RESUMO
Here, we report the identification and characterization of two novel viruses associated with bird's-foot trefoil. Virus sequences related to those of enamoviruses (ssRNA (+); Luteoviridae; Enamovirus) and nucleorhabdoviruses (ssRNA (-); Rhabdoviridae; Nucleorhabdovirus) were detected in Lotus corniculatus transcriptome data. The genome of the tentatively named "bird's-foot trefoil-associated virus 1" (BFTV-1) is a 13,626-nt-long negative-sense ssRNA. BFTV-1 encodes six predicted gene products in the antigenome orientation in the canonical order 3'-N-P-P3-M-G-L-5'. The genome of the proposed "bird's-foot trefoil-associated virus 2" (BFTV-2) is 5,736 nt long with a typical 5Î-PO-P1-2-IGS-P3-P5-3' enamovirus genome structure. Phylogenetic analysis indicated that BFTV-1 is closely related to datura yellow vein nucleorhabdovirus and that BFTV-2 clusters into a monophyletic lineage of legume-associated enamoviruses. This subclade of highly related and co-divergent legume-associated viruses provides insights into the evolutionary history of the enamoviruses.
Assuntos
Genoma Viral/genética , Luteoviridae/classificação , Luteoviridae/genética , Rhabdoviridae/classificação , Rhabdoviridae/genética , Sequência de Bases , Evolução Molecular , Lotus/genética , Lotus/virologia , Luteoviridae/isolamento & purificação , Filogenia , Doenças das Plantas/virologia , RNA Viral/genética , Rhabdoviridae/isolamento & purificação , Análise de Sequência de DNARESUMO
Transposable elements represent a large proportion of the eukaryotic genomes. Long Terminal Repeat (LTR) retrotransposons are very abundant and constitute the predominant family of transposable elements in plants. Recent studies have identified chromoviruses to be a widely distributed lineage of Gypsy elements. These elements contain chromodomains in their integrases, which suggests a preference for insertion into heterochromatin. In turn, this preference might have contributed to the patterning of heterochromatin observed in host genomes. Despite their potential importance for our understanding of plant genome dynamics and evolution, the regulatory mechanisms governing the behavior of chromoviruses and their activities remain largely uncharacterized. Here, we report a detailed analysis of the spatio-temporal activity of a plant chromovirus in the endogenous host. We examined LORE1a, a member of the endogenous chromovirus LORE1 family from the model legume Lotus japonicus. We found that this chromovirus is stochastically de-repressed in plant populations regenerated from de-differentiated cells and that LORE1a transposes in the male germline. Bisulfite sequencing of the 5' LTR and its surrounding region suggests that tissue culture induces a loss of epigenetic silencing of LORE1a. Since LTR promoter activity is pollen specific, as shown by the analysis of transgenic plants containing an LTR::GUS fusion, we conclude that male germline-specific LORE1a transposition in pollen grains is controlled transcriptionally by its own cis-elements. New insertion sites of LORE1a copies were frequently found in genic regions and show no strong insertional preferences. These distinctive novel features of LORE1 indicate that this chromovirus has considerable potential for generating genetic and epigenetic diversity in the host plant population. Our results also define conditions for the use of LORE1a as a genetic tool.
Assuntos
Elementos de DNA Transponíveis/genética , Células Germinativas Vegetais/metabolismo , Lotus/genética , Lotus/virologia , Vírus de Plantas/genética , Regeneração/genética , Elementos Alu/genética , Mapeamento Cromossômico , Citosina/metabolismo , Metilação de DNA/genética , Variação Genética , Mutagênese Insercional , Especificidade de Órgãos/genética , Regiões Promotoras Genéticas/genética , Sequências Repetidas Terminais/genética , Transcrição GênicaRESUMO
Virus-induced gene silencing (VIGS) has become an important reverse genetics tool for functional genomics. VIGS vectors based on Pea early browning virus (PEBV, genus Tobravirus) and Bean pod mottle virus (genus Comovirus) are available for the legume species Pisum sativum and Glycine max, respectively. With the aim of extending the application of the PEBV VIGS vector to other legumes, we examined susceptibility of 99 accessions representing 24 legume species including 21 accessions of Medicago truncatula and 38 accessions Lotus japonicus. Infectivity of PEBV was tested by agro-inoculation with a vector carrying the complete beta-glucuronidase (GUS) coding sequence. In situ histochemical staining analysis indicated that 4 of 21 M. truncatula and three of three Lathyrus odorata accessions were infected systemically by GUS tagged PEBV, while none of 38 L. japonicus accessions displayed GUS staining of either inoculated or uninoculated leaves. Agro-inoculation of plants representing PEBV-GUS susceptible M. truncatula and L. odorata accessions with PEBV carrying a fragment of Phytoene desaturase (PDS) resulted in development of a bleaching phenotype suggesting a down-regulation of PDS expression. In M. truncatula this was supported by quantification of PDS mRNA levels by real-time PCR.
Assuntos
Inativação Gênica , Vetores Genéticos , Glucuronidase/metabolismo , Lathyrus/virologia , Medicago truncatula/virologia , Vírus de Plantas , Vírus de RNA , RNA Mensageiro/metabolismo , Regulação da Expressão Gênica de Plantas , Glucuronidase/genética , Lathyrus/genética , Lathyrus/metabolismo , Lotus/genética , Lotus/metabolismo , Lotus/virologia , Medicago truncatula/genética , Medicago truncatula/metabolismo , Folhas de Planta/genética , Folhas de Planta/metabolismo , Folhas de Planta/virologia , Vírus de Plantas/genética , Vírus de Plantas/metabolismo , Vírus de Plantas/patogenicidade , Plantas Geneticamente Modificadas , Vírus de RNA/genética , Vírus de RNA/metabolismo , Vírus de RNA/patogenicidade , RNA Mensageiro/genéticaRESUMO
A virus collection was used to identify a pathogen suitable for laboratory use with the model legume Lotus japonicus. Several Lotus species or L. japonicus accessions were tested and various degrees of susceptibility to the Arabis mosaic virus derived from barley (ArMV-ba) were found. Virus multiplication and persistence in Lotus tissue were examined, as well as plant responses to it. Sensitivity to the virus among the accessions and species is discussed in light of their geographical origin.
