RESUMO
MicroRNAs are regulatory RNAs that are scarcely described in Baculoviruses. In this work we predicted a microRNA in silico, denominated agmnpv-miR-4, encoded in the genome of Anticarsia gemmatalis Nucleopolyhedrovirus (AgMNPV), which is homologous to the already validated bmnpv-miR-4 from Bombyx mori Nucleopolyhedrovirus (BmNPV). Considering information known for bmnpv-miR-4 such as seed sequence, coding location in the genome and putative target binding, we searched for the coding sequence of agmnpv-miR-4 in AgMNPV genome. A precursor sequence of agmnpv-miR-4 was predicted, and we identified a putative 23â¯nt mature microRNA, agmnpv-miR-4, coded in the complementary strand of AgMNPV-2D between positions 49,450 and 49,472. We validated agmnpv-miR-4 by Northern blot from HighFive cells and A. gemmatalis larve extracts infected with AgMNPV.
RESUMO
Citrus psorosis (CPsV) and Mirafiori lettuce big-vein virus (MiLBVV) belong to the family Ophioviridae, plant viruses with filamentous nucleocapsids and segmented genomes of negative polarity, causing the worldwide distributed citrus psorosis and lettuce big-vein diseases, respectively. To gain insight into the replication cycle of these viruses, the subcellular localization of the viral coat proteins (CP) was studied. Immunoblot analysis of fractionated extracts derived from natural and experimental infected hosts indicated that the CP of CPsV occurs in the soluble cytoplasmic fraction. The cytoplasmic localization of this protein was confirmed by confocal microscopy of fluorescent protein (FP)-tagged CP following its expression in either CPsV-infected and healthy Citrus sinensis plants or in Nicotiana benthamiana plants. The same localization was observed for FP-tagged CP of MiLBVV. The CPs of CPsV and MiLBBV can undergo homologous and heterologous interactions as revealed by fluorescent lifetime imaging microscopy and co-immunoprecipitation analysis. A putative leucine zipper motif that is conserved among ophiovirus CP sequences may account for these interactions.
Assuntos
Proteínas do Capsídeo/metabolismo , Citrus/virologia , Citoplasma/metabolismo , Doenças das Plantas/virologia , Vírus de Plantas/metabolismo , Vírus de RNA/metabolismo , Motivos de Aminoácidos , Sequência de Aminoácidos , Proteínas do Capsídeo/química , Proteínas do Capsídeo/genética , Sequência Conservada , Expressão Gênica , Ordem dos Genes , Genes Reporter , Vetores Genéticos/genética , Dados de Sequência Molecular , Folhas de Planta/virologia , Vírus de Plantas/genética , Plantas Geneticamente Modificadas , Ligação Proteica , Transporte Proteico , Vírus de RNA/genética , Alinhamento de SequênciaRESUMO
The lack of naturally occurring resistance to Citrus psorosis virus (CPsV) has demanded exploitation of a transgenic approach for the development of CPsV-resistant sweet orange plants. Transgenic sweet orange plants producing intron-hairpin RNA transcripts (ihpRNA) corresponding to viral cp, 54K or 24K genes were generated and analyzed at the molecular and phenotypic levels. Two independent CPsV challenge assays demonstrated that expression of ihpRNA derived from the cp gene (ihpCP) provided a high level of virus resistance, while those derived from 54K and 24K genes (ihp54K and ihp24K) provided partial or no resistance. The presence of small interfering RNA molecules (siRNAs) in the ihpCP transgenic sweet orange plants prior to virus challenge, indicated that CPsV resistance was due to pre-activated RNA silencing, but siRNAs accumulation level was not directly correlated to the degree of the triggered virus resistance among the different lines. However, pre-activation of the RNA-silencing machinery and a certain minimum accumulation level of siRNA molecules targeting the viral genome are key factors for creating virus-resistant plants. This is the first report of resistance in citrus plants against a negative-strand RNA virus as CPsV.
