Abstract
Plants are a promising platform to produce biopharmaceutical proteins, however the toxic nature of some proteins inhibits their accumulation. We previously created a replicating geminiviral expression system based on bean yellow dwarf virus (BeYDV) that enables very high-level production of recombinant proteins. To study the role of replication in this system, we generated vectors that allow separate and controlled expression of BeYDV Rep and RepA proteins. We show that the ratio of Rep and RepA strongly affects the efficiency of replication. Rep, RepA, and vector replication all elicit the plant hypersensitive response, resulting in cell death. We find that a modest reduction in expression of Rep and RepA reduces plant leaf cell death which, despite reducing the accumulation of viral replicons, increases target protein accumulation. A single nucleotide change in the 5’ untranslated region (UTR) reduced Rep/RepA expression, reduced cell death, and enhanced the production of monoclonal antibodies. We also find that replicating vectors achieve optimal expression with lower agrobacterium concentrations than nonreplicating vectors, further reducing cell death. Viral UTRs are also shown to contribute substantially to cell death, while a native plant-derived 5’ UTR does not.
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Claims 14
Claim · model-written summary
The Rep to RepA ratio strongly affected vector replication efficiency.
- Taxon
- Nicotiana benthamiana
Nicotiana benthamianat0 - Gene
- Rep and RepAe0
- Outcome
- efficiency of replicatione0
Not reportedDirection
Organisms pictured · Representative pictures of the matched species, not figures from the paper.
Verbatim
e0“the ratio of Rep and RepA strongly affects the efficiency of replication”
t0“Nicotiana benthamiana”
Claim · model-written summary
Rep expression elicited the plant hypersensitive response and cell death.
- Taxon
- Nicotiana benthamianaNicotiana benthamianat0
- Treatment
- Repe0
- Gene
- Repe0
- Outcome
- plant hypersensitive response, resulting in cell deathe0
Not reportedComparatorDirection
Organisms pictured · Representative pictures of the matched species, not figures from the paper.
Verbatim
e0“Rep, RepA, and vector replication all elicit the plant hypersensitive response, resulting in cell death”
t0“Nicotiana benthamiana”
Claim · model-written summary
RepA expression elicited the plant hypersensitive response and cell death.
- Taxon
- Nicotiana benthamianaNicotiana benthamianat0
- Treatment
- RepAe0
- Gene
- RepAe0
- Outcome
- plant hypersensitive response, resulting in cell deathe0
Not reportedComparatorDirection
Organisms pictured · Representative pictures of the matched species, not figures from the paper.
Verbatim
e0“Rep, RepA, and vector replication all elicit the plant hypersensitive response, resulting in cell death”
t0“Nicotiana benthamiana”
Claim · model-written summary
Vector replication elicited the plant hypersensitive response and cell death.
- Taxon
- Nicotiana benthamianaNicotiana benthamianat0
- Treatment
- vector replicatione0
- Outcome
- plant hypersensitive response, resulting in cell deathe0
Not reportedComparatorDirection
Organisms pictured · Representative pictures of the matched species, not figures from the paper.
Verbatim
e0“Rep, RepA, and vector replication all elicit the plant hypersensitive response, resulting in cell death”
t0“Nicotiana benthamiana”
Claim · model-written summary
Modestly reducing Rep and RepA expression reduced leaf cell death.
- Taxon
- Nicotiana benthamianaNicotiana benthamianat0
- Treatment
- modest reduction in expression of Rep and RepAe0
- Gene
- Rep and RepAe0
- Outcome
- plant leaf cell deathe0
- Direction
- reducese0
Not reportedComparator
Organisms pictured · Representative pictures of the matched species, not figures from the paper.
Verbatim
e0“a modest reduction in expression of Rep and RepA reduces plant leaf cell death”
t0“Nicotiana benthamiana”
Claim · model-written summary
Modestly reducing Rep and RepA expression reduced viral replicon accumulation.
- Taxon
- Nicotiana benthamianaNicotiana benthamianat0
- Treatment
- modest reduction in expression of Rep and RepAe0
- Outcome
- accumulation of viral repliconse0
- Direction
- reducinge0
Not reportedComparator
Organisms pictured · Representative pictures of the matched species, not figures from the paper.
Verbatim
e0“a modest reduction in expression of Rep and RepA reduces plant leaf cell death which, despite reducing the accumulation of viral replicons, increases target protein accumulation”
t0“Nicotiana benthamiana”
Claim · model-written summary
Modestly reducing Rep and RepA expression increased target protein accumulation.
