RESUMO
Infections by plant virus generally cause disease symptoms by interfering with cellular processes. Here we demonstrated that infection of Nicotiana tabacum (N.t) by plant viruses representative of the Tobamoviridae, Potyviridae, and Potexviridae families altered accumulation of certain microRNAs (miRNAs). A correlation was observed between symptom severity and alteration in levels of miRNAs 156, 160, 164,166, 169, and 171 that is independent of viral posttranscriptional gene silencing suppressor activity. Hybrid transgenic plants that produced tobacco mosaic virus (TMV) movement protein (MP) plus coat protein (CP)(T42W) (a variant of CP) exhibited disease-like phenotypes, including abnormal plant development. Grafting studies with a plant line in which both transgenes are silenced confirmed that the disease-like phenotypes are due to the coexpression of CP and MP. In hybrid MPxCP(T42W) plants and TMV-infected plants, miRNAs 156, 164, 165, and 167 accumulated to higher levels compared with nontransgenic and noninfected tissues. Bimolecular fluorescence complementation assays revealed that MP interacts with CP(T42W) in vivo and leads to the hypothesis that complexes formed between MP and CP caused increases in miRNAs that result in disease symptoms. This work presents evidence that virus infection and viral proteins influence miRNA balance without affecting posttranscriptional gene silencing and contributes to the hypothesis that viruses exploit miRNA pathways during pathogenesis.
Assuntos
Proteínas do Capsídeo/metabolismo , MicroRNAs/metabolismo , Nicotiana/crescimento & desenvolvimento , Nicotiana/virologia , Doenças das Plantas/virologia , Proteínas do Movimento Viral em Plantas/metabolismo , Vírus do Mosaico do Tabaco/fisiologia , Inativação Gênica , Fenótipo , Folhas de Planta/citologia , Folhas de Planta/virologia , Plantas Geneticamente Modificadas , Ligação Proteica , Nicotiana/genéticaRESUMO
Replication of Potato virus X (PVX) was reduced in transgenic protoplasts that accumulated wild-type coat protein (CPWT) of Tobacco mosaic virus (TMV) or a mutant CP, CP(T42W), that produced highly ordered states of aggregation, including pseudovirions. This reaction is referred to as heterologous CP-mediated resistance. However, protoplasts expressing a CP mutant that abolished aggregation and did not produce pseudovirions, CPT28W, did not reduce PVX replication. Similarly, in transgenic tobacco plants producing TMV CPWT or CP(T42W), there was a delay in local cell-to-cell spread of PVX infection that was not observed in CP(T28W) plants or in non-transgenic plants. The results suggest that the quaternary structure of the TMV CP regulates the mechanism(s) of heterologous CP-mediated resistance. Similarly, transgenic protoplasts that produced PVX CP conferred transient protection against infection by TMV RNA. Transgenic plants that accumulated PVX CP reduced the cell-to-cell spread of infection and resulted in a delay in systemic infection following inoculation with TMV or TMV RNA. Heterologous CP-mediated resistance was characterized by a brief delay in systemic infection, whilst homologous CP-mediated resistance conferred reduced or no systemic infection.
Assuntos
Proteínas do Capsídeo/metabolismo , Nicotiana/virologia , Doenças das Plantas/virologia , Plantas Geneticamente Modificadas , Potexvirus/patogenicidade , Vírus do Mosaico do Tabaco/patogenicidade , Proteínas do Capsídeo/genética , Linhagem Celular , Potexvirus/genética , Potexvirus/metabolismo , Nicotiana/genética , Vírus do Mosaico do Tabaco/genética , Vírus do Mosaico do Tabaco/metabolismo , Replicação ViralRESUMO
The p24 protein, one of the three proteins implicated in local movement of potato virus X (PVX), was expressed in transgenic tobacco plants (Nicotiana tabacum Xanthi D8 NN). Plants with the highest level of p24 accumulation exhibited a stunted and slightly chlorotic phenotype. These transgenic plants facilitate the cell-to-cell movement of a mutant of PVX that contained a frameshift mutation in p24. Upon inoculation with tobacco mosaic virus (TMV), the size of necrotic local lesions was significantly smaller in p24+ plants than in nontransgenic, control plants. Systemic resistance to tobamoviruses was also evidenced after inoculation of p24+ plants with Ob, a virus that evades the hypersensitive response provided by the N gene. In the latter case, no systemic symptoms were observed, and virus accumulation remained low or undetectable by Western immunoblot analysis and back-inoculation assays. In contrast, no differences were observed in virus accumulation after inoculation with PVX, although more severe symptoms were evident on p24-expressing plants than on control plants. Similarly, infection assays conducted with potato virus Y showed no differences between control and transgenic plants. On the other hand, a considerable delay in virus accumulation and symptom development was observed when transgenic tobacco plants containing the movement protein (MP) of TMV were inoculated with PVX. Finally, a movement defective mutant of TMV was inoculated on p24+ plants or in mixed infections with PVX on nontransgenic plants. Both types of assays failed to produce TMV infections, implying that TMV MP is not interchangeable with the PVX MPs.
Assuntos
Potexvirus/genética , Potexvirus/fisiologia , Vírus do Mosaico do Tabaco/patogenicidade , Tobamovirus/patogenicidade , Proteínas Virais/genética , Proteínas Virais/fisiologia , Animais , Anticorpos Antivirais , Sequência de Bases , Primers do DNA/genética , Mutação da Fase de Leitura , Expressão Gênica , Teste de Complementação Genética , Movimento , Fases de Leitura Aberta , Fenótipo , Plantas Geneticamente Modificadas , Plantas Tóxicas , Coelhos , NicotianaRESUMO
ABSTRACT Seven crop and eight weed species from 12 agricultural locations in Trinidad and Tobago were assayed for the presence of whitefly-transmitted geminiviruses (WTGs) by using dot blot hybridization and polymerase chain reaction (PCR) amplification of the N-terminal coat protein sequence with degenerate primers. The amplified fragments were cloned and analyzed by restriction enzyme digestion to determine fragment length polymorphism among the cloned fragments. Representative clones were then sequenced and subjected to phylogenetic analysis to determine the sequence similarity to known WTGs. WTGs were found in every location sampled and in 10 of the 15 species investigated: Lycopersicon esculentum(tomato), Capsicum annuum (pepper), Capsicum frutescens (sweet pepper), Abelmoschus esculentus (okra), Phaseolus vulgaris (beans), Alternanthera tenella, Desmodium frutescens, Euphorbia heterophylla, Malva alceifolia, and Sida acuta. The geminiviruses infecting these plants were closely related to potato yellow mosaic virus from Venezuela (PYMV-VE) and tomato leaf curl virus from Panama (ToLCV-PA). However, in pepper, sweet pepper, okra, Alternanthera tenella, Euphorbia heterophylla, Des-modium frutescens, and in one sample of tomato, a PYMV-VE-related virus was found in mixed infections with a virus related to pepper huasteco virus. Full-length infectious DNA-A and DNA-B of a tomato-infecting geminivirus from Trinidad and Tobago were cloned and sequenced. DNA-A appears to be a recombinant derived from PYMV-VE or ToLCV-PA, and Sida golden mosaic from Honduras. The implications of these findings in the control of WTGs are discussed.