Florian Brioudes

Last Name

Brioudes

First Name

Florian

ORCID

0000-0001-6665-307X

Organisational unit

03876 - Voinnet, Olivier / Voinnet, Olivier

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Publications 1 - 7 of 7

Movement and differential consumption of short interfering RNA duplexes underlie mobile RNA interference
Devers, Emanuel; Brosnan, Christopher A.; Sarazin, Alexis; et al. (2020)
Nature Plants
In RNA interference (RNAi), the RNase III Dicer processes long double-stranded RNA (dsRNA) into short interfering RNA (siRNA), which, when loaded into ARGONAUTE (AGO) family proteins, execute gene silencing1. Remarkably, RNAi can act non-cell autonomously2,3: it is graft transmissible4,5,6,7, and plasmodesmata-associated proteins modulate its cell-to-cell spread8,9. Nonetheless, the molecular mechanisms involved remain ill defined, probably reflecting a disparity of experimental settings. Among other caveats, these almost invariably cause artificially enhanced movement via transitivity, whereby primary RNAi-target transcripts are converted into further dsRNA sources of secondary siRNA5,10,11. Whether siRNA mobility naturally requires transitivity and whether it entails the same or distinct signals for cell-to-cell versus long-distance movement remains unclear, as does the identity of the mobile signalling molecules themselves. Movement of long single-stranded RNA, dsRNA, free/AGO-bound secondary siRNA or primary siRNA have all been advocated12,13,14,15; however, an entity necessary and sufficient for all known manifestations of plant mobile RNAi remains to be ascertained. Here, we show that the same primary RNAi signal endows both vasculature-to-epidermis and long-distance silencing movement from three distinct RNAi sources. The mobile entities are AGO-free primary siRNA duplexes spreading length and sequence independently. However, their movement is accompanied by selective siRNA depletion reflecting the AGO repertoires of traversed cell types. Coupling movement with this AGO-mediated consumption process creates qualitatively distinct silencing territories, potentially enabling unlimited spatial gene regulation patterns well beyond those granted by mere gradients.
A contemporary reassessment of the enhanced transient expression system based on the tombusviral silencing suppressor protein P19
Jay, Florence; Brioudes, Florian; Voinnet, Olivier (2023)
The Plant Journal
Transient transgenic expression accelerates pharming and facilitates protein studies in plants. One embodiment of the approach involves leaf infiltration of Agrobacterium strains whose T-DNA is engineered with the gene(s) of interest. However, gene expression during 'agro-infiltration' is intrinsically and universally impeded by the onset of post-transcriptional gene silencing (PTGS). Nearly 20 years ago, a simple method was developed, whereby co-expression of the tombusvirus-encoded P19 protein suppresses PTGS and thus enhances transient gene expression. Yet, how PTGS is activated and suppressed by P19 during the process has remained unclear to date. Here, we address these intertwined questions in a manner also rationalizing how vastly increased protein yields are achieved using a minimal viral replicon as a transient gene expression vector. We also explore, in side-by-side analyses, why some proteins do not accumulate to the expected high levels in the assay, despite vastly increased mRNA levels. We validate that enhanced co-expression of multiple constructs is achieved within the same transformed cells, and illustrate how the P19 system allows rapid protein purification for optimized downstream in vitro applications. Finally, we assess the suitability of the P19 system for subcellular localization studies - an originally unanticipated, yet increasingly popular application - and uncover shortcomings of this specific implement. In revisiting the P19 system using contemporary knowledge, this study sheds light onto its hitherto poorly understood mechanisms while further illustrating its versatility but also some of its limits.
A pectin acetyl‐transferase facilitates secondary plasmodesmata formation and RNA silencing movement between plant cells
Jay, Florence; Brioudes, Florian; Novaković, Lazar; et al. (2025)
The Plant Journal
Some silencing small (s)RNAs, comprising micro (mi)RNAs and small-interfering (si)RNAs, move between plant cells to orchestrate gene expression and defense. Besides possible redundancy or embryo lethality, a prevalent challenge in genetic studies of mobile silencing is to discriminate bona fide alterations to sRNA movement from impaired cell-autonomous sRNA activity within silencing-recipient cells. Without such clarifications, cell-to-cell mobility factors are yet to be unequivocally identified. Consequently, known properties of sRNA movement, including contextuality and directionality, remain poorly explained. Circumstantial evidence and synthetic biology pinpoint plasmodesmata (PDs) – the pores traversing plant cell walls (CWs) – as the likely channels involved. Yet, how plants control the number of primary and secondary PDs developing respectively before and after CW formation remains largely unknown. Here, we address these intertwined issues in Arabidopsis using a forward screen for compromised epidermis-to-mesophyll movement of an artificial (a)miRNA. We identify a pectin acetyl-transferase mutation that, we demonstrate, reduces amiRNA physical trafficking but also impedes siRNA, GFP, and viral movement by decreasing the frequency of leaf secondary PDs. sRNA movement at leaf interfaces involving primary PDs remains unaffected, however, as does miRNA and GFP cell-to-cell mobility in roots, hinting at how movement's contextuality and directionality might be achieved. We also show that reducing de-esterified pectin depolymerization decreases leaves' symplasmic connectivity, whereas defective pectin biogenesis increases PD number. Combining genetics with antibody-based pectin probing and atomic force microscopy helps delineate a mechanistically coherent framework whereby pectin esterification and/or abundance impact CW loosening, a process required for CW extension during which secondary PDs form to enable macromolecular trafficking.
