Expression and Purification of Heterologous Proteins in Plant Tissue Using a Geminivirus Vector System

Hefferon, Kathleen; Kipp, Peter; Moon, Yong-Sun

doi:10.1159/000078654

Cited by 9 publications

(4 citation statements)

References 49 publications

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“…GUS reporter gene expression in NGA cell culture increased 2.4‐fold when the REP‐supplying construct was used to complement the replication‐competent reporter construct. This is consistent with previous reports of geminivirus‐based vectors (11−13, 33, 34). Although the magnitude of the increase in expression is lower than in these previous reports, the basal protein yield is greater in the work presented here.…”

Section: Resultssupporting

confidence: 93%

Agrobacterium-Mediated Viral Vector-Amplified Transient Gene Expression in Nicotiana glutinosa Plant Tissue Culture

Collens

Mason

Curtis

2008

Biotechnol Progress

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A viral vector based on the bean yellow dwarf virus was investigated for its potential to increase transient gene expression. An intron-containing GUS reporter gene and the cis-acting viral regulatory elements were incorporated in the viral vector and could be complemented by the viral replication-associated proteins provided on a secondary vector. All vectors were delivered to Nicotiana glutinosa plant cell suspension or hairy root cultures via auxotrophic Agrobacterium tumefaciens. Cell culture generated greater yield of reporter gene expression than did root culture, as a result of the limitation imposed on roots to express the protein only in surface tissue containing actively dividing cells. Reporter gene expression increased for cell culture when the reporter gene construct was co-delivered with the construct supplying both viral replication associated proteins (REP and REPA); gene expression decreased when the construct supplying only the viral REP protein was co-delivered. Reporter protein expression increased from 0.091% for the reporter construct alone to 0.22% total soluble protein (% TSP) when the viral Rep-supplying vector was co-delivered with the reporter gene construct. Reporter protein was generated 3 days after the initiation of bacterial co-culture, providing for rapid generation of heterologous protein in cell culture.

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Section: Resultssupporting

confidence: 93%

Agrobacterium-Mediated Viral Vector-Amplified Transient Gene Expression in Nicotiana glutinosa Plant Tissue Culture

Collens

Mason

Curtis

2008

Biotechnol Progress

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“…Extrachromosomal amplification from geminivirus-based constructs has been exploited for the production of valuable peptides and proteins (28,43,51) or to analyze the function of Rep in replication (30).…”

Section: Tomato Yellow Leaf Curl Sardinia Virus (Tylcsv) or Tomato Yementioning

confidence: 99%

A Versatile Transreplication-Based System To Identify Cellular Proteins Involved in Geminivirus Replication

