Highly efficient multiplex editing: one-shot generation of 8× Nicotiana benthamiana and 12× Arabidopsis mutants.

Genome editing by RNA-guided nucleases, such as SpCas9, has been used in numerous different plant species. However, to what extent multiple independent loci can be targeted simultaneously by multiplexing has not been well documented. Here, we developed a toolkit, based on a highly intron-optimized zCas9i gene, which allows assembly of nuclease constructs expressing up to 32 single guide RNAs (sgRNAs). We used this toolkit to explore the limits of multiplexing in two major model species, and report on the isolation of transgene-free octuple (8×) Nicotiana benthamiana and duodecuple (12×) Arabidopsis thaliana mutant lines in a single generation (T1 and T2 , respectively). We developed novel counter-selection markers for N. benthamiana, most importantly Sl-FAST2, comparable to the well-established Arabidopsis seed fluorescence marker, and FCY-UPP, based on the production of toxic 5-fluorouracil in the presence of a precursor. Targeting eight genes with an array of nine different sgRNAs and relying on FCY-UPP for selection of non-transgenic T1 , we identified N. benthamiana mutant lines with astonishingly high efficiencies: All analyzed plants carried mutations in all genes (approximately 112/116 target sites edited). Furthermore, we targeted 12 genes by an array of 24 sgRNAs in A. thaliana. Efficiency was significantly lower in A. thaliana, and our results indicate Cas9 availability is the limiting factor in such higher-order multiplexing applications. We identified a duodecuple mutant line by a combination of phenotypic screening and amplicon sequencing. The resources and results presented provide new perspectives for how multiplexing can be used to generate complex genotypes or to functionally interrogate groups of candidate genes.

EDS1 complexes are not required for PRR responses and execute TNL-ETI from the nucleus in Nicotiana benthamiana.

Heterodimeric complexes incorporating the lipase-like proteins EDS1 with PAD4 or SAG101 are central hubs in plant innate immunity. EDS1 functions encompass signal relay from TIR domain-containing intracellular NLR-type immune receptors (TNLs) towards RPW8-type helper NLRs (RNLs) and, in Arabidopsis thaliana, bolstering of signaling and resistance mediated by cell-surface pattern recognition receptors (PRRs). Increasing evidence points to the activation of EDS1 complexes by small molecule binding. We used CRISPR/Cas-generated mutant lines and agroinfiltration-based complementation assays to interrogate functions of EDS1 complexes in Nicotiana benthamiana. We did not detect impaired PRR signaling in N. benthamiana lines deficient in EDS1 complexes or RNLs. Intriguingly, in assays monitoring functions of SlEDS1-NbEDS1 complexes in N. benthamiana, mutations within the SlEDS1 catalytic triad could abolish or enhance TNL immunity. Furthermore, nuclear EDS1 accumulation was sufficient for N. benthamiana TNL (Roq1) immunity. Reinforcing PRR signaling in Arabidopsis might be a derived function of the TNL/EDS1 immune sector. Although Solanaceae EDS1 functionally depends on catalytic triad residues in some contexts, our data do not support binding of a TNL-derived small molecule in the triad environment. Whether and how nuclear EDS1 activity connects to membrane pore-forming RNLs remains unknown.

An EDS1-SAG101 Complex Is Essential for TNL-Mediated Immunity in Nicotiana benthamiana.

Heterodimeric complexes containing the lipase-like protein ENHANCED DISEASE SUSCEPTIBILITY1 (EDS1) are regarded as central regulators of plant innate immunity. In this context, a complex of EDS1 with PHYTOALEXIN DEFICIENT4 (PAD4) is required for basal resistance and signaling downstream of immune receptors containing an N-terminal Toll-interleukin-1 receptor-like domain (TNLs) in Arabidopsis (Arabidopsis thaliana). Here we analyze EDS1 functions in the model Solanaceous plant Nicotiana benthamiana (Nb). Stable Nb mutants deficient in EDS1 complexes are not impaired in basal resistance, a finding which contradicts a general role for EDS1 in immunity. In Nb, PAD4 demonstrated no detectable immune functions, but TNL-mediated resistance responses required EDS1 complexes incorporating a SENESCENCE ASSOCIATED GENE101 (SAG101) isoform. Intriguingly, SAG101 is restricted to those genomes also encoding TNL receptors, and we propose it may be required for TNL-mediated immune signaling in most plants, except the Brassicaceae. Transient complementation in Nb was used for accelerated mutational analyses while avoiding complex biotic interactions. We identify a large surface essential for EDS1-SAG101 immune functions that extends from the N-terminal lipase domains to the C-terminal EDS1-PAD4 domains and might mediate interaction partner recruitment. Furthermore, this work demonstrates the value of genetic resources in Nb, which will facilitate elucidation of EDS1 functions.

Targeting specificity of nuclear-encoded organelle proteins with a self-assembling split-fluorescent protein toolkit.

A large number of nuclear-encoded proteins are targeted to the organelles of endosymbiotic origin, namely mitochondria and plastids. To determine the targeting specificity of these proteins, fluorescent protein tagging is a popular approach. However, ectopic expression of fluorescent protein fusions commonly results in considerable background signals and often suffers from the large size and robust folding of the reporter protein, which may perturb membrane transport. Among the alternative approaches that have been developed in recent years, the self-assembling split-fluorescent protein (sasplit-FP) technology appears particularly promising to analyze protein targeting specificity in vivo Here, we improved the sensitivity of this technology and systematically evaluated its utilization to determine protein targeting to plastids and mitochondria. Furthermore, to facilitate high-throughput screening of candidate proteins we developed a Golden Gate-based vector toolkit (PlaMinGo). As a result of these improvements, dual targeting could be detected for a number of proteins that had earlier been characterized as being targeted to a single organelle only. These results were independently confirmed with a plant phenotype complementation approach based on the immutans mutant.This article has an associated First Person interview with the first author of the paper.

