Publications
60 results found
Ibrahim T, Khandare V, Mirkin FG, et al., 2023, AlphaFold2-multimer guided high-accuracy prediction of typical and atypical ATG8-binding motifs., PLoS Biol, Vol: 21
Macroautophagy/autophagy is an intracellular degradation process central to cellular homeostasis and defense against pathogens in eukaryotic cells. Regulation of autophagy relies on hierarchical binding of autophagy cargo receptors and adaptors to ATG8/LC3 protein family members. Interactions with ATG8/LC3 are typically facilitated by a conserved, short linear sequence, referred to as the ATG8/LC3 interacting motif/region (AIM/LIR), present in autophagy adaptors and receptors as well as pathogen virulence factors targeting host autophagy machinery. Since the canonical AIM/LIR sequence can be found in many proteins, identifying functional AIM/LIR motifs has proven challenging. Here, we show that protein modelling using Alphafold-Multimer (AF2-multimer) identifies both canonical and atypical AIM/LIR motifs with a high level of accuracy. AF2-multimer can be modified to detect additional functional AIM/LIR motifs by using protein sequences with mutations in primary AIM/LIR residues. By combining protein modelling data from AF2-multimer with phylogenetic analysis of protein sequences and protein-protein interaction assays, we demonstrate that AF2-multimer predicts the physiologically relevant AIM motif in the ATG8-interacting protein 2 (ATI-2) as well as the previously uncharacterized noncanonical AIM motif in ATG3 from potato (Solanum tuberosum). AF2-multimer also identified the AIM/LIR motifs in pathogen-encoded virulence factors that target ATG8 members in their plant and human hosts, revealing that cross-kingdom ATG8-LIR/AIM associations can also be predicted by AF2-multimer. We conclude that the AF2-guided discovery of autophagy adaptors/receptors will substantially accelerate our understanding of the molecular basis of autophagy in all biological kingdoms.
Adachi H, Sakai T, Harant A, et al., 2023, An atypical NLR protein modulates the NRC immune receptor network in Nicotiana benthamiana., PLoS Genet, Vol: 19
The NRC immune receptor network has evolved in asterid plants from a pair of linked genes into a genetically dispersed and phylogenetically structured network of sensor and helper NLR (nucleotide-binding domain and leucine-rich repeat-containing) proteins. In some species, such as the model plant Nicotiana benthamiana and other Solanaceae, the NRC (NLR-REQUIRED FOR CELL DEATH) network forms up to half of the NLRome, and NRCs are scattered throughout the genome in gene clusters of varying complexities. Here, we describe NRCX, an atypical member of the NRC family that lacks canonical features of these NLR helper proteins, such as a functional N-terminal MADA motif and the capacity to trigger autoimmunity. In contrast to other NRCs, systemic gene silencing of NRCX in N. benthamiana markedly impairs plant growth resulting in a dwarf phenotype. Remarkably, dwarfism of NRCX silenced plants is partially dependent on NRCX paralogs NRC2 and NRC3, but not NRC4. Despite its negative impact on plant growth when silenced systemically, spot gene silencing of NRCX in mature N. benthamiana leaves doesn't result in visible cell death phenotypes. However, alteration of NRCX expression modulates the hypersensitive response mediated by NRC2 and NRC3 in a manner consistent with a negative role for NRCX in the NRC network. We conclude that NRCX is an atypical member of the NRC network that has evolved to contribute to the homeostasis of this genetically unlinked NLR network.
