Review: Plant Binary Vectors of Ti Plasmid in <i>Agrobacterium tumefaciens</i> with a Broad Host-Range Replicon of pRK2, pRi, pSa or pVS1

Abstract

This review chronicles the development of the plant binary vectors of Ti plasmid in Agrobacterium tumefaciens during the last 30 years. A binary vector strategy was designed in 1983 to separate the T-DNA region in a small plasmid from the virulence genes in avirulent T-DNA-less Ti plasmid. The small plant vectors with the T-DNA region have been simply now called binary Ti vectors. A binary Ti vector consist of a broad host-range replicon for propagation in A. tumeraciens, an antibiotic resistance gene for bacterial selection and the T-DNA region that would be transferred to the plant genome via the bacterial virulence machinery. The T-DNA region delimited by the right and left border sequences contains an antibiotic resistance gene for plant selection, reporter gene, and/or any genes of interest. The ColEI replicon was also added to the plasmid backbone to enhance the propagation in Escherichia coli. A general trend in the binary vector development has been to increase the plasmid stability during a long co-cultivation period of A. tumefaciens with the target host plant tissues. A second trend is to understand the molecular mechanism of broad host-range replication, and to use it to reduce the size of plasmid for ease in cloning and for higher plasmid yield in E. coli. The broad host-range replicon of VS1 was shown to be a choice of replicon over those of pRK2, pRi and pSA because of the superior stability and of small well-defined replicon. Newly developed plant binary vectors pLSU has the small size of plasmid backbone (4566 bp) consisting of VS1 replicon (2654 bp), ColE1 replicon (715 bp), a bacterial kanamycin (999 bp) or tetracycline resistance gene, and the T-DNA region (152 bp).

Transient expression assays in grapevine: a step towards genetic improvement

“…These efficient and reliable cloning systems are well suited for highthroughput analysis of plant genes. Moreover, a large set of Gateway â -compatible destination vectors are available for many applications (Karimi et al, 2002;Murai, 2013). Binary vectors most often used for transient expression assays in grapevine are pBIN19 and derivative vectors (Le Henanff et al, 2009;Li et al, 2001 andLi et al, 2004;Santos-Rosa et al, 2008;Visser et al, 2012), pCambia Xu et al, 2010) and varied Gateway â destination vectors (Jelly et al, 2012;Li et al, 2011;Urso et al, 2013).…”

A Novel Ternary Vector System United with Morphogenic Genes Enhances CRISPR/Cas Delivery in Maize

“…In fact, the coexistence of pVS1-and pRK2derived binary vectors appeared to increase the stability of pRK2 derivatives (Murai, 2013). Second, pRK2 per se is more stable than pSa (Murai, 2013). Third, the copy number difference between pRK2 (approximately 10 copies per cell) and pVS1 (approximately 20 copies per cell) is much smaller than that between pSa (approximately two copies) and pVS1 .…”

“…First, pRK2 and pVS1 belong to different incompatibility groups, which means that they can stably coexist in the same cell. In fact, the coexistence of pVS1-and pRK2derived binary vectors appeared to increase the stability of pRK2 derivatives (Murai, 2013). Second, pRK2 per se is more stable than pSa (Murai, 2013).…”

“…2B), the propagation and stability of pGreen3 in Agrobacterium are able to be maintained. In addition, pVS1-derived vectors per se are very stable (Murai, 2013;Anand et al, 2018). Thus, the virulence helper pVS1-VIR2 and the pGreen3 binary vectors constitute a novel and stable ternary vector system.…”

“…The first-generation pGreen vectors were unstable in Escherichia coli and in Agrobacterium due to inefficient transcription termination of the kanamycin-resistance gene (KmR). Inserting the ampicillin-resistance gene terminator behind the KmR open reading frame improved the stability in E. coli of the modified plasmids, named pGreen2 (Hellens et al, 2000;Thole et al, 2007;Murai, 2013). The pGreen and pGreen2 vectors were based on the pSa replication system in Agrobacterium, whereas our Figure 2.…”