This is the report from a BSPP Undergraduate ‘Vacation’ Bursary.
Click here to read more/apply for one yourself.
Wheat (genus Triticum) is a major cereal crop grown in many countries, predominantly China, India and Russia, providing food for over 3 billion people worldwide. Wheat production has nearly quadrupled since the 1960s, as a result of technological improvements including improved storage proteins reducing spoilage and genetic improvements such as grain size, plant height, nutritional value and disease resistance.
However, a number of threats to wheat production remain. One of these is the disease wheat stem rust, caused by the fungus Puccinia graminis f. sp. tritici (Pgt), which can result in nearly 100% yield losses under favourable conditions. A new strain of stem rust, Ug99, emerged in Uganda in 1998. Ug99 is virulent to many stem rust resistance genes and continues to spread. Currently stem rust is not present in the UK following a historic eradication regime. However, 80% of cultivated wheat varieties in the UK are susceptible to stem rust, therefore emerging risks such as Ug99 pose a significant threat to domestic wheat production. The identification of new resistance and susceptibility genes in wheat is important.
During my 10-week placement in Professor Diane Saunders’ laboratory at the John Innes Centre, I performed infection assays on various TILLING (Targeting Induced Local Lesions in Genomes) mutant and wild-type plants of the cadenza wheat cultivar (hexaploid bread wheat, Triticum aestivum). TILLING mutants were identified that had a different disruption in wheat genes shown previously to be linked to susceptibility to the related wheat yellow rust pathogen (Puccinia striiformis f. sp. tritici). The aim was to determine if the disruption of these genes would have a similar effect in reducing wheat stem rust infection. The first gene selected was TaBCAT1 on the 4A homoeolog of wheat. TaBCAT1 regulates the levels of branched-chain amino acids (BCAAs), which act as precursors to several important defence molecules. Previous research at the Saunders Laboratory revealed that TaBCAT1 disruption mutants have increased levels of BCAAs and salicylic acid, a phytohormone involved in activating systemic acquired resistance (SAR) in response to pathogen attack. This leads to the possibility that disruptions to TaBCAT1 will provide resistance to stem rust infection from the increased presence of defence molecules and the priming of SAR allowing for a more rapid response to infection. The second gene was TaICL on the 2B homoeolog of wheat. TaICL is responsible for encoding the enzyme isocitrate lyase, a unique enzyme involved in the glyoxylate cycle, that is upregulated during rust infection especially on susceptible host plants. Disruption of TaICL leads to an accumulation of aconitic acid that was previously shown to also reduce wheat yellow rust infection. During the infection assays, 5 repeats were used for 4 mutant lines with 5 wild type plants as a control to ensure any differences observed could be analysed for significance rather than chance.
Following the infection of the TILLING mutants with activated UK-01 Pgt urediniospores, the infection was allowed to progress for 19 days until sporulation was clearly visible. The second leaves of each plant were then harvested and imaged. I visually scored the leaves for their healthy surface area using the pliman package in RStudio, creating colour palettes for the process. The scoring results were then analysed statistically to determine their significance. A Dunnett’s test revealed that there were no significant differences in healthy leaf surface area between the TILLING mutants and wild type plants. To confirm the results, another infection assay will be repeated in the future.
In addition, I also began to explore the second unique enzyme involved in the glyoxylate cycle – malate synthase which is encoded by the gene TaMLS1 and similarly is upregulated during yellow rust infection. TILLING TaMLS1 disruption mutants will also be investigated through an infection assay to determine if TaMLS1 disruption decreases susceptibility to stem rust. To prepare for this, alongside the infection assay I optimised primers for detecting the gene disruptions in TaMLS1 mutant plants. The primers were designed using Geneious Prime and tested using PCR on cadenza wild type DNA and DreamTaqMM2X. Following this, electrophoresis in a 1% agarose gel was used to separate the amplified DNA before imaging. If multiple bands were observed, I used a gradient PCR to increase the specificity of the primers by increasing their annealing temperature. If the primers were still not specific to the region of interest, they were redesigned and tested again. Once the primers were specific, the PCR products were sent for sequencing to ensure the correct region is amplified. In this way, primers for three different TaMLS1 mutants were optimised.
I would like to thank Professor Diane Saunders and PhD student Sarah Bailey for their support and guidance as well as BSPP for their generous funding for this summer research project.
Tomos Hughes
University of Nottingham
Cover image: Wild type UK-01 infection assay. Red-brown sporulating areas are clearly visible alongside yellow chlorotic areas afflicted by stem rust infection.
This is the report from a BSPP Undergraduate ‘Vacation’ Bursary.
Click here to read more/apply for one yourself.
