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β-1,3-glucanase class III promotes spread of PVY(NTN) and improves in planta protein production.

Dobnik D, Baebler S, Kogovšek P, Pompe-Novak M, Stebih D, Panter G, Janež N, Morisset D, Zel J, Gruden K - Plant Biotechnol Rep (2013)

Bottom Line: Differences in viral spread were observed between transgenic lines overexpressing Glu-III and non-transgenic lines, with stronger and faster viral spread in transgenic Désirée, and some multiplication in transgenic Santé.In addition, the ability of Glu-III to improve in planta protein production after agroinfiltration was tested.The results have shown that Glu-III overexpression enables faster spreading of vectors between cells and better protein production, which could be beneficial in improving in planta protein production system using viral vectors.

View Article: PubMed Central - PubMed

Affiliation: Department of Biotechnology and Systems Biology, National Institute of Biology, Večna pot 111, 1000 Ljubljana, Slovenia.

ABSTRACT
Glucanases are enzymes regulating the size exclusion limit and permeability of plasmodesmata and play a role in biotic stress. In plant genomes, they are encoded as relatively large gene families divided into four classes. Most studies of plant virus interactions have focused on glucanases from classes I and II. In our study, we have evaluated the role of the β-1,3-glucanase class III (Glu-III) gene in the potato-potato virus Y(NTN) (PVY(NTN)) interaction and implemented the findings to plant biotechnology application. Potato cultivars Désirée and Santé, which are tolerant and extremely resistant to PVY(NTN), respectively, were stably transformed with Agrobacterium tumefaciens harbouring constructs for Glu-III overexpression. Localization of Glu-III protein in patches within the cell wall was determined by tagging the Glu-III protein with green fluorescent protein. Transgenic and non-transgenic plants were challenged with PVY(NTN) and its multiplication and spreading was followed. Differences in viral spread were observed between transgenic lines overexpressing Glu-III and non-transgenic lines, with stronger and faster viral spread in transgenic Désirée, and some multiplication in transgenic Santé. In addition, the ability of Glu-III to improve in planta protein production after agroinfiltration was tested. The results have shown that Glu-III overexpression enables faster spreading of vectors between cells and better protein production, which could be beneficial in improving in planta protein production system using viral vectors.

No MeSH data available.


Related in: MedlinePlus

Glu-III overexpression increases production of heterologous protein by using virus based vectors. Area of GFP fluorescence (average fluorescence spot area) was determined in two separate experiments on the leaves of N. benthamiana agroinfiltrated with bacteria harboring the construct for GFP production alone or constructs for GFP and Glu-III production (a, b). Relative amount of NVCP (c) was determined in the leaves of N. benthamiana agroinfiltrated with bacteria harboring constructs for NVCP production alone or constructs for NVCP and Glu-III production. Error bars standard error. Asterisk statistically significant difference (p < 0.01)
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Fig3: Glu-III overexpression increases production of heterologous protein by using virus based vectors. Area of GFP fluorescence (average fluorescence spot area) was determined in two separate experiments on the leaves of N. benthamiana agroinfiltrated with bacteria harboring the construct for GFP production alone or constructs for GFP and Glu-III production (a, b). Relative amount of NVCP (c) was determined in the leaves of N. benthamiana agroinfiltrated with bacteria harboring constructs for NVCP production alone or constructs for NVCP and Glu-III production. Error bars standard error. Asterisk statistically significant difference (p < 0.01)

Mentions: To confirm that Glu-III overexpression and its presumed localization at Pd are actually responsible for the effect on viral spread observed in our experiments (Fig. 3), we tested the impact of Glu-III overexpression on the capacity of the plants for heterologous protein production, using PVX- and TMV-based viral vectors. The latter are organized as a set of pro-vectors that, after agroinfiltration, enable local and systemic production of heterologous protein (Marillonnet et al. 2004). Two model proteins were used to monitor the effect of Glu-III overexpression on heterologous protein production: GFP and Norovirus coat protein (NVCP) that is able to form virus-like particles (VLPs). The average area producing GFP was from 35 to 45 % higher in leaves infiltrated with the combination of pro-vectors for GFP and Glu-III production than under agroinfiltration with pro-vectors for GFP alone (p = 0.002) (Fig. 3a, b). The NVCP content was 36 % higher in leaves infiltrated with the combination of pro-vectors for NVCP and Glu-III production than in those agroinfiltrated with pro-vectors for NVCP alone (p = 0.0002) (Fig. 3c).Fig. 3


β-1,3-glucanase class III promotes spread of PVY(NTN) and improves in planta protein production.

