Plasmodesmata: genetic control of cell-to-cell communication during plant defence

Lead Research Organisation: John Innes Centre
Department Name: Crop Genetics

Abstract

Like animals, plants suffer all manner of diseases caused by various fungal, bacterial and viral pathogens. When a fungal or bacterial pathogen attacks a plant, the battlefront occurs initially at a single cell. One means by which a plant can fight off the pathogen is to initiate a process that results in death of the cell that is being attacked, preventing the pathogen from gaining nutrients from this cell and spreading. This process involves the generation of many toxic molecules and it is crucial that the cells surrounding the attacked cell do not suffer the same fate.

Plant cells are connected to their neighbouring cells by channels called plasmodesmata. These channels are like open doorways into neighbouring cells and small molecules can simply pass from cell to cell. The question then is how a dying cell stops toxic molecules leaking out into its neighbours? Does the cell close the doors into its neighbours? We know that plasmodesmata can open and close to control the movement of signals between cells when a plant is exposed to cold or when the plant undergoes a developmental transition. We want to investigate whether these doors close when a pathogen attacks and how this happens.

Recently, many new proteins that are located at plasmodesmata have been identified. One of these is a receptor for fungal chitin and causes plasmodesmata to close when a fungus is detected. I will examine why plasmodesmata close when a pathogen is perceived by microscopic analysis of living tissue that is under attack from different pathogens. I will also determine how plasmodesmata close by biochemical and genetic identification of proteins that required for this response.

By establishing when and how plasmodesmata open and close when a pathogen attacks we will be able to understand a very important mechanism that a plant uses to fight disease. The last objective of this programme will begin to explore this phenomenon in rice with the aim of understanding how this global food crop uses this mechanism to fight off pathogens. Understanding these mechanisms will enable us to exploit them to help plants fight disease, placing us in a strong position to combat the impact of increased prevalence of plant disease that is likely to arise from global climate changes.

Technical Summary

A comprehensive understanding of the complexities of plant-pathogen interactions is crucial to the fight against the increased spread and severity of plant disease posed by a changing global climate. When a pathogen is present, whole tissues and organs must respond to the threat of invasion in a co-ordinated fashion. Most plant cells are connected to their neighbours by dynamic channels called plasmodesmata (PD) which allow and regulate the flux of molecules and information between cells.

I have observed that the pathogen associated molecular patterns (PAMPs) chitin and the flagellin derivative flg22 induce a reduction in molecular flux via PD. Chitin and flg22 also induce dramatic changes in the concentration of small molecules such as reactive oxygen species and calcium ions within the attacked cell and therefore it is possible that an attacked cell isolates itself to concentrate these essential defence molecules at the site of attack. To test this hypothesis I will employ live cell imaging, using a variety of inert and biologically relevant probes, to map the generation of symplastic domains around an infection site.

Chitin-triggered changes to molecular flux are mediated by the plasmodesmata-located protein LYM2. lym2 mutants do not respond to chitin with respect to changes to molecular flux and thus will allow comparative analysis. Further, interactors of LYM2 will be identified to dissect the mechanism by which PD close following chitin perception. For LYM2-interactors, and other PD-located defence associated proteins, overexpressors and genetic knockouts will be analysed with respect to PAMP-triggered PD closure and pathogen susceptibility to determine their role in the regulation of molecular flux. With an outlook to translating this research to agriculturally significant species I will also examine PAMP-induced changes to molecular flux in rice and determine which of the five member rice LYP protein family associates with plasmodesmata.

Planned Impact

With the threat of increasing carbon dioxide levels and temperatures across the globe, the relationships between plant pathogens and their hosts are poised to change in ways that will increase the prevalence and severity of plant disease. By impacting agricultural and native species, plant diseases manifest as disasters such as famine and environmental damage. Biotic pathogens cause huge agricultural losses each year and the security of these and related industries is dependent in part on more complete understanding of the interactions that cause disease and the applications of this knowledge.

