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Kazuhiro Fujiwara - One of the best experts on this subject based on the ideXlab platform.

  • removal of Bacterial Suspension water occupying the intercellular space of detached leaves after agroinfiltration improves the yield of recombinant hemagglutinin in a nicotiana benthamiana transient gene expression system
    Biotechnology and Bioengineering, 2016
    Co-Authors: Naomichi Fujiuchi, Nobuyuki Matoba, Ryo Matsuda, Kazuhiro Fujiwara
    Abstract:

    The use of detached leaves instead of whole plants provides an alternative means for recombinant protein production based on Agrobacterium tumefaciens-mediated transient gene overexpression. However, the process for high-level protein production in detached leaves has not yet been established. In this study, we focused on leaf handling and maintenance conditions immediately after infiltration with Agrobacterium Suspension (agroinfiltration) to improve recombinant protein expression in detached Nicotiana benthamiana leaves. We demonstrated that the residual water of Bacterial Suspension in detached leaves had significant impact on the yield of recombinant influenza hemagglutinin (HA). Immediately after agroinfiltration, detached leaves were stored in a dehumidified chamber to allow Bacterial Suspension water occupying intercellular space to be removed by transpiration. We varied the duration of this water removal treatment from 0.7 to 4.4 h, which resulted in leaf fresh weights ranging from 0.94 to 1.28 g g−1 relative to weights measured just before agroinfiltration. We used these relative fresh weights (RFWs) as an indicator of the amount of residual water. The detached leaves were then incubated in humidified chambers for 6 days. We found that the presence of residual water significantly decreased HA yield, with a clear inverse correlation observed between HA yield and RFW. We next compared HA yields in detached leaves with those obtained from intact leaves by whole-plant expression performed at the same time. The maximum HA yield obtained from a detached leaf with a RFW of approximately 1.0, namely, 800 μg gFW−1, was comparable to the mean HA yield of 846 μg gFW−1 generated in intact leaves. Our results indicate the necessity of removing Bacterial Suspension water from agroinfiltrated detached leaves in transient overexpression systems and point to a critical factor enabling the detached-leaf system as a viable recombinant protein factory. Biotechnol. Bioeng. 2015;9999: 1–6. © 2015 Wiley Periodicals, Inc.

  • removal of Bacterial Suspension water occupying the intercellular space of detached leaves after agroinfiltration improves the yield of recombinant hemagglutinin in a nicotiana benthamiana transient gene expression system
    Biotechnology and Bioengineering, 2016
    Co-Authors: Naomichi Fujiuchi, Nobuyuki Matoba, Ryo Matsuda, Kazuhiro Fujiwara
    Abstract:

    The use of detached leaves instead of whole plants provides an alternative means for recombinant protein production based on Agrobacterium tumefaciens-mediated transient gene overexpression. However, the process for high-level protein production in detached leaves has not yet been established. In this study, we focused on leaf handling and maintenance conditions immediately after infiltration with Agrobacterium Suspension (agroinfiltration) to improve recombinant protein expression in detached Nicotiana benthamiana leaves. We demonstrated that the residual water of Bacterial Suspension in detached leaves had significant impact on the yield of recombinant influenza hemagglutinin (HA). Immediately after agroinfiltration, detached leaves were stored in a dehumidified chamber to allow Bacterial Suspension water occupying intercellular space to be removed by transpiration. We varied the duration of this water removal treatment from 0.7 to 4.4 h, which resulted in leaf fresh weights ranging from 0.94 to 1.28 g g(-1) relative to weights measured just before agroinfiltration. We used these relative fresh weights (RFWs) as an indicator of the amount of residual water. The detached leaves were then incubated in humidified chambers for 6 days. We found that the presence of residual water significantly decreased HA yield, with a clear inverse correlation observed between HA yield and RFW. We next compared HA yields in detached leaves with those obtained from intact leaves by whole-plant expression performed at the same time. The maximum HA yield obtained from a detached leaf with a RFW of approximately 1.0, namely, 800 μg gFW(-1), was comparable to the mean HA yield of 846 μg gFW(-1) generated in intact leaves. Our results indicate the necessity of removing Bacterial Suspension water from agroinfiltrated detached leaves in transient overexpression systems and point to a critical factor enabling the detached-leaf system as a viable recombinant protein factory.

