Biology subjects

Dagdas, Y.

Publications and source records attributed to Dagdas, Y..

At least 37 records · Page 2Linked to original sources

bioRxiv2024

The Arabidopsis thaliana TRAPPIII subunit AtTRAPPC8/AtTRS85 is involved in ER functioning and autophagy

TRAPP (transport protein particle) tethering complexes are known for their function as Rab-GTPase exchange factors (GEFs). Two versions of the complex are considered functionally separate: TRAPPII, an activator of GTPases of the Rab11 family (RabA in plants) which functions in post-Golgi sorting, and TRAPPIII, activating the Rab1 family (RabD in plants) which regulates ER-to-Golgi trafficking and autophagy. In Arabidopsis thaliana, the TRAPPIII complex has been identified and its subunit composition established, but little is known about its functions. Here, we found that binary subunit interactions of the plant TRAPPIII complex are analogous to those of metazoan TRAPPIII, with the two large subunits TRAPPC8 and -C11 linking the TRAPP core and the small C12-C13 dimer. To gain insight into the functions of TRAPPIII in plants, we characterized two A. thaliana trappc8 mutants. The mutants display abnormalities in plant morphology, in particular in flower and seed development. They also have autophagic defects, constitutive ER stress response, and elevated levels of the ER lipid dolichol - an indispensable cofactor of protein glycosylation. These results show that plant TRAPPC8 is involved in multiple trafficking steps in the cells and they suggest a novel link between ER membrane turnover and dolichol levels. HIGHLIGHTArabidopsis thaliana TRAPPC8 is necessary for correct functioning of the ER, in particular for its lipid homeostasis. Dysruption of TRAPPC8 leads to defects in secretion, autophagosome formation, and plant development.

plant biology↗

bioRxiv2023

Autophagy restricts fungal accommodation in the roots ofArabidopsis thaliana

Endophytic colonization of Arabidopsis thaliana by the beneficial root endophyte Serendipita indica is characterized by an initial biotrophic phase followed by a restricted host cell death-associated phase. This latter phase involves regulated cell death (RCD) for fungal accommodation. However, the host molecular pathways that limit S. indica colonization and govern symbiosis remain largely unknown. Our study demonstrates that autophagy, a major cellular degradation pathway, is activated during S. indica colonization and is required to restrict fungal colonization in Arabidopsis. Independent Arabidopsis knockout (KO) mutants deficient in autophagosome formation are more susceptible to deoxyadenosine (dAdo), a cell death inducer produced by two secreted S. indica effectors at the onset of the cell death-associated phase. In the atg5 autophagy mutant background, impaired dAdo uptake prevents dAdo-induced and symbiosis-mediated cell death. Based on our data, we propose that autophagy-mediated pro-survival responses in the host are crucial for maintaining a balanced symbiotic interaction between S. indica and Arabidopsis. In a NutshellOur study reveals that during colonization of Arabidopsis thaliana roots by the beneficial root endophyte Serendipita indica, autophagy, a key cellular degradation pathway, is activated to limit fungal colonization. Autophagy-deficient Arabidopsis mutants are more susceptible to deoxyadenosine (dAdo), a cell death inducer produced by S. indica. We propose that autophagy-mediated pro-survival responses are essential for maintaining a balanced symbiotic interaction between S. indica and Arabidopsis.

