Biology subjects

Sophien Kamoun

Publications and source records attributed to Sophien Kamoun.

12 recordsLinked to original sources

bioRxiv2016

Editing of the urease gene by CRISPR-Cas in the diatom Thalassiosira pseudonana

Background: CRISPR-Cas is a recent and powerful edition to the molecular toolbox which allows programmable genome editing. It has been used to modify genes in a wide variety of organisms, but only two alga to date. Here we present a methodology to edit the genome of T. pseudonana, a model centric diatom with both ecological significance and high biotechnological potential, using CRISPR-Cas.\n\nResults: A single construct wa assembled using Golden Gate cloning. Two sgRNAs were used to introduce a precise 37nt deletion early in the coding region of the urease gene. A high percentage of bi-allelic mutations ([≤] 61.5%) were observed in clones with the CRISPR-Cas construct. Growth of bi-allelic mutants in urea led to a significant reduction in growth rate and cell size compared to growth in nitrate.\n\nConclusions: CRISPR-Cas can precisely and efficiently edit the genome of T. pseudonana. The use of Golden Gate cloning to assemble CRISPR-Cas constructs gives additional flexibility to the CRISPR-Cas method and facilitates modifications to target alternative genes or species.

Genomics↗

bioRxiv2016

Emergence of wheat blast in Bangladesh was caused by a South American lineage of Magnaporthe oryzae

In February 2016, a new fungal disease was spotted in wheat fields across eight districts in Bangladesh. The epidemic spread to an estimated 15,741 hectares, about 16% of cultivated wheat area in Bangladesh, with yield losses reaching up to 100%. Within weeks of the onset of the epidemic, we performed transcriptome sequencing of symptomatic leaf samples collected directly from Bangladeshi fields. Population genomics analyses revealed that the outbreak was caused by a wheatLinfecting South American lineage of the blast fungus Magnaporthe oryzae. We show that genomic surveillance can be rapidly applied to monitor plant disease outbreaks and provide valuable information regarding the identity and origin of the infectious agent.

Plant Biology↗

bioRxiv2016

The potato NLR immune receptor R3a does not contain non-canonical integrated domains

A recent study by Kroj et al. (New Phytologist, 2016) surveyed nucleotide binding-leucine rich repeat (NLR) proteins from plant genomes for the presence of extraneous integrated domains that may serve as decoys or sensors for pathogen effectors. They reported that a FAM75 domain of unknown function occurs near the C-terminus of the potato late blight NLR protein R3a. Here, we investigated in detail the domain architecture of the R3a protein, its potato paralog R3b, and their tomato ortholog I2. We conclude that the R3a, R3b, and I2 proteins do not carry additional domains besides the classic NLR modules, and that the FAM75 domain match is likely a false positive among computationally predicted NLR-integrated domains.

Plant Biology↗

bioRxiv2016

Cell re-entry assays do not support models of pathogen- independent translocation of AvrM and AVR3a effectors into plant cells

The cell re-entry assay is widely used to evaluate pathogen effector protein uptake into plant cells. The assay is based on the premise that effector proteins secreted out of a leaf cell would translocate back into the cytosol of the same cell via a yet unknown host-derived uptake mechanism. Here, we critically assess this assay by expressing domains of the effector proteins AvrM-A of Melampsora lini and AVR3a of Phytophthora infestans fused to a signal peptide and fluorescent proteins in Nicotiana benthamiana. We found that the secreted fusion proteins do not re-enter plant cells from the apoplast and that the assay is prone to false-positives. We therefore emit a cautionary note on the use of the cell re-entry assay for protein trafficking studies.

Plant Biology↗

bioRxiv2015

Arabidopsis late blight: Infection of a nonhost plant by Albugo laibachii enables full colonization by Phytophthora infestans

The oomycete pathogen Phytophthora infestans causes potato late blight, and as a potato and tomato specialist pathogen, is seemingly poorly adapted to infect plants outside the Solanaceae. Here, we report the unexpected finding that P. infestans can infect Arabidopsis thaliana when another oomycete pathogen, Albugo laibachii, has colonized the host plant. The behaviour and speed of P. infestans infection in Arabidopsis pre-infected with A. laibachii resemble P. infestans infection of susceptible potato plants. Transcriptional profiling of P. infestans genes during infection revealed a significant overlap in the sets of secreted-protein genes that are induced in P. infestans upon colonisation of potato and susceptible Arabidopsis, suggesting major similarities in P. infestans gene expression dynamics on the two plant species. Furthermore, we found haustoria of A. laibachii and P. infestans within the same Arabidopsis cells. This Arabidopsis - A. laibachii - P. infestans tripartite interaction opens up various possibilities to dissect the molecular mechanisms of P. infestans infection and the processes occurring in co-infected Arabidopsis cells.

