Deconstruction of the (Paleo)Polyploid Grapevine Genome Based on the Analysis of Transposition Events Involving NBS Resistance Genes

Plants have followed a reticulate type of evolution and taxa have frequently merged via allopolyploidization. A polyploid structure of sequenced genomes has often been proposed, but the chromosomes belonging to putative component genomes are difficult to identify. The 19 grapevine chromosomes are evolutionary stable structures: their homologous triplets have strongly conserved gene order, interrupted by rare translocations. The aim of this study is to examine how the grapevine nucleotide-binding site (NBS)-encoding resistance (NBS-R) genes have evolved in the genomic context and to understand mechanisms for the genome evolution. We show that, in grapevine, i) helitrons have significantly contributed to transposition of NBS-R genes, and ii) NBS-R gene cluster similarity indicates the existence of two groups of chromosomes (named as Va and Vc) that may have evolved independently. Chromosome triplets consist of two Va and one Vc chromosomes, as expected from the tetraploid and diploid conditions of the two component genomes. The hexaploid state could have been derived from either allopolyploidy or the separation of the Va and Vc component genomes in the same nucleus before fusion, as known for Rosaceae species. Time estimation indicates that grapevine component genomes may have fused about 60 mya, having had at least 40–60 mya to evolve independently. Chromosome number variation in the Vitaceae and related families, and the gap between the time of eudicot radiation and the age of Vitaceae fossils, are accounted for by our hypothesis

Lox-dependent gene expression in transgenic plants obtained via Agrobacterium-mediated transformation

Lox sites of the Cre/lox recombination system from bacteriophage P1 were analyzed for their ability to affect on transgene expression when inserted upstream from a gene coding sequence adjacent to the right border (RB) of T-DNA. Wild and mutated types of lox sites were tested for their effect upon bar gene expression in plants obtained via Agrobacterium-mediated and biolistic transformation methods. Lox-mediated expression of bar gene, recognized by resistance of transgenic plants to PPT, occurred only in plants obtained via Agrobacterium-mediated transformation. RT-PCR analysis confirms that PPT-resistant phenotype of transgenic plants obtained via Agrobacterium-mediated transformation was caused by activation of bar gene. The plasmid with promoterless gus gene together with the lox site adjacent to the RB was constructed and transferred to Nicotiana tabacum as well. Transgenic plants exhibited GUS activity and expression of gus gene was detected in plant leaves. Expression of bar gene from the vectors containing lox site near RB allowed recovery of numerous PPT-resistant transformants of such important crops as Beta vulgaris, Brassica napus, Lactuca sativa and Solanum tuberosum. Our results demonstrate that the lox site sequence adjacent to the RB can be used to control bar gene expression in transgenic plants.Проанализирована способность lox-сайтов Cre/lox системы рекомбинации бактериофага Р1 влиять на экспрессию трансгенов при расположении этой последовательности непосредственно возле правого бордера (RB) перед кодирующей последовательностью гена. Нативная и мутированная последовательность lox-сайта были размещены в векторах для трансформации возле гена bar и проведена генетическая трансформация растений с помощью агробактерии и биолистическим методом. Lox-опосредованная экспрессия гена bar, обусловливающая устойчивость растений к фосфинотрицину, наблюдалась только у растений, которые получены с помощью агробактериальной трансформации. Методом РТ-ПЦР анализа подтверждено, что в трансгенных растениях, устойчивых к фосфинотрицину, происходит транскрипция гена bar. Сконструирован вектор, в котором ген gus и предшествующий ему lox-сайт размещены вблизи правого бордера, и проведена трансформация табака этим вектором. Экспрессия гена gus задетектирована в листьях трансгенных растений. Векторы, у которых последовательность lox-сайта предшествует гену bar возле правого бордера (RB-lox-bar), успешно использованы для получения устойчивых к фосфинотрицину трансгенных растений таких видов, как Beta vulgaris, Brassica napus, Lactuca sativa и Solanum tuberosum. Наши результаты подтверждают возможность использования последовательности lox-сайта возле правого бордера для контроля экспрессии гена bar в трансгенных растениях

Nuclear import of CaMV P6 is required for infection and suppression of the RNA silencing factor DRB4

Replication of Cauliflower mosaic virus (CaMV), a plant double-stranded DNA virus, requires the viral translational transactivator protein P6. Although P6 is known to form cytoplasmic inclusion bodies (viroplasms) so far considered essential for virus biology, a fraction of the protein is also present in the nucleus. Here, we report that monomeric P6 is imported into the nucleus through two importin-α-dependent nuclear localization signals, and show that this process is mandatory for CaMV infectivity and is independent of translational transactivation and viroplasm formation. One nuclear function of P6 is to suppress RNA silencing, a gene regulation mechanism with antiviral roles, commonly counteracted by dedicated viral suppressor proteins (viral silencing suppressors; VSRs). Transgenic P6 expression in Arabidopsis is genetically equivalent to inactivating the nuclear protein DRB4 that facilitates the activity of the major plant antiviral silencing factor DCL4. We further show that a fraction of P6 immunoprecipitates with DRB4 in CaMV-infected cells. This study identifies both genetic and physical interactions between a VSR to a host RNA silencing component, and highlights the importance of subcellular compartmentalization in VSR function