microbiology↗

bioRxiv2023

A RabGAP-Rab GTPase pair regulates plant autophagy and immunity

Plants rely on autophagy and membrane trafficking to tolerate stress, combat infections, and maintain cellular homeostasis. However, the molecular interplay between autophagy and membrane trafficking is poorly understood. Using an AI-assisted approach, we identified Rab3GAP-like (Rab3GAPL) as an important membrane trafficking node that controls plant autophagy negatively. Rab3GAPL suppresses autophagy by binding to ATG8, the core autophagy adaptor, and deactivating Rab8a, a small GTPase essential for autophagosome formation and defense-related secretion. Rab3GAPL from Nicotiana benthamiana, but not its mutated form deficient in ATG8 binding, reduced autophagic flux in N. benthamiana and Arabidopsis. Furthermore, Rab3GAPL-knockout mutants of the liverwort Marchantia polymorpha exhibited enhanced autophagic flux under both normal and heat stress conditions, suggesting that Rab3GAPLs negative regulatory role in autophagy is conserved in land plants. Beyond autophagy regulation, Rab3GAPL modulates focal immunity against the oomycete pathogen Phytophthora infestans by preventing defense-related secretion. Altogether, our results suggest that Rab3GAPL acts as a molecular rheostat to coordinate autophagic flux and defense-related secretion by restraining Rab8a-mediated trafficking. This unprecedented interplay between a RabGAP-Rab pair and ATG8 sheds new light on the intricate membrane transport mechanisms underlying plant autophagy and immunity.

plant biology↗

bioRxiv2022

mRNA decapping machinery targets LBD3/ASL9 transcripts to allow developmental changes in Arabidopsis

Multicellular organisms perceive and transduce multiple cues to optimize development. Key transcription factors drive developmental changes, but RNA processing also contributes to tissue development. Here, we report that multiple decapping deficient mutants share developmental defects in apical hook, primary and lateral root growth. More specifically, LATERAL ORGAN BOUNDARIES DOMAIN 3 (LBD3)/ASYMMETRIC LEAVES 2-LIKE 9 (ASL9) transcripts accumulate in decapping deficient plants and can be found in complexes with decapping components. Accumulation of ASL9 inhibits apical hook, primary root growth and lateral root formation. Interestingly, exogenous auxin application restores lateral roots formation in both ASL9 over-expressors and mRNA decay-deficient mutants. Likewise, mutations in the cytokinin transcription factors type-B ARABIDOPSIS RESPONSE REGULATORS (B-ARRs) ARR10 and ARR12 restore the developmental defects caused by over-accumulation of capped ASL9 transcript upon ASL9 overexpression. Most importantly, loss-of-function of asl9 partially restores apical hook and lateral root formation in decapping deficient mutants. Thus, the mRNA decay machinery directly targets ASL9 transcripts for decay, possibly to interfere with cytokinin/auxin responses, during development.

developmental biology↗

bioRxiv2022

PAT mRNA decapping factors function specifically and redundantly during development in Arabidopsis

Evolutionarily conserved PAT1 proteins activate mRNA decay through binding mRNA and recruiting decapping enzymes and other factors hence optimize transcriptional reprogramming during development. Here, we generated multiple mutants of pat1 (Protein Associated with Topoisomerase II), path1 and path2 and inspected their growth and leaf morphology phenotype. pat triple mutants exhibit extreme stunted growth and all mutants with pat1 exhibit leaf serration while mutants with pat1 and path1 all display short petioles. All 3 PATs can be found localized to Prossessing Bodies (PBs) upon auxin treatment and RNA-seq analysis indicate that all 3 PATs redundantly regulate auxin responses. Moreover, shade avoidance and NAC genes are misregulated in pat1path1 double and pat triple mutants suggesting PAT1 and PATH1 function in petiole elongation and leaf patterning. In conclusion, PAT proteins exhibit both specific and overlapping functions during different stages of plant growth and our observations underpin the importance of the mRNA decay machinery for proper development.

plant biology↗

bioRxiv2022

Shuffled ATG8 interacting motifs form an ancestral bridge between UFMylation and C53-mediated autophagy

