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Species tree inference from gene family trees is becoming increasingly popular because it can account for discordance between the species tree and the corresponding gene family trees. In particular, methods that can account for multiple-copy gene families exhibit potential to leverage paralogy as informative signal. At present, there does not exist any widely adopted inference method for this purpose. Here, we present SpeciesRax, the first maximum likelihood method that can infer a rooted species tree from a set of gene family trees and can account for gene duplication, loss, and transfer events. By explicitly modeling events by which gene trees can depart from the species tree, SpeciesRax leverages the phylogenetic rooting signal in gene trees. SpeciesRax infers species tree branch lengths in units of expected substitutions per site and branch support values via paralogy-aware quartets extracted from the gene family trees. Using both empirical and simulated data sets we show that SpeciesRax is at least as accurate as the best competing methods while being one order of magnitude faster on large data sets at the same time. We used SpeciesRax to infer a biologically plausible rooted phylogeny of the vertebrates comprising 188 species from 31,612 gene families in 1 h using 40 cores. SpeciesRax is available under GNU GPL at https://github.com/BenoitMorel/GeneRax and on BioConda.

Susceptibility to fungal pathogens and plant immunity in rice and wheat can be modulated by the presence of intraspecific neighbours and does not require the neighbours to be infected., As part of a trend towards diversifying cultivated areas, varietal mixtures are subject to renewed interest as a means to manage diseases. Besides the epidemiological effects of varietal mixtures on pathogen propagation, little is known about the effect of intraspecific plant–plant interactions and their impact on responses to disease. In this study, genotypes of rice (Oryza sativa) or durum wheat (Triticum turgidum) were grown with different conspecific neighbours and manually inoculated under conditions preventing pathogen propagation. Disease susceptibility was measured together with the expression of basal immunity genes as part of the response to intra-specific neighbours. The results showed that in many cases for both rice and wheat susceptibility to pathogens and immunity was modified by the presence of intraspecific neighbours. This phenomenon, which we term ‘neighbour-modulated susceptibility’ (NMS), could be caused by the production of below-ground signals and does not require the neighbours to be infected. Our results suggest that the mechanisms responsible for reducing disease in varietal mixtures in the field need to be re-examined.

Next generation sequencing (NGS) technologies have led to a ubiquity of molecular sequence data. This data avalanche is particularly challenging in metagenetics, which focuses on taxonomic identification of sequences obtained from diverse microbial environments. Phylogenetic placement methods determine how these sequences fit into an evolutionary context. Previous implementations of phylogenetic placement algorithms, such as the evolutionary placement algorithm (EPA) included in RAxML, or PPLACER, are being increasingly used for this purpose. However, due to the steady progress in NGS technologies, the current implementations face substantial scalability limitations. Herein, we present EPA-NG, a complete reimplementation of the EPA that is substantially faster, offers a distributed memory parallelization, and integrates concepts from both, RAxML-EPA and PPLACER. EPA-NG can be executed on standard shared memory, as well as on distributed memory systems (e.g., computing clusters). To demonstrate the scalability of EPA-NG, we placed \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{upgreek} \usepackage{mathrsfs} \setlength{\oddsidemargin}{-69pt} \begin{document} }{}$1$\end{document} billion metagenetic reads from the Tara Oceans Project onto a reference tree with 3748 taxa in just under \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{upgreek} \usepackage{mathrsfs} \setlength{\oddsidemargin}{-69pt} \begin{document} }{}$7$\end{document} h, using 2048 cores. Our performance assessment shows that EPA-NG outperforms RAxML-EPA and PPLACER by up to a factor of \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{upgreek} \usepackage{mathrsfs} \setlength{\oddsidemargin}{-69pt} \begin{document} }{}$30$\end{document} in sequential execution mode, while attaining comparable parallel efficiency on shared memory systems. We further show that the distributed memory parallelization of EPA-NG scales well up to 2048 cores. EPA-NG is available under the AGPLv3 license: https://github.com/Pbdas/epa-ng.

BGPI : Equipe 4 : Interactions Céréales Agents Pathogènes (ICAP); Few resistance genes providing defence against the major fungal diseases septoria tritici blotch (STB), septoria nodorum blotch, leaf rust (LR), and an emerging wheat blast disease have been identified in durum wheat. We identified sixteen fungal disease-associated QTL through genome-wide association mapping of 180 inbred lines sampled from a durum wheat Composite Cross-population. Two STB resistance-associated QTL mapped to chromosome 3A, one of which colocalizes with Stb6, a known resistance gene previously identified in bread wheat. This partial resistance could be conferred by a new allele of Stb6 or another paralogous gene. The second locus is associated with a reduction in pycnidia density, a recently identified and poorly understood form of resistance. A resistance QTL strongly associated with LR, and colocalizing with Lr61, was observed in a 3.24 Mbp region on chromosome 6B. QTL mapping of LR resistance following treatment by chitin used in the context of inducer treatment was also investigated. Using a combination of resistance alleles at these loci could confer durable resistance to multiple fungal diseases and aid durum wheat breeders in their fight against these fungal pathogens.

ModelTest-NG is a reimplementation from scratch of jModelTest and ProtTest, two popular tools for selecting the best-fit nucleotide and amino acid substitution models, respectively. ModelTest-NG is one to two orders of magnitude faster than jModelTest and ProtTest but equally accurate and introduces several new features, such as ascertainment bias correction, mixture, and free-rate models, or the automatic processing of single partitions. ModelTest-NG is available under a GNU GPL3 license at https://github.com/ddarriba/modeltest , last accessed September 2, 2019. Ministerio de Economía, Industria y Competitividad (España) | Ref. TIN2016-75845-P Xunta de Galicia | Ref. ED431C 2017/04 Klaus Tschira Foundation and DFG | Ref. STA-860/6

