Nucleoside decoys - metabolic interference in plant defence

Lead Research Organisation: University of Warwick
Department Name: School of Life Sciences

Abstract

Plant disease resistance (R) genes are widely deployed in plant breeding to help mitigate global crop losses to pests and pathogens which exceed 30%. Unfortunately, resistance is often overcome in the field as pathogens evolve ever sophisticated methods of deploying multi-functional "effectors" - key elements of the pathogens armoury the work collectively to avoid detection and suppress host immunity.
Despite having cloned R proteins more than 25 years ago, until 12 months ago we had little idea how these functioned. R proteins come in two flavours, TNLs and CNLs, both containing common key functional domains, the central nucleotide binding (N) domain and carboxyl terminal "leucine rich repeat" (L) region.
The recent major research breakthrough showed that the amino terminal TIR, (Toll Interleukin 1) domain of "T"NL disease resistance proteins dimerises to generate a complex capable of cleaving NADH or NADPH, key energy sources for cells. Critically, this "NADase" activity was essential to activate disease resistance. Notably, although animal and bacteria TIR domains have similar enzymatic activities, the products appear to differ. Plants and bacteria produce a compound called v-cADPR (variant cyclic ADP Ribose).
For a number of years we have studied the metabolic transition from defence to disease, specifically looking at how the bacterial plant pathogen Pseudomonas syringae overcomes host defences. The most exciting finding of comprehensive untargeted metabolite profiling of infected tissue was the identification of a totally novel molecule that accumulated rapidly in leaves that were infected with Pseudomonas that was going to cause disease but not a non-disease causing mutant. Remarkably, we have recently confirmed that this molecule, which we call "540" based on its molecular mass, is of identical molecular mass to v-cADPR formed by activated TNL disease resistance proteins. Critically, it has a different retention time, as also does the bacterial TIR produced v-cADPR suggesting some very subtle structural variations probably confer quite different specificities. To date, it is unclear whether either the TNL produced plant or bacterial v-cADPR have any biological activity.
Importantly, we had previously published two key pieces of evidence.
First, disease causing bacteria, but not disarmed bacteria induce a very specific locus of 6 truncated TNL genes (tTNs). This is at first counterintuitive. Why induce R genes?
Secondly, we know disease causing bacteria rapidly suppress a defense response - called a reactive oxygen burst - in the chloroplast. This is necessary for disease progression. A consequence of this is elevated NADP+ - an NADase substrate. These are really rapid events, occurring before TNL proteins are activated.
Putting this evidence together; the rapid accumulation of 540 co-incident with suppression of the oxidative burst and induction of the tTNs, we theorise that bacterial effectors both suppress the oxidative burst and simultaneously induce the tTNs to mop up accumulating NADP+, which would otherwise activate TNLs. We also cannot rule out the tTNs can also bind to, and interfere with, functional TNL TIR domains preventing activation.

Our previous work characterised 540 as a highly labile "cyclic phosphoriboside". In collaboration with an Australian group we elucidated the structure of a prokaryotic v-cADPR, which is subtly different from 540. We have recently developed a method to stabilize 540 and will determine the NMR structure and collaborate on the plant v-cADPR structure with US researchers.
Concomitantly, our work programme will fully characterise the tTNs using genetics, gene-editing, biochemistry and structural approaches. Finally we will determine the dynamics of NAD/P accumulation/loss during disease and defence development using state-of-the-art genetically encoded reporters. This multidisciplinary project benefits from collaborations in Australia, Hong Kong and the USA.

Technical Summary

TNL resistance proteins use proximity induced dimerization to form functional NADases. NADase activity is essential to trigger the plant HR and leads to production of 'variant cyclic ADP ribose" (v-cADPR). Bacterial TIRs similarly produce v-cADPR, though with a different HPLC retention time. By untargeted metabolite profiling of Arabidopsis leaves infected with virulent Pseudomonas, we identified another v-cADPR, "540", with accurate mass identical to the plant and bacterial v-cADPR but with different retention time. 540 is also induced in leaves of tomato and Nicotiana benthamiana following virulent pathogen challenge. This implies subtle structural variations may confer different biological function.

