To facilitate their spread, plant viruses have developed several methods for dispersal including ... more To facilitate their spread, plant viruses have developed several methods for dispersal including insect and seed transmission. While insect transmission requires virus stability against insect digestion, seed-transmitted viruses have to overcome barriers to entry into embryos. Bean pod mottle virus (BPMV) is transmitted through seed at levels typically below 0.1%, but co-infection with Soybean mosaic virus (SMV) enhanced the seed transmission rate of BPMV in one experiment. In contrast, the rate of SMV seed transmission was not affected by BPMV co-infection. In a second preliminary study, the rate of SMV transmission was lower in an isoline of Williams 82 that contained a null mutation for the Kunitz trypsin inhibitor gene than in Williams 82. In this preliminary study, we observed that factors such as protease inhibitor expression and dual infection may affect the frequency of seed transmission of BPMV and SMV.
The multifunctional triple gene block protein 1 (TGB1) of the Potexvirus Alternanthera mosaic vir... more The multifunctional triple gene block protein 1 (TGB1) of the Potexvirus Alternanthera mosaic virus (AltMV) has been reported to have silencing suppressor, cell-to-cell movement, and helicase functions. Yeast two hybrid screening using an Arabidopsis thaliana cDNA library with TGB1 as bait, and co-purification with TGB1 inclusion bodies identified several host proteins which interact with AltMV TGB1. Host protein interactions with TGB1 were confirmed by biomolecular fluorescence complementation, which showed positive TGB1 interaction with mitochondrial ATP synthase delta' chain subunit (ATP synthase delta'), light harvesting chlorophyll-protein complex I subunit A4 (LHCA4), chlorophyll a/b binding protein 1 (LHB1B2), chloroplast-localized IscA-like protein (ATCPISCA), and chloroplast β-ATPase. However, chloroplast β-ATPase interacts only with TGB1L88, and not with weak silencing suppressor TGB1P88. This selective interaction indicates that chloroplast β-ATPase is not require...
We have developed plant virus-based vectors for virus-induced gene silencing (VIGS) and protein e... more We have developed plant virus-based vectors for virus-induced gene silencing (VIGS) and protein expression, based on Alternanthera mosaic virus (AltMV), for infection of a wide range of host plants including Nicotiana benthamiana and Arabidopsis thaliana by either mechanical inoculation of in vitro transcripts or via agroinfiltration. In vivo transcripts produced by co-agroinfiltration of bacteriophage T7 RNA polymerase resulted in T7-driven AltMV infection from a binary vector in the absence of the Cauliflower mosaic virus 35S promoter. An artificial bipartite viral vector delivery system was created by separating the AltMV RNA-dependent RNA polymerase and Triple Gene Block (TGB)123-Coat protein (CP) coding regions into two constructs each bearing the AltMV 5′ and 3′ non-coding regions, which recombined in planta to generate a full-length AltMV genome. Substitution of TGB1 L(88)P, and equivalent changes in other potexvirus TGB1 proteins, affected RNA silencing suppression efficacy and suitability of the vectors from protein expression to VIGS.
Different isolates of Alternanthera mosaic virus (AltMV; Potexvirus), including four infectious c... more Different isolates of Alternanthera mosaic virus (AltMV; Potexvirus), including four infectious clones derived from AltMV-SP, induce distinct systemic symptoms in Nicotiana benthamiana. Virus accumulation was enhanced at 15 1C compared to 25 1C; severe clone AltMV 3-7 induced systemic necrosis (SN) and plant death at 15 1C. No interaction with potexvirus resistance gene Rx was detected, although SN was ablated by silencing of SGT1, as for other cases of potexvirus-induced necrosis. Substitution of AltMV 3-7 coat protein (CP SP) with that from AltMV-Po (CP Po) eliminated SN at 15 1C, and ameliorated symptoms in Alternanthera dentata and soybean. Substitution of only two residues from CP Po [either MN(13,14)ID or LA(76,77)IS] efficiently ablated SN in N. benthamiana. CP SP but not CP Po interacted with Arabidopsis boron transporter protein AtBOR1 by yeast two-hybrid assay; N. benthamiana homolog NbBOR1 interacted more strongly with CP SP than CP Po in bimolecular fluorescence complementation, and may affect recognition of CP as an elicitor of SN.