BGPI : équipe 4 Interactions Céréales Agents Pathogènes (ICAP); International audience; Despite the importance of plant-plant interactions on crop yield and plant community dynamics, our understanding of the genetic and molecular bases underlying natural variation of plant-plant interactions is largely limited in comparison with other types of biotic interactions. By listing 63 quantitative trait loci (QTL) mapping and global gene expression studies based on plants directly challenged by other plants, we explored whether the genetic architecture and the function of the candidate genes underlying natural plant-plant interactions depend on the type of interactions between two plants (competition versus commensalism versus reciprocal helping versus asymmetry). The 16 transcriptomic studies are unevenly distributed between competitive interactions (n = 12) and asymmetric interactions (n = 4, all focusing on response to parasitic plants). By contrast, 17 and 30 QTL studies were identified for competitive interactions and asymmetric interactions (either weed suppressive ability or response to parasitic plants), respectively. Surprisingly, no studies have been carried out on the identification of genetic and molecular bases underlying natural variation in positive interactions. The candidate genes underlying natural plant-plant interactions can be classified into seven categories of plant function that have been identified in artificial environments simulating plant-plant interactions either frequently (photosynthesis, hormones), only recently (cell wall modification and degradation, defense pathways against pathogens) or rarely (ABC transporters, histone modification and meristem identity/life history traits). Finally, we introduce several avenues that need to be explored in the future to obtain a thorough understanding of the genetic and molecular bases underlying plant-plant interactions within the context of realistic community complexity.

BGPI : équipe 4; International audience; Plants are constantly exposed to a variety of biotic and abiotic stresses that reduce their fitness and performance. At the molecular level, the perception of extracellular stimuli and the subsequent activation of defense responses require a complex interplay of signaling cascades, in which protein phosphorylation plays a central role. Several studies have shown that some members of the Leucine-Rich Repeat Receptor-Like Kinase (LRR-RLK) family are involved in stress and developmental pathways. We report here a systematic analysis of the role of the members of this gene family by mutant phenotyping in the monocotyledon model plant rice, Oryza sativa. We have then targeted 176 of the ∼320 LRR-RLK genes (55.7%) and genotyped 288 mutant lines. Position of the insertion was confirmed in 128 lines corresponding to 100 LRR-RLK genes (31.6% of the entire family). All mutant lines harboring homozygous insertions have been screened for phenotypes under normal conditions and under various abiotic stresses. Mutant plants have been observed at several stages of growth, from seedlings in Petri dishes to flowering and grain filling under greenhouse conditions. Our results show that 37 of the LRR-RLK rice genes are potential targets for improvement especially in the generation of abiotic stress tolerant cereals.

BGPI : équipe 4; Plants are often facing several stresses simultaneously. Understanding how they reactand the way pathogens adapt to such combinational stresses is poorly documented.Here, we developed an experimental system mimicking field intermittent drought onrice followed by inoculation by the pathogenic fungus Magnaporthe oryzae. Thisexperimental system triggers an enhancement of susceptibility that could be correlatedwith the dampening of several aspects of plant immunity, namely the oxidative burst andthe transcription of several pathogenesis-related genes. Quite strikingly, the analysisof fungal transcription by RNASeq analysis under drought reveals that the fungus isgreatly modifying its virulence program: genes coding for small secreted proteins weremassively repressed in droughted plants compared to unstressed ones whereas genescoding for enzymes involved in degradation of cell-wall were induced. We also showthat drought can lead to the partial breakdown of several major resistance genes byaffecting R plant gene and/or pathogen effector expression.We propose a model wherea yet unknown plant signal can trigger a change in the virulence program of the pathogento adapt to a plant host that was affected by drought prior to infection.

BGPI : équipe 4; International audience; Receptor-like kinases (RLKs) play key roles in disease resistance, in particular basal immunity. They recognize patterns produced by the pathogen invasion and often work as complexes in the plasma membrane. Among these RLKs, there is increasing evidence in several plant species of the key role of Wall-associated kinases (WAKs) in disease resistance. We recently showed using rice (Oryza sativa) loss-of-function mutants of three transcriptionaly co-regulated OsWAK genes that individual OsWAKs are positively required for quantitative resistance to the rice blast fungus, Magnaporthe oryzae. This finding was unexpected since WAK genes belong to large gene families where functional redundancy is expected. Here we provide evidence that this may be due to complex physical interaction between OsWAK proteins.

Elevated constitutive expression of components of the defence arsenal is associated with quantitative resistance to the rice blast fungus, a phenomenon called preformed defence. While the role of many disease regulators in inducible defence systems has been extensively studied, little attention has been paid so far to genes that regulate preformed defence. In this study, we show by microarray analysis across rice diversity that the preformed defence phenomenon impacts on a large number of defence-related genes without apparently affecting other biological processes. Using a guilt-by-association strategy, we identified two positive regulators that promote constitutive expression of known defence markers and partial resistance to rice blast. The HSF23 gene encodes for a putative member of the heat shock transcription factor family, while CaMBP encodes for a putative Calmodulin-binding protein. Both HSF23 and CaMBP strongly affect preformed defence and also plant growth. Additionally, we identified the OB-fold gene as a negative regulator of blast resistance, which could be involved in RNA stabilization. The OB-fold mutants do not suffer from obvious developmental defects. Taken together, our results prove that our strategy of combining analysis of gene expression diversity with guilt-by-association is a powerful way to identify disease resistance regulators in rice.

With the aim of identifying novel regulators of host and nonhost resistance to fungi in rice, we carried out a systematic mutant screen of mutagenized lines. Two mutant wrky22 knockout lines revealed clear-cut enhanced susceptibility to both virulent and avirulent Magnaporthe oryzae strains and altered cellular responses to nonhost Magnaporthe grisea and Blumeria graminis fungi. In addition, the analysis of the pathogen responses of 24 overexpressor OsWRKY22 lines revealed enhanced resistance phenotypes on infection with virulent M. oryzae strain, confirming that OsWRKY22 is involved in rice resistance to blast. Bioinformatic analyses determined that the OsWRKY22 gene belongs to a well-defined cluster of monocot-specific WRKYs. The co-regulatory analysis revealed no significant co-regulation of OsWRKY22 with a representative panel of OsWRKYs, supporting its unique role in a series of transcriptional responses. In contrast, inquiring a subset of biotic stress-related Affymetrix data, a large number of resistance and defence-related genes were found to be putatively co-expressed with OsWRKY22. Taken together, all gathered experimental evidence places the monocot-specific OsWRKY22 gene at the convergence point of signal transduction circuits in response to both host and nonhost fungi encountering rice plants.

