Biology subjects

Pilot, G.

Publications and source records attributed to Pilot, G..

6 recordsLinked to original sources

bioRxiv2024

Characterization and whole genome sequencing of Saccharomyces cerevisiae strains lacking several amino acid transporters: tools for studying amino acid transport

Saccharomyces cerevisiae mutants have been used since the early 1980s as a tool to characterize genes from other organisms by functional complementation. This approach has been extremely successful in cloning and studying transporters, for instance, plant amino acid, sugar, urea, ammonium, peptide, sodium, and potassium were characterized using yeast mutants lacking these functions. Over the years, new strains lacking even more endogenous transporters have been developed, enabling the characterization of transport properties of heterologous proteins in a more precise way. Furthermore, these strains provide the added advantage of characterization of a transporter belonging to a family of proteins in isolation, and thus can be used to study the relative contribution of redundant transporters to the whole function. We focused on amino acid transport; starting with the yeast strain 22{Delta}8AA, developed to clone plant amino acid transporters in the early 2000s. We recently deleted two additional amino acid permeases, Gnp1 and Agp1, creating 22{Delta}10. In the present work, five additional permeases (Bap3, Tat1, Tat2, Agp3, Bap2) were deleted from 22{Delta}10 genome in up to a combination of three at a time. Unexpectedly, the amino acid transport properties of the new strains were not very different from the parent, suggesting that these amino acid permeases play a minor role in amino acid uptake in our conditions. The inability to grow on a few amino acids as the sole nitrogen sources did not correlate with lower uptake activity, questioning the well-accepted relationship between lack of growth and loss of transport properties. Finally, in order to verify the mutations and the integrity of 22{Delta}10 genome, we performed whole-genome sequencing of 22{Delta}10 using long-read PacBio sequencing technology. We successfully assembled 22{Delta}10s genome de novo, identified all expected mutations and precisely characterized the nature of the deletions of the ten amino acid transporters. The sequencing data and genome will serve as a resource to researchers interested in using these strains as a tool for amino acid transport study.

molecular biology↗

bioRxiv2024

The Arabidopsis amino acid transporter UmamiT20 confers susceptibility to B. cinerea

O_LIInduction of SWEET sugar transporters by bacterial pathogens via transcription activator-like (TAL) effectors is necessary for successful blight infection of rice, cassava and cotton, - likely providing sugars for bacterial propagation. C_LIO_LIHere, we show that infection of Arabidopsis by the necrotrophic fungus Botrytis cinerea causes increased accumulation of amino acid transporter UmamiT20 mRNA in leaves. UmamiT20 protein accumulates in leaf veins surrounding the lesions after infection. Consistent with a role during infection, umamiT20 knock-out mutants were less susceptible to B. cinerea. C_LIO_LIFunctional assays demonstrate that UmamiT20 mediates amino acid transport of a wide range of amino acid substrates. C_LIO_LIPathogen-induced UmamiT20 mRNA and protein accumulation support the hypothesis that transporter-mediated pathogen susceptibility is not unique to SWEETs in bacterial blight of rice but also for a necrotrophic fungus and implicate nutrients other than sucrose, i.e., amino acids, in nutrition or nutrient signaling related to immunity. We hypothesize that stacking of mutations in different types of susceptibility-related nutrient carriers to interfere with access to several nutrients may enable engineering robust pathogen resistance in a wide range of plant-pathogen systems. C_LI Lay AbstractPathogens infect plants to gain access to their nutrient resources, enabling the pathogens to cause disease and reproduce efficiently. Here we find that an amino acid transporter constitutes a susceptibility factor for the fungal pathogen B. cinerea.

plant biology↗

bioRxiv2022

Elicitors of plant defense induce the accumulation of amino acids that suppress both bacterial virulence and growth

Plant immunity relies on the perception of Microbe-Associate Molecular Patterns (MAMPs) from invading microbes to induce defense responses that suppress attempted infections. It has been proposed that MAMP-triggered immunity (MTI) suppresses bacterial infections by suppressing the onset of bacterial virulence. However, the mechanisms by which plants exert this action are poorly understood. Here we uncover that MAMP-perception in Arabidopsis induces the accumulation of free amino acids (AA) in a salicylic acid (SA) dependent manner. When co-infiltrated with Glutamine and Serine, two of the MAMP-induced highly accumulating amino acids, Pseudomonas syringae pv. tomato DC3000 expressed low levels of virulence genes and failed to produce robust infections in otherwise susceptible plants. When applied exogenously, Glutamine and Serine suppressed bacterial virulence and bacterial growth directly without the need for MAMP-perception and SA-signaling, bypassing MAMP-elicited defense. In addition, an increased level of endogenous Glutamine in the leaf apoplast of a gain-of-function mutant of Glutamine Dumper-1 rescued the partially compromised bacterial virulence and growth suppressing phenotype of the SA-induced deficient-2 (sid2) mutant. Our data suggest that MTI suppresses bacterial infections by the direct suppressing effect of accumulating amino acids on the onset of bacterial virulence.

