Periodic Reporting for period 1 - PowR (Powdery Mildew Resistance)
Periodo di rendicontazione: 2023-11-01 al 2025-10-31
The Marie Curie project titled: PowR (Powdery Mildew Resistance), aims at defining ESCRT-independent extracellular vesicle secretion in barley immunity against powdery mildew infection. The eradication of hunger is a central pillar of the United Nations Sustainable Development Goals. A major step in achieving this goal involves sustainable increases in crop productivity. One of the major threats to global food security is yield loss caused by plant diseases. Among these, fungal pathogens represent a particularly severe and persistent challenge, as they affect a wide range of staple and economically important crops and are often difficult to control through chemical or agronomic means alone. Powdery mildew fungi are a prominent group of obligate biotrophic pathogens that infect many important crop species, like wheat and barley, and cause significant reductions in biomass and yield by suppressing host immune responses and living on nutrients from living host cells. In barley (Hordeum vulgare), infection by the powdery mildew fungus, Blumeria hordei (Bh), can result in yield losses of up to 40%, posing a substantial threat to both food production and agricultural sustainability, making disease mitigation of direct relevance to food security and economic stability. Consequently, there is a pressing need for a deeper mechanistic understanding of plant immunity and pathogen virulence strategies at the molecular and cellular levels, which can inform the rational design of durable resistance traits and reduce reliance on chemical control.
Plants possess a multilayered immune system that detects and responds to pathogen attack. The first layer of defence, pattern-triggered immunity (PTI), is activated when conserved pathogen-associated molecular patterns are recognised by the plant. PTI initiates a cascade of signalling events that act to block pathogen entry. A hallmark of PTI in interactions with filamentous pathogens is the formation of papillae—cell wall appositions deposited at the site of attempted penetration. These papillae, along with later-forming encasements that surround invasive pathogen structures, function as physical and biochemical barriers that limit penetration and nutrient transfer. Timely and efficient formation of papillae is therefore a critical determinant of penetration resistance. The formation of these immune structures depends heavily on intracellular membrane vesicle trafficking, which deliver papillae components to the site of attack together with extracellular vesicles (EVs). EVs are membrane-bound vesicles released into the apoplast and are thought to carry proteins, lipids, and potentially RNA molecules that contribute to defence. Despite their importance, the molecular pathways governing EV formation and secretion during plant immunity remain poorly understood. In particular, the origin of papilla-associated EVs and the trafficking routes responsible for their delivery are largely unknown.
Adapted pathogens such as Blumeria hordei have evolved sophisticated strategies to overcome plant immunity. Central to this is the secretion of effector proteins into host cells. Bh secretes 600 effector proteins, many of which enter the host cytosol and manipulate host cellular processes to suppress immunity and promote pathogen growth. This project proposed to use these effectors as powerful molecular probes, in addition to studying their role in EV formation. Because effectors tend to target key regulatory nodes in host pathways, identifying their host targets can reveal critical components of plant cellular machinery that might otherwise remain elusive. In this sense, pathogen effectors provide a unique opportunity to dissect complex biological processes such as membrane trafficking and vesicle secretion.
In the Bh–barley pathosystem, several host proteins required for penetration resistance have been identified, including the PENETRATION (PEN) proteins. The syntaxin PEN1, also known as SYP121, in Arabidopsis thaliana and its barley orthologue ROR2 are essential for timely papilla formation. Notably, PEN1 and ROR2 are excellent markers for papilla-associated EVs, and ROR2 was used in this project to understand the processes of vesicular secretion.
Prior to the conception of this project, recent work in the host laboratory had demonstrated that dominant-negative approaches targeting classical ESCRT (Endosomal Sorting Complex Required for Transport) machinery components impair encasement formation, but leave papilla EV formation largely unaffected. These findings strongly suggest the existence of a yet unexplored ESCRT-independent multivesicular body (MVB)-like pathway delivering papilla EVs. Thus, the overarching objective of this project was to define the molecular mechanisms by which barley secretes extracellular vesicles into papillae during immune responses against the adapted fungal pathogen, Blumeria hordei.
