Choreographing plant disease; pathogen effector manipulation of organelles – the view from inside

Project Outline

Biotic stress contributes disproportionately to crop losses, accounting for in excess of 25% of global yield. Thus, developing novel approaches to restricting pathogen infections of crops and consequently improving yields must be a key future objective for food security.

Plants deploy two main immune defence pathways. Plants initially perceive pathogens via surface receptors that recognise non-self, or Pathogen Associated Molecular Patterns or PAMPs. This is termed PAMP Triggered Immunity, or PTI. Pathogens deliver effectors into the cell which both suppress PTI and reconfigure host cells to provide a favourable environment for pathogen multiplication. R proteins are primarily intracellular receptors which perceive these pathogen effectors to confer Effector Triggered Immunity (ETI) by either direct interaction, or indirectly initiating the hypersensitive response (HR), a form of programmed cell death. Recent studies suggest that these pathways are not exclusive and mutually potentiate immune responses.

Unfortunately, R-gene mediated resistance, the primary target of plant breeders, is often overcome in the field. This is due to strong selective pressure driving pathogens to rapidly adapt and evolve sophisticated ways of avoiding detection.

The majority of work in the plant-pathogen field focusses on PTI and ETI and few groups look at HOW the pathogen manipulates the host at the cellular level. Effectors collaborate to cause disease, with each providing an incremental contribution to successful infections. Effectors target different organelles, not just the nucleus and cell membrane. Currently our knowledge of how pathogens target cellular organelles and how these inter-organellular interactions are choregraphed to ultimately result in successful infections are poorly understood. Several years ago the Grant lab, using the Arabidopsis thaliana – Pseudomonas syringae pathosystem, demonstrated that chloroplasts are key targets of pathogen effectors and hence the field of “chloroplast immunity” developed. We later showed a role for the Endoplasmic Reticulum (ER) in plant immunity. However, our unpublished results implicate that all major organelles contribute to plant disease progression.

In collaboration with the McKenna lab we have been developing a suite of state-of-the-art imaging resources that will allow us to undertake a detailed, quantitative study of inter-organelle dynamics during P. syrinage infection. Moreover, we have the capacity to visualise across scales, from whole plant imaging of propagating signalling events to subcellular changes in organelle dynamics. We have a large number of dual organelle reporter lines which will allow us to determine how multiple organelles interact with each other and their dynamics during host-pathogen interactions. Additionally, we have several reporter lines for different signalling molecules, including secondary messengers such as Ca2+, targeted to different organelles. This will allow us to not only determine how organelles interact but also the molecular signalling between different organelles during pathogen interactions. This work is underpinned by several advanced imaging technologies from long-term imaging of whole plants to structured illumination microscopy providing unprecedented spatial and temporal resolution. These techniques are readily used in the supervisor’s laboratory.

The successful candidate will receive training and become proficient in plant pathology and cell biology including techniques such as molecular cloning including multigene assembly, confocal and structured illumination microscopy (SIM).

Techniques

• Microbiology & plant pathology
• Plant Molecular Biology
• Targeted analytical chemistry (mass spectrometry)
• Genetically encoded reporter analysis & whole plant imaging
• Proteomics (including proximity labelling) and RNA-seq
• Advanced Live cell microscopy (confocal, SIM)
• Quantitative Image Analysis

Saving the Brussel sprout – understanding infection dynamics and the hydrathode

Project Outline

The Xanthomonas genus includes diverse phytopathogens that collectively cause disease in more than 350 plant species. Xanthomonas campestris pv. campestris (Xcc), causes black rot on crucifers and is now considered the most important disease of brassicas worldwide. This seed-borne pathogen has been spread around the world via the seed trade. Black rot of crucifers is a regular occurrence in the main brassica production areas of the UK, notably Lincolnshire and Cornwall, and can cause significant economic losses. Xcc is not easy to study as it is a hydathode-infecting vascular pathogen meaning it enters through specialised cells in the leaf then invades the vasculature (plumbing) of the plant causing wilting (“clogging – think of a fat berg!).

Objectives

(i) To understand dynamics of Xanthomonas infection, disease development and transmission (tools already available) and (ii) to engineer resistance Xcc using a novel approach by expressing bacteriocins specifically in specialised hydrathode cells (you develop this strategy).

