Periodic Reporting for period 1 - DYNWALL (Dynamic cell wall remodeling during plant-microbe interaction)
Reporting period: 2023-02-01 to 2025-07-31
Vascular wilt diseases affect virtually all plants on Earth, causing high devastation of crops and species in natural ecosystems. Rising temperatures are facilitating the spread of these pathogens to new geographical areas, including central and northern Europe. These threads result from the blockage of the vascular system caused by the proliferation inside these tissues of mainly soil-borne microbes that enter the root through its outer epidermal cells and crosses all root layers until reaching the xylem (Image 1). Here, the microbial proliferation blocks water and nutrient transport, resulting in wilting symptoms characterized by yellow and dry leaves, which eventually causes plant death (Image 2). These infection strategy, that gave them the name of “silent killers”, together with their soil-borne and resilient nature, make the chemical, cultural and biological controls generally ineffective to limit their infection. Indeed, most studies of these infections have been conducted in aerial plant tissues, yet the essential root-colonization stage remains poorly understood, largely due to the difficulty in accessing this organ. In fact, details of the precise root infection strategies and plant defense mechanisms are still unclear for many root vascular-plant pathosystems. The work of our group and others have shown that these vascular pathogens mainly live in the root apoplast, the space outside the plant plasma membranes almost completely occupied by the cell walls. We have shown that the plant cell wall properties and its changes during microbial colonization are essential in the outcome of the interaction and the plant response to the infection.
One of the most aggressive root vascular pathogens is the fungus Fusarium oxysporum (Fo), the biggest threat to worldwide banana production and the cause of high yield losses in crops like potato, that has a great potential in ensuring food security in developing nations, and tomato, the most grown vegetable in the world. Importantly, though pathogenic Fos can be devastating, most Fo strains are actually non-pathogenic and many establish beneficial interactions with the plant, that can reduce disease caused by root vascular pathogens. A main factor for the Fo pathogenicity is the ability to modify the plant cell walls of internal root cell layers. Thus, the overall objectives of our project are (1) understanding how the dynamic remodeling of root cell walls during fungal infection determines the outcome of the interaction and (2) using this knowledge to increase plant resistance to vascular pathogens while maintaining their interaction with beneficial microbes.
Conveniently, pathogen and non-pathogen Fos also infect the model plant Arabidopsis thaliana, making this pathosystem, Arabidopsis-Fo, ideal to reach our goals in a reasonable time-frame. The know-how generated in this project will be transferred to crops as new agricultural practices that will help to achieve food safety worldwide without adversely affecting the ecosystems.
One of the most aggressive root vascular pathogens is the fungus Fusarium oxysporum (Fo), the biggest threat to worldwide banana production and the cause of high yield losses in crops like potato, that has a great potential in ensuring food security in developing nations, and tomato, the most grown vegetable in the world. Importantly, though pathogenic Fos can be devastating, most Fo strains are actually non-pathogenic and many establish beneficial interactions with the plant, that can reduce disease caused by root vascular pathogens. A main factor for the Fo pathogenicity is the ability to modify the plant cell walls of internal root cell layers. Thus, the overall objectives of our project are (1) understanding how the dynamic remodeling of root cell walls during fungal infection determines the outcome of the interaction and (2) using this knowledge to increase plant resistance to vascular pathogens while maintaining their interaction with beneficial microbes.
Conveniently, pathogen and non-pathogen Fos also infect the model plant Arabidopsis thaliana, making this pathosystem, Arabidopsis-Fo, ideal to reach our goals in a reasonable time-frame. The know-how generated in this project will be transferred to crops as new agricultural practices that will help to achieve food safety worldwide without adversely affecting the ecosystems.
We have significantly advanced our understanding of how dynamic root cell wall remodeling during fungal infection dictates interaction outcomes. Our main achievements include:
1. Elucidating a crucial Fo pathogenicity mechanism: We discovered that pathogenic Fusarium oxysporum (Fo) traverses internal root apoplast layers, requiring manipulation of suberin deposition for xylem entry, a vital virulence step previously unknown (see more details in “Results beyond the state of the art”). We also characterized root vascular reprogramming post-Fo infection (see more details in “Results beyond the state of the art”). Advanced microscopy enabled these findings.
2. Establishing methodologies: We developed methods to identify and quantify dynamic root cell wall changes during Fo colonization and are creating novel high-resolution techniques.
3. Identifying a novel Fo virulence factor family: These factors modify pectin acetylation, a specific plant cell wall decoration (see more details in “Results beyond the state of the art”).
4. Revealing the plant's role in pectin acetylation: Plant regulation of pectin acetylation significantly impacts Fo infection. A single mutation in a plant protein involved conferred stable Fo resistance without affecting normal development. Ongoing research will assess its impact on beneficial microbe interactions for potential biotechnological applications.
In addition, our ongoing research focuses on:
• Deciphering how pectin-acetylated fragments induce plant defense and characterizing the sensing, signaling, and response mechanisms.
• Studying dynamic root cell wall changes during beneficial-Fo colonization.
• Identifying new pathogenic-Fo virulence factors for internal cell layer entry using a novel RNA translation sequencing methodology at specific infection stages.
