Effective methods for isolation and purification of extracellular vesicles from plants

Plant extracellular vesicles(EVs)play critical roles in the cross-kingdom trafficking of molecules from hosts to interacting microbes,most notably in plant defense responses.However,the isolation of pure,intact EVs from plants remains challenging.A variety of methods have been utilized to isolate plant EVs from apoplastic washing fluid(AWF).Here,we compare published plant EV isolation methods,and provide our recommended method for the isolation and purification of plant EVs.This method includes a detailed protocol for clean AWF collection from Arabidopsis thaliana leaves,followed by EV isolation via differential centrifugation.To further separate and purify specific subclasses of EVs from heterogeneous vesicle populations,density gradient ultracentrifugation and immunoaffinity capture are then utilized.We found that immunoaffinity capture is the most precise method for specific EV subclass isolation when suitable specific EV biomarkers and their corresponding antibodies are available.Overall,this study provides a guide for the selection and optimization of EV isolation methods for desired downstream applications.

A Bunyavirus-Inducible Ubiquitin Ligase Targets RNA Polymerase IV for Degradation during Viral Pathogenesis in Rice

The ubiquitin-proteasome system(UPS)is an important post-translational regulatory mechanism that controls many cellular functions in eukaryotes.Here,we show that stable expression of P3 protein encoded by Rice grassy stunt virus(RGSV),a negative-strand RNA virus in the Bunyavirales,causes developmental abnormities similar to the disease symptoms caused by RGSV,such as dwarfing and excess tillering,in transgenic rice plants.We found that both transgenic expression of P3 and RGSV infection induce ubiquitination and UPS-dependent degradation of rice NUCLEAR RNA POLYMERASE D1a(OsNRPD1a),one of two orthologs of the largest subunit of plant-specific RNA polymerase IV(Pol IV),which is required for RNA-directed DNA methylation(RdDM).Furthermore,we identified a P3-inducible U-box type E3 ubiquitin ligase,designated as P3-inducible protein 1(P3IP1),which interacts with OsNRPD1a and mediates its ubiquitination and UPS-dependent degradation in vitro and in vivo.Notably,both knockdown of OsNRPD1 and overexpression of P3IP1 in rice plants induced developmental phenotypes similar to RGSV disease symptomss.Taken together,our findings reveal a novel virulence mechanism whereby plant pathogens target host RNA Pol IV for UPS-dependent degradation to induce disease symptoms.Our study also identified an E3 ubiquitin ligase,which targets the RdDM compotent NRPD1 for UPS-mediated degradation in rice.