RESUMEN
Stress granules (SGs) are dynamic RNA-protein complexes localized in the cytoplasm that rapidly form under stress conditions and disperse when normal conditions are restored. The formation of SGs depends on the Ras-GAP SH3 domain-binding protein (G3BP). Formations, interactions and functions of plant and human SGs are strikingly similar, suggesting a conserved mechanism. However, functional analyses of plant G3BPs are missing. Thus, members of the Arabidopsis thaliana G3BP (AtG3BP) protein family were investigated in a complementation assay in a human G3BP knock-out cell line. It was shown that two out of seven AtG3BPs were able to complement the function of their human homolog. GFP-AtG3BP fusion proteins co-localized with human SG marker proteins Caprin-1 and eIF4G1 and restored SG formation in G3BP double KO cells. Interaction between AtG3BP-1 and -7 and known human G3BP interaction partners such as Caprin-1 and USP10 was also demonstrated by co-immunoprecipitation. In addition, an RG/RGG domain exchange from Arabidopsis G3BP into the human G3BP background showed the ability for complementation. In summary, our results support a conserved mechanism of SG function over the kingdoms, which will help to further elucidate the biological function of the Arabidopsis G3BP protein family.
Asunto(s)
Arabidopsis/metabolismo , Gránulos Citoplasmáticos/metabolismo , Estrés Fisiológico , Proteínas de Arabidopsis/química , Proteínas de Arabidopsis/metabolismo , Línea Celular Tumoral , Proteínas Fluorescentes Verdes/metabolismo , Humanos , Fenotipo , Filogenia , Unión Proteica , Dominios ProteicosRESUMEN
Turnip mosaic virus (TuMV), belonging to the genus Potyvirus (family Potyviridae), has a large host range and consists of a single-stranded positive sense RNA genome encoding 12 proteins, including the P1 protease. This protein which is separated from the polyprotein by cis cleavage at its respective C-terminus, has been attributed with different functions during potyviral infection of plants. P1 of Turnip mosaic virus (P1-TuMV) harbors an FGSF-motif and FGSL-motif at its N-terminus. This motif is predicted to be a binding site for the host Ras GTPase-activating protein-binding protein (G3BP), which is a key factor for stress granule (SG) formation in mammalian systems and often targeted by viruses to inhibit SG formation. We therefore hypothesized that P1-TuMV might interact with G3BP to control and regulate plant SGs to optimize cellular conditions for the production of viral proteins. Here, we analyzed the co-localization of the Arabidopsis thaliana G3BP-2 with the P1 of two TuMV isolates, namely UK 1 and DEU 2. Surprisingly, P1-TuMV-DEU 2 co-localized with AtG3BP-2 under abiotic stress conditions, whereas P1-TuMV-UK 1 did not. AtG3BP-2::RFP showed strong SGs formation after stress, while P1-UK 1::eGFP maintained a chloroplastic signal under stress conditions, the signal of P1-DEU 2::eGFP co-localized with that of AtG3BP-2::RFP. This indicates a specific interaction between P1-DEU 2 and the AtG3BP family which is not solely based on the canonical interaction motifs.
Asunto(s)
Proteínas de Arabidopsis/metabolismo , Arabidopsis/virología , Potyvirus/metabolismo , Proteínas Virales/metabolismo , Secuencias de Aminoácidos , Arabidopsis/metabolismo , Brassica napus/virología , Raphanus/virologíaRESUMEN
The Arabidopsis thaliana genome encodes several genes that are known or predicted to participate in the formation of stress granules (SG). One family of genes encodes for Ras GTPase-activating protein-binding protein (G3BP)-like proteins. Seven genes were identified, of which one of the members was already shown to interact with plant virus proteins in a previous study. A phylogenetic and tissue-specific expression analysis, including laser-dissected phloem, by qRT-PCRs was performed and the sub-cellular localization of individual AtG3BP::EYFP fluorescent fusion proteins expressed in Nicotiana benthamiana epidermal cells was observed. Individual AtG3BP-protein interactions in planta were studied using the bimolecular fluorescence complementation approach in combination with confocal imaging in living cells. In addition, the early and late induction of G3BP-like expression upon Turnip mosaic virus infection was investigated by RNAseq and qRT-PCR. The results showed a high divergence of transcription frequency in the different plant tissues, promiscuous protein-protein interaction within the G3BP-like gene family, and a general induction by a viral infection with TuMV in A. thaliana. The information gained from these studies leads to a better understanding of stress granules, in particular their molecular mode of action in the plant and their role in plant virus infection.