Biology subjects

Michaud, D.

Publications and source records attributed to Michaud, D..

13 recordsLinked to original sources

bioRxiv2025

GRAS salts and eastern hemlock extract: a dual approach to sustainable plant disease management‎‎

Several studies assessed the potential of salts and plant extracts as sustainable, eco-friendly phytosanitary products in plant protection. Here, we examined the efficacy of Generally Recognized as Safe (GRAS) salts sodium benzoate (SBE) and sodium bicarbonate (SBI) used alone or in combination with a twig extract from eastern hemlock (EH) against the bacterial pathogen Pseudomonas syringae pv. tomato DC3000 (Pst DC3000). The antimicrobial activity of the three products, and their ability to bolster host plant natural defenses, were assessed using the Arabidopsis-Pst DC3000 pathosystem as a model. RT-qPCR and leaf staining assays on a transgenic Arabidopsis line engineered to express reporter protein {beta}-glucuronidase under the control of pathogen-inducible promoter PR1 showed both GRAS salts and the EH extract to trigger the expression of salicylic acid (SA)-inducible genes. Salt- and EH extract-treated plants exhibited callose deposits in leaf tissue, further pointing to the induction of the SA signaling pathway and suggesting the establishment of a physical barrier in the leaf apoplast upon treatment. Direct toxicity measurements and a bacterial infection assay in growth chamber showed both GRAS salts and the EH extract to inhibit Pst DC3000 growth and Arabidopsis leaf colonization. Toxicity assays also revealed complementary, additive effects between the two salts and the EH extract in their detrimental effect against the pathogen, as evidenced by reduced minimum inhibitory concentrations when used in combination. Interestingly, SBE and SBI strongly delayed leaf senescence following infection, either alone or combined with the EH extract. Together, these findings confirm the potential of SBE, SBI and the EH twig extract as both toxic compounds against Pst DC3000 and natural defense stimulators in Arabidopsis. They also highlight the potential of GRAS salt and plant extract combinations as a sustainable alternative to chemical pesticides in plant pathogen management.

plant biology↗

bioRxiv2025

Gene editing of Nicotiana benthamiana architecture for space-efficient production of recombinant proteins in controlled environments

Indoor vertical farming (VF) offers several practical advantages for the cultivation of plant protein bio-factories, including plant uniformity, product consistency, water/nutrient recycling and production cycles on a year-round basis. Much progress has been achieved in recent years toward the development of innovative systems for artificial lighting, automated irrigation, plant handling, environment control and space use optimization in VF systems. Here, we used a CRISPR-Cas9 gene editing approach to generate mutant lines of transient protein expression host Nicotiana benthamiana presenting a compact, space-efficient phenotype compared to the so-called LAB strain commonly used for protein production. Our strategy consisted of altering apical dominance by suppressing the biosynthesis of strigolactone, a negative regulator of axillary bud outgrowth-promoting cytokinins. Strigolactone-depleted lines were generated by knocking-down the expression of either Carotenoid cleavage dioxygenase 7 (CCD7) or Carotenoid cleavage dioxygenase 8 (CCD8), two key enzymes of the metabolic pathway leading to strigolactone synthesis. Knocking-down the genes of either enzyme had no impact on the overall growth rate of the plant but drastically influenced its leaf proteome, auxin/cytokinin ratio and overall architecture. More specifically, the {Delta}CCD mutants exhibited altered glycolytic and malate-processing enzyme fluxes driving the production of pyruvate and cytokinins in leaf tissue, an axillary growth-oriented development pattern and, most importantly, a spatial footprint reduced by 45% to 50% compared to the LAB strain. Most importantly, recombinant protein yields per plant were maintained in the mutant lines, as here illustrated for the model protein GFP and for rituximab, a chimeric monoclonal antibody of confirmed clinical value in humans. Our data demonstrate the usefulness of {Delta}CCD7 and {Delta}CCD8 knockout leading to strigolactone depletion for the generation of compact, space-efficient N. benthamiana lines well suited to VF systems intended for biopharmaceutical production.

plant biology↗

bioRxiv2025

Comparative Machine Learning Analysis of Saliva and Plaque Microbiomes in Children with Type 1 Diabetes