Contreras MP, Pai H, Tumtas Y, et al., 2022, Sensor NLR immune proteins activate oligomerization of their NRC helpers in response to plant pathogens, EMBO JOURNAL, ISSN: 0261-4189
Coatsworth P, Gonzalez-Macia L, Collins ASP, et al., 2022, Continuous monitoring of chemical signals in plants under stress, NATURE REVIEWS CHEMISTRY, Vol: 7, Pages: 7-25
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- Citations: 3
Zess EK, Dagdas YF, Peers E, et al., 2022, Regressive evolution of an effector following a host jump in the Irish potato famine pathogen lineage, PLOS PATHOGENS, Vol: 18, ISSN: 1553-7366
Ibrahim T, Khandare V, Mirkin FG, et al., 2022, AF2-multimer guided high accuracy prediction of typical and atypical ATG8 binding motifs
<jats:title>Abstract</jats:title><jats:p>Macroautophagy/autophagy is an intracellular degradation process central to cellular homeostasis and defense against pathogens in eukaryotic cells. Regulation of autophagy relies on hierarchical binding of autophagy cargo receptors and adaptors to ATG8/LC3 protein family members. Interactions with ATG8/LC3 are typically facilitated by a conserved, short linear sequence, referred to as the ATG8/LC3 interacting motif/region (AIM/LIR), present in autophagy adaptors and receptors as well as pathogen virulence factors targeting host autophagy machinery. Since the canonical AIM/LIR sequence can be found in many proteins, identifying functional AIM/LIR motifs has proven challenging. Here we show that protein modelling using Alphafold-Multimer (AF2-multimer) identifies both canonical and atypical AIM/LIR motifs with a high level of accuracy. AF2-multimer can be modified to detect additional functional AIM/LIR motifs by using protein sequences with mutations in primary AIM/LIR residues. By combining protein modelling data from AF2-multimer with phylogenetic analysis of protein sequences and protein-protein interaction assays, we demonstrate that AF2-multimer predicts the physiologically relevant AIM motif in the ATG8-interacting protein 2 (ATI-2) as well as the previously uncharacterized non-canonical AIM motif in ATG3 from potato (<jats:italic>Solanum tuberosum</jats:italic>). AF2-multimer also identified the AIM/LIR motifs in pathogen-encoded virulence factors that target ATG8 members in their plant and human hosts, revealing that cross-kingdom ATG8-LIR/AIM associations can also be predicted by AF2-multimer. We conclude that the AF2-guided discovery of autophagy adaptors/receptors will substantially accelerate our understanding of the molecular basis of autophagy in all biological kingdoms.</jats:p>
Leong JX, Raffeiner M, Spinti D, et al., 2022, A bacterial effector counteracts host autophagy by promoting degradation of an autophagy component, EMBO JOURNAL, Vol: 41, ISSN: 0261-4189
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- Citations: 9
Contreras MP, Pai H, Tumtas Y, et al., 2022, Sensor NLR immune proteins activate oligomerization of their NRC helper
<jats:title>Abstract</jats:title><jats:p>Nucleotide-binding domain and leucine-rich repeat (NLR) immune receptors are important components of plant and metazoan innate immunity that can function as individual units or as pairs or networks. Upon activation, NLRs form multiprotein complexes termed resistosomes or inflammasomes. Whereas metazoan paired NLRs, such as NAIP/NLRC4, activate into hetero-complexes, the molecular mechanisms underpinning activation of plant paired NLRs, especially whether they associate in resistosome hetero-complexes is unknown. In asterid plant species, the NLR required for cell death (NRC) immune receptor network is composed of multiple resistance protein sensors and downstream helpers that confer immunity against diverse plant pathogens. Here, we show that pathogen effector-activation of the NLR proteins Rx (confers virus resistance) and Bs2 (confers bacterial resistance) leads to oligomerization of the helper NLR NRC2. Activated Rx does not oligomerize or enter into a stable complex with the NRC2 oligomer and remains cytoplasmic. In contrast, activated NRC2 oligomers accumulate in membrane-associated puncta. We propose an activation-and-release model for NLRs in the NRC immune receptor network. This points to a distinct activation model compared to mammalian paired NLRs.</jats:p>
Adachi H, Sakai T, Harant A, et al., 2021, An atypical NLR protein modulates the NRC immune receptor network
<jats:title>ABSTRACT</jats:title><jats:p>The NRC immune receptor network has evolved in asterid plants from a pair of linked genes into a genetically dispersed and phylogenetically structured network of sensor and helper NLR (nucleotide-binding domain and leucine-rich repeat-containing) proteins. In some species, such as the model plant <jats:italic>Nicotiana benthamiana</jats:italic> and other Solanaceae, the NRC network forms up to half of the NLRome, and NRCs are scattered throughout the genome in gene clusters of varying complexities. Here, we describe NRCX, an atypical, but essential member of the NRC family that lacks canonical features of these NLR helper proteins, such as a functional N-terminal MADA motif and the capacity to trigger autoimmunity. In contrast to other NRCs, systemic gene silencing of <jats:italic>NRCX</jats:italic> markedly impairs plant growth resulting in a dwarf phenotype. Remarkably, dwarfism of <jats:italic>NRCX</jats:italic> silenced plants is partially dependent on NRCX paralogs NRC2 and NRC3, but not NRC4. Despite its negative impact on plant growth when silenced systemically, transient RNA interference of <jats:italic>NRCX</jats:italic> in mature <jats:italic>N. benthamiana</jats:italic> leaves doesn’t result in visible cell death phenotypes. However, alteration of <jats:italic>NRCX</jats:italic> expression modulates the hypersensitive response mediated by NRC2 and NRC3 in a manner consistent with a negative role for NRCX in the NRC network. We conclude that NRCX is an atypical member of the NRC network that has evolved to contribute to the homeostasis of this genetically unlinked NLR network.</jats:p>
