The SGS3 protein involved in PTGS finds a family

BACKGROUND: Post transcriptional gene silencing (PTGS) is a recently discovered phenomenon that is an area of intense research interest. Components of the PTGS machinery are being discovered by genetic and bioinformatics approaches, but the picture is not yet complete. RESULTS: The gene for the PTGS impaired Arabidopsis mutant sgs3 was recently cloned and was not found to have similarity to any other known protein. By a detailed analysis of the sequence of SGS3 we have defined three new protein domains: the XH domain, the XS domain and the zf-XS domain, that are shared with a large family of uncharacterised plant proteins. This work implicates these plant proteins in PTGS. CONCLUSION: The enigmatic SGS3 protein has been found to contain two predicted domains in common with a family of plant proteins. The other members of this family have been predicted to be transcription factors, however this function seems unlikely based on this analysis. A bioinformatics approach has implicated a new family of plant proteins related to SGS3 as potential candidates for PTGS related functions

The DNA- and RNA-binding protein FACTOR of DNA METHYLATION 1 requires XH domain-mediated complex formation for its function in RNA-directed DNA methylation

Studies have identified a sub-group of SGS3-LIKE proteins including FDM1–5 and IDN2 as key components of RNA-directed DNA methylation pathway (RdDM). Although FDM1 and IDN2 bind RNAs with 5\u27 overhangs, their functions in the RdDM pathway remain to be examined. Here we show that FDM1 interacts with itself and with IDN2. Gel filtration suggests that FDM1 may exist as a homodimer in a heterotetramer complex in vivo. The XH domain of FDM1 mediates the FDM1–FDM1 and FDM1–IDN2 interactions. Deletion of the XH domain disrupts FDM1 complex formation and results in loss-of-function of FDM1. These results demonstrate that XH domainmediated complex formation of FDM1 is required for its function in RdDM. In addition, FDM1 binds unmethylated but not methylated DNAs through its coiled-coil domain. RNAs with 5\u27 overhangs does not compete with DNA for binding by FDM1, indicating that FDM1 may bind DNA and RNA simultaneously. These results provide insight into how FDM1 functions in RdDM

IDN2 and Its Paralogs Form a Complex Required for RNA–Directed DNA Methylation

IDN2/RDM12 has been previously identified as a component of the RNA–directed DNA methylation (RdDM) machinery in Arabidopsis thaliana, but how it functions in RdDM remains unknown. By affinity purification of IDN2, we co-purified two IDN2 paralogs IDP1 and IDP2 (IDN2 PARALOG 1 and 2). The coiled-coil domain between the XS and XH domains of IDN2 is essential for IDN2 homodimerization, whereas the IDN2 C-terminal XH domain but not the coiled-coil domain is required for IDN2 interaction with IDP1 and IDP2. By introducing the wild-type IDN2 sequence and its mutated derivatives into the idn2 mutant for complementation testing, we demonstrated that the previously uncharacterized IDN2 XH domain is required for the IDN2-IDP1/IDP2 complex formation as well as for IDN2 function. IDP1 is required for de novo DNA methylation, siRNA accumulation, and transcriptional gene silencing, whereas IDP2 has partially overlapping roles with IDP1. Unlike IDN2, IDP1 and IDP2 are incapable of binding double-stranded RNA, suggesting that the roles of IDP1 and IDP2 are different from those of IDN2 in the IDN2-IDP1/IDP2 complex and that IDP1 and IDP2 are essential for the functioning of the complex in RdDM

Evidence for Antisense Transcription Associated with MicroRNA Target mRNAs in Arabidopsis