1. Elucidating a crucial Fo pathogenicity mechanism: We discovered that pathogenic Fusarium oxysporum (Fo) traverses internal root apoplast layers, requiring manipulation of suberin deposition for xylem entry, a vital virulence step previously unknown (see more details in “Results beyond the state of the art”). We also characterized root vascular reprogramming post-Fo infection (see more details in “Results beyond the state of the art”). Advanced microscopy enabled these findings.
2. Establishing methodologies: We developed methods to identify and quantify dynamic root cell wall changes during Fo colonization and are creating novel high-resolution techniques.
3. Identifying a novel Fo virulence factor family: These factors modify pectin acetylation, a specific plant cell wall decoration (see more details in “Results beyond the state of the art”).
4. Revealing the plant's role in pectin acetylation: Plant regulation of pectin acetylation significantly impacts Fo infection. A single mutation in a plant protein involved conferred stable Fo resistance without affecting normal development. Ongoing research will assess its impact on beneficial microbe interactions for potential biotechnological applications.
In addition, our ongoing research focuses on:
• Deciphering how pectin-acetylated fragments induce plant defense and characterizing the sensing, signaling, and response mechanisms.
• Studying dynamic root cell wall changes during beneficial-Fo colonization.
• Identifying new pathogenic-Fo virulence factors for internal cell layer entry using a novel RNA translation sequencing methodology at specific infection stages.
We consider three of our novel findings significantly advance the field:
1) Fo xylem entry via passage cells and suberin modulation: Pathogenic Fo reaches the xylem by traversing the apoplast of xylem-pole cells. In mature root regions with a formed Casparian strip, Fo enters suberin-lacking endodermal passage cells. Notably, Fo infection triggers premature suberin production, reducing passage cell numbers and expanding the suberized area. Altering endodermal suberization, chemically or genetically, impacts Fo vascular access. We confirmed Fo requires suberin degradation for xylem colonization using a mutant lacking a key enzyme. Contrary to previous assumptions, our data indicate suberin, not lignin, is the primary endodermal barrier against Fo.
2) ABA-mediated disruption of root vascular development and growth by Fo: Despite their agricultural importance, the impact of vascular pathogens like Fo on root growth and vasculature is poorly understood. Using Arabidopsis-Fo, we show Fo dramatically alters root vascular patterning, inducing root-apical-meristem abnormalities and disrupting developmental programs. Protophloem differentiation is impaired, and defective xylem differentiation occurs systemically, beyond direct fungal contact. Abscisic acid (ABA) signaling, known for its role in xylem development, directly contributes to these defects. Plants with impaired endodermal ABA signaling showed partial rescue of Fo-induced meristem abnormalities and altered Fo xylem colonization, revealing a systemic root response linking immune trade-offs and vascular patterning.
3) Pectin acetylesterases (PAEs) are essential for Fo virulence: Using Arabidopsis-Fo, we identified two putative FoPAEs linked to pathogenicity. We characterized one FoPAE structurally and biochemically, with the second ongoing. Single knockout mutants in each PAE exhibited reduced virulence in Arabidopsis. While in vitro growth was similar to wild-type under control and stress, mutant growth was significantly reduced on pectin as a sole carbon source. This identifies FoPAEs as critical virulence factors in host-pathogen interactions and potential targets for vascular crop disease control.
1) Fo xylem entry via passage cells and suberin modulation: Pathogenic Fo reaches the xylem by traversing the apoplast of xylem-pole cells. In mature root regions with a formed Casparian strip, Fo enters suberin-lacking endodermal passage cells. Notably, Fo infection triggers premature suberin production, reducing passage cell numbers and expanding the suberized area. Altering endodermal suberization, chemically or genetically, impacts Fo vascular access. We confirmed Fo requires suberin degradation for xylem colonization using a mutant lacking a key enzyme. Contrary to previous assumptions, our data indicate suberin, not lignin, is the primary endodermal barrier against Fo.
2) ABA-mediated disruption of root vascular development and growth by Fo: Despite their agricultural importance, the impact of vascular pathogens like Fo on root growth and vasculature is poorly understood. Using Arabidopsis-Fo, we show Fo dramatically alters root vascular patterning, inducing root-apical-meristem abnormalities and disrupting developmental programs. Protophloem differentiation is impaired, and defective xylem differentiation occurs systemically, beyond direct fungal contact. Abscisic acid (ABA) signaling, known for its role in xylem development, directly contributes to these defects. Plants with impaired endodermal ABA signaling showed partial rescue of Fo-induced meristem abnormalities and altered Fo xylem colonization, revealing a systemic root response linking immune trade-offs and vascular patterning.
3) Pectin acetylesterases (PAEs) are essential for Fo virulence: Using Arabidopsis-Fo, we identified two putative FoPAEs linked to pathogenicity. We characterized one FoPAE structurally and biochemically, with the second ongoing. Single knockout mutants in each PAE exhibited reduced virulence in Arabidopsis. While in vitro growth was similar to wild-type under control and stress, mutant growth was significantly reduced on pectin as a sole carbon source. This identifies FoPAEs as critical virulence factors in host-pathogen interactions and potential targets for vascular crop disease control.