Imperial College London

Dr Tolga Bozkurt

Faculty of Natural SciencesDepartment of Life Sciences

Senior Lecturer
 
 
 
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Contact

 

+44 (0)20 7594 5381o.bozkurt

 
 
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Location

 

6167Sir Alexander Fleming BuildingSouth Kensington Campus

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Summary

 

Publications

Publication Type
Year
to

50 results found

Duggan C, Moratto E, Savage Z, Hamilton E, Adachi H, Wu C-H, Leary AY, Tumtas Y, Rothery SM, Maqbool A, Nohut S, Martin TR, Kamoun S, Bozkurt Oet 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.

Journal article

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.

Journal article

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, 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.

Journal article

Petre B, Contreras MP, Bozkurt TO, Schattat MH, Sklenar J, Schornack S, Abd-El-Haliem A, Castells-Graells R, Lozano-Duran R, Dagdas YF, Menke FLH, Jones AME, Vossen JH, Robatzek S, Kamoun S, Win Jet 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.

Journal article

Leong JX, Raffeiner M, Spinti D, Langin G, Franz-Wachtel M, Guzman AR, Kim J-G, Pandey P, Minina AE, Macek B, Hafrén A, Bozkurt TO, Mudgett MB, Börnke F, Hofius D, Üstün Set al., 2021, Self-ubiquitination of a pathogen type-III effector traps and blocks the autophagy machinery to promote disease

<jats:title>Abstract</jats:title><jats:p>Beyond its role in cellular homeostasis, autophagy is considered to play anti- and pro-microbial roles in host-microbe interactions, both in animals and plants. One of the prominent roles of anti-microbial autophagy in animals 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. However, the extent to which xenophagy contributes to plant-bacteria interactions remains unknown. Here, we provide evidence that NBR1/Joka2-dependent selective autophagy functions in plant defence by degrading the bacterial type-III effector (T3E) XopL from <jats:italic>Xanthomonas campestris</jats:italic> pv. <jats:italic>vesicatoria (Xcv)</jats:italic>. We show how XopL associates with the autophagy machinery and undergoes self-ubiquitination, subsequently triggering its own degradation by NBR1/Joka2-mediated selective autophagy. Intriguingly, <jats:italic>Xcv</jats:italic> is also able to suppress autophagy in a T3E-dependent manner by utilizing the same T3E XopL that interacts and degrades the autophagy component SH3P2 via its E3 ligase activity. Thus, XopL is able to escape its own degradation and promote pathogenicity of <jats:italic>Xcv</jats:italic> by inhibiting autophagy through SH3P2 depletion. Together, we reveal a novel phenomenon how NBR1/Joka2 contributes to anti-bacterial autophagy and provide a unique mechanism how a T3E undergoes self-modification to act as a bait to trap host cellular degradation machineries.</jats:p><jats:sec><jats:title>Significant statement</jats:title><jats:p>Autophagy has anti- and pro-microbial roles in host-microbe interactions. Its anti-microbial role is derived from its ability to degrade intracellular pathogens, termed “xenophagy”. The contribution of

