RESUMO
BACKGROUND: Occurrence of extrachromosomal dsRNA elements has been described in the red-yeast Xanthophyllomyces dendrorhous, with numbers and sizes that are highly variable among strains with different geographical origin. The studies concerning to the encapsidation in viral-like particles and dsRNA-curing have suggested that some dsRNAs are helper viruses, while others are satellite viruses. However, the nucleotide sequences and functions of these dsRNAs are still unknown. In this work, the nucleotide sequences of four dsRNAs of the strain UCD 67-385 of X. dendrorhous were determined, and their identities and genome structures are proposed. Based on this molecular data, the dsRNAs of different strains of X. dendrorhous were analyzed. RESULTS: The complete sequences of L1, L2, S1 and S2 dsRNAs of X. dendrorhous UCD 67-385 were determined, finding two sequences for L1 dsRNA (L1A and L1B). Several ORFs were uncovered in both S1 and S2 dsRNAs, but no homologies were found for any of them when compared to the database. Instead, two ORFs were identified in each L1A, L1B and L2 dsRNAs, whose deduced amino acid sequences were homologous with a major capsid protein (5'-ORF) and a RNA-dependent RNA polymerase (3'-ORF) belonging to the Totiviridae family. The genome structures of these dsRNAs are characteristic of Totiviruses, with two overlapped ORFs (the 3'-ORF in the -1 frame with respect to the 5'-ORF), with a slippery site and a pseudoknot in the overlapped regions. These structures are essential for the synthesis of the viral polymerase as a fusion protein with the viral capsid protein through -1 ribosomal frameshifting. In the RNase protection analysis, all the dsRNAs in the four analyzed X. dendrorhous strains were protected from enzymatic digestion. The RT-PCR analysis revealed that, similar to strain UCD 67-385, the L1A and L1B dsRNAs coexist in the strains VKM Y-2059, UCD 67-202 and VKM Y-2786. Furthermore, determinations of the relative amounts of L1 dsRNAs using two-step RT-qPCR revealed a 40-fold increment of the ratio L1A/L1B in the S2 dsRNA-cured strain compared to its parental strain. CONCLUSIONS: Three totiviruses, named as XdV-L1A, XdV-L1B and XdV-L2, were identified in the strain UCD 67-385 of X. dendrorhous. The viruses XdV-L1A and XdV-L1B were also found in other three X. dendrorhous strains. Our results suggest that the smaller dsRNAs (named XdRm-S1 and XdRm-S2) of strain UCD 67-385 are satellite viruses, and particularly that XdRm-S2 is a satellite of XdV-L1A.
Assuntos
Basidiomycota/virologia , RNA Viral/genética , RNA Viral/isolamento & purificação , Totivirus/classificação , Totivirus/isolamento & purificação , Sequência de Aminoácidos , Sequência de Bases , Análise por Conglomerados , Dados de Sequência Molecular , Fases de Leitura Aberta , Filogenia , RNA de Cadeia Dupla/genética , RNA de Cadeia Dupla/isolamento & purificação , Vírus Satélites/classificação , Vírus Satélites/genética , Vírus Satélites/isolamento & purificação , Análise de Sequência de DNA , Homologia de Sequência de Aminoácidos , Totivirus/genéticaRESUMO
BACKGROUND: Strains of the astaxanthin producing yeast Xanthophyllomyces dendrorhous have been isolated from different cold regions around the earth, and the presence of double stranded RNA (dsRNA) elements was described in some isolates. This kind of viruses is widely distributed among yeasts and filamentous fungi and, although generally are cryptic in function, their studies have been a key factor in the knowledge of important fungi. In this work, the characterization and genetic relationships among dsRNA elements were determined in strains representatives of almost all regions of the earth where X. dendrorhous have been isolated. RESULTS: Almost all strains of X. dendrorhous analyzed carry one, two or four dsRNA elements, of molecular sizes in the range from 0.8 to 5.0 kb. Different dsRNA-patterns were observed in strains with different geographic origin, being L1 (5.0 kb) the common dsRNA element. By hybridization assays a high genomic polymorphism was observed among L1 dsRNAs of different X. dendrorhous strains. Contrary, hybridization was observed between L1 and L2 dsRNAs of strains from same or different regions, while the dsRNA elements of minor sizes (M, S1, and S2) present in several strains did not show hybridization with neither L1 or L2 dsRNAs. Along the growth curve of UCD 67-385 (harboring four dsRNAs) an increase of L2 relative to L1 dsRNA was observed, while the S1/L1 ratio remains constant, as well as the M/L1 ratio of Patagonian strain. Strains cured of S2 dsRNA were obtained by treatment with anisomycin, and comparison of its dsRNA contents with uncured strain, revealed an increase of L1 dsRNA while the L2 and S1 dsRNA remain unaltered. CONCLUSION: The dsRNA elements of X. dendrorhous are highly variable in size and sequence, and the dsRNA pattern is specific to the geographic region of isolation. Each L1 and L2 dsRNA are viral elements able to self replicate and to coexist into a cell, and L1 and S2 dsRNAs elements could be part of a helper/satellite virus system in X. dendrorhous.
Assuntos
Basidiomycota/virologia , Polimorfismo Genético , Vírus de RNA/genética , RNA de Cadeia Dupla/genética , Animais , Basidiomycota/isolamento & purificação , Geografia , Hibridização de Ácido NucleicoRESUMO
Foi realizada a analise bioquimica, antigenica e alergenica de extratos de esporos e micelios de Pisolithus tinctorius. Os esporos foram coletados de basidiocarpos em plantaçöes de Encalyptus spp e os micelios obtidos de cultura em meio de MNM. Baseado na curva de crescimento do fungo, obteve-se massa miceliana aos 10, 20, 30 e 40 dias de incubaçäo, que correspondem respectivamente ao início, meio, final da fase log e início da fase de declínio...