AlphaFold2-multimer guided high-accuracy prediction of typical and atypical ATG8-binding motifs

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

Modulation of plant autophagy during pathogen attack

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.Fil: Leary, Alexandre Y. Imperial College London; Reino UnidoFil: Sanguankiattichai, Nattapong. University of Oxford; Reino UnidoFil: Duggan, Cian. Imperial College London; Reino UnidoFil: Tumtas, Yasin. Imperial College London; Reino UnidoFil: Pandey, Pooja. Imperial College London; Reino UnidoFil: Segretin, Maria Eugenia. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular "Dr. Héctor N. Torres"; ArgentinaFil: Salguero Linares, Jose. Imperial College London; Reino UnidoFil: Savage, Zachary D. Imperial College London; Reino UnidoFil: Yow, Rui Jin. Imperial College London; Reino UnidoFil: Bozkurt, Tolga O.. Imperial College London; Reino Unid

Improving pancreatic islet in vitro functionality and transplantation efficiency by using heparin mimetic peptide nanofiber gels

Pancreatic islet transplantation is a promising treatment for type 1 diabetes. However, viability and functionality of the islets after transplantation are limited due to loss of integrity and destruction of blood vessel networks. Thus, it is important to provide a proper mechanically and biologically supportive environment for enhancing both in vitro islet culture and transplantation efficiency. Here, we demonstrate that heparin mimetic peptide amphiphile (HM-PA) nanofibrous network is a promising platform for these purposes. The islets cultured with peptide nanofiber gel containing growth factors exhibited a similar glucose stimulation index as that of the freshly isolated islets even after 7 days. After transplantation of islets to STZ-induced diabetic rats, 28 day-long monitoring displayed that islets that were transplanted in HM-PA nanofiber gels maintained better blood glucose levels at normal levels compared to the only islet transplantation group. In addition, intraperitoneal glucose tolerance test revealed that animals that were transplanted with islets within peptide gels showed a similar pattern with the healthy control group. Histological assessment showed that islets transplanted within peptide nanofiber gels demonstrated better islet integrity due to increased blood vessel density. This work demonstrates that using the HM-PA nanofiber gel platform enhances the islets function and islet transplantation efficiency both in vitro and in vivo. © 2015 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved

Dynamic localization of a helper NLR at the plant-pathogen interface underpins pathogen recognition

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

An oomycete effector 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 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 labeled 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

Chloroplasts alter their morphology and accumulate at the pathogen interface during infection by Phytophthora infestans

Upon immune activation, chloroplasts switch off photosynthesis, produce antimicrobial 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 chloroplast movement in response to pathogen attack. Here, we report that during infection with the Irish potato famine pathogen Phytophthora infestans, chloroplasts accumulate at the pathogen interface, associating with the specialized membrane that engulfs the pathogen haustorium. The chemical inhibition of actin polymerization reduces the accumulation of chloroplasts at pathogen haustoria, suggesting that 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.Fil: Savage, Zachary. Imperial College London; Reino UnidoFil: Duggan, Cian. Imperial College London; Reino UnidoFil: Toufexi, Alexia. Imperial College London; Reino UnidoFil: Pandey, Pooja. Imperial College London; Reino UnidoFil: Liang, Yuxi. Imperial College London; Reino UnidoFil: Segretin, Maria Eugenia. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular "Dr. Héctor N. Torres"; ArgentinaFil: Yuen, Lok Him. Imperial College London; Reino UnidoFil: Gaboriau, David C. A.. Imperial College London; Reino UnidoFil: Leary, Alexandre Y.. Imperial College London; Reino UnidoFil: Tumtas, Yasin. Imperial College London; Reino UnidoFil: Khandare, Virendrasinh. Imperial College London; Reino UnidoFil: Ward, Andrew D.. Science and Technology Facilities Council; Reino UnidoFil: Botchway, Stanley W.. Science and Technology Facilities Council; Reino UnidoFil: Bateman, Benji C.. Science and Technology Facilities Council; Reino UnidoFil: Pan, Indranil. Alan Turing Institute; Reino Unido. Imperial College London; Reino UnidoFil: Schattat, Martin. Martin Luther Universitat Halle-Wittenberg; AlemaniaFil: Sparkes, Imogen. University of Bristol; Reino UnidoFil: Bozkurt, Osman Tolga. Imperial College London; Reino Unid

Reusable bacteria immobilized electrospun nanofibrous web fro decolorization of methylene blue dye in wastewatewr treatment

Cataloged from PDF version of article.In our study, an electrospun cellulose acetate nanofibrous web (CA-NFW) was found to be quite effective in immobilizing bacterial cells. Here, decolorization of methylene blue (MB) dye in aqueous medium was achieved by using three types of bacteria (Aeromonas eucrenophila, Clavibacter michiganensis and Pseudomonas aeruginosa) immobilized on the CA-NFW. The decolorization time (0-48 h) and different MB dye concentrations (20-500 mg L-1) were studied to elucidate the maximum MB dye removal by the bacteria immobilized CA-NFWs. The effective dye decolorization was achieved within 24 hours and MB dye removal was similar to 95%. Interestingly, MB dye decolorization performance of bacteria immobilized CA-NFWs was quite close to that of free bacteria. We have also tested the reusability of bacteria immobilized NFWs after four cycles and similar to 45% of the dye decolorization capacity was obtained at the end of the 4th cycle. These results are quite promising and therefore suggest that bacteria immobilized electrospun NFWs could be quite applicable for the decolorization of dyes in wastewater due to their versatility and reusability

Modulation of plant autophagy during pathogen attack

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