Zess EK, Dagdas YF, Peers E, et al., 2021, Regressive evolution of an effector following a host jump in the Irish Potato Famine Pathogen Lineage
<jats:title>Abstract</jats:title><jats:p>In order to infect a new host species, the pathogen must evolve to enhance infection and transmission in the novel environment. Although we often think of evolution as a process of accumulation, it is also a process of loss. Here, we document an example of regressive evolution in the Irish potato famine pathogen (<jats:italic>Phytophthora infestans</jats:italic>) lineage, providing evidence that a key sequence motif in the effector PexRD54 has degenerated following a host jump. We began by looking at PexRD54 and PexRD54-like sequences from across <jats:italic>Phytophthora</jats:italic> species. We found that PexRD54 emerged in the common ancestor of <jats:italic>Phytophthora</jats:italic> clade 1b and 1c species, and further sequence analysis showed that a key functional motif, the C-terminal ATG8-interacting motif (AIM), was also acquired at this point in the lineage. A closer analysis showed that the <jats:italic>P. mirabilis</jats:italic> PexRD54 (PmPexRD54) AIM appeared unusual, the otherwise-conserved central residue mutated from a glutamate to a lysine. We aimed to determine whether this PmPexRD54 AIM polymorphism represented an adaptation to the <jats:italic>Mirabilis jalapa</jats:italic> host environment. We began by characterizing the <jats:italic>M. jalapa</jats:italic> ATG8 family, finding that they have a unique evolutionary history compared to previously characterized ATG8s. Then, using co-immunoprecipitation and isothermal titration calorimetry assays, we showed that both full-length PmPexRD54 and the PmPexRD54 AIM peptide bind very weakly to the <jats:italic>M. jalapa</jats:italic> ATG8s. Through a combination of binding assays and structural modelling, we showed that the identity of the residue at the position of the PmPexRD54 AIM polymorphism can underpin high-affinity binding to plant ATG8s. Finally, we conc
Bozkurt O, Savage Z, Duggan C, 2021, Chloroplasts alter their morphology and accumulate at the pathogen interface during infection by Phytophthora infestans, The Plant Journal, Vol: 107, Pages: 1771-1787, ISSN: 0960-7412
Upon immune activation, chloroplasts switch off photosynthesis, produce anti-microbial compounds, and associate with the nucleus through tubular extensions called stromules. Although it is well-established that chloroplasts alter their position in response to light, little is known about the dynamics of chloroplasts movement in response to pathogen attack. Here, we report that chloroplasts accumulate at the pathogen interface during infection by the Irish potato famine pathogen Phytophthora infestans, associating with the specialized membrane that engulfs the pathogen haustorium. Chemical inhibition of actin polymerization reduces the accumulation of chloroplasts at the pathogen haustoria, suggesting this process is partially dependent on the actin cytoskeleton. However, chloroplast accumulation at haustoria does not necessarily rely on movement of the nucleus to this interface and is not affected by light conditions. Stromules are typically induced during infection, embracing haustoria and facilitating chloroplast interactions, to form dynamic organelle clusters. We found that infection-triggered stromule formation relies on BRASSINOSTEROID INSENSITIVE 1-ASSOCIATED KINASE 1 (BAK1) mediated surface immune signaling, whereas chloroplast repositioning towards haustoria does not. Consistent with the defense-related induction of stromules, effector mediated suppression of BAK1 mediated immune signaling reduced stromule formation during infection. On the other hand, immune recognition of the same effector stimulated stromules, presumably via a different pathway. These findings implicate chloroplasts in a polarized response upon pathogen attack and point to more complex functions of these organelles in plant-pathogen interactions.
Duggan C, Moratto E, Savage Z, et al., 2021, Dynamic localization of a helper NLR at the plant-pathogen interface underpins pathogen recognition, Proceedings of the National Academy of Sciences of USA, Vol: 118, Pages: 1-12, ISSN: 0027-8424
Plants employ sensor-helper pairs of NLR immune receptors to recognize pathogen effectors and activate immune responses (1). Yet the subcellular localization of NLRs pre- and post-activation during pathogen infection remains poorly understood. Here we show that NRC4, from the ‘NRC’ solanaceous helper NLR family (1), undergoes dynamic changes in subcellular localization by shuttling to and from the plant-pathogen haustorium interface established during infection by the Irish potato famine pathogen Phytophthora infestans. Specifically, prior to activation, NRC4 accumulates at the extra-haustorial membrane (EHM), presumably to mediate response to perihaustorial effectors, that are recognized by NRC4- dependent sensor NLRs. However not all NLRs accumulate at the EHM, as the closely related helper NRC2, and the distantly related ZAR1, did not accumulate at the EHM. NRC4 required an intact N-terminal coiled coil domain to accumulate at the EHM, whereas the functionally conserved MADA motif implicated in cell death activation and membrane insertion was dispensable for this process. Strikingly, a constitutively autoactive NRC4 mutant did not accumulate at the EHM and showed punctate distribution that mainly associated with the plasma membrane, suggesting that post-activation, NRC4 may undergo a conformation switch to form clusters that do not preferentially associate with the EHM. When NRC4 is activated by a sensor NLR during infection however, NRC4 forms puncta mainly at the EHM and to a lesser extent at the plasma membrane. We conclude that following activation at the EHM, NRC4 may spread to other cellular membranes from its primary site of activation to trigger immune responses.
