Biology subjects

Marillonnet, S.

Publications and source records attributed to Marillonnet, S..

12 recordsLinked to original sources

bioRxiv2026

CAN1-mediated autophagosome size regulation fine-tunes phosphate starvation tolerance in Arabidopsis thaliana

Inorganic phosphate (Pi) availability determines root development and plant performance. In Arabidopsis thaliana, external Pi is sensed by root tips and Pi limitation triggers ER stress-induced autophagy, yet the physiological impact and mechanisms controlling autophagosome biogenesis remain unclear. Here, we identify CANE1 (COMPONENT OF AUTOPHAGIC NETWORK) as a novel plant-specific regulator of autophagosome size in local Pi sensing. CANE1 associates with the plasma membrane-ER tethering protein VAP27-1 and binds to Pi-responsive ATG8 isoforms through conserved interaction motifs. Loss of CANE1 augments ER stress resistance by reducing autophagosome size in Pi-deprived root tips. Our findings establish CANE1 as a determinant of autophagosome size at the ER membrane. Thus, CANE1 functions as a molecular link between Pi-dependent ER stress signaling and fine-tuning of autophagic capacity.

plant biology↗

bioRxiv2025

Cytokinin-mediated trichome initiation in Nicotiana benthamiana upon Agrobacterium tumefaciens infiltration

Infiltration of Agrobacterium tumefaciens in leaves of Nicotiana benthamiana has become a standard method for transient expression in plants. Like many Solanaceae species, N. benthamiana develops on its leaf surface capitate glandular trichomes that produce and secrete acylsugars. We observed that A. tumefaciens strain GV3101 harboring the disarmed Ti plasmid pMP90 induces de novo formation of GTs and increased production of acylsugars when infiltrated in N. benthamiana leaves. In contrast, other strains, such as AGL1 or LBA4404, do not induce GTs. We demonstrate that the presence of the gene coding for the trans-zeatin synthase (tzs) present on pMP90 is responsible for the de novo trichome initiation and that infiltration of cytokinins such as trans-zeatin or benzylaminopurine is sufficient to induce GTs, establishing a direct link between cytokinins and trichome initiation. While these results provide an opportunity to better understand the development of GTs, they make us aware that Agrobacterium infiltration has developmental, biochemical and physiological consequences. To avoid or minimize potential interference of Agrobacterium on the outcome of the infiltration experiments, we recommend testing several strains when establishing a transient assay. Plain Language summaryAgrobacterium infiltration of tobacco leaves is a method of choice for transient expression in plants. Agrobacterium strains that produce cytokinins induce glandular trichome initiation and acylsugar production, providing a new angle for studying trichome development and highlighting side effects of Agro-infiltration that should be considered when designing transient assays.

plant biology↗

bioRxiv2024

Nonuple atg8 mutant provides genetic evidence for functional specialization of ATG8 isoforms in Arabidopsis thaliana

Autophagy sustains cellular health by recycling damaged or excess components through autophagosomes. It is mediated by conserved ATG proteins, which coordinate autophagosome biogenesis and selective cargo degradation. Among these, the ubiquitin-like ATG8 protein plays a central role by linking cargo to the growing autophagosomes through interacting with selective autophagy receptors. Unlike most ATG proteins, the ATG8 gene family is significantly expanded in vascular plants, but its functional specialization remains poorly understood. Using transcriptional and translational reporters in Arabidopsis thaliana, we revealed that ATG8 isoforms are differentially expressed across tissues and form distinct autophagosomes within the same cell. To explore ATG8 specialization, we generated the nonuple{Delta} atg8 mutant lacking all nine ATG8 isoforms. The mutant displayed hypersensitivity to carbon and nitrogen starvation, coupled with defects in bulk and selective autophagy as shown by biochemical and ultrastructural analyses. Complementation experiments demonstrated that ATG8A could rescue both carbon and nitrogen starvation phenotypes, whereas ATG8H could only complement carbon starvation. Proximity labeling proteomics further identified isoform-specific interactors under nitrogen starvation, underscoring their functional divergence. These findings provide genetic evidence for functional specialization of ATG8 isoforms in plants and lay the foundation for investigating their roles in diverse cell types and stress conditions.