HASTY, the Arabidopsis EXPORTIN5 ortholog, regulates cell-to-cell and vascular microRNA movement
Brioudes, Florian; Jay, Florence; Sarazin, Alexis; et al. (2021)
The EMBO Journal
Plant microRNAs (miRNAs) guide cytosolic post-transcriptional gene silencing of sequence-complementary transcripts within the producing cells, as well as in distant cells and tissues. Here, we used an artificial miRNA-based system (amiRSUL) in Arabidopsis thaliana to explore the still elusive mechanisms of inter-cellular miRNA movement via forward genetics. This screen identified many mutant alleles of HASTY (HST), the ortholog of mammalian EXPORTIN5 (XPO5) with a recently reported role in miRNA biogenesis in Arabidopsis. In both epidermis-peeling and grafting assays, amiRSUL levels were reduced much more substantially in miRNA-recipient tissues than in silencing-emitting tissues. We ascribe this effect to HST controlling cell-to-cell and phloem-mediated movement of the processed amiRSUL, in addition to regulating its biogenesis. While HST is not required for the movement of free GFP or siRNAs, its cell-autonomous expression in amiRSUL-emitting tissues suffices to restore amiRSUL movement independently of its nucleo-cytosolic shuttling activity. By contrast, HST is dispensable for the movement and activity of amiRSUL within recipient tissues. Finally, HST enables movement of endogenous miRNAs that display mostly unaltered steady-state levels in hst mutant tissues. We discuss a role for HST as a hitherto unrecognized regulator of miRNA movement in relation to its recently assigned nuclear function at the nexus of MIRNA transcription and miRNA processing.
Suppression of both intra- and intercellular RNA silencing by the tombusviral P19 protein requires its small RNA binding property
Brioudes, Florian; Jay, Florence; Voinnet, Olivier (2022)
New Phytologist
A comment on Garnelo Gomez et al. (2021) 'The viral silencing suppressor P19 interacts with the receptorlike kinases BAM1 and BAM2 and suppresses the cell to-cell movement of RNA silencing independently of its ability to bind sRNA'
Chemical enhancers of post-transcriptional gene-silencing in Arabidopsis
Jay, Florence; Vitel, Maxime; Brioudes, Florian; et al. (2019)
RNA
RNAi mediated by small-interfering RNAs (siRNAs) operates via transcriptional (TGS) and posttranscriptional gene silencing (PTGS). In Arabidopsis thaliana, TGS relies on DICER-LIKE-3 (DCL3)-dependent 24-nt siRNAs loaded into AGO4-clade ARGONAUTE effector proteins. PTGS operates via DCL4-dependent 21-nt siRNAs loaded into AGO1-clade proteins. We set up and validated a medium-throughput, semi-automatized procedure enabling chemical screening, in a 96-well in vitro format, of Arabidopsis transgenic seedlings expressing an inverted-repeat construct from the phloem companion cells. The ensuing quantitative PTGS phenotype was exploited to identify molecules, which, upon topical application, either inhibit or enhance siRNA biogenesis/activities. The vast majority of identified modifiers were enhancers, among which Sortin1, Isoxazolone, and [5-(3,4-dichlorophenyl)furan-2-yl]-piperidine-1-ylmethanethione (DFPM) provided the most robust and consistent results, including upon their application onto soil-grown plants in which their effect was nonautonomous and long lasting. The three molecules increased the RNAi potency of the inverted-repeat construct, in large part by enhancing 21-nt siRNA accumulation and loading into AGO1, and concomitantly reducing AGO4 and DCL3 levels in planta. A similar, albeit not identical effect, was observed on 22-nt siRNAs produced from a naturally occurring inverted-repeat locus, demonstrating that the molecules also enhance endogenous PTGS. In standardized assays conducted in seedling extracts, the three enhancers selectively increased DCL4-mediated processing of in vitro-synthesized double-stranded RNAs, indicating the targeting of a hitherto unknown PTGS component probably independent of the DCL4-cofactor DOUBLE-STRANDED RNA-BINDING 4 (DRB4). This study establishes the proof-of-concept that RNAi efficacy can be modulated by chemicals in a whole organism. Their potential applications and the associated future research are discussed.
TCTP and CSN4 control cell cycle progression and development by regulating CULLIN1 neddylation in plants and animals
Betsch, Léo; Boltz, Véronique; Brioudes, Florian; et al. (2019)
PLoS Genetics
Translationally Controlled Tumor Protein (TCTP) controls growth by regulating the G1/S transition during cell cycle progression. Our genetic interaction studies show that TCTP fulfills this role by interacting with CSN4, a subunit of the COP9 Signalosome complex, known to influence CULLIN-RING ubiquitin ligases activity by controlling CULLIN (CUL) neddylation status. In agreement with these data, downregulation of CSN4 in Arabidopsis and in tobacco cells leads to delayed G1/S transition comparable to that observed when TCTP is downregulated. Loss-of-function of AtTCTP leads to increased fraction of deneddylated CUL1, suggesting that AtTCTP interferes negatively with COP9 function. Similar defects in cell proliferation and CUL1 neddylation status were observed in Drosophila knockdown for dCSN4 or dTCTP, respectively, demonstrating a conserved mechanism between plants and animals. Together, our data show that CSN4 is the missing factor linking TCTP to the control of cell cycle progression and cell proliferation during organ development and open perspectives towards understanding TCTP’s role in organ development and disorders associated with TCTP miss-expression.

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Florian Brioudes

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