The plant immune system
Fundamental to all immune systems is an effective means of perceiving both harmful and beneficial microbes. In plants and animals this is achieved through a suite of receptor proteins located either at the cell membrane or inside the cell [1, 2]. Cell surface-localized receptor proteins evolved to identify molecules that are not normally found in the host (‘non-self’ molecules), as well as molecules that indicate host cell damage due to infection (‘infectious-self’ molecules) [3]. Activated receptors trigger signaling cascades involving several classes of proteins, such as enzymes and transcription factors, resulting in anti-microbial defence programs [4]. As in animals, plant cells recognize ‘non-self’ and ‘infectious-self’ molecules. For example, carbohydrates found specifically in microbial cell walls, such as fungal chitin and bacterial lipopolysaccharides and peptidoglycans, are sensed as ‘non-self’ molecules and trigger innate immune pathways [3]. These molecules are also referred to as pathogen-associated molecular patterns (PAMPs) and are generally essential to microbial lifestyles and therefore well conserved across entire groups of microbes [5]. Comparatively, ‘infectious-self’ molecules can be thought of as damage-associated molecular patterns (DAMPs) and include plant cell wall fragments and small peptide hormones that are released during infection or wounding [3]. Put simply, recognition of PAMPs and/or DAMPs signals ‘danger’ and initiates defence programmes in plant cells that are generally sufficient to halt ingress of most microbes [3].
All known PAMP and DAMP receptors in plants are plasma membrane-resident receptor kinases or receptor-like proteins that bind ligands outside the cell and transduce the signal inside the cell by forming larger complexes with additional proteins [2]. The earliest experimentally tractable responses following receptor complex activation in the model plant A. thaliana include release of secondary messengers such as Ca2+ and reactive oxygen species (ROS) [4]. Signal transduction is achieved by phosphorylation relays mediated by different sub-classes of protein kinases that result in profound physiological changes culminating in pattern-triggered immunity (PTI) that effectively protects plants against a broad range of pathogens [6, 7]. Accordingly, pathogen virulence strategies typically involve shutting down immune systems by disabling key signaling proteins. For example, the plasma membrane-associated protein BOTRYTIS INDUCED KINASE 1 (BIK1) is a direct target of at least two unrelated effector proteins from different bacterial pathogens [8-10]. As a direct convergent substrate of several different P/DAMP receptors [9, 11-13], BIK1 is an essential component of the plant immune system. P/DAMP perception triggers hyper-phosphorylation and activation of BIK1 [8, 9], allowing it to phosphorylate downstream substrates including a transmembrane NADPH oxidase resulting in an oxidative ROS burst [14, 15]. Plants lacking functional BIK1 and highly similar proteins (such as PBS1-LIKE KINASE 1; PBL1), either through genetic mutation (bik1 pbl1 mutants) or pathogen manipulation (cleavage, modification, and probably others), display altered P/DAMP-triggered ROS production and are more susceptible to bacterial and fungal pathogens [8-10, 16]. We and others found that transgenic over-expression of BIK1 (BIK1-OE) results in A. thaliana plants with heightened PTI signaling [17,18]. Furthermore, BIK1 is poly-ubiquitinated in vivo and continuously degraded by the ubiquitin-proteasome system to buffer its potent signaling potential, thereby ensuring an optimal immune response [17,18].
All known PAMP and DAMP receptors in plants are plasma membrane-resident receptor kinases or receptor-like proteins that bind ligands outside the cell and transduce the signal inside the cell by forming larger complexes with additional proteins [2]. The earliest experimentally tractable responses following receptor complex activation in the model plant A. thaliana include release of secondary messengers such as Ca2+ and reactive oxygen species (ROS) [4]. Signal transduction is achieved by phosphorylation relays mediated by different sub-classes of protein kinases that result in profound physiological changes culminating in pattern-triggered immunity (PTI) that effectively protects plants against a broad range of pathogens [6, 7]. Accordingly, pathogen virulence strategies typically involve shutting down immune systems by disabling key signaling proteins. For example, the plasma membrane-associated protein BOTRYTIS INDUCED KINASE 1 (BIK1) is a direct target of at least two unrelated effector proteins from different bacterial pathogens [8-10]. As a direct convergent substrate of several different P/DAMP receptors [9, 11-13], BIK1 is an essential component of the plant immune system. P/DAMP perception triggers hyper-phosphorylation and activation of BIK1 [8, 9], allowing it to phosphorylate downstream substrates including a transmembrane NADPH oxidase resulting in an oxidative ROS burst [14, 15]. Plants lacking functional BIK1 and highly similar proteins (such as PBS1-LIKE KINASE 1; PBL1), either through genetic mutation (bik1 pbl1 mutants) or pathogen manipulation (cleavage, modification, and probably others), display altered P/DAMP-triggered ROS production and are more susceptible to bacterial and fungal pathogens [8-10, 16]. We and others found that transgenic over-expression of BIK1 (BIK1-OE) results in A. thaliana plants with heightened PTI signaling [17,18]. Furthermore, BIK1 is poly-ubiquitinated in vivo and continuously degraded by the ubiquitin-proteasome system to buffer its potent signaling potential, thereby ensuring an optimal immune response [17,18].
Plant immune homeostasis
As immune responses redirect resources from growth and development, unbuffered signaling can cause considerable fitness costs and can be illustrated by a ‘damage-response’ relationship articulated by Casadevall and Pirofski [19] (Figure 1). This is best exemplified by the severe inhibition of plant growth observed upon prolonged exposure to pathogen elicitors (Figure 2) [3]. Although attenuation of immune signaling is therefore essential to plant growth, very few negative regulators have been described. Through a genetic screen to identify negative regulators of PTI, we found that the Ca2+-dependent protein kinase (CPK) CPK28 contributes to the proteasomal turnover of BIK1 [17]. Plants lacking functional CPK28 (cpk28 mutants) display heightened immune signaling and over-accumulate BIK1 protein, while transgenic over-expression of CPK28 (CPK28-OE) results in plants with drastically reduced BIK1 protein accumulation and immune signaling [17]. CPK28 directly interacts with and phosphorylates BIK1 [17, 20], suggesting that BIK1 is regulated through a complex interplay between different post-translational modifications that contribute to immune homeostasis. Our current understanding of plant immunity lacks important details about how immune signaling pathways are regulated and buffered as well as how immune signals are integrated with growth and development programs. Modern technologies make it possible to tackle these key unknowns with unprecedented molecular detail in vivo – offering the exciting opportunity to make novel discoveries about signal transduction mechanisms.
Fig 1 - Concept of immune homeostasis
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Fig 2 - Growth of Arabidopsis seedlings in media containing immunogenic peptides
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Our current projects focus on understanding how CPK28 is regulated - by Ca2+, binding partners, and post-translational modifications. We are also investigating the regulation of BIK1 by post-translational modifications, particularly phosphorylation and ubiquitination. Additional projects are in place to study immune homeostasis and the trade-off between growth and immunity. We use a wide-range of techniques but rely heavily on molecular methods to understand immune signalling cascades. This includes the generation and analysis of transgenic and mutant plants, structure/function analysis using site-directed mutagenesis, discovery of protein binding partners and post-translational modifications using mass spectrometry, and forward-genetic screens assisted by next-generation sequencing.
References
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Our research is generously supported by the following operational and infrastructure grants
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Research Initiation Grant (2016 - 2020) |
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John R Evans Leader's Fund (2017 - 2019)
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Ontario Research Fund - Research Infrastructure (2017-2019)
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