RESUMO
Major themes in pathogen evolution are emergence, evolution of virulence, host adaptation and the processes that underlie them. RNA viruses are of particular interest due to their rapid evolution. The in vivo molecular evolution of an RNA plant virus was demonstrated here using a necrotic isolate of cowpea mild mottle virus (CPMMV) and a susceptible soybean genotype submitted to serial inoculations. We show that the virus lost the capacity to cause necrosis after six passages through the host plant. When a severe bottleneck was imposed, virulence reduction occurred in the second passage. The change to milder symptoms had fitness benefits for the virus (higher RNA accumulation) and for its vector, the whitefly Bemisia tabaci. Genetic polymorphisms were highest in ORF1 (viral replicase) and were independent of the symptom pattern. Recombination was a major contributor to this diversity - even with the strong genetic bottleneck, recombination events and hot spots were detected within ORF1. Virulence reduction was associated with different sites in ORF1 associated to recombination events in both experiments. Overall, the results demonstrate that the reduction in virulence was a consequence of the emergence of new variants, driven by recombination. Besides providing details of the evolutionary mechanisms behind a reduction in virulence and its effect under viral and vector fitness, we propose that this recombination-driven switch in virulence allows the pathogen to rapidly adapt to a new host and, potentially, switch back.
Assuntos
Carlavirus , Hemípteros , Vírus de RNA , Vigna , Animais , Carlavirus/genética , Vírus de DNA/genética , Hemípteros/genética , RNA , Vírus de RNA/genética , Recombinação Genética , Virulência/genéticaRESUMO
Biological invasions of vectorborne diseases can be devastating. Bioclimatic modeling provides an opportunity to assess and predict areas at risk from complex multitrophic interactions of pathogens, highlighting areas in need of increased monitoring effort. Here, we model the distribution of an economically critical vectorborne plant pathogen 'Candidatus Phytoplasma aurantifolia', the etiological agent of Witches' Broom Disease of Lime. This disease is a significant limiting factor on acid lime production (Citrus aurantifolia, Swingle) in the Middle East and threatens its production globally. We found that temperature, humidity, and the vector populations significantly determine disease distribution. Following this, we used bioclimatic modeling to predict potential novel sites of infections. The model outputs identified potential novel sites of infection in the citrus producing regions of Brazil and China. We also used our model to explore sites in Oman where the pathogen may not be infectious, and suggest nurseries be established there. Recent major turbulence in the citrus agricultural economy has highlighted the importance of this work and the need for appropriate and targeted monitoring programs to safeguard lime production.
Assuntos
Citrus aurantiifolia/microbiologia , Clima , Modelos Biológicos , Phytoplasma/fisiologia , Animais , Hemípteros/microbiologia , Insetos Vetores/microbiologia , Omã , Doenças das Plantas , Medição de RiscoRESUMO
A novel satellite virus of 1,228 bp in length was found in a single cassava plant. Bioinformatic analyses show that it has two open reading frames (ORFs) in its genome, probably encoding a coat protein of 156 and a putative protein of 90 amino acids.
RESUMO
Infectious cDNA clones are an important tool to study the molecular and cellular process of RNA virus infection. In vitro and in vivo transcription systems are the two main strategies used in the generation of infectious cDNA clones for RNA viruses. This study describes the first generation of a full-length infectious cDNA clone of Cowpea mild mottle virus (CPMMV), a Carlavirus. The full-length genome was synthesized by Overlap Extension PCR of two overlapping fragments and cloned in a pUC-based vector under control of the SP6 RNA polymerase promoter. After in vitro run-off transcription, the produced RNA was mechanically inoculated into soybean plants cv. CD206. The systemic infection was confirmed by RT-PCR and further sequencing of amplified cDNA fragments. To simplify the transfection process, the complete genome was subcloned into a binary vector under control of the 35S promoter of cauliflower mosaic virus by the Gibson Assembly protocol. The resulting clones were inoculated by particle bombardment onto soybean seedlings and the recovery of the virus was confirmed 2 weeks later by RT-PCR. Our results indicate the constructs of the full-length cDNA of CPMMV are fully infectious in both in vitro and in vivo transcription strategies.
Assuntos
Carlavirus/genética , DNA Complementar , Genoma Viral , Clonagem Molecular , Ordem dos Genes , Fases de Leitura Aberta , Doenças das Plantas/virologia , Glycine max/virologiaRESUMO
Cassava frogskin disease (CFSD) is a particular threat in cassava because symptoms remain hidden until harvest and losses can be total. The information related to the etiological agent of this disease is contradictory, because some authors believe it is caused by phytoplasmas while others believe that it is caused by a virus. In order to refine detection protocols and to characterize organisms associated with CFSD in Brazil, 32 symptomatic and 20 asymptomatic cassava plants were collected in Minas Gerais state. Total DNA was extracted and used for nested polymerase chain reaction (PCR) to detect phytoplasmas. Because endophytic Bacillus spp. led to false positives, primers were designed to facilitate the detection of phytoplasma in the presence of bacteria. In addition, double-stranded (ds)RNA was extracted from tubers and used in reverse-transcription PCR for the detection of the RNA-dependent RNA polymerase gene from Cassava frogskin virus segment 4. The detected phytoplasma was identified as belonging to the group 16SrIII-A by restriction fragment length polymorphism (RFLP), sequencing, and RFLP in silico. This is the first report of a phytoplasma belonging to the 16SrIII-A group associated with cassava plants, the first molecular characterization of a phytoplasma associated with CFSD in Brazil, and a first report of phytoplasma and a dsRNA virus (possible reovirus) co-infecting cassava plants with CFSD symptoms.