Optimized Cas9 expression systems for highly efficient Arabidopsis genome editing facilitate isolation of complex alleles in a single generation.

Genetic resources for the model plant Arabidopsis comprise mutant lines defective in almost any single gene in reference accession Columbia. However, gene redundancy and/or close linkage often render it extremely laborious or even impossible to isolate a desired line lacking a specific function or set of genes from segregating populations. Therefore, we here evaluated strategies and efficiencies for the inactivation of multiple genes by Cas9-based nucleases and multiplexing. In first attempts, we succeeded in isolating a mutant line carrying a 70 kb deletion, which occurred at a frequency of ~ 1.6% in the T2 generation, through PCR-based screening of numerous individuals. However, we failed to isolate a line lacking Lhcb1 genes, which are present in five copies organized at two loci in the Arabidopsis genome. To improve efficiency of our Cas9-based nuclease system, regulatory sequences controlling Cas9 expression levels and timing were systematically compared. Indeed, use of DD45 and RPS5a promoters improved efficiency of our genome editing system by approximately 25-30-fold in comparison to the previous ubiquitin promoter. Using an optimized genome editing system with RPS5a promoter-driven Cas9, putatively quintuple mutant lines lacking detectable amounts of Lhcb1 protein represented approximately 30% of T1 transformants. These results show how improved genome editing systems facilitate the isolation of complex mutant alleles, previously considered impossible to generate, at high frequency even in a single (T1) generation.

Receptor-like cytoplasmic kinases of different subfamilies differentially regulate SOBIR1/BAK1-mediated immune responses in Nicotiana benthamiana.

Cell-surface receptors form the front line of plant immunity. The leucine-rich repeat (LRR)-receptor-like kinases SOBIR1 and BAK1 are required for the functionality of the tomato LRR-receptor-like protein Cf-4, which detects the secreted effector Avr4 of the pathogenic fungus Fulvia fulva. Here, we show that the kinase domains of SOBIR1 and BAK1 directly phosphorylate each other and that residues Thr522 and Tyr469 of the kinase domain of Nicotiana benthamiana SOBIR1 are required for its kinase activity and for interacting with signalling partners, respectively. By knocking out multiple genes belonging to different receptor-like cytoplasmic kinase (RLCK)-VII subfamilies in N. benthamiana:Cf-4, we show that members of RLCK-VII-6, -7, and -8 differentially regulate the Avr4/Cf-4-triggered biphasic burst of reactive oxygen species. In addition, members of RLCK-VII-7 play an essential role in resistance against the oomycete pathogen Phytophthora palmivora. Our study provides molecular evidence for the specific roles of RLCKs downstream of SOBIR1/BAK1-containing immune complexes.

Targeted gene deletion with SpCas9 and multiple guide RNAs in Arabidopsis thaliana: four are better than two.

BACKGROUND: In plant genome editing, RNA-guided nucleases such as Cas9 from Streptococcus pyogenes (SpCas9) predominantly induce small insertions or deletions at target sites. This can be used for inactivation of protein-coding genes by frame shift mutations. However, in some cases, it may be advantageous to delete larger chromosomal segments. This is achieved by simultaneously inducing double strand breaks upstream and downstream of the segment to be deleted. Experimental approaches for the deletion of larger chromosomal segments have not been systematically evaluated. RESULTS: We designed three pairs of guide RNAs for deletion of a ~ 2.2 kb chromosomal segment containing the Arabidopsis WRKY30 locus. We tested how the combination of guide RNA pairs and co-expression of the exonuclease TREX2 affect the frequency of wrky30 deletions in editing experiments. Our data demonstrate that compared to one pair of guide RNAs, two pairs increase the frequency of chromosomal deletions. The exonuclease TREX2 enhanced mutation frequency at individual target sites and shifted the mutation profile towards larger deletions. However, TREX2 did not elevate the frequency of chromosomal segment deletions. CONCLUSIONS: Multiplex editing with at least two pairs of guide RNAs (four guide RNAs in total) elevates the frequency of chromosomal segment deletions at least at the AtWRKY30 locus, and thus simplifies the selection of corresponding mutants. Co-expression of the TREX2 exonuclease can be used as a general strategy to increase editing efficiency in Arabidopsis without obvious negative effects.

Peripheral infrastructure vectors and an extended set of plant parts for the Modular Cloning system.

Standardized DNA assembly strategies facilitate the generation of multigene constructs from collections of building blocks in plant synthetic biology. A common syntax for hierarchical DNA assembly following the Golden Gate principle employing Type IIs restriction endonucleases was recently developed, and underlies the Modular Cloning and GoldenBraid systems. In these systems, transcriptional units and/or multigene constructs are assembled from libraries of standardized building blocks, also referred to as phytobricks, in several hierarchical levels and by iterative Golden Gate reactions. Here, a toolkit containing further modules for the novel DNA assembly standards was developed. Intended for use with Modular Cloning, most modules are also compatible with GoldenBraid. Firstly, a collection of approximately 80 additional phytobricks is provided, comprising e.g. modules for inducible expression systems, promoters or epitope tags. Furthermore, DNA modules were developed for connecting Modular Cloning and Gateway cloning, either for toggling between systems or for standardized Gateway destination vector assembly. Finally, first instances of a "peripheral infrastructure" around Modular Cloning are presented: While available toolkits are designed for the assembly of plant transformation constructs, vectors were created to also use coding sequence-containing phytobricks directly in yeast two hybrid interaction or bacterial infection assays. The presented material will further enhance versatility of hierarchical DNA assembly strategies.