UFMylation mediates the covalent modification of substrate proteins with UFM1 (Ubiquitin-fold modifier 1) and regulates the selective degradation of endoplasmic reticulum (ER) via autophagy (ER-phagy) to maintain ER homeostasis. Specifically, collisions of the ER-bound ribosomes trigger ribosome UFMylation, which in turn activates C53-mediated autophagy that clears the toxic incomplete polypeptides. C53 has evolved non-canonical shuffled ATG8 interacting motifs (sAIMs) that are essential for ATG8 interaction and autophagy initiation. Why these non-canonical motifs were selected during evolution, instead of canonical ATG8 interacting motifs remains unknown. Here, using a phylogenomics approach, we show that UFMylation is conserved across the eukaryotes and secondarily lost in fungi and some other species. Further biochemical assays have confirmed those results and showed that the unicellular algae, Chlamydomonas reinhardtii has a functional UFMylation machinery, overturning the assumption that this process is linked to multicellularity. Our conservation analysis also revealed that UFM1 co-evolves with the sAIMs in C53, reflecting a functional link between UFM1 and the sAIMs. Using biochemical and structural approaches, we confirmed the interaction of UFM1 with the C53 sAIMs and found that UFM1 and ATG8 bound to the sAIMs in a different mode. Conversion of sAIMs into canonical AIMs prevented binding of UFM1 to C53, while strengthening ATG8 interaction. This led to the autoactivation of the C53 pathway and sensitized Arabidopsis thaliana to ER stress. Altogether, our findings reveal an ancestral toggle switch embodied in the sAIMs that regulates C53-mediated autophagy to maintain ER homeostasis.

plant biology↗

bioRxiv2022

Plant autophagosomes mature into amphisomes prior to their delivery to the central vacuole

Autophagosomes are double-membraned vesicles that traffic harmful or unwanted cellular macromolecules to the vacuole for recycling. Although autophagosome biogenesis has been extensively studied, mechanisms of autophagosome maturation, i.e., delivery and fusion with the vacuole, remain largely unknown in plants. Here, we have identified an autophagy adaptor, CFS1, that directly interacts with the autophagosome marker ATG8 and localizes on both membranes of the autophagosome. Autophagosomes form normally in Arabidopsis thaliana cfs1 mutants, but their delivery to the vacuole is disrupted. CFS1s function is evolutionarily conserved in plants as it also localizes to the autophagosomes and plays a role in autophagic flux in the liverwort Marchantia polymorpha. CFS1 regulates autophagic flux by connecting autophagosomes with the ESCRT-I component VPS23, leading to the formation of amphisomes. Disrupting the VPS23-CFS1 interaction affects autophagic flux and renders plants sensitive to starvation stress. Altogether, our results reveal a deeply conserved mechanism of vacuolar delivery in plants that is mediated by amphisomes.

plant biology↗

bioRxiv2022

Autophagy promotes programmed cell death and corpse clearance in specific cell types of the Arabidopsis root cap

Autophagy is a conserved quality control pathway that mediates the degradation of unnecessary or dysfunctional cellular components by targeting them to the lysosomes or central vacuoles. Autophagy has been implicated in the regulation or execution of regulated cell death processes in a wide range of eukaryotes. However, its function in developmentally controlled programmed cell death (dPCD) in plants remains little studied and controversial. Here, we investigated the role of autophagy in dPCD using the Arabidopsis root cap as an accessible and genetically tractable model system. We show that autophagic flux is induced prior to dPCD execution in both root cap tissues, the columella and the lateral root cap (LRC), and impaired in autophagy-deficient mutants. These mutants show a strongly delayed cell death and an absence of corpse clearance in the columella during and after their shedding into the rhizosphere. However, autophagy deficiency does not affect dPCD execution or corpse clearance in LRC cells at the distal end of the root cap. Our results demonstrate that autophagy promotes dPCD in a highly cell-type specific manner, and present the root cap as a powerful model system to study organ-specific autophagy in vivo.

plant biology↗

bioRxiv2021

A NET4-RabG3 couple mediate the link between actin and the tonoplast and is essential for normal actin cytoskeletal remodelling in stomatal closure to flg22

