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1.
Viruses ; 12(1)2020 Jan 08.
Artigo em Inglês | MEDLINE | ID: mdl-31936267

RESUMO

Potyviruses represent the largest group of known plant RNA viruses and include many agriculturally important viruses, such as Plum pox virus, Soybean mosaic virus, Turnip mosaic virus, and Potato virus Y. Potyviruses adopt polyprotein processing as their genome expression strategy. Among the 11 known viral proteins, the nuclear inclusion protein b (NIb) is the RNA-dependent RNA polymerase responsible for viral genome replication. Beyond its principal role as an RNA replicase, NIb has been shown to play key roles in diverse virus-host interactions. NIb recruits several host proteins into the viral replication complexes (VRCs), which are essential for the formation of functional VRCs for virus multiplication, and interacts with the sumoylation pathway proteins to suppress NPR1-mediated immunity response. On the other hand, NIb serves as a target of selective autophagy as well as an elicitor of effector-triggered immunity, resulting in attenuated virus infection. These contrasting roles of NIb provide an excellent example of the complex co-evolutionary arms race between plant hosts and potyviruses. This review highlights the current knowledge about the multifunctional roles of NIb in potyvirus infection, and discusses future research directions.

2.
Phytopathology ; 110(1): 187-193, 2020 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-31516080

RESUMO

Potyviral helper component protease (HC-Pro), as a major determinant of symptom expression in susceptible plants, is a likely target candidate in the production of attenuated strains for cross-protection. In this study, single or double mutations of Lys (K) to Glu (E) in the Lys-Ile-Thr-Cys motif and Arg (R) to Ile (I) in the Phe-Arg-Asn-Lys motif of the HC-Pro from the severe papaya leaf distortion mosaic virus strain DF (PLDMV-DF) reduced symptom expression and virus accumulation in infected papaya (Carica papaya) plants. The papaya plants infected with the attenuated double mutant of PLDMV-EI presented as symptomless. PLDMV-EI provided effective protection against PLDMV-DF infection in three papaya cultivars and had no effect on plant growth and development. Our result showed that PLDMV-EI is a promising mild strain for the practical use of cross-protection in the field.


Assuntos
Motivos de Aminoácidos , Carica , Peptídeo Hidrolases , Potyvirus , Motivos de Aminoácidos/genética , Carica/virologia , Mutação/genética , Peptídeo Hidrolases/genética , Potyvirus/enzimologia , Potyvirus/genética
3.
J Virol Methods ; : 113795, 2019 Dec 03.
Artigo em Inglês | MEDLINE | ID: mdl-31809783

RESUMO

Two reverse-transcription loop-mediated isothermal amplification (RT-LAMP) assays were developed for the detection of areca palm necrotic ringspot virus (ANRSV) and areca palm necrotic spindle-spot virus (ANSSV), respectively. These two emerging viruses both induce necrotic symptoms in areca palms. The coat protein (CP) gene of ANRSV and the 9 K gene of ANSSV were used to design the respective RT-LAMP primers for the assays. Each set of four primers designed for each of these viruses was found to be highly specific in the detection of the respective targeted virus. The optimal incubation conditions for the RT-LAMP assays were 63 °C for 40 min for ANRSV and at 61 °C for 40 min for ANSSV. The sensitivity of the RT-LAMP method for each of these viruses was 10-fold greater than that of the corresponding conventional reverse-transcription polymerase chain reaction (RT-PCR). The RT-LAMP assays may be useful for the rapid early detection of ANSSV and ANRSV in commercial areca palm production.

