RESUMEN
Cucumber mosaic virus (CMV) is a destructive pathogen affecting Capsicum annuum (pepper) production. The pepper Cmr1 gene confers resistance to most CMV strains, but is overcome by CMV-P1 in a process dependent on the CMV-P1 RNA1 helicase domain (P1 helicase). Here, to identify host factors involved in CMV-P1 infection in pepper, a yeast two-hybrid library derived from a C. annuum 'Bukang' cDNA library was screened, producing a total of 76 potential clones interacting with the P1 helicase. Beta-galactosidase filter lift assay, PCR screening, and sequencing analysis narrowed the candidates to 10 genes putatively involved in virus infection. The candidate host genes were silenced in Nicotiana benthamiana plants that were then inoculated with CMV-P1 tagged with the green fluorescent protein (GFP). Plants silenced for seven of the genes showed development comparable to N. benthamiana wild type, whereas plants silenced for the other three genes showed developmental defects including stunting and severe distortion. Silencing formate dehydrogenase and calreticulin-3 precursor led to reduced virus accumulation. Formate dehydrogenase-silenced plants showed local infection in inoculated leaves, but not in upper (systemic) leaves. In the calreticulin-3 precursor-silenced plants, infection was not observed in either the inoculated or the upper leaves. Our results demonstrate that formate dehydrogenase and calreticulin-3 precursor are required for CMV-P1 infection.
Asunto(s)
Capsicum/genética , Cucumovirus/enzimología , Genes de Plantas , ARN Helicasas/metabolismo , Agrobacterium/metabolismo , Calreticulina/genética , Cucumovirus/genética , ADN Complementario/metabolismo , Formiato Deshidrogenasas/genética , Regulación de la Expresión Génica de las Plantas , Biblioteca de Genes , Silenciador del Gen , Proteínas Fluorescentes Verdes/metabolismo , Enfermedades de las Plantas/genética , Hojas de la Planta/metabolismo , Reacción en Cadena de la Polimerasa , Unión Proteica , Estructura Terciaria de Proteína , Análisis de Secuencia de ADN , Técnicas del Sistema de Dos Híbridos , beta-Galactosidasa/metabolismoRESUMEN
The Cmr1 gene in peppers confers resistance to Cucumber mosaic virus isolate-P0 (CMV-P0). Cmr1 restricts the systemic spread of CMV strain-Fny (CMV-Fny), whereas this gene cannot block the spread of CMV isolate-P1 (CMV-P1) to the upper leaves, resulting in systemic infection. To identify the virulence determinant of CMV-P1, six reassortant viruses and six chimeric viruses derived from CMV-Fny and CMV-P1 cDNA clones were used. Our results demonstrate that the C-terminus of the helicase domain encoded by CMV-P1 RNA1 determines susceptibility to systemic infection, and that the helicase domain contains six different amino acid substitutions between CMV-Fny and CMV-P1(.) To identify the key amino acids of the helicase domain determining systemic infection with CMV-P1, we then constructed amino acid substitution mutants. Of the mutants tested, amino acid residues at positions 865, 896, 957, and 980 in the 1a protein sequence of CMV-P1 affected the systemic infection. Virus localization studies with GFP-tagged CMV clones and in situ localization of virus RNA revealed that these four amino acid residues together form the movement determinant for CMV-P1 movement from the epidermal cell layer to mesophyll cell layers. Quantitative real-time PCR revealed that CMV-P1 and a chimeric virus with four amino acid residues of CMV-P1 accumulated more genomic RNA in inoculated leaves than did CMV-Fny, indicating that those four amino acids are also involved in virus replication. These results demonstrate that the C-terminal region of the helicase domain is responsible for systemic infection by controlling virus replication and cell-to-cell movement. Whereas four amino acids are responsible for acquiring virulence in CMV-Fny, six amino acid (positions at 865, 896, 901, 957, 980 and 993) substitutions in CMV-P1 were required for complete loss of virulence in 'Bukang'.
Asunto(s)
Cucumovirus/genética , ARN Helicasas/química , ARN Viral/genética , Agrobacterium/virología , Secuencia de Aminoácidos , Aminoácidos/química , Capsicum/virología , ADN Complementario/metabolismo , Resistencia a la Enfermedad/genética , Ensayo de Inmunoadsorción Enzimática/métodos , Proteínas Fluorescentes Verdes/metabolismo , Hibridación in Situ , Datos de Secuencia Molecular , Mutación , Enfermedades de las Plantas/virología , Estructura Terciaria de Proteína , ARN Viral/metabolismo , Virosis/virologíaRESUMEN
A phenyl propanoid derivative, dillapional(1) was found to be a antimicrobial principle of the stems of Foeniculum vulgare (Umbelliferae) with MIC values of 125, 250 and 125/ against Bacillus subtilis, Aspergillus niger and Cladosporium cladosporioides, respectively. A coumarin derivative, scopoletin(2) was also isolated as marginally antimicrobial agent along with inactive compounds, dillapiol(3), bergapten(4), imperatorin(5) and psolaren(6) from this plant. The isolates 1-6 were not active against the Escherichia coli.