Biology subjects

Pumplin, N.

Publications and source records attributed to Pumplin, N..

3 recordsLinked to original sources

bioRxiv2023

Evidence for an RNAi-independent role of DICER-LIKE2 in conferring growth inhibition and basal antiviral resistance

Higher plants encode four DICER-LIKE (DCL) enzymes responsible for the production of small non-coding RNAs which function in RNA interference (RNAi). Different RNAi pathways in plants effect transposon silencing, antiviral defense and endogenous gene regulation. DCL2 acts genetically redundantly with DCL4 to confer basal antiviral defense, but in other settings, DCL2 has the opposite function of DCL4, at least in formal genetic terms. For example, knockout of DCL4 causes growth defects that are suppressed by inactivation of DCL2. Current models maintain that the biochemical basis of both of these effects is RNAi via DCL2-dependent small interfering RNAs (siRNAs). Here, we report that neither DCL2-mediated antiviral resistance nor growth defects can be explained by silencing effects of DCL2-dependent siRNAs. Both functions are defective in genetic backgrounds that maintain high levels of DCL2-dependent siRNAs, either through specific point mutations in DCL2 or simply by reducing DCL2 dosage in plants heterozygous for dcl2 knockout alleles. Intriguingly, however, all functions of DCL2 depend on it having some level of catalytic activity. We discuss this requirement for catalytic activity, but not for the resulting siRNAs, in the light of recent findings that reveal a function of DCL2 in activation of innate immunity in response to cytoplasmic double-stranded RNA.

plant biology↗

bioRxiv2020

ASYMMETRIC EXPRESSION OF ARGONAUTES IN ARABIDOPSIS REPRODUCTIVE TISSUES

AO_SCPLOWBSTRACTC_SCPLOWDuring sexual reproduction, development of a totipotent zygote from the fusion of highly differentiated gametes is accompanied by dynamic regulation of gene expression. This notably involves RNA silencing operated by Argonautes (AGO) effector proteins. While AGOs roles during Arabidopsis somatic life have been extensively investigated, less is known about their expression during reproduction, which proceeds via double-fertilization of an egg and a central cell, leading respectively to the embryo and a supportive tissue known as endosperm. Using full-locus translational reporters for all ten Arabidopsis AGOs, we uncover cell-specific expression patterns and AGO-intrinsic subcellular localizations in reproductive tissues. However, while some Arabidopsis AGOs are enriched in both male and female gametes, i.e. sperm and egg cells, they are comparably low-expressed in accessory, i.e. vegetative and central cells. Likewise, following fertilization, several AGOs are expressed in the early embryo, yet below detection in the early endosperm. Thus, there is pre- and post-fertilization asymmetry between the embryo and endosperm lineages. Later during embryo development, AGO9, AGO5 and AGO7 are restricted to the apical embryonic meristem in contrast to AGO1, AGO4, AGO6 and AGO10. Beside shedding light onto potential roles for RNA silencing during Arabidopsis reproduction, the plant material generated here should constitute a valuable asset enabling functional AGOs studies in many tissues beyond those involved in reproduction. Summary statementArabidopsis genome encodes ten Argonautes proteins showing distinct expression pattern as well as intracellular localisation during sexual reproduction.

developmental biology↗

bioRxiv2018

Functional characterization of Arabidopsis ARGONAUTE 3 in reproductive tissue

Arabidopsis encodes ten ARGONAUTE (AGO) effectors of RNA silencing, canonically loaded with either 21-22nt small RNAs (sRNA) to mediate post-transcriptional-gene-silencing (PTGS) or 24nt sRNAs to promote RNA-directed-DNA-methylation. Using full-locus constructs, we characterized the expression, biochemical properties, and possible modes of action of AGO3. Although AGO3 arose from a recent duplication at the AGO2 locus, their expression differs drastically, with AGO2 being expressed in both male and female gametes whereas AGO3 accumulates in aerial vascular terminations and specifically in chalazal seed integuments. Accordingly, AGO3 down-regulation alters gene expression in siliques. Similar to AGO2, AGO3 binds sRNAs with a strong 5-adenosine bias, but unlike most Arabidopsis AGOs - AGO2 included - it binds efficiently both 24nt and 21nt sRNAs. AGO3 immunoprecipitation experiments in siliques revealed that these sRNAs mostly correspond to genes and intergenic regions. in a manner reflecting their respective accumulation from their loci-of-origin. AGO3 localizes to the cytoplasm and co-fractionates with polysomes to possibly mediate PTGS via translation inhibition.\n\nSignificance statementThe regulation of gene expression by small RNAs is key for proper plant development and defense. Here, we characterize Arabidopsis AGO3 expression pattern, microRNA regulation and biochemical properties during sexual reproduction.

plant biology↗