The genome of Lolium latent virus (LoLV; genus Lolavirus, family Alphaflexiviridae) is encapsidat... more The genome of Lolium latent virus (LoLV; genus Lolavirus, family Alphaflexiviridae) is encapsidated by two carboxy-coterminal coat protein (CP) variants (about 28 and 33 kDa), in equimolar proportions. The CP ORF contains two 5′-proximal AUGs encoding Met 1 and Met 49, respectively promoting translation of the 33 and 28 kDa CP variants. The 33 kDa CP N-terminal domain includes a 42 aa sequence encoding a putative chloroplast transit peptide, leading to protein cleavage and alternative derivation of the approximately 28 kDa CP. Mutational analysis of the two in-frame start codons and of the putative proteolytic-cleavage site showed that the N-terminal sequence is crucial for efficient cell-to-cell movement, functional systemic movement, homologous CP interactions and particle formation, but is not required for virus replication. Blocking production of the 28 kDa CP by internal initiation shows no major outcome, whereas additional mutation to prevent proteolytic cleavage at the chloro...
Lolium latent virus (LoLV) was recently detected in the USA for the first time in ryegrass hybrid... more Lolium latent virus (LoLV) was recently detected in the USA for the first time in ryegrass hybrids (Lolium perenne 9 Lolium multiflorum). The genome of one USA isolate, LoLV-US1, has now been fully sequenced. The positive strand genomic RNA is 7674 nucleotides (nt) long and is organized in five open reading frames (ORFs) encoding the replication-associated protein, the movement-associated triple gene block proteins and the coat protein (CP). The genome organization is similar to that of viruses in the genera Potexvirus and Foveavirus; however, analysis of the complete LoLV genomic sequence, phylogenetic analyses of the deduced amino acid (aa) sequences of the polymerase and the CP, presence of a putative ORF 6, and the in vivo detection of two CPs in equimolar amounts, highlight features peculiar to LoLV. These characteristics indicate that LoLV forms a monotypic group separate from existing genera and unassigned species within the family Flexiviridae, for which we propose the genus name Lolavirus. One-step RT-PCR was developed for quick and reliable LoLV detection.
To facilitate their spread, plant viruses have developed several methods for dispersal including ... more To facilitate their spread, plant viruses have developed several methods for dispersal including insect and seed transmission. While insect transmission requires virus stability against insect digestion, seed-transmitted viruses have to overcome barriers to entry into embryos. Bean pod mottle virus (BPMV) is transmitted through seed at levels typically below 0.1%, but co-infection with Soybean mosaic virus (SMV) enhanced the seed transmission rate of BPMV in one experiment. In contrast, the rate of SMV seed transmission was not affected by BPMV co-infection. In a second preliminary study, the rate of SMV transmission was lower in an isoline of Williams 82 that contained a null mutation for the Kunitz trypsin inhibitor gene than in Williams 82. In this preliminary study, we observed that factors such as protease inhibitor expression and dual infection may affect the frequency of seed transmission of BPMV and SMV.
The multifunctional triple gene block protein 1 (TGB1) of the Potexvirus Alternanthera mosaic vir... more The multifunctional triple gene block protein 1 (TGB1) of the Potexvirus Alternanthera mosaic virus (AltMV) has been reported to have silencing suppressor, cell-to-cell movement, and helicase functions. Yeast two hybrid screening using an Arabidopsis thaliana cDNA library with TGB1 as bait, and co-purification with TGB1 inclusion bodies identified several host proteins which interact with AltMV TGB1. Host protein interactions with TGB1 were confirmed by biomolecular fluorescence complementation, which showed positive TGB1 interaction with mitochondrial ATP synthase delta' chain subunit (ATP synthase delta'), light harvesting chlorophyll-protein complex I subunit A4 (LHCA4), chlorophyll a/b binding protein 1 (LHB1B2), chloroplast-localized IscA-like protein (ATCPISCA), and chloroplast β-ATPase. However, chloroplast β-ATPase interacts only with TGB1L88, and not with weak silencing suppressor TGB1P88. This selective interaction indicates that chloroplast β-ATPase is not require...