Naomichi Fujiuchi - One of the best experts on this subject based on the ideXlab platform.

  • removal of Bacterial Suspension water occupying the intercellular space of detached leaves after agroinfiltration improves the yield of recombinant hemagglutinin in a nicotiana benthamiana transient gene expression system
    Biotechnology and Bioengineering, 2016
    Co-Authors: Naomichi Fujiuchi, Nobuyuki Matoba, Ryo Matsuda, Kazuhiro Fujiwara
    Abstract:

    The use of detached leaves instead of whole plants provides an alternative means for recombinant protein production based on Agrobacterium tumefaciens-mediated transient gene overexpression. However, the process for high-level protein production in detached leaves has not yet been established. In this study, we focused on leaf handling and maintenance conditions immediately after infiltration with Agrobacterium Suspension (agroinfiltration) to improve recombinant protein expression in detached Nicotiana benthamiana leaves. We demonstrated that the residual water of Bacterial Suspension in detached leaves had significant impact on the yield of recombinant influenza hemagglutinin (HA). Immediately after agroinfiltration, detached leaves were stored in a dehumidified chamber to allow Bacterial Suspension water occupying intercellular space to be removed by transpiration. We varied the duration of this water removal treatment from 0.7 to 4.4 h, which resulted in leaf fresh weights ranging from 0.94 to 1.28 g g−1 relative to weights measured just before agroinfiltration. We used these relative fresh weights (RFWs) as an indicator of the amount of residual water. The detached leaves were then incubated in humidified chambers for 6 days. We found that the presence of residual water significantly decreased HA yield, with a clear inverse correlation observed between HA yield and RFW. We next compared HA yields in detached leaves with those obtained from intact leaves by whole-plant expression performed at the same time. The maximum HA yield obtained from a detached leaf with a RFW of approximately 1.0, namely, 800 μg gFW−1, was comparable to the mean HA yield of 846 μg gFW−1 generated in intact leaves. Our results indicate the necessity of removing Bacterial Suspension water from agroinfiltrated detached leaves in transient overexpression systems and point to a critical factor enabling the detached-leaf system as a viable recombinant protein factory. Biotechnol. Bioeng. 2015;9999: 1–6. © 2015 Wiley Periodicals, Inc.

  • removal of Bacterial Suspension water occupying the intercellular space of detached leaves after agroinfiltration improves the yield of recombinant hemagglutinin in a nicotiana benthamiana transient gene expression system
    Biotechnology and Bioengineering, 2016
    Co-Authors: Naomichi Fujiuchi, Nobuyuki Matoba, Ryo Matsuda, Kazuhiro Fujiwara
    Abstract:

    The use of detached leaves instead of whole plants provides an alternative means for recombinant protein production based on Agrobacterium tumefaciens-mediated transient gene overexpression. However, the process for high-level protein production in detached leaves has not yet been established. In this study, we focused on leaf handling and maintenance conditions immediately after infiltration with Agrobacterium Suspension (agroinfiltration) to improve recombinant protein expression in detached Nicotiana benthamiana leaves. We demonstrated that the residual water of Bacterial Suspension in detached leaves had significant impact on the yield of recombinant influenza hemagglutinin (HA). Immediately after agroinfiltration, detached leaves were stored in a dehumidified chamber to allow Bacterial Suspension water occupying intercellular space to be removed by transpiration. We varied the duration of this water removal treatment from 0.7 to 4.4 h, which resulted in leaf fresh weights ranging from 0.94 to 1.28 g g(-1) relative to weights measured just before agroinfiltration. We used these relative fresh weights (RFWs) as an indicator of the amount of residual water. The detached leaves were then incubated in humidified chambers for 6 days. We found that the presence of residual water significantly decreased HA yield, with a clear inverse correlation observed between HA yield and RFW. We next compared HA yields in detached leaves with those obtained from intact leaves by whole-plant expression performed at the same time. The maximum HA yield obtained from a detached leaf with a RFW of approximately 1.0, namely, 800 μg gFW(-1), was comparable to the mean HA yield of 846 μg gFW(-1) generated in intact leaves. Our results indicate the necessity of removing Bacterial Suspension water from agroinfiltrated detached leaves in transient overexpression systems and point to a critical factor enabling the detached-leaf system as a viable recombinant protein factory.

