Natural and CRISPR-induced genetic variation for plant immunity

Our understanding of the genetic basis of a trait primarily relies on analysing heritable phenotypic diversity. For instance, different accessions of Arabidopsis thaliana (Arabidopsis) can either be resistant or susceptible to a given strain of Albugo candida, an oomycete that causes the white rust disease. The virulent Albugo candida race Exeter1 (AcEx1) can grow on most Arabidopsis accessions. Using the resistant Arabidopsis Oy-0, I mapped and cloned the gene responsible for AcEx1 resistance: White Rust Resistance 4A (WRR4A). Arabidopsis Col-0 also contains WRR4A but does not resist AcEx1. I found that WRR4ACol-0 has an early stop codon, which is responsible for the recognition specificity of some effector candidates from Albugo candida. This example illustrates how natural diversity can be used to identify Resistance-genes and reveal components of the plant immune system. However, natural diversity is not always available. Clustered and regularly interspaces short palindromic repeats (CRISPR) from bacterial genomes defines an immune system, re-invented for genome editing. I optimized a CRISPR-Cas9 method to generate null alleles in Arabidopsis. Using this method, I produced a double mutant of two immunity-related gene candidates that are in tandem in the genome: AtNRG1A and AtNRG1B. I confirmed the 7-year-old hypothesis that NRG1A and NRG1B are redundantly required for signalling downstream of multiple Resistance-genes, mainly from the TIR-NLR immune receptor family. So far very few genes required for immunity upon Resistance-protein activation were defined. This second example illustrates that CRISPR can be used to generate variation to unravel redundant genetic pathways. The widespread adoption of CRISPR tools is likely to lead to a better understanding of the plant immune system. Ultimately, it will result in solutions to deploy genetics-based resistance to protect our crops from disease, reducing the need for chemicals

Diverse NLR immune receptors activate defence via the RPW8-NLR NRG1

Most land plant genomes carry genes that encode RPW8-NLR Resistance (R) proteins. Angiosperms carry two RPW8-NLR subclasses: ADR1 and NRG1. ADR1s act as 'helper' NLRs for multiple TIR- and CC-NLR R proteins in Arabidopsis. In angiosperm families, NRG1 co-occurs with TIR-NLR Resistance (R) genes. We tested whether NRG1 is required for signalling of multiple TIR-NLRs. Using CRISPR mutagenesis, we obtained an nrg1a-nrg1b double mutant in two Arabidopsis accessions, and an nrg1 mutant in Nicotiana benthamiana. These mutants are compromised in signalling of all TIR-NLRs tested, including WRR4A, WRR4B, RPP1, RPP2, RPP4 and the pairs RRS1/RPS4, RRS1B/RPS4B, CHS1/SOC3 and CHS3/CSA1. In Arabidopsis, NRG1 is required for the hypersensitive cell death response (HR) and full oomycete resistance, but not for salicylic acid induction or bacterial resistance. By contrast, nrg1 loss of function does not compromise the CC-NLR R proteins RPS5 and MLA. RPM1 and RPS2 (CC-NLRs) function is slightly compromised in an nrg1 mutant. Thus, NRG1 is required for full TIR-NLR function and contributes to the signalling of some CC-NLRs. Some NRG1-dependent R proteins also signal partially via the NRG1 sister clade, ADR1. We propose that some NLRs signal via NRG1 only, some via ADR1 only and some via both or neither

Plant NLRs get by with a little help from their friends

Many plant NLR (nucleotide-binding, leucine-rich repeat) immune receptors require other NLRs for their function. In pairs of chromosomally adjacent sensor/helper NLRs, the sensor typically carries an integrated domain (ID) that mimics the authentic target of a pathogen effector. The RPW8-NLR clade supports the function of many diverse plant NLRs, particularly those with a TIR N-terminal domain, in concert with a family of EP-domain containing signalling partners. The NRC clade of NLRs are required for the function of many unlinked sensor NLRs in Solanaceous plants. We evaluate recent advances in paired NLR biology in the context of the structure and possible mechanisms of the first defined plant inflammasome containing ZAR1