Journal article

Klionsky DJ, Abdel-Aziz AK, Abdelfatah S, Abdellatif M, Abdoli A, Abel S, Abeliovich H, Abildgaard MH, Abudu YP, Acevedo-Arozena A, Adamopoulos IE, Adeli K, Adolph TE, Adornetto A, Aflaki E, Agam G, Agarwal A, Aggarwal BB, Agnello M, Agostinis P, Agrewala JN, Agrotis A, Aguilar P, Ahmad ST, Ahmed ZM, Ahumada-Castro U, Aits S, Aizawa S, Akkoc Y, Akoumianaki T, Akpinar HA, Al-Abd AM, Al-Akra L, Al-Gharaibeh A, Alaoui-Jamali MA, Alberti S, Alcocer-Gomez E, Alessandri C, Ali M, Al-Bari MAA, Aliwaini S, Alizadeh J, Almacellas E, Almasan A, Alonso A, Alonso GD, Altan-Bonnet N, Altieri DC, Alves S, da Costa CA, Alzaharna MM, Amadio M, Amantini C, Amaral C, Ambrosio S, Amer AO, Ammanathan V, An Z, Andersen SU, Andrabi SA, Andrade-Silva M, Andres AM, Angelini S, Ann D, Anozie UC, Ansari MY, Antas P, Antebi A, Anton Z, Anwar T, Apetoh L, Apostolova N, Araki T, Araki Y, Arasaki K, Araujo WL, Araya J, Arden C, Arevalo M-A, Arguelles S, Arias E, Arikkath J, Arimoto H, Ariosa AR, Armstrong-James D, Arnaune-Pelloquin L, Aroca A, Arroyo DS, Arsov I, Artero R, Asaro DML, Aschner M, Ashrafizadeh M, Ashur-Fabian O, Atanasov AG, Au AK, Auberger P, Auner HW, Aurelian L, Autelli R, Avagliano L, Avalos Y, Aveic S, Aveleira CA, AvinWittenberg T, Aydin Y, Ayton S, Ayyadevara S, Azzopardi M, Baba M, Backer JM, Backues SK, Bae D-H, Bae O-N, Bae SH, Baehrecke EH, Baek A, Baek S-H, Baek SH, Bagetta G, Bagniewska-Zadworna A, Bai H, Bai J, Bai X, Bai Y, Bairagi N, Baksi S, Balbi T, Baldari CT, Balduini W, Ballabio A, Ballester M, Balazadeh S, Balzan R, Bandopadhyay R, Banerjee S, Banerjee S, Bao Y, Baptista MS, Baracca A, Barbati C, Bargiela A, Barila D, Barlow PG, Barmada SJ, Barreiro E, Barreto GE, Bartek J, Bartel B, Bartolome A, Barve GR, Basagoudanavar SH, Bassham DC, Jr RCB, Basu A, Batoko H, Batten I, Baulieu EE, Baumgarner BL, Bayry J, Beale R, Beau I, Beaumatin F, Bechara LRG, Beck GR, Beers MF, Begun J, Behrends C, Behrens GMN, Bei R, Bejarano E, Bel S, Behl C, Belaid A, Belgareh-Touzeet 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.

Journal article

Oikawa K, Fujisaki K, Shimizu M, Takeda T, Saitoh H, Hirabuchi A, Hiraka Y, Białas A, Langner T, Kellner R, Bozkurt TO, Cesari S, Kroj T, Maidment JHR, Banfield MJ, Kamoun S, Terauchi Ret 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>

Journal article

Petre B, Contreras MP, Bozkurt TO, Schattat MH, Sklenar J, Schornack S, Abd-El-Haliem A, Castells-Graells R, Lozano-Duran R, Dagdas YF, Menke FLH, Jones AME, Vossen JH, Robatzek S, Kamoun S, Win Jet al., 2020, Host-interactor screens of Phytophthora infestans 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>

Working paper

Gao C, Xu H, Huang J, Sun B, Zhang F, Savage Z, Duggan C, Yan T, Wu C-H, Wang Y, Vleeshouwers VGAA, Kamoun S, Bozkurt TO, Dong Set 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.

Journal article

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.

Journal article

Adachi H, Contreras MP, Harant A, Wu C-H, Derevnina L, Sakai T, Duggan C, Moratto E, Bozkurt TO, Maqbool A, Win J, Kamoun Set 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.

Journal article

Leary AY, Savage Z, Tumtas Y, Bozkurt Tet al., 2019, Contrasting and emerging roles of autophagy in plant immunity, CURRENT OPINION IN PLANT BIOLOGY, Vol: 52, Pages: 46-53, ISSN: 1369-5266

Journal article

Bozkurt TO, Pandey P, Tumtas Y, Duggan C, Leary AY, Savage Z, Toufexi A, Contreras MP, Segretin ME, Dagdas YF, Kamoun Set 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

Conference paper

Zess EK, Jensen C, Cruz-Mireles N, De la Concepcion JC, Sklenar J, Stephani M, Imre R, Roitinger E, Hughes R, Belhaj K, Mechtler K, Menke FLH, Bozkurt T, Banfield MJ, Kamoun S, Maqbool A, Dagdas YFet 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.

Journal article

Adachi H, Contreras M, Harant A, Wu C-H, Derevnina L, Sakai T, Duggan C, Moratto E, Bozkurt TO, Maqbool A, Win J, Kamoun Set 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>

Working paper

Pennington HG, Rhian J, Kwon S, Bonciani G, Thieron H, Chandler T, Luong P, Morgan S, Przydacz M, Bozkurt T, Wallington E, Kwaaitaal M, Panstruga R, Cota E, Spanu Pet 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.