We report here the molecular and phenotypic features of a library of 31,562 insertion lines generated in the model japonica cultivar Nipponbare of rice (Oryza sativa L.), called Oryza Tag Line (OTL). Sixteen thousand eight hundred and fourteen T-DNA and 12,410 Tos17 discrete insertion sites have been characterized in these lines. We estimate that 8686 predicted gene intervals--i.e. one-fourth to one-fifth of the estimated rice nontransposable element gene complement--are interrupted by sequence-indexed T-DNA (6563 genes) and/or Tos17 (2755 genes) inserts. Six hundred and forty-three genes are interrupted by both T-DNA and Tos17 inserts. High quality of the sequence indexation of the T2 seed samples was ascertained by several approaches. Field evaluation under agronomic conditions of 27,832 OTL has revealed that 18.2% exhibit at least one morphophysiological alteration in the T1 progeny plants. Screening 10,000 lines for altered response to inoculation by the fungal pathogen Magnaporthe oryzae allowed to observe 71 lines (0.7%) developing spontaneous lesions simulating disease mutants and 43 lines (0.4%) exhibiting an enhanced disease resistance or susceptibility. We show here that at least 3.5% (four of 114) of these alterations are tagged by the mutagens. The presence of allelic series of sequence-indexed mutations in a gene among OTL that exhibit a convergent phenotype clearly increases the chance of establishing a linkage between alterations and inserts. This convergence approach is illustrated by the identification of the rice ortholog of AtPHO2, the disruption of which causes a lesion-mimic phenotype owing to an over-accumulation of phosphate, in nine lines bearing allelic insertions.

In Arabidopsis, gene expression studies and analysis of knock-out (KO) mutants have been instrumental in building an integrated view of disease resistance pathways. Such an integrated view is missing in rice where shared tools, including genes and mutants, must be assembled. This work provides a tool kit consisting of informative genes for the molecular characterization of the interaction of rice with the major fungal pathogen Magnaporthe oryzae. It also provides for a set of eight KO mutants, all in the same genotypic background, in genes involved in key steps of the rice disease resistance pathway. This study demonstrates the involvement of three genes, OsWRKY28, rTGA2.1 and NH1, in the establishment of full basal resistance to rice blast. The transcription factor OsWRKY28 acts as a negative regulator of basal resistance, like the orthologous barley gene. Finally, the up-regulation of the negative regulator OsWRKY28 and the down-regulation of PR gene expression early during M. oryzae infection suggest that the fungus possesses infection mechanisms that enable it to block host defences.

UMR BGPI Equipe 4; iseases caused by fungal and bacterial pathogens like Magnaporthe oryzae and Xanthomonas oryzae pv. oryzae are responsible for considerable yield loss. Up to now, in rice, the modification of the expression of more than 60 genes from diverse origins has shown beneficial effects with respect to disease resistance. In this paper, we review this large set of data to identify the best genes and strategies to achieve disease resistance by transgenic approaches. Altered expression of genes involved in signal transduction and transcription may lead to many unwanted side effects, like lesion mimic phenotypes. Moreover, modification of resistance to abiotic stress has been neglected and should be carefully examined in the future. Genes like resistance genes and pathogenesis-related genes can confer broad spectrum and high levels of resistance to several pathogens. Preformed expression of defense is often observed but does not necessarily lead to detrimental effects. Although examples of gene pyramiding are scarce, they suggest that this is a very promising strategy. More field evaluation of the transgenic plants is required to draw final conclusions on the usefulness of these genes for improving disease resistance.

BGPI : équipe 4; International audience; The cuticle covers the surface of the polysaccharide cell wall of leaf epidermal cells and forms an essential diffusion barrier between plant and environment. Homologs of the ATP-binding cassette (ABC) transporter AtABCG32/HvABCG31 clade are necessary for the formation of a functional cuticle in both monocots and dicots. Here we characterize the osabcg31 knockout mutant and hairpin RNA interference (RNAi)-down-regulated OsABCG31 plant lines having reduced plant growth and a permeable cuticle. The reduced content of cutin in leaves and structural alterations in the cuticle and at the cuticle-cell wall interface in plants compromised in OsABCG31 expression explain the cuticle permeability. Effects of modifications of the cuticle on plant-microbe interactions were evaluated. The cuticular alterations in OsABCG31-compromised plants did not cause deficiencies in germination of the spores or the formation of appressoria of Magnaporthe oryzae on the leaf surface, but a strong reduction of infection structures inside the plant. Genes involved in pathogen resistance were constitutively up-regulated in OsABCG31-compromised plants, thus being a possible cause of the resistance to M. oryzae and the dwarf growth phenotype. The findings show that in rice an abnormal cuticle formation may affect the signaling of plant growth and defense.

BGPI : équipe 4; International audience; Plant immune receptors of the class of nucleotide-binding and leucine-rich repeat domain (NLR) proteins can contain additional domains besides canonical NB-ARC (nucleotide-binding adaptor shared by APAF-1, R proteins, and CED-4 (NB-ARC)) and leucine-rich repeat (LRR) domains. Recent research suggests that these additional domains act as integrated decoys recognizing effectors from pathogens. Proteins homologous to integrated decoys are suspected to be effector targets and involved in disease or resistance. Here, we scrutinized 31 entire plant genomes to identify putative integrated decoy domains in NLR proteins using the Interpro search. The involvement of the Zinc Finger-BED type (ZBED) protein containing a putative decoy domain, called BED, in rice (Oryza sativa) resistance was investigated by evaluating susceptibility to the blast fungus Magnaporthe oryzae in rice over-expression and knock-out mutants. This analysis showed that all plants tested had integrated various atypical protein domains into their NLR proteins (on average 3.5% of all NLR proteins). We also demonstrated that modifying the expression of the ZBED gene modified disease susceptibility. This study suggests that integration of decoy domains in NLR immune receptors is widespread and frequent in plants. The integrated decoy model is therefore a powerful concept to identify new proteins involved in disease resistance. Further in-depth examination of additional domains in NLR proteins promises to unravel many new proteins of the plant immune system.