Members of the NETWORKED (NET) family are involved in actin-membrane interactions. They tether the cells plasma membrane (PM) to the actin network. Moreover, in a similar manner, they are also involved in the tethering of membrane bound organelles to the actin cytoskeleton; the endoplasmic reticulum (ER) and the ER to the PM. This raises the question as to whether NET proteins are involved in actin cytoskeletal remodelling. Here we show that two members of the NET family, NET4A and NET4B, are essential for normal guard cell actin reorganization, which is a process critical for stomatal closure in plant immunity. NET4 proteins interact with F-actin and with members of the Rab7 GTPase RABG3 family through two distinct domains, allowing for simultaneous localization to actin filaments and the tonoplast. NET4 proteins interact with GTP-bound, active RABG3 members, suggesting their function as downstream effectors. We also show that RABG3b is critical for stomatal closure induced by microbial patterns. Taken together, we conclude that the actin cytoskeletal remodelling during stomatal closure depends on a molecular link between actin filaments and the tonoplast, which is mediated by the NET4-RABG3b interaction. We propose that stomatal closure to microbial patterns involves the coordinated action of immune signalling events and proper actin cytoskeletal remodelling.

plant biology↗

bioRxiv2021

Bacterial-type plant ferroxidases tune local phosphate sensing in root development

Fluctuating bioavailability of inorganic phosphate (Pi), often caused by complex Pi-metal interactions, guide root tip growth and root system architecture for maximizing the foraged soil volume. Two interacting genes in Arabidopsis thaliana, PDR2 (P5-type ATPase) and LPR1 (multicopper oxidase), are central to external Pi monitoring by root tips, which is modified by iron (Fe) co-occurrence. Upon Pi deficiency, the PDR2-LPR1 module facilitates cell type-specific Fe accumulation and cell wall modifications in root meristems, inhibiting intercellular communication and thus root growth. LPR1 executes local Pi sensing, whereas PDR2 restricts LPR1 function. We show that native LPR1 displays specific ferroxidase activity and requires a conserved acidic triad motif for high-affinity Fe2+ binding and root growth inhibition under limiting Pi. Our data indicate that substrate availability tunes LPR1 function and implicate PDR2 in maintaining Fe homeostasis. LPR1 represents the prototype of an ancient ferroxidase family, which evolved very early upon bacterial colonization of land. During plant terrestrialization, horizontal gene transfer transmitted LPR1-type ferroxidase from soil bacteria to the common ancestor of Zygnematophyceae algae and embryophytes, a hypothesis supported by homology modeling, phylogenomics, and activity assays of bacterial LPR1-type multicopper oxidases.

plant biology↗

bioRxiv2020

Host-interactor screens of Phytophthora infestans RXLR proteins reveal vesicle trafficking as a major effector-targeted process

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 (co-IP) and liquid chromatography tandem mass spectrometry (LC-MS/MS). 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. We anticipate that the interactome dataset we generated will serve as a useful community resource for functional studies of P. infestans effectors and of effector-targeted host processes.

plant biology↗

bioRxiv2020

The Irish potato famine pathogen subverts host vesicle trafficking to channel starvation-induced autophagy to the pathogen interface

Eukaryotic cells deploy autophagy to eliminate invading microbes. In turn, pathogens have evolved effector proteins to counteract antimicrobial autophagy. How and why adapted pathogens co-opt autophagy for their own benefit is poorly understood. The Irish famine pathogen Phythophthora infestans secretes the effector protein PexRD54 that selectively activates an unknown plant autophagy pathway, while antagonizing antimicrobial autophagy. Here we show that PexRD54 induces autophagosome formation by bridging small GTPase Rab8a-decorated vesicles with autophagic compartments labelled by the core autophagy protein ATG8CL. Rab8a is required for pathogen-triggered and starvation-induced but not antimicrobial autophagy, revealing that specific trafficking pathways underpin selective autophagy. We discovered that Rab8a contributes to basal immunity against P. infestans, but PexRD54 diverts a sub-population of Rab8a vesicles to lipid droplets that associate with autophagosomes. These are then diverted towards pathogen feeding structures that are accommodated within the host cells. We propose that PexRD54 mimics starvation-induced autophagy by channeling host endomembrane trafficking towards the pathogen interface possibly to acquire nutrients. This work reveals that effectors can interconnect independent host compartments to stimulate complex cellular processes that benefit the pathogen. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=200 SRC="FIGDIR/small/000117v1_ufig1.gif" ALT="Figure 1"> View larger version (83K): org.highwire.dtl.DTLVardef@bfc9fforg.highwire.dtl.DTLVardef@166b5f8org.highwire.dtl.DTLVardef@34d581org.highwire.dtl.DTLVardef@ad397f_HPS_FORMAT_FIGEXP M_FIG C_FIG

