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Extracellular vesicles (EV) are membrane particles released by cells into their environment and are considered to be key players in intercellular communication. EV are produced by all domains of life but limited knowledge about EV in plants is available, although their implication in plant defense has been suggested. We have characterized sunflower EV and tested whether they could interact with fungal cells. EV were isolated from extracellular fluids of seedlings and characterized by transmission electron microscopy and proteomic analysis. These nanovesicles appeared to be enriched in cell wall remodeling enzymes and defense proteins. Membrane-labeled EV were prepared and their uptake by the phytopathogenic fungus Sclerotinia sclerotiorum was verified. Functional tests further evaluated the ability of EV to affect fungal growth. Spores treated with plant EV showed growth inhibition, morphological changes, and cell death. Conclusive evidence on the existence of plant EV is presented and we demonstrate their ability to interact with and kill fungal cells. Our results introduce the concept of cell-to-cell communication through EV in plants.

Plant primary microRNA (miRNA) transcripts (pri-miRNAs) are not just a source of miRNAs but can also encode regulatory peptides (miPEPs) that enhance the accumulation, and so the effect, of the corresponding mature miRNAs—an observation that may have agronomical applications. miRNA transcripts MicroRNAs (miRNAs) are known primarily for inhibiting the expression of their target genes at the level of the messenger RNA. They arise from processing of much larger primary transcripts (pri-miRNAs). Jean-Philippe Combier and colleagues provide data suggesting that pri-miRNAs — in plants at least — are not just a source of miRNAs but can also encode regulatory peptides (miPEPs). In a further twist, the miPEPs arising from pri-miRNAs seem to enhance the accumulation, and hence the effect, of the associated mature miRNAs. The authors demonstrate the role of two such peptides, miPEP171b and miPEP165a, in plant root development. They also identify an additional five active miPEPs, hinting at the generality of this phenomenon. These observations may have agronomical applications, as synthetic miPEP171b and miPEP165a generated by these researchers and introduced into plants followed the same molecular path and had similar effects on root development as their natural counterparts. MicroRNAs (miRNAs) are small regulatory RNA molecules that inhibit the expression of specific target genes by binding to and cleaving their messenger RNAs or otherwise inhibiting their translation into proteins 1 . miRNAs are transcribed as much larger primary transcripts (pri-miRNAs), the function of which is not fully understood. Here we show that plant pri-miRNAs contain short open reading frame sequences that encode regulatory peptides. The pri-miR171b of Medicago truncatula and the pri-miR165a of Arabidopsis thaliana produce peptides, which we term miPEP171b and miPEP165a, respectively, that enhance the accumulation of their corresponding mature miRNAs, resulting in downregulation of target genes involved in root development. The mechanism of miRNA-encoded peptide (miPEP) action involves increasing transcription of the pri-miRNA. Five other pri-miRNAs of A. thaliana and M. truncatula encode active miPEPs, suggesting that miPEPs are widespread throughout the plant kingdom. Synthetic miPEP171b and miPEP165a peptides applied to plants specifically trigger the accumulation of miR171b and miR165a, leading to reduction of lateral root development and stimulation of main root growth, respectively, suggesting that miPEPs might have agronomical applications.

Plant cell wall proteins (CWPs) play critical roles during plant development and in response to stresses. Proteomics has revealed their great diversity. With nearly 1000 identified CWPs, the Arabidopsis thaliana cell wall proteome is the best described to date and it covers the main plant organs and cell suspension cultures. Other monocot and dicot plants have been studied as well as bryophytes, such as Physcomitrella patens and Marchantia polymorpha. Although these proteomes were obtained using various flowcharts, they can be searched for the presence of members of a given protein family. Thereby, a core cell wall proteome which does not pretend to be exhaustive, yet could be defined. It comprises: (i) glycoside hydrolases and pectin methyl esterases, (ii) class III peroxidases, (iii) Asp, Ser and Cys proteases, (iv) non-specific lipid transfer proteins, (v) fasciclin arabinogalactan proteins, (vi) purple acid phosphatases and (vii) thaumatins. All the conserved CWP families could represent a set of house-keeping CWPs critical for either the maintenance of the basic cell wall functions, allowing immediate response to environmental stresses or both. Besides, the presence of non-canonical proteins devoid of a predicted signal peptide in cell wall proteomes is discussed in relation to the possible existence of alternative secretion pathways.