Agriculture is dependent on the use of chemicals for the management of pathogens. Many chemicals have limited longevity due to the emergence of resistance, and public opposition to chemicals is also increasing. By investigating a poorly described strategy for defence responses, a long term benefit from this research programme will be the novel approaches for the generation of pathogen resistance. This could be through the selection of improved crops or via genetic modification (GM) technologies. Although public opinion relating to this technology is currently divided, it still offers valuable possibilities for generating pathogen resistance, and novel developments allow genetic modifications in the absence of transgenes and selection markers.

This research will accordingly have benefits for a wide cross-section of society. The direct benefits will lie with the agricultural industry: primarily farmers and plant biotechnology companies. The research outputs will also be of benefit to policy makers and the general public, from schools through to general interest groups, given the social relevance of discussion around the optimal paths (GM or selected breeding) for delivering novel traits in crops. The PI has significant experience in delivering scientific output to the general public and will engage with Oxford Brookes University science outreach activities such as the Science Bazaar and the annual Oxfordshire Science festival, as well as with organisations such as Science Oxford and the Science Media Centre.

Results from this project will be first communicated in original research articles, presented at national and international conferences and then summarized in more general publications. The PI and PDRA will submit abstracts for poster or oral presentation at scientific meetings (e.g. International Plasmodesmata Conference 2014, Molecular-Plant Microbe Interactions Congress 2016). The PI and PDRA will also present this research regularly within departmental seminar programmes and research group lab meetings. The PDRA will be involved in all relevant collaborations and be able to meet national and international visitors to the site.

The PI will have overall responsibility for ensuring the delivery of this impact plan. The PDRA will be expected to contribute substantially to these activities and will be given appropriate training.

Publications

10 25 50
 
Description Chitin, a fungal cell wall component, induces closure of plasmodesmata (the membrane-lined pores that connect neighbouring plant cells). We have identified several components of the signalling cascade from chitin perception to plasmodesmal closure; we discovered that chitin-triggered plasmodesmal closure requires LysM receptor kinases and phosphorylation of an NADPH oxidase by two calcium dependent protein kinase, and that chitin induces localised callose deposition at plasmodesmata (the mechanism by which plasmodesmata physically close). Significantly, these processes are independent and specific relative to other chitin triggered signalling cascades that occur elsewhere in the cell. We have also determined that the signalling pathway that induces plasmodesmal closure in response to peptidoglycan (a bacterial cell wall component) shares components with chitin-responses.

We have screened rice LysM receptor proteins for any that localise to plasmodesmata and therefore might mediate similar plasmodesmal closure responses in rice. We have identified that at least one, is located at plasmodesmata and will form the basis for characterisation of the contribution of chitin-triggered plasmodesmal closure to rice immune responses.

Flagellin-induced plasmodesmal closure is independent of chitin-induced plasmodesmal closure and we have identified a calcium responsive protein that is located at plasmodesmata and mediates plasmodesmal closure, again via localised callose deposition. We have determined that this response occurs within 15 minutes of a cell's exposure to flagellin, making it one of the earliest defence responses.

We have examined host defence responses that are dependent on plasmodesmal closure and determined that defence hormone signalling (salicylic acid and jasmonic acid) critically relies on the plasmodesmal connections between cells. Further, we have identified that bacterial pathogens can suppress host plasmodesmal responses via the activity of an, as yet, unknown effector protein.
Exploitation Route We have identified novel components of plasmodesmal function and host defence responses. We except these will be used by many academic researchers in both fields of research.
Sectors Agriculture

Food and Drink

 
Description European Research Council Consolidator Grant
Amount € 2,162,500 (EUR)
Funding ID 725459 "INTERCELLAR" 
Organisation European Commission 
Sector Public
Country European Union (EU)
Start 05/2017 
End 05/2023
 
Description Norwich Research Park Doctoral Tranining Programme
Amount £76,228 (GBP)
Funding ID FAULKNER_J16WDTP 
Organisation Biotechnology and Biological Sciences Research Council (BBSRC) 
Sector Public
Country United Kingdom
Start 09/2016 
End 09/2020
 