Our data is indicative of a complex immune suppression mechanism, potentially operational against the whole class of TNLs. It involves 540 and a cluster of 6 truncated TNLs (tTNs) induced 4hpi by Pseudomonas, identified from our infection transcriptome dataset. The tTN promoters contain highly conserved motifs, one conferring effector specific induction. We hypothesise effectors induce tTNs to convert NADP+, which accumulates at Photosystem I following suppression of chloroplastic ROS ~4hpi, into 540. This sequestration likely precedes activation of functional TNLs.

We will determine the structure of 540 via NMR (as we did for bacterial v-cADPR), and address 540s biological significance - is it a "dead-end" product? In collaboration with leading US groups we will compare this to TNL produced v-cADPR.
In parallel we will comprehensively characterise the tTNs using genetics, biochemistry and structural approaches. We will collaborate with the Kobe lab in Brisbane to solve tTN TIR structures and use CRISPR to delete the entire tTN locus. We will also investigate whether tTNs additionally bind to, and interfere with, TNLs.
Finally we explore the metabolic changes during both disease and defense using genetically encoded reporter for both NADH and NADPH.

Publications

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Description Nucleoside decoys - metabolic interference in plant defence
Amount £650,292 (GBP)
Funding ID BB/V01627X/1 
Organisation Biotechnology and Biological Sciences Research Council (BBSRC) 
Sector Public
Country United Kingdom
Start 06/2021 
End 07/2024
 
Description Developing novel chloroplast localised NAD/P sensors 
Organisation University of Hong Kong
Country Hong Kong 
Sector Academic/University 
PI Contribution We are collaborating to undertake measure a set of unique sensors developed by Dr Boon Lim, and expert in energy balances in plants, to specifically understand how dynamics of NAD/P change during basal immunity, effector triggered immunity and suppression of effector triggered immunity.
Collaborator Contribution Dr Boon Leong LIM, an Associate Professor in gthe School of Biological Sciences, University of Hong Kong is specifically developing a unique cytosol and chloroplast localised Apollo sensor for measuring NADP during pathogen responses - this being developed in wildtype , NADK2 overexpressor and the nadk3 mutant . He is also selecting and supplying iNAP sensors for ratiometric measurements of NADP/H in chloroplasts and cytosolic locations to monitor these metabolite dynamics during plant disease and defense
Impact This involves development of novel sensors, never before deployed in plants and the application of sophisticated imaging technologies, confocal with 2-photon system and a GaAsP detector, and a plant-pathogen infection system amenable to both syncronous infections and the ability to discriminate PTI responses from pathogen effector activity.
Start Year 2020
 
Description Identification and chartacterisation of variant cyclic ADP Ribose during effector triggered immunity and impact of key immune loci in its accumulation 
Organisation Colorado State University
Country United States 
Sector Academic/University 
PI Contribution We run a large number of pathogen and pharmacologically challenged plant samples to look at a variety of specific compounds relate to novel nucleosides by quantitative mass spectrometry using special multiple reaction monitoring methods we have developed in Chemistry at Warwick
Collaborator Contribution Marc's lab design and construct a large range of vectors for transient expression in Nicotiana bentamiana, including mutated TIR domains to allow us to determine (i) novel nucleosides, (ii) their accumulation dynamics in dififerent genetic backgrounds and (iii) the impact of targetted mutations in the TIR structural domain on nucleoside profiles.
Impact Generated novel data- confidential - that provides new insights into the role of nucleosides, notably v-cADPRs in plant immunity. this includes providing the tools to identify these structural variants. Molecular plant pathology Molecular biology Structural modelling Biochemistry Analytical and structural chemistry
Start Year 2021
 
Description Structural elucidation 
Organisation University of Queensland
Country Australia 
Sector Academic/University 
PI Contribution The travel award enabled the PI to meet ad initiate a collaboration with one of the world leading structural biologists in plant defence responses - Bostjan Kobe, who is an expert in on TIR domains
Collaborator Contribution We identified a novel set of effector activated genes containing TIR domains. This counter-intuitive finding led to development of a new hypothesis we have asked Prof. Kobe to collaborate on
Impact While not directly related to the work undertaken on the grant, the ability to work in an environment where Prof. Kobe was, meant I was familiar with his work and was able to meet more recently to discuss the possibilities of collaboration (March 2017). The project is multidisciplinary - we are currently in the process of developing the constructs to pursue this. This collaboration would not have happened had the PI not been at CSIRO in Brisbane on BBSRC travel monies.
Start Year 2017