Biology subjects

Kretschmer, C.

Publications and source records attributed to Kretschmer, C..

9 recordsLinked to original sources

bioRxiv2026

Molecular and phenotypic footprints of climate in native Arabidopsis thaliana

Climate change poses a major threat to humanity by driving biodiversity loss and reducing crop yields1,2. To understand the molecular and developmental impacts of rising temperatures, plant science has relied heavily on the model organism Arabidopsis thaliana. Despite decades of research, its development under fully natural conditions remains poorly understood, and only [~]30% of genes have experimental functional annotations, largely because many functions are subtle or manifest only in specific laboratory or ecological contexts3. Here, we address this gap with a landscape transcriptomic approach that integrates intensive phenotyping and transcriptomic profiling of naturally occurring plants in their native habitats4. Across two contrasting field sites and five growing seasons (2021-2025), we phenotyped more than 3,000 A. thaliana plants and generated >1,600 matching transcriptomes. The resulting >30,000 quantitative trait measurements provide a unique opportunity to link climate fluctuations with plant traits and gene expression. Seasons characterized by extreme temperature anomalies directly influenced plant traits, and climatic variables together explained up to 17% of phenotypic variation. In situ transcriptomes carried clear temperature and local environmental signatures, closely matching temperature-response programs known from the laboratory. Leveraging paired per-plant transcriptomes and phenotypes, we applied machine learning to predict regulators of climate-relevant and other plant traits under natural conditions. The models recovered canonical thermomorphogenesis regulators, including PHYTOCHROME INTERACTING FACTOR 4 (PIF4)5,6, providing ecological evidence that temperature signaling pathways defined in controlled environments operate in the wild, and expanded this regulatory landscape by identifying hormonal receptors, signaling components, and previously uncharacterized genes, some of which we functionally validated. Together, this work demonstrates that landscape transcriptomics, by integrating natural field transcriptomes with phenotypes, and thus, capturing environmental and regulatory states, enables the predictive identification of genetic regulators of temperature responses and broader plant traits. This makes landscape transcriptomics a scalable framework for climate-aware functional genomics in plants.

plant biology↗

bioRxiv2023

Receptor-like cytoplasmic kinases belonging to different subfamilies mediate immune responses downstream of the Cf-4 resistance protein in Nicotiana benthamiana

Cell-surface receptors, which are either receptor-like proteins (RLPs) or receptor-like kinases (RLKs), form the first layer of the plant innate immune system. The LRR-RLKs SUPPRESSOR OF BIR1-1 (SOBIR1) and BRASSINOSTEROID INSENSITIVE 1-ASSOCIATED KINASE 1 (BAK1) are the common regulatory co-receptors of LRR-RLPs. The tomato LRR-RLP Cf-4, which specifically detects the apoplastic effector Avr4 that is secreted by the pathogenic intercellular fungus Cladosporium fulvum, requires SOBIR1 and BAK1 to mediate resistance against this fungus. It is proposed that phosphorylation events take place between the cytoplasmic kinase domains of SOBIR1 and BAK1, which lead to the initiation of various downstream signaling outputs such as the rapid phosphorylation of receptor-like cytoplasmic kinases (RLCKs), the accumulation of reactive oxygen species (ROS), the activation of mitogen-activated protein kinase (MAPK) cascades, and in some cases, the hypersensitive response (HR). Here, by performing in vitro phosphorylation assays, we show that SOBIR1 directly phosphorylates BAK1, whereas BAK1, in its turn, directly phosphorylates SOBIR1, which results in the full activation of the Cf-4-containing immune complex. We found that threonine 522, present in the activation segment of the SOBIR1 kinase domain of Nicotiana benthamiana (Nb), is required for its intrinsic kinase activity, and is essential for the Avr4/Cf-4/SOBIR1/BAK1-triggered ROS burst, MAPK activation and the HR. Tyrosine residue Y469 was found to be crucial for the Avr4/Cf-4- triggered activation of MAPKs and the HR, but not for ROS production and NbSOBIR1 intrinsic kinase activity. RLCKs are well-recognized to act as the initial cytoplasmic transducers, bridging cell-surface receptor complexes with their downstream signaling partners. By knocking out multiple genes belonging to different RLCK-VII subfamilies in N. benthamiana plants stably expressing Cf-4, we show that members of the RLCK-VII subfamilies 6, 7 and 8 are required for the Avr4/Cf-4-triggered ROS production. Typically, the Avr4 protein triggers a biphasic ROS burst in leaf discs obtained from N. benthamiana:Cf-4 plants, in which the first transitory response is followed by a second, sustained ROS burst. Interestingly, the intensities of the first and second phase of the ROS burst are affected in different ways in the various rlck-vii knock-out plants, indicating that there are different downstream ROS regulatory mechanisms, involving different RLCKs in N. benthamiana. Further studies show that members from RLCK-VII-6, 7 and 8 also play an essential role in regulating the ROS production triggered by flg22, chitin, and the nlp20/RLP23 and the pg13/RLP42 combinations.

plant biology↗

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↗

bioRxiv2021

Differential requirement for the EDS1 catalytic triad in A. thaliana and N. benthamiana

O_LIHeterodimeric 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 A. thaliana, bolstering of signaling and resistance mediated by cell-surface pattern recognition receptors (PRRs). Biochemical activities underlying these mechanistic frameworks remain unknown. C_LIO_LIWe used CRISPR/Cas-generated mutant lines and agroinfiltration-based complementation assays to interrogate functions of EDS1 complexes in N. benthamiana. C_LIO_LIWe do not detect impaired PRR signaling in N. benthamiana lines deficient in EDS1 complexes or RNLs. Intriguingly, mutations within the catalytic triad of Solanaceae EDS1 can abolish or enhance TNL immunity in N. benthamiana. Furthermore, nuclear EDS1 accumulation is sufficient for N. benthamiana TNL (Roq1) immunity. C_LIO_LIReinforcing PRR signaling in Arabidopsis might be a derived function of the TNL/EDS1 immune sector. Dependency of Solanaceae but not A. thaliana EDS1 on catalytic triad residues raises the possibility that a TNL-derived small molecule binds to the Solanaceae EDS1 lipase-like domain, and that EDS1 lipase-like domain pocket contributions to TNL immune responses vary between lineages. Whether and how nuclear EDS1 activity connects to membrane pore-forming RNLs remains unknown. C_LI

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

Determining targeting specificity of nuclear-encoded organelle proteins with the 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 have improved this technology with respect to sensitivity and systematically evaluated its utilization to determine protein targeting to plastids and mitochondria. Furthermore, to facilitate high throughput screening of candidate proteins we have developed a Golden Gate-based vector toolkit, named PlaMiNGo (Plastid and/or Mitochondria targeted proteins N-terminally fused to GFP11 tags via Golden Gate cloning). As a result of these improvements, dual targeting could be detected for a number of proteins, which had earlier been characterized as being targeted to a single organelle only. These results were independently confirmed with a plant phenotype complementation approach thus demonstrating the sensitivity and robustness of the sasplit-FP-based method to analyze the targeting specificity of nuclear-encoded proteins. HighlightSeveral mono-specific proteins showed dual targeting to plastids and mitochondria with the self-assembling split-GFP system. A Golden Gate-based vector toolkit was constructed to facilitate easy cloning and subsequent determination of protein targeting specificity.

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↗