plant biology↗

bioRxiv2021

Detailed characterization of the UMAMITs provides insight into their evolution, functional properties as amino acid transporters and role in the plant

Amino acid transporters play a critical role in distributing amino acids within the cell compartments and between the plant organs. Despite this importance, relatively few amino acid transporter genes have been characterized and their role elucidated with certainty. Two main families of proteins encode amino acid transporters in plants: the Amino Acid-Polyamine-Organocation superfamily, containing mostly importers, and the Usually Multiple Acids Move In and out Transporter family, apparently encoding exporters, totaling about 100 genes in Arabidopsis alone. Knowledge on UMAMITs is scarce, focused on six Arabidopsis genes and a handful of genes from other species. To get insight into the role of the members of this family and provide data to be used for future characterization, we studied the evolution of the UMAMITs in plants, and determined the functional properties, the structure, and the localization of the 44 Arabidopsis UMAMITs. Our analysis showed that the AtUMAMIT are essentially localized at the tonoplast or the plasma membrane, and that most of them are able to export amino acids from the cytosol, confirming a role in intra- and inter-cellular amino acid transport. As an example, this set of data was used to hypothesize the role of a few AtUMAMITs in the plant and the cell.

plant biology↗

bioRxiv2021

A split GFP system to enhance spatial and temporal sensitivity of translating ribosome affinity purification (TRAP)

Translating ribosome affinity purification (TRAP) utilizes transgenic plants expressing a ribosomal protein fused to a tag for affinity purification of ribosomes and the mRNAs that they are translating. These actively translated mRNAs (translatome) can be interrogated by qPCR or RNAseq. Condition- or cell-specific promoters can be utilized to isolate the translatome of specific cell types, at different growth stages and/or in response to environmental variables. While advantageous for revealing differential expression, this approach may not provide sufficient sensitivity when activity of the condition/cell-specific promoter is weak, when ribosome turnover is low in the cells of interest, or when the targeted cells are ephemeral. In these situations, expressing tagged ribosomes under the control of these specific promoters may not yield sufficient polysomes for downstream analysis. Here, we describe a new TRAP system that employs two transgenes: one is constitutively expressed and encodes a ribosomal protein fused to one fragment of a split GFP; the second is controlled by a stimulus-specific promoter and encodes the second GFP fragment fused to an affinity purification tag. In cells where both transgenes are active, the purification tag is attached to ribosomes by bi-molecular folding and assembly of the split GFP fragments. This approach provides increased sensitivity and better temporal resolution because it labels pre-existing ribosomes and does not depend on rapid ribosome turnover. We describe the optimization and key parameters of this system, and then apply it to a plant-pathogen interaction in which spatial and temporal resolution are difficult to achieve with current technologies. SignificanceTranslating ribosome affinity purification (TRAP) has been modified to allow with increased sensitivity the isolation of RNA from sets of cells in which the activity of condition/cell-specific promoters is weak, ribosome turnover is low, or cells whose nature is ephemeral. Based on the use of a split linker constituted of the GFP driven by a pathogen-inducible promoter, this new TRAP system enabled efficient isolation of translated RNA from pathogen-infected leaf cells.

molecular biology↗

bioRxiv2020

Induction of GLUTAMINE DUMPER1 reveals a link between amino acid export, abscisic acid, and immune responses

Amino acid homeostasis in plants is finely tuned to match developmental needs and response to adverse environments. Over-expression of the single-transmembrane domain protein GLUTAMINE DUMPER1 (GDU1) leads to increased amino acid export, reduced growth and constitutive induction of immune responses. We used an inducible gene expression system to tease apart the primary and secondary effects caused by GDU1, and demonstrated that the primary effect is increasing amino acid export, followed by increased amino acid content and abscisic acid (ABA) response, and a subsequent activation of defense responses. The GDU1-mediated hypersensitivity to ABA partially depended on the E3 ubiquitin ligase LOSS-OF-GDU1 2 (LOG2), a known GDU1 interactor. More importantly, the lysine catabolite pipecolic acid played a pivotal role in the GDU1-induced defense responses. This work unravels a novel relationship between amino acid transport, ABA and defense responses, potentially mediated by the GDU1-LOG2 complex, critical for understanding how plants respond to amino acid imbalance. ONE SENTENCE SUMMARYGenetically induced disturbance of amino acid homeostasis sequentially triggers responses to abiotic stresses and plant defenses to pathogens in Arabidopsis through undefined sensing mechanisms

plant biology↗