RESUMEN
MicroRNAs (miRNAs) are small (approximately 21 nt), noncoding RNAs that negatively regulate target mRNAs at the posttranscriptional level that are involved in development. In plants, virus-induced disease symptoms often result in developmental abnormalities resembling perturbation of miRNA-mediated function. Here, we report that expression in transgenic plants of a geminivirus-encoded AC4 protein from African cassava mosaic virus Cameroon Strain (ACMV), a suppressor of posttranscriptional gene silencing, was correlated with decreased accumulation of host miRNAs and increased development abnormalities in Arabidopsis. Down-regulation of miRNA correlated with an up-regulation of target mRNA level. In vitro binding assays revealed the ability of AC4 of ACMV (A-AC4) but not East African cassava mosaic Cameroon virus AC2 to bind single-stranded forms of miRNAs and short interfering RNAs but not double-stranded RNA forms. Normally, a labile intermediate during the miRNA biogenesis/RNA-induced silencing complex assembly, miRNA*, was below the level of detection, indicating that AC4 might interfere at a point downstream of the miRNA duplex unwinding process. The association of AC4 with miRNA was demonstrated by the association of A-AC4-GFP fusion protein, extracted from Arabidopsis protoplasts, with 2'-O-methyloligonucleotide complementary to miR159 (miR159*) and by the presence of miRNA with the A-AC4-GFP fusion protein after immunoprecipitation with antibody against GFP. In both assays, A-AC4 protein and miRNA complexes were copurified. These results provide direct evidence that AC4 is a unique virus-encoded posttranscriptional gene-silencing suppressor protein that binds to and presumably inactivates mature miRNAs and thus blocks the normal miRNA-mediated regulation of target mRNAs, resulting in developmental defects in Arabidopsis.
Asunto(s)
Arabidopsis/crecimiento & desarrollo , Arabidopsis/genética , Silenciador del Gen , MicroARNs/metabolismo , Virus del Mosaico/genética , Enfermedades de las Plantas/virología , Proteínas Virales/metabolismo , Northern Blotting , Cartilla de ADN , Proteínas Fluorescentes Verdes , Inmunoprecipitación , Enfermedades de las Plantas/genética , Plantas Modificadas Genéticamente , Unión Proteica , Especificidad de la Especie , Proteínas Virales/genéticaRESUMEN
Short-interfering RNAs (siRNAs), the molecular markers of posttranscriptional gene silencing (PTGS), are powerful tools that interfere with gene expression and counter virus infection both in plants and animals. Here, we report the effect of temperature on geminivirus-induced gene silencing by quantifying virus-derived siRNAs and by evaluating their distribution along the virus genome for isolates of five species of cassava geminiviruses in cassava (Manihot esculenta, Crantz) and Nicotiana benthamiana. Cassava geminivirus-induced RNA silencing increased by raising the temperature from 25 degrees C to 30 degrees C, with the appearance of less symptomatic newly developed leaves, irrespective of the nature of the virus. Consequently, nonrecovery-type geminiviruses behaved like recovery-type viruses under high temperature. Next, we evaluated the distribution of virus-derived siRNAs on the respective virus genome at three temperatures (25 degrees C, 25 degrees C-30 degrees C, and 30 degrees C). For recovery-type viruses, siRNAs accumulated at moderately higher levels during virus-induced PTGS at higher temperatures, and there was no change in the distribution of the siRNA population along the virus genome. For nonrecovery-type viruses, siRNAs accumulated at strikingly higher levels than those observed for infections with recovery-type viruses at high temperature. As determined for an RNA virus, temperature influences gene silencing for single-stranded DNA geminiviruses. It is possible that other mechanisms besides gene silencing also control geminivirus accumulation at high temperatures. The findings presented here should be taken into consideration when implementing PTGS-based strategies to control plant virus accumulation.
