Abstract
Evolution has seen an arms race between plants and invading pathogens. Pathogens inject plant cells with proteins to block plant cell protective responses. Plant cells have responded through the evolution of resistance proteins that detect these bacterial proteins and counter their activity. An important function of the resistance protein is to permit plant cells to suicide thus restricting further spread of the pathogen. Understanding resistance protein function and, importantly, manipulating this function to improve plant defences to pathogens can help to feed millions of people globally. Despite their key role in protecting plants from invading pathogens, relatively little is known of the specifics of how resistance proteins function, in part due to difficulties in generating resistance proteins for analysis in the test tube. Here we describe the generation of a critical portion of a resistance protein for test tube based analysis. We observe a distinct and exciting biochemical activity, the generation of adenosine from adenine nucleotides (a nucleotidase activity). In a further surprise, the resistance protein was observed to bind DNA in the test tube, a telling result given that many resistance proteins are hypothesized to function in the nucleus. Our test tube based experiments therefore provide a new insight into resistance protein function. Here we propose to exploit our new method for generating resistance protein fragments for test tube analysis First, we will investigate the specifics of the resistance protein nucleotidase activity in the test tube. We will ask (i) what range of nucleotide like molecules is the resistance protein able to target, (ii) how and where does the resistance protein attack its target molecules, and (iii) which portions of the resistance protein are important for this activity. These important studies will reveal how the resistance protein is able to function in the plant and provide tools for protecting plants in our further studies. Second, we will investigate the molecular basis of DNA binding by resistance proteins in the test tube. We will ask (i) what DNA structures are targeted and bound by resistance proteins, and (ii) what portions of the resistance protein are important for binding DNA. It will also be interesting to investigate the extent to which nucleotidase and DNA binding activities are mutually dependent in the test tube as this can reveal a deeper insight into a unified resistance protein function. Together these experiments will provide key insight into resistance protein function within the cell and further tools for the plant protection experiments to come. Third, we will utilize our test tube experiments as a starting point for a crop protection strategy. We will permit the production of resistance protein fragments in tobacco leaves, thus causing cell death. We will investigate whether those portions of the resistance protein identified as being required for nucleotidase or DNA binding activity in the test tube are also required to direct cell death in tobacco leaves. This important experiment will unify our test tube experiments with what actually occurs in the plant. Next we will engineer plants in which a resistance protein fragment is only produced when the plant detects an invading pathogen. We anticipate that specific cells in these plants will only die when exposed to the pathogen. This will block any further spread of the pathogen and therefore protect the remainder of the plant. This strategy for protecting plants from pathogens has two clear benefits 1) It uses plant proteins that do not generate toxic products, 2) We can utilize knowledge from our test tube based experiments to generate resistance proteins in which the activity has been fine tuned to compensate for effects that would otherwise compromise plant fitness. Importantly, this strategy for plant protection should be broadly applicable across a broad spectrum of crops.
Technical Summary
Plant R-proteins trigger disease resistance in response to pathogen (avirulence) proteins. R-proteins typically consist of a nucleotide binding (NB) domain adjacent to one or more ARC domains and a Leucine Rich Repeat (LRR). The LRR is involved in pathogen sensing and is thought to transmit this signal, via the ARC domain, to the NB domain. The role of the NB domain is uncertain but it is presumed to specify an ATPase, based on analysis of a refolded recombinant protein. Knowledge of how R-proteins function in signalling invasion by pathogens has been hampered by the lack of information on their in vitro properties. We have recently purified a rice R-protein NB domain, discovering two novel activities. First, the NB domain is a nucleotidase generating adenosine from ATP, ADP, and AMP. Second, the NB domain binds to double-stranded DNA. NB domain signalling is therefore more complex and diverse than previously suspected. We will characterize the nucleotidase activity in wild-type and mutant R-proteins to define the substrate range utilized, nucleotide cleavage site, kinetic parameters, and catalytic residues. Proteins will be assayed for nucleotidase activity using HPLC and TLC. NB domains and mutant derivatives will be assessed for binding to a range of specific DNA structures. Binding will be assayed using electrophoresis techniques. This investigation of the character of NB domain interactions with protein will provide key information on the R-protein signalling process. The NB domain of a model R-protein is sufficient to cause cell death and we will investigate nucleotidase activity and DNA binding for their role in this process by expression of proteins in Nicotiana. NB domains will be investigated as crop protection tools through expression from a pathogen responsive promoter in Arabidopsis. Mutants will enable us to tune activity in response to promoter leakage. Plants will be examined for a resistance to pathogen proliferation.
Planned Impact
WHO WILL BENEFIT FROM THIS RESEARCH? Two direct beneficiaries can be identified in the commercial sector. Funding will provide the opportunity to advance experiments to a stage at which commercialization can be realistically pursued through these partners. 1) Companies (e.g. Celsis) that provided microbial testing to the pharmaceutical, personal care, home care, consumer product, and food and beverage industries can benefit from our discovery of a broadly active nucleotidase enzyme. 2) Alternative commercial beneficiaries are companies with R&D programs in the area of crop protection (e.g. BASF Plant Science, Monsanto, and Syngenta). Indirect beneficiaries of this technology are other academics (see Academic Beneficiaries) and researchers in molecular biology for whom an enzyme that clears reactions of adenine nucleotides could be a useful research tool. HOW WILL THEY BENEFIT FROM THIS RESEARCH? 1) Companies that provide microbial testing services utilizing ATP detection generally pre-clear blank samples for analysis using apyrases that generate AMP from ADP and ATP. This blank determines the sensitivity limit of the test and it is well established that some non-ATP substrates can give undesirably high background signals. Our R-protein enzyme is active on AMP, ADP and ATP and may also have nucleotidase activity on guanine nucleotides. The enzyme therefore presents an excellent tool to lower the limit of detection of microbial contamination through more effective blank sample pre-clearing. The microbial testing industry was estimated to be worth $1.65 billion in 2005, hence a new technological development would provide a company a competitive edge and a powerful incentive to utilize this research. With an appropriate commercial partner and further research to improve the tool, economic impact could be realised over a short timescale. 2) Our proof of principle studies will provide the knowledge tools for further investigation and introduction into crop stocks for commercialization. Given the vast agricultural losses globally through pathogen attack, a crop protection strategy that bestows even moderate protection would be a priceless resource. The strategy we propose is generically applicable and could be adapted for diverse crops. Should the approach prove successful it would be reasonable to infer a time scale to commercialization of several years from field-testing, to licence, to release. WHAT WILL BE DONE TO ENSURE THEY BENEFIT FROM THE RESEARCH? Communication and engagement with potential commercial partners will proceed when our own IP position is established through the Technology Transfer Office. Networking at industry-relevant meetings will enable us to formally and informally meet with industry representatives. On identifying suitable commercial partners and a prioritized lead product we will apply for follow-on funding to support this work and seek advice from regional development agencies and Durham University support teams to put in place robust IP agreements and a management structure for any collaboration. Research Staff in plant sciences at Durham University have long-established interactions with industry (particularly Profs. Slabas and Lindsey). There is, therefore, the perfect expertise to aid the development of these technologies from biochemistry through to transgenic plant products. Further details are provided in the Impact Plan.
