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RH3 enhances antiviral defense by facilitating small RNA loading into Argonaute 2

ABSTRACT: To explore the sRNA loading mechanisms and the involvement of chloroplasts in RNA silencing, we conducted an IP-MS analysis to identify chloroplast-localized proteins associated with AGO2.

ORGANISM(S): Arabidopsis Thaliana

SUBMITTER: Xiaoming Zhang

PROVIDER: PXD051119 | iProX | Mon Apr 01 00:00:00 GMT+01:00 2024

REPOSITORIES: iProX

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RH3 enhances antiviral defense by facilitating small RNA loading into Argonaute 2 at endoplasmic reticulum–chloroplast membrane contact sites

Project description:To explore the sRNA loading mechanisms and the involvement of chloroplasts in RNA silencing, we conducted an IP-MS analysis to identify chloroplast-localized proteins associated with AGO2.

2023-07-14 | PXD043780 |

Toss GERALT into chloroplast to make it green: an age-dependent regulator of chloroplast biogenesis and chlorophyll biosynthesis.

Project description:The studies were performed using Arabidopsis thaliana: wt Col (N6000) and SALK_116579 (geralt, GERMINATION ALBINO TRANSIENT) mutant. At2g47590 gene encodes GERALT protein. This protein was not characterized so far. It is localized in chloroplasts, influences chloroplast biogenesis and chlorophyll biosynthesis and its role decreases with the plant age.

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RH3 enhances antiviral defense by facilitating small RNA loading into Argonaute 2 at endoplasmic reticulum-chloroplast membrane contact sites.

Project description: Not available

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Loss-of-function mutation of Plastid Terminal Oxidase (ZmPTOX) cause zebra leaf and white kernel by affecting plastid development in maize

Project description:Zebra leaf is a special phenotype of variegated leaf in monocotyledonous plant. Here, we characterized a mutant zebra10 (zb10) in maize, which displays defective chloroplast development in white section of leaves at the seedling stage and pale-white kernels. Map based cloning revealed that ZB10 encodes plastid terminal oxidase (ZmPTOX) which is localized in chloroplasts. ZmPTOX protein is highest expressed in leaves and also highly expressed in endosperm. Expression and metabolism analysis showed that function-loss of PTOX interferes with carotenoid synthesis and chloroplast biogenesis by affecting phytoene desaturase activity. In-depth observation showed that crossband formation of zebra leaf could be related to photoperiodic rhythm, and expression of alternative oxidases 2 (ZmAOX2) could be regulated by circadian rhythm and light intensity in zb10. Further studies revealed ectopic expression of ZmAOX2 protein can function in chloroplasts and rescue the defective phenotype of im. We conclude that ZmPTOX plays an vital role in carotenoid synthesis and plastid biogenesis in maize. ZmAOX2 involve in formation of zebra crossbands with diurnal rhythm and green-revertible process of leaves in zb10.

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An improved nuclei isolation protocol from leaf tissue for single-cell transcriptomics

Project description:We developed an alternative method for isolating nuclei to be utilized in scRNA-seq experiments from Zea mays leaves with reduced chloroplast contamination. By effectively removing chloroplasts during the FACS step of our protocol, using the autofluorescence from the chloroplasts, we achieved improved alignment of reads to the genome and transcriptome. Our enhanced protocol offers a valuable solution for applying snRNA-seq in tissues with a high content of chloroplasts.

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Drosophila melanogaster

Project description:miRNA* loading on Ago2

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SIB1-SEC23A undergo ER to chloroplast relocalization to mediate immunity in Arabidopsis thaliana

Project description:Inter-organellar communication has emerged as a critical factor in maintaining cellular homeostasis under stress conditions. Chloroplasts function not only as central organelles for energy production but are also increasingly recognized as stress sensors and signal integrators. SIGMA FACTOR-BINDING PROTEIN 1 (SIB1), encoded by a nuclear gene, has been identified as a positive regulator of plant immunity, with localization in both chloroplasts and the nucleus. In this study, we identified Arabidopsis SEC23A, a COPII complex component known to mediate membrane trafficking between the endoplasmic reticulum (ER) and the Golgi apparatus, as a novel interactor of SIB1. Our findings reveal that under normal conditions, both SIB1 and SEC23A localize to the ER, while SIB1 is also localized in the nucleus. Upon ER stress and/or treatment with the immunity inducer salicylate, both proteins are relocated from the ER to the chloroplasts. Notably, SEC23A, similar to SIB1, also functions as a positive regulator of disease resistance. In response to pathogen infection, SIB1 and SEC23A downregulate expression of chloroplast- and nucleus-encoded genes associated with photosynthesis while enhancing expression of defense-related genes. Together, our findings reveal a previously uncharacterized pathway of ER–chloroplast communication mediated by SIB1 and SEC23A during plant stress responses and immunity, providing novel insights into the intricate regulatory networks that govern inter-organellar communication under stress in plants.