International audience; Our view of genes involved in rice disease resistance is far from complete. Here we used a gene-for-gene relationship corresponding to the interaction between atypical avirulence gene ACE1 from Magnaporthe grisea and rice resistance gene Pi33 to better characterize early rice defence responses induced during such interaction. Rice genes differentially expressed during early stages of Pi33/ACE1 interaction were identified using DNA chip-based differential hybridization and QRT-PCR survey of the expression of known and putative regulators of disease resistance. One hundred genes were identified as induced or repressed during rice defence response, 80% of which are novel, including resistance gene analogues. Pi33/ACE1 interaction also triggered the up-regulation of classical PR defence genes and a massive down-regulation of chlorophyll a/b binding genes. Most of these differentially expressed genes were induced or repressed earlier in Pi33/ACE1 interaction than in the gene-for-gene interaction involving Nipponbare resistant cultivar. Besides demonstrating that an ACE1/Pi33 interaction induced classical and specific expression patterns, this work provides a list of new genes likely to be involved in rice disease resistance.

Universite de Montpellier, Unite Mixte de Recherche Diversite, Adaptation, et Developpement des Plantes, 34095 Montpellier cedex 5, France (G.N.K., I.B., P.G.); Centre de Cooperation Internationale en Recherche Agronomique pour le Developpement, Unite Mixte de Recherche Amelioration Genetique et Adaptation des Plantes Mediterraneennes et Tropicales, 34398 Montpellier cedex 5, France (G.N.K., P.K.P., F.R., M.B., D.M., E.G.); Department of Biotechnology, Guru Nanak Dev University, Amritsar 143 005, India (P.K.P.); Department of Plant Systems Biology, Flanders Institute for Biotechnology, 9052 Ghent, Belgium (B.P., T.B.); Department of Plant Biotechnology and Bioinformatics, Ghent University, 9052 Ghent, Belgium (B.P., T.B.); Institut National de la Recherche Agronomique, Unite Mixte de Recherche Biologie et Genetique des Interactions Plante-Parasite, 34398 Montpellier, France (P.B., J.-B.M.); Laboratoire Mixte International Rice Functional Genomics and Plant Biotechnology, Institut de Recherche pour le Developpement, University of Science and Technology of Hanoi, Agricultural Genetics Institute, 00 000 Hanoi, Vietnam (C.D.M., V.N.D., P.G.); International Center for Tropical Agriculture, 6713 Cali, Colombia (M.G.S., I.M.); Consiglio Nazionale delle Ricerche, Institute of Agricultural Biology and Biotechnology, 20133 Milan, Italy (A.-M.G.);and Institut de Recherche pour le Developpement, Unite Mixte de Recherche Interactions Plantes Microorganismes et Environnement, 34398 Montpellier cedex, France (C.B.)

International audience; Second-site mutagenesis was performed on the argonaute1-33 (ago1-33) hypomorphic mutant, which exhibits reduced sense transgene posttranscriptional gene silencing (S-PTGS). Mutations in FIERY1, a positive regulator of the cytoplasmic 5'-to-3' EXORIBONUCLEASE4 (XRN4), and in SUPERKILLER3 (SKI3), a member of the SKI complex that threads RNAs directly to the 3'-to-5' exoribonuclease of the cytoplasmic exosome, compensated AGO1 partial deficiency and restored S-PTGS with 100% efficiency. Moreover, xrn4 and ski3 single mutations provoked the entry of nonsilenced transgenes into S-PTGS and enhanced S-PTGS on partially silenced transgenes, indicating that cytoplasmic 5'-to-3' and 3'-to-5' RNA degradation generally counteract S-PTGS, likely by reducing the amount of transgene aberrant RNAs that are used by the S-PTGS pathway to build up small interfering RNAs that guide transgene RNA cleavage by AGO1. Constructs generating improperly terminated transgene messenger RNAs (mRNAs) were not more sensitive to ski3 or xrn4 than regular constructs, suggesting that improperly terminated transgene mRNAs not only are degraded from both the 3' end but also from the 5' end, likely after decapping. The facts that impairment of either 5'-to-3' or 3'-to-5' RNA degradation is sufficient to provoke the entry of transgene RNA into the S-PTGS pathway, whereas simultaneous impairment of both pathways is necessary to provoke the entry of endogenous mRNA into the S-PTGS pathway, suggest poor RNA quality upon the transcription of transgenes integrated at random genomic locations.

Transgene-induced post-transcriptional gene silencing (PTGS) results from specific degradation of RNAs that are homologous with the transgene transcribed sequence. This phenomenon, also known as cosuppression in plants and quelling in fungi, resembles RNA interference (RNAi) in animals. Indeed, cosuppression/quelling/RNAi require related PAZ/PIWI proteins (AGO1/QDE-2/RDE-1), indicating that these mechanisms are related. Unlike Neurospora crassa qde-2 and Caenorhabditis elegans rde-1 mutants, which are morphologically normal, the 24 known Arabidopsis ago1 mutants display severe developmental abnormalities and are sterile. Here, we report the isolation of hypomorphic ago1 mutants, including fertile ones. We show that these hypomorphic ago1 mutants are defective for PTGS, like null sgs2, sgs3, and ago1 mutants, suggesting that PTGS is more sensitive than development to perturbations in AGO1. Conversely, a mutation in ZWILLE/PINHEAD, another member of the Arabidopsis AGO1 gene family, affects development but not PTGS. Similarly, mutations in ALG-1 and ALG-2, two members of the C. elegans RDE-1 gene family, affect development but not RNAi, indicating that the control of PTGS/RNAi and development by PAZ/PIWI proteins can be uncoupled. Finally, we show that hypomorphic ago1 mutants are hypersensitive to virus infection, confirming the hypothesis that in plants PTGS is a mechanism of defense against viruses.

