Biology subjects

Tissier, A. F.

Publications and source records attributed to Tissier, A. F..

11 recordsLinked to original sources

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

Metabolome and transcriptome profiling of root chicory provide insights into laticifer development and specialized metabolism

Chicory roots produce inulin, a dietary fiber, as well as large quantities of bitter sesquiterpene lactones (STLs), which have valuable biological activities. In an effort to understand the compartmentalization of metabolism within chicory roots and the molecular basis of the development of laticifers that produce the chicory latex, we performed metabolomics and transcriptomics profiling. GC-MS and LC-MS identified a total of 22 580 features of which 135 were differentially abundant between cell types. Further analysis indicated that the major STLs accumulated primarily in the latex. Gene expression of known STL pathway genes indicates a compartmentalization of the biosynthesis across multiple tissues, with implications regarding the trafficking of pathway intermediates. Phytohormone measurements and gene expression analysis point to a major role for jasmonate signaling in the development and differentiation of laticifers. Furthermore, inulin accumulates mostly outside the laticifers but expression of inulin metabolic genes also point to a complex distribution and trafficking of inulin or inulin precursors across different root compartments. Altogether, the data presented here constitute a unique resource to investigate several biological processes in chicory roots, including laticifer development, STL biosynthesis and transport and inulin biosynthesis regulation. Significance statementA combination of transcriptomics, targeted and untargeted metabolomics of different tissues of chicory roots was generated. These data constitute a resource basis for the investigation of various processes taking place in chicory taproots, including sesquiterpene lactone biosynthesis, laticifer development and inulin biosynthesis and trafficking.

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↗

bioRxiv2023

Heat stress induces a developmental shift from type-V to type-IV trichome dependent on jasmonate signaling in tomato

Cultivated tomato (Solanum lycopersicum) and related wild species develop several types of trichomes, both glandular and non-glandular, on their aerial parts. Among these, type-IV trichomes are responsible for the synthesis and secretion of acylsugars, which act as defense compounds against herbivores. In contrast to related wild species such as S. pennellii, type-IV trichomes are present only in the juvenile stages of cultivated tomato plants and absent in later stages of development. By submitting tomato plants to high temperatures during the day (37 {degrees}C), we observe that non-glandular type-V trichomes are replaced by type-IV trichomes. This is accompanied by a massive increase in acylsugar production. On the other hand, heat treatment does not affect type VI-trichomes, which produce mono- and sesquiterpenes, but the production of monoterpenes is increased while that of sesquiterpenes is suppressed. Furthermore, tomato jai1 mutants deficient in jasmonate (JA) perception do not exhibit this developmental switch from type-V to type-IV trichomes. The implication of JA signaling in this process was further supported by an increase in JA-isoleucine and in the expression of genes involved in JA-signalling within hours of heat stress application. These results establish a unique system to study how environmental factors affect developmental fate decisions in plants while opening opportunities to understand mechanisms controlling type-IV trichome initiation and development.

plant biology↗

bioRxiv2023

Metabolic arms race between a plant and a fungal pathogen

In this work, we uncover a metabolite interaction between barley and the fungal pathogen Bipolaris sorokiniana (Bs), involving hordedanes, a previously undescribed set of labdane-related diterpenoids with antimicrobial properties. Bs infection of barley roots elicits hordedane synthesis from a 600-kb gene cluster. Heterologous reconstruction of the synthesis pathway in yeast produced several hordedanes, including one of the most advanced products 19-b-hydroxy-hordetrienoic acid (19-OH-HTA). Barley mutants in the diterpene synthase genes of the cluster are unable to produce hordedanes but, unexpectedly, show reduced Bs colonization. Accordingly, 19-OH-HTA enhances both germination and growth of Bs, while it inhibits other fungi, and Bs chemically modifies 19-OH-HTA. Thus, plant and pathogen molecular interactions extend beyond protein-protein recognition and the simple detoxification of plant antimicrobial metabolites. One-Sentence SummaryA fungal pathogen uses barley diterpenoid phytoalexins to facilitate root colonization.