Bozkurt O, 2021, An oomycete effector subverts host vesicle trafficking to channel starvation-induced autophagy to the pathogen interface, eLife, Vol: 10, Pages: 1-35, ISSN: 2050-084X
Eukaryotic cells deploy autophagy to eliminate invading microbes. In turn, pathogens have evolved effector proteins to counteract antimicrobial autophagy. How adapted pathogens co opt autophagy for their own benefit is poorly understood. The Irish famine pathogen Phytophthora infestans secretes the effector protein PexRD54 that selectively activates an unknown plant autophagy pathway that antagonizes antimicrobial autophagy at the pathogen interface. Here we show that PexRD54 induces autophagosome formation by bridging vesicles decorated by the small GTPase Rab8a with autophagic compartments labelled by the core autophagy protein ATG8CL. Rab8a is required for pathogen-triggered and starvation induced but not antimicrobial autophagy, revealing specific trafficking pathways underpin selective autophagy. By subverting Rab8a mediated vesicle trafficking, PexRD54 utilizes lipid droplets to facilitate biogenesis of autophagosomes diverted to pathogen feeding sites. Altogether, we show that PexRD54 mimics starvation-induced autophagy to subvert endomembrane trafficking at the host-pathogen interface, revealing how effectors bridge distinct host compartments to expedite colonization.
Petre B, Contreras MP, Bozkurt TO, et al., 2021, Host-interactor screens of Phytophthora infestans RXLR proteins reveal vesicle trafficking as a major effector-targeted process, The Plant Cell, Vol: 33, Pages: 1447-1471, ISSN: 1040-4651
Pathogens modulate plant cell structure and function by secreting effectors into host tissues. Effectors typically function by associating with host molecules and modulating their activities. This study aimed to identify the host processes targeted by the RXLR class of host-translocated effectors of the potato blight pathogen Phytophthora infestans. To this end, we performed an in planta protein-protein interaction screen by transiently expressing P. infestans RXLR effectors in Nicotiana benthamiana leaves followed by co-immunoprecipitation and liquid chromatography tandem mass spectrometry. This screen generated an effector-host protein interactome matrix of 59 P. infestans RXLR effectors x 586 N. benthamiana proteins. Classification of the host interactors into putative functional categories revealed over 35 biological processes possibly targeted by P. infestans. We further characterized the PexRD12/31 family of RXLR-WY effectors, which associate and co-localize with components of the vesicle trafficking machinery. One member of this family, PexRD31, increased the number of FYVE positive vesicles in N. benthamiana cells. FYVE positive vesicles also accumulated in leaf cells near P. infestans hyphae, indicating that the pathogen may enhance endosomal trafficking during infection. This interactome data set will serve as a useful resource for functional studies of P. infestans effectors and of effector-targeted host processes.
Leong JX, Raffeiner M, Spinti D, et al., 2021, A bacterial effector counteracts host autophagy by promoting degradation of an autophagy component
<jats:title>Abstract</jats:title><jats:p>Beyond its role in cellular homeostasis, autophagy plays anti- and pro-microbial roles in host-microbe interactions, both in animals and plants. One prominent role of anti-microbial autophagy is to degrade intracellular pathogens or microbial molecules, in a process termed xenophagy. Consequently, microbes evolved mechanisms to hijack or modulate autophagy to escape elimination. Although well-described in animals, the extent to which xenophagy contributes to plant-bacteria interactions remains unknown. Here, we provide evidence that <jats:italic>Xanthomonas campestris</jats:italic> pv. <jats:italic>vesicatoria (Xcv)</jats:italic> suppresses host autophagy by utilizing type-III effector XopL. XopL interacts with and degrades the autophagy component SH3P2 via its E3 ligase activity to promote infection. Intriguingly, XopL is targeted for degradation by defense-related selective autophagy mediated by NBR1/Joka2, revealing a complex antagonistic interplay between XopL and the host autophagy machinery. Our results implicate plant antimicrobial autophagy in depletion of a bacterial virulence factor and unravels an unprecedented pathogen strategy to counteract defense-related autophagy.</jats:p>
Klionsky DJ, Abdel-Aziz AK, Abdelfatah S, et al., 2021, Guidelines for the use and interpretation of assays for monitoring autophagy (4th edition), Autophagy, Vol: 17, Pages: 1-382, ISSN: 1554-8627
In 2008, we published the first set of guidelines for standardizing research in autophagy. Since then, this topic has received increasing attention, and many scientists have entered the field. Our knowledge base and relevant new technologies have also been expanding. Thus, it is important to formulate on a regular basis updated guidelines for monitoring autophagy in different organisms. Despite numerous reviews, there continues to be confusion regarding acceptable methods to evaluate autophagy, especially in multicellular eukaryotes. Here, we present a set of guidelines for investigators to select and interpret methods to examine autophagy and related processes, and for reviewers to provide realistic and reasonable critiques of reports that are focused on these processes. These guidelines are not meant to be a dogmatic set of rules, because the appropriateness of any assay largely depends on the question being asked and the system being used. Moreover, no individual assay is perfect for every situation, calling for the use of multiple techniques to properly monitor autophagy in each experimental setting. Finally, several core components of the autophagy machinery have been implicated in distinct autophagic processes (canonical and noncanonical autophagy), implying that genetic approaches to block autophagy should rely on targeting two or more autophagy-related genes that ideally participate in distinct steps of the pathway. Along similar lines, because multiple proteins involved in autophagy also regulate other cellular pathways including apoptosis, not all of them can be used as a specific marker for bona fide autophagic responses. Here, we critically discuss current methods of assessing autophagy and the information they can, or cannot, provide. Our ultimate goal is to encourage intellectual and technical innovation in the field.