BackgroundType 1 diabetes (T1D) is associated with microbial dysbiosis. While most research has focused on the gut microbiome, limited data address the role of the oral microbiome in T1D. The oral and gut microbiomes overlap substantially, and the oral cavity may influence the gut microbial composition. Saliva and dental plaque represent two distinct oral niches with unique microbial communities, but it remains unclear which better reflects systemic disease states such as T1D. This study compared the performance of salivary and plaque microbiomes in classifying pediatric T1D status. MethodsPaired saliva and plaque samples were collected from 46 children (23 with T1D and 23 healthy controls). Microbial DNA was extracted and sequenced to target the 16S rRNA gene. The data were processed via QIIME 2 for taxonomic classification and centered log-ratio transformation. Alpha diversity, microbial abundance, and clustering analyses were performed to compare the oral microbiome between the T1D and control groups. Random forest classifiers were used to evaluate and compare the predictive accuracy of saliva- and plaque-based models, both with and without clinical metadata. ResultsSaliva samples presented lower alpha diversity than plaque samples did but presented significantly greater bacterial loads and total microbial abundances. Saliva-based models outperformed plaque-based models, achieving a classification accuracy of 94.2% with or without clinical metadata, compared with 73.3% accuracy for plaque-based models. ROC curve analysis further supported this difference, with saliva models reaching an AUC of approximately 0.94 versus 0.75 for plaque, indicating superior discriminative performance. UMAP clustering revealed more distinct separation of the T1D and control groups in terms of the salivary profiles than in the plaque profiles. Feature importance analysis revealed both unique and shared taxa predictive of T1D in each niche. The incorporation of clinical and demographic metadata did not enhance model performance, underscoring the robustness and predictive strength of microbiome data alone. ConclusionThe salivary microbiome is a more effective biospecimen than dental plaque for detecting T1D-associated microbial profiles in children. It offers superior classification accuracy and greater sensitivity in distinguishing T1D status, supporting salivas potential as a noninvasive, scalable medium for future microbiome-based monitoring.

microbiology↗

bioRxiv2025

Tumor-myeloid crosstalk drives therapy resistance in localized bladder cancer

Neoadjuvant cisplatin-based chemotherapy results in pathologic complete response for only a minority of patients with muscle-invasive bladder cancer (MIBC), and mechanisms of resistance and the effects of chemotherapy on the MIBC microenvironment remain incompletely understood. Here, we defined the single-cell and spatial transcriptomes of cancer and immune cells from MIBC patients with resistance to cisplatin-based chemotherapy. Tumors with persistent MIBC after chemotherapy harbored cancer cells expressing epithelial-to-mesenchymal programs that were associated with worse overall survival in independent cisplatin-treated bladder cancer cohorts. These cisplatin-resistant tumor cells were infiltrated by macrophages that upregulated tumor permissive programs defined by increased PARP14 expression in spatially resolved multicellular niches. Macrophage reprogramming through PARP14 inhibition sensitized tumors to cisplatin via downregulation of tumor cell pathways implicated in resistance. Our results demonstrate that cancer cells and macrophages cooperate to promote cisplatin resistance and identify macrophage-directed PARP14 inhibition as a novel therapeutic strategy to sensitize MIBC to cisplatin.

cancer biology↗

bioRxiv2024

Molecular changes in agroinfiltrated leaves of Nicotiana benthamiana expressing suppressor of silencing P19 and coronavirus-like particles

The production of coronavirus disease 2019 vaccines can be achieved by transient expression of the Spike (S) protein of Severe Acute Respiratory Syndrome Coronavirus 2 in agroinfiltrated leaves of Nicotiana benthamiana. Relying on bacterial vector Agrobacterium tumefaciens, this process is favored by the co-expression of viral silencing suppressor P19. Upon expression, the S protein enters the cell secretory pathway, before being trafficked to the plasma membrane where formation of coronavirus-like particles (CoVLPs) occurs. We previously characterized effects of influenza virus hemagglutinin forming VLPs through similar processes. However, leaf samples were only collected after six days of expression and it remains unknown whether influenza VLPs and CoVLPs induce similar responses. Here, time course sampling was used to profile responses of N. benthamiana leaf cells expressing P19 only, or P19 along with the S protein. The latter triggered early, but transient activation of the unfolded protein response and waves of transcription factor genes involved in immunity. Accordingly, defense genes were induced with different expression kinetics, including those promoting lignification, terpene biosynthesis, and oxidative stress. Crosstalk between stress hormone pathways also occurred, notably leading to the repression of jasmonic acid biosynthesis genes after agroinfiltration, and dampening of salicylic acid-inducible responses upon S protein accumulation. Overall, influenza VLP- and CoVLP-induced responses broadly overlapped, suggesting nanoparticle production to have the most effects on plant immunity, regardless of the virus surface proteins expressed. Taking advantage of RNAseq inferences, we finally show the co-expression of Kunitz trypsin inhibitors to reduce CoVLP-induced defense and leaf symptoms, with no adverse effect on plant productivity.

