Functional analysis of Arabidopsis immune-related MAPKs uncovers a role for MPK3 as negative regulator of inducible defences

Background : Mitogen-activated protein kinases (MAPKs) are key regulators of immune responses in animals and plants. In Arabidopsis, perception of microbe-associated molecular patterns (MAMPs) activates the MAPKs MPK3, MPK4 and MPK6. Increasing information depicts the molecular events activated by MAMPs in plants, but the specific and cooperative contributions of the MAPKs in these signalling events are largely unclear.[br/] Results: In this work, we analyse the behaviour of MPK3, MPK4 and MPK6 mutants in early and late immune responses triggered by the MAMP flg22 from bacterial flagellin. A genome-wide transcriptome analysis reveals that 36% of the flg22-upregulated genes and 68% of the flg22-downregulated genes are affected in at least one MAPK mutant. So far MPK4 was considered as a negative regulator of immunity, whereas MPK3 and MPK6 were believed to play partially redundant positive functions in defence.[br/] Our work reveals that MPK4 is required for the regulation of approximately 50% of flg22-induced genes and we identify a negative role for MPK3 in regulating defence gene expression, flg22-induced salicylic acid accumulation and disease resistance to Pseudomonas syringae. Among the MAPK-dependent genes, 27% of flg22-upregulated genes and 76% of flg22-downregulated genes require two or three MAPKs for their regulation. The flg22-induced MAPK activities are differentially regulated in MPK3 and MPK6 mutants, both in amplitude and duration, revealing a highly interdependent network.[br/] Conclusions : These data reveal a new set of distinct functions for MPK3, MPK4 and MPK6 and indicate that the plant immune signalling network is choreographed through the interplay of these three interwoven MAPK pathways

A high quality Arabidopsis transcriptome for accurate transcript-level analysis of alternative splicing

Alternative splicing generates multiple transcript and protein isoforms from the same gene and thus is important in gene expression regulation. To date, RNA-sequencing (RNA-seq) is the standard method for quantifying changes in alternative splicing on a genome-wide scale. Understanding the current limitations of RNA-seq is crucial for reliable analysis and the lack of high quality, comprehensive transcriptomes for most species, including model organisms such as Arabidopsis, is a major constraint in accurate quantification of transcript isoforms. To address this, we designed a novel pipeline with stringent filters and assembled a comprehensive Reference Transcript Dataset for Arabidopsis (AtRTD2) containing 82,190 non-redundant transcripts from 34 212 genes. Extensive experimental validation showed that AtRTD2 and its modified version, AtRTD2-QUASI, for use in Quantification of Alternatively Spliced Isoforms, outperform other available transcriptomes in RNA-seq analysis. This strategy can be implemented in other species to build a pipeline for transcript-level expression and alternative splicing analyses

The Tudor SND1 protein is an m6A RNA reader essential for replication of Kaposi’s sarcoma-associated herpesvirus

N6-methyladenosine (m6A) is the most abundant internal RNA modification of cellular mRNAs. m6A is recognised by YTH domain-containing proteins, which selectively bind to m6A-decorated RNAs regulating their turnover and translation. Using an m6A-modified hairpin present in the Kaposi’s sarcoma associated herpesvirus (KSHV) ORF50 RNA, we identified seven members from the ‘Royal family’ as putative m6A readers, including SND1. RIP-seq and eCLIP analysis characterised the SND1 binding profile transcriptome-wide, revealing SND1 as an m6A reader. We further demonstrate that the m6A modification of the ORF50 RNA is critical for SND1 binding, which in turn stabilises the ORF50 transcript. Importantly, SND1 depletion leads to inhibition of KSHV early gene expression showing that SND1 is essential for KSHV lytic replication. This work demonstrates that members of the ‘Royal family’ have m6A-reading ability, greatly increasing their epigenetic functions beyond protein methylation

Thousands of Rab GTPases for the Cell Biologist

Rab proteins are small GTPases that act as essential regulators of vesicular trafficking. 44 subfamilies are known in humans, performing specific sets of functions at distinct subcellular localisations and tissues. Rab function is conserved even amongst distant orthologs. Hence, the annotation of Rabs yields functional predictions about the cell biology of trafficking. So far, annotating Rabs has been a laborious manual task not feasible for current and future genomic output of deep sequencing technologies. We developed, validated and benchmarked the Rabifier, an automated bioinformatic pipeline for the identification and classification of Rabs, which achieves up to 90% classification accuracy. We cataloged roughly 8.000 Rabs from 247 genomes covering the entire eukaryotic tree. The full Rab database and a web tool implementing the pipeline are publicly available at www.RabDB.org. For the first time, we describe and analyse the evolution of Rabs in a dataset covering the whole eukaryotic phylogeny. We found a highly dynamic family undergoing frequent taxon-specific expansions and losses. We dated the origin of human subfamilies using phylogenetic profiling, which enlarged the Rab repertoire of the Last Eukaryotic Common Ancestor with Rab14, 32 and RabL4. Furthermore, a detailed analysis of the Choanoflagellate Monosiga brevicollis Rab family pinpointed the changes that accompanied the emergence of Metazoan multicellularity, mainly an important expansion and specialisation of the secretory pathway. Lastly, we experimentally establish tissue specificity in expression of mouse Rabs and show that neo-functionalisation best explains the emergence of new human Rab subfamilies. With the Rabifier and RabDB, we provide tools that easily allows non-bioinformaticians to integrate thousands of Rabs in their analyses. RabDB is designed to enable the cell biology community to keep pace with the increasing number of fully-sequenced genomes and change the scale at which we perform comparative analysis in cell biology

