The role of the symplast in host-pathogen interactions – how does the symplastic, intercellular exchange of molecules regulate the outcomes of defence and infection?

Project Information

INTERCELLAR

Grant agreement ID: 725459

DOI

10.3030/725459

Project closed

EC signature date 17 March 2017

Start date 1 June 2017

End date 31 May 2023

Funded under

• EXCELLENT SCIENCE - European Research Council (ERC)

Total cost

€ 2 162 500,00

EU contribution

€ 2 162 500,00

Coordinated by

JOHN INNES CENTRE
United Kingdom

Periodic Reporting for period 4 - INTERCELLAR (The role of the symplast in host-pathogen interactions – how does the symplastic, intercellular exchange of molecules regulate the outcomes of defence and infection?)

Reporting period: 2021-12-01 to 2023-05-31

Summary of the context and overall objectives of the project

Cell-to-cell communication is fundamental to all multicellular organisms. In plants, the cytoplasm of adjacent cells is connected via intercellular ‘tunnels’ that cross the cell wall called plasmodesmata. These establish direct connections between cells and allow for the exchange of molecular resources and information to enable the co-ordination of responses to environmental and developmental signals. Plasmodesmata function as molecular ‘sluice gates’ to regulate the flux of soluble molecules between cells. We observed that whether plasmodesmata are open or closed underpins whether tissues can execute a full immune response, but it is not understood how independent immune-competent cells communicate and co-ordinate their responses which are both critical for organism-level responses.

This led us to ask how the connectivity of cells regulates multicellular immunity and, in this project, to address the specific questions: what immune responses are dependent upon plasmodesmal connectivity between cells? and what do host plants gain from regulation of their plasmodesmata during pathogen infection?

We have performed a series of high resolution experiments that firstly addressed how plasmodesmata underpin immune responses. We have shown that whether cells are symplastically connected to their neighbours affects the spatiotemporal profile of the response. We find that if plasmodesmata are closed independently of a pathogen, this alone triggers stress and induces the production of defence hormones to enhance plant resistance to biotrophic pathogens. Calcium responses are a key element of immunity and when we examine how calcium waves spread through a plant we found that unexpectedly, they do not travel through plasmodesmata. This changed common perception about these dramatic and critical response and will force the research community to explore new hypotheses for how signals spread through plants.

We have also found that pathogens target and manipulate plasmodesmata, and that many of their effector proteins that are secreted into host cells can move between cells through plasmodesmata. These effectors can interfere with the host immune response, suggesting that effectors advance ahead of the infection front to disable immunity before the pathogen gets to specific cells. We also found that pathogens can open the plasmodesmata up to facilitate this, raising intriguing questions of how a protein from an organism that does not have plasmodesmata can specifically manipulate these plant-specific structures.

Cell to cell connectivity is critical to both the host and the invading microbe, presenting a key battleground in infection. Our data identifies that for the host, whether plasmodesmata are open or closed regulates immune execution and carbon distribution and this must be balanced to optimise the outcomes of both growth and immunity. Pathogens can subvert these host processes and access resources to enable through own growth. Thus, all these factors are under complex spatiotemporal regulation to determine whether host or microbe controls the cellular network.

Work performed from the beginning of the project to the end of the period covered by the report and main results achieved so far

The overall results of the grant are that: (i) plasmodesmata regulate the spatiotemporal profile of host immune responses to MAMPs, i.e. they regulate the timing and location of gene responses but not the identity of the genes that respond; (ii) that calcium responses are not transmitted by plasmodesmata through the symplast but rather by diffusion and flow of amino acids in the apoplast. This finding was disseminated widely in the popular press (BBC Science Focus, Chemistry world); (iii) that Colletotrichum and Hylaoperonospora both produce suites of effectors that move cell-to-cell in plant tissues, some of which induce changes to plasmodesmal function despite not directly targeting them; (iv) ChEC111 targets host nuclei where it interacts with ribosomal proteins and induces nucleolar stress. ChEC111 is involved in host immune manipulation; (v) ChEC108 targets plasmodesmata and moves cell-to-cell. Like other pathogens from different kingdoms, it targets a host protein that has HMA-domains and behaves like a susceptibility factor; (vi) core plasmodesmal proteins conserved from moss through to Arabidopsis are involved immune responses to chitin, possibly by defining the plasmodesmal membrane domain that recruits specific machinery to execute immune responses; (vii) plasmodesmal immune responses converge on common signalling machinery providing a nexus point for modelling how cell-to-cell connectivity impacts immune success; (viii) the narrow escape solution is a good model for predicting cell-to-cell molecular movement through plasmodesmata; (ix) that plasmodesmal regulation in immunity is age-dependent and likely a regulatory factor of the growth defence trade-off.

Progress beyond the state of the art and expected potential impact (including the socio-economic impact and the wider societal implications of the project so far)

Current understanding of the plant immune system and the mechanisms by which immune responses are executed is primarily limited to understand how single cells respond. With respect to the recognition of microbe-associated molecular patterns by cell surface receptors it has been the dogma that a single receptor perceives a single ligand but we identified that chitin is perceived independently by a plasma membrane receptor (CERK1) and a plasmodesmal receptor (LYM2). Our work in this project has suggested that while these receptors and downstream signalling pathways are independent, and that plasmodesmal responses are executed in an age-dependent manner. We also determined that plasmodesmal responses underpin the timing and location of transcriptional responses, although not the composition of the response components. This opens new understanding of plamsodesmal regulation of immunity and allows new hypotheses to be developed.

We have generated lines in which we can inducibly close plasmodesmata. We used these to establish a stimulus-independent understanding of the effect of plasmodesmal regulation that will apply to any context in which plasmodesmata close. We found that plasmodesmal closure itself activates stress responses which leads to the hypothesis that this also occurs in developmental contexts during which cells and tissues are isolated. We used these lines to demonstrate that calcium responses are transmitted between cells and tissue independently of plasmodesmata, confounding the current models and demanding new hypotheses. The quantitative image analysis tools we developed for this work transform the resolution of data that can be extracted from live imaging of rapid responses.

It is becoming an increasingly frequent observation that microbes target and manipulate host plasmodesmata. We identified Colletotrichum effectors that move between cells. For effectors of particular interest we have identified the targets and determined that they each manipulate or interfere with host immune execution, suggesting that cell-to-cell mobility facilitates more extensive immune suppression by the pathogen. The tools and methods we used to perform this screen will establish a framework that can be deployed for any microbe and opens a new field of effector biology. Combining the outputs of independent effector analysis with manipulation of plasmodesmata will build an understanding of the multi-component mechanisms by which pathogens exploit the host symplast.

The spread of GFP from between cells in this epidermis demonstrates intercellular communication

The spread of a calcium wave through a plant is mediated by apoplastic movement of amino acids