Antisense transcription is a pervasive phenomenon, but its source and functional significance is largely unknown. We took an expression-based approach to explore microRNA (miRNA)-related antisense transcription by computational analyses of published whole-genome tiling microarray transcriptome and deep sequencing small RNA (smRNA) data. Statistical support for greater abundance of antisense transcription signatures and smRNAs was observed for miRNA targets than for paralogous genes with no miRNA cleavage site. Antisense smRNAs were also found associated with MIRNA genes. This suggests that miRNA-associated “transitivity” (production of small interfering RNAs through antisense transcription) is more common than previously reported. High-resolution (3 nt) custom tiling microarray transcriptome analysis was performed with probes 400 bp 5′ upstream and 3′ downstream of the miRNA cleavage sites (direction relative to the mRNA) for 22 select miRNA target genes. We hybridized RNAs labeled from the smRNA pathway mutants, including hen1-1, dcl1-7, hyl1-2, rdr6-15, and sgs3-14. Results showed that antisense transcripts associated with miRNA targets were mainly elevated in hen1-1 and sgs3-14 to a lesser extent, and somewhat reduced in dcl11-7, hyl11-2, or rdr6-15 mutants. This was corroborated by semi-quantitative reverse transcription PCR; however, a direct correlation of antisense transcript abundance in MIR164 gene knockouts was not observed. Our overall analysis reveals a more widespread role for miRNA-associated transitivity with implications for functions of antisense transcription in gene regulation. HEN1 and SGS3 may be links for miRNA target entry into different RNA processing pathways

Annotation and Expression of IDN2-like and FDM-like genes in sexual and aposporous hypericum perforatum L. Accessions

The protein IDN2, together with the highly similar interactors FDM1 and FDM2, is required for RNA-directed DNA methylation (RdDM) and siRNA production. Epigenetic regulation of gene expression is required to restrict cell fate determination in A. thaliana ovules. Recently, three transcripts sharing high similarity with the A. thaliana IDN2 and FDM1-2 were found to be differentially expressed in ovules of apomictic Hypericum perforatum L. accessions. To gain further insight into the expression and regulation of these genes in the context of apomixis, we investigated genomic, transcriptional and functional aspects of the gene family in this species. The H. perforatum genome encodes for two IDN2-like and 7 FDM-like genes. Differential and heterochronic expression of FDM4-like genes was found in H. perforatum pistils. The involvement of these genes in reproduction and seed development is consistent with the observed reduction of the seed set and high variability in seed size in A. thaliana IDN2 and FDM-like knockout lines. Differential expression of IDN2-like and FDM-like genes in H. perforatum was predicted to affect the network of potential interactions between these proteins. Furthermore, pistil transcript levels are modulated by cytokinin and auxin but the effect operated by the two hormones depends on the reproductive phenotype

Protein interactions between RNA silencing maintaining proteins and viral silencing suppressors