Oikawa K, Fujisaki K, Shimizu M, et al., 2020, The blast pathogen effector AVR-Pik binds and stabilizes rice heavy metal-associated (HMA) proteins to co-opt their function in immunity
<jats:title>Abstract</jats:title><jats:p>Plant intracellular nucleotide-binding domain and leucine-rich repeat-containing (NLR) immune receptors have a complex architecture. They can include noncanonical integrated domains that are thought to have evolved from host targets of pathogen effectors to serve as pathogen baits. However, the functions of host proteins with similarity to NLR integrated domains and the extent to which they are targeted by pathogen effectors remain largely unknown. Here, we show that the blast fungus effector AVR-Pik binds a subset of related rice proteins containing a heavy metal-associated (HMA) domain, one of the domains that has repeatedly integrated into plant NLR immune receptors. We find that AVR-Pik binding stabilizes the rice HMA proteins OsHIPP19 and OsHIPP20. Knockout of <jats:italic>OsHIPP20</jats:italic> causes enhanced disease resistance towards the blast pathogen, indicating that <jats:italic>OsHIPP20</jats:italic> is a susceptibility gene (<jats:italic>S</jats:italic>-gene). We propose that AVR-Pik has evolved to bind HMA domain proteins and co-opt their function to suppress immunity. Yet this binding carries a trade-off, it triggers immunity in plants carrying NLR receptors with integrated HMA domains.</jats:p>
Petre B, Contreras MP, Bozkurt TO, et al., 2020, Host-interactor screens of <i>Phytophthora infestans</i> RXLR proteins reveal vesicle trafficking as a major effector-targeted process, Publisher: Cold Spring Harbor Laboratory
<jats:title>ABSTRACT</jats:title><jats:p>Pathogens modulate plant cell structure and function by secreting effectors into host tissues. Effectors typically function by associating with host molecules and modulating their activities. This study aimed to identify the host processes targeted by the RXLR class of host-translocated effectors of the potato blight pathogen <jats:italic>Phytophthora infestans.</jats:italic> To this end, we performed an <jats:italic>in planta</jats:italic> protein-protein interaction screen by transiently expressing <jats:italic>P. infestans</jats:italic> RXLR effectors in <jats:italic>Nicotiana benthamiana</jats:italic> leaves followed by co-immunoprecipitation (co-IP) and liquid chromatography tandem mass spectrometry (LC-MS/MS). This screen generated an effector-host protein interactome matrix of 59 <jats:italic>P. infestans</jats:italic> RXLR effectors x 586 <jats:italic>N. benthamiana</jats:italic> proteins. Classification of the host interactors into putative functional categories revealed over 35 biological processes possibly targeted by <jats:italic>P. infestans.</jats:italic> We further characterized the PexRD12/31 family of RXLR-WY effectors, which associate and co-localize with components of the vesicle trafficking machinery. One member of this family, PexRD31, increased the number of FYVE positive vesicles in <jats:italic>N. benthamiana</jats:italic> cells. FYVE positive vesicles also accumulated in leaf cells near <jats:italic>P. infestans</jats:italic> hyphae, indicating that the pathogen may enhance endosomal trafficking during infection. We anticipate that the interactome dataset we generated will serve as a useful community resource for functional studies of <jats:italic>P. infestans</jats:italic> effectors and of effector-targeted host processes.</jats:p>
Gao C, Xu H, Huang J, et al., 2020, Pathogen manipulation of chloroplast function triggers a light-dependent immune recognition, Proceedings of the National Academy of Sciences of the United States of America, Vol: 117, Pages: 9613-9620, ISSN: 0027-8424
In plants and animals, nucleotide-binding leucine-rich repeat (NLR) proteins are intracellular immune sensors that recognize and eliminate a wide range of invading pathogens. NLR-mediated immunity is known to be modulated by environmental factors. However, how pathogen recognition by NLRs is influenced by environmental factors such as light remains unclear. Here, we show that the agronomically important NLR Rpi-vnt1.1 requires light to confer disease resistance against races of the Irish potato famine pathogen Phytophthora infestans that secrete the effector protein AVRvnt1. The activation of Rpi-vnt1.1 requires a nuclear-encoded chloroplast protein, glycerate 3-kinase (GLYK), implicated in energy production. The pathogen effector AVRvnt1 binds the full-length chloroplast-targeted GLYK isoform leading to activation of Rpi-vnt1.1. In the dark, Rpi-vnt1.1–mediated resistance is compromised because plants produce a shorter GLYK—lacking the intact chloroplast transit peptide—that is not bound by AVRvnt1. The transition between full-length and shorter plant GLYK transcripts is controlled by a light-dependent alternative promoter selection mechanism. In plants that lack Rpi-vnt1.1, the presence of AVRvnt1 reduces GLYK accumulation in chloroplasts counteracting GLYK contribution to basal immunity. Our findings revealed that pathogen manipulation of chloroplast functions has resulted in a light-dependent immune response.