4.
Phytopathology ; 109(5): 887-894, 2019 May.
Artigo em Inglês | MEDLINE | ID: mdl-30133353

RESUMO

Areca palm (Areca catechu), one of the two most important commercial crops in Hainan, China, has been severely damaged by a variety of pathogens and insects. Here, we report a new disease, tentatively referred to as areca palm necrotic ringspot disease (ANRSD), which is highly epidemic in the main growing regions in Hainan. Transmission electron microscopy observation and small RNA deep sequencing revealed the existence of a viral agent of the family Potyviridae in a diseased areca palm plant (XC1). The virus was tentatively named areca palm necrotic ringspot virus (ANRSV). Subsequently, the positive-sense single-stranded genome of ANRSV isolate XC1 was completely determined. The genome annotation revealed the existence of two cysteine proteinases in tandem (HC-Pro1 and HC-Pro2) in the genomic 5' terminus of ANRSV. Sequence comparison and phylogenetic analysis suggested the taxonomic classification of ANRSV into the recently proposed genus Arepavirus in the family Potyviridae. Given the close relationship of ANRSV with another newly reported arepavirus (areca palm necrotic spindle-spot virus), the exact taxonomic status of ANRSV needs to be further investigated. In this study, a reverse transcription polymerase chain reaction assay for ANRSV-specific detection was developed and a close association between ANRSV and ANRSD was found.


Assuntos
Areca/virologia , Filogenia , Doenças das Plantas/virologia , Potyviridae/patogenicidade , China , Genoma Viral , Potyviridae/classificação , RNA Viral
6.
Virus Genes ; 54(6): 833-839, 2018 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-30218292

RESUMO

We used green fluorescent protein (GFP)-tagged Papaya leaf distortion mosaic virus (PLDMV-GFP) to track PLDMV infection by fluorescence. The virus-derived small interfering RNAs (vsiRNAs) of PLDMV-GFP were characterized from papaya plants by next-generation sequencing. The foreign GFP gene inserted into the PLDMV genome was also processed as a viral gene into siRNAs by components involved in RNA silencing. The siRNAs derived from PLDMV-GFP accumulated preferentially as 21- and 22-nucleotide (nt) lengths, and most of the 5'-terminal ends were biased towards uridine (U) and adenosine (A). The single-nucleotide resolution map revealed that vsiRNAs were heterogeneously distributed throughout the PLDMV-GFP genome, and vsiRNAs derived from the sense strand were more abundant than those from the antisense strand. The hotspots were mainly distributed in the P1 and GFP coding region of the antisense strand. In addition, 979 papaya genes targeted by the most abundant 1000 PLDMV-GFP vsiRNAs were predicted and annotated using GO and KEGG classification. Results suggest that vsiRNAs play key roles in PLDMV-papaya interactions. These data on the characterization of PLDMV-GFP vsiRNAs will help to provide insight into the function of vsiRNAs and their host target regulation patterns.


Assuntos
Carica/virologia , Potyvirus/isolamento & purificação , RNA Interferente Pequeno/genética , RNA Viral/genética , Carica/genética , Carica/crescimento & desenvolvimento , Genoma Viral/genética , Proteínas de Fluorescência Verde/genética , Sequenciamento de Nucleotídeos em Larga Escala , Doenças das Plantas/genética , Doenças das Plantas/virologia , Potyvirus/genética , Potyvirus/patogenicidade , Interferência de RNA
7.
Arch Virol ; 163(12): 3471-3475, 2018 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-30136252

RESUMO

A novel virus, tentatively named "areca palm necrotic spindle-spot virus" (ANSSV), was identified in Areca catechu L. in Hainan, China, and its complete genomic sequence was determined. Its positive-sense single-stranded RNA genome is comprised of 9,437 nucleotides (nt), excluding the poly (A) tail, and contains one large open reading frame encoding a polyprotein of 3,019 amino acids (aa). A Blastp search showed that the polyprotein of ANSSV shared a maximum of 31%-32% aa sequence identity (with 86%-95% coverage) with all seven known macluraviruses. Nucleotide sequence comparison of the ORF of ANSSV to those of macluraviruses revealed identities ranging from 41.0% to 44.6%, which is less than the inter-genus identity values for the family Potyviridae. Phylogenetic analysis based on either the aa or nt sequence of the polyprotein did not cluster ANSSV into any established or unassigned genus of the family Potyviridae. Therefore, we suggest that ANSSV is the first member of a previously unrecognized genus of the family Potyviridae.


