Oligomerization of a plant helper NLR requires cell-surface and intracellular immune receptor activation.

Plant disease resistance involves both detection of microbial molecular patterns by cell-surface pattern recognition receptors and detection of pathogen effectors by intracellular NLR immune receptors. NLRs are classified as sensor NLRs, involved in effector detection, or helper NLRs required for sensor NLR signaling. TIR-domain-containing sensor NLRs (TNLs) require helper NLRs NRG1 and ADR1 for resistance, and helper NLR activation of defense requires the lipase-domain proteins EDS1, SAG101, and PAD4. Previously, we found that NRG1 associates with EDS1 and SAG101 in a TNL activation-dependent manner [X. Sun et al., Nat. Commun. 12, 3335 (2021)]. We report here how the helper NLR NRG1 associates with itself and with EDS1 and SAG101 during TNL-initiated immunity. Full immunity requires coactivation and mutual potentiation of cell-surface and intracellular immune receptor-initiated signaling [B. P. M. Ngou, H.-K. Ahn, P. Ding, J. D. G. Jones, Nature 592, 110-115 (2021), M. Yuan et al., Nature 592, 105-109 (2021)]. We find that while activation of TNLs is sufficient to promote NRG1-EDS1-SAG101 interaction, the formation of an oligomeric NRG1-EDS1-SAG101 resistosome requires the additional coactivation of cell-surface receptor-initiated defense. These data suggest that NRG1-EDS1-SAG101 resistosome formation in vivo is part of the mechanism that links intracellular and cell-surface receptor signaling pathways.

Pathogen effector recognition-dependent association of NRG1 with EDS1 and SAG101 in TNL receptor immunity.

Plants utilise intracellular nucleotide-binding, leucine-rich repeat (NLR) immune receptors to detect pathogen effectors and activate local and systemic defence. NRG1 and ADR1 "helper" NLRs (RNLs) cooperate with enhanced disease susceptibility 1 (EDS1), senescence-associated gene 101 (SAG101) and phytoalexin-deficient 4 (PAD4) lipase-like proteins to mediate signalling from TIR domain NLR receptors (TNLs). The mechanism of RNL/EDS1 family protein cooperation is not understood. Here, we present genetic and molecular evidence for exclusive EDS1/SAG101/NRG1 and EDS1/PAD4/ADR1 co-functions in TNL immunity. Using immunoprecipitation and mass spectrometry, we show effector recognition-dependent interaction of NRG1 with EDS1 and SAG101, but not PAD4. An EDS1-SAG101 complex interacts with NRG1, and EDS1-PAD4 with ADR1, in an immune-activated state. NRG1 requires an intact nucleotide-binding P-loop motif, and EDS1 a functional EP domain and its partner SAG101, for induced association and immunity. Thus, two distinct modules (NRG1/EDS1/SAG101 and ADR1/EDS1/PAD4) mediate TNL receptor defence signalling.

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.

Generation and Analysis of Xenopus laevis Models of Retinal Degeneration Using CRISPR/Cas9.

Xenopus laevis have proven to be a useful system for rapid generation and analysis of transgenic models of human retinal disease. However, experimental approaches in this system were limited by lack of a robust knockdown or knockout technology. Here we describe a protocol for generation of Cas9-edited X. laevis embryos. The technique introduces point mutations into the genome of X. laevis resulting in in-frame and out-of-frame insertions and deletions that allow modeling of both dominant and recessive human diseases and efficiently generates gene knockdown and knockout. Our techniques can produce high-frequency gene editing in X. laevis, permitting analysis in the F0 generation.

Modeling Dominant and Recessive Forms of Retinitis Pigmentosa by Editing Three Rhodopsin-Encoding Genes in Xenopus Laevis Using Crispr/Cas9.

The utility of Xenopus laevis, a common research subject for developmental biology, retinal physiology, cell biology, and other investigations, has been limited by lack of a robust gene knockout or knock-down technology. Here we describe manipulation of the X. laevis genome using CRISPR/Cas9 to model the human disorder retinitis pigmentosa, and to introduce point mutations or exogenous DNA sequences. We introduced and characterized in-frame and out-of-frame insertions and deletions in three genes encoding rhodopsin by co-injection of Cas9 mRNA, eGFP mRNA, and single guide RNAs into fertilized eggs. Deletions were characterized by direct sequencing and cloning; phenotypes were assessed by assays of rod opsin in retinal extracts, and confocal microscopy of cryosectioned and immunolabeled contralateral eyes. We obtained germline transmission of editing to F1 offspring. In-frame deletions frequently caused dominant retinal degeneration associated with rhodopsin biosynthesis defects, while frameshift phenotypes were consistent with knockout. We inserted eGFP or point mutations into rhodopsin genes by co-injection of repair fragments with homology to the Cas9 target sites. Our techniques can produce high frequency gene editing in X. laevis, permitting analysis in the F0 generation, and advancing the utility of X. laevis as a subject for biological research and disease modeling.

Purification of High Molecular Weight Genomic DNA from Powdery Mildew for Long-Read Sequencing.

The powdery mildew fungi are a group of economically important fungal plant pathogens. Relatively little is known about the molecular biology and genetics of these pathogens, in part due to a lack of well-developed genetic and genomic resources. These organisms have large, repetitive genomes, which have made genome sequencing and assembly prohibitively difficult. Here, we describe methods for the collection, extraction, purification and quality control assessment of high molecular weight genomic DNA from one powdery mildew species, Golovinomyces cichoracearum. The protocol described includes mechanical disruption of spores followed by an optimized phenol/chloroform genomic DNA extraction. A typical yield was 7 µg DNA per 150 mg conidia. The genomic DNA that is isolated using this procedure is suitable for long-read sequencing (i.e., > 48.5 kbp). Quality control measures to ensure the size, yield, and purity of the genomic DNA are also described in this method. Sequencing of the genomic DNA of the quality described here will allow for the assembly and comparison of multiple powdery mildew genomes, which in turn will lead to a better understanding and improved control of this agricultural pathogen.