RESUMO
NSP-interacting kinase (NIK1) is a receptor-like kinase identified as a virulence target of the begomovirus nuclear shuttle protein (NSP). We found that NIK1 undergoes a stepwise pattern of phosphorylation within its activation-loop domain (A-loop) with distinct roles for different threonine residues. Mutations at Thr-474 or Thr-468 impaired autophosphorylation and were defective for kinase activation. In contrast, a mutation at Thr-469 did not impact autophosphorylation and increased substrate phosphorylation, suggesting an inhibitory role for Thr-469 in kinase function. To dissect the functional significance of these results, we used NSP-expressing virus infection as a mechanism to interfere with wild type and mutant NIK1 action in plants. The NIK1 knockout mutant shows enhanced susceptibility to virus infections, a phenotype that could be complemented with ectopic expression of a 35S-NIK1 or 35S-T469A NIK1 transgenes. However, ectopic expression of an inactive kinase or the 35S-T474A NIK1 mutant did not reverse the enhanced susceptibility phenotype of knockout lines, demonstrating that Thr-474 autophosphorylation was needed to transduce a defense response to geminiviruses. Furthermore, mutations at Thr-474 and Thr-469 residues antagonistically affected NIK-mediated nuclear relocation of the downstream effector rpL10. These results establish that NIK1 functions as an authentic defense receptor as it requires activation to elicit a defense response. Our data also suggest a model whereby phosphorylation-dependent activation of a plant receptor-like kinase enables the A-loop to control differentially auto- and substrate phosphorylation.
Assuntos
Antivirais/farmacologia , Treonina/química , Alanina/química , Sequência de Aminoácidos , Arabidopsis/metabolismo , Proteínas de Arabidopsis , Núcleo Celular/metabolismo , Geminiviridae/genética , Dados de Sequência Molecular , Mutação , Fosforilação , Mutação Puntual , Estrutura Terciária de Proteína , Proteína Ribossômica L10 , Proteínas Ribossômicas/química , Homologia de Sequência de Aminoácidos , Transdução de Sinais , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Nicotiana/metabolismo , Vírus/metabolismoRESUMO
We performed an inventory of soybean NAC transcription factors, in which 101 NAC domain-containing proteins were annotated into 15 different subgroups, showing a clear relationship between structure and function. The six previously described GmNAC proteins (GmNAC1 to GmNAC6) were located in the nucleus and a transactivation assay in yeast confirmed that GmNAC2, GmNAC3, GmNAC4 and GmNAC5 function as transactivators. We also analyzed the expression of the six NAC genes in response to a variety of stress conditions. GmNAC2, GmNAC3 and GmNAC4 were strongly induced by osmotic stress. GmNAC3 and GmNAC4 were also induced by ABA, JA and salinity but differed in their response to cold. Consistent with an involvement in cell death programs, the transient expression of GmNAC1, GmNAC5 and GmNAC6 in tobacco leaves resulted in cell death and enhanced expression of senescence markers. Our results indicate that the described soybean NACs are functionally non-redundant transcription factors involved in response to abiotic stresses and in cell death events in soybean.
Assuntos
Glycine max/metabolismo , Proteínas de Plantas/metabolismo , Fatores de Transcrição/metabolismo , Sequência de Aminoácidos , Núcleo Celular/metabolismo , Células Cultivadas , Sequência Conservada , Regulação da Expressão Gênica de Plantas , Pressão Osmótica , Filogenia , Folhas de Planta/metabolismo , Proteínas de Plantas/genética , Estresse Fisiológico , Nicotiana/metabolismo , Fatores de Transcrição/genéticaRESUMO
The NSP-interacting kinase (NIK) receptor-mediated defense pathway has been identified recently as a virulence target of the geminivirus nuclear shuttle protein (NSP). However, the NIK1-NSP interaction does not fit into the elicitor-receptor model of resistance, and hence the molecular mechanism that links this antiviral response to receptor activation remains obscure. Here, we identified a ribosomal protein, rpL10A, as a specific partner and substrate of NIK1 that functions as an immediate downstream effector of NIK1-mediated response. Phosphorylation of cytosolic rpL10A by NIK1 redirects the protein to the nucleus where it may act to modulate viral infection. While ectopic expression of normal NIK1 or a hyperactive NIK1 mutant promotes the accumulation of phosphorylated rpL10A within the nuclei, an inactive NIK1 mutant fails to redirect the protein to the nuclei of co-transfected cells. Likewise, a mutant rpL10A defective for NIK1 phosphorylation is not redirected to the nucleus. Furthermore, loss of rpL10A function enhances susceptibility to geminivirus infection, resembling the phenotype of nik1 null alleles. We also provide evidence that geminivirus infection directly interferes with NIK1-mediated nuclear relocalization of rpL10A as a counterdefensive measure. However, the NIK1-mediated defense signaling neither activates RNA silencing nor promotes a hypersensitive response but inhibits plant growth and development. Although the virulence function of the particular geminivirus NSP studied here overcomes this layer of defense in Arabidopsis, the NIK1-mediated signaling response may be involved in restricting the host range of other viruses.