Assuntos
Areca/virologia , Genoma Viral , Doenças das Plantas/virologia , Potyviridae/genética , Potyviridae/isolamento & purificação , Sequência de Bases , China , Dados de Sequência Molecular , Fases de Leitura Aberta , Filogenia , Potyviridae/classificação , Análise de Sequência de DNA
8.
Protein Expr Purif ; 146: 17-22, 2018 06.
Artigo em Inglês | MEDLINE | ID: mdl-29373846

RESUMO

Plant methionine sulfoxide reductase B1 (MsrB1) protects the photosynthetic apparatus from oxidative damage by scavenging reactive oxygen species to repair Met-oxidized proteins in response to abiotic stresses and biotic attack. Papaya MsrB1 (PaMsrB1) was identified previously to interact with papaya ringspot virus NIa-Pro, and this interaction inhibits the import of PaMsrB1 into the chloroplast. Further functional characterization of PaMsrB1 requires the production of a biologically active purified recombinant protein. In this report, PaMsrB1 as a fusion protein containing an N-terminal maltose-binding protein (MBP) was expressed in Escherichia coli Rosetta (DE3) cells and purified. Production of soluble fusion protein was greater when the cells were cultured at 16 °C than at 37 °C. The Factor Xa protease digested MBP-PaMsrB1 fusion protein and subsequently purified recombinant PaMsrB1 specifically reduced the R-diastereomer of methionine sulfoxide (MetSO) and Dabsyl-MetSO to Met in the presence of dithiothreitol. Eight chloroplast-localized and five non-chloroplast-localized candidate proteins that interact with PaMsrB1 were isolated by affinity chromatography and liquid chromatography coupled to tandem mass spectrometry. The results provide a platform to further understand the anti-oxidative defense mechanism of PaMsrB1.


Assuntos
Carica/enzimologia , Metionina Sulfóxido Redutases/metabolismo , Mapas de Interação de Proteínas , Sequência de Aminoácidos , Carica/química , Carica/genética , Carica/metabolismo , Cloroplastos/genética , Cloroplastos/metabolismo , Escherichia coli/genética , Expressão Gênica , Metionina Sulfóxido Redutases/química , Metionina Sulfóxido Redutases/genética , Oxirredução , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Solubilidade
9.
Plant J ; 92(5): 846-861, 2017 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-28941316

RESUMO

Plasmodesmata (PD), unique to the plant kingdom, are structurally complex microchannels that cross the cell wall to establish symplastic communication between neighbouring cells. Viral intercellular movement occurs through PD. To better understand the involvement of PD in viral infection, we conducted a quantitative proteomic study on the PD-enriched fraction from Nicotiana benthamiana leaves in response to infection by Turnip mosaic virus (TuMV). We report the identification of a total of 1070 PD protein candidates, of which 100 (≥2-fold increase) and 48 (≥2-fold reduction) are significantly differentially accumulated in the PD-enriched fraction, when compared with protein levels in the corresponding healthy control. Among the differentially accumulated PD protein candidates, we show that an α-expansin designated NbEXPA1, a cell wall loosening protein, is PD-specific. TuMV infection downregulates NbEXPA1 mRNA expression and protein accumulation. We further demonstrate that NbEXPA1 is recruited to the viral replication complex via the interaction with NIb, the only RNA-dependent RNA polymerase of TuMV. Silencing of NbEXPA1 inhibits plant growth and TuMV infection, whereas overexpression of NbEXPA1 promotes viral replication and intercellular movement. These data suggest that NbEXPA1 is a host factor for potyviral infection. This study not only generates a PD-proteome dataset that is useful in future studies to expound PD biology and PD-mediated virus-host interactions but also characterizes NbEXPA1 as the first PD-specific cell wall loosening protein and its essential role in potyviral infection.


