Arabidopsis apoplastic fluid contains sRNA- and circular RNA-protein complexes that are located outside extracellular vesicles

Hana Zand Karimi, Patricia Baldrich, Brian D. Rutter, Lucía Borniego, Kamil K. Zajt, Blake C. Meyers, Roger W. Innes

Research output: Contribution to journalArticlepeer-review

58 Scopus citations

Abstract

Previously, we have shown that apoplastic wash fluid (AWF) purified from Arabidopsis leaves contains small RNAs (sRNAs). To investigate whether these sRNAs are encapsulated inside extracellular vesicles (EVs), we treated EVs isolated from Arabidopsis leaves with the protease trypsin and RNase A, which should degrade RNAs located outside EVs but not those located inside. These analyses revealed that apoplastic RNAs are mostly located outside and are associated with proteins. Further analyses of these extracellular RNAs (exRNAs) revealed that they include both sRNAs and long noncoding RNAs (lncRNAs), including circular RNAs (circRNAs). We also found that exRNAs are highly enriched in the posttranscriptional modification N6-methyladenine (m6A). Consistent with this, we identified a putative m6A-binding protein in AWF, GLYCINE-RICH RNA-BINDING PROTEIN 7 (GRP7), as well as the sRNA-binding protein ARGONAUTE2 (AGO2). These two proteins coimmunoprecipitated with lncRNAs, including circRNAs. Mutation of GRP7 or AGO2 caused changes in both the sRNA and lncRNA content of AWF, suggesting that these proteins contribute to the secretion and/or stabilization of exRNAs. We propose that exRNAs located outside of EVs mediate host-induced gene silencing, rather than RNA located inside EVs.

Original languageEnglish
Pages (from-to)1863-1881
Number of pages19
JournalPlant Cell
Volume34
Issue number5
DOIs
StatePublished - May 2022

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