scholarly journals Robust Agrobacterium-Mediated Transient Expression in Two Duckweed Species (Lemnaceae) Directed by Non-replicating, Replicating, and Cell-to-Cell Spreading Vectors

2021 ◽  
Vol 9 ◽  
Author(s):  
Anton Peterson ◽  
Olena Kishchenko ◽  
Yuzhen Zhou ◽  
Maksym Vasylenko ◽  
Anatoli Giritch ◽  
...  
Keyword(s):  
Transient Expression ◽  
Potato Virus ◽  
Mammalian Cells ◽  
Fluorescent Protein ◽  
Scale Up ◽  
Potato Virus X ◽  
High Yield ◽  
Protein Accumulation ◽  
Expression Systems ◽  
Potato Leaf

Plant-based transient expression systems have recognized potential for use as rapid and cost-effective alternatives to expression systems based on bacteria, yeast, insect, or mammalian cells. The free-floating aquatic plants of the Lemnaceae family (duckweed) have compact architecture and can be vegetatively propagated on low-cost nutrient solutions in aseptic conditions. These features provide an economically feasible opportunity for duckweed-based production of high-value products via transient expression of recombinant products in fully contained, controlled, aseptic and bio-safe conditions in accordance with the requirements for pharmaceutical manufacturing and environmental biosafety. Here, we demonstrated Agrobacterium-mediated high-yield transient expression of a reporter green fluorescent protein using deconstructed vectors based on potato virus X and sweet potato leaf curl virus, as well as conventional binary vectors, in two representatives of the Lemnaceae (Spirodela polyrhiza and Landoltia punctata). Aseptically cultivated duckweed populations yielded reporter protein accumulation of >1 mg/g fresh biomass, when the protein was expressed from a deconstructed potato virus X-based vector, which is capable of replication and cell-to-cell movement of the replicons in duckweed. The expression efficiency demonstrated here places duckweed among the most efficient host organisms for plant-based transient expression systems, with the additional benefits of easy scale-up and full containment.

scholarly journals Induction of the Cellular E2F-1 Promoter by the Adenovirus E4-6/7 Protein

2000 ◽  
Vol 74 (5) ◽  
pp. 2084-2093 ◽  
Author(s):  
Joel Schaley ◽  
Robert J. O'Connor ◽  
Laura J. Taylor ◽  
Dafna Bar-Sagi ◽  
Patrick Hearing
Keyword(s):  
Dna Binding ◽  
Binding Site ◽  
Transient Expression ◽  
Binding Sites ◽  
Promoter Region ◽  
Fluorescent Protein ◽  
Protein Accumulation ◽  
Promoter Regions ◽  
Single Binding Site

ABSTRACT The adenovirus type 5 (Ad5) E4-6/7 protein interacts directly with different members of the E2F family and mediates the cooperative and stable binding of E2F to a unique pair of binding sites in the Ad5 E2a promoter region. This induction of E2F DNA binding activity strongly correlates with increased E2a transcription when analyzed using virus infection and transient expression assays. Here we show that while different adenovirus isolates express an E4-6/7 protein that is capable of induction of E2F dimerization and stable DNA binding to the Ad5 E2a promoter region, not all of these viruses carry the inverted E2F binding site targets in their E2a promoter regions. The Ad12 and Ad40 E2a promoter regions bind E2F via a single binding site. However, these promoters bind adenovirus-induced (dimerized) E2F very weakly. The Ad3 E2a promoter region binds E2F very poorly, even via a single binding site. A possible explanation of these results is that the Ad E4-6/7 protein evolved to induce cellular gene expression. Consistent with this notion, we show that infection with different adenovirus isolates induces the binding of E2F to an inverted configuration of binding sites present in the cellular E2F-1 promoter. Transient expression of the E4-6/7 protein alone in uninfected cells is sufficient to induce transactivation of the E2F-1 promoter linked to chloramphenicol acetyltransferase or green fluorescent protein reporter genes. Further, expression of the E4-6/7 protein in the context of adenovirus infection induces E2F-1 protein accumulation. Thus, the induction of E2F binding to the E2F-1 promoter by the E4-6/7 protein observed in vitro correlates with transactivation of E2F-1 promoter activity in vivo. These results suggest that adenovirus has evolved two distinct mechanisms to induce the expression of the E2F-1 gene. The E1A proteins displace repressors of E2F activity (the Rb family members) and thus relieve E2F-1 promoter repression; the E4-6/7 protein complements this function by stably recruiting active E2F to the E2F-1 promoter to transactivate expression.