Assuntos
Doenças das Plantas/virologia , Proteínas de Plantas/metabolismo , Plasmodesmos/metabolismo , Potyvirus/metabolismo , Tabaco/microbiologia , Potyvirus/fisiologia , Proteômica , Tabaco/metabolismo , Replicação Viral
10.
Virology ; 510: 99-103, 2017 10.
Artigo em Inglês | MEDLINE | ID: mdl-28715654

RESUMO

A novel Rhizobium radiobacter (synonym Agrobacterium tumefaciens)-mediated approach was developed to generate stable infectious clones of plant viruses. This method uses R. radiobacter for both cloning and inoculation of infectious clones, bypassing the requirement of cloning in E. coli to avoid the instability. Only three steps are included in this method: (i) construct viral genome-encoding plasmids in vitro by one-step Gibson assembly; (ii) transform the assembled DNA products into R. radiobacter; (iii) inoculate plants with the R. radiobacter clones containing the viral genome. Stable infectious clones were obtained from two potyviruses papaya ringspot virus (PRSV) and papaya leaf distortion mosaic virus (PLDMV) using this method, whereas attempts utilizing "classical" E. coli cloning system failed repeatedly. This method is simple and efficient, and is promising for a wide application in generation of infectious clones of plant virus, especially for those which are instable in E. coli.


Assuntos
Agrobacterium tumefaciens/genética , Clonagem Molecular , Potyvirus/genética , Genética Reversa , Virologia/métodos , Plantas/virologia , Transformação Genética
11.
Viruses ; 7(12): 6241-50, 2015 Dec 01.
Artigo em Inglês | MEDLINE | ID: mdl-26633465

RESUMO

Papaya leaf distortion mosaic virus (PLDMV) is becoming a threat to papaya and transgenic papaya resistant to the related pathogen, papaya ringspot virus (PRSV). The generation of infectious viral clones is an essential step for reverse-genetics studies of viral gene function and cross-protection. In this study, a sequence- and ligation-independent cloning system, the In-Fusion(®) Cloning Kit (Clontech, Mountain View, CA, USA), was used to construct intron-less or intron-containing full-length cDNA clones of the isolate PLDMV-DF, with the simultaneous scarless assembly of multiple viral and intron fragments into a plasmid vector in a single reaction. The intron-containing full-length cDNA clone of PLDMV-DF was stably propagated in Escherichia coli. In vitro intron-containing transcripts were processed and spliced into biologically active intron-less transcripts following mechanical inoculation and then initiated systemic infections in Carica papaya L. seedlings, which developed similar symptoms to those caused by the wild-type virus. However, no infectivity was detected when the plants were inoculated with RNA transcripts from the intron-less construct because the instability of the viral cDNA clone in bacterial cells caused a non-sense or deletion mutation of the genomic sequence of PLDMV-DF. To our knowledge, this is the first report of the construction of an infectious full-length cDNA clone of PLDMV and the splicing of intron-containing transcripts following mechanical inoculation. In-Fusion cloning shortens the construction time from months to days. Therefore, it is a faster, more flexible, and more efficient method than the traditional multistep restriction enzyme-mediated subcloning procedure.


Assuntos
Carica/virologia , DNA Complementar/genética , DNA Viral/genética , Doenças das Plantas/virologia , Potyvirus/genética , Potyvirus/fisiologia , Genética Reversa/métodos , Clonagem Molecular , Escherichia coli/genética , Vetores Genéticos , Genoma Viral , Instabilidade Genômica , Plasmídeos
12.
Genome Announc ; 3(5)2015 Sep 10.
Artigo em Inglês | MEDLINE | ID: mdl-26358610

RESUMO

The complete genome sequence (10,326 nucleotides) of a papaya ringspot virus isolate infecting genetically modified papaya in Hainan Island of China was determined through reverse transcription (RT)-PCR. The virus shares 92% nucleotide sequence identity with the isolate that is unable to infect PRSV-resistant transgenic papaya.