Calcium signals are crucial for the activation and coordination of signaling cascades leading to the establishment of plant defense mechanisms. Here, we studied the contribution of CML8, an Arabidopsis calmodulin-like protein in response to Ralstonia solanacearum and to pathogens with different lifestyles, such as Xanthomonas campestris pv. campestris and Phytophtora capsici. We used pathogenic infection assays, gene expression, RNA-seq approaches, and comparative analysis of public data on CML8 knockdown and overexpressing Arabidopsis lines to demonstrate that CML8 contributes to defense mechanisms against pathogenic bacteria and oomycetes. CML8 gene expression is finely regulated at the root level and manipulated during infection with Ralstonia, and CML8 overexpression confers better plant tolerance. To understand the processes controlled by CML8, genes differentially expressed at the root level in the first hours of infection have been identified. Overexpression of CML8 also confers better tolerance against Xanthomonas and Phytophtora, and most of the genes differentially expressed in response to Ralstonia are differentially expressed in these different pathosystems. Collectively, CML8 acts as a positive regulator against Ralstonia solanaceraum and against other vascular or root pathogens, suggesting that CML8 is a multifunctional protein that regulates common downstream processes involved in the defense response of plants to several pathogens.

Several examples previously established that the first ORF after the transcription start site corresponded to a translated ORF coding a miPEP able to increase pri-miRNA expression (Chen et al., 2020; Couzigou et al., 2016, 2017; Lauressergues et al., 2015; Sharma et al., 2020; Zhang et al., 2020). [...]we selected the first ORF of each identified pri-miRNA as a high confident candidate to produce functional miPEPs and synthesized the 87 miPEPs corresponding to the 87 conserved A. thaliana miRNAs, from miR156a to miR399f. [...]we validated that BvmiPEP164b and BomiPEP397a were able to increase the expression of their respective pri-miRNA while decreasing the expression of the corresponding target genes (Fig. 1l, m). While some countries try to strongly restrict the use of chemicals, more and more plants are starting to be resistant to these chemicals. Since miPEPs are very specific (Lauressergues et al., 2015), we can imagine the use of a cocktail of several peptides to improve the development of crops and their resistance to stresses (pathogens, starvation…) while reducing weed growth.

Glomeromycotina is a lineage of early diverging fungi that establish arbuscular mycorrhizal (AM) symbiosis with land plants. Despite their major ecological role, the genetic basis of their obligate mutualism remains largely unknown, hindering our understanding of their evolution and biology. We compared the genomes of Glomerales (Rhizophagus irregularis, Rhizophagus diaphanus, Rhizophagus cerebriforme) and Diversisporales (Gigaspora rosea) species, together with those of saprotrophic Mucoromycota, to identify gene families and processes associated with these lineages and to understand the molecular underpinning of their symbiotic lifestyle. Genomic features in Glomeromycotina appear to be very similar with a very high content in transposons and protein-coding genes, extensive duplications of protein kinase genes, and loss of genes coding for lignocellulose degradation, thiamin biosynthesis and cytosolic fatty acid synthase. Most symbiosis-related genes in R. irregularis and G. rosea are specific to Glomeromycotina. We also confirmed that the present species have a homokaryotic genome organisation. The high interspecific diversity of Glomeromycotina gene repertoires, affecting all known protein domains, as well as symbiosis-related orphan genes, may explain the known adaptation of Glomeromycotina to a wide range of environmental settings. Our findings contribute to an increasingly detailed portrait of genomic features defining the biology of AM fungi.