plant biology↗

bioRxiv2024

Expression of a constitutively active nitrate reductase increases SARS-CoV-2 Spike protein production in Nicotiana benthamiana leaves that otherwise show traits of senescence

The production of coronavirus disease 2019 vaccines can be achieved by transient expression of the Spike (S) protein of Severe Acute Respiratory Syndrome Coronavirus 2 in agroinfiltrated leaves of Nicotiana benthamiana, a process promoted by the co-expression of viral silencing suppressor P19. Upon expression, the S protein enters the cell secretory pathway, before being trafficked to the plasma membrane where formation of coronavirus-like particles (CoVLPs) occurs. We recently used RNAseq and time course sampling to characterize molecular responses of N. benthamiana leaf cells expressing P19 only, or P19 in combination with recombinant S protein. This revealed expression of the viral proteins to deeply affect the physiological status of plant cells, including through the activation of immune responses. Here, transcriptomics shows that the production of CoVLPs also induces leaf senescence, as revealed by the upregulation of senescence-associated genes, activation of senescence-related proteases, and downregulation of genes involved in basic metabolic functions like photosynthesis or nitrogen uptake and assimilation. CoVLP production also upregulated asparagine synthetase genes and led to consequent accumulation of asparagine, a nitrogen-rich amino acid is known to facilitate the reallocation of nitrogen resources from senescent to young growing organs. Hypothesizing these combined host responses to restrain foreign protein accumulation, an attempt was made to support nitrogen reduction in CoVLP-producing leaves by co-expressing a constitutively active, light-insensitive form of the nitrate reductase. We show this strategy to increase S protein accumulation in leaf tissues, thereby suggesting that boosting nitrogen metabolism of agroinfiltrated leaves improves recombinant protein yields in N. benthamiana.

plant biology↗

bioRxiv2024

Tomato cystatin SlCYS8 as a trigger of drought tolerance and tuber yield in potato

Current climate change scenarios predict an increased incidence of drought episodes likely to affect potato crops worldwide. Potato exhibits a low-density, shallow root system that makes it particularly vulnerable to water shortage and any successful attempt to implement drought tolerance in cultivated potato varieties is potentially relevant from an agronomic standpoint. In this study, we assessed the potential of tomato cystatin SlCYS8 to promote drought tolerance in SlCYS8-expressing potato lines by induction of stress-related pleiotropy. Up to now, protease inhibitors of the cystatin protein superfamily have been mostly considered as biotechnological tools to engineer pest or pathogen resistance in crops, but several recent studies have also revealed a possible link between abiotic stress tolerance and these regulatory proteins in leaf tissue. SlCYS8-expressing plantlets grown on culture medium containing the drought mimic polyethylene glycol (PEG) exhibited an elevated root-to-shoot ratio, an indicator of drought tolerance in potato. A similar conclusion could be drawn with greenhouse-grown acclimated plants, confirming a relative root growth-promoting effect for the recombinant inhibitor upon water deficit. SlCYS8-potato lines also showed a high tuber yield compared to the control line under both limiting and non-limiting water regimes, suggesting an improved efficiency of the primary metabolism and the avoidance of a growth- stress response tradeoff in the modified lines. Accordingly, SlCYS8 expression was associated with a stress response-oriented proteome in leaves likely explained by pleiotropic effects of the recombinant cystatin driving the constitutive expression of stress-related proteins and the upregulation of primary metabolism-associated proteins. Overall, these data suggest the potential of cystatins as molecular triggers of tuber biomass production and drought resilience in potato. Complementary studies will be warranted to assess tuber yield of the SlCYS8-lines under different water regimes in field conditions.

plant biology↗

bioRxiv2023

Forest tree extracts induce resistance to Pseudomonas syringae pv. tomato in Arabidopsis

BackgroundThe widespread use of conventional pesticides to control plant fungal and bacterial pathogens poses significant risks to human health and the environment, and there is an urgent need for safer and more sustainable alternatives in agricultural management. Studies have shown that plant extracts can be effective in controlling plant diseases either by directly targeting the pathogens or by reinforcing the host plants own defenses. Here, we examined the potential of ethanolic extracts from forest tree species eastern hemlock, English oak, eastern red cedar and red pine for their antibacterial activity against Pseudomonas syringae pv. tomato (Pst) strain DC3000 and the ability of these forestry by-products to trigger effective defense responses in the model plant Arabidopsis thaliana. ResultsThe four tree extracts exhibited direct toxic effects against Pst DC3000, as notably observed for the English oak extract inhibiting bacterial growth and showing bactericidal activity at relatively low concentrations. Using an Arabidopsis line expressing reporter protein {beta}-glucuronidase under the control of a salicylic acid-inducible pathogenesis-related protein gene promoter, the extracts were shown also to induce defense-related genes expression in leaf tissue. RT-qPCR assays with DNA primers for different gene markers further confirmed this conclusion and highlighted gene-inducing effects for the tree extracts triggering, at different rates, the expression of salicylic acid- and oxidative stress-responsive genes. The extracts direct antibacterial effects, combined with their defense gene-inducing effects in planta, resulted in a strong host plant-protecting effect against Pst DC3000 associated with bacterial growth rates reduced by [~]75 to 98% seven days post-infection, depending on the extract. ConclusionsThese findings show the effectiveness of tree extracts as eventual plant protectants against the plant bacterial pathogen Pst. In a broader perspective, they suggest the potential of these forestry by-products as a source of bioactive compounds useful in plant protection and as a sustainable, eco-friendly alternative to conventional synthetic pesticides for the management of economically important plant pathogens.