Transient expression of HPV16 E7 peptide (aa 44–60) and HPV16 L2 peptide (aa 108–120) on chimeric potyvirus-like particles using Potato virus X-based vector

2008 ◽  
Vol 58 (1) ◽  
pp. 154-161 ◽  
Author(s):  
Noemi Čeřovská ◽  
Hana Hoffmeisterová ◽  
Tamara Pečenková ◽  
Tomáš Moravec ◽  
Helena Synková ◽  
...  
Keyword(s):  
Transient Expression ◽  
Potato Virus ◽  
Potato Virus X

scholarly journals Phenotypes and Functional Effects Caused by Various Viral RNA Silencing Suppressors in Transgenic Nicotiana benthamiana and N. tabacum

2008 ◽  
Vol 21 (2) ◽  
pp. 178-187 ◽  
Author(s):  
Shahid Aslam Siddiqui ◽  
Cecilia Sarmiento ◽  
Erkki Truve ◽  
Harry Lehto ◽  
Kirsi Lehto
Keyword(s):  
Mosaic Virus ◽  
Potato Virus ◽  
Nicotiana Benthamiana ◽  
Rna Silencing ◽  
Fluorescent Protein ◽  
Potato Virus X ◽  
Mottle Virus ◽  
African Cassava Mosaic Virus ◽  
Rice Yellow Mottle Virus ◽  
Green Fluorescent

RNA silencing suppressor genes derived from six virus genera were transformed into Nicotiana benthamiana and N. tabacum plants. These suppressors were P1 of Rice yellow mottle virus (RYMV), P1 of Cocksfoot mottle virus, P19 of Tomato bushy stunt virus, P25 of Potato virus X, HcPro of Potato virus Y (strain N), 2b of Cucumber mosaic virus (strain Kin), and AC2 of African cassava mosaic virus (ACMV). HcPro caused the most severe phenotypes in both Nicotiana spp. AC2 also produced severe effects in N. tabacum but a much milder phenotype in N. benthamiana, although both HcPro and AC2 affected the leaf tissues of the two Nicotiana spp. in similar ways, causing hyperplasia and hypoplasia, respectively. P1-RYMV caused high lethality in the N. benthamiana plants but only mild effects in the N. tabacum plants. Phenotypic alterations produced by the other transgenes were minor in both species. Interestingly, the suppressors had very different effects on crucifer-infecting Tobamovirus (crTMV) infections. AC2 enhanced both spread and brightness of the crTMV-green fluorescent protein (GFP) lesions, whereas 2b and both P1 suppressors enhanced spread but not brightness of these lesions. P19 promoted spread of the infection into new foci within the infiltrated leaf, whereas HcPro and P25 suppressed the spread of crTMV-GFP lesions.

Efficient transient expression of human GM-CSF protein in Nicotiana benthamiana using potato virus X vector

2006 ◽  
Vol 72 (4) ◽  
pp. 756-762 ◽  
Author(s):  
Fengyong Zhou ◽  
Ming-Li Wang ◽  
Henrik H. Albert ◽  
Paul H. Moore ◽  
Yun J. Zhu
Keyword(s):  
Transient Expression ◽  
Potato Virus ◽  
Nicotiana Benthamiana ◽  
Potato Virus X ◽  
Gm Csf

scholarly journals The hydrophobic segment of Potato virus X TGBp3 is a major determinant of the protein intracellular trafficking