Description The role of CML41 in flagellin induced plasmodesmata closure 
Organisation University of Adelaide
Department School of Agriculture, Food and Wine
Country Australia 
Sector Academic/University 
PI Contribution We provide expertise and technologies on the analysis of plasmodesmata in response to pathogens, live cell imaging and image analysis
Collaborator Contribution The Gilliham lab identified CML41 and performed gene expression analysis
Impact Publication: Xu B, Cheval C, Laohavisit A, Hocking B, Chiasson D, Olsson TSG, Shirasu K, Faulkner C, Gilliham M A calmodulin-like protein regulates plasmodesmal closure during bacterial immune responses. New Phytol. 2017 Jul;215(1):77-84. doi: 10.1111/nph.14599. Epub 2017 May 17.
Start Year 2015
 
Title Bootstrap analysis tool for non-normal biological data 
Description Phytologists rely on experimentally perturbing plants and monitoring the responses. Frequentist statistics are used to ascertain the probability that an observed difference between conditions was due to chance (a p value). When data are not normal and have differing variances, we propose data sets are better analysed by a bootstrap method that tests the null hypothesis that means (or medians) are the same between two conditions, instead of the commonly used Mann-Whitney-Wilcoxon test. We illustrate this with data from the cell-to-cell movement of GFP through plasmodesmata. We found that that with hypothetical distributions similar to cell-to-cell movement data, the Mann-Whitney-Wilcoxon produces a false positive rate of 17% while the bootstrap method maintains a false positive at the set rate of 5% under the same circumstances. Here we present this finding, as well as our rationale, an explanation of the bootstrap method and an R script for easy use. We have further demonstrated its use on published datasets from independent laboratories. 
Type Of Technology Software 
Year Produced 2020 
Open Source License? Yes  
Impact none to date 
 
Title Find Plasmodesmata 
Description This tool can automatically detect spots in confocal z-stacks. It measures the voxel dimensions and intensity of these structures. 
Type Of Technology Software 
Year Produced 2016 
Open Source License? Yes  
Impact We use this software to automate detection of aniline blue fluorescence in confocal z-stacks. This allows us to quantify callose deposition at plasmodesmata, which we have shown serves as a proxy for plasmodesmal functional state. This tool has allowed us to greatly expand our analysis across many genotypes. 
URL https://github.com/JIC-CSB/find-plasmodesmata
 
Title faulknerfalcons/Cheval-2020-PNAS: Analysis Scripts for Cheval, Samwald, Johnston, et al., 2020, PNAS 
Description Chitin perception in plasmodesmata characterizes submembrane immune-signaling specificity in plants Cheval, Samwald, Johnston, et al., 2020, PNAS, DOI: 10.1073/pnas.1907799117 A collection of R scripts to exemplify how we analysed our bombardment and FRAP data in the manuscript. Contents Combinatorics statistics R script (.R) FRAP Example raw data (.csv) Example decay curves (.csv) Example sanity checking figures (.png) R scripts to analyse data (.R) Sample data provided by Samwald, S (https://orcid.org/0000-0003-4401-8862). Raw data, collated by figure, for use with the combinatorics script is available as a supplemental file at PNAS (distributed under Creative Commons Attribution License 4.0 (CC BY)). 
Type Of Technology Software 
Year Produced 2020 
Open Source License? Yes  
URL https://zenodo.org/record/3727531
 
Title faulknerfalcons/Cheval-2020-PNAS: Analysis Scripts for Cheval, Samwald, Johnston, et al., 2020, PNAS 
Description Chitin perception in plasmodesmata characterizes submembrane immune-signaling specificity in plants Cheval, Samwald, Johnston, et al., 2020, PNAS, DOI: 10.1073/pnas.1907799117 A collection of R scripts to exemplify how we analysed our bombardment and FRAP data in the manuscript. Contents Combinatorics statistics R script (.R) FRAP Example raw data (.csv) Example decay curves (.csv) Example sanity checking figures (.png) R scripts to analyse data (.R) Sample data provided by Samwald, S (https://orcid.org/0000-0003-4401-8862). Raw data, collated by figure, for use with the combinatorics script is available as a supplemental file at PNAS (distributed under Creative Commons Attribution License 4.0 (CC BY)). 
Type Of Technology Software 
Year Produced 2020 
Open Source License? Yes  
URL https://zenodo.org/record/3727532
 