Asunto(s)
Geminiviridae/fisiología , Silenciador del Gen/fisiología , ARN de Planta/metabolismo , Regulación de la Expresión Génica de las Plantas , Manihot/fisiología , Manihot/virología , Enfermedades de las Plantas/virología , ARN Interferente Pequeño , ARN Viral , Temperatura , Factores de Tiempo , Nicotiana/fisiología , Nicotiana/virologíaRESUMEN
Geminiviruses are single-stranded circular DNA viruses that cause economically significant diseases in a wide range of crop plants worldwide. In plants, post-transcriptional gene silencing (PTGS) acts as a natural anti-viral defense system and plays a role in genome maintenance and development. During the past decade there has been considerable evidence of PTGS suppression by viruses, which is often required to establish infection in plants. In particular, nuclear-replicating geminiviruses, which have no double-stranded RNA phase in their replication cycle, can induce and suppress the PTGS and become targets for PTGS. Here, we summarize recent developments in determining how these viruses trigger PTGS and how they suppress the induced PTGS, as well as how we can use the system to control these viruses in plants better and manipulate the system to study functional genomics in crop plants.
Asunto(s)
Geminiviridae/genética , Plantas/genética , Interferencia de ARN , Silenciador del Gen , Desarrollo de la Planta , Enfermedades de las Plantas/virología , Plantas/virología , Plantas Modificadas Genéticamente/crecimiento & desarrollo , Transcripción GenéticaRESUMEN
Posttranscriptional gene silencing (PTGS) in plants is a natural defense mechanism against virus infection. In mixed infections, virus synergism is proposed to result from suppression of the host defense mechanism by the viruses. Synergistic severe mosaic disease caused by simultaneous infection with isolates of the Cameroon strain of African cassava mosaic virus (ACMV-[CM]) and East African cassava mosaic Cameroon virus (EACMCV) in cassava and tobacco is characterized by a dramatic increase in symptom severity and a severalfold increase in viral-DNA accumulation by both viruses compared to that in singly infected plants. Here, we report that synergism between ACMV-[CM] and EACMCV is a two-way process, as the presence of the DNA-A component of ACMV-[CM] or EACMCV in trans enhanced the accumulation of viral DNA of EACMCV and ACMV-[CM], respectively, in tobacco BY-2 protoplasts. Furthermore, transient expression of ACMV-[CM] AC4 driven by the Cauliflower mosaic virus 35S promoter (p35S-AC4) enhanced EACMCV DNA accumulation by approximately 8-fold in protoplasts, while p35S-AC2 of EACMCV enhanced ACMV-[CM] DNA accumulation, also by approximately 8-fold. An Agrobacterium-based leaf infiltration assay determined that ACMV-[CM] AC4 and EACMCV AC2, the putative synergistic genes, were able to suppress PTGS induced by green fluorescent protein (GFP) and eliminated the short interfering RNAs associated with PTGS, with a correlated increase in GFP mRNA accumulation. In addition, we have identified AC4 of Sri Lankan cassava mosaic virus and AC2 of Indian cassava mosaic virus as suppressors of PTGS, indicating that geminiviruses evolved differently in regard to interaction with the host. The specific and different roles played by these AC2 and AC4 proteins of cassava geminiviruses in regulating anti-PTGS activity and their relation to synergism are discussed.
Asunto(s)
Proteínas de Unión al ADN/metabolismo , Geminiviridae/genética , Geminiviridae/metabolismo , Manihot/virología , Interferencia de ARN , Proteínas Virales/metabolismo , ADN Viral/genética , ADN Viral/metabolismo , Proteínas de Unión al ADN/genética , Geminiviridae/patogenicidad , Regulación Viral de la Expresión Génica , Genes Virales/genética , Interacciones Huésped-Parásitos , Enfermedades de las Plantas/genética , Enfermedades de las Plantas/virología , Regiones Promotoras Genéticas/genética , Protoplastos/virología , ARN Mensajero/genética , ARN Mensajero/metabolismo , Nicotiana/citología , Nicotiana/virología , Proteínas Virales/genética , VirulenciaRESUMEN
Viruses are both inducers and targets of posttranscriptional gene silencing (PTGS), a natural defense mechanism in plants. Here we report molecular evidence of the ability of single-stranded DNA (ssDNA) viruses to induce PTGS in infected plants irrespective of the severity of or recovery from the symptoms. Our results reveal that five distinct species of cassava-infecting geminiviruses were capable of triggering PTGS by producing two classes of virus-specific short interfering RNAs (siRNAs) of 21 to 26 nucleotides in two plant hosts, tobacco (Nicotiana benthamiana) and cassava (Manihot esculenta, Crantz). However, the efficacy of virus-induced PTGS varied depending on the intrinsic features of the virus and its interaction with the plant host. We found that symptom recovery over time in plants infected with the isolates of African cassava mosaic virus (ACMV-[CM]) or Sri Lankan cassava mosaic virus was associated with a much higher level of virus-derived siRNA accumulation compared to plants infected with viruses that do not show symptom recovery. Furthermore, we determined that the C terminus of AC1 that overlaps with the N terminus of AC2 early viral genes involved in virus replication were the primary targets for ACMV-[CM]-induced PTGS, whereas the C terminus of BC1 was targeted for the East African cassava mosaic Cameroon virus. In addition, our results reveal the possibility for double-stranded RNA formation during transcription in ssDNA viruses, which explains in part how these viruses can trigger PTGS in plants.