13.
Virus Genes ; 50(1): 97-103, 2015 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-25416301

RESUMO

The interaction of papaya eukaryotic translation initiation factor 3 subunit G (CpeIF3G) with Papaya ringspot virus (PRSV) NIa-Pro was validated using a bimolecular fluorescence complementation assay in papaya protoplasts based on the previous yeast two-hybrid assay results. The C-terminal (residues 133-239) fragment of PRSV NIa-Pro and the central domain (residues 59-167) of CpeIF3G were required for effective interaction between NIa-Pro and CpeIF3G as shown by a Sos recruitment yeast two-hybrid system with several deletion mutants of NIa-Pro and CpeIF3G. The central domain of CpeIF3G, which contains a C2HC-type zinc finger motif, is required to bind to other eIFs of the translational machinery. In addition, quantitative real-time reverse transcription PCR assay confirmed that PRSV infection leads to a 2- to 4.5-fold up-regulation of CpeIF3G mRNA in papaya. Plant eIF3G is involved in various stress response by enhancing the translation of resistance-related proteins. It is proposed that the NIa-Pro-CpeIF3G interaction may impair translation preinitiation complex assembly of defense proteins and interfere with host defense.


Assuntos
Carica/virologia , Endopeptidases/metabolismo , Fator de Iniciação 3 em Eucariotos/metabolismo , Interações Hospedeiro-Patógeno , Potyvirus/enzimologia , Proteínas Virais/metabolismo , Análise Mutacional de DNA , Iniciação Traducional da Cadeia Peptídica , Ligação Proteica , Mapeamento de Interação de Proteínas , Técnicas do Sistema de Duplo-Híbrido
14.
Viruses ; 6(10): 3893-906, 2014 Oct 21.
Artigo em Inglês | MEDLINE | ID: mdl-25337891

RESUMO

Papaya ringspot virus (PRSV), Papaya leaf distortion mosaic virus (PLDMV), and Papaya mosaic virus (PapMV) produce similar symptoms in papaya. Each threatens commercial production of papaya on Hainan Island, China. In this study, a multiplex reverse transcription PCR assay was developed to detect simultaneously these three viruses by screening combinations of mixed primer pairs and optimizing the multiplex RT-PCR reaction conditions. A mixture of three specific primer pairs was used to amplify three distinct fragments of 613 bp from the P3 gene of PRSV, 355 bp from the CP gene of PLDMV, and 205 bp from the CP gene of PapMV, demonstrating the assay's specificity. The sensitivity of the multiplex RT-PCR was evaluated by showing plasmids containing each of the viral target genes with 1.44 × 103, 1.79 × 103, and 1.91 × 102 copies for the three viruses could be detected successfully. The multiplex RT-PCR was applied successfully for detection of three viruses from 341 field samples collected from 18 counties of Hainan Island, China. Rates of single infections were 186/341 (54.5%), 93/341 (27.3%), and 3/341 (0.9%), for PRSV, PLDMV, and PapMV, respectively; 59/341 (17.3%) of the samples were co-infected with PRSV and PLDMV, which is the first time being reported in Hainan Island. This multiplex RT-PCR assay is a simple, rapid, sensitive, and cost-effective method for detecting multiple viruses in papaya and can be used for routine molecular diagnosis and epidemiological studies in papaya.


Assuntos
Carica/virologia , Reação em Cadeia da Polimerase Multiplex/métodos , Doenças das Plantas/virologia , Potexvirus/isolamento & purificação , Potyvirus/isolamento & purificação , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , Primers do DNA/genética , Reação em Cadeia da Polimerase Multiplex/normas , Potexvirus/genética , Potyvirus/genética , RNA Viral/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa/normas , Análise de Sequência de RNA
15.
J Virol Methods ; 204: 93-100, 2014 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-24769198

RESUMO

Papaya ringspot virus (PRSV) and Papaya leaf distortion mosaic virus (PLDMV), which causes disease symptoms similar to PRSV, threaten commercial production of both non-transgenic-papaya and PRSV-resistant transgenic papaya in China. A reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay to detect PLDMV was developed previously. In this study, the development of another RT-LAMP assay to distinguish among transgenic, PRSV-infected and PLDMV-infected papaya by detection of PRSV is reported. A set of four RT-LAMP primers was designed based on the highly conserved region of the P3 gene of PRSV. The RT-LAMP method was specific and sensitive in detecting PRSV, with a detection limit of 1.15×10(-6)µg of total RNA per reaction. Indeed, the reaction was 10 times more sensitive than one-step RT-PCR. Field application of the RT-LAMP assay demonstrated that samples positive for PRSV were detected only in non-transgenic papaya, whereas samples positive for PLDMV were detected only in commercialized PRSV-resistant transgenic papaya. This suggests that PRSV remains the major limiting factor for non-transgenic-papaya production, and the emergence of PLDMV threatens the commercial transgenic cultivar in China. However, this study, combined with the earlier development of an RT-LAMP assay for PLDMV, will provide a rapid, sensitive and cost-effective diagnostic power to distinguish virus infections in papaya.


