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Agroinfiltration-based efficient transient protein expression in leguminous plants.

ABSTRACT: Transient protein expression is an effective tool to rapidly unravel novel gene functions, such as transcriptional activity of promoters and sub-cellular localization of proteins. However, transient expression is not applicable to some species and varieties because of insufficient expression levels. We recently developed one of the strongest agroinfiltration-based transient protein expression systems for plant cells, termed 'Tsukuba system.' About 4 mg/g fresh weight of protein expression in Nicotiana benthamiana was obtained using this system. The vector pBYR2HS, which contains a geminiviral replication system and a double terminator, can be used in various plant species and varieties, including lettuces, eggplants, tomatoes, hot peppers, and orchids. In this study, we assessed the applicability of the Tsukuba system to several species of legumes, including Lotus japonicus, soybean Glycine max, and common bean Phaseolus vulgaris. The GFP protein was transiently expressed in the seedpods of all examined legume species, however, protein expression in leaves was observed only in P. vulgaris. Taken together, our system is an effective tool to examine gene function rapidly in several legume species based on transient protein expression.

SUBMITTER: Suzaki T

PROVIDER: S-EPMC6847781 | biostudies-literature | 2019

REPOSITORIES: biostudies-literature

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Agroinfiltration-based efficient transient protein expression in leguminous plants.

Suzaki Takuya T Tsuda Mai M Ezura Hiroshi H Day Brad B Miura Kenji K

Plant biotechnology (Tokyo, Japan) 20190101 2

Transient protein expression is an effective tool to rapidly unravel novel gene functions, such as transcriptional activity of promoters and sub-cellular localization of proteins. However, transient expression is not applicable to some species and varieties because of insufficient expression levels. We recently developed one of the strongest agroinfiltration-based transient protein expression systems for plant cells, termed 'Tsukuba system.' About 4 mg/g fresh weight of protein expression in <i> ...[more]

PMID: 31768113

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Project description:BackgroundAgroinfiltration is a simple and effective method of delivering transgenes into plant cells for the rapid production of recombinant proteins and has become the preferred transient expression platform to manufacture biologics in plants. Despite its popularity, few studies have sought to improve the efficiency of agroinfiltration to further increase protein yields. This study aimed to increase agroinfiltration-based transient gene expression in Nicotiana benthamiana by improving all levels of transgenesis.ResultsUsing the benchmark pEAQ-HT deconstructed virus vector system and the GUS reporter enzyme, physical, chemical, and molecular features were independently assessed for their ability to enhance Agrobacterium-mediated transformation and improve protein production capacities. Optimal Agrobacterium strain, cell culture density and co-cultivation time for maximal transient GUS (β-glucuronidase) expression were established. The effects of chemical additives in the liquid infiltration media were investigated and acetosyringone (500 μM), the antioxidant lipoic acid (5 μM), and a surfactant Pluronic F-68 (0.002%) were all shown to significantly increase transgene expression. Gene products known to suppress post-transcriptional gene silencing, activate cell cycle progression and confer stress tolerance were also assessed by co-expression. A simple 37 °C heat shock to plants, 1-2 days post infiltration, was shown to dramatically increase GUS reporter levels. By combining the most effective features, a dual vector delivery system was developed that provided approximately 3.5-fold higher levels of absolute GUS protein compared to the pEAQ-HT platform.ConclusionsIn this paper, different strategies were assessed and optimised with the aim of increasing plant-made protein capacities in Nicotiana benthamiana using agroinfiltration. Chemical additives, heat shock and the co-expression of genes known to suppress stress and gene silencing or stimulate cell cycle progression were all proven to increase agroinfiltration-based transient gene expression. By combining the most effective of these elements a novel expression platform was developed capable of producing plant-made protein at a significantly higher level than a benchmark hyper-expression system.

Dataset Information

Agroinfiltration-based efficient transient protein expression in leguminous plants.

Publications

Agroinfiltration-based efficient transient protein expression in leguminous plants.

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