plant biology↗

bioRxiv2023

Production of 130 diterpenoids by combinatorial biosynthesis in yeast

Diterpenoids form a diverse group of natural products, many of which are or could become pharmaceuticals or industrial chemicals. However, low concentration, presence in complex mixtures and challenging synthesis often limit their exploitation. The modular character of diterpene biosynthesis and the substrate flexibility of the enzymes involved make combinatorial biosynthesis a promising approach. Here, we report on the assembly in yeast of 130 diterpenoids by pairwise combinations of ten diterpene synthases producing (+)-copalyl diphosphate-derived backbones and four cytochrome P450 enzymes (CYPs); 80 of these diterpenoids have not yet been reported. The CYPs accepted the majority of substrates they were given but remained regioselective. Our results bode well for the systematic exploration of diterpenoid chemical space using combinatorial assembly in yeast.

synthetic biology↗

bioRxiv2022

Bioenergetics of the secondary metabolites production in photosynthetic glandular trichomes

Several commercially important secondary metabolites are produced and accumulated in high amounts by glandular trichomes, giving the prospect of using them as metabolic cell factories. Due to extremely high metabolic fluxes through glandular trichomes, previous research focused on how such flows are achieved. The question regarding their bioenergetics became even more interesting with the discovery of photosynthetic activity in some glandular trichomes. Despite recent advances, how primary metabolism contributes to the high metabolic fluxes in glandular trichomes is still not fully elucidated. Using computational methods and available multi-omics data, we first developed a quantitative framework to investigate the possible role of photosynthetic energy supply in terpenoid production and next tested experimentally the simulation-driven hypothesis. With this work, we provide the first reconstruction of specialised metabolism in Type-VI photosynthetic glandular trichomes of Solanum lycopersicum. Our model predicted that increasing light intensities results in a shift of carbon partitioning from catabolic to anabolic reactions driven by the energy availability of the cell. Moreover, we show the benefit of shifting between isoprenoid pathways under different light regimes, leading to a production of different classes of terpenes. Our computational predictions were confirmed in vivo, demonstrating a significant increase in production of monoterpenoids while the sesquiterpenes remained unchanged under higher light intensities. The outcomes of this research provide quantitative measures to assess the beneficial role of chloroplast in glandular trichomes for enhanced production of secondary metabolites and can guide the design of new experiments that aim at modulating terpenoid production. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=200 SRC="FIGDIR/small/510054v2_ufig1.gif" ALT="Figure 1"> View larger version (9K): org.highwire.dtl.DTLVardef@2ec6f4org.highwire.dtl.DTLVardef@107d0c1org.highwire.dtl.DTLVardef@1a545corg.highwire.dtl.DTLVardef@98747b_HPS_FORMAT_FIGEXP M_FIG C_FIG Please check the journals author guidelines for whether a graphical abstract, key points, new findings, or other items are required for display in the Table of Contents.

plant 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↗

bioRxiv2022

A fungal endophyte-generated nucleoside signal regulates host cell death and promotes root colonization

The intracellular colonization of plant roots by the beneficial fungal endophyte Serendipita indica follows a biphasic strategy. After an early biotrophic phase, the interaction transitions to a host cell death phase restricted to the epidermal and cortex layers of the root. Host cell death contributes to the successful accommodation of the fungus during the beneficial interaction in Arabidopsis thaliana. How host cell death is initiated and controlled is largely unknown. Here we show that two fungal enzymes, the ecto-5-nucleotidase SiE5NT and the nuclease SiNucA, act synergistically in the plant apoplast at the onset of cell death to produce deoxyadenosine (dAdo), a potent cell death inducer in animal systems. The uptake of extracellular dAdo, but not the structurally related adenosine (Ado), activates a previously undescribed cell death mechanism in A. thaliana. Mutation of the equilibrative nucleoside transporter ENT3 in A. thaliana results in resistance to cell death triggered by extracellular dAdo and reduced fungal-mediated cell death during root colonization. A library screen of A. thaliana T-DNA insertion lines identified a toll/interleukin-1 receptor nucleotide-binding leucine-rich repeat (TIR-NLR) protein as an additional intracellular component in dAdo-triggered cell death. Mutation of this previously uncharacterised TIR-NLR, which we have named ISI (induced by S. indica), affects host cell death, fungal colonization and growth promotion, suggesting a key role in the regulation of root cell death and plant-microbe interaction. Our data show that the combined activity of two fungal apoplastic enzymes leads to the production of a metabolite that, upon uptake, triggers TIR-NLR-modulated plant cell death, providing a link to immunometabolism in plants. Short summaryEfficient intraradical colonization by the beneficial fungal endophyte Serendipita indica requires restricted host cell death. How this symbiotic host cell death is initiated and controlled is largely unknown. Here we show that two fungal enzymes, the ecto-5-nucleotidase SiE5NT and the nuclease SiNucA, act synergistically in the apoplast at the onset of cell death to produce deoxyadenosine (dAdo), a potent cell death inducer in animal systems. Uptake of extracellular dAdo activates a previously undescribed cell death mechanism in plants. Mutation of the A. thaliana equilibrative nucleoside transporter ENT3 leads to resistance to cell death triggered by uptake of extracellular dAdo and to reduced fungal-mediated cell death during colonization. A library screen of A. thaliana T-DNA insertion lines identified a TIR-NLR protein as an additional intracellular component in dAdo-triggered cell death, providing a link to immunometabolism in plants. In a nutshellRegulated host cell death is part of the plant defense strategy against pathogens, but it is also involved in the accommodation of certain beneficial microbes in the roots. We have identified extracellular metabolites and intracellular metabolic signals that contribute to colonization by beneficial root fungal endophytes and uncovered a conserved cell death mechanism likely co-opted for establishing plant-endophyte symbiosis.

