PSTVd infection in Nicotiana benthamiana plants has a minor yet detectable effect on CG methylation

Viroids are small circular RNAs infecting a wide range of plants. They do not code for any protein or peptide and therefore rely on their structure for their biological cycle. Observed phenotypes of viroid infected plants are thought to occur through changes at the transcriptional/translational level of the host. A mechanism involved in such changes is RNA-directed DNA methylation (RdDM). Till today, there are contradictory works about viroids interference of RdDM. In this study, we investigated the epigenetic effect of viroid infection in Nicotiana benthamiana plants. Using potato spindle tuber viroid (PSTVd) as the triggering pathogen and via bioinformatic analyses, we identified endogenous gene promoters and transposable elements targeted by 24 nt host siRNAs that differentially accumulated in PSTVd-infected and healthy plants. The methylation status of these targets was evaluated following digestion with methylation-sensitive restriction enzymes coupled with PCR amplification, and bisulfite sequencing. In addition, we used Methylation Sensitive Amplification Polymorphism (MSAP) followed by sequencing (MSAP-seq) to study genomic DNA methylation of 5-methylcytosine (5mC) in CG sites upon viroid infection. In this study we identified a limited number of target loci differentially methylated upon PSTVd infection. These results enhance our understanding of the epigenetic host changes as a result of pospiviroid infection

Identification of Domains and Factors Involved in MINIYO Nuclear Import

© 2019 Contreras, Kallemi, González-García, Lazarova, Sánchez-Serrano, Sanmartín and Rojo.The transition of stem cells from self-renewal into differentiation is tightly regulated to assure proper development of the organism. Arabidopsis MINIYO (IYO) and its mammalian orthologue RNA polymerase II associated protein 1 (RPAP1) are essential factors for initiating stem cell differentiation in plants and animals. Moreover, there is evidence suggesting that the translocation of IYO and RPAP1 from the cytosol into the nucleus functions as a molecular switch to initiate this cell fate transition. Identifying the determinants of IYO subcellular localization would allow testing if, indeed, nuclear IYO migration triggers cell differentiation and could provide tools to control this crucial developmental transition. Through transient and stable expression assays in Nicotiana benthamiana and Arabidopsis thaliana, we demonstrate that IYO contains two nuclear localization signals (NLSs), located at the N- and C-terminus of the protein, which mediate the interaction with the NLS-receptor IMPA4 and the import of the protein into the nucleus. Interestingly, IYO also interacts with GPN GTPases, which are involved in selective nuclear import of RNA polymerase II. This interaction is prevented when the G1 motif in GPN1 is mutated, suggesting that IYO binds specifically to the nucleotide-bound form of GPN1. In contrast, deleting the NLSs in IYO does not prevent the interaction with GPN1, but it interferes with import of GPN1 into the nucleus, indicating that IYO and GPN1 are co-transported as a complex that requires the IYO NLSs for import. This work unveils key domains and factors involved in IYO nuclear import, which may prove instrumental to determine how IYO and RPAP1 control stem cell differentiation.This work was supported by the Spanish Ministry of Economy & Competitivity and FEDER funds (BIO2018-094257-B MICINN/FEDER to ER, MS and JJSS). RC and AL were recipient of a FPI scholarships from the Spanish Ministry of Economy and Competitivity. PK was recipient of an ERASMUS fellowship. We acknowledge support of the publication fee by the CSIC Open Access Publication Support Initiative through its Unit of Information Resources for Research (URICI)

Identification of Domains and Factors Involved in MINIYO Nuclear Import

[EN] The transition of stem cells from self-renewal into differentiation is tightly regulated to assure proper development of the organism. Arabidopsis MINIYO (IYO) and its mammalian orthologue RNA polymerase II associated protein 1 (RPAP1) are essential factors for initiating stem cell differentiation in plants and animals. Moreover, there is evidence suggesting that the translocation of IYO and RPAP1 from the cytosol into the nucleus functions as a molecular switch to initiate this cell fate transition. Identifying the determinants of IYO subcellular localization would allow testing if, indeed, nuclear IYO migration triggers cell differentiation and could provide tools to control this crucial developmental transition. Through transient and stable expression assays in Nicotiana benthamiana and Arabidopsis thaliana, we demonstrate that IYO contains two nuclear localization signals (NLSs), located at the N- and C-terminus of the protein, which mediate the interaction with the NLS-receptor IMPA4 and the import of the protein into the nucleus. Interestingly, IYO also interacts with GPN GTPases, which are involved in selective nuclear import of RNA polymerase II. This interaction is prevented when the G1 motif in GPN1 is mutated, suggesting that IYO binds specifically to the nucleotide-bound form of GPN1. In contrast, deleting the NLSs in IYO does not prevent the interaction with GPN1, but it interferes with import of GPN1 into the nucleus, indicating that IYO and GPN1 are co-transported as a complex that requires the IYO NLSs for import. This work unveils key domains and factors involved in IYO nuclear import, which may prove instrumental to determine how IYO and RPAP1 control stem cell differentiation.This work was supported by the Spanish Ministry of Economy & Competitivity and FEDER funds (BIO2018-094257-B MICINN/FEDER to ER, MS and JJSS). RC and AL were recipient of a FPI scholarships from the Spanish Ministry of Economy and Competitivity. PK was recipient of an ERASMUS fellowship. We acknowledge support of the publication fee by the CSIC Open Access Publication Support Initiative through its Unit of Information Resources for Research (URICI).Contreras, R.; Kallemi, P.; González-García, MP.; Lazarova, A.; Sánchez-Serrano, JJ.; Sanmartín, M.; Rojo, E. (2019). Identification of Domains and Factors Involved in MINIYO Nuclear Import. Frontiers in Plant Science. 10:1-12. https://doi.org/10.3389/fpls.2019.010441121