Eucalypts are the most planted hardwoods worldwide. The availability of the Eucalyptus grandis genome highlighted many genes awaiting functional characterization, lagging behind because of the lack of efficient genetic transformation protocols. In order to efficiently generate knock-out mutants to study the function of eucalypts genes, we implemented the powerful CRISPR/Cas9 gene editing technology with the hairy roots transformation system. As proofs-of-concept, we targeted two wood-related genes: Cinnamoyl-CoA Reductase1 (CCR1), a key lignin biosynthetic gene and IAA9A an auxin dependent transcription factor of Aux/IAA family. Almost all transgenic hairy roots were edited but the allele-editing rates and spectra varied greatly depending on the gene targeted. Most edition events generated truncated proteins, the prevalent edition types were small deletions but large deletions were also quite frequent. By using a combination of FT-IR spectroscopy and multivariate analysis (partial least square analysis (PLS-DA)), we showed that the CCR1-edited lines, which were clearly separated from the controls. The most discriminant wave-numbers were attributed to lignin. Histochemical analyses further confirmed the decreased lignification and the presence of collapsed vessels in CCR1-edited lines, which are characteristics of CCR1 deficiency. Although the efficiency of editing could be improved, the method described here is already a powerful tool to functionally characterize eucalypts genes for both basic research and industry purposes.

Background Recent genome-wide studies of many species reveal the existence of a myriad of RNAs differing in size, coding potential and function. Among these are the long non-coding RNAs, some of them producing functional small peptides via the translation of short ORFs. It now appears that any kind of RNA presumably has a potential to encode small peptides. Accordingly, our team recently discovered that plant primary transcripts of microRNAs ( pri-miRs ) produce small regulatory peptides (miPEPs) involved in auto-regulatory feedback loops enhancing their cognate microRNA expression which in turn controls plant development. Here we investigate whether this regulatory feedback loop is present in Drosophila melanogaster . Results We perform a survey of ribosome profiling data and reveal that many pri-miRNAs exhibit ribosome translation marks. Focusing on miR-8, we show that pri-miR-8 can produce a miPEP-8. Functional assays performed in Drosophila reveal that miPEP-8 affects development when overexpressed or knocked down. Combining genetic and molecular approaches as well as genome-wide transcriptomic analyses, we show that miR-8 expression is independent of miPEP-8 activity and that miPEP-8 acts in parallel to miR-8 to regulate the expression of hundreds of genes. Conclusion Taken together, these results reveal that several Drosophila pri-miRs exhibit translation potential. Contrasting with the mechanism described in plants, these data shed light on the function of yet undescribed primary-microRNA -encoded peptides in Drosophila and their regulatory potential on genome expression.

BACKGROUND: During the last fifteen years, cell wall proteomics has become a major research field with the publication of more than 50 articles describing plant cell wall proteomes. The WallProtDB database has been designed as a tool to facilitate the inventory, the interpretation of cell wall proteomics data and the comparisons between cell wall proteomes. RESULTS: WallProtDB (http://www.polebio.lrsv.ups-tlse.fr/WallProtDB/) presently contains 2170 proteins and ESTs identified experimentally in 36 cell wall proteomics studies performed on 11 different plant species. Two criteria have to be met for entering WallProtDB. First one is related to the identification of proteins. Only proteins identified in plant with available genomic or ESTs data are considered to ensure unambiguous identification. Second criterion is related to the difficulty to obtain clean cell wall fractions. Indeed, since cell walls constitute an open compartment difficult to isolate, numerous proteins predicted to be intracellular and/or having functions inside the cell have been identified in cell wall extracts. Then, except proteins predicted to be plasma membrane proteins, only proteins having a predicted signal peptide and no known intracellular retention signal are included in the database. In addition, WallProtDB contains information about the strategies used to obtain cell wall protein extracts and to identify proteins by mass spectrometry and bioinformatics. Mass spectrometry data are included when available. All the proteins of WallProtDB are linked to ProtAnnDB, another database, which contains structural and functional bioinformatics annotations of proteins as well as links to other databases (Aramemnon, CAZy, Planet, Phytozome). A list of references in the cell wall proteomics field is also provided. CONCLUSIONS: WallProtDB aims at becoming a cell wall proteome reference database. It can be updated at any time on request and provide a support for sharing cell wall proteomics data and literature references with researchers interested in plant cell wall biology.