RNA-hiljennys on sekvenssispesifinen RNA:n hajotusmenetelmä, jota kasvit käyttävät geeniekspression säätelyyn ja puolustukseen virustartuntoja vastaan. Virukset ovat kehittäneet RNA-hiljennystä vastaan RNA-hiljennyksenestäjiä, jotka voivat estää ja häiritä hiljennysreaktion kulkua monin tavoin. Virusproteiinit voivat esimerkiksi sitoutua hiljennysreaktion keskeisiä vaiheita ylläpitäviin proteiineihin tai hiljennyksen signaloinnista vastaaviin molekyyleihin. Tässä työssä tutkittiin crini- ja potyvirusten tunnettujen RNA-hiljennyksenestäjäproteiinien vuorovaikutuksia neljän RNA-hiljennystä ylläpitävän kasviproteiinin kanssa. Proteiinien vuorovaikutuksia testattiin hiivan kaksihybridi-menetelmällä (YTHS) ja kahden molekyylin välistä fluoresenssikomplementaatiota (BiFC) käyttäen. Jälkimmäisen menetelmän avulla voidaan tutkittua proteiinivuorovaikutusta havainnoida soluympäristössä. Hiivavektoreihin kloonattujen geenien proteiinintuotto hiivasoluissa tarkastettiin western blot -menetelmällä. BiFC-menetelmässä keskityttiin pääosin hiivan kaksihybridi-menetelmällä havaittujen proteiinien välisten vuorovaikutusten tarkasteluun. Tutkimuksessa havaittiin kolme aikaisemmin tuntematonta proteiinien välistä vuorovaikutusta. Tämän lisäksi tutkittujen virusproteiinien todettiin ensimmäistä kertaa sitoutuvan hiljennystä ylläpitäviin kasviproteiineihin. Vastaavien kasviproteiinien on aikaisemmin todettu olevan muiden hiljennyksenestäjien kohteena. Koska kyseisten hiljennystä ylläpitävien proteiinien toiminnot tiedetään varsin tarkasti, voidaan kolmen havaitun proteiinivuorovaikutuksen ajatella häiritsevän RNA-hiljennystä kyseisten kasviproteiinien toimintaa estämällä.RNA silencing is a sequence specific RNA degradation mechanism which is used by plants to regulate gene expression and to combat virus infections. However, viruses have developed so called silencing suppressors, which can prevent and interfere silencing reaction by many ways. For example, virus proteins can bind to maintaining proteins of the silencing reaction or to molecules which are responsible for signaling of the silencing reaction. This thesis focused on the study of protein-protein-interactions between known silencing suppressors of crini- and potyviruses and four maintaining plant proteins of RNA silencing. Protein-protein-interactions were studied using the yeast two-hybrid system (YTHS) and the bimolecular fluorescence complementation assay (BiFC). The latter method enables visualization of the studied protein interactions in plant cells. Protein expression of the cloned genes in yeast vectors were studied by using western blot. BiFC analysis was focused on protein interactions which were found by YTHS. This study detected three previously unknown protein interactions. Two virus proteins were found for the first time to bind directly to silencing maintaining proteins that are known to be targets of other silencing suppressors. Because the functions of these silencing maintaining proteins are known, it is possible that the three interactions described in this study interfere RNA silencing by impeding the functions of the plant proteins

