a-stepwise-guide-to-isolation-analysis-leaf-surface-apoplastic-rna

A Stepwise Guide to the Isolation and Analysis of Leaf Surface and Apoplastic RNA Using Arabidopsis Rosettes

The surface of plant leaves and intercellular space within leaves (the apoplast) serve as key interfaces for molecular exchange between plants and their ...

Indiana University

In plant chloroplasts, certain ribosomal proteins (RPs) and ribosome biogenesis factors (RBFs) are present in nucleoids, implying an association between nucleoids and ribosome biogenesis. In Arabidopsis, the YqeH-type GTPase Brassinazole-Insensitive Pale Green2 (BPG2) is a chloroplast nucleoid-associated RBF. Here, we investigated the relationship between nucleoids and BPG2-involved ribosome biogenesis steps by exploring how BPG2 targets ribosomes. Our findings demonstrate that BPG2 interacts with an essential plastid RP, uS10c, in chloroplast nucleoids in a ribosomal RNA (rRNA)-independent manner. We also discovered that uS10c is a haploinsufficient gene, as the heterozygous deletion of this gene leads to variegated shoots and chlorophyll aggregation. uS10c is integrated into 30S ribosomal particles when rRNA is relatively exposed and also exists in polysome fractions. In contrast, BPG2 exclusively associates with 30S ribosomal particles. Notably, the interaction between BPG2 and 30S particles is influenced by the absence of uS10c, resulting in BPG2 diffusing in chloroplasts instead of targeting nucleoids. Further, our results reveal that the loss of BPG2 function and the heterozygous deletion of uS10c impair the processing of 16S and 23S-4.5S rRNAs, reduce plastid protein accumulation, and trigger the plastid signaling response. Together, these findings indicate that the uS10c-BPG2 module mediates ribosome biogenesis in chloroplast nucleoids.

The uS10c-BPG2 module mediates ribosomal RNA processing in chloroplast nucleoids.

From Enemy to Ally: Harnessing CPR1 for Soybean Defense Strategies.

Transgenic expression of a double-stranded RNA in plants can induce silencing of homologous mRNAs in fungal pathogens. Although such host-induced gene silencing is well documented, the molecular mechanisms by which RNAs can move from the cytoplasm of plant cells across the plasma membrane of both the host cell and fungal cell are poorly understood. Indirect evidence suggests that this RNA transfer may occur at a very early stage of the infection process, prior to breach of the host cell wall, suggesting that silencing RNAs might be secreted onto leaf surfaces. To assess whether Arabidopsis plants possess a mechanism for secreting RNA onto leaf surfaces, we developed a protocol for isolating leaf surface RNA separately from intercellular (apoplastic) RNA. This protocol yielded abundant leaf surface RNA that displayed an RNA banding pattern distinct from apoplastic RNA, suggesting that it may be secreted directly onto the leaf surface rather than exuded through stomata or hydathodes. Notably, this RNA was not associated with either extracellular vesicles or protein complexes; however, RNA species longer than 100 nucleotides could be pelleted by ultracentrifugation. Furthermore, pelleting was inhibited by the divalent cation chelator EGTA, suggesting that these RNAs may form condensates on the leaf surface. These leaf surface RNAs are derived almost exclusively from Arabidopsis, but come from diverse genomic sources, including rRNA, tRNA, mRNA, intergenic RNA, microRNAs, and small interfering RNAs, with tRNAs especially enriched. We speculate that endogenous leaf surface RNA plays an important role in the assembly of distinct microbial communities on leaf surfaces.

Diverse plant RNAs coat  leaves and are distinct from apoplastic RNAs.

Yin and Yang: A pair of miRNA modules antagonistically regulate xylem development.

A maize architect: An epiallele of a PfkB-type carbohydrate kinase affects plant growth and development.

Melatonin a day: Mitigates saline-alkali stress away!

The complex immune puzzle: A deeper dive into the MORC1-mediated broad-spectrum defense signaling pathway.

Extracellular vesicles (EVs) secreted by mammalian cells are highly heterogeneous in content and function. Whether this is also true for EVs secreted by plant cells is not yet known. To address this, we used high-resolution density gradient ultracentrifugation and total internal fluorescence microscopy (TIRF-M) to purify and distinguish distinct subpopulations of Arabidopsis EVs. The EV marker protein TETRASPANIN 8 (TET8) was detected specifically in medium-density EVs. TET8 and PENETRATION 1 (PEN1) were confirmed to be secreted in mostly separate EV populations using TIRF-M, while PEN1 was co-secreted with PENETRATION 3 (PEN3) much more often. Secretion of EV subpopulations marked by TET8, PEN1 and RPM1-INTERACTING PROTEIN 4 (RIN4) into the apoplast and onto the leaf surface was induced by phytohormones, changes in temperature and infection with fungal pathogens. Treatment of Arabidopsis seedlings with plant EVs delayed the progression of fungal infection by altering fungal germ tube development and fungal morphology. Significantly, extracellular RNAs, including miRNAs and siRNAs, did not co-fractionate with TET8-labeled EVs, and instead, co-fractionated with extravesicular ARGONAUTE proteins in high-density fractions. Together, these data indicate that Arabidopsis EVs are highly heterogeneous and contribute to immunity but are unlikely to mediate cross-kingdom RNA interference.

Arabidopsis Produces Distinct Subpopulations of Extracellular Vesicles That Respond Differentially to Biotic Stress, Altering Growth and Infectivity of a Fungal Pathogen.

Article	Year
The uS10c-BPG2 module mediates ribosomal RNA processing in chloroplast nucleoids. Nucleic acids research\| PubMed ID: 38686791	2024
From Enemy to Ally: Harnessing CPR1 for Soybean Defense Strategies. Molecular plant-microbe interactions : MPMI\| PubMed ID: 39630415	2024
Diverse plant RNAs coat leaves and are distinct from apoplastic RNAs. Proceedings of the National Academy of Sciences of the United States of America\| PubMed ID: 39752527	2025
Yin and Yang: A pair of miRNA modules antagonistically regulate xylem development. The Plant cell\| PubMed ID: 39820553	2025
A maize architect: An epiallele of a PfkB-type carbohydrate kinase affects plant growth and development. The Plant cell\| PubMed ID: 39899468	2025
Melatonin a day: Mitigates saline-alkali stress away! The Plant cell\| PubMed ID: 39980350	2025
The complex immune puzzle: A deeper dive into the MORC1-mediated broad-spectrum defense signaling pathway. The Plant cell\| PubMed ID: 40171615	2025
Arabidopsis Produces Distinct Subpopulations of Extracellular Vesicles That Respond Differentially to Biotic Stress, Altering Growth and Infectivity of a Fungal Pathogen. Journal of extracellular vesicles\| PubMed ID: 40415221	2025
By Hijacking a Host Chaperone, the Bacterial Effector XopAE Disarms Cassava Immunity. Molecular plant-microbe interactions : MPMI\| PubMed ID: 40580438	2025

Meenu Singla-Rastogi

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