Introduction of transgene DNA may lead to specific degradation of RNAs that are homologous to the transgene transcribed sequence through phenomena named post-transcriptional gene silencing (PTGS) in plants, quelling in fungi, and RNA interference (RNAi) in animals. It was shown previously that PTGS, quelling, and RNAi require a set of related proteins (SGS2, QDE-1, and EGO-1, respectively). Here we report the isolation of Arabidopsis mutants impaired in PTGS which are affected at the Argonaute1 ( AGO1 ) locus. AGO1 is similar to QDE-2 required for quelling and RDE-1 required for RNAi. Sequencing of ago1 mutants revealed one amino acid essential for PTGS that is also present in QDE-2 and RDE-1 in a highly conserved motif. Taken together, these results confirm the hypothesis that these processes derive from a common ancestral mechanism that controls expression of invading nucleic acid molecules at the post-transcriptional level. As opposed to rde-1 and qde-2 mutants, which are viable, ago1 mutants display several developmental abnormalities, including sterility. These results raise the possibility that PTGS, or at least some of its elements, could participate in the regulation of gene expression during development in plants.

It has been known for more than a decade that increasing the gene copy number does not necessarily lead to increased gene activity. Plants have developed efficient mechanisms such as post-transcriptional gene silencing (PTGS) to regulate abnormal gene expression in a sequence-specific fashion. PTGS of (trans)genes can be inhibited by non-homologous viruses, and PTGS-impaired mutants can be hypersensitive to such viruses, indicating that in plants this mechanism is triggered to protect against viral invasion. Genetic analysis of a related phenomenon, quelling, in Neurospora has led to the identification of two genes encoding proteins that share homologies with RNA-dependent RNA polymerases and with DNA helicases. This finding reinforces previous models in which PTGS involves RNA molecules complementary to the RNA species targeted for degradation. Insight into the mechanisms of PTGS may also be obtained in other distant organisms such as Caenorhabditis elegans in which a related phenomenon, RNA interference, has been genetically studied, leading to the identification of two genes encoding proteins sharing homologies with a translation factor and an RNase D.

Summary A novel myb oncogene homologue (AtMYB30) has been isolated by differential screening of a cDNA library prepared from Xanthomonas campestris pv. campestris (X. campestris)-inoculated Arabidopsis thaliana cells cultured in the presence of cycloheximide. AtMYB30 is a single-copy gene, and the encoded protein contains a MYB domain highly homologous to other plant and animal MYB proteins. Analyses of transcript levels in A. thaliana plants, or in cultured A. thaliana cells infected with either virulent or avirulent strains of the pathogens X. campestris and Pseudomonas syringae pv. tomato, showed that maximal levels of transcription of this gene occurred during the hypersensitive response. Furthermore, in A. thaliana mutants affected in the control of cell death initiation (lsd3, lsd4 and lsd5), constitutive expression or expression in lesion-positive plants was observed, while in suppressors of the mutations lsd5 and lsd4, AtMYB30 transcripts did not accumulate. However, AtMYB30 expression could not be detected in the lsd1 mutant, which was hyper-responsive to cell death initiators and unable to limit the extent of cell death, whatever the environmental conditions. The results presented here suggest a strong correlation between AtMYB30 and genetically controlled cell death, with a role in the initiation of cell death rather than in the limitation of its extent. Our results further indicate that the lsd mutants constitute an appropriate genetic model for studying the role of this gene in hypersensitive cell death, and their relation to different steps of the pathway(s) leading to cell death.

The recent development of gene transfer methods for almost all eukaryotes has revealed that transgenes can undergo silencing after integration in the genome. Host genes can also be silenced as a consequence of the presence of a homologous transgene, thus limiting the potential application of genetic transformation. Despite this limitation, transgene-induced gene silencing events were considered originally as anecdotal phenomena. However, as more and more similarities were found between transgene-induced gene silencing and natural epigenetic phenomena, considerable interest has been devoted to this subject (for recent reviews see Depicker and Van Montagu, 1997; Stam et al., 1997b). Epigenetics is commonly defined as ‘the study of mitotically and/or meiotically heritable changes in the function of a gene that cannot be explained by changes in its DNA sequence’ (Russo et al., 1996). For a long time, DNA was considered as the only target for epigenetic modifications. Epigenetic changes corresponding to changes in chromatin structure and affecting transcription have been reported in almost all eukaryotes: yeast, fungi, Drosophila, plants and mammals (Dorer, 1997; Foss and Selker, 1991; Rossignol and Faugeron, 1994; Ye and Signer, 1996). However, recent studies have suggested that, besides DNA, other molecules can be modified in a manner that resembles epigenetic DNA changes. First, it was shown that proteins can be converted into molecules of aberrant conformation called prions in yeast and mammals (Lacroute, 1971; Prusiner, 1982). More recently, the involvement of RNA was hypothesized to explain post-transcriptional silencing in plants, fungi and nematodes (Cogoni et al., 1996; Fire et al., 1998; Napoli et al., 1990). This review will focus on transgene-induced silencing phenomena in plants. The number of copies of a transgene that integrate into the genome of a transformed plant and

The transcription of the tobacco Tnt1 retrotransposon was previously shown to be induced, in tobacco and in heterologous species, by microbial elicitors and by pathogen infections. We report here that the expression of the Tnt1 promoter is also activated in heterologous species such as tomato and Arabidopsis by wounding, freezing and by other abiotic factors known to induce the plant defence response, such as salicylic acid, CuCl2, or oxidative stress. A similar regulation is observed in tobacco for most treatments. The induction of the Tnt1 promoter expression by wounding remains localized around injury points. In CuCl2-treated Arabidopsis plants, the transcription of Tnt1 is correlated with accumulation of the phytoalexin camalexin and with the expression of the EL13 defence gene. The interest of the Tnt1 promoter as a sensitive indicator of the plant defence responses is discussed.

