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RNA viral vectors for improved Agrobacterium-mediated transient expression of heterologous proteins in Nicotiana benthamiana cell suspensions and hairy roots.

Larsen JS, Curtis WR - BMC Biotechnol. (2012)

Bottom Line: In hairy roots, a TRV vector capable of systemic movement increased GUS accumulation by 150-fold relative to the analogous PVX vector.For the first time, replicating PVX vectors and a non-replicating CPMV-HT vector were successfully applied toward transient heterologous protein expression in cell suspensions.A replicating TRV vector achieved transient GUS expression levels in hairy roots more than an order of magnitude higher than the highest level previously reported with a viral vector delivered by A. tumefaciens.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Chemical Engineering, The Pennsylvania State University, University Park, Pennsylvania 16802, USA.

ABSTRACT

Background: Plant cell suspensions and hairy root cultures represent scalable protein expression platforms. Low protein product titers have thus far limited the application of transient protein expression in these hosts. The objective of this work was to overcome this limitation by harnessing A. tumefaciens to deliver replicating and non-replicating RNA viral vectors in plant tissue co-cultures.

Results: Replicating vectors derived from Potato virus X (PVX) and Tobacco rattle virus (TRV) were modified to contain the reporter gene β-glucuronidase (GUS) with a plant intron to prevent bacterial expression. In cell suspensions, a minimal PVX vector retaining only the viral RNA polymerase gene yielded 6.6-fold more GUS than an analogous full-length PVX vector. Transient co-expression of the minimal PVX vector with P19 of Tomato bushy stunt virus or HC-Pro of Tobacco etch virus to suppress post-transcriptional gene silencing increased GUS expression by 44 and 83%, respectively. A non-replicating vector containing a leader sequence from Cowpea mosaic virus (CPMV-HT) modified for enhanced translation led to 70% higher transient GUS expression than a control treatment. In hairy roots, a TRV vector capable of systemic movement increased GUS accumulation by 150-fold relative to the analogous PVX vector. Histochemical staining for GUS in TRV-infected hairy roots revealed the capacity for achieving even higher productivity per unit biomass.

Conclusions: For the first time, replicating PVX vectors and a non-replicating CPMV-HT vector were successfully applied toward transient heterologous protein expression in cell suspensions. A replicating TRV vector achieved transient GUS expression levels in hairy roots more than an order of magnitude higher than the highest level previously reported with a viral vector delivered by A. tumefaciens.

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Schematic representation of the vector T-DNA regions. Control elements are illustrated in black boxes and coding sequences are shown in white or blue boxes. Overhead arrows represent duplicated subgenomic coat protein promoters driving expression of the inserted transgene. All vectors contain a backbone sequence derived from pBIN19 except for the PVX vectors, which are based on pGreen0000. 35S, dual enhanced Cauliflower mosaic virus (CaMV) 35S promoter; 35ST, CaMV 35S terminator; TEV 5’, Tobacco etch virus 5’ untranslated region; CPMV 5’, Cowpea mosaic virus 5’ untranslated region modified with mutations A115G and U162C; LB and RB, left and right T-DNA borders; RdRp, RNA-dependent RNA polymerase; TGB, triple gene block; CP, coat protein gene; Rz, self-cleaving ribozyme; NOST, nopaline synthase terminator; MP, movement protein gene; VSPT, soybean vegetative storage protein B 3’ untranslated region; NPTII, expression cassette conferring kanamycin resistance; LIR and SIR, long and short intergenic regions of the Bean yellow dwarf virus genome; C1/C2, Bean yellow dwarf virus open reading frames encoding replication initiation proteins REP and REPA.
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Figure 1: Schematic representation of the vector T-DNA regions. Control elements are illustrated in black boxes and coding sequences are shown in white or blue boxes. Overhead arrows represent duplicated subgenomic coat protein promoters driving expression of the inserted transgene. All vectors contain a backbone sequence derived from pBIN19 except for the PVX vectors, which are based on pGreen0000. 35S, dual enhanced Cauliflower mosaic virus (CaMV) 35S promoter; 35ST, CaMV 35S terminator; TEV 5’, Tobacco etch virus 5’ untranslated region; CPMV 5’, Cowpea mosaic virus 5’ untranslated region modified with mutations A115G and U162C; LB and RB, left and right T-DNA borders; RdRp, RNA-dependent RNA polymerase; TGB, triple gene block; CP, coat protein gene; Rz, self-cleaving ribozyme; NOST, nopaline synthase terminator; MP, movement protein gene; VSPT, soybean vegetative storage protein B 3’ untranslated region; NPTII, expression cassette conferring kanamycin resistance; LIR and SIR, long and short intergenic regions of the Bean yellow dwarf virus genome; C1/C2, Bean yellow dwarf virus open reading frames encoding replication initiation proteins REP and REPA.

Mentions: The constructs used in this study are summarized in Figure 1. The GUS reporter consists of the uidA gene modified by the insertion of the potato PIV2 intron to prevent bacterial expression as described previously for the construction of pBY031-I1 and pGPTVK-GI [5]. Binary vectors pPSP19 and pRep110 contain the Tobacco bushy stunt virus 19 kDa gene product (P19) and the native Bean yellow dwarf virus replication proteins, respectively [2]. Vector pHCPro contains the TEV P1/HC-Pro polyprotein as a HindIII fragment containing the dual 35S expression cassette from pRTL2-0027 in the pGA482 binary vector [33].