Plant Biology↗

bioRxiv2015

In planta expression screens of candidate effector proteins from the wheat yellow rust fungus reveal processing bodies as a pathogen-targeted plant cell compartment

Rust fungal pathogens of wheat (Triticum spp.) affect crop yields worldwide. The molecular mechanisms underlying the virulence of these pathogens remain elusive, due to the limited availability of suitable molecular genetic research tools. Notably, the inability to perform high-throughput analyses of candidate virulence proteins (also known as effectors) impairs progress. We previously established a pipeline for the fast-forward screens of rust fungal effectors in the model plant Nicotiana benthamiana. This pipeline involves selecting candidate effectors in silico and performing cell biology and protein-protein interaction assays in planta to gain insight into the putative functions of candidate effectors. In this study, we used this pipeline to identify and characterize sixteen candidate effectors from the wheat yellow rust fungal pathogen Puccinia striiformis f sp tritici. Nine candidate effectors targeted a specific plant subcellular compartment or protein complex, providing valuable information on their putative functions in plant cells. One candidate effector, PST02549, accumulated in processing bodies (P-bodies), protein complexes involved in mRNA decapping, degradation, and storage. PST02549 also associates with the P-body-resident ENHANCER OF mRNA DECAPPING PROTEIN 4 (EDC4) from N. benthamiana and wheat. Our work identifies P-bodies as a novel plant cell compartment targeted by pathogen effectors.

Plant Biology↗

bioRxiv2015

Tomato I2 immune receptor can be engineered to confer partial resistance to the oomycete Phytophthora infestans in addition to the fungus Fusarium oxysporum

Plants and animals rely on immune receptors, known as nucleotide-binding domain and leucine-rich repeat containing proteins (NB-LRR or NLR), 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 to 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 NLRs from other plant species.

Plant Biology↗

bioRxiv2015

The two-speed genomes of filamentous pathogens: waltz with plants

Fungi and oomycetes include deep and diverse lineages of eukaryotic plant pathogens. The last 10 years have seen the sequencing of the genomes of a multitude of species of these so-called filamentous plant pathogens. Already, fundamental concepts have emerged. Filamentous plant pathogen genomes tend to harbor large repertoires of genes encoding virulence effectors that modulate host plant processes. Effector genes are not randomly distributed across the genomes but tend to be associated with compartments enriched in repetitive sequences and transposable elements. These findings have led to the \"two-speed genome\" model in which filamentous pathogen genomes have a bipartite architecture with gene sparse, repeat rich compartments serving as a cradle for adaptive evolution. Here, we review this concept and discuss how plant pathogens are great model systems to study evolutionary adaptations at multiple time scales. We will also introduce the next phase of research on this topic.

Genomics↗

bioRxiv2015

RUST FUNGAL EFFECTORS MIMIC HOST TRANSIT PEPTIDES TO TRANSLOCATE INTO CHLOROPLASTS

Parasite effector proteins target various host cell compartments to alter host processes and promote infection. How effectors cross membrane-rich interfaces to reach these compartments is a major question in effector biology. Growing evidence suggests that effectors use molecular mimicry to subvert host cell machinery for protein sorting. We recently identified CTP1 (chloroplast-targeted protein 1), a candidate effector from the poplar leaf rust fungus Melampsora larici-populina that carries a predicted transit peptide and accumulates in chloroplasts. Here, we show that the CTP1 transit peptide is necessary and sufficient for accumulation in the stroma of chloroplasts, and is cleaved after translocation. CTP1 is part of a Melampsora-specific family of polymorphic secreted proteins whose members translocate and are processed in chloroplasts in a N-terminal signal-dependent manner. Our findings reveal that fungi have evolved effector proteins that mimic plant-specific sorting signals to traffic within plant cells.

Plant Biology↗

bioRxiv2015

The NLR helper protein NRC3 but not NRC1 is required for Pto-mediated cell death in Nicotiana benthamiana

Intracellular immune receptors of the nucleotide-binding leucine-rich repeat (NB-LRR or NLR) proteins often function in pairs, with \"helper\" proteins required for the activity of \"sensors\" that mediate pathogen recognition. The NLR helper NRC1 (NB-LRR protein required for HR-associated cell death 1) has been described as a signalling hub required for the cell death mediated by both cell surface and intracellular immune receptors in the model plant Nicotiana benthamiana. However, this work predates the availability of the N. benthamiana genome and whether NRC1 is indeed required for the reported phenotypes has not been confirmed. Here, we investigated the NRC family of solanaceous plants using a combination of genome annotation, phylogenetics, gene silencing and genetic complementation experiments. We discovered that a paralog of NRC1, we termed NRC3, is required for the hypersensitive cell death triggered by the disease resistance protein Pto but not Rx and Mi-1.2. NRC3 may also contribute to the hypersensitive cell death triggered by the receptor-like protein Cf-4. Our results highlight the importance of applying genetic complementation to validate gene function in RNA silencing experiments.