Plants possess a multilayered immune system that detects and responds to pathogen attack. The first layer of defence, pattern-triggered immunity (PTI), is activated when conserved pathogen-associated molecular patterns are recognised by the plant. PTI initiates a cascade of signalling events that act to block pathogen entry. A hallmark of PTI in interactions with filamentous pathogens is the formation of papillae—cell wall appositions deposited at the site of attempted penetration. These papillae, along with later-forming encasements that surround invasive pathogen structures, function as physical and biochemical barriers that limit penetration and nutrient transfer. Timely and efficient formation of papillae is therefore a critical determinant of penetration resistance. The formation of these immune structures depends heavily on intracellular membrane vesicle trafficking, which deliver papillae components to the site of attack together with extracellular vesicles (EVs). EVs are membrane-bound vesicles released into the apoplast and are thought to carry proteins, lipids, and potentially RNA molecules that contribute to defence. Despite their importance, the molecular pathways governing EV formation and secretion during plant immunity remain poorly understood. In particular, the origin of papilla-associated EVs and the trafficking routes responsible for their delivery are largely unknown.
Adapted pathogens such as Blumeria hordei have evolved sophisticated strategies to overcome plant immunity. Central to this is the secretion of effector proteins into host cells. Bh secretes 600 effector proteins, many of which enter the host cytosol and manipulate host cellular processes to suppress immunity and promote pathogen growth. This project proposed to use these effectors as powerful molecular probes, in addition to studying their role in EV formation. Because effectors tend to target key regulatory nodes in host pathways, identifying their host targets can reveal critical components of plant cellular machinery that might otherwise remain elusive. In this sense, pathogen effectors provide a unique opportunity to dissect complex biological processes such as membrane trafficking and vesicle secretion.
In the Bh–barley pathosystem, several host proteins required for penetration resistance have been identified, including the PENETRATION (PEN) proteins. The syntaxin PEN1, also known as SYP121, in Arabidopsis thaliana and its barley orthologue ROR2 are essential for timely papilla formation. Notably, PEN1 and ROR2 are excellent markers for papilla-associated EVs, and ROR2 was used in this project to understand the processes of vesicular secretion.
Prior to the conception of this project, recent work in the host laboratory had demonstrated that dominant-negative approaches targeting classical ESCRT (Endosomal Sorting Complex Required for Transport) machinery components impair encasement formation, but leave papilla EV formation largely unaffected. These findings strongly suggest the existence of a yet unexplored ESCRT-independent multivesicular body (MVB)-like pathway delivering papilla EVs. Thus, the overarching objective of this project was to define the molecular mechanisms by which barley secretes extracellular vesicles into papillae during immune responses against the adapted fungal pathogen, Blumeria hordei.
In order to achieve the objectives of the project, the following workflow was implemented, which has yielded concrete scientific outputs and conceptual advances.
1. Identification of Bh effectors suppressing papilla EV formation. For an initial screen, based on expression data from publicly available datasets, twenty effectors were shortlisted, which expressed well during early timepoints of infection. The screen resulted in four such effectors, namely CSEP0077, CSEP0128, CSEP0168 and CSEP0214, which were taken as starting points for downstream interaction studies.
2. Discovery of novel plant trafficking components, using the effectors identified as baits in yeast two-hybrid screens. The library screen with CSEP0214 yielded the VPS18 protein as a target, which is an important component of the membrane trafficking pathway. Further in-planta interaction assays and functional validation through RNAi, expression of dominant-negative mutant proteins and microscopy, established the role of CSEP0214 as a core, conserved powdery mildew effector and key player in the establishment of disease by hijacking the plant membrane trafficking pathways by blocking VPS18.