plant biology↗

bioRxiv2020

A cross-kingdom conserved ER-phagy receptor maintains endoplasmic reticulum homeostasis during stress

Eukaryotes have evolved various quality control mechanisms to promote proteostasis in the ER. Selective removal of certain ER domains via autophagy (termed as ER-phagy) has emerged as a major quality control mechanism. However, the degree to which ER-phagy is employed by other branches of ER-quality control remains largely elusive. Here, we identify a cytosolic protein, C53, that is specifically recruited to autophagosomes during ER-stress, in both plant and mammalian cells. C53 interacts with ATG8 via a distinct binding epitope, featuring a shuffled ATG8 interacting motif (sAIM). C53 senses proteotoxic stress in the ER lumen by forming a tripartite receptor complex with the ER-associated ufmylation ligase UFL1 and its membrane adaptor DDRGK1. The C53/UFL1/DDRGK1 receptor complex is activated by stalled ribosomes and induces the degradation of internal or passenger proteins in the ER. Consistently, the C53 receptor complex and ufmylation mutants are highly susceptible to ER stress. Thus, C53 forms an ancient quality control pathway that bridges selective autophagy with ribosome-associated quality control at the ER.

cell biology↗

bioRxiv2020

EXO70D isoforms mediate selective autophagic degradation of Type-A ARR proteins to regulate cytokinin sensitivity

The phytohormone cytokinin influences many aspects of plant growth and development, several of which also involve the cellular process of autophagy, including leaf senescence, nutrient re-mobilization, and developmental transitions. The Arabidopsis type-A Response Regulators (type-A ARR) are negative regulators of cytokinin signaling that are transcriptionally induced in response to cytokinin. Here, we describe a mechanistic link between cytokinin signaling and autophagy, demonstrating that plants modulate cytokinin sensitivity through autophagic regulation of type-A ARR proteins. Type-A ARR proteins were degraded by autophagy in an AUTOPHAGY-RELATED (ATG)5-dependent manner. EXO70D family members interacted with Type-A ARR proteins, likely in a phosphorylation-dependent manner, and recruited them to autophagosomes via interaction with the core autophagy protein, ATG8. Consistently, loss-of-function exo70D1,2,3 mutants compromised targeting of type-A ARRs to autophagic vesicles, have elevated levels of type-A ARR proteins, and are hyposensitive to cytokinin. Disruption of both type-A ARRs and EXO70D1,2,3 compromised survival in carbon-deficient conditions, suggesting interaction between autophagy and cytokinin responsiveness in response to stress. These results indicate that the EXO70D proteins act as selective autophagy receptors to target type-A ARR cargos for autophagic degradation, demonstrating that cytokinin signaling can be modulated by selective autophagy.

plant biology↗

bioRxiv2019

mRNA decapping machinery targets transcripts of the LBD3/ASL9 transcription factor to authorize formation of apical hook and lateral roots in Arabidopsis

