Biology subjects

Ordon, J.

Publications and source records attributed to Ordon, J..

7 recordsLinked to original sources

bioRxiv2023

Simultaneous tracking of near-isogenic bacterial strains in synthetic Arabidopsis microbiota by chromosomally-integrated barcodes

DNA amplicon-based microbiota profiling currently relies on polymorphisms in microbial marker genes to estimate species diversity and abundance. However, this technique cannot resolve genetic differences among microbial individuals of the same species. We report here the development of modular bacterial tags (MoBacTags) encoding DNA barcodes. These tags facilitate tracking of near-isogenic bacterial commensals in synthetic communities (SynComs), which allow assessment of the contributions of individual bacterial genes to root microbiota establishment in Arabidopsis thaliana. Chromosomally-integrated DNA barcodes are co-amplified with endogenous marker genes of the community by integrating corresponding primer binding sites into the barcode. We generated MoBacTag-labeled strains of wild-type Pseudomonas capeferrum WCS358 and of pqqF and cyoB mutants with known defects in gluconic acid-mediated host immunosuppression and validated reduced root colonization of both mutants in a 15-member synthetic microbiota. We detected a reduced SynCom load on roots in the presence of the WCS358:pqqF mutant, but not WCS358:cyoB, revealing distinct pqqF and cyoB activities in a community context. Using MoBacTags, we also show that WCS358 pqqF mutant-specific colonization and community establishment is not trans-complemented by wild-type WCS358. Given that gluconic acid production in P. capeferrum is indirectly abolished in the pqqF mutant by disruption of pyrroloquinoline quinone (PQQ) biosynthesis, we propose that drastic changes in the root-associated community result from depletion of the cofactor PQQ, which might serve as a common good during root microbiota establishment. Our proof-of-principle experiments illustrate how MoBacTags can be applied to assess scaling of individual bacterial genetic determinants in the plant microbiota.

microbiology↗

bioRxiv2023

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

BackgroundIn 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 fragment to be deleted. Experimental approaches for deletion induction have not been systematically evaluated. ResultsWe designed three pairs of guide RNAs for deletion of the Arabidopsis WRKY30 locus (~2.2 kb). 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 deletions. ConclusionsMultiplex editing with at least two pairs of guide RNAs (four guide RNAs in total) elevates the frequency of chromosomal deletions, 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.

plant biology↗

bioRxiv2020

Disentangling cause and consequence: Genetic dissection of the DANGEROUS MIX2 risk locus, and activation of the DM2h NLR in autoimmunity

Nucleotide-binding domain-leucine-rich repeat-type immune receptors (NLRs) protect plants against pathogenic microbes through intracellular detection of effector proteins. However, this comes at a cost, as NLRs can also induce detrimental autoimmunity in genetic interactions with foreign alleles. This may occur when independently evolved genomes are combined in inter- or intraspecific crosses, or when foreign alleles are introduced by mutagenesis or transgenesis. Most autoimmunity-inducing NLRs are encoded within highly variable NLR gene clusters with no known immune functions, which were termed autoimmune risk loci. Whether risk NLRs differ from sensor NLRs operating in natural pathogen resistance and how risk NLRs are activated in autoimmunity is unknown. Here, we analyzed the DANGEROUS MIX2 risk locus, a major autoimmunity hotspot in Arabidopsis thaliana. By gene editing and heterologous expression, we show that a single gene, DM2h, is necessary and sufficient for autoimmune induction in three independent cases of autoimmunity in accession Landsberg erecta. We focus on autoimmunity provoked by an EDS1-YFPNLS fusion protein to functionally characterize DM2h and determine features of EDS1-YFPNLS activating the immune receptor. Our data suggest that risk NLRs function reminiscent of sensor NLRs, while autoimmunity-inducing properties of EDS1-YFPNLS are in this context unrelated to the proteins functions as immune regulator. We propose that autoimmunity may, at least in some cases, be caused by spurious, stochastic interactions of foreign alleles with co-incidentally matching risk NLRs.

plant biology↗

bioRxiv2020

One-shot generation of duodecuple (12x) mutant Arabidopsis: Highly efficient routine editing in model species

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 sgRNAs. We used this toolkit to explore the limits of multiplexing in two major model species, and report on isolation of transgene-free octuple Nicotiana benthamiana and duodecuple (12x) 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 production of toxic 5-fluorouracyl in 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 (~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 identify 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.

plant biology↗

bioRxiv2019

An EDS1-SAG101 complex functions in TNL-mediated immunity in Solanaceae

EDS1 (Enhanced disease susceptibility 1) forms mutually exclusive heterodimers with its interaction partners PAD4 (Phytoalexin-deficient 4) and SAG101 (Sensecence-associated gene 101). Collectively, these complexes are required for resistance responses mediated by nucleotide-binding leucine-rich repeat-type immune receptors (NLRs) possessing an N-terminal Toll-interleukin-1 receptor-like domain (TNLs). Here, immune functions of EDS1 complexes were comparatively analyzed in a mixed species approach relying on Nicotiana benthamiana (Nb), Solanum lycopersicum (Sl) and Arabidopsis thaliana (At). Genomes of most Solanaceae plants including Nb and Sl encode for two SAG101 isoforms, which engage into distinct complexes with EDS1. By a combination of genome editing and transient complementation, we show that one of these EDS1-SAG101 complexes, and not an EDS1-PAD4 complex as previously described in At, is necessary and sufficient for all tested TNL-mediated immune responses in Nb. Intriguingly, not this EDS1-SAG101 module, but mainly Solanaceae EDS1-PAD4 execute immune functions when transferred to At, and TNL functions are not restored in Nb mutant lines by expression of At EDS1 complexes. We conclude that EDS1 complexes do not represent a complete functional module, but co-evolve with additional factors, most likely protein interaction partners, for their function in TNL signaling networks of individual species. In agreement, we identify a large surface on SlEDS1 complexes required for immune activities, which may function in partner recruitment. We highlight important differences in TNL signaling networks between At and Nb, and genetic resources in the Nb system will be instrumental for future elucidation of EDS1 molecular functions.

plant biology↗

bioRxiv2018

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.

plant biology↗

bioRxiv2017

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. This combinatorial assembly strategy meets the increasingly complex demands in biotechnology and bioengineering, and also represents a cost-efficient and versatile alternative to previous molecular cloning techniques. For Modular Cloning, a collection of commonly used Plant Parts was previously released together with the Modular Cloning toolkit itself, which largely facilitated the adoption of this cloning system in the research community. Here, a collection of approximately 80 additional phytobricks is provided. These phytobricks comprise e.g. modules for inducible expression systems, different promoters or epitope tags, which will increase the versatility of Modular Cloning-based DNA assemblies. Furthermore, 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. Additionally, DNA modules and assembly strategies for connecting Modular Cloning with Gateway Cloning are presented, which may serve as an interface between available resources and newly adopted hierarchical assembly strategies. The presented material will be provided as a toolkit to the plant research community and will further enhance the usefulness and versatility of Modular Cloning.

plant biology↗