Assuntos
Proteínas de Arabidopsis/metabolismo , Núcleo Celular/metabolismo , Imunidade Inata/fisiologia , Proteínas Nucleares/fisiologia , Vírus de Plantas/imunologia , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Ribossômicas/metabolismo , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/fisiologia , Begomovirus/imunologia , Células Cultivadas , Citosol/metabolismo , Geminiviridae/imunologia , Solanum lycopersicum/genética , Solanum lycopersicum/imunologia , Solanum lycopersicum/metabolismo , Modelos Biológicos , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Fosforilação , Doenças das Plantas/imunologia , Doenças das Plantas/virologia , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Proteínas de Plantas/fisiologia , Plantas Geneticamente Modificadas , Ligação Proteica , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/fisiologia , Transporte Proteico , Proteína Ribossômica L10 , Proteínas Ribossômicas/genética , Proteínas Ribossômicas/fisiologia , Especificidade por Substrato , TransfecçãoRESUMO
SUMMARY: In contrast to the accumulated data on nuclear transport mechanisms of macromolecules, little is known concerning the regulated release of nuclear-exported complexes and their subsequent trans-cytoplasmic movement. The bipartite begomovirus nuclear shuttle protein (NSP) facilitates the nuclear export of viral DNA and cooperates with the movement protein (MP) to transport viral DNA across the plant cell wall. Here, we identified a cellular NSP-interacting GTPase (NIG) with biochemical properties consistent with a nucleocytoplasmic transport role. We show that NIG is a cytosolic GTP-binding protein that accumulates around the nuclear envelope and possesses intrinsic GTPase activity. NIG interacts with NSP in vitro and in vivo (under transient expression), and redirects the viral protein from the nucleus to the cytoplasm. We propose that NIG acts as a positive contributor to geminivirus infection by modulating NSP nucleocytoplasmic shuttling and hence facilitating MP-NSP interaction in the cortical cytoplasm. In support of this, overexpression of NIG in Arabidopsis enhances susceptibility to geminivirus infection. In addition to highlighting the relevance of NIG as a cellular co-factor for NSP function, our findings also have implications for general nucleocytoplasmic trafficking of cellular macromolecules.
Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/genética , Begomovirus/genética , GTP Fosfo-Hidrolases/metabolismo , Proteínas do Movimento Viral em Plantas/metabolismo , Transporte Ativo do Núcleo Celular , Arabidopsis/metabolismo , Arabidopsis/virologia , Proteínas de Arabidopsis/genética , Begomovirus/metabolismo , Núcleo Celular/metabolismo , DNA Viral/genética , Microscopia Confocal , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Doenças das Plantas/virologia , Folhas de Planta/genética , Folhas de Planta/metabolismo , Proteínas do Movimento Viral em Plantas/genética , Plantas Geneticamente Modificadas/genética , Plantas Geneticamente Modificadas/metabolismo , Plasmídeos , Protoplastos/metabolismo , RNA de Plantas/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Nicotiana/genética , Nicotiana/metabolismo , Técnicas do Sistema de Duplo-HíbridoRESUMO
NRPs (N-rich proteins) were identified as targets of a novel adaptive pathway that integrates endoplasmic reticulum (ER) and osmotic stress signals based on coordinate regulation and synergistic up-regulation by tunicamycin and polyethylene glycol treatments. This integrated pathway diverges from the molecular chaperone-inducing branch of the unfolded protein response (UPR) in several ways. While UPR-specific targets were inversely regulated by ER and osmotic stresses, NRPs required both signals for full activation. Furthermore, BiP (binding protein) overexpression in soybean prevented activation of the UPR by ER stress inducers, but did not affect activation of NRPs. We also found that this integrated pathway transduces a PCD signal generated by ER and osmotic stresses that result in the appearance of markers associated with leaf senescence. Overexpression of NRPs in soybean protoplasts induced caspase-3-like activity and promoted extensive DNA fragmentation. Furthermore, transient expression of NRPs in planta caused leaf yellowing, chlorophyll loss, malondialdehyde production, ethylene evolution, and induction of the senescence marker gene CP1. This phenotype was alleviated by the cytokinin zeatin, a potent senescence inhibitor. Collectively, these results indicate that ER stress induces leaf senescence through activation of plant-specific NRPs via a novel branch of the ER stress response.