Estudio funcional de genes involucrados en la simbiosis fijadora de nitrógeno

Las leguminosas son capaces de establecer una simbiosis con bacterias que convierten el nitrógeno atmosférico a formas utilizables para la planta. Esta fijación de nitrógeno ocurre en órganos postembrionarios formados en la raíz llamados nódulos. El establecimiento de esta simbiosis involucra la participación de mecanismos de señalización que derivan en la activación de diferentes factores de transcripción. Los miembros de la familia del factor nuclear Y (NF-Y) se han implicado en diferentes etapas de la simbiosis del nódulo de la raíz. Anteriormente hemos demostrado que NF-YA1 y NF-YC1 juegan un papel crucial durante la nodulación, modulando la expresión de los genes del ciclo celular de transición G2/M en poroto (Phaseolus vulgaris). Un ensayo de inmunoprecipitación de cromatina (ChIP) seguido de PCR mostró un enriquecimiento de la región del promotor de una ciclina P en raíces que expresan FLAG-NF-YC1 en comparación con las raíces transformadas con el vector vacío, lo que sugiere que PvNF-YC1 se une directamente a esta región promotora y podría modular su expresión. El gen ortólogo de M. truncatula codifica para una ciclina tipo P4 que se expresa principalmente en la zona meristemática del nódulo y podría estar implicado en la reactivación del ciclo celular que inicia la organogénesis del nódulo. En el presente trabajo de tesis, para dilucidar la función biológica del gen PvCYCP4-1 en el contexto de la simbiosis fijadora de nitrógeno, se utilizó una estrategia de RNAi para reducir sus niveles de mRNA en raíces de poroto seguida de una caracterización del fenotípo. El silenciamiento de PvCYCP4-1 produjo a una marcada reducción en la cantidad y el tamaño de los nódulos formados por Rhizobium etli y afectó negativamente la frecuencia de los eventos de infección. Estos datos sugirieron que PvCYCP4-1 podría desempeñar un papel en la activación de las divisiones de células corticales necesarias para el desarrollo de nódulos y la infección de rizobios. Por otro lado, para investigar el papel de las moléculas de señalización bacterianas en la activación de los genes de respuesta a la simbiosis, hemos realizado un análisis del transcriptoma de raíces de P. vulgaris inoculadas con cepas mutantes de R. etli defectuosas en la síntesis de Nod Factor, exopolisacáridos o lipopolisacáridos. Se identificó un gen asociado a la regulación de la expresión génica mediada por pequeños RNAs, que codifica una proteína con dominio XH/XS. Este gen es ortólogo al gen IDN2 de Arabidopsis thaliana, una proteína de unión a RNA de doble cadena involucrada en la ruta de metilación del DNA dirigida por RNA (RdDM). Para obtener información sobre el papel de PvIDN2 durante la infección de la raíz y la organogénesis de los nódulos, se llevó cabo un análisis funcional de PvIDN2 utilizando RNAi. El análisis fenotípico mostró que las plantas con niveles reducidos de PvIDN2 exhibieron una reducción en el número de nódulos formados por R. etli en comparación con las raíces de control. Además, la frecuencia de los eventos de infección se vio afectada por el silenciamiento de PvIDN2. Estos resultados sugieren que PvIDN2 podría desempeñar un papel tanto en la infección de rizobios como en la organogénesis de los nódulos en las raíces de P. vulgaris. Utilizando la técnica de inmunoprecipitación de DNA metilado (MeDIP) pudimos demostrar que el silenciamiento génico postranscripcional de PvIDN2 afecta los niveles de metilación de genes que se encuentran asociados a los picos de acumulación de hetsiRNAs, permitiendo establecer una relación entre PvIDN2 y la ruta del RdDM. El conjunto de resultados obtenidos en este trabajo de tesis contribuyen a comprender en mayor detalle los mecanismos moleculares que gobiernan el establecimiento de una simbiosis fijadora de nitrógeno eficiente entre P. vulgaris y R. etli, además de que sienta las bases para futuros proyectos de investigación que permitan optimizar la fijación de nitrógeno en una planta de alto valor social e interés agronómico.Facultad de Ciencias Exacta

Enhanced metabolic engineering of lipid biosynthesis in leaves and seeds with the use of viral silencing-suppressor proteins

The use of transgenic pathways is a cornerstone of basic and applied research into plant biology. However, transgenes can fail over time and obtaining elite plant materials that perform well over numerous generations is an intensive process. Such failures in transgene performance are associated with the generation of small RNA (sRNA) in the host plant that trigger silencing. This mechanism is related to host defence pathways against invading nucleic acids including those from viral genomes. To counteract this silencing mechanism, plant viruses have evolved and encode for viral silencing-suppressor proteins (VSP) to block the silencing machinery of the host. This thesis tests the hypothesis that VSPs are also capable of enhancing transgene performance in stably-transformed plants. The effects of a number of VSPs on transgenic pathways were assessed transiently in Nicotiana benthamiana leaves and in long-term population studies spanning five and four generations in Arabidopsis thaliana and Brassica napus seeds, respectively. Overall this study shows that VSPs are able to enhance the performance of transgenic pathways in both leaves and seeds. The transient leaf assay in N.benthamiana leaves is a well-established tool and allows a rapid examination of transgenic pathways in a short period of time. One limitation of the assay format is an inability to both silence endogenous pathways and permit maximal overexpression of transgenes. This study demonstrates extensive manipulation of lipid pathways in N.benthamiana leaves by introducing an alternative VSP, V2, which stops the co-suppression of transgenes and allows simultaneous silencing of endogenes. A combination of V2, silencing of NbFAD2 and overexpression of GhCPFAS and AtDGAT1 resulted in high levels of a novel fatty acid, dihydrosterculic acid, in leaf oil. The V2-based assay was used to silence NbFAD7 and shunt linoleic acid into a three step transgenic pathway to synthesise arachidonic acid (AA), an ω-6 long chain polyunsaturated fatty acid (LCPUFA). Lipid head group fractionations of infiltrated leaf extracts showed that leaf cells rapidly shuffle novel fatty acids between various soluble and membrane-bound lipid pools. The assay was also used to investigate the effect of silencing a number of key lipid biosynthesis genes which included NbSAD1, NbFATA, NbFATB, NbFAD3, NbFAD6, NbLPCAT, NbGPAT9, NbLPAAT4 and NbLPAAT6 on lipid fluxes in N.benthamiana leaves. Various VSPs were co-expressed with a three step transgenic pathway for the synthesis of AA in A.thaliana and B.napus seeds. The expression of the VSP was limited to oil synthesis in the seed and in A.thaliana the results showed that transgenic populations co-expressing V2 or p19 contained higher levels of AA. A p19 line contained 40% of AA in T3 seeds although such high levels came at the expense of oil content. Similar constructs were also transformed into B.napus. Unlike A.thaliana, B.napus displayed a bottleneck in AA biosynthesis at an intermediate step, indicating differences in the biochemical capacity of B.napus and A.thaliana for the production of LCPUFA. Finally, a range of resources and experiments were designed for the characterisation of V2 protein. These resources resulted in the production and verification of a polyclonal antibody recognising V2. The experiments were aimed at quantifying V2 protein in various transgenic plant materials to assess the role of V2 in inhibiting co-suppression of transgenes however, the results were not conclusive