plant biology↗

bioRxiv2023

Heterologous expression of influenza hemagglutinin leads to early and transient activation of the unfolded protein response in Nicotiana benthamiana

The unfolded protein response (UPR) allows cells to cope with endoplasmic reticulum (ER) stress induced by the accumulation of misfolded proteins in the ER. Due to its sensitivity to Agrobacterium tumefaciens, model plant Nicotiana benthamiana is widely employed for the transient expression of recombinant proteins of biopharmaceutical interest, including therapeutic antibodies and virus surface proteins used for vaccine production. As such, study of the plant UPR is of practical significance, since enforced expression of complex secreted proteins often results in ER stress. After 6 days of expression, we recently reported that influenza hemagglutinin (HA) induces accumulation of UPR proteins. Since the upregulation of corresponding UPR genes was not detected at this time point, accumulation of UPR proteins was hypothesized to either be independent of transcriptional regulation, or associated with early but transient UPR gene upregulation. Using time course sampling, we here show that HA expression does result in early and transient activation of the UPR, as inferred from unconventional splicing of NbbZIP60 transcripts and induction of UPR genes with varied functions. The transient nature of HA-induced UPR suggests that this response was sufficient to cope with ER stress provoked by expression of the secreted protein, as opposed to an antibody that triggered a stronger and more sustained UPR. As defense-related genes were induced after the peak of UPR activation and correlated with high increase in HA protein accumulation, we hypothesize that these immune responses, rather than the UPR, were responsible for the onset of necrotic symptoms on HA-expressing leaves. One-sentence summaryAgrobacterium-mediated expression of influenza hemagglutinin results in early and transient activation of the unfolded protein response, preventing deleterious effects caused by unresolved endoplasmic reticulum stress.

plant biology↗

bioRxiv2023

Molecular responses of agroinfiltrated Nicotiana benthamiana leaves expressing suppressor of silencing P19 and influenza virus-like particles

The production of influenza vaccines in plants is achieved through transient Agrobacterium-mediated expression of viral hemagglutinins (HAs). These proteins are produced and matured through the secretory pathway of plant cells, before being trafficked to the plasma membrane where they induce formation of virus-like particles (VLPs). Production of VLPs unavoidably impacts plant cells, as do viral suppressors of RNA silencing (VSRs) that are often co-expressed to increase protein yields. However, little information is available on host molecular responses to these foreign proteins. The present work provides a comprehensive overview of transcriptomic, metabolic, and signaling changes occurring in Nicotiana benthamiana leaf cells transiently expressing the VSR P19, or co-expressing P19 and an influenza HA. Our data identifies generic responses to Agrobacterium-mediated expression of foreign proteins, including shutdown of chloroplast gene expression, activation of oxidative stress responses, and reinforcement of the plant cell wall through lignification. Our results also indicate that P19 expression promotes salicylic acid (SA) signaling, a process apparently antagonized by co-expression of HA. As the latter induces specific signatures, with effects on lipid metabolism, lipid distribution, and oxylipin signaling, dampening of P19 responses suggests crosstalk between SA and oxylipin pathways. Consistent with the upregulation of oxidative stress-related genes and proteins, we finally show that reduction of oxidative stress damage through exogenous application of ascorbic acid improves plant biomass quality during production of VLPs. One-sentence summaryAgrobacterium-mediated expression of influenza virus-like particles induces a unique molecular signature in Nicotiana benthamiana leaf cells.

plant biology↗

bioRxiv2022

Hidden potential of the supporting scaffold as a structural module for plant cystatin design

