Periodic Reporting for period 3 - 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: 2020-06-01 to 2021-11-30
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 aim to exploit mutants that cannot regulate their plasmodesmata in response to pathogen perception to identify and characterise the symplast-dependent.
In the context of plant-microbe interactions, immune responses do not occur in isolation of infection processes executed by the attacking pathogen. We noted that for some pathogens, while plasmodesmata would normally close upon their perception, the plasmodesmata were open during infection which suggested to us that pathogens seek to keep plasmodesmata open. This raises the questions of what a pathogen gains from keeping access to the host symplast open and how it does so? To answer these, we are examining the proteins that are secreted into the plant host by the fungal pathogen Colletotrichum higginsianum to identify and characterise those that can move through the symplast or that target and modify plasmodesmata. Ultimately, we will combine the information yielded by our study of the symplastic context of host immune responses and of pathogen infection mechanisms to establish an understanding of how the battle for resources is played out in multicellular plant tissues. This will establish a new multicellular understanding of the battle between plants and pathogenic microbes.
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 aim to exploit mutants that cannot regulate their plasmodesmata in response to pathogen perception to identify and characterise the symplast-dependent.
In the context of plant-microbe interactions, immune responses do not occur in isolation of infection processes executed by the attacking pathogen. We noted that for some pathogens, while plasmodesmata would normally close upon their perception, the plasmodesmata were open during infection which suggested to us that pathogens seek to keep plasmodesmata open. This raises the questions of what a pathogen gains from keeping access to the host symplast open and how it does so? To answer these, we are examining the proteins that are secreted into the plant host by the fungal pathogen Colletotrichum higginsianum to identify and characterise those that can move through the symplast or that target and modify plasmodesmata. Ultimately, we will combine the information yielded by our study of the symplastic context of host immune responses and of pathogen infection mechanisms to establish an understanding of how the battle for resources is played out in multicellular plant tissues. This will establish a new multicellular understanding of the battle between plants and pathogenic microbes.
During this reporting period we have worked on (i) developing the methods and controls for spatiotemporal analysis of host immune responses; (ii) identifying markers specific to plasmodesmal responses; (iii) developing tools for manipulation of plasmodesmata; (iv) identifying Colletotrichum higginsianum effectors that exploit and manipulate the symplast; (v) developing simple models that describe symplastic communication. To this end we have developed new methods for in vitro plant growth and elicitor treatment that reduces variation induced by environmental factors and wounding, placing us in a strong position to experimentally dissect how gene expression and metabolic processes change in plasmodesmal mutants. We have also finalised testing of lines in which we can spatially induce plasmodesmal callose synthesis (and thus plasmodesmal aperture). We have performed a simple gene expression analysis to compare mutants in plasmodesmal responses to chitin (lym2) with wild-type plants (Col-0) and mutants in plasma membrane chitin perception (cerk1). In these whole seedling samples, we were able to identify responses associate with each receptor and subsequently developed a new hypothesis that plasmodesmal responses ‘tune’ cellular processes. Our screen of Colletotrichum effectors has identified proteins that are symplast mobile, and that can both modify and target plasmodesmata. We have many effectors to exploit in investigating how pathogens manipulate and exploit plasmodesmata. We have also defined a simple model for symplastic transport that will be further developed.
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, that their impact on immune responses is not. We have developed new methods for plant tissue culture and elicitor treatment to increase the power of our analysis to determine how both pathways underpin spatio-temporal regulation of immune responses across a tissue. We are currently working on this in both the chitin-triggered and infection context and aim to fully characterise how plasmodesmata underpin transcriptional and metabolic responses in immunity.
We have developed and tested lines in which we can inducibly close plasmodesmata. We are initially testing these for transcriptional responses to plasmodesmal closure to identify how tissues respond when their intercellular connectivity is disconnected. This will establish a stimulus-independent understanding of the effect of plasmodesmal regulation that will apply to any context in which plasmodesmata close. We will use these lines to determine how plasmodesmal closure perturbs immune responses to both microbe-associated molecular patterns and overall resistance (or susceptibility) to a range of pathogens to gain understanding of how this gives advantage to both host and pathogen.
It is becoming an increasingly frequent observation that microbes target and manipulate host plasmodesmata. To understand what a microbe gains from access to the host symplast, we performed a screen of Colletotrichum effectors and identified effectors that move between cells. The targets of these effectors will identify processes that a pathogen targets in non-infected cells and determine the benefits of maintaining symplastic connectivity. 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. For this candidate list of Colletotrichum effectors, we will investigate their biochemical targets and influence on gene expression analysis. Combining the outputs of independent effector analysis will build an understanding of the multi-component mechanisms by which this pathogen exploits the host symplast.
We have developed and tested lines in which we can inducibly close plasmodesmata. We are initially testing these for transcriptional responses to plasmodesmal closure to identify how tissues respond when their intercellular connectivity is disconnected. This will establish a stimulus-independent understanding of the effect of plasmodesmal regulation that will apply to any context in which plasmodesmata close. We will use these lines to determine how plasmodesmal closure perturbs immune responses to both microbe-associated molecular patterns and overall resistance (or susceptibility) to a range of pathogens to gain understanding of how this gives advantage to both host and pathogen.
It is becoming an increasingly frequent observation that microbes target and manipulate host plasmodesmata. To understand what a microbe gains from access to the host symplast, we performed a screen of Colletotrichum effectors and identified effectors that move between cells. The targets of these effectors will identify processes that a pathogen targets in non-infected cells and determine the benefits of maintaining symplastic connectivity. 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. For this candidate list of Colletotrichum effectors, we will investigate their biochemical targets and influence on gene expression analysis. Combining the outputs of independent effector analysis will build an understanding of the multi-component mechanisms by which this pathogen exploits the host symplast.