Morilla

Castillo

Preiß

et al. 2006

J Virol

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A versatile green fluorescent protein (GFP) expression cassette containing the replication origins of the monopartite begomovirus Tomato yellow leaf curl Sardinia virus (TYLCSV) is described. Transgenic Nicotiana benthamiana plants containing one copy of the cassette stably integrated into their genome were superinfected with TYLCSV, which mobilized and replicated the cassette as an episomal replicon. The expression of the reporter gene (the GFP gene) was thereby modified. Whereas GFP fluorescence was dimmed in the intercostal areas, an increase of green fluorescence in veins of all leaves placed above the inoculation site, as well as in transport tissues of roots and stems, was observed. The release of episomal trans replicons from the transgene and the increase in GFP expression were dependent on the cognate geminiviral replication-associated protein (Rep) and required interaction between Rep and the intergenic region of TYLCSV. This expression system is able to monitor the replication status of TYLCSV in plants, as induction of GFP expression is only produced in those tissues where Rep is present. To further confirm this notion, the expression of a host factor required for geminivirus replication, the proliferating cellular nuclear antigen (PCNA) was transiently silenced. Inhibition of PCNA prevented GFP induction in veins and reduced viral DNA. We propose that these plants could be widely used to easily identify host factors required for geminivirus replication by virus-induced gene silencing.Geminiviruses belong to a large family of plant viruses with circular, single-stranded DNA (ssDNA) genomes packaged within geminate particles (9). The Geminiviridae family (58) is divided into four genera according to their genome organization and biological properties. The genus Begomovirus includes members that are transmitted by whiteflies, infect only dicotyledonous plants, and may have bipartite (A and B components), like Tomato golden mosaic virus (TGMV), or monopartite genomes, like Tomato yellow leaf curl Sardinia virus (TYLCSV) or Tomato yellow leaf curl virus (TYLCV).Although among plant virus a minority, geminiviruses have a great economical impact, affecting many different crops worldwide (42). TYLCSV and TYLCV are two distinct species causing tomato yellow leaf curl disease, one of the most important threats to tomato crops in many tropical and subtropical regions of the world (45). Both viruses possess genomes of about 2.7 kb in size, which encode at least six proteins and contain an intergenic region (IR) that comprises the origin of replication and viral promoters. The open reading frames (ORFs) in the complementary sense orientation encoding a replication-associated protein (Rep, also known as AC1, AL1, C1, or L1), a transcription activator protein (TrAP, also known as AC2, AL2, C2, or L2), and a replication enhancer protein (REn) partially overlap; a small ORF, C4, is located within the Rep ORF but in a different reading frame (25). Rep and REn are required for efficient viral DNA replication, although...

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“…Future research should shed some light regarding the underlying molecular mechanisms behind CP expression of BeYDV. From a biotechnology perspective, such knowledge may serve as a powerful tool to enhance or direct the translation of foreign proteins in plants to more desirable levels within BeYDV‐based expression vector systems [46,47]. Such a system is currently being used to produce foreign proteins from a plant virus expression vector [48].…”

Section: Discussionmentioning

confidence: 99%

“…Tobacco protoplasts were prepared as described by Hefferon & Fan [47]. Protoplasts were electroporated with constructs p35SGFP, p35SRep–GFP, p35SRepA–GFP and pSKBYD1.4, and visualized under a Bio‐Rad MRC 600 confocal microscope adapted to a Nikon Optiphat microscope, with a ×40 Fluor.…”

Section: Methodsmentioning

confidence: 99%

Multi‐tasking of nonstructural gene products is required for bean yellow dwarf geminivirus transcriptional regulation

2006

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Geminiviruses belong to a family of plant viruses that can be classified into four distinct genera on the basis of genomic organization, vector transmissibility and host range. These include the mastreviruses, which possess monopartite genomes, are transmitted by leafhoppers and infect monocotyledonous plants. Exceptions to the rule are the Australian-derived tobacco yellow dwarf virus and South African-derived bean yellow dwarf virus (BeYDV), two distantly related mastreviruses that infect dicotyledenous plants [1].BeYDV consists of a single-stranded circular DNA molecule of 2.6 kb in length, and contains four ORFs encoding three different genes. The coding region is divided bidirectionally by long intergenic regions (LIR) and short intergenic regions (SIR). The MP and CP genes are expressed from the virion sensestrand, while the replication-associated protein (Rep) is produced from overlapping ORFs C1 and C2 from the complementary sense-strand. An intron spans the region overlapping C1 and C2 and this is spliced during Rep expression. Both Rep, which functions as the replication-associated protein, and RepA, the gene product of ORF C1, are produced during virus infection [1][2][3].

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Expression and Purification of Heterologous Proteins in Plant Tissue Using a Geminivirus Vector System

Cited by 9 publications

References 49 publications

Agrobacterium-Mediated Viral Vector-Amplified Transient Gene Expression in Nicotiana glutinosa Plant Tissue Culture

Agrobacterium-Mediated Viral Vector-Amplified Transient Gene Expression in Nicotiana glutinosa Plant Tissue Culture

A Versatile Transreplication-Based System To Identify Cellular Proteins Involved in Geminivirus Replication

Multi‐tasking of nonstructural gene products is required for bean yellow dwarf geminivirus transcriptional regulation

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