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2-Cys peroxiredoxins and the chaperone cpHSP70 act in concert in chloroplast biogenesis in Arabidopsis seedlings

Project description:The redox balance between NADPH-dependent thioredoxin reductase C (NTRC) and 2-Cys peroxiredoxins (PRXs) help chloroplast photosynthetic performance acclimate rapidly to environmental cues. The Arabidopsis (Arabidopsis thaliana) 2cpab mutant, which lacks chloroplast 2-Cys PRXs A and B, shows impaired embryogenesis and cotyledon development. This phenotype indicates that these enzymes have a relevant function at early stages of plant development, although this function is poorly understood. Here, we show that the albino cotyledons of the 2cpab mutant have unstructured chloroplasts and decreased chloroplast lipid contents, revealing a key function of 2-Cys PRXs in cotyledon chloroplast differentiation. These phenotypes are mimicked by NTRC overexpression, whereas loss of NTRC function has no effect. RNA-Seq analyses showed transcriptomic changes resembling the response to proteotoxic stress in 2cpab seedlings, which was confirmed by the high sensitivity of 2cpab seedlings to heat stress. In de-etiolating seedlings 2-Cys PRXs interact with chloroplast-localized chaperone heat shock protein 70 (cpHSP70); moreover, Arabidopsis seedlings simultaneously lacking 2-Cys PRXs and cpHSP70-1 exhibit a dramatic alteration of seedling development. Based on these results, we propose that 2-Cys PRXs contribute to cotyledon chloroplast differentiation by affecting organellar proteostasis. This function is exerted in concert with chaperone cpHSP70 and is essential for seedling establishment.

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Ago1 vs Ago2-IP small RNA deep-sequencing with age in Drosophila

Project description:Drosophila miRNAs show distinct change in isoform distribution pattern with age. Some miRNAs show accumulation of the short isoforms, while other miRNAs show the accumulation of the long isoforms with age. The increase of the long isoforms of some miRNAs reflects increased 2'-O-methylated miRNA isoforms with age. The increase in 2'-O-methylated miRNA isoforms reflected increased Ago2-loading, but not Ago1-loading of specific miRNA isoforms with age. This raised a question on whether there is global shift in small RNA loading pattern between Ago1 and Ago2 with age. To investigate change in small RNA loading pattern between Ago1 and Ago2 with age, we performed small RNA deep-sequencing of Ago1 vs Ago2-IP small RNAs at 3d and 30d in Drosophila. This analysis revealed global increase of miRNA loading into Ago2, but not into Ago1 with age.

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Loqs-PD and R2D2 define independent pathways for RISC generation in Drosophila

Project description:In Drosophila, siRNAs are classified as endo- or exo-siRNAs based on their origin. Both are processed from double-stranded RNA precursors by Dcr-2, then loaded into the Argonaute protein Ago2. While exo-siRNAs serve to defend the cell against viruses, endo-siRNAs restrict the spread of selfish DNA in somatic cells, analogous to piRNAs in the germ line. Endo- and exo-siRNAs display a differential requirement for double-stranded RNA binding domain proteins (dsRBPs): R2D2 is needed to load exo-siRNAs into Ago2 while the PD isoform of Loquacious (Loqs-PD) stimulates Dcr-2 during the nucleolytic processing of hairpin-derived endo-siRNAs. In cell culture assays, R2D2 antagonizes Loqs-PD in endo-siRNA silencing and Loqs-PD is an inhibitor of RNA interference. Loqs-PD can interact via the C-terminus unique to this isoform with the DExH/D-helicase domain of Drosophila Dcr-2, where binding of R2D2 has also been localized. Separation of the two pathways is not complete; rather, the dicing and Ago2-loading steps appear uncoupled, analogous to the corresponding steps in miRNA biogenesis. Analysis of deep sequencing data further demonstrates that in r2d2 mutant flies, siRNAs can be loaded into Ago2 but not all siRNA classes are equally proficient for this. Thus, the canonical Ago2-RISC loading complex can be bypassed under certain circumstances. Examination of small RNAs from two different mutant strains as well as the corresponding heterozygous controls

2011-02-10 | E-GEOD-26230 | biostudies-arrayexpress

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