the synthesis of many pro-defence molecules, chloroplasts are vital for plant immune responses. Chloroplasts have been elucidated as a major target for pathogen effectors, corroborating their importance for defence. Several chloroplast-associated candidate genes important for wheat responses to Pst have been identified using transcriptomic data produced in the Saunders’ lab. Under the supervision of Francesca Minter, I worked towards functionally characterising some of these genes.
My first aim was to generate loss-of-function mutants for our genes of interest. Utilising the readily available sequence data and seed stocks for Target Induced Local Lesions in Genome (TILLING) mutant populations of tetraploid Kronos wheat, I selected lines with homeolog-specific single nucleotide polymorphisms (SNPs) within the candidate genes. After ordering, sterilising, and planting the seeds, I extracted DNA and carried out Kompetative Allele Specific PCR (KASP)TM genotyping. Results showed that I had successfully obtained homozygous mutants for the B homeolog of one target gene (AAbb). As mutation to a single homeolog could be masked by redundancy in the other homeolog, the AAbb plants will be crossed with an aaBB line to obtain double homozygous (aabb) mutants with complete loss-of-function. To assess the contribution of the candidate gene to wheat susceptibility, the loss-of-function mutants will be infected with Pst and then phenotyped.
My second aim was to fuse target genes with GFP, facilitating localisation in the model plant Nicotiana benthamiana. For this, I planned to produce a level 1 transcriptional unit using Golden Gate cloning. As RNA-seq data shows candidate genes to be most strongly expressed at 0- and 3-dpi, I harvested wheat tissue at these time points following inoculation with Pst. RNA was then extracted and cDNA synthesised, providing a template for amplification of our target genes. I designed primers which would attach the extensions required for insertion into the level 0 acceptor plasmid, using these in the PCR. As our genes proved difficult to amplify, I spent time independently troubleshooting and following logical steps to optimise the PCR. I initially tried using a GC-rich buffer for amplicons with a high GC-content. Next, I added DMSO to prevent primers from forming secondary structures. I then used a temperature gradient for primer pairs which had a large difference in Tm. When these optimisation methods proved unsuccessful, I tried performing a Touchdown PCR (lowering the annealing temperature with each cycle). Visualisation of the PCR product using gel electrophoresis and UV illumination showed that Touchdown PCR successfully amplified two target genes. I then cut bands of the correct length and purified the DNA using a gel extraction. The product obtained will later be used in a level 0 cloning reaction and subsequently to produce the desired level 1 construct.
I am extremely grateful to BSPP for awarding me this bursary, giving me the opportunity to undertake an exciting project at a world-leading research institute. Through the project, I developed key wet lab and bioinformatic skills and gained an insight into working in academic research. I would like to thank Dr Diane Saunders for hosting my project, Francesca Minter for her patience, encouragement and support, and other lab members for being so welcoming.