3. Structural and molecular understanding of effector–target interactions and engineering effector-insensitive host targets by mapping the domains and amino acid residues involved in effector–target interactions. By exploiting amino acid differences between host and non-host orthologues, the project aimed to modify barley target proteins to render them insensitive to effector binding. However, the target identified in the screen with CSEP0214 is a highly conserved plant protein across different plant species. While on one hand, this is a highly promising target for modification, it was not possible to exploit differences between host and non-host plant species. Therefore, we made deletions of VPS18 and CSEP0214 to narrow down the regions of interaction. This indicated that the conserved RING domain of VPS18 and the CFEM domain of CSEP0214 are important for interaction between this effector-target pair. Further work is ongoing using AlphaFold3 structural prediction to pinpoint VPS18 interacting residues to be modified to make it effector insensitive.
4. Demonstration of ceramide involvement in plant EV pathways, by directed targeting of the ceramide synthesis pathway. Parallel work in mammalian systems indicates roles of ceramides in ESCRT-independent EV formation. RNAi based knockdown and chemical inhibition of ceramide synthase, and further, of the glucosylceramide synthase indicate that ceramides and glucosylceramides are important components of EVs formed by an ESCRT-independent pathway during papilla formation.
1. Identification of Bh effectors suppressing papilla EV formation. For an initial screen, based on expression data from publicly available datasets, twenty effectors were shortlisted, which expressed well during early timepoints of infection. The screen resulted in four such effectors, namely CSEP0077, CSEP0128, CSEP0168 and CSEP0214, which were taken as starting points for downstream interaction studies.
2. Discovery of novel plant trafficking components, using the effectors identified as baits in yeast two-hybrid screens. The library screen with CSEP0214 yielded the VPS18 protein as a target, which is an important component of the membrane trafficking pathway. Further in-planta interaction assays and functional validation through RNAi, expression of dominant-negative mutant proteins and microscopy, established the role of CSEP0214 as a core, conserved powdery mildew effector and key player in the establishment of disease by hijacking the plant membrane trafficking pathways by blocking VPS18.
3. Structural and molecular understanding of effector–target interactions and engineering effector-insensitive host targets by mapping the domains and amino acid residues involved in effector–target interactions. By exploiting amino acid differences between host and non-host orthologues, the project aimed to modify barley target proteins to render them insensitive to effector binding. However, the target identified in the screen with CSEP0214 is a highly conserved plant protein across different plant species. While on one hand, this is a highly promising target for modification, it was not possible to exploit differences between host and non-host plant species. Therefore, we made deletions of VPS18 and CSEP0214 to narrow down the regions of interaction. This indicated that the conserved RING domain of VPS18 and the CFEM domain of CSEP0214 are important for interaction between this effector-target pair. Further work is ongoing using AlphaFold3 structural prediction to pinpoint VPS18 interacting residues to be modified to make it effector insensitive.
4. Demonstration of ceramide involvement in plant EV pathways, by directed targeting of the ceramide synthesis pathway. Parallel work in mammalian systems indicates roles of ceramides in ESCRT-independent EV formation. RNAi based knockdown and chemical inhibition of ceramide synthase, and further, of the glucosylceramide synthase indicate that ceramides and glucosylceramides are important components of EVs formed by an ESCRT-independent pathway during papilla formation.
This project addresses a critical challenge at the intersection of plant biology, pathogen evolution, and global food security. By uncovering an ESCRT-independent EV secretion pathway central to barley immunity against Blumeria hordei, it advances fundamental understanding of plant defence mechanisms while delivering knowledge with clear translational potential. Through the strategic use of pathogen effectors as molecular tools, and innovative exploration of lipid-mediated vesicle formation, the project outcomes is positioned to make significant contributions to both basic science and applied agriculture. Ultimately, the outcomes will support the development of disease-resistant crops, contributing to sustainable food production in the face of growing global demand.