A conserved RxLR effector interacts with host RABA-type GTPases to inhibit vesicle-mediated secretion of antimicrobial proteins

Plant pathogens of the oomycete genus Phytophthora produce virulence factors, known as RxLR effector proteins that are transferred into host cells to suppress disease resistance. Here, we analyse the function of the highly conserved RxLR24 effector of Phytophthora brassicae. RxLR24 was expressed early in the interaction with Arabidopsis plants and ectopic expression in the host enhanced leaf colonization and zoosporangia formation. Co‐immunoprecipitation (Co‐IP) experiments followed by mass spectrometry identified different members of the RABA GTPase family as putative RxLR24 targets. Physical interaction of RxLR24 or its homologue from the potato pathogen Phytophthora infestans with different RABA GTPases of Arabidopsis or potato, respectively, was confirmed by reciprocal Co‐IP. In line with the function of RABA GTPases in vesicular secretion, RxLR24 co‐localized with RABA1a to vesicles and the plasma membrane. The effect of RxLR24 on the secretory process was analysed with fusion constructs of secreted antimicrobial proteins with a pH‐sensitive GFP tag. PATHOGENESIS RELATED PROTEIN 1 (PR‐1) and DEFENSIN (PDF1.2) were efficiently exported in control tissue, whereas in the presence of RxLR24 they both accumulated in the endoplasmic reticulum. Together our results imply a virulence function of RxLR24 effectors as inhibitors of RABA GTPase‐mediated vesicular secretion of antimicrobial PR‐1, PDF1.2 and possibly other defence‐related compounds

Plant‐parasitic nematode secreted peptides hijack a plant secretory pathway

International audienceSmall plant signalling peptides play key roles in plant development and in plant–microbe interactions. The plant CLAVATA3 (CLV3)/ENDOSPERM SURROUNDING REGION (ESR) (CLE) peptides have been shown to orchestrate shoot meristem differentiation and to be involved in root growth and vascular development (Yamaguchi et al., 2016). CLE peptides have also been implicated in symbiosis and parasitism. Intriguingly, plant-parasitic nematodes (PPNs) (Mitchum et al., 2012; Gheysen & Mitchum, 2019), endomycorrhizal fungi (Le Marquer et al., 2019), and other fungi and bacteria (Ronald & Joe, 2018) have been shown to secrete molecules mimicking plant peptides within plant roots, to promote infection. The soybean cyst nematode (CN) Heterodera glycines produces CLE propeptides that are delivered to the cytoplasm of plant cells, but ultimately function in the apoplast, consistent with their proposed role as ligand mimics of plant CLE peptides (Wang et al., 2010). A cryptic endoplasmic reticulum (ER) translocation signal has been identified in the variable domain (VD) of HgCLE2, but the precise translocation motif responsible for directing the CLE peptide to the apoplast has not been deciphered (Wang et al., 2010). In this issue of New Phytologist, Wang et al. (2021; pp. 563–574) present very interesting new data explaining how PPN peptide effectors exploit a conserved host post-translational mechanism of trafficking through the ER to the apoplast, using a specific translocation signal: the variable Domain I translocation signal (VDIT) (Fig. 1a). This discovery reminds us that studies of the molecular dialogue between plants and micro- or macro-organisms may improve our understanding of fundamental plant cellular processes, in this case, the protein secretion system. Wang et al. (2021) obtained functional evidence, by assessing the secretion of CLV3 or avirulence AVR9 peptides fused to different full-length or truncated versions of nematode CLEs, to identify the minimal motif required for the ER translocation and secretion of the injected propeptide. They then used an elegant split-GFP approach to validate the passage of the effector from the cytoplasm to the ER, Golgi apparatus and, finally, the extracellular space

Mécanismes moléculaires de tolérance au stress (étude fonctionnelle chez arabidopsis de Tudor-SN, une protéine conservée dans l évolution impliquée dans le métabolisme des ARNs)