Bozkurt TO, Kamoun S, 2020, The plant–pathogen haustorial interface at a glance, Journal of Cell Science, Vol: 133, Pages: 1-6, ISSN: 0021-9533
Many filamentous pathogens invade plant cells through specialized hyphae called haustoria. These infection structures are enveloped by a newly synthesized plant-derived membrane called the extrahaustorial membrane (EHM). This specialized membrane is the ultimate interface between the plant and pathogen, and is key to the success or failure of infection. Strikingly, the EHM is reminiscent of host-derived membrane interfaces that engulf intracellular metazoan parasites. These perimicrobial interfaces are critical sites where pathogens facilitate nutrient uptake and deploy virulence factors to disarm cellular defenses mounted by their hosts. Although the mechanisms underlying the biogenesis and functions of these host–microbe interfaces are poorly understood, recent studies have provided new insights into the cellular and molecular mechanisms involved. In this Cell Science at a Glance and the accompanying poster, we summarize these recent advances with a specific focus on the haustorial interfaces associated with filamentous plant pathogens. We highlight the progress in the field that fundamentally underpin this research topic. Furthermore, we relate our knowledge of plant–filamentous pathogen interfaces to those generated by other plant-associated organisms. Finally, we compare the similarities between host–pathogen interfaces in plants and animals, and emphasize the key questions in this research area.
Adachi H, Contreras MP, Harant A, et al., 2020, An N-terminal motif in NLR immune receptors is functionally conserved across distantly related plant species, eLife, Vol: 8, Pages: 1-31, ISSN: 2050-084X
The molecular codes underpinning the functions of plant NLR immune receptors are poorly understood. We used in vitro Mu transposition to generate a random truncation library and identify the minimal functional region of NLRs. We applied this method to NRC4—a helper NLR that functions with multiple sensor NLRs within a Solanaceae receptor network. This revealed that the NRC4 N-terminal 29 amino acids are sufficient to induce hypersensitive cell death. This region is defined by the consensus MADAxVSFxVxKLxxLLxxEx (MADA motif) that is conserved at the N-termini of NRC family proteins and ~20% of coiled-coil (CC)-type plant NLRs. The MADA motif matches the N-terminal α1 helix of Arabidopsis NLR protein ZAR1, which undergoes a conformational switch during resistosome activation. Immunoassays revealed that the MADA motif is functionally conserved across NLRs from distantly related plant species. NRC-dependent sensor NLRs lack MADA sequences indicating that this motif has degenerated in sensor NLRs over evolutionary time.