2005 ◽  
Vol 86 (8) ◽  
pp. 2379-2391 ◽  
Author(s):  
M. V. Schepetilnikov ◽  
U. Manske ◽  
A. G. Solovyev ◽  
A. A. Zamyatnin ◽  
J. Schiemann ◽  
...  
Keyword(s):  
Potato Virus ◽  
Intracellular Trafficking ◽  
Intracellular Transport ◽  
Fluorescent Protein ◽  
Cell Movement ◽  
Potato Virus X ◽  
Movement Proteins ◽  
C Terminus ◽  
Hydrophilic Region ◽  
Green Fluorescent

Potato virus X (PVX) encodes three movement proteins, TGBp1, TGBp2 and TGBp3. The 8 kDa TGBp3 is a membrane-embedded protein that has an N-terminal hydrophobic sequence segment and a hydrophilic C terminus. TGBp3 mutants with deletions in the C-terminal hydrophilic region retain the ability to be targeted to cell peripheral structures and to support limited PVX cell-to-cell movement, suggesting that the basic TGBp3 functions are associated with its N-terminal transmembrane region. Fusion of green fluorescent protein to the TGBp3 N terminus abrogates protein activities in intracellular trafficking and virus movement. The intracellular transport of TGBp3 from sites of its synthesis in the rough endoplasmic reticulum (ER) to ER-derived peripheral bodies involves a non-conventional COPII-independent pathway. However, integrity of the C-terminal hydrophilic sequence is required for entrance to this non-canonical route.

scholarly journals Cell-to-Cell Movement of Potato virus X: The Role of p12 and p8 Encoded by the Second and Third Open Reading Frames of the Triple Gene Block

2001 ◽  
Vol 14 (10) ◽  
pp. 1158-1167 ◽  
Author(s):  
Atsushi Tamai ◽  
Tetsuo Meshi
Keyword(s):  
Potato Virus ◽  
Fluorescent Protein ◽  
Microprojectile Bombardment ◽  
Triple Gene Block ◽  
Cell Movement ◽  
Potato Virus X ◽  
Open Reading Frames ◽  
Gene Block ◽  
Infected Cells ◽  
Green Fluorescent

Potato virus X (PVX) requires three proteins, p25, p12, and p8, encoded by the triple gene block plus the coat protein (CP) for cell-to-cell movement. When each of these proteins was co-expressed with a cytosolic green fluorescent protein (GFP) in the epidermal cells of Nicotiana benthamiana by the microprojectile bombardment-mediated gene delivery method, only p12 enhanced diffusion of co-expressed GFP, indicating an ability to alter plasmodesmal permeability. p25, p12, and CP, expressed transiently in the initially infected cells, transcomplemented the corresponding movement-defective mutants to spread through two or more cell boundaries. Thus, these proteins probably move from cell to cell with the genomic RNA. In contrast, p8 only functioned intracellularly and was not absolutely required for cell-to-cell movement. Since overexpression of p12 overcame the p8 deficiency, p8 appears to facilitate the functioning of p12, presumably by mediating its intracellular trafficking. Considering the likelihood that p12 and p8 are membrane proteins, it is suggested that intercellular as well as intracellular movement of PVX involves a membrane-mediated process.

scholarly journals Plastocyanin Transit Peptide Interacts with Potato virus X Coat Protein, While Silencing of Plastocyanin Reduces Coat Protein Accumulation in Chloroplasts and Symptom Severity in Host Plants

2009 ◽  
Vol 22 (12) ◽  
pp. 1523-1534 ◽  
Author(s):  
Y. Qiao ◽  
H. F. Li ◽  
S. M. Wong ◽  
Z. F. Fan
Keyword(s):  
Coat Protein ◽  
Potato Virus ◽  
Symptom Severity ◽  
Host Plants ◽  
Transit Peptide ◽  
Potato Virus X ◽  
Precursor Protein ◽  
Bimolecular Fluorescence Complementation ◽  
Protein Accumulation