Ryo Matsuda - One of the best experts on this subject based on the ideXlab platform.

  • removal of Bacterial Suspension water occupying the intercellular space of detached leaves after agroinfiltration improves the yield of recombinant hemagglutinin in a nicotiana benthamiana transient gene expression system
    Biotechnology and Bioengineering, 2016
    Co-Authors: Naomichi Fujiuchi, Nobuyuki Matoba, Ryo Matsuda, Kazuhiro Fujiwara
    Abstract:

    The use of detached leaves instead of whole plants provides an alternative means for recombinant protein production based on Agrobacterium tumefaciens-mediated transient gene overexpression. However, the process for high-level protein production in detached leaves has not yet been established. In this study, we focused on leaf handling and maintenance conditions immediately after infiltration with Agrobacterium Suspension (agroinfiltration) to improve recombinant protein expression in detached Nicotiana benthamiana leaves. We demonstrated that the residual water of Bacterial Suspension in detached leaves had significant impact on the yield of recombinant influenza hemagglutinin (HA). Immediately after agroinfiltration, detached leaves were stored in a dehumidified chamber to allow Bacterial Suspension water occupying intercellular space to be removed by transpiration. We varied the duration of this water removal treatment from 0.7 to 4.4 h, which resulted in leaf fresh weights ranging from 0.94 to 1.28 g g−1 relative to weights measured just before agroinfiltration. We used these relative fresh weights (RFWs) as an indicator of the amount of residual water. The detached leaves were then incubated in humidified chambers for 6 days. We found that the presence of residual water significantly decreased HA yield, with a clear inverse correlation observed between HA yield and RFW. We next compared HA yields in detached leaves with those obtained from intact leaves by whole-plant expression performed at the same time. The maximum HA yield obtained from a detached leaf with a RFW of approximately 1.0, namely, 800 μg gFW−1, was comparable to the mean HA yield of 846 μg gFW−1 generated in intact leaves. Our results indicate the necessity of removing Bacterial Suspension water from agroinfiltrated detached leaves in transient overexpression systems and point to a critical factor enabling the detached-leaf system as a viable recombinant protein factory. Biotechnol. Bioeng. 2015;9999: 1–6. © 2015 Wiley Periodicals, Inc.

  • removal of Bacterial Suspension water occupying the intercellular space of detached leaves after agroinfiltration improves the yield of recombinant hemagglutinin in a nicotiana benthamiana transient gene expression system
    Biotechnology and Bioengineering, 2016
    Co-Authors: Naomichi Fujiuchi, Nobuyuki Matoba, Ryo Matsuda, Kazuhiro Fujiwara
    Abstract:

    The use of detached leaves instead of whole plants provides an alternative means for recombinant protein production based on Agrobacterium tumefaciens-mediated transient gene overexpression. However, the process for high-level protein production in detached leaves has not yet been established. In this study, we focused on leaf handling and maintenance conditions immediately after infiltration with Agrobacterium Suspension (agroinfiltration) to improve recombinant protein expression in detached Nicotiana benthamiana leaves. We demonstrated that the residual water of Bacterial Suspension in detached leaves had significant impact on the yield of recombinant influenza hemagglutinin (HA). Immediately after agroinfiltration, detached leaves were stored in a dehumidified chamber to allow Bacterial Suspension water occupying intercellular space to be removed by transpiration. We varied the duration of this water removal treatment from 0.7 to 4.4 h, which resulted in leaf fresh weights ranging from 0.94 to 1.28 g g(-1) relative to weights measured just before agroinfiltration. We used these relative fresh weights (RFWs) as an indicator of the amount of residual water. The detached leaves were then incubated in humidified chambers for 6 days. We found that the presence of residual water significantly decreased HA yield, with a clear inverse correlation observed between HA yield and RFW. We next compared HA yields in detached leaves with those obtained from intact leaves by whole-plant expression performed at the same time. The maximum HA yield obtained from a detached leaf with a RFW of approximately 1.0, namely, 800 μg gFW(-1), was comparable to the mean HA yield of 846 μg gFW(-1) generated in intact leaves. Our results indicate the necessity of removing Bacterial Suspension water from agroinfiltrated detached leaves in transient overexpression systems and point to a critical factor enabling the detached-leaf system as a viable recombinant protein factory.