Leary AY, Savage Z, Tumtas Y, et al., 2019, Contrasting and emerging roles of autophagy in plant immunity, CURRENT OPINION IN PLANT BIOLOGY, Vol: 52, Pages: 46-53, ISSN: 1369-5266
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- Citations: 33
Bozkurt TO, Pandey P, Tumtas Y, et al., 2019, Subversion of plant immunity at the host-pathogen interface, 18th Congress of International-Society-for-Molecular-Plant-Microbe-Interactions (IS-MPMI), Publisher: AMER PHYTOPATHOLOGICAL SOC, Pages: 242-242, ISSN: 0894-0282
Zess EK, Jensen C, Cruz-Mireles N, et al., 2019, N-terminal beta-strand underpins biochemical specialization of an ATG8 isoform, PLoS Biology, Vol: 17, Pages: 1-27, ISSN: 1544-9173
Autophagy-related protein 8 (ATG8) is a highly conserved ubiquitin-like protein that modulates autophagy pathways by binding autophagic membranes and a number of proteins, including cargo receptors and core autophagy components. Throughout plant evolution, ATG8 has expanded from a single protein in algae to multiple isoforms in higher plants. However, the degree to which ATG8 isoforms have functionally specialized to bind distinct proteins remains unclear. Here, we describe a comprehensive protein–protein interaction resource, obtained using in planta immunoprecipitation (IP) followed by mass spectrometry (MS), to define the potato ATG8 interactome. We discovered that ATG8 isoforms bind distinct sets of plant proteins with varying degrees of overlap. This prompted us to define the biochemical basis of ATG8 specialization by comparing two potato ATG8 isoforms using both in vivo protein interaction assays and in vitro quantitative binding affinity analyses. These experiments revealed that the N-terminal β-strand—and, in particular, a single amino acid polymorphism—underpins binding specificity to the substrate PexRD54 by shaping the hydrophobic pocket that accommodates this protein’s ATG8-interacting motif (AIM). Additional proteomics experiments indicated that the N-terminal β-strand shapes the broader ATG8 interactor profiles, defining interaction specificity with about 80 plant proteins. Our findings are consistent with the view that ATG8 isoforms comprise a layer of specificity in the regulation of selective autophagy pathways in plants.
Adachi H, Contreras M, Harant A, et al., 2019, An N-terminal motif in NLR immune receptors is functionally conserved across distantly related plant species, Publisher: Cold Spring Harbor Laboratory
<jats:p>The molecular codes underpinning the functions of plant NLR immune receptors are poorly understood. We used <jats:italic>in vitro</jats:italic> Mu transposition to generate a random truncation library and identify the minimal functional region of NLRs. We applied this method to NRC4—a helper NLR that functions with multiple sensor NLRs within a Solanaceae receptor network. This revealed that the NRC4 N-terminal 29 amino acids are sufficient to induce hypersensitive cell death. This region is defined by the consensus MADAxVSFxVxKLxxLLxxEx (MADA motif) that is conserved at the N-termini of NRC family proteins and ~20% of coiled-coil (CC)-type plant NLRs. The MADA motif matches the N-terminal α1 helix of Arabidopsis NLR protein ZAR1, which undergoes a conformational switch during resistosome activation. Immunoassays revealed that the MADA motif is functionally conserved across NLRs from distantly related plant species. NRC-dependent sensor NLRs lack MADA sequences indicating that this motif has degenerated in sensor NLRs over evolutionary time.</jats:p>
Pennington HG, Rhian J, Kwon S, et al., 2019, The fungal ribonuclease-like effector protein CSEP0064/BEC1054 represses plant immunity and interferes with degradation of host ribosomal RNA, PLoS Pathogens, Vol: 15, ISSN: 1553-7366
The biotrophic fungal pathogen Blumeria graminis causes the powdery mildew disease of cereals and grasses. We present the first crystal structure of a B. graminis effector of pathogenicity (CSEP0064/BEC1054), demonstrating it has a ribonuclease (RNase)-like fold. This effector is part of a group of RNase-like proteins (termed RALPHs) which comprise the largest set of secreted effector candidates within the B. graminis genomes. Their exceptional abundance suggests they play crucial functions during pathogenesis. We show that transgenic expression of RALPH CSEP0064/BEC1054 increases susceptibility to infection in both monocotyledonous and dicotyledonous plants. CSEP0064/BEC1054 interacts in planta with the pathogenesis-related protein PR10. The effector protein associates with total RNA and weakly with DNA. Methyl jasmonate (MeJA) levels modulate susceptibility to aniline-induced host RNA fragmentation. In planta expression of CSEP0064/BEC1054 reduces the formation of this RNA fragment. We propose CSEP0064/BEC1054 is a pseudoenzyme that binds to host ribosomes, thereby inhibiting the action of plant ribosome-inactivating proteins (RIPs) that would otherwise lead to host cell death, an unviable interaction and demise of the fungus.