Transgressive segregation reveals mechanisms of Arabidopsis immunity to Brassica-infecting races of white rust (Albugo candida)

Arabidopsis thaliana accessions are universally resistant at the adult leaf stage to white rust (Albugo candida) races that infect the crop species Brassica juncea and Brassica oleracea. We used transgressive segregation in recombinant inbred lines to test if this apparent species-wide (nonhost) resistance in A. thaliana is due to natural pyramiding of multiple Resistance (R) genes. We screened 593 inbred lines from an Arabidopsis multiparent advanced generation intercross (MAGIC) mapping population, derived from 19 resistant parental accessions, and identified two transgressive segregants that are susceptible to the pathogen. These were crossed to each MAGIC parent, and analysis of resulting F2 progeny followed by positional cloning showed that resistance to an isolate of A. candida race 2 (Ac2V) can be explained in each accession by at least one of four genes encoding nucleotide-binding, leucine-rich repeat (NLR) immune receptors. An additional gene was identified that confers resistance to an isolate of A. candida race 9 (AcBoT) that infects B. oleracea. Thus, effector-triggered immunity conferred by distinct NLR-encoding genes in multiple A. thaliana accessions provides species-wide resistance to these crop pathogens

Transgressive segregation reveals mechanisms of Arabidopsis immunity to Brassica-infecting races of white rust (Albugo candida)

Arabidopsis thaliana accessions are universally resistant at the adult leaf stage to white rust (Albugo candida) races that infect the crop species Brassica juncea and Brassica oleracea. We used transgressive segregation in recombinant inbred lines to test if this apparent species-wide (nonhost) resistance in A. thaliana is due to natural pyramiding of multiple Resistance (R) genes. We screened 593 inbred lines from an Arabidopsis multiparent advanced generation intercross (MAGIC) mapping population, derived from 19 resistant parental accessions, and identified two transgressive segregants that are susceptible to the pathogen. These were crossed to each MAGIC parent, and analysis of resulting F 2 progeny followed by positional cloning showed that resistance to an isolate of A. candida race 2 (Ac2V) can be explained in each accession by at least one of four genes encoding nucleotide-binding, leucine-rich repeat (NLR) immune receptors. An additional gene was identified that confers resistance to an isolate of A. candida race 9 (AcBoT) that infects B. oleracea. Thus, effector-triggered immunity conferred by distinct NLR-encoding genes in multiple A. thaliana accessions provides species-wide resistance to these crop pathogens

Evolutionary trade-offs at the Arabidopsis WRR4A resistance locus underpin alternate Albugo candida race recognition specificities

The oomycete Albugo candida causes white rust of Brassicaceae, including vegetable and oilseed crops, and wild relatives such as Arabidopsis thaliana. Novel White Rust Resistance (WRR) genes from Arabidopsis enable new insights into plant/parasite co-evolution. WRR4A from Arabidopsis accession Columbia (Col-0) provides resistance to many but not all white rust races, and encodes a nucleotide-binding, leucine-rich repeat immune receptor. Col-0 WRR4A resistance is broken by AcEx1, an isolate of A. candida. We identified an allele of WRR4A in Arabidopsis accession Øystese-0 (Oy-0) and other accessions that confers full resistance to AcEx1. WRR4A Oy-0 carries a C-terminal extension required for recognition of AcEx1, but reduces recognition of several effectors recognized by the WRR4A Col-0 allele. WRR4A Oy-0 confers full resistance to AcEx1 when expressed in the oilseed crop Camelina sativa