Journal article

Savage Z, Duggan C, Toufexi A, Pandey P, Liang Y, Segretin ME, Yuen LH, Gaboriau DCA, Leary AY, Tumtas Y, Khandare V, Ward AD, Botchway SW, Bateman BC, Pan I, Schattat M, Sparkes I, Bozkurt TOet al., 2019, Chloroplasts alter their morphology and accumulate at the pathogen interface during infection by Phytophthora infestans, 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>

Working paper

Dagdas Y, Pandey P, Tumtas Y, Nattapong S, Khaoula B, Cian D, Alexandre L, Maria S, Mauricio C, Zachary S, Virendrasinh K, Sophien K, Bozkurt OTet 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.

Journal article

Leary AY, Sanguankiattichai N, Duggan C, Tumtas Y, Pandey P, Segretin ME, Linares JS, Savage ZD, Yow RJ, Bozkurt TOet 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.

Journal article

Wu C-H, Abd-El-Haliem A, Bozkurt TO, Belhaj K, Terauchi R, Vossen JH, Kamoun Set 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.

Journal article

Dagvadorj B, Ozketen AC, Andac A, Duggan C, Bozkurt TO, Akkaya MSet al., 2017, A Puccinia striiformis f. sp tritici secreted protein activates plant immunity at the cell surface, SCIENTIFIC REPORTS, Vol: 7, ISSN: 2045-2322

Pathogens secrete effector proteins to suppress host immunity, mediate nutrient uptake and subsequently enable parasitism. However, on non-adapted hosts, effectors can be detected as non-self by host immune receptors and activate non-host immunity. Nevertheless, the molecular mechanisms of effector triggered non-host resistance remain unknown. Here, we report that a small cysteine-rich protein PstSCR1 from the wheat rust pathogen Puccinia striiformis f. sp. tritici (Pst) activates immunity in the non-host solanaceous model plant Nicotiana benthamiana. PstSCR1 homologs were found to be conserved in Pst, and in its closest relatives, Puccinia graminis f. sp. tritici and Puccinia triticina. When PstSCR1 was expressed in N. benthamiana with its signal peptide, it provoked the plant immune system, whereas no stimulation was observed when it was expressed without its signal peptide. PstSCR1 expression in N. benthamiana significantly reduced infection capacity of the oomycete pathogens. Moreover, apoplast-targeted PstSCR1 triggered plant cell death in a dose dependent manner. However, in Brassinosteroid insensitive 1-Associated Kinase 1 (SERK3/BAK1) silenced N. benthamiana, cell death was remarkably decreased. Finally, purified PstSCR1 protein activated defence related gene expression in N. benthamiana. Our results show that a Pst-secreted protein, PstSCR1 can activate surface mediated immunity in non-adapted hosts and contribute to non-host resistance.

Journal article

Dagdas YF, Pandey P, Sanguankiattichai N, Tumtas Y, Belhaj K, Duggan C, Segretin ME, Kamoun S, Bozkurt TOet al., 2017, Host autophagosomes are diverted to a plant-pathogen interface

<jats:title>Abstract</jats:title><jats:p>Filamentous plant pathogens and symbionts invade their host cells but remain enveloped by host-derived membranes. The mechanisms underlying the biogenesis and functions of these host-microbe interfaces are poorly understood. Recently, we showed that PexRD54, an effector from the Irish potato famine pathogen <jats:italic>Phytophthora infestans</jats:italic>, binds host protein ATG8CL to stimulate autophagosome formation and deplete the selective autophagy receptor Joka2 from ATG8CL complexes. Here, we show that during <jats:italic>P. infestans</jats:italic> infection, ATG8CL autophagosomes are diverted to the pathogen interface. Our findings are consistent with the view that the pathogen coopts host selective autophagy for its own benefit.</jats:p>

Journal article

Maqbool A, Hughes RK, Dagdas YF, Tregidgo N, Zess E, Belhaj K, Round A, Bozkurt TO, Kamoun S, Banfield MJet al., 2016, Structural Basis of Host Autophagy-related Protein 8 (ATG8) Binding by the Irish Potato Famine Pathogen Effector Protein PexRD54, Journal of Biological Chemistry, Vol: 291, Pages: 20270-20282, ISSN: 1083-351X