Study of the interactions between Arabis mosaic virus and its host plants

Arabis mosaic virus (ArMV) belongs to the plant virus genus Nepovirus of the Secoviridae. In the wine producing areas southwest of Germany, including Neustadt an der Weinstrasse (NW), ArMV is, along with the Grapevine fanleaf virus (GFLV) and the Raspberry ringspot virus (RpRSV), two other nepoviruses, a causative agent of the grapevine fanleaf disease, one of the most widespread and damaging virus diseases affecting grapevine. ArMV is transmitted by the nematode vector Xiphinema diversicaudatum, and has a wide natural host range. Nepoviruses have two single-stranded positive sense genomic RNAs, which are linked to a VPg at their 5’ ends, and polyadenylated at their 3’ends. ArMV isolates from different hosts and geographical origins were mechanically inoculated onto Chenopodium quinoas. The symptoms obtained with ArMV-NW were very mild, whereas ArMV-Lilac and –Lv produced symptoms of different severity. To characterize the symptom determinant(s) encoded by ArMV, fragments corresponding to genes from both RNAs 1 and 2 of full-length infectious clones of ArMV-NW were exchanged by their counterpart of the ArMV-Lv or -Lilac isolates and tested by mechanical inoculations onto Chenopodium quinoa for their infectivity and functionality. The results obtained from the first set of clones showed the N-terminal protein of the protein 2A, the movement protein and the protein 1A are involved in the symptoms development. In Nicotiana benthamiana, the establishment rates of infection between ArMV-NW and -Lv differed, however the recovery phenomenon took place around the same time for both isolates, resulting in a disappearance of symptoms in ArMV-Lv-infected plants and a similarly low accumulation of viral RNAs for both isolates. Moreover, the ArMV-NW-recovered plants were not resistant to a secondary infection with ArMV-Lv. Post-Transcriptional Gene Silencing (PTGS) is an important antiviral defense system in plants. However, numerous viruses have developed a counter-defense strategy, by coding for a protein acting as a suppressor of gene silencing. So far, no suppressor of gene silencing has been identified for nepoviruses. The use of wild-type and GFP-transgenic Nicotiana benthamiana 16C for coinfiltration experiments via Agrobacterium tumefaciens of constructs containing the GFP and the different genes encoded by ArMV RNAs 1 or 2 allowed to identify the implication of NTB, VPg-Pro and/or VPg-Pro-Pol in the suppression of RNA silencing