We have developed plant virus-based vectors for virus-induced gene silencing (VIGS) and protein e... more We have developed plant virus-based vectors for virus-induced gene silencing (VIGS) and protein expression, based on Alternanthera mosaic virus (AltMV), for infection of a wide range of host plants including Nicotiana benthamiana and Arabidopsis thaliana by either mechanical inoculation of in vitro transcripts or via agroinfiltration. In vivo transcripts produced by co-agroinfiltration of bacteriophage T7 RNA polymerase resulted in T7-driven AltMV infection from a binary vector in the absence of the Cauliflower mosaic virus 35S promoter. An artificial bipartite viral vector delivery system was created by separating the AltMV RNA-dependent RNA polymerase and Triple Gene Block (TGB)123-Coat protein (CP) coding regions into two constructs each bearing the AltMV 5′ and 3′ non-coding regions, which recombined in planta to generate a full-length AltMV genome. Substitution of TGB1 L(88)P, and equivalent changes in other potexvirus TGB1 proteins, affected RNA silencing suppression efficacy and suitability of the vectors from protein expression to VIGS.
Different isolates of Alternanthera mosaic virus (AltMV; Potexvirus), including four infectious c... more Different isolates of Alternanthera mosaic virus (AltMV; Potexvirus), including four infectious clones derived from AltMV-SP, induce distinct systemic symptoms in Nicotiana benthamiana. Virus accumulation was enhanced at 15 1C compared to 25 1C; severe clone AltMV 3-7 induced systemic necrosis (SN) and plant death at 15 1C. No interaction with potexvirus resistance gene Rx was detected, although SN was ablated by silencing of SGT1, as for other cases of potexvirus-induced necrosis. Substitution of AltMV 3-7 coat protein (CP SP) with that from AltMV-Po (CP Po) eliminated SN at 15 1C, and ameliorated symptoms in Alternanthera dentata and soybean. Substitution of only two residues from CP Po [either MN(13,14)ID or LA(76,77)IS] efficiently ablated SN in N. benthamiana. CP SP but not CP Po interacted with Arabidopsis boron transporter protein AtBOR1 by yeast two-hybrid assay; N. benthamiana homolog NbBOR1 interacted more strongly with CP SP than CP Po in bimolecular fluorescence complementation, and may affect recognition of CP as an elicitor of SN.
The genome of Lolium latent virus (LoLV; genus Lolavirus, family Alphaflexiviridae) is encapsidat... more The genome of Lolium latent virus (LoLV; genus Lolavirus, family Alphaflexiviridae) is encapsidated by two carboxy-coterminal coat protein (CP) variants (about 28 and 33 kDa), in equimolar proportions. The CP ORF contains two 5′-proximal AUGs encoding Met 1 and Met 49, respectively promoting translation of the 33 and 28 kDa CP variants. The 33 kDa CP N-terminal domain includes a 42 aa sequence encoding a putative chloroplast transit peptide, leading to protein cleavage and alternative derivation of the approximately 28 kDa CP. Mutational analysis of the two in-frame start codons and of the putative proteolytic-cleavage site showed that the N-terminal sequence is crucial for efficient cell-to-cell movement, functional systemic movement, homologous CP interactions and particle formation, but is not required for virus replication. Blocking production of the 28 kDa CP by internal initiation shows no major outcome, whereas additional mutation to prevent proteolytic cleavage at the chloro...
Lolium latent virus (LoLV) was recently detected in the USA for the first time in ryegrass hybrid... more Lolium latent virus (LoLV) was recently detected in the USA for the first time in ryegrass hybrids (Lolium perenne 9 Lolium multiflorum). The genome of one USA isolate, LoLV-US1, has now been fully sequenced. The positive strand genomic RNA is 7674 nucleotides (nt) long and is organized in five open reading frames (ORFs) encoding the replication-associated protein, the movement-associated triple gene block proteins and the coat protein (CP). The genome organization is similar to that of viruses in the genera Potexvirus and Foveavirus; however, analysis of the complete LoLV genomic sequence, phylogenetic analyses of the deduced amino acid (aa) sequences of the polymerase and the CP, presence of a putative ORF 6, and the in vivo detection of two CPs in equimolar amounts, highlight features peculiar to LoLV. These characteristics indicate that LoLV forms a monotypic group separate from existing genera and unassigned species within the family Flexiviridae, for which we propose the genus name Lolavirus. One-step RT-PCR was developed for quick and reliable LoLV detection.
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