Asunto(s)
Virus ADN/genética , Virus ADN/fisiología , Manihot/virología , Enfermedades de las Plantas/virología , Interferencia de ARN , ARN Interferente Pequeño/metabolismo , Secuencia de Bases , Virus ADN/metabolismo , Geminiviridae/genética , Regulación Viral de la Expresión Génica , Datos de Secuencia Molecular , Hojas de la Planta/virología , Nicotiana/virología , Proteínas Virales/genética , Proteínas Virales/metabolismoRESUMEN
Geminiviruses are ssDNA viruses that infect a range of economically important crop species. We have developed a pathogen-derived transgenic approach to generate high levels of resistance against these pathogens in a susceptible cultivar of cassava (Manihot esculenta). Integration of the AC1 gene (which encodes the replication-associated protein) from African cassava mosaic virus imparted resistance against the homologous virus and provided strong cross-protection against two heterologous species of cassava-infecting geminiviruses. Short-interfering RNAs specific to the AC1 transgene were identified in the two most resistant transgenic plant lines prior to virus challenge. Levels of AC1 mRNA were suppressed in these plants. When challenged with geminiviruses, accumulation of viral DNA was reduced by up to 98% compared to controls, providing evidence that integration of AC1 initiates protection against viral infection via a post-transcriptional gene silencing mechanism. The robust cross-resistance reported has important implications for field deployment of transgenic strategies to control geminiviruses.
Asunto(s)
Regulación Viral de la Expresión Génica , Manihot/genética , Enfermedades de las Plantas/genética , Virus de Plantas/genética , ARN Interferente Pequeño/genética , Northern Blotting , ADN de Cadena Simple/genética , ADN Viral/genética , Geminiviridae/genética , Geminiviridae/crecimiento & desarrollo , Silenciador del Gen , Inmunidad Innata/genética , Manihot/crecimiento & desarrollo , Manihot/virología , Enfermedades de las Plantas/virología , Virus de Plantas/crecimiento & desarrollo , Plantas Modificadas Genéticamente , Plásmidos/genética , ARN Interferente Pequeño/metabolismo , Transgenes/genética , Proteínas Virales/genética , Replicación Viral/genéticaRESUMEN
Gene silencing mediated by double-stranded RNA is a sequence-specific RNA degradation mechanism highly conserved in eukaryotes that serves as an antiviral defense pathway in both plants and Drosophila. Short interfering RNAs (siRNAs), the 21- to 23-nt double-stranded intermediates of this natural defense mechanism, are becoming powerful tools for reducing gene expression and countering viral infection in a variety of mammalian cells. Here we report the use of siRNAs to target reporter gene expression and viral DNA accumulation in cultured plant cells. Transient expression of reporter genes encoding either GFP or red fluorescent protein from Discosoma was specifically reduced by 58% and 47%, respectively, at 24 h after codelivery of cognate siRNAs in BY2 protoplasts. In contrast to mammalian systems, the siRNA-induced silencing of GFP expression was transitive as indicated by the presence of siRNAs representing parts of the target RNA outside the region homologous to the triggering siRNA. Codelivery of an siRNA designed to target the mRNA encoding the replication-associated protein (AC1) of the geminivirus African cassava mosaic virus (ACMV) from Cameroon blocked AC1 mRNA accumulation by approximately 91% and inhibited accumulation of the ACMV genomic DNA by approximately 66% at 36 and 48 h after transfection. As with siRNA-induced reporter gene silencing, the siRNA targeting ACMV AC1 was specific and did not affect the replication of East African cassava mosaic Cameroon virus. This report demonstrates the occurrence of siRNA-mediated suppression of gene expression in cultured plant cells and that siRNA can interfere with and suppress accumulation of a nuclear-replicated DNA virus.