Assuntos
Carica/virologia , Técnicas de Amplificação de Ácido Nucleico/métodos , Potyvirus/classificação , Potyvirus/isolamento & purificação , Transcrição Reversa , Viroses/virologia , China , Análise Custo-Benefício , Primers do DNA/genética , Técnicas de Amplificação de Ácido Nucleico/economia , Potyvirus/genética , RNA Viral/genética , Sensibilidade e Especificidade , Tempo , Fatores de Tempo
16.
J Virol Methods ; 195: 174-9, 2014 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-24100065

RESUMO

Papaya leaf distortion mosaic virus (PLDMV) can infect transgenic papaya resistant to a related pathogen, Papaya ringspot virus (PRSV), posing a substantial threat to papaya production in China. Current detection methods, however, are unable to be used for rapid detection in the field. Here, a reverse-transcription loop-mediated isothermal amplification (RT-LAMP) assay was developed for the detection of PLDMV, using a set of four RT-LAMP primers designed based on the conserved sequence of PLDMV CP. The RT-LAMP method detected specifically PLDMV and was highly sensitive, with a detection limit of 1.32×10(-6) µg of total RNA per reaction. Indeed, the reaction was 10 times more sensitive than one-step RT-PCR, while also requiring significantly less time and equipment. The effectiveness of RT-LAMP and one-step RT-PCR in detecting the virus were compared using 90 field samples of non-transgenic papaya and 90 field samples of commercialized PRSV-resistant transgenic papaya from Hainan Island. None of the non-transgenic papaya tested positive for PLDMV using either method. In contrast, 19 of the commercialized PRSV-resistant transgenic papaya samples tested positive by RT-LAMP assay, and 6 of those tested negative by RT-PCR. Therefore, the PLDMV-specific RT-LAMP is a simple, rapid, sensitive, and cost-effective tool in the field diagnosis and control of PLDMV.


Assuntos
Carica/virologia , Técnicas de Amplificação de Ácido Nucleico/métodos , Doenças das Plantas/virologia , Potyvirus/isolamento & purificação , Virologia/métodos , China , Análise Custo-Benefício , Primers do DNA/genética , Técnicas de Amplificação de Ácido Nucleico/economia , Potyvirus/genética , Transcrição Reversa , Sensibilidade e Especificidade , Fatores de Tempo , Virologia/economia
17.
Anal Biochem ; 437(2): 172-7, 2013 Jun 15.
Artigo em Inglês | MEDLINE | ID: mdl-23499974

RESUMO

Construction of plasmids is the basic and pivotal technology in molecular biology. The common method for constructing plasmids is to cut DNA fragments by restriction enzymes and then join the resulting fragments using ligase. We present here a modified Golden Gate cloning method for modular construction of plasmids. Unlike the original Golden Gate cloning system for cloning from entry vector to expression vector, this method can be used to construct plasmids immediately from linear DNA fragments. After polymerase chain reaction (PCR) amplification for flanking with BsaI sites, multiple linear DNA components (modules) can be parallel assembled into a circle plasmid by a single restriction-ligation reaction using the method. This method is flexible to construct different types of plasmids because the modules can be freely selected and assembled in any combination. This method was applied successfully to construct a prokaryotic expression plasmid from four modules and a plant expression plasmid from five modules (fragments). The results suggest that this method provides a simple and flexible platform for modular construction of plasmids.