Savage Z, Duggan C, Toufexi A, et al., 2019, Chloroplasts alter their morphology and accumulate at the pathogen interface during infection by<i>Phytophthora infestans</i>, Publisher: Cold Spring Harbor Laboratory
<jats:title>Abstract</jats:title><jats:p>Upon immune activation, chloroplasts switch off photosynthesis, produce anti-microbial compounds, and associate with the nucleus through tubular extensions called stromules. Although it is well-established that chloroplasts alter their position in response to light, little is known about the dynamics of chloroplasts movement in response to pathogen attack. Here, we report that chloroplasts accumulate at the pathogen interface during infection by the Irish potato famine pathogen<jats:italic>Phytophthora infestans</jats:italic>, associating with the specialized membrane that engulfs the pathogen haustorium. Chemical inhibition of actin polymerization reduces the accumulation of chloroplasts at the pathogen haustoria, suggesting this process is partially dependent on the actin cytoskeleton. However, chloroplast accumulation at haustoria does not necessarily rely on movement of the nucleus to this interface and is not affected by light conditions. Stromules are typically induced during infection, embracing haustoria and interconnecting chloroplasts, to form dynamic organelle clusters. We found that infection-triggered stromule formation relies on BRASSINOSTEROID INSENSITIVE 1-ASSOCIATED KINASE 1 (BAK1) mediated surface immune signaling, whereas chloroplast repositioning towards haustoria does not. Consistent with the defense-related induction of stromules, effector mediated suppression of BAK1 mediated immune signaling reduced stromule formation during infection. On the other hand, immune recognition of the same effector stimulated stromules, presumably via a different pathway. These findings implicate chloroplasts in a polarized response upon pathogen attack and point to more complex functions of these organelles in plant-pathogen interactions.</jats:p>
Dagdas Y, Pandey P, Tumtas Y, et al., 2018, Host autophagy machinery is diverted to the pathogen interface tomediate focal defense responses against the Irish potato faminepathogen, eLife, Vol: 7, ISSN: 2050-084X
During plant cell invasion, the oomycete Phytophthora infestans remains enveloped byhost-derived membranes whose functional properties are poorly understood. P. infestans secretesa myriad of effector proteins through these interfaces for plant colonization. Recently we showedthat the effector protein PexRD54 reprograms host-selective autophagy by antagonisingantimicrobial-autophagy receptor Joka2/NBR1 for ATG8CL binding (Dagdas et al., 2016). Here, weshow that during infection, ATG8CL/Joka2 labelled defense-related autophagosomes are divertedtoward the perimicrobial host membrane to restrict pathogen growth. PexRD54 also localizes toautophagosomes across the perimicrobial membrane, consistent with the view that the pathogenremodels host-microbe interface by co-opting the host autophagy machinery. Furthermore, weshow that the host-pathogen interface is a hotspot for autophagosome biogenesis. Notably,overexpression of the early autophagosome biogenesis protein ATG9 enhances plant immunity.Our results implicate selective autophagy in polarized immune responses of plants and point tomore complex functions for autophagy than the widely known degradative roles.
Leary AY, Sanguankiattichai N, Duggan C, et al., 2018, Modulation of plant autophagy during pathogen attack, Journal of Experimental Botany, Vol: 69, Pages: 1325-1333, ISSN: 0022-0957
In plants, the highly conserved catabolic process of autophagy has long been known as a means of maintaining cellular homeostasis and coping with abiotic stress conditions. Accumulating evidence has linked autophagy to immunity against invading pathogens, regulating plant cell death, and antimicrobial defences. In turn, it appears that phytopathogens have evolved ways not only to evade autophagic clearance but also to modulate and co-opt autophagy for their own benefit. In this review, we summarize and discuss the emerging discoveries concerning how pathogens modulate both host and self-autophagy machineries to colonize their host plants, delving into the arms race that determines the fate of interorganismal interaction.
Wu C-H, Abd-El-Haliem A, Bozkurt TO, et al., 2017, NLR network mediates immunity to diverse plant pathogens, Proceedings of the National Academy of Sciences of the United States of America, Vol: 114, Pages: 8113-8118, ISSN: 0027-8424
Both plants and animals rely on nucleotide-binding domain and leucine-rich repeat-containing (NLR) proteins to respond to invading pathogens and activate immune responses. An emerging concept of NLR function is that “sensor” NLR proteins are paired with “helper” NLRs to mediate immune signaling. However, our fundamental knowledge of sensor/helper NLRs in plants remains limited. In this study, we discovered a complex NLR immune network in which helper NLRs in the NRC (NLR required for cell death) family are functionally redundant but display distinct specificities toward different sensor NLRs that confer immunity to oomycetes, bacteria, viruses, nematodes, and insects. The helper NLR NRC4 is required for the function of several sensor NLRs, including Rpi-blb2, Mi-1.2, and R1, whereas NRC2 and NRC3 are required for the function of the sensor NLR Prf. Interestingly, NRC2, NRC3, and NRC4 redundantly contribute to the immunity mediated by other sensor NLRs, including Rx, Bs2, R8, and Sw5. NRC family and NRC-dependent NLRs are phylogenetically related and cluster into a well-supported superclade. Using extensive phylogenetic analysis, we discovered that the NRC superclade probably emerged over 100 Mya from an NLR pair that diversified to constitute up to one-half of the NLRs of asterids. These findings reveal a complex genetic network of NLRs and point to a link between evolutionary history and the mechanism of immune signaling. We propose that this NLR network increases the robustness of immune signaling to counteract rapidly evolving plant pathogens.
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