Dobnik D, Baebler S, Kogovšek P, Pompe-Novak M, Stebih D, Panter G, Janež N, Morisset D, Zel J, Gruden K - Plant Biotechnol Rep (2013)

Glu-III overexpression increases production of heterologous protein by using virus based vectors. Area of GFP fluorescence (average fluorescence spot area) was determined in two separate experiments on the leaves of N. benthamiana agroinfiltrated with bacteria harboring the construct for GFP production alone or constructs for GFP and Glu-III production (a, b). Relative amount of NVCP (c) was determined in the leaves of N. benthamiana agroinfiltrated with bacteria harboring constructs for NVCP production alone or constructs for NVCP and Glu-III production. Error bars standard error. Asterisk statistically significant difference (p < 0.01)
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3824212&req=5

Fig3: Glu-III overexpression increases production of heterologous protein by using virus based vectors. Area of GFP fluorescence (average fluorescence spot area) was determined in two separate experiments on the leaves of N. benthamiana agroinfiltrated with bacteria harboring the construct for GFP production alone or constructs for GFP and Glu-III production (a, b). Relative amount of NVCP (c) was determined in the leaves of N. benthamiana agroinfiltrated with bacteria harboring constructs for NVCP production alone or constructs for NVCP and Glu-III production. Error bars standard error. Asterisk statistically significant difference (p < 0.01)
Mentions: To confirm that Glu-III overexpression and its presumed localization at Pd are actually responsible for the effect on viral spread observed in our experiments (Fig. 3), we tested the impact of Glu-III overexpression on the capacity of the plants for heterologous protein production, using PVX- and TMV-based viral vectors. The latter are organized as a set of pro-vectors that, after agroinfiltration, enable local and systemic production of heterologous protein (Marillonnet et al. 2004). Two model proteins were used to monitor the effect of Glu-III overexpression on heterologous protein production: GFP and Norovirus coat protein (NVCP) that is able to form virus-like particles (VLPs). The average area producing GFP was from 35 to 45 % higher in leaves infiltrated with the combination of pro-vectors for GFP and Glu-III production than under agroinfiltration with pro-vectors for GFP alone (p = 0.002) (Fig. 3a, b). The NVCP content was 36 % higher in leaves infiltrated with the combination of pro-vectors for NVCP and Glu-III production than in those agroinfiltrated with pro-vectors for NVCP alone (p = 0.0002) (Fig. 3c).Fig. 3

Bottom Line: Differences in viral spread were observed between transgenic lines overexpressing Glu-III and non-transgenic lines, with stronger and faster viral spread in transgenic Désirée, and some multiplication in transgenic Santé.In addition, the ability of Glu-III to improve in planta protein production after agroinfiltration was tested.The results have shown that Glu-III overexpression enables faster spreading of vectors between cells and better protein production, which could be beneficial in improving in planta protein production system using viral vectors.

View Article: PubMed Central - PubMed

Affiliation: Department of Biotechnology and Systems Biology, National Institute of Biology, Večna pot 111, 1000 Ljubljana, Slovenia.

ABSTRACT
Glucanases are enzymes regulating the size exclusion limit and permeability of plasmodesmata and play a role in biotic stress. In plant genomes, they are encoded as relatively large gene families divided into four classes. Most studies of plant virus interactions have focused on glucanases from classes I and II. In our study, we have evaluated the role of the β-1,3-glucanase class III (Glu-III) gene in the potato-potato virus Y(NTN) (PVY(NTN)) interaction and implemented the findings to plant biotechnology application. Potato cultivars Désirée and Santé, which are tolerant and extremely resistant to PVY(NTN), respectively, were stably transformed with Agrobacterium tumefaciens harbouring constructs for Glu-III overexpression. Localization of Glu-III protein in patches within the cell wall was determined by tagging the Glu-III protein with green fluorescent protein. Transgenic and non-transgenic plants were challenged with PVY(NTN) and its multiplication and spreading was followed. Differences in viral spread were observed between transgenic lines overexpressing Glu-III and non-transgenic lines, with stronger and faster viral spread in transgenic Désirée, and some multiplication in transgenic Santé. In addition, the ability of Glu-III to improve in planta protein production after agroinfiltration was tested. The results have shown that Glu-III overexpression enables faster spreading of vectors between cells and better protein production, which could be beneficial in improving in planta protein production system using viral vectors.

No MeSH data available.


Related in: MedlinePlus