To study Xcc dymamics we have developed a dual reporter vector that allows us to “tag” a highly virulent Xcs strain with the ilux bacterial bioluminescence reporter (has 7 higher emission intensity compared to conventional lux reporters; Gregor et al. 2018 PNAS doi/10.1073/pnas.1715946115) and the fluorescent protein mScarlet (Canty et al. 2018 PLoS One 13, e0208075) which has a strong quantum yield on the far-red spectrum yet imaging is not confounded by (i) interference from natural chlorophyll fluorescence and (ii) absorbance from the polysaccharide coat of Xcc which precludes the use of traditional GFP reporters.

The project aims to get spatial, temporal imaging data from both Brassica and the model plant Arabidopsis thaliana using different races of Xcc that are economically important; we have recently sequenced over 400 Xcc isolated and pathotested 5 key races on Brassica lines to identify resistance genes. A. thaliana will enable us to deploy key immune compromised mutants.  Collectively these tools will allow high resolution imaging and assessment of infection dynamic and will be complemented by chlorophyll fluorescence imaging that we have pioneered for bacterial infection studies (de Torres Zabala et al. 2015 Nature Plants). Importantly, this objective aims to address the transmission of Xcc to the seed by imaging. Infected seed is the major issue in the Brassica seed industry, thus there exists potential to develop a non-destructive diagnostic platform to support UK industry.

The second part of the project is to develop in A, thaliana a proof of concept, by engineering the specialised hydrathode cells to specifically express bacteriocins – highly active antibacterial proteins that can specifically target Xcc. We have already identified a number of potential bacteriocins through our large scale Xcc sequencing.

So, while you may not be a fan of Brussel sprouts, the tools and technologies developed here are applicable to kale, rocket, watercress and strawberries.

You will be exposed to a range of technique including whole plant real time imaging (chlorophyll fluorescence and ilux imaging), confocal imaging, microbiology, genetics, pant molecular biology and synthetic biology (GoldenGate Cloning) was well as quantitative image analysis.

See our review Greer et al. 2023 Front Microbiol doi: 10.3389/fmicb.2023.1209258 for an overview of the importance of Brassica’s in Food Security and the disease threats.

References

Contreras et al. 2023, EMBO Reports DOI 10.15252/embr.202357495

Chai et al. 2023 Current Opinion in Plant Biology DOI 10.1016/j.pbi.2022.102334

Techniques

• Microbiology & plant pathology
• Plant Molecular Biology
• Imaging, whole plant and confocal
• Synthetic biology – re-engineering the hydrathode

TIRrifically complex but TIRribly exciting – unravelling metabolic immunity

Project Outline

This project delves into one of the hottest and most fascinating recent discoveries in our journey to understanding plant disease defense responses. It has potential o make a massive contribution to enhancing food security as plant disease resistance genes underpin all major breeding programmes in elite crops. The project builds on some pioneering discoveries that underline the complexity of pathogen plant interactions. For nearly 30 years we have known the structure of plant disease resistance proteins but for more than a quarter of a century we were unable to understand how they functioned to trigger disease resistance.

This project focusses on the Toll-Interlukin 1 (TIR) class of plant disease resistance proteins (TNLs) which form a tetrameric resistosome. The TIR domain has NADase activity which results in the production of variant cADP-ribose (vcADPR). This NADase activity is essential for defence and remarkably, pathogens deploy TIR domains to produce different v-cADPRs that interfere with plant immune signalling. This project will dissect how these contrasting TIR domain products impact the immune outcome. It involves collaborators in the USA, Hong Kong and Australia and deploys a range of pre-existing tools and techniques including real time imaging, RNAseq, CRISPR, genetically encoded reporters and targeted mass spectrometry to achieve this objective. Warwick has excellent facilities for this research including state-of-the-art imaging suite and one of the UKs leading Mass Spectrometry facilities.

If you want to work at the cutting edge of plant immunity and develop interdisciplinary skill sets for a future research career, then you may want to consider this project.