Assuntos
Citrus/virologia , Interações Hospedeiro-Patógeno , Vírus de Plantas/metabolismo , Plantas Geneticamente Modificadas/virologia , Interferência de RNA , Southern Blotting , Proteínas do Capsídeo/química , Proteínas do Capsídeo/metabolismo , Ensaio de Imunoadsorção Enzimática , Dados de Sequência Molecular , Vírus de Plantas/genética , RNA Interferente PequenoRESUMO
Citrus psorosis virus (CPsV), genus Ophiovirus, family Ophioviridae, is the causal agent of a serious disease affecting citrus trees in many countries. The viral genome consists of three ssRNAs of negative polarity. Post-transcriptional gene silencing (PTGS), a mechanism of plant defence against viruses, can be induced by transgenic expression of virus-derived sequences encoding hairpin RNAs. Since the production of transgenic citrus lines and their evaluation would take years, a herbaceous model plant, Nicotiana benthamiana, was used to test hairpin constructs. The expression of self-complementary hairpin RNA fragments from the coat protein (cp) and 54K genes of the Argentine CPsV 90-1-1 isolate conferred resistance on N. benthamiana plants, indicating that these constructs are good candidates for the transformation of citrus plants. The degree of resistance obtained varied depending on the viral sequence chosen. The analysis of the levels of small interfering RNA accumulation and viral RNAs indicated that the construct derived from cp gene was better at inducing PTGS than that originating from the 54K gene. The dependence of PTGS induction on the degree of identity between the target and the inducer transgene sequences was tested using sequences derived from CPV4, a more distant isolate of CPsV, as PTGS targets. Efficient silencing induction was also obtained to this isolate through the expression of the cp-derived hairpin. This is the first report of transgenic-resistant plants within the context of this serious citrus disease.
Assuntos
Imunidade Inata/imunologia , Nicotiana/genética , Nicotiana/virologia , Doenças das Plantas/imunologia , Vírus de Plantas/genética , RNA Interferente Pequeno/genética , Proteínas Virais/genética , Sequência de Bases , Proteínas do Capsídeo/genética , Regulação da Expressão Gênica de Plantas , Inativação Gênica , Genes Virais/genética , Imunidade Inata/genética , Dados de Sequência Molecular , Peso Molecular , Doenças das Plantas/genética , Doenças das Plantas/virologia , Vírus de Plantas/isolamento & purificação , Plantas Geneticamente Modificadas , RNA Viral/genética , Alinhamento de Sequência , Fatores de TempoRESUMO
Citrus psorosis is a serious viral disease affecting citrus trees in many countries. Its causal agent is Citrus psorosis virus (CPsV), the type member of genus Ophiovirus. CPsV infects most important citrus varieties, including oranges, mandarins and grapefruits, as well as hybrids and citrus relatives used as rootstocks. Certification programs have not been sufficient to control the disease and no sources of natural resistance have been found. Pathogen-derived resistance (PDR) can provide an efficient alternative to control viral diseases in their hosts. For this purpose, we have produced 21 independent lines of sweet orange expressing the coat protein gene of CPsV and five of them were challenged with the homologous CPV 4 isolate. Two different viral loads were evaluated to challenge the transgenic plants, but so far, no resistance or tolerance has been found in any line after 1 year of observations. In contrast, after inoculation all lines showed characteristic symptoms of psorosis in the greenhouse. The transgenic lines expressed low and variable amounts of the cp gene and no correlation was found between copy number and transgene expression. One line contained three copies of the cp gene, expressed low amounts of the mRNA and no coat protein. The ORF was cytosine methylated suggesting a PTGS mechanism, although the transformant failed to protect against the viral load used. Possible causes for the failed protection against the CPsV are discussed.
Assuntos
Proteínas do Capsídeo/genética , Citrus/genética , Vírus de Plantas/genética , Plantas Geneticamente Modificadas/genética , Transformação Genética/genética , Northern Blotting , Southern Blotting , Citrus/crescimento & desenvolvimento , Citrus/virologia , Ensaio de Imunoadsorção Enzimática , Imunidade Inata/genética , Modelos Genéticos , Doenças das Plantas/genética , Doenças das Plantas/virologia , Plantas Geneticamente Modificadas/crescimento & desenvolvimento , Plantas Geneticamente Modificadas/virologia , Plasmídeos/genética , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
Citrus Psorosis in Argentina is a serious disease. Citrus is produced in two regions located in the northeast (NE) and northwest (NW) area of the country. These two areas have different climates and soil types, and therefore different citrus species and varieties are cultivated. In the NE region, Psorosis is epidemic, and in the NW region, the disease was described on several occasions since 1938, but it is not observed commonly in the orchards. Recently, trees with symptoms of Psorosis were observed in the Tucumán and Salta Provinces located in the NW region. Epidemiological studies in Argentina and Texas suggested that the disease is spread naturally by an unknown vector. The causal agent of the disease is the Citrus psorosis virus (CPsV), which can be detected by TAS-ELISA, RT-PCR and indicator plants. A new more rapid TAS-ELISA-HRP (horseradish peroxidase) is described which is more reliable, faster and more sensitive than the currently used for this virus, the TAS-ELISA-AP (alkaline phosphatase). Psorosis was detected by this improved method in few trees in the orchards of the Tucumán Province, in the NW citrus region, although natural spread does not seem to occur.