plant biology↗

bioRxiv2021

A barley gene cluster for the biosynthesis of diterpenoid phytoalexins

Phytoalexins are specialized metabolites that are induced upon pathogen infection and contribute to the defense arsenal of plants. Maize and rice produce multiple diterpenoid phytoalexins and there is evidence from genomic sequences that other monocots may also produce diterpenoid phytoalexins. Here we report on the identification and characterization of a gene cluster in barley (Hordeum vulgare cv. Golden Promise) that is involved in the production of a set of labdane-related diterpenoids upon infection of roots by the fungal pathogen Bipolaris sorokiniana. The cluster is localized on chromosome 2, covers over 600 kb and comprises genes coding for a (+)-copalyl diphosphate synthase (HvCPS2), a kaurene synthase like (HvKSL4) and several cytochrome P450 oxygenases (CYPs). Expression of HvCPS2 and HvKSL4 in yeast and Nicotiana benthamiana resulted in the production of a single major product, whose structure was determined to be of the cleistanthane type and was named hordediene. Co-expression of HvCPS2, HvKSL4 and one of the CYPs from the cluster (CYP89E31) afforded two additional products, hordetriene and 11-hydroxy-hordetriene. Both of these compounds could be detected in extracts of barley roots infected by B. sorokiniana, validating the function of these genes in planta. Furthermore, diterpenoids with multiple oxidations and with molecular masses of 316, 318 and 332 were induced in infected barley roots and secreted in the medium, indicating that additional oxidases, possibly from the same genomic cluster are involved in the production of these phytoalexins. Our results provide the basis for further investigation of the role of this gene cluster in the defense of barley against pathogens and more generally in the interaction with the microbiome.

plant biology↗

bioRxiv2020

Two novel 7-epi-zingiberene derivatives with biological activity from Solanum habrochaites are produced by a single cytochrome P450 monooxygenase

Secretions from glandular trichomes potentially protect the plant against a variety of aggressors. In the tomato genus, wild species constitute a rich source of chemical diversity produced at the leaf surface by glandular trichomes. Previously, 7-epi-zingiberene produced in several accessions of Solanum habrochaites was found to confer resistance to whiteflies (Bemisia tabaci) and other insect pests. Here, we identify two derivatives of 7-epi-zingiberene from S. habrochaites that had not been reported as yet. We identified them as 9-hydroxy-zingiberene and 9-hydroxy-10,11-epoxyzingiberene. Using a combination of genetics and transcriptomics we identified a single cytochrome P450 oxygenase, ShCYP71D184 that carries out two successive oxidations to generate the two sesquiterpenoids. Bioactivity assays showed that only 9-hydroxy-10,11-epoxyzingiberene exhibits substantial toxicity against B. tabaci. In addition, both 9-hydroxy-zingiberene and 9-hydroxy-10,11-epoxyzingiberene display substantial growth inhibitory activities against a range of microorganisms, including Bacillus subtilis, Phytophtora infestans and Botrytis cinerea. Our work shows that trichome secretions from wild tomato species can provide protection against a wide variety of organisms. In addition, the availability of the genes encoding the enzymes for the pathway of 7-epi-zingiberene derivatives makes it possible to introduce this trait in cultivated tomato by precision breeding.

plant biology↗