plant biology↗

bioRxiv2023

Efficient scar-free knock-ins of several kilobases by engineered CRISPR/Cas endonucleases

In plants and mammals, non-homologous end-joining is the dominant pathway to repair DNA double strand breaks, making it challenging to generate knock-in events. Using a transient assay in Nicotiana benthamiana we identified two groups of exonucleases, respectively from the Herpes Virus and from the bacteriophage T7 families, hat confer up to 38-fold increase in HDR frequencies when fused to Cas9. We achieved precise and scar-free insertion of several kilobases of DNA both in transient and stable transformation systems. In Arabidopsis thaliana, fusion of Cas9 to a Herpes Virus family exonuclease leads to 10-fold higher frequencies of knock-ins. Our results open perspectives for the routine production of knock-in and gene replacement events in plants. One-Sentence SummaryFusions of CRISPR endonucleases to specific 5'-exonucleases leads to significant increase in scar-free multikilobase knock-ins.

molecular biology↗

bioRxiv2022

Molecular cloning and recombinant protein expression of 9-Lipoxygenase gene from Solanum tuberosum in Pichia pastoris yeast cells

Synthetic fungicides have effectively controlled plant pathogenic fungi but their continued use has been detrimental to natural biological systems, and sometimes resulted into development of fungal resistance. They also have undesirable effects on non-target organisms and foster environmental and human health concerns thus new biodegradable alternatives have to be investigated. Lipoxygenases (LOX) are ubiquitous non-heme Iron containing dioxygenases that catalyze the addition of molecular oxygen to Polyunsaturated Fatty Acids (PUFAs) such as Linoleic acid to form Oxylipins that possess anti-microbial activity.The aim of this study was to generate a recombinant 9-Lipoxygenase protein for chemo enzymatic synthesis of Oxylipin based biodegradable fungicides. Golden gate assembly, a molecular cloning method that allows assembly of many DNA fragments into a complete piece using Type II s restriction enzymes and T4 DNA ligase was used to clone the complete 9-LOX gene into the expression plasmid vector pPICZB. Protein expression in Pichia pastoris yeast cells was induced by addition of absolute methanol every after 24h for up to four days. Analysis of protein expression from cell lysates was achieved by SDS-PAGE and Western blotting probed with anti-histidine antibody which showed putative protein bands of 97kDa representing recombinant 9-LOX protein. It is recommended that optimization studies on the yeast kex2 convertase and the - secretion factor can be done to enable secretion of recombinant Solanum tuberosum 9-LOX protein since the protein in this study was recovered from cell lysates.

molecular biology↗

bioRxiv2022

DNA double strand breaks lead to de novo transcription and translation of damage-induced long RNAs in planta

DNA double strand breaks (DSBs) are lethal threats that need to be repaired. Although many of the proteins involved in the early steps of DSB repair have been characterized, recent reports indicate that damage induced long and small RNAs also play an important role in DSB repair. Here, using a Nicotiana benthamiana transgenic line originally designed as a reporter for targeted knock-ins, we show that DSBs generated by Cas9 induce the transcription of long stable RNAs (damage-induced long RNAs - dilRNAs) that are translated into proteins. Using an array of single guide RNAs we show that the initiation of transcription takes place in the vicinity of the DSB. Single strand DNA nicks are not able to induce transcription, showing that cis DNA damage-induced transcription is specific for DSBs. Our results support a model in which a default and early event in the processing of DSBs is transcription into RNA which, depending on the genomic and genic context, can undergo distinct fates, including translation into protein, degradation or production of small RNAs. Our results have general implications for understanding the role of transcription in the repair of DSBs and, reciprocally, reveal DSBs as yet another way to regulate gene expression.

molecular biology↗

bioRxiv2020

A modular two yeast species secretion system for the production and preparative application of fungal peroxygenases