Multicellular organisms perceive and transduce multiple cues to optimize developmental reprogramming and cell state switching. Key transcription factors drive developmental changes, but transitions also require the attenuation of previous states. Here, we demonstrate that the mRNA levels of the LATERAL ORGAN BOUNDARIES DOMAIN 3 (LBD3) / ASYMMETRIC LEAVES 2-LIKE 9 (ASL9) transcription factor are directly regulated by mRNA decapping. Capped ASL9 transcripts accumulate in decapping deficient plants and ASL9 mRNAs are found together with decapping components. Accumulation of ASL9 inhibits apical hook and lateral roots formation and interestingly, exogenous auxin application restores lateral roots formation in both ASL9 overexpressor and mRNA decay-deficient mutants. Moreover, mutations in the cytokinin transcription factors type-B ARABIDOPSIS RESPONSE REGULATORS (B-ARRs) ARR10 and ARR12 restore the developmental defects in apical hooking and lateral root formation caused by over-accumulation of capped ASL9 transcript upon ASL9 overexpression. Thus, the mRNA decay machinery directly targets ASL9 transcripts for decay to balance cytokinin/auxin responses during developmental reprogramming.

plant biology↗

bioRxiv2019

Autophagy mediates temporary reprogramming and dedifferentiation in plant somatic cells

Somatic cells acclimate to changes in the environment by temporary reprogramming. Much has been learned about transcription factors that induce these cell-state switches in both plants and animals, but how cells rapidly modulate their proteome remains elusive. Here, we show rapid induction of autophagy during temporary reprogramming in plants triggered by phytohormones, immune and danger signals. Quantitative proteomics following sequential reprogramming revealed that autophagy is required for timely decay of previous cellular states and for tweaking the proteome to acclimate to the new conditions. Signatures of previous cellular programs thus persist in autophagy deficient cells, affecting cellular decision-making. Concordantly, autophagy deficient cells fail to acclimatize to dynamic climate changes. Similarly, they have defects in dedifferentiating into pluripotent stem cells, and redifferentiation during organogenesis. These observations indicate that autophagy mediates cell state switches that underlie somatic cell reprogramming in plants and possibly other organisms, and thereby promotes phenotypic plasticity.

plant biology↗

bioRxiv2018

N-terminal β-strand underpins biochemical specialization of an ATG8 isoform

ATG8 is a highly-conserved ubiquitin-like protein that modulates autophagy pathways by binding autophagic membranes and numerous 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 followed by mass spectrometry, 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 {beta}-strand--and, in particular, a single amino acid polymorphism--underpins binding specificity to the substrate PexRD54 by shaping the hydrophobic pocket that accommodates this proteins ATG8 interacting motif. Additional proteomics experiments indicated that the N-terminal {beta}-strand shapes the 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.

plant biology↗

bioRxiv2018

The anti-apoptosis ubiquitin E3 ligase XIAP promotes autophagosome-lysosome fusion during autophagy

The Inhibitor of Apoptosis Protein (IAP) family members are well-known endogenous regulators of apoptosis. Whether these proteins regulate other degradation pathways is unclear. Here, we discovered that the IAP member X-linked IAP (XIAP) is crucial for macroautophagy. Loss of XIAP in mouse and human cells inhibited starvation-induced degradation of LC3 proteins and an autophagy substrate p62. It also led to the accumulation of mature autophagosomes, suggesting that XIAP controls autophagic flux by mediating autolysosome formation. Xiap{triangleup}RING/{triangleup}RING cells phenocopy the autophagy defects of Xiap-/- cells, suggesting that the ubiquitinating activity mediated by the catalytic RING domain is critical for autophagic flux. We found that XIAP physically interacts with Syntaxin 17, a regulator of autophagosome-lysosome fusion. Syntaxin 17-positive mature autophagosomes positive accumulate in the cytoplasm of starved Xiap-/- cells, suggesting that XIAP might regulate its dissociation from autophagosomes after fusion. XIAP selectively interacts with GABARAP among LC3 family members via the LIR-Docking Site (LDS). Together, our data suggest that XIAP-mediated ubiquitination regulates key autophagy regulators to promote autophagosome-lysosome fusion.

cell biology↗