Periodic Reporting for period 1 - SC-Foxy (Resolving temporal immune responses of Arabidopsis roots to infection by Fusarium oxysporum at the single-cell level)
Période du rapport: 2023-03-10 au 2025-03-09
While there is tremendous knowledge about the plant immune system, and how the plant defends itself against pathogens, we lack spatial resolution. Plant immune responses are typically recorded via transcriptomics (RNA-seq or qRT-PCR using pooled total RNA/DNA), in vitro assays (Calcium burst in cuvette, kinase phosphorylation in-gel, ROS-burst in cuvette), cell death assays in heterologous system (leaves with infiltrated effectors or pathogens) or with markers using leaves of transiently transformed and expressed proteins. In all cases, the spatial resolution is lost, and the infection is far from natural (the pathogen or its effectors are infiltrated in leaves, or physical damage is done by hand).
To close this knowledge gap, we are using fluorescent markers for a wide array of plant immune pathways in live-imaging experiments. We allow Fusarium oxysporum, an important plant pathogen, to infect the plant by itself and in the tissue it naturally infects, without any infiltration, or dripping onto a certain tissue, by growing plant and pathogen in parallel in one sealed experimental dish. Once the fungus has infected the plants, we live-image to infection process and the immune responses of the plant via the different markers live and in real-time on a microscope. By doing so, we can map which immune responses are triggered at which time point, in which tissues, and in which cell types with cellular resolution, and while simultaneously imaging progression of the infection.
This assay has allowed us to create a map of certain immune pathways, and show how cells in direct contact with the infection site respond, how cells further away respond, and which tissues don't respond.
Further, we have started to analyze the plant's responses in a more natural environment, using 'real world' agricultural soil, rather than just laboratory medium or potting soil.
This spatial immunity approach is new and highly promising to understand the plant immune system on an individual cell level.
To close this knowledge gap, we are using fluorescent markers for a wide array of plant immune pathways in live-imaging experiments. We allow Fusarium oxysporum, an important plant pathogen, to infect the plant by itself and in the tissue it naturally infects, without any infiltration, or dripping onto a certain tissue, by growing plant and pathogen in parallel in one sealed experimental dish. Once the fungus has infected the plants, we live-image to infection process and the immune responses of the plant via the different markers live and in real-time on a microscope. By doing so, we can map which immune responses are triggered at which time point, in which tissues, and in which cell types with cellular resolution, and while simultaneously imaging progression of the infection.
This assay has allowed us to create a map of certain immune pathways, and show how cells in direct contact with the infection site respond, how cells further away respond, and which tissues don't respond.
Further, we have started to analyze the plant's responses in a more natural environment, using 'real world' agricultural soil, rather than just laboratory medium or potting soil.
This spatial immunity approach is new and highly promising to understand the plant immune system on an individual cell level.
We have monitored the plant's immune response to infection with several of our ~200 markers.
So far, the main results are:
- The phytohormones jasmonic acid (JA), salicylic acid (SA) and ethylene (ET) define two distinct response zones to infection: SA and high ET together define a hypersensitive response zone in the cells closest to the infection site in the tissue under attack (the vasculature). These cells will undergo programmed cell death, to halt progression of the infection. JA and low ET together describe a defense response zones in a group of vascular cells immediately behind the hypersensitive response zone. We believe that this zone is the second line of defense, should programmed cell death not suffice.
- Chemical defense metabolites, or antimicrobial compounds, also contribute to the plant's defense against Fusarium. We show that specifically two Trp-derived compounds, camalexin and hycanite (4-OH-ICN) are produced in infected tissues. hycanite is primarily produced and stored in the cells in and around the hypersensitive response zone, indicating that these toxic compounds will be released into the rhizosphere upon programmed cell death, adn thus be dumped onto the pathogen. Camalexin is primarily produced in the defense response zone and acts locally against the pathogen, but also systemically to launch systemic resistance.
- Autophagy not only contributes to the plant immune system by proteolysis, but also contains a second pathway, which protects vacuolar integrity should cell wall damage occur. This connection between cell wall and vacuole is important, since these two compartments contribute to cell wall growth and expansion.
- The plant's immune response is completely different when a plant is grown on natural agricultural soil, instead of potting soil or laboratory medium. This indicates that many observations made in labs are probably not transferable to the field.
So far, the main results are:
- The phytohormones jasmonic acid (JA), salicylic acid (SA) and ethylene (ET) define two distinct response zones to infection: SA and high ET together define a hypersensitive response zone in the cells closest to the infection site in the tissue under attack (the vasculature). These cells will undergo programmed cell death, to halt progression of the infection. JA and low ET together describe a defense response zones in a group of vascular cells immediately behind the hypersensitive response zone. We believe that this zone is the second line of defense, should programmed cell death not suffice.
- Chemical defense metabolites, or antimicrobial compounds, also contribute to the plant's defense against Fusarium. We show that specifically two Trp-derived compounds, camalexin and hycanite (4-OH-ICN) are produced in infected tissues. hycanite is primarily produced and stored in the cells in and around the hypersensitive response zone, indicating that these toxic compounds will be released into the rhizosphere upon programmed cell death, adn thus be dumped onto the pathogen. Camalexin is primarily produced in the defense response zone and acts locally against the pathogen, but also systemically to launch systemic resistance.
- Autophagy not only contributes to the plant immune system by proteolysis, but also contains a second pathway, which protects vacuolar integrity should cell wall damage occur. This connection between cell wall and vacuole is important, since these two compartments contribute to cell wall growth and expansion.
- The plant's immune response is completely different when a plant is grown on natural agricultural soil, instead of potting soil or laboratory medium. This indicates that many observations made in labs are probably not transferable to the field.
The maps we have created for our spatial immunity project are beyond the state of the art. Such spatial resolution is typically not described, and one reason why previous observations are incomplete.
The observation that plants react completely different in natural soil than in the lab, goes way beyond the state of the art, as it questions how lab-observations could possibly be transferred to field-grown crop plants.
The observation that plants react completely different in natural soil than in the lab, goes way beyond the state of the art, as it questions how lab-observations could possibly be transferred to field-grown crop plants.