- Taxon
- Nicotiana benthamianaNicotiana benthamianat0
- Treatment
- modest reduction in expression of Rep and RepAe0
- Outcome
- target protein accumulatione0
- Direction
- increasese0
Not reportedComparator
Organisms pictured · Representative pictures of the matched species, not figures from the paper.
Verbatim
e0“a modest reduction in expression of Rep and RepA reduces plant leaf cell death which, despite reducing the accumulation of viral replicons, increases target protein accumulation”
t0“Nicotiana benthamiana”
Claim · model-written summary
A single 5’ UTR nucleotide change reduced Rep/RepA expression.
- Taxon
- Nicotiana benthamianaNicotiana benthamianat0
- Treatment
- A single nucleotide change in the 5’ untranslated region (UTR)e0
- Outcome
- Rep/RepA expressione0
- Direction
- reducede0
Not reportedComparator
Organisms pictured · Representative pictures of the matched species, not figures from the paper.
Verbatim
e0“A single nucleotide change in the 5’ untranslated region (UTR) reduced Rep/RepA expression, reduced cell death, and enhanced the production of monoclonal antibodies”
t0“Nicotiana benthamiana”
Claim · model-written summary
A single 5’ UTR nucleotide change reduced cell death.
- Taxon
- Nicotiana benthamianaNicotiana benthamianat0
- Treatment
- A single nucleotide change in the 5’ untranslated region (UTR)e0
- Outcome
- cell deathe0
- Direction
- reducede0
Not reportedComparator
Organisms pictured · Representative pictures of the matched species, not figures from the paper.
Verbatim
e0“A single nucleotide change in the 5’ untranslated region (UTR) reduced Rep/RepA expression, reduced cell death, and enhanced the production of monoclonal antibodies”
t0“Nicotiana benthamiana”
Claim · model-written summary
A single 5’ UTR nucleotide change enhanced monoclonal antibody production.
- Taxon
- Nicotiana benthamianaNicotiana benthamianat0
- Treatment
- A single nucleotide change in the 5’ untranslated region (UTR)e0
- Outcome
- production of monoclonal antibodiese0
- Direction
- enhancede0
Not reportedComparator
Organisms pictured · Representative pictures of the matched species, not figures from the paper.
Verbatim
e0“A single nucleotide change in the 5’ untranslated region (UTR) reduced Rep/RepA expression, reduced cell death, and enhanced the production of monoclonal antibodies”
t0“Nicotiana benthamiana”
Claim · model-written summary
Replicating vectors achieved optimal expression at lower agrobacterium concentrations than nonreplicating vectors.
- Taxon
- Nicotiana benthamianaNicotiana benthamianat0
- Outcome
- optimal expressione0
- Direction
- lowere0
- Comparator
- nonreplicating vectorse0
Organisms pictured · Representative pictures of the matched species, not figures from the paper.
Verbatim
e0“replicating vectors achieve optimal expression with lower agrobacterium concentrations than nonreplicating vectors”
t0“Nicotiana benthamiana”
Claim · model-written summary
Lower agrobacterium concentrations further reduced cell death.
- Taxon
- Nicotiana benthamianaNicotiana benthamianat0
- Treatment
- lower agrobacterium concentrationse0
- Outcome
- cell deathe0
- Direction
- reducinge0
Not reportedComparator
Organisms pictured · Representative pictures of the matched species, not figures from the paper.
Verbatim
e0“replicating vectors achieve optimal expression with lower agrobacterium concentrations than nonreplicating vectors, further reducing cell death”
t0“Nicotiana benthamiana”
Claim · model-written summary
Viral UTRs contributed substantially to cell death.
- Taxon
- Nicotiana benthamianaNicotiana benthamianat0
- Gene
- Viral UTRse0
- Outcome
- cell deathe0
Organisms pictured · Representative pictures of the matched species, not figures from the paper.
Verbatim
e0“Viral UTRs are also shown to contribute substantially to cell death”
t0“Nicotiana benthamiana”
Claim · model-written summary
A native plant-derived 5’ UTR did not contribute to cell death.
- Taxon
- Nicotiana benthamianaNicotiana benthamianat0
- Gene
- native plant-derived 5’ UTRe1
- Outcome
- cell deathe1
Organisms pictured · Representative pictures of the matched species, not figures from the paper.
Verbatim
e0“a native plant-derived 5’ UTR does not”
e1“Viral UTRs are also shown to contribute substantially to cell death, while a native plant-derived 5’ UTR does not”
t0“Nicotiana benthamiana”
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