Title faulknerfalcons/Johnston-2020-Bootstrap: Scripts and data for Johnston and Faulkner 'A bootstrap approach is a superior statistical method for the comparison of non-normal data with differing variances.' 
Description A bootstrap approach is a superior statistical method for the comparison of non-normal data with differing variances. Johnston and Faulkner, New Phytologist (2020) doi: 10.1111/nph.17159 Scripts for using our proposed bootstrap method: medianBootstrap.R contains functions to replace wilcox.test() with medianBootstrap() medianBootstrapToolbox.R contains an extended set of functions, allowing for the plotting of the null distribution, replacement of one-way ANOVAs, and a meanBootstrap(). A primer on how to use the medianBootstrap script is provided as a webpage (medianBootstrap.html) and R markdown (medianBootstrap.Rmd). All the scripts and data used within the manuscript and figures used under a CC BY 4.0 licence. 
Type Of Technology Software 
Year Produced 2020 
Open Source License? Yes  
URL https://zenodo.org/record/4326967
 
Description Interactive Hands on experiment - Norwich Science Festival, PDRA Cecilia Cheval 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach Regional
Primary Audience Public/other audiences
Results and Impact Cecilia participated in the experimental stand hosted by JIC at the Norwich Sceince Festival. This involved demonstrations and hands-on experiments and discussions with membres of the public across broad age ranges.
Year(s) Of Engagement Activity 2017
 
Description Invited Talk - ETH, Zurich, Switzerland 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact I presented our work relating to plasmodesmal function during immunity and infection. I also spent time discussing research projects with postdocs and faculy members. Discussions with the Rodriguez-Villalon lab led to the development of new strategies for experiments in their project.
Year(s) Of Engagement Activity 2019
 
Description Invited speaker - 36th New Phytologist Symposium, "Cell biology at the plant-microbe interface", Munich, Germany 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact I presented our reserach at this symposium and from discussions aftrewards identified novel areas for future research and collaboration
Year(s) Of Engagement Activity 2015
 
Description Invited speaker - Cold Spring Harbor Asia "Latest Advances in Plant Development and Environmental Response" meeting, Japan 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact I presented my research at this workshop to a large international audience. My presentation sparked some debate on the relevance of cell to cell communication which continued throughout the duration of the meeting at informal opportunities.
Year(s) Of Engagement Activity 2016
 
Description Invited speaker - Plant Signaling and Behaviour Symposium, St Petersburg, Russia 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact I presented our research to scientists and held many discussions with other participants, identifying new potential collaborators
Year(s) Of Engagement Activity 2016
 
Description Invited speaker - Vienna Biocenter PhD Symposium "Communication - let's talk about it", Austria 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Postgraduate students
Results and Impact I was an invited speaker at the Vienna Biocenter PhD student symposium. The majority of PhD students in attendance were from non-plant biology backgrounds and my presentation sparked a lot of interest in the differences between plants and their own experimental systems. The symposium covered a range of topics from intracellular communication to inter-organism communication and therefore allowed crosstalk of science from protein structure to language deveolpment.
Year(s) Of Engagement Activity 2015
 
Description Invited speaker and session chair - International Congress of Molecular Plant Microbe Interactions 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact The International Congress of Molecular Plant Microbe Interactions serves to disseminate cutting edge research developments in the field and was attended . I communicated our work relating to receptor specialisation at plasmodesmata and several audience members expressed that this expanded their views and understanding of plant responses to chitin.
Year(s) Of Engagement Activity 2019
 
Description Invited speaker and session chair - International Plant Molecular Biology Congress, Brazil 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact I co-ordinated the session, and presented my research, on symplastic and vascular transport. This opportunity allowed me to frame the topics included in the meeting and highlight key research I think will define future advances in the field.
Year(s) Of Engagement Activity 2015
 