Background Nitrogen often increases disease susceptibility, a phenomenon that can be observed under controlled conditions and called NIS, for Nitrogen-Induced Susceptibility. NIS has long been reported in the case of rice blast disease caused by the fungus Magnaporthe oryzae. We used an experimental system that does not strongly affect plant development to address the question of NIS polymorphism across rice diversity and further explored this phenomenon in wheat. We tested the two major types of resistance, namely quantitative/partial resistance and resistance driven by known resistance genes. Indeed there are conflicting reports on the effects of NIS on the first one and none on the last one. Finally, the genetics of NIS is not well documented and only few loci have been identified that may control this phenomenon. Results Our data indicate that NIS is a general phenomenon affecting resistance to blast fungus in these two cereals. We show that the capacity of rice to display NIS is highly polymorphic and does not correlate with difference related to indica/japonica sub-groups. We also tested the robustness of three different major resistance genes under high nitrogen. Nitrogen partially breaks down resistance triggered by the Pi1 gene. Cytological examination indicates that penetration rate is not affected by high nitrogen whereas growth of the fungus is increased inside the plant. Using the CSSL mapping population between Nipponbare and Kasalath, we identified a Kasalath locus on chromosome 1, called NIS1, which dominantly increases susceptibility under high nitrogen. We discuss the possible relationships between Nitrogen Use Efficiency (NUE), disease resistance regulation and NIS. Conclusions This work provides evidences that robust forms of partial resistance exist across diversity and can be easily identified with our protocol. This work also suggests that under certain environmental circumstances, complete resistance may breakdown, irrelevantly of the capacity of the fungus to mutate. These aspects should be considered while breeding for robust forms of resistance to blast disease.

BGPI : équipes 4 et 6; Effector proteins are key elements in plant-fungal interactions. The rice blast fungus Magnaporthe oryzae secretes numerous effectors that are suspected to be translocated inside plant cells. However, their cellular targets and the mechanisms of translocation are still unknown. Here, we have identified the open reading frame (ORF3) corresponding to the M. oryzae avirulence gene AVR1-CO39 that interacts with the rice resistance gene Pi-CO39 and encodes a small secreted protein without homology to other proteins. We demonstrate that AVR1-CO39 is specifically expressed and secreted at the plant-fungal interface during the biotrophic phase of infection. Live-cell imaging with M. oryzae transformants expressing a translational fusion between AVR1-CO39 and the monomeric red fluorescent protein (mRFP) indicated that AVR1-CO39 is translocated into the cytoplasm of infected rice cells. Transient expression of an AVR1-CO39 isoform without a signal peptide in rice protoplasts triggers a Pi-CO39-specific hypersensitive response, suggesting that recognition of AVR1-CO39 by the Pi-CO39 gene product occurs in the cytoplasm of rice cells. The native AVR1-CO39 protein enters the secretory pathway of rice protoplasts as demonstrated by the ER localization of AVR1-CO39:mRFP:HDEL translational fusions, and is correctly processed as shown by Western blotting. However, this secreted AVR1-CO39 isoform triggers a Pi-CO39-specific hypersensitive response and accumulates inside rice protoplasts as shown by Western blotting and localization of AVR1- CO39:mRFP translational fusions. This indicates that AVR1-CO39 is secreted by rice protoplasts and re-enters into the cytoplasm by unknown mechanisms, suggesting that translocation of AVR1-CO39 into rice cells occurs independently of fungal factors.

Resistance (R) proteins recognize pathogen avirulence (Avr) proteins by direct or indirect binding and are multidomain proteins generally carrying a nucleotide binding (NB) and a leucine-rich repeat (LRR) domain. Two NB-LRR protein-coding genes from rice (Oryza sativa), RGA4 and RGA5, were found to be required for the recognition of the Magnaporthe oryzae effector AVR1-CO39. RGA4 and RGA5 also mediate recognition of the unrelated M. oryzae effector AVR-Pia, indicating that the corresponding R proteins possess dual recognition specificity. For RGA5, two alternative transcripts, RGA5-A and RGA5-B, were identified. Genetic analysis showed that only RGA5-A confers resistance, while RGA5-B is inactive. Yeast two-hybrid, coimmunoprecipitation, and fluorescence resonance energy transfer–fluorescence lifetime imaging experiments revealed direct binding of AVR-Pia and AVR1-CO39 to RGA5-A, providing evidence for the recognition of multiple Avr proteins by direct binding to a single R protein. Direct binding seems to be required for resistance as an inactive AVR-Pia allele did not bind RGA5-A. A small Avr interaction domain with homology to the Avr recognition domain in the rice R protein Pik-1 was identified in the C terminus of RGA5-A. This reveals a mode of Avr protein recognition through direct binding to a novel, non-LRR interaction domain.

Plants produce cytokinin (CK) hormones for controlling key developmental processes like source/sink distribution, cell division or programmed cell-death. Some plant pathogens have been shown to produce CKs but the function of this mimicry production by non-tumor inducing pathogens, has yet to be established. Here we identify a gene required for CK biosynthesis, CKS1, in the rice blast fungus Magnaporthe oryzae. The fungal-secreted CKs are likely perceived by the plant during infection since the transcriptional regulation of rice CK-responsive genes is altered in plants infected by the mutants in which CKS1 gene was deleted. Although cks1 mutants showed normal in vitro growth and development, they were severely affected for in planta growth and virulence. Moreover, we showed that the cks1 mutant triggered enhanced induction of plant defenses as manifested by an elevated oxidative burst and expression of defense-related markers. In addition, the contents of sugars and key amino acids for fungal growth were altered in and around the infection site by the cks1 mutant in a different manner than by the control strain. These results suggest that fungal-derived CKs are key effectors required for dampening host defenses and affecting sugar and amino acid distribution in and around the infection site., Author Summary The role of plant-like hormonal compounds produced by fungal pathogens during infection has not been elucidated. Here we identified a conserved gene in most fungi, required for cytokinin production by the rice blast fungus and for its full virulence. Fungal-derived cytokinins are likely potent inhibitors of plant immunity. They are also needed to maintain elevated sugar contents at the site of infection and to drain or consume essential amino acids at, and around, the infection site. Thus, cytokinins represent the second example, after the bacterially-produced coronatine, of plant hormones hijacked by pathogens to successfully invade plant tissues. These findings also suggest that this invasion strategy could be widely conserved among fungi.