Assuntos
Clonagem Molecular/métodos , Plasmídeos/genética , Sequência de Bases
18.
Virology ; 434(1): 78-87, 2012 Dec 05.
Artigo em Inglês | MEDLINE | ID: mdl-23040510

RESUMO

A chloroplast-localized papaya methionine sulfoxide reductase B1 (PaMsrB1) interacting with Papaya ringspot virus (PRSV) NIa-Pro was identified using a Sos recruitment two-hybrid system (SRS). SRS analysis of several deletion mutants of PRSV NIa-Pro and PaMsrB1 demonstrated that the C-terminal (residues 133-239) fragment of PRSV NIa-Pro and residues 112-175 of PaMsrB1 were necessary for this interaction between PRSV NIa-Pro and PaMsrB1. MsrB1 can repair Met-oxidized proteins damaged by reactive oxygen species (ROS). We confirmed that PRSV infection leads to ROS accumulation and a slight upregulation of level PaMsrB1 mRNA in papaya. This interaction between PaMsrB1 with PRSV NIa-Pro may disturb the import of PaMsrB1 into the chloroplasts. These results suggest that this specific interaction could interfere with PaMsrB1 into the chloroplasts to scavenge ROS caused by PRSV infection. This may be a novel mechanism of PRSV towards the host defense.


Assuntos
Carica/virologia , Endopeptidases/metabolismo , Interações Hospedeiro-Patógeno , Metionina Sulfóxido Redutases/metabolismo , Potyvirus/enzimologia , Potyvirus/patogenicidade , Proteínas Virais/metabolismo , Sequência de Aminoácidos , Dados de Sequência Molecular , Ligação Proteica , Mapeamento de Interação de Proteínas , Análise de Sequência de DNA , Deleção de Sequência , Técnicas do Sistema de Duplo-Híbrido
19.
Anal Biochem ; 430(1): 65-7, 2012 Nov 01.
Artigo em Inglês | MEDLINE | ID: mdl-22885236

RESUMO

A parallel assembly method for multiple site-directed mutagenesis of plasmids was developed here based on Golden Gate cloning. It takes advantage of type IIs restriction enzymes and T4 DNA ligase to assemble multiple DNA fragments into a plasmid by a defined order. This method can accommodate multiple plasmid mutagenesis at any desired position with all three sequence modification types (substitution, deletion, and insertion) simultaneously. Furthermore, it can be used to create otherwise difficult-to-make mutants-larger deletions and insertions and mutagenesis on larger plasmids. The processes of mutagenesis can be completed quickly by a single restriction-ligation reaction.


Assuntos
Mutagênese Sítio-Dirigida/métodos , Plasmídeos/genética , Bacteriófago T4/enzimologia , DNA Ligases/metabolismo , Desoxirribonucleases de Sítio Específico do Tipo II/metabolismo , Escherichia coli/genética , Plasmídeos/metabolismo , Reação em Cadeia da Polimerase
20.
PLoS One ; 7(5): e38186, 2012.
Artigo em Inglês | MEDLINE | ID: mdl-22675447

RESUMO

With the wide use of double-stranded RNA interference (RNAi) for the analysis of gene function in plants, a high-throughput system for making hairpin RNA (hpRNA) constructs is in great demand. Here, we describe a novel restriction-ligation approach that provides a simple but efficient construction of intron-containing hpRNA (ihpRNA) vectors. The system takes advantage of the type IIs restriction enzyme BsaI and our new plant RNAi vector pRNAi-GG based on the Golden Gate (GG) cloning. This method requires only a single PCR product of the gene of interest flanked with BsaI recognition sequence, which can then be cloned into pRNAi-GG at both sense and antisense orientations simultaneously to form ihpRNA construct. The process, completed in one tube with one restriction-ligation step, produced a recombinant ihpRNA with high efficiency and zero background. We demonstrate the utility of the ihpRNA constructs generated with pRNAi-GG vector for the effective silencing of various individual endogenous and exogenous marker genes as well as two genes simultaneously. This method provides a novel and high-throughput platform for large-scale analysis of plant functional genomics.


Assuntos
Vetores Genéticos , Íntrons , Plantas/genética , Interferência de RNA , RNA de Plantas/genética , RNA Interferente Pequeno/genética , Clonagem Molecular , Ordem dos Genes , Inativação Gênica , Genes de Plantas , Genes Reporter , Genômica/métodos
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