The importance of metal binding for the function of rice proteins that interact with pathogen effectors

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. In recent studies, rice heavy metal-associated plant proteins (HPPs), including the heavy metal-associated isoprenylated plant proteins (HIPPs), were shown to be targets of effector proteins from the rice blast pathogen Magnaporthe oryzae, presumably to promote infection. HPPs/HIPPs form a large diverse family of proteins in crops and other plants, but little is known about their physiological function and role in disease. HPPs/HIPPs possess heavy metal-associated domains (HMAs), 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 data on metal binding by HPPs/HIPPs. Preliminary in vitro studies in Newcastle have shown that HIPP19 has a preference for copper over zinc. One of the cysteines in the CXXC-motif is replaced with a serine in HIPP19, but other cysteines are present, and site-directed mutagenesis is being used to identify the location of metal binding. To study metal binding further in this family of proteins a carefully chosen selection of rice HPPs/HIPPs HMA domains, both with and without the full CXXC motif, will be over-expressed in bacteria. As studies progress, this choice will be assisted by protein bioinformatics (Prof. Dan Rigden, Liverpool) to predict HMAs that bind different metals, and also those without metal-binding capability, as controls. In vitro characterization of metal binding will be achieved using an array of approaches. Full length HPPs/HIPPs will also be produced and analysed. This has proved challenging for the one example tested and will be assisted by covariance analysis (Liverpool) to better define complete folding units and the full extent of regions to clone.

Once metal binding has been demonstrated in vitro, its influence on the structure and function of HPPs/HIPPs will be investigated with Prof. Mark Banfield (John Innes Centre). This will include protein crystallography, testing how metal binding influences interactions with M. oryzae 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 HMAs that act as bait domains to directly detect the presence of effectors, will also be tested.

This project fits within the remit of the 'Agriculture and Food Security' BBSRC strategic framework. As global demand for food is rising the threat posed by pathogens that can dramatically reduce crop harvests is a major concern. The rice blast pathogen Magnaporthe oryzae is the most devastating disease of rice, estimated to destroy enough of this crop to feed 212-742 million people annually. One approach to address such plant diseases is to investigate the molecular basis of communication between pathogen and host as outlined in this project. In particular, an understanding of how pathogens target host cell processes for their own benefit is key. Evidence to date suggests that rice HPPs and HIPPs are the targets of M. oryzae effectors. HPPs and HIPPs are also present in a number of other major crops, including wheat, therefore understanding their function, and how this can be influenced by pathogen effectors, has wide ranging impact on food security. As HMAs are also found in some plant immune receptors, this work may lead to the development of receptors that can detect a wider range of effectors. This will contribute to efforts to protect the world's most important crops from plant diseases.