RNA viral vectors for improved Agrobacterium-mediated transient expression of heterologous proteins in Nicotiana benthamiana cell suspensions and hairy roots.

Larsen JS, Curtis WR - BMC Biotechnol. (2012)

Schematic representation of the vector T-DNA regions. Control elements are illustrated in black boxes and coding sequences are shown in white or blue boxes. Overhead arrows represent duplicated subgenomic coat protein promoters driving expression of the inserted transgene. All vectors contain a backbone sequence derived from pBIN19 except for the PVX vectors, which are based on pGreen0000. 35S, dual enhanced Cauliflower mosaic virus (CaMV) 35S promoter; 35ST, CaMV 35S terminator; TEV 5’, Tobacco etch virus 5’ untranslated region; CPMV 5’, Cowpea mosaic virus 5’ untranslated region modified with mutations A115G and U162C; LB and RB, left and right T-DNA borders; RdRp, RNA-dependent RNA polymerase; TGB, triple gene block; CP, coat protein gene; Rz, self-cleaving ribozyme; NOST, nopaline synthase terminator; MP, movement protein gene; VSPT, soybean vegetative storage protein B 3’ untranslated region; NPTII, expression cassette conferring kanamycin resistance; LIR and SIR, long and short intergenic regions of the Bean yellow dwarf virus genome; C1/C2, Bean yellow dwarf virus open reading frames encoding replication initiation proteins REP and REPA.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3403893&req=5

Figure 1: Schematic representation of the vector T-DNA regions. Control elements are illustrated in black boxes and coding sequences are shown in white or blue boxes. Overhead arrows represent duplicated subgenomic coat protein promoters driving expression of the inserted transgene. All vectors contain a backbone sequence derived from pBIN19 except for the PVX vectors, which are based on pGreen0000. 35S, dual enhanced Cauliflower mosaic virus (CaMV) 35S promoter; 35ST, CaMV 35S terminator; TEV 5’, Tobacco etch virus 5’ untranslated region; CPMV 5’, Cowpea mosaic virus 5’ untranslated region modified with mutations A115G and U162C; LB and RB, left and right T-DNA borders; RdRp, RNA-dependent RNA polymerase; TGB, triple gene block; CP, coat protein gene; Rz, self-cleaving ribozyme; NOST, nopaline synthase terminator; MP, movement protein gene; VSPT, soybean vegetative storage protein B 3’ untranslated region; NPTII, expression cassette conferring kanamycin resistance; LIR and SIR, long and short intergenic regions of the Bean yellow dwarf virus genome; C1/C2, Bean yellow dwarf virus open reading frames encoding replication initiation proteins REP and REPA.
Mentions: The constructs used in this study are summarized in Figure 1. The GUS reporter consists of the uidA gene modified by the insertion of the potato PIV2 intron to prevent bacterial expression as described previously for the construction of pBY031-I1 and pGPTVK-GI [5]. Binary vectors pPSP19 and pRep110 contain the Tobacco bushy stunt virus 19 kDa gene product (P19) and the native Bean yellow dwarf virus replication proteins, respectively [2]. Vector pHCPro contains the TEV P1/HC-Pro polyprotein as a HindIII fragment containing the dual 35S expression cassette from pRTL2-0027 in the pGA482 binary vector [33].

Bottom Line: In hairy roots, a TRV vector capable of systemic movement increased GUS accumulation by 150-fold relative to the analogous PVX vector.For the first time, replicating PVX vectors and a non-replicating CPMV-HT vector were successfully applied toward transient heterologous protein expression in cell suspensions.A replicating TRV vector achieved transient GUS expression levels in hairy roots more than an order of magnitude higher than the highest level previously reported with a viral vector delivered by A. tumefaciens.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Chemical Engineering, The Pennsylvania State University, University Park, Pennsylvania 16802, USA.

ABSTRACT

Background: Plant cell suspensions and hairy root cultures represent scalable protein expression platforms. Low protein product titers have thus far limited the application of transient protein expression in these hosts. The objective of this work was to overcome this limitation by harnessing A. tumefaciens to deliver replicating and non-replicating RNA viral vectors in plant tissue co-cultures.

Results: Replicating vectors derived from Potato virus X (PVX) and Tobacco rattle virus (TRV) were modified to contain the reporter gene β-glucuronidase (GUS) with a plant intron to prevent bacterial expression. In cell suspensions, a minimal PVX vector retaining only the viral RNA polymerase gene yielded 6.6-fold more GUS than an analogous full-length PVX vector. Transient co-expression of the minimal PVX vector with P19 of Tomato bushy stunt virus or HC-Pro of Tobacco etch virus to suppress post-transcriptional gene silencing increased GUS expression by 44 and 83%, respectively. A non-replicating vector containing a leader sequence from Cowpea mosaic virus (CPMV-HT) modified for enhanced translation led to 70% higher transient GUS expression than a control treatment. In hairy roots, a TRV vector capable of systemic movement increased GUS accumulation by 150-fold relative to the analogous PVX vector. Histochemical staining for GUS in TRV-infected hairy roots revealed the capacity for achieving even higher productivity per unit biomass.

Conclusions: For the first time, replicating PVX vectors and a non-replicating CPMV-HT vector were successfully applied toward transient heterologous protein expression in cell suspensions. A replicating TRV vector achieved transient GUS expression levels in hairy roots more than an order of magnitude higher than the highest level previously reported with a viral vector delivered by A. tumefaciens.

Show MeSH
Related in: MedlinePlus