RESUMO
Rice crops in South and Southeast Asian countries suffer critical yield losses due to rice tungro disease caused by joint infection with rice tungro bacilliform virus (RTBV) and rice tungro spherical virus (RTSV). Previously, for generating RNA interference-based transgenic resistance against tungro viruses, RTBV ORF IV was used as a transgene to develop RTBV resistance in a popular high-yielding scented rice variety. The transgene from this line was then introgressed into five popular high-yielding but tungro-susceptible rice varieties by marker-assisted backcross breeding with a view to combine the resistant trait with the agronomic traits. The present work includes a resistance assay of the BC3F5 lines of these varieties under glasshouse conditions. Out of a total of 28 lines tested, each consisting of 12 individual plants, eight lines showed significant amelioration in height reduction and 100- to 1000-fold reduction in RTBV titers. The RNAi-mediated resistance was clearly manifested by the presence of virus-derived small RNA (vsRNA) specific for RTBV ORF IV in the transgenic backcrossed lines.
Assuntos
Resistência à Doença , Oryza/imunologia , Doenças das Plantas/virologia , Plantas Geneticamente Modificadas/imunologia , Tungrovirus/fisiologia , Proteínas Virais/genética , Índia , Oryza/genética , Oryza/virologia , Doenças das Plantas/imunologia , Plantas Geneticamente Modificadas/genética , Plantas Geneticamente Modificadas/virologia , Interferência de RNA , Transgenes , Tungrovirus/genética , Tungrovirus/isolamento & purificação , Proteínas Virais/metabolismo , Waikavirus/genética , Waikavirus/metabolismoRESUMO
Rice tungro disease is caused by a complex of two viruses, Rice tungro bacilliform virus (RTBV) and Rice tungro spherical virus (RTSV). To examine the RNAi-based defence response in rice during tungro disease, we characterized the virus-derived small RNAs and miRNAs by Deep Sequencing. We found that, while 21 nt/22 nt (nucleotide) siRNAs are predominantly produced in a continuous, overlapping and asymmetrical manner from RTBV, siRNA accumulation from RTSV were negligible. Additionally, 54 previously known miRNAs from rice, predicted to be regulating genes involved in plant defence, hormone signaling and developmental pathways were differentially expressed in the infected samples, compared to the healthy ones. This is the first study of sRNA profile of tungro virus complex from infected rice plants. The biased response of the host antiviral machinery against the two viruses and the differentially-expressed miRNAs are novel observations, which entail further studies.
Assuntos
Regulação da Expressão Gênica de Plantas/imunologia , Regulação Viral da Expressão Gênica , Oryza/genética , RNA Interferente Pequeno/genética , Tungrovirus/genética , Waikavirus/genética , Perfilação da Expressão Gênica , Sequenciamento de Nucleotídeos em Larga Escala , Interações Hospedeiro-Patógeno , MicroRNAs/antagonistas & inibidores , MicroRNAs/genética , MicroRNAs/metabolismo , Oryza/imunologia , Oryza/virologia , Doenças das Plantas/genética , Doenças das Plantas/imunologia , Doenças das Plantas/virologia , Imunidade Vegetal/genética , Proteínas de Plantas/genética , Proteínas de Plantas/imunologia , RNA Interferente Pequeno/metabolismo , RNA Viral/antagonistas & inibidores , RNA Viral/genética , RNA Viral/metabolismo , Tungrovirus/metabolismo , Waikavirus/metabolismoRESUMO
Maize chlorotic dwarf virus (MCDV), a member of the genus Waikavirus, family Secoviridae, has a 11784 nt (+)ssRNA genome that encodes a 389kDa proteolytically processed polyprotein. We show that the N-terminal 78kDa polyprotein (R78) of MCDV acts as a suppressor of RNA silencing in a well-established assay system. We further demonstrate that R78 is cleaved by the viral 3C-like protease into 51 and 27kDa proteins (p51 and p27), and that p51 is responsible for silencing suppressor activity. Silencing suppressor activity of R78 is conserved in three divergent MCDV strains (MCDV-Severe, MCDV-M1, and MCDV-Tennessee), as well as the waikavirus Bellflower vein chlorosis virus, but was not detected for orthologous protein of Rice tungro spherical virus (RTSV-A) or the similarly-positioned protein from the sequivirus Parsnip yellow fleck virus (PYFV). This is the first identification of a virus suppressor of RNA silencing encoded by a waikavirus.
Assuntos
Genoma Viral/genética , Interferência de RNA/fisiologia , Waikavirus/genética , Waikavirus/metabolismo , Zea mays/virologia , Doenças das Plantas/virologia , Proteínas Virais/metabolismoRESUMO
The genome of Maize chlorotic dwarf virus (MCDV; genus Waikavirus; family Sequiviridae) consists of a monopartite positive-sense RNA genome encoding a single large polyprotein. Antibodies were produced to His-fusions of three undefined regions of the MCDV polyprotein: the N-terminus of the polyprotein (R78), a region between coat proteins (CPs) and the nucleotide-binding site (NBS) (R37), and a region between the NBS and a 3C-like protease (R69). The R78 antibodies react with proteins of 50 kDa (P50), 35 kDa (P35), and 25 kDa (P25) in virus preparations, and with P35 in plant extracts. In extracts of the leafhopper vector Graminella nigrifrons fed on MCDV-infected plants, the R78 antibodies reacted with P25 but not with P50 and P35. The R69 antibodies bound proteins of approximately 36 kDa (P36), 30 kDa (P30), and 26 kDa (P26) in virus preparations, and P36 and P26 in plant extracts. Antibodies to R37 reacted with a 26-kDa protein in purified virus preparations, but not in plant extracts. Neither the R69 nor the R37 antibodies bound any proteins in G. nigrifrons. Thus, in addition to the three CPs, cysteine protease and RNA-dependent RNA polymerase, the MCDV polyprotein is apparently post-transitionally cleaved into P50, P35, P25, P36, P30, and P26.