Filamentous plant pathogens deliver effector proteins to host cells to promote infection. The Phytophthora infestans RXLR-type effector PexRD54 binds potato ATG8 via its ATG8 family-interacting motif (AIM) and perturbs host-selective autophagy. However, the structural basis of this interaction remains unknown. Here, we define the crystal structure of PexRD54, which includes a modular architecture, including five tandem repeat domains, with the AIM sequence presented at the disordered C terminus. To determine the interface between PexRD54 and ATG8, we solved the crystal structure of potato ATG8CL in complex with a peptide comprising the effector's AIM sequence, and we established a model of the full-length PexRD54-ATG8CL complex using small angle x-ray scattering. Structure-informed deletion of the PexRD54 tandem domains reveals retention of ATG8CL binding in vitro and in planta This study offers new insights into structure/function relationships of oomycete RXLR effectors and how these proteins engage with host cell targets to promote disease.

Journal article

Dagdas YF, Belhaj K, Maqbool A, Chaparro-Garcia A, Pandey P, Petre B, Tabassum N, Cruz-Mireles N, Hughes RK, Sklenar J, Win J, Menke F, Findlay K, Banfield MJ, Kamoun S, Bozkurt TOet al., 2016, An effector of the Irish potato famine pathogen antagonizes a host autophagy cargo receptor, eLife, Vol: 5, ISSN: 2050-084X

Plants use autophagy to safeguard against infectious diseases. However, how plant pathogens interfere with autophagy-related processes is unknown. Here, we show that PexRD54, an effector from the Irish potato famine pathogen Phytophthora infestans, binds host autophagy protein ATG8CL to stimulate autophagosome formation. PexRD54 depletes the autophagy cargo receptor Joka2 out of ATG8CL complexes and interferes with Joka2's positive effect on pathogen defense. Thus, a plant pathogen effector has evolved to antagonize a host autophagy cargo receptor to counteract host defenses.

Journal article

Giannakopoulou A, Steele JFC, Eugenia Segretin M, Bozkurt TO, Zhou J, Robatzek S, Banfield MJ, Pais M, Kamoun Set al., 2015, Tomato 12 Immune Receptor Can Be Engineered to Confer Partial Resistance to the Oomycete Phytophthora infestans in Addition to the Fungus Fusarium oxysporum, Molecular Plant-Microbe Interactions, Vol: 28, Pages: 1316-1329, ISSN: 0894-0282

Plants and animals rely on immune receptors, known as nucleotide-binding domain and leucine-rich repeat (NLR)-containing proteins, to defend against invading pathogens and activate immune responses. How NLR receptors respond to pathogens is inadequately understood. We previously reported single-residue mutations that expand the response of the potato immune receptor R3a to AVR3aEM, a stealthy effector from the late blight oomycete pathogen Phytophthora infestans. I2, another NLR that mediates resistance to the wilt-causing fungus Fusarium oxysporum f. sp. lycopersici, is the tomato ortholog of R3a. We transferred previously identified R3a mutations to I2 to assess the degree to which the resulting I2 mutants have an altered response. We discovered that wild-type I2 protein responds weakly to AVR3a. One mutant in the N-terminal coiled-coil domain, I2I141N, appeared sensitized and displayed markedly increased response to AVR3a. Remarkably, I2I141N conferred partial resistance to P. infestans. Further, I2I141N has an expanded response spectrum to F. oxysporum f. sp. lycopersici effectors compared with the wild-type I2 protein. Our results suggest that synthetic immune receptors can be engineered to confer resistance to phylogenetically divergent pathogens and indicate that knowledge gathered for one NLR could be exploited to improve NLR from other plant species.

Journal article

Dagdas YF, Bozkurt TO, 2015, Fungal sex receptors recalibrated to detect host plants, Cell Host & Microbe, Vol: 18, Pages: 637-638, ISSN: 1934-6069

Secreted peroxidases are well-known components of damage-induced defense responses in plants. A recent study in Nature ( Turrà et al., 2015) has revealed that these enzymes can inadvertently serve as reporters of wounded sites and constitute an “Achilles heel,” allowing adapted pathogens to track and enter host tissue.