Potato virus X coat protein (PVXCP) is, through communication with host proteins, involved in processes such as virus movement and symptom development. Here, we report that PVXCP also interacts with the precursor of plastocyanin, a protein involved in photosynthesis, both in vitro and in vivo. Yeast two-hybrid analysis indicated that PVXCP interacted with only the plastocyanin transit peptide. In subsequent bimolecular fluorescence complementation assays, both proteins were collocated within chloroplasts. Western blot analyses of chloroplast fractions showed that PVXCP could be detected in the envelope, stroma, and lumen fractions. Transmission electron microscopy demonstrated that grana were dilated in PVX-infected Nicotiana benthamiana. Furthermore, virus-induced gene silencing of plastocyanin by prior infection of N. benthamiana using a Tobacco rattle virus vector reduced the severity of symptoms that developed following subsequent PVX infection as well as the accumulation of PVXCP in isolated chloroplasts. However, PVXCP could not be detected in pea chloroplasts in an in vitro re-uptake assay using the plastocyanin precursor protein. Taken together, these data suggest that PVXCP interacts with the plastocyanin precursor protein and that silencing the expression of this protein leads to reduced PVXCP accumulation in chloroplasts and ameliorates symptom severity in host plants.

scholarly journals Antiviral and Antiviroid Activity of MAP-Containing Extracts from Mirabilis jalapa Roots

Plant Disease ◽  
1999 ◽  
Vol 83 (12) ◽  
pp. 1116-1121 ◽  
Author(s):  
Jorge M. Vivanco ◽  
Maddalena Querci ◽  
Luis F. Salazar
Keyword(s):  
Potato Virus ◽  
Leaf Roll ◽  
Potato Virus X ◽  
Antiviral Effect ◽  
Potato Spindle Tuber Viroid ◽  
Antiviral Protein ◽  
Potato Leaf ◽  
Mirabilis Jalapa ◽  
Spot Hybridization ◽  
Mirabilis Antiviral Protein

Extracts of Mirabilis jalapa (Nyctaginaceae), containing a ribosome inactivating protein (RIP) called Mirabilis antiviral protein (MAP), were tested against infection by potato virus X, potato virus Y, potato leaf roll virus, and potato spindle tuber viroid. Root extracts of M. jalapa sprayed on test plants 24 h before virus or viroid inoculation inhibited infection by almost 100%, as corroborated by infectivity assays and the nucleic acid spot hybridization test. Antiviral activity of MAP extracts was observed against mechanically transmitted viruses but not against aphid-transmitted viruses. Purified MAP showed the same antiviral effect as the crude extracts.

Potato viruses and resistance genes in potato

2012 ◽  
Vol 60 (3) ◽  
pp. 283-298 ◽  
Author(s):  
R. Ahmadvand ◽  
A. Takács ◽  
J. Taller ◽  
I. Wolf ◽  
Z. Polgár
Keyword(s):  
Potato Virus ◽  
Leaf Roll ◽  
Potato Virus X ◽  
Potato Leaf ◽  
Potato Virus S ◽  
Potato Virus A ◽  
The World ◽  
Potato Virus M ◽  
Cultivated Potatoes

Potato (Solanum tuberosum L.) is the fourth most important food crop in the world. It is the most economically valuable and well-known member of the plant family Solanaceae. Potato is the host of many pathogens, including fungi, bacteria, Phytoplasmas, viruses, viroids and nematodes, which cause reductions in the quantity and quality of yield. Apart from the late blight fungus [Phytophthora infestans (Mont.) de Bary] viruses are the most important pathogens, with over 40 viruses and virus-like pathogens infecting cultivated potatoes in the field, among which Potato virus Y (PVY), Potato leaf roll virus (PLRV), Potato virus X (PVX), Potato virus A (PVA), Potato virus S (PVS) and Potato virus M (PVM) are some of the most important viruses in the world. In this review, their characteristics and types of resistance to them will be discussed.

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