Description Invited talk - James Hutton Institute UK 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact I presented a reserach talk on my work to project leaders and postgraduate students at the James Hutton Institute. I also had 1-2-1 meetings with postgraduate students and discussed research and career progression options.
Year(s) Of Engagement Activity 2015
 
Description Invited talk - Distinguished Seminar Series, Plant Stress Centre, Shanghai, China 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact I presented my research to researchers at the Plant Stress Centre (Shanghai, China) and held 1 to 1 meetins with project leaders and postgraduate students to discuss research ideas and collaboration opportunities
Year(s) Of Engagement Activity 2015
 
Description Invited talk - University of Durham, UK 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Professional Practitioners
Results and Impact I presented my work in the research seminar series at the university Durham and spent time in one to one meetings with students and faculty. One meeting lead to an insect specialist sending us samples to assay for insect chitin responses
Year(s) Of Engagement Activity 2017
 
Description Invited talk - University of Tubingen, Germany 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Postgraduate students
Results and Impact After my talk at the Vienna Biocenter Symposium, I was invited by the Masters students in Tubingen to present my research in their seminar series. I also spent time with the stuident hosts and discussed research and career development topics.
Year(s) Of Engagement Activity 2016
 
Description JIC International Summer School 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Undergraduate students
Results and Impact I presented a talk to the students taking part in the JIC International Summer School about my scientific interests and career progression.
Year(s) Of Engagement Activity 2016
URL https://opportunities.jic.ac.uk/summerprogramme/
 
Description Main organiser - EMBO workshop on Intercellular Communication in Development and Disease, Berlin, Germany 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact As main organiser of this workshop I secured funding from EMBO and shaped the programme of this workshop. We invited speakers from broad geographical locations and topic areas. The workshop feedback was overwhelmingly positive with a number of participants describing how they had established new collaborations from attending this workshop. The workshop has been passed on to a new group of organisers to arrange for a repeat event in 2020.
Year(s) Of Engagement Activity 2017
 
Description Pint of Science 2017 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach Local
Primary Audience Public/other audiences
Results and Impact Pint of Science is an outreach event held in pubs across the country with the aim of taking reseearch andscientific ideas to the general public. I presented in the York Tavern were I estimate 35-50 people attended. The audience were members of the general public.
Year(s) Of Engagement Activity 2017
URL https://pintofscience.co.uk/
 
Description School Visit - Year 11 mock interview day, Sir John Leman High School - PDRA Cecilia Cheval 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach Regional
Primary Audience Schools
Results and Impact Cecilia attended this Career and interview day as a STEM ambassador to share her experiences as a bench scientist and career development with local high school students. She acted in mock interviews to give students experience of what to expect if applying for jobs in the science and research sector.
Year(s) Of Engagement Activity 2015
 
Description Talk - invited speaker "Mechanisms of Plant Perception - from the endogenous to the exogenous", University of Lausanne, Switzerland 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Professional Practitioners
Results and Impact I attended this symposium as an invited speaker. Discussion related to future possibile research directions and career development with postdocs and students.
Year(s) Of Engagement Activity 2019
 
Description Women of the Future Conference 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach Regional
Primary Audience Schools
Results and Impact Approximately 300 female year 10 students attended the conference at the John Innes Centre. I presented a workshop to small groups on my research and career and answered student questions.
Year(s) Of Engagement Activity 2015
URL http://nextgenstemm.org.uk/wotf15/
 
Description Year 10 Science Camp 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Schools
Results and Impact I spoke to year 10 students who visited JIC to participate in the summer science camp. Students were mostly from schools across Norfolk but others from further across the country. I spoke both about my scientific interest, what my job entails and how I pursued a career in science.
Year(s) Of Engagement Activity 2016
URL https://www.jic.ac.uk/year10/
 
Description Year 10 Science Camp 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Schools
Results and Impact Year 10 students from across the country attend 'science camp' for a week. They are exposed to research in laboratories as well as science career options. I spoke to students in a workshop session about my career path and research.
Year(s) Of Engagement Activity 2015
URL https://www.jic.ac.uk/year10/index.htm