Pour s adapter aux contraintes environnementales, les plantes modulent continuellement leur croissance et leur différenciation. Au niveau des racines, il est intéressant d étudier ces mécanismes dans les apex puisque les activités d expansion et de division cellulaire s y concentrent. Dans une approche visant à isoler des transcrits abondants dans des apex racinaires adaptés au stress hydrique, un transcrit codant une protéine conservée dans l évolution, Tudor-Sn (TSN), a été isolé. TSN est composée de quatre domaines Staphylococcus Nucléase, dont l activité nucléolytique est dépendante du calcium, et d un domaine TUDOR d interaction protéine-protéine. Chez les animaux, TSN a été copurifiée dans le RNA Induced Silencing Complex (RISC) et plus récemment proposée pour dégrader des ARN doubles brins potentiellement précurseurs de miARNs. Cependant, aucun phénotype d un mutant tsn n a été décrit à ce jour pour tester ces hypothèses. Lors de ce travail de thèse, nous avons pu observer chez Arabidopsis que TSN est présente dans tous les organes et qu elle accumule préférentiellement dans les cellules de la racine initiant leur élongation. A l aide d immunolocalisations et de fusions traductionnelles avec la GFP, nous avons localisé TSN dans le cytosol, répartie de manière granuleuse. Afin de déterminer sa fonction, un mutant perte de fonction pour les deux gènes TSN1 et TSN2 a été généré par croisement de lignées d intersections ADN-T. Le double mutant TSN1-1 TSN2-1 présente in vitro un défaut de croissance racinaire lié à un défaut d expansion cellulaire, une hypersensiblilité au stress salin ainsi que des phénotypes d hypersensibilités aux stress dans la plante adulte. Ces observations ont pu être validées par complémentation avec les loci génomiques de TSN1 ou TSN2. De plus, aucun phénotype morphologique ou moléculaire correspondant à la perte d activité RISC n a été détecté chez TSN1-1 TSN2-1. Finalement, une analyse transcriptomique a révélé une sous-représentation de transcrits codant presque exclusivement des protéines destinées à être secrétées. Nous avons commencé à étudier la stabililté et la régulation de ces transcrits, qui représentent de nouveaux outils pour étudier le rôle de TSN dans le métabolisme des ARNs chez les eucaryotes.To adapt to their environmental conditions, plants are continuously modulating their growth and differentiation. At the root system level, root apices are ideal to dissect these mechanisms because they host most of cell expansion and cell division activities. In a strategy to identify abundant mRNAs in stress-adapted root tips, a transcript encoding an evolutionary conserved protein, Tudor- SN (TSN), was isolated. TSN is composed of four calcium-dependant Staphylococcus Nuclease domains, and by a protein-protein interaction domain, the TUDOR domain. In animals, TSN was copurified with the RNA Induced Silencing Complex (RISC) and more recently proposed to degrade double stranded RNAs able to act as miRNAs precusors. Nevertheless, no TSN mutant phenotype has been described yet to test these hypotheses. IN this PhD work, we detected TSN protein in all organs of Arabidopsis plants, accumulating preferentially in elongating roots cells. Using immunolocalization and GFP fusions, we localized TSN in the cytosol as a granular signal. To determine TSN functions, a tsn null mutant was generated by crossing T-DNA insertion lines mutated in either TSN1 or TSN2. In vitro, the TSN-1 TSN2-1 double mutant has a root growth-retardation phenotype associated with a defect in cell elongation and a hypersensitivity to salt stress. Stress-sensitivity was also present in soil-grown mature plants. These observations were validated by complementation with the TSN1 or TSN2 genomic loci. Moreover, no morphological nor molecular phenotypes related to RISC dysfunction were observed in tsn1-1 tsn 2-1. Finally, a transcriptomic approach revealed an under-representation of mRNAs encoding almost exclusively for secreted proteins. We have begun studying the stability and the regulation of these transcripts which represent new tools to study the role TSN in RNA metabolism in eukaryotes.ORSAY-PARIS 11-BU Sciences (914712101) / SudocSudocFranceF

Arbuscular mycorrhizal fungi possess a CLAVATA3/embryo surrounding region‐related gene that positively regulates symbiosis

International audienceThe arbuscular mycorrhizal (AM) symbiosis is a beneficial association established between land plants and the members of a subphylum of fungi, the Glomeromycotina. How the two symbiotic partners regulate their association is still enigmatic. Secreted fungal peptides are candidates for regulating this interaction.We searched for fungal peptides with similarities with known plant signalling peptides.We identified CLAVATA (CLV)/EMBRYO SURROUNDING REGION (ESR)-RELATED PROTEIN (CLE) genes in phylogenetically distant AM fungi: four Rhizophagus species and one Gigaspora species. These CLE genes encode a signal peptide for secretion and the conserved CLE C-terminal motif. They seem to be absent in the other fungal clades. Rhizophagus irregularis and Gigaspora rosea CLE genes (RiCLE1 and GrCLE1) are transcriptionally induced in symbiotic vs asymbiotic conditions. Exogenous application of synthetic RiCLE1 peptide on Medicago truncatula affects root architecture, by slowing the apical growth of primary roots and stimulating the formation of lateral roots. In addition, pretreatment of seedlings with RiCLE1 peptide stimulates mycorrhization.Our findings demonstrate for the first time that in addition to plants and nematodes, AM fungi also possess CLE genes. These results pave the way for deciphering new mechanisms by which AM fungi modulate plant cellular responses during the establishment of AM symbiosis