Plant Biology↗

bioRxiv2014

Phytophthora infestans RXLR-WY effector AVR3a associates with a Dynamin-Related Protein involved in endocytosis of a plant pattern recognition receptor

Perception of pathogen associated molecular patterns (PAMPs) by cell surface localized pattern recognition receptors (PPRs), activates plant basal defense responses in a process known as PAMP/PRR-triggered immunity (PTI). In turn, pathogens deploy effector proteins that interfere with different steps in PTI signaling. However, our knowledge of PTI suppression by filamentous plant pathogens, i.e. fungi and oomycetes, remains fragmentary. Previous work revealed that BAK1/SERK3, a regulatory receptor of several PRRs, contributes to basal immunity against the Irish potato famine pathogen Phytophthora infestans. Moreover BAK1/SERK3 is required for the cell death induced by P. infestans elicitin INF1, a protein with characteristics of PAMPs. The P. infestans host-translocated RXLR-WY effector AVR3a is known to supress INF1-mediated defense by binding the E3 ligase CMPG1. In contrast, AVR3aKl-Y147del, a deletion mutant of the C-terminal tyrosine of AVR3a, fails to bind CMPG1 and suppress INF1 cell death. Here we studied the extent to which AVR3a and its variants perturb additional BAK1/SERK3 dependent PTI responses using the plant PRR FLAGELLIN SENSING 2 (FLS2). We found that all tested variants of AVR3a, including AVR3aKl-Y147del, suppress early defense responses triggered by the bacterial flagellin-derived peptide flg22 and reduce internalization of activated FLS2 from the plasma membrane without disturbing its nonactivated localization. Consistent with this effect of AVR3a on FLS2 endocytosis, we discovered that AVR3a associates with the Dynamin-Related Protein DRP2, a plant GTPase implicated in receptor-mediated endocytosis. Interestingly, DRP2 is required for ligand-induced FLS2 internalization but does not affect internalization of the growth receptor BRASSINOSTEROID INSENSITIVE 1 (BRI1). Furthermore, overexpression of DRP2 suppressed accumulation of reactive oxygen species triggered by PAMP treatment. We conclude that AVR3a associates with a key cellular trafficking and membrane-remodeling complex involved in immune receptor-mediated endocytosis and signaling. AVR3a is a multifunctional effector that can suppress BAK1/SERK3 mediated immunity through at least two different pathways.\n\nAUTHOR SUMMARYPlants have a basal layer of immunity to mount defense responses against invading pathogens; in turn, pathogens deploy effector proteins to subvert plant immunity and manipulate host processes to enable parasitic infection. The Irish potato famine pathogen Phytophthora infestans has a large set of effectors that target multiple host cellular sites. The best-characterized P. infestans effector AVR3a supports enhanced infection and suppresses the cell death induced by the P. infestans protein INF1-elicitin. Previous work demonstrated that the plant immunity co-receptor BAK1/SERK3 contributes to basal immunity to P. infestans and that the RXLR-WY effector of P. infestans suppresses BAK1/SERK3-mediated immunity by binding the E3 ligase protein CMPG1. Here we show that AVR3a suppresses additional defense responses mediated by BAK1/SERK3 independently of CMPG1. AVR3a reduces the endocytosis of the plant receptor FLS2, which recognizes the flagellin-derived peptide flg22 in a BAK1/SERK3 dependent manner. Furthermore, we demonstrate that AVR3a associates with the Dynamin-Related Protein DRP2, a plant GTPase involved in receptor-mediated endocytosis that is required for FLS2 internalization. Our work revealed that AVR3a is a multifunctional effector that perturbs cellular trafficking initiated at the cell periphery by at least two mechanisms, and that this effector associates with a key cellular trafficking and membrane-remodeling complex involved in immune receptor-mediated endocytosis and signaling.

Plant Biology↗

bioRxiv2014

Crowdsourced analysis of ash and ash dieback through the Open Ash Dieback project: A year 1 report on datasets and analyses contributed by a self-organising community.

Ash dieback is a fungal disease of ash trees caused by Hymenoscyphus pseudoalbidus that has swept across Europe in the last two decades and is a significant threat to the ash population. This emergent pathogen has been relatively poorly studied and little is known about its genetic make-up. In response to the arrival of this dangerous pathogen in the UK we took the unusual step of providing an open access database and initial sequence datasets to the scientific community for analysis prior to performing an analysis of our own. Our goal was to crowdsource genomic and other analyses and create a community analysing this pathogen. In this report on the evolution of the community and data and analysis obtained in the first year of this activity, we describe the nature and the volume of the contributions and reveal some preliminary insights into the genome and biology of H. pseudoalbidus that emerged. In particular our nascent community generated a first-pass genome assembly containing abundant collapsed AT-rich repeats indicating a typically complex genome structure. Our open science and crowdsourcing effort has brought a wealth of new knowledge about this emergent pathogen within a short time-frame. Our community endeavour highlights the positive impact that open, collaborative approaches can have on fast, responsive modern science.

Genomics↗