References

From the lab

• Manik et al., Science 377, eadc8969 (2022). DOI: 10.1126/science.adc8969
• Bayless et al., Sci. Adv. 9, eade8487 (2023)
• Jacob et al. 2023 PNAS doi.org/10.1073/pnas.2220921120

Reviews

• Chai et al. 2023, MPMI Vol. doi.org/10.1094/MPMI-05-23-0073-HH
• Chai et al. 2023 Current Opinion in Plant Biology 2023 doi.org/10.1016/j.pbi.2022.102334

Techniques

• Microbiology & plant pathology
• Plant Molecular Biology
• Targeted analytical chemistry (mass spectrometry)
• Genetically encoded reporter analysis & whole plant imaging
• RNA-seq
• CRISPR gene editing

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Who was first? Dissecting synergism and antagonism in plant disease resistance signalling

Project Outline

Plant disease resistance (R) genes are widely deployed in plant breeding and confer protection against a vast array of plant pathogens. Unfortunately, R-gene mediated resistance is often overcome in the field as strong selective pressure drives pathogens to rapidly adapt and evolve sophisticated ways of avoiding detection. This is primarily achieved through a suite of host adapted “effectors”.

Plants initially perceive pathogens via surface receptors that recognise non-self. This is termed PTI (Pathogen associated molecular Pattern Triggered Immunity). Pathogen effectors both suppress PTI and reconfigure host cells to provide a favourable environment for pathogen multiplication. R proteins are primarily intracellular receptors which perceive these pathogen effectors to confer effector triggered immunity (ETI) by either direct interaction, or indirectly initiating the hypersensitive response (HR), a form of programmed cell death. R proteins come in two flavours dictated by whether they have a coiled-coil or TIR (Toll-like, Interleukin-1 receptor; TNL) N-terminal domain. Many of these “NLRs are termed “sensor” NLRs and rely on “Helper” NLRs (of the CNL class) to mediate signalling. For nearly 30 years following the discovery of plant disease resistance genes, our understanding of how activated R proteins function was fragmentary until structural biology gave us our first insights into how both classes of activated R proteins “work”. Briefly, CNLs for pentameric and TNLs tetrameric “resistosomes”. Moreover TNL resistosome formation results in TIR dimerization, and generation of NADase activity resulting in novel signalling nucleosides being generation.

While recent studies have shown PTI and ETI work synergistically, potentiating responses no one has yet explored cross-talk between TNLs and CNLs.

Using a unique combination of real time whole imaging approaches that capture temporal and spatial information on physiological changes during plant pathogen interactions elicited in the Arabidopsis thaliana – Pseudomonas syringae pathosystem. This system allows synchronous activation of ETI. Using this approach we have found that simultaneous delivery of combinations of CNLs or TNLs activating effectors lead to remarkable alterations (both synergistic and antagonistic depending on the effector combination) in these parameters which, importantly, faithfully report the ultimate output of an effective ETI interaction, the hypersensitive response (HR).

We hypothesise that we are modifying dynamics of resistosome formation by altering a “limited supply” of key components. Thus, we are in an ideal position to really dissect this process using our extensive collection of Arabidopsis mutants in the “helpers” (combinatorial across the classes) and specific R genes combined with P. syringae isolates designed to deliver different combinations of effectors in both wild type and mutant backgrounds.

You will be exposed to a range of technique including whole plant real time imaging (chlorophyll fluorescence and biophoton generation), RNA-seq, microbiology, CRISPR, genetically encoded reporters and targeted mass spectrometry (to report discriminant ETI compounds).

If you want to work at the cutting edge of plant immunity, are highly motivated and have a passion for plant molecular biology and imaging this project will allow you to develop a range of interdisciplinary skill sets for your future research career.

While this is a novel area and no publications currently exist on this subject (license to operate) please see references below for the current state of the field.

References

Contreras et al. 2023, EMBO Reports DOI 10.15252/embr.202357495

Chai et al. 2023 Current Opinion in Plant Biology DOI 10.1016/j.pbi.2022.102334

Techniques

• Microbiology & plant pathology
• Plant Molecular Biology
• Targetted analytical chemistry (mass spectrometry)
• Genetically encoded reporter analysis & whole plant imaging
• Proteomics and RNA-seq
• Quantitative Image Analysis