Nobuyuki Matoba - One of the best experts on this subject based on the ideXlab platform.

  • removal of Bacterial Suspension water occupying the intercellular space of detached leaves after agroinfiltration improves the yield of recombinant hemagglutinin in a nicotiana benthamiana transient gene expression system
    Biotechnology and Bioengineering, 2016
    Co-Authors: Naomichi Fujiuchi, Nobuyuki Matoba, Ryo Matsuda, Kazuhiro Fujiwara
    Abstract:

    The use of detached leaves instead of whole plants provides an alternative means for recombinant protein production based on Agrobacterium tumefaciens-mediated transient gene overexpression. However, the process for high-level protein production in detached leaves has not yet been established. In this study, we focused on leaf handling and maintenance conditions immediately after infiltration with Agrobacterium Suspension (agroinfiltration) to improve recombinant protein expression in detached Nicotiana benthamiana leaves. We demonstrated that the residual water of Bacterial Suspension in detached leaves had significant impact on the yield of recombinant influenza hemagglutinin (HA). Immediately after agroinfiltration, detached leaves were stored in a dehumidified chamber to allow Bacterial Suspension water occupying intercellular space to be removed by transpiration. We varied the duration of this water removal treatment from 0.7 to 4.4 h, which resulted in leaf fresh weights ranging from 0.94 to 1.28 g g−1 relative to weights measured just before agroinfiltration. We used these relative fresh weights (RFWs) as an indicator of the amount of residual water. The detached leaves were then incubated in humidified chambers for 6 days. We found that the presence of residual water significantly decreased HA yield, with a clear inverse correlation observed between HA yield and RFW. We next compared HA yields in detached leaves with those obtained from intact leaves by whole-plant expression performed at the same time. The maximum HA yield obtained from a detached leaf with a RFW of approximately 1.0, namely, 800 μg gFW−1, was comparable to the mean HA yield of 846 μg gFW−1 generated in intact leaves. Our results indicate the necessity of removing Bacterial Suspension water from agroinfiltrated detached leaves in transient overexpression systems and point to a critical factor enabling the detached-leaf system as a viable recombinant protein factory. Biotechnol. Bioeng. 2015;9999: 1–6. © 2015 Wiley Periodicals, Inc.

  • removal of Bacterial Suspension water occupying the intercellular space of detached leaves after agroinfiltration improves the yield of recombinant hemagglutinin in a nicotiana benthamiana transient gene expression system
    Biotechnology and Bioengineering, 2016
    Co-Authors: Naomichi Fujiuchi, Nobuyuki Matoba, Ryo Matsuda, Kazuhiro Fujiwara
    Abstract:

    The use of detached leaves instead of whole plants provides an alternative means for recombinant protein production based on Agrobacterium tumefaciens-mediated transient gene overexpression. However, the process for high-level protein production in detached leaves has not yet been established. In this study, we focused on leaf handling and maintenance conditions immediately after infiltration with Agrobacterium Suspension (agroinfiltration) to improve recombinant protein expression in detached Nicotiana benthamiana leaves. We demonstrated that the residual water of Bacterial Suspension in detached leaves had significant impact on the yield of recombinant influenza hemagglutinin (HA). Immediately after agroinfiltration, detached leaves were stored in a dehumidified chamber to allow Bacterial Suspension water occupying intercellular space to be removed by transpiration. We varied the duration of this water removal treatment from 0.7 to 4.4 h, which resulted in leaf fresh weights ranging from 0.94 to 1.28 g g(-1) relative to weights measured just before agroinfiltration. We used these relative fresh weights (RFWs) as an indicator of the amount of residual water. The detached leaves were then incubated in humidified chambers for 6 days. We found that the presence of residual water significantly decreased HA yield, with a clear inverse correlation observed between HA yield and RFW. We next compared HA yields in detached leaves with those obtained from intact leaves by whole-plant expression performed at the same time. The maximum HA yield obtained from a detached leaf with a RFW of approximately 1.0, namely, 800 μg gFW(-1), was comparable to the mean HA yield of 846 μg gFW(-1) generated in intact leaves. Our results indicate the necessity of removing Bacterial Suspension water from agroinfiltrated detached leaves in transient overexpression systems and point to a critical factor enabling the detached-leaf system as a viable recombinant protein factory.