Jasmonates are important phytohormones regulating reproductive development. We used two recessive rice Tos17 alleles of OsJAR1, osjar1-2 and osjar1-3, to study the biological function of jasmonates in rice anthesis. The florets of both osjar1 alleles stayed open during anthesis because the lodicules, which control flower opening in rice, were not withering on time. Furthermore, dehiscence of the anthers filled with viable pollen, was impaired, resulting in lower fertility. In situ hybridization and promoter GUS transgenic analysis confirmed OsJAR1 expression in these floral tissues. Flower opening induced by exogenous applied methyl jasmonate was impaired in osjar1 plants and was restored in a complementation experiment with transgenics expressing a wild type copy of OsJAR1 controlled by a rice actin promoter. Biochemical analysis showed that OsJAR1 encoded an enzyme conjugating jasmonic acid (JA) to at least Ile, Leu, Met, Phe, Trp and Val and both osjar1 alleles had substantial reduction in content of JA-Ile, JA-Leu and JA-Val in florets. We conclude that OsJAR1 is a JA-amino acid synthetase that is required for optimal flower opening and closing and anther dehiscence in rice. © 2014 Springer Science+Business Media Dordrecht. Molecular Sequence Numbers: GENBANK: AF234312, AF294978, FI494725, FI494726, FI494727, FI494728

UMR BGPI - Equipe 4; International audience; P>Blast disease (causal agent Magnaporthe oryzae) has presented as a new and serious field disease of wheat in South America. Here, we investigated the responses of wheat to both adapted and nonadapted isolates of the blast fungus Magnaporthe, examining cellular defence and transcriptional changes. Resistance towards the nonadapted isolate was associated with the formation of appositions, here termed halos, beneath attempted Magnaporthe grisea penetration sites that wheat-adapted, M. oryzae isolates were able to breach. Transcriptome analysis indicated extensive transcriptional reprogramming following inoculation with both wheat-adapted and nonadapted isolates of Magnaporthe. Functional annotation of many of the differentially expressed transcripts classified into the categories: cell rescue and defence, plant metabolism, cellular transport and regulation of transcription (although a significant number of transcripts remain unclassified). Defence-related transcripts induced in common by adapted and nonadapted isolates were differentially regulated in response to M. oryzae and M. grisea isolates over time. Differential expression of genes involved in cellular transport indicated the importance of this process in plant defence. Functional characterisation of these transcripts and their role in defence may eventually lead to the identification of broad-spectrum resistance mechanisms in wheat towards Magnaporthe.

Large amounts of major resistance genes, QTL (Quantitative Trait Locus) and expression data dealing with rice blast resistance have been described in the past. We extensively reviewed this information and inserted it into the OryGenesDB database, creating the ARCHIPELAGO resource. This represents more than 1,500 genes differentially expressed upon infection by Magnaporthe oryzae, 1,000 resistance gene analogs, 88 mapped resistance genes, 341 QTLs and 165 metaQTLs. Further analysis of the metaQTL data allowed the identification of robust or broad spectrum metaQTLs. Cross referencing defense gene expression and metaQTL information identified some candidate genes for metaQTL but does not demonstrate preferential co-localization of defense-related genes in metaQTLs. In contrast, disease regulators are statistically associated with disease QTLs. At the genomic level, we can observe that some regions of the rice genome are richer than others for defense-related genes. This resource opens new possibilities for marker-assisted selection and QTL cloning.

UMR BGPI Equipe 4; International audience; The completion of the genome sequences of both rice and Magnaporthe oryzae has strengthened the position of rice blast disease as a model to study plant–pathogen interactions in monocotyledons. Genetic studies of blast resistance in rice were established in Japan as early as 1917. Despite such long-term study, examples of cultivars with durable resistance are rare, partly due to our limited knowledge of resistance mechanisms. A rising number of blast resistance genes and quantitative trait loci (QTL) have been genetically described, and some have been characterized during the last 20 years. Using the rice genome sequence, can we now go a step further toward a better understanding of the genetics of blast resistance by combining all these results? Is such knowledge appropriate and sufficient to improve breeding for durable resistance? A review of bibliographic references identified 85 blast resistance genes and approximately 350 QTL, which we mapped on the rice genome. These data provide a useful update on blast resistance genes as well as new insights to help formulate hypotheses about the molecular function of blast QTL, with special emphasis on QTL for partial resistance. All these data are available from the OrygenesDB database.

UMR BGPI Equipe 4; International audience; Large amounts of expression data dealing with biotic stresses in rice have been produced in the past 5 years. Here, we extensively review approximately 70 publications and gather together information on more than 2,500 genes of the rice defense arsenal. This information was integrated into the OryGenesDB database. Several genes (e.g., metallothioneins and PBZ1) appear to be hallmarks of rice–pathogen interactions. Cross-referencing this information with the rice kinome highlighted some defense genes and kinases as possible central nodes of regulation. Cross referencing defense gene expression and quantitative trait loci (QTL) information identified some candidate genes for QTL. Overall, pathogenesis-related genes and disease regulators were found to be statistically associated with disease QTL. At the genomic level, we observed that some regions are richer than others and that some chromosomes (e.g., 11 and 12), which contain a lot of resistance gene analogs, have a low content of defense genes. Finally, we show that classical defense genes and defense-related genes such as resistance genes are preferentially organized in clusters. These clusters are not always coregulated and individual paralogs can show specific expression patterns. Thus, the rice defense arsenal has an ARCHIPELAGO-like genome structure at the macro and micro level. This resource opens new possibilities for marker-assisted selection and QTL cloning.