Fungal unspecific peroxygenases (UPOs) are biocatalysts of outstanding interest. Providing access to novel UPOs using a modular secretion system was the central goal of this work. UPOs represent an enzyme class, catalysing versatile oxyfunctionalisation reactions on a broad substrate scope. They are occurring as secreted, glycosylated proteins bearing a haem-thiolate active site and solely rely on hydrogen peroxide as the oxygen source. Fungal peroxygenases are widespread throughout the fungal kingdom and hence a huge variety of UPO gene sequences is available. However, the heterologous production of UPOs in a fast-growing organism suitable for high throughput screening has only succeeded once--enabled by an intensive directed evolution campaign. Here, we developed and applied a modular Golden Gate-based secretion system, allowing the first yeast production of four active UPOs, their one-step purification and application in an enantioselective conversion on a preparative scale. The Golden Gate setup was designed to be broadly applicable and consists of the three module types: i) a signal peptide panel guiding secretion, ii) UPO genes, and iii) protein tags for purification and split-GFP detection. We show that optimal signal peptides could be selected for successful UPO secretion by combinatorial testing of 17 signal peptides for each UPO gene. The modular episomal system is suitable for use in Saccharomyces cerevisiae and was transferred to episomal and chromosomally integrated expression cassettes in Pichia pastoris. Shake flask productions in Pichia pastoris yielded up to 24 mg/L secreted UPO enzyme, which was employed for the preparative scale conversion of a phenethylamine derivative reaching 98.6 % ee. Our results demonstrate a rapid workflow from putative UPO gene to preparative scale enantioselective biotransformations.

synthetic biology↗

bioRxiv2020

Addition of Multiple Introns to a Cas9 Gene Results in Dramatic Improvement in Efficiency for Generation of Gene Knockouts in Plants

The recent discovery of the mode of action of the CRISPR/Cas9 system has provided biologists with a useful tool for generating site-specific mutations in genes of interest. In plants, site-targeted mutations are usually obtained by stably transforming a Cas9 expression construct into the plant genome. The efficiency with which mutations are obtained in genes of interest can vary considerably depending on specific features of the constructs, including the source and nature of the promoters and terminators used for expression of the Cas9 gene and the guide RNA, and the sequence of the Cas9 nuclease itself. To optimize the efficiency with which mutations could be obtained in target genes in Arabidopsis thaliana with the Cas9 nuclease, we have investigated several features of its nucleotide and/or amino acid sequence, including the codon usage, the number of nuclear localization signals (NLS) and the presence or absence of introns. We found that the Cas9 gene codon usage had some effect on Cas9 activity and that two NLSs work better than one. However, the most important impact on the efficiency of the constructs was obtained by addition of 13 introns into the Cas9 coding sequence, which dramatically improved editing efficiencies of the constructs; none of the primary transformants obtained with a Cas9 lacking introns displayed a knockout mutant phenotype, whereas between 70% and 100% of primary transformants generated with intronized Cas9 displayed mutant phenotypes. The intronized Cas9 was also found to be effective in other plants such as Nicotiana benthamiana and Catharanthus roseus.

molecular 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↗

bioRxiv2018

Golden Mutagenesis: An efficient multi-site saturation mutagenesis approach by Golden Gate cloning with automated primer design

Site-directed methods for the generation of genetic diversity are essential tools in the field of directed enzyme evolution. The Golden Gate cloning technique has been proven to be an efficient tool for a variety of cloning setups. The utilization of restriction enzymes which cut outside of their recognition domain allows the assembly of multiple gene fragments obtained by PCR amplification without altering the open reading frame of the reconstituted gene. We have developed a protocol, termed Golden Muta-genesis that allows the rapid, straightforward, reliable and inexpensive construction of mutagenesis libraries. One to five amino acid positions within a coding sequence could be altered simultaneously using a protocol which can be performed within one day. To facilitate the implementation of this technique, a software library and web application for automated primer design and for the graphical evaluation of the randomization success based on the sequencing results was developed. This allows facile primer design and application of Golden Mutagenesis also for laboratories, which are not specialized in molecular biology.

molecular 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↗