description
- Plant diseases are a continuous threat to global food production and security. Many plant pathogens use effector proteins to interfere with cellular processes in the host promoting colonisation and growth. Rice heavy metal-associated plant proteins (HPPs), including the heavy metal-associated isoprenylated plant proteins (HIPPs), are targets of effector proteins from the rice blast pathogen Magnaporthe oryzae, presumably to promote infection. HPPs/HIPPs form a diverse family of proteins in crops and other plants, but little is known about their function and role in disease. HPPs/HIPPs possess heavy metal-associated (HMA) domains, which in proteins that bind metals typically have an N-terminal CXXC (C=cysteine) motif. The hypothesis we will test is that metal binding by HPPs/HIPPs is important for their cellular functions and perturbation by pathogen effectors. There is limited information about the role of metals for the structure and function of HPPs/HIPPs. To study metal binding in this protein family, a carefully chosen selection of rice HPPs/HIPPs HMA domains, both with and without the full CXXC motif, will be produced. In vitro characterisation of metal binding will be achieved with an array of spectroscopic and biophysical approaches. As studies progress, this choice will be assisted by protein bioinformatics. The structures of HMA domains, either determined by protein crystallography or modelling, will be used in conjunction with new deep learning-based methods to predict metal-binding capability and specificity. Full length HPPs/HIPPs will also be produced and analysed. Some HPPs/HIPPs are relatively small possessing approximately 120 residues, and over-express in E. coli. However, purification has proved challenging. The availability of AlphaFold2 models could assist by better defining the boundaries of folded units and allow the elimination of unnecessary terminal regions. Once metal binding has been demonstrated in vitro, its influence on the structure and function of HPPs/HIPPs will be investigated. This will include protein crystallography (the structure of HIPP19-HMA with an M. oryzae effector has been determined), testing how metal binding influences interactions with effector proteins and the ability of effectors to perturb ROS production by HPPs/HIPPs. Introducing metal binding into rice immune receptor proteins (e.g. Pik), which have HMA domains that act as bait domains to directly detect the presence of effectors, will also be tested. The interdisciplinary team involved in this project will teach a range of skills. This will include modern molecular biology techniques as well as how to purify proteins, particularly HPPs/HIPPs. Many approaches will be used to investigate metal binding, mostly under strict anaerobic conditions. The influence of metals on interactions with effectors will be studied including using X-ray crystallography, which will also provide detailed information about how metal binding alters the structures of HPPs/HIPPs. In vivo techniques will be used to investigate the role of metals on the function of HPPs/HIPPs and their involvement in pathogenesis. A range of protein bioinformatics will complement these studies. This project fits within BBSRCs Tacking Strategic Challenges objective under the Bioscience for Sustainable Agriculture and Food (previously Agriculture and Food Security) priority. The rice blast pathogen is the most devastating disease of rice, estimated to destroy enough of this crop to feed 212-742 million people annually. This disease can be addressed by investigating the molecular basis of pathogen-host communication as outlined in this project. HPPs/HIPPs are present in other crops including wheat and this work therefore has wide-ranging impact on food security. Such studies can contribute to efforts to protect the world's most important crops from plant diseases.