The eukaryotic defense response posttranscriptional gene silencing (PTGS) is directed by short-interfering RNAs and thwarts invading nucleic acids via the RNA slicing activity of conserved ARGONAUTE (AGO) proteins. PTGS can be counteracted by exogenous or endogenous suppressors, including the cytoplasmic exoribonuclease XRN4, which also degrades microRNA (miRNA)-guided mRNA cleavage products but does not play an obvious role in development. Here, we show that the nuclear exoribonucleases XRN2 and XRN3 are endogenous PTGS suppressors. We also identify excised MIRNA loops as templates for XRN2 and XRN3 and show that XRN3 is critical for proper development. Independently, we identified the nucleotidase/phosphatase FIERY1 (FRY1) as an endogenous PTGS suppressor through a suppressor screen in a hypomorphic ago1 genetic background. FRY1 is one of six Arabidopsis thaliana orthologs of yeast Hal2. Yeast hal2 mutants overaccumulate 3′-phosphoadenosine 5′-phosphate, which suppresses the 5′→3′ exoribonucleases Xrn1 and Rat1. fry1 mutant plants recapitulate developmental and molecular characteristics of xrn mutants and likely restore PTGS in ago1 hypomorphic mutants by corepressing XRN2, XRN3, and XRN4, thus increasing RNA silencing triggers. We anticipate that screens incorporating partially compromised silencing components will uncover additional PTGS suppressors that may not be revealed using robust silencing systems.

Posttranscriptional gene silencing (PTGS) in plants results from the degradation of mRNAs and shows phenomenological similarities with quelling in fungi and RNAi in animals. Here, we report the isolation of sgs2 and sgs3 Arabidopsis mutants impaired in PTGS. We establish a mechanistic link between PTGS, quelling, and RNAi since the Arabidopsis SGS2 protein is similar to an RNA-dependent RNA polymerase like N. crassa QDE-1, controlling quelling, and C. elegans EGO-1, controlling RNAi. In contrast, SGS3 shows no significant similarity with any known or putative protein, thus defining a specific step of PTGS in plants. Both sgs2 and sgs3 mutants show enhanced susceptibility to virus, definitively proving that PTGS is an antiviral defense mechanism that can also target transgene RNA for degradation.

Cell death is associated with the development of the plant disease resistance hypersensitive reaction (HR). Arabidopsis lsd mutants that spontaneously exhibit cell death reminiscent of the HR were identified previously. To study further the regulatory context in which cell death acts during disease resistance, one of these mutants, lsd5, was used to isolate new mutations that suppress its cell death phenotype. Using a simple lethal screen, nine lsd5 cell death suppressors, designated phx (for the mythological bird Phoenix that rises from its ashes), were isolated. These mutants were characterized with respect to their response to a bacterial pathogen and oomycete parasite. The strongest suppressors—phx2, 3, 6, and 11-1—showed complex, differential patterns of disease resistance modifications. These suppressors attenuated disease resistance to avirulent isolates of the biotrophic Peronospora parasitica pathogen, but only phx2 and phx3 altered disease resistance to avirulent strains of Pseudomonas syringae pv tomato. Therefore, some of these phx mutants define common regulators of cell death and disease resistance. In addition, phx2 and phx3 exhibited enhanced disease susceptibility to different virulent pathogens, confirming probable links between the disease resistance and susceptibility pathways.

Activation of retrotransposons by stresses and external changes is common in all eukaryotic systems, including plants. The transcription of the tobacco Tnt1 retrotransposon was studied in its natural host as well as in Arabidopsis and tomato. It is activated by factors of microbial origin, by external stresses, and by viral, bacterial, and fungal attacks. Tnt1 expression is linked with the biological responses of the plant to the elicitor or to the pathogen attack and in particular with the early steps of the metabolic pathways leading to the activation of plant defense genes. In most cases, the basic features of Tnt1 regulation in tobacco are maintained in tomato and Arabidopsis, but some host-specific regulations were shown. The U3 region of the Tnt1 LTR contains the major cis-acting components of Tnt1 transcriptional activation in association with the plant defense responses. Furthermore, the Tnt1 U3 region, and especially the tandemly repeated BII boxes, contains several sequences similar to well-characterized motifs involved in the activation of several plant defense genes. The possible origin of Tnt1 regulatory sequences as well as the biological implications of Tnt1 activation by pathogen attacks are discussed.

Transcriptional regulation of the tobacco retrotransposon Tnt1 was monitored in Arabidopsis thaliana and Lyco-persicon esculentum. In these two heterologous host species, Tnt1 expression is developmentally regulated, and its tissue-specific pattern of transcription is similar to that observed in tobacco, with the exception that Tnt1 is expressed in their flowers. Induction of Tnt1 by microbial elicitors, previously found in tobacco, is maintained in Arabidopsis and tomato. Tnt1 activation is not strictly dependent upon a Hypersensitive Response-like necrotic response, but also occurs in a plant-pathogen compatible interaction triggered by viral or bacterial infections. The putative role of Tnt1 in generating genetic variability in its host plant is discussed.

Many interactions between plants and their parasites begin with specific recognition. The nature of this recognition, and of subsequent signal transduction by both host and parasite have profound impact on the outcome of the interaction. Plants have evolved effective mechanisms to recognize pathogenic microbes and halt their biotrophic or necrotrophic growth. Active plant defense mechanisms obviously force adaptive selection for microbe variants which can evade the plant’s recognition capabilities. This evolutionary tug of war has led to a complex set of both plant and microbe genes whose interaction is required for a successful resistance reaction. As well as a potentially large array of recognition functions, a number of subsequent functions must exist which are necessary to establish a completely effective resistant phenotype.

Numerous studies covering some aspects of SARS-CoV-2 data analyses are being published on a daily basis, including a regularly updated phylogeny on nextstrain.org. Here, we review the difficulties of inferring reliable phylogenies by example of a data snapshot comprising all virus sequences available on May 5, 2020 from gisaid.org. We find that it is difficult to infer a reliable phylogeny on these data due to the large number of sequences in conjunction with the low number of mutations. We further find that rooting the inferred phylogeny with some degree of confidence either via the bat and pangolin outgroups or by applying novel computational methods on the ingroup phylogeny does not appear to be possible. Finally, an automatic classification of the current sequences into sub-classes based on statistical criteria is also not possible, as the sequences are too closely related. We conclude that, although the application of phylogenetic methods to disentangle the evolution and spread of COVID-19 provides some insight, results of phylogenetic analyses, in particular those conducted under the default settings of current phylogenetic inference tools, as well as downstream analyses on the inferred phylogenies, should be considered and interpreted with extreme caution.