C Speers - One of the best experts on this subject based on the ideXlab platform.

  • first report of goss s Bacterial wilt and leaf blight on corn caused by clavibacter michiganensis subsp nebraskensis in indiana
    Plant Disease, 2009
    Co-Authors: G Ruhl, Kiersten A Wise, T Creswell, A Leonberger, C Speers
    Abstract:

    In August of 2008, leaves of hybrid corn (Zea mays L.) and popcorn from Pulaski and Jasper counties in northwest Indiana were submitted to the Purdue Plant and Pest Diagnostic Lab with symptoms characteristic of a Bacterial disease. Symptomatic leaves had large, tan-to-gray necrotic lesions with dark freckling present within the lesions. Shiny Bacterial exudate was present on the surface of many of the lesions. Microscopic observation revealed no fungal structures within the lesions, and Bacterial streaming was observed from the cut edge of symptomatic tissue under ×100 magnification with phase contrast. A commercially available ELISA test (Agdia Inc., Elkhart, IN) determined that samples were negative for Pantoea stewartii, the causal agent of Stewart's Bacterial leaf blight and wilt. A Bacterial Suspension was prepared from symptomatic tissue and streaked onto King's B medium and subcultured on semiselective CNS medium (1,2). Axenic, peach-colored colonies present on the CNS medium tested gram positive with a KOH test. Analysis of fatty acid methyl esters (MIDI Inc, Newark, DE) indicated that the strain was very similar (0.611) to Clavibacter michiganensis. Amplification of the 500-bp 16S rRNA region of the Bacterial gene and subsequent BLAST alignments of the resulting sequence indicated a 99% match for C. michiganensis subsp. nebraskensis (GenBank Accession Nos. AM410697 and U09763; D16S2 gene Bacterial library, version 2.10; MIDI Inc,). Koch's postulates were used to confirm pathogenicity of the isolated bacteria on corn inbred B73. Eighteen plants were mechanically inoculated at growth stage V1 to V2 with a Bacterial Suspension of approximately 1 × 108 CFU/ml prepared from cultures grown on CNS for 10 days at 28°C (2). Inoculum was rubbed onto leaves dusted with Carborundum and 0.1 ml of the Bacterial Suspension was injected into stems with a hypodermic needle. Nine control plants were inoculated with sterile water. Plants were kept at greenhouse conditions (24°C) with supplemental 400W high-pressure sodium light. Within 5 to 8 days, leaves and stems of all 18 inoculated plants developed water-soaked, necrotic lesions. No symptoms were observed in control plants. Bacteria were reisolated from symptomatic plants on CNS medium as described above, and gram-positive colonies were obtained. Reisolated strains were identical to C. michiganensis subsp. nebraskensis by D16S2 DNA sequence analysis, confirming the causal agent of the disease. Disease incidence in affected fields ranged from 20 to 60% and significant yield loss was reported. This confirmation is of regulatory importance because of potential export restrictions of Indiana-grown seed corn and popcorn to select countries. To our knowledge, this is the first report of Goss's Bacterial wilt and leaf blight on corn in Indiana. References: (1) D. C. Gross and A. K. Vidaver. Phytopathology 69:82, 1979. (2) L. M. Shepherd. M.S. thesis. Iowa State University. Ames, 1999.