Loups, chiens et sociétés du Paléolithique supérieur

Le processus de domestication du loup vraisemblablement initié au cours du Paléolithique supérieur, pose aujourd’hui encore de nombreuses questions en termes notamment d’ancienneté du phénomène et de sa diffusion au sein des différents groupes de chasseurs-cueilleurs se succédant au cours de cette période. Pour tenter d’apporter de nouveaux éléments de discussion, de nouvelles analyses pluridisciplinaires ont récemment été entreprises sur la Grotte Maldidier, l’Abri Pataud, l’Abri du Morin, l’Abri Mège, Rochereil, la Grotte-Abri du Moulin à Troubat et le Pont d’Ambon. Ce travail, à visée diachronique, permet de lancer une discussion concernant le statut sauvage ou domestique des grands Canidés dans ces contextes et plus généralement dans les séries du Paléolithique supérieur du Sud-Ouest de la France. Pour cette analyse, nous avons notamment croisé biométrie et archéozoologie. Les données biométriques obtenues à partir des restes de Canidés participent à la diagnose taxinomique. Les études archéozoologique et taphonomique de l’ensemble des vestiges fauniques associés à ces restes permettent quant à elle de documenter la prédation humaine et animale. L’ensemble de ces données nous permet donc de contextualiser la mise en place de cette innovation zootechnique majeure qu’est la domestication ; et plus globalement, de questionner l’évolution des relations Hommes-Canidés à travers les différents techno-complexes du Paléolithique supérieur

Complete Genome and Phylogeny of Puumala Hantavirus Isolates Circulating in France

Puumala virus (PUUV) is the agent of nephropathia epidemica (NE), a mild form of hemorrhagic fever with renal syndrome (HFRS) in Europe. NE incidence presents a high spatial variation throughout France, while the geographical distribution of the wild reservoir of PUUV, the bank vole, is rather continuous. A missing piece of the puzzle is the current distribution and the genetic variation of PUUV in France, which has been overlooked until now and remains poorly understood. During a population survey, from 2008 to 2011, bank voles were trapped in eight different forests of France located in areas known to be endemic for NE or in area from where no NE case has been reported until now. Bank voles were tested for immunoglobulin (Ig)G ELISA serology and two seropositive animals for each of three different areas (Ardennes, Jura and Orleans) were then subjected to laboratory analyses in order to sequence the whole S, M and L segments of PUUV. Phylogenetic analyses revealed that French PUUV isolates globally belong to the central European (CE) lineage although isolates from Ardennes are clearly distinct from those in Jura and Orleans, suggesting a different evolutionary history and origin of PUUV introduction in France. Sequence analyses revealed specific amino acid signatures along the N protein, including in PUUV from the Orleans region from where NE in humans has never been reported. The relevance of these mutations in term of pathophysiology is discussed.Peer reviewe

Induced proximity of a TIR signaling domain on a plant-mammalian NLR chimera activates defense in plants

Plant and animal intracellular nucleotide-binding, leucine-rich repeat (NLR) immune receptors detect pathogen-derived molecules and activate defense. Plant NLRs can be divided into several classes based upon their N-terminal signaling domains, including TIR (Toll-like, Interleukin-1 receptor, Resistance protein)- and CC (coiled-coil)-NLRs. Upon ligand detection, mammalian NAIP and NLRC4 NLRs oligomerize, forming an inflammasome that induces proximity of its N-terminal signaling domains. Recently, a plant CC-NLR was revealed to form an inflammasome-like hetero-oligomer. To further investigate plant NLR signaling mechanisms, we fused the N-terminal TIR domain of several plant NLRs to the N terminus of NLRC4. Inflammasome-dependent induced proximity of the TIR domain in planta initiated defense signaling. Thus, induced proximity of a plant TIR domain imposed by oligomerization of a mammalian inflammasome is sufficient to activate authentic plant defense. Ligand detection and inflammasome formation is maintained when the known components of the NLRC4 inflammasome is transferred across kingdoms, indicating that NLRC4 complex can robustly function without any additional mammalian proteins. Additionally, we found NADase activity of a plant TIR domain is necessary for plant defense activation, but NADase activity of a mammalian or a bacterial TIR is not sufficient to activate defense in plants