Journal article

Wu C-H, Belhaj K, Bozkurt TO, Birk MS, Kamoun Set al., 2015, Helper NLR proteins NRC2a/b and NRC3 but not NRC1 are required for Pto-mediated cell death and resistance in Nicotiana benthamiana, New Phytologist, Vol: 209, Pages: 1344-1352, ISSN: 1469-8137

Journal article

Chaparro-Garcia A, Schwizer S, Sklenar J, Yoshida K, Petre B, Bos JIB, Schornack S, Jones AME, Bozkurt TO, Kamoun Set al., 2015, Phytophthora infestans RXLR-WY Effector AVR3a Associates with Dynamin-Related Protein 2 Required for Endocytosis of the Plant Pattern Recognition Receptor FLS2, PLOS One, Vol: 10, ISSN: 1932-6203

Pathogens utilize effectors to suppress basal plant defense known as PTI (Pathogen-associatedmolecular pattern-triggered immunity). However, our knowledge of PTI suppressionby filamentous plant pathogens, i.e. fungi and oomycetes, remains fragmentary. Previouswork revealed that the co-receptor BAK1/SERK3 contributes to basal immunity against thepotato pathogen Phytophthora infestans. Moreover BAK1/SERK3 is required for the celldeath induced by P. infestans elicitin INF1, a protein with characteristics of PAMPs. TheP. infestans host-translocated RXLR-WY effector AVR3a is known to supress INF1-mediatedcell death by binding the plant E3 ligase CMPG1. In contrast, AVR3aKI-Y147del, a deletionmutant of the C-terminal tyrosine of AVR3a, fails to bind CMPG1 and does notsuppress INF1-mediated cell death. Here, we studied the extent to which AVR3a and itsvariants perturb additional BAK1/SERK3-dependent PTI responses in N. benthamianausing the elicitor/receptor pair flg22/FLS2 as a model. We found that all tested variants ofAVR3a suppress defense responses triggered by flg22 and reduce internalization of activatedFLS2. Moreover, we discovered that AVR3a associates with the Dynamin-RelatedProtein 2 (DRP2), a plant GTPase implicated in receptor-mediated endocytosis. Interestingly,silencing of DRP2 impaired ligand-induced FLS2 internalization but did not affectinternalization of the growth receptor BRI1. Our results suggest that AVR3a associateswith a key cellular trafficking and membrane-remodeling complex involved in immune receptor-mediated endocytosis. We conclude that AVR3a is a multifunctional effector thatcan suppress BAK1/SERK3-mediated immunity through at least two different pathways.

Journal article

Ilyas M, Hoerger AC, Bozkurt TO, van den Burg HA, Kaschani F, Kaiser M, Belhaj K, Smoker M, Joosten MHAJ, Kamoun S, van der Hoorn RALet al., 2015, Functional divergence of two secreted immune proteases of tomato, Current Biology, Vol: 25, Pages: 2300-2306, ISSN: 1879-0445

Rcr3 and Pip1 are paralogous secreted papain-like proteases of tomato. Both proteases are inhibited by Avr2 from the fungal pathogen Cladosporium fulvum, but only Rcr3 acts as a co-receptor for Avr2 recognition by the tomato Cf-2 immune receptor [1–4]. Here, we show that Pip1-depleted tomato plants are hyper-susceptible to fungal, bacterial, and oomycete plant pathogens, demonstrating that Pip1 is an important broad-range immune protease. By contrast, in the absence of Cf-2, Rcr3 depletion does not affect fungal and bacterial infection levels but causes increased susceptibility only to the oomycete pathogen Phytophthora infestans. Rcr3 and Pip1 reside on a genetic locus that evolved over 36 million years ago. These proteins differ in surface-exposed residues outside the substrate-binding groove, and Pip1 is 5- to 10-fold more abundant than Rcr3. We propose a model in which Rcr3 and Pip1 diverged functionally upon gene duplication, possibly driven by an arms race with pathogen-derived inhibitors or by coevolution with the Cf-2 immune receptor detecting inhibitors of Rcr3, but not of Pip1.

Journal article

Oliva RF, Cano LM, Raffaele S, Win J, Bozkurt TO, Belhaj K, Oh S-K, Thines M, Kamoun Set al., 2015, A Recent Expansion of the RXLR Effector Gene Avrblb2 Is Maintained in Global Populations of Phytophthora infestans Indicating Different Contributions to Virulence, MOLECULAR PLANT-MICROBE INTERACTIONS, Vol: 28, Pages: 901-912, ISSN: 0894-0282

Journal article

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