Protein engineering approaches have been proposed to improve the inhibitory properties of plant cystatins towards herbivorous pest digestive Cys proteases, typically involving sequence alterations in the inhibitory loops and/or N-terminal trunk of the protein interacting with specific amino acid residues of the target protease. In this study, we assessed whether the loops-supporting frame, or scaffold, would represent a valuable structural module for cystatin function improvement. Twenty hybrid cystatins were designed in silico, consisting of the N-terminal trunk and two inhibitory loops of a given donor cystatin grafted onto the scaffold of an alternative, recipient cystatin. Synthetic genes for the hybrids were expressed in E. coli, and the resulting proteins assessed for their potency to inhibit model Cys protease papain and the digestive Cys proteases of Colorado potato beetle (Leptinotarsa decemlineata) used as an insect pest model. In line with the occurrence of positively selected amino acids presumably influencing inhibitory activity in the scaffold region of plant cystatins, grafting the N-terminal trunk and inhibitory loops of a given cystatin onto the scaffold of an alternative cystatin generally had an effect on the inhibitory potency of these function-related elements against Cys proteases. For instance, hybrid cystatins including the three structural elements of model tomato cystatin SlCYS8 grafted on the scaffold of cystatins from other plant families showed Ki values altered by up to 3-fold for papain, and inhibitory efficiencies increased by up to 8-fold against L. decemlineata cathepsin L-like proteases, compared to wild-type SlCYS8 bearing the original scaffold. Our data point to a significant influence of the cystatin scaffold on the inhibitory activity of the N-terminal trunk and protease inhibitory loops. They also suggest the potential of this structural element as a module for plant cystatin design to generate functional variability against Cys proteases, including the digestive proteases of herbivorous pests.

plant biology↗

bioRxiv2021

Harnessing the functional diversity of plant cystatins to design inhibitor variants highly active against herbivorous arthropod digestive proteases

Protein engineering approaches have been proposed to improve the inhibitory properties of plant cystatins against herbivorous arthropod digestive proteases. These approaches typically involve the site-directed mutagenesis of functionally relevant amino acids, the production and selection of improved inhibitory variants by molecular phage display procedures, or the design of bi/multifunctional translational fusions integrating one or several cystatin inhibitory domains. Here, we propose a new approach where the function-related structural elements of a cystatin are substituted by the corresponding elements of an alternative cystatin. Cys protease inhibitory assays were first performed with 20 representative plant cystatins and model Cys proteases, including herbivorous arthropod digestive proteases, to appreciate the extent of functional variability among plant cystatin protein family members. The most, and less, potent of these cystatins were then used as donors of structural elements to create hybrids of tomato cystatin SlCYS8 used as a model recipient inhibitor. Our data confirm the wide variety of cystatin protease inhibitory profiles among plant taxa. They also demonstrate the usefulness of these proteins as a pool of discrete structural elements for the design of cystatin variants with improved potency against herbivorous pest digestive Cys proteases.

plant biology↗

bioRxiv2018

pH gradient mitigation in the leaf cell secretory pathway alters the defense response of Nicotiana benthamiana to agroinfiltration

Partial neutralization of the Golgi lumen pH by ectopic expression of influenza virus M2 proton channel stabilizes acid-labile and protease-susceptible recombinant proteins in the plant cell secretory pathway. Here, we assessed the impact of M2 channel expression on the proteome of Nicotiana benthamiana leaf tissue infiltrated with the bacterial gene vector Agrobacterium tumefaciens, keeping in mind the key role of pH homeostasis on secreted protein processing and the involvement of protein secretion processes in plant cells upon microbial challenge. The proteomes of leaves agroinfiltrated with an empty vector or with an M2 channel-encoding vector were compared with the proteome of non-infiltrated leaves using a iTRAQ quantitative proteomics procedure. Leaves infiltrated with the empty vector had a low soluble protein content compared to non-infiltrated leaves, associated with a strong decrease of photosynthesis-associated proteins (including Rubisco) and a parallel increase of stress-related secreted proteins (including pathogenesis-related proteins, protease inhibitors and molecular chaperones). M2 expression partly compromised these alterations of the proteome to restore original soluble protein and Rubisco contents, associated with higher levels of translation-associated (ribosomal) proteins and reduced levels of stress-related proteins in the apoplast. Proteome changes in M2-expressing leaves were determined both transcriptionally and post-transcriptionally, to alter the steady-state levels of proteins not only along the secretory pathway but also in other cellular compartments including the chloroplast, the cytoplasm, the nucleus and the mitochondrion. These data illustrate the cell-wide influence of Golgi lumen pH homeostasis on the leaf proteome of N. benthamiana plants responding to microbial challenge. They underline in practice the relevance of carefully considering the eventual off-target effects of accessory proteins used to modulate specific cellular or metabolic functions in plant protein biofactories.

plant biology↗