Identification and Characterization of Plant Genes Involved in<i>Agrobacterium</i>-Mediated Plant Transformation by Virus-Induced Gene Silencing

Anand, Ajith; Vaghchhipawala, Zarir; Ryu, Choong‐Min; Li, Kang; Wang, Keri; Pozo, Olga del; Martin, Gregory B.; Mysore, Kirankumar S.

doi:10.1094/mpmi-20-0041

Cited by 78 publications

(125 citation statements)

References 67 publications

Supporting

Mentioning

118

Contrasting

Unclassified

Order By: Relevance

“…WT, the wild type. results were confirmed by quantifying the relative amount of T-DNA integrated into the genome by qPCR as described (Anand et al, 2007a(Anand et al, , 2007b. The amount of PCR products specific to the uidA gene, determined by qPCR, was two-to fourfold more in XRCC4 RNAi lines and threefold less in overexpressor lines compared with Col-0 plants ( Figure 4D).…”

Section: Xrcc4 Downregulated and Overexpressing Arabidopsis Lines Arementioning

confidence: 60%

“…To investigate the role of XRCC4 in Agrobacterium-mediated plant transformation, we used a well-established virus-induced gene silencing (VIGS)-based reverse genetics system (Anand et al, 2007a(Anand et al, , 2007b(Anand et al, , 2008. A partial cDNA of N. benthamiana XRCC4 (see Supplemental Figure 1A online) cloned into the vector pTRV2 was used to silence the XRCC4 gene in N. benthamiana plants.…”

Section: Resultsmentioning

confidence: 99%

“…The T-DNA integration assay was performed as described previously (Mysore et al, 2000a;Anand et al, 2007b;Li et al, 2005) on the genomic DNA extracted from suspension cell lines generated from the calli produced on nonselective CIM by leaf disks of N. benthamiana control and Nb-XRCC4 silenced lines or Arabidopsis root segments of Col-0, XRCC4 RNAi lines (XR4i-3, XR4i-25, and XR4i-31), and overexpressing lines (XR4OE-3 and XR4OE-4) infected with either disarmed strain Agrobacterium GV2260 (for N. benthamiana) or GV3101 (for Arabidopsis) carrying pBISN1.…”

Section: T-dna Integration and Qpcr Assaysmentioning

confidence: 99%

“…The intensity of the signals was quantified using the LabWorks Image Acquisition and Analysis Software (UVP). Quantification of integrated T-DNA was also performed by qPCR as outlined (Anand et al, 2007b) using uidA (59-ATGAAGATGCGGACTTA-CGTGGCA-39 and 59-ATCTGCCCAGTCGAGCATCTCTTC-39) and EF-1a (59-TTCACCCTTGGTGTCAAGCA-39 and 59-TTTCATCGTACCTGGCCTTG-CA-39) primers. Normalization of templates was performed using EF-1a amplification levels.…”

Section: T-dna Integration and Qpcr Assaysmentioning

confidence: 99%

See 3 more Smart Citations

Agrobacterium May Delay Plant Nonhomologous End-Joining DNA Repair via XRCC4 to Favor T-DNA Integration

et al. 2012

Self Cite

View full text Add to dashboard Cite

Agrobacterium tumefaciens is a soilborne pathogen that causes crown gall disease in many dicotyledonous plants by transfer of a portion of its tumor-inducing plasmid (T-DNA) into the plant genome. Several plant factors that play a role in Agrobacterium attachment to plant cells and transport of T-DNA to the nucleus have been identified, but the T-DNA integration step during transformation is poorly understood and has been proposed to occur via nonhomologous end-joining (NHEJ)-mediated doublestrand DNA break (DSB) repair. Here, we report a negative role of X-RAY CROSS COMPLEMENTATION GROUP4 (XRCC4), one of the key proteins required for NHEJ, in Agrobacterium T-DNA integration. Downregulation of XRCC4 in Arabidopsis and Nicotiana benthamiana increased stable transformation due to increased T-DNA integration. Overexpression of XRCC4 in Arabidopsis decreased stable transformation due to decreased T-DNA integration. Interestingly, XRCC4 directly interacted with Agrobacterium protein VirE2 in a yeast two-hybrid system and in planta. VirE2-expressing Arabidopsis plants were more susceptible to the DNA damaging chemical bleomycin and showed increased stable transformation. We hypothesize that VirE2 titrates or excludes active XRCC4 protein available for DSB repair, thus delaying the closure of DSBs in the chromosome, providing greater opportunity for T-DNA to integrate.

show abstract

Section: Xrcc4 Downregulated and Overexpressing Arabidopsis Lines Arementioning

confidence: 60%

Section: Resultsmentioning

confidence: 99%

Section: T-dna Integration and Qpcr Assaysmentioning

confidence: 99%

Section: T-dna Integration and Qpcr Assaysmentioning

confidence: 99%

See 2 more Smart Citations

Agrobacterium May Delay Plant Nonhomologous End-Joining DNA Repair via XRCC4 to Favor T-DNA Integration

et al. 2012

Self Cite

View full text Add to dashboard Cite

show abstract

“…It has been suggested that VIP1 (VirE2 interacting protein 1) protein can target T-DNA to host plant chromatin by interaction with histones (Li et al, 2005a;Loyter et al, 2005). Using viralinduced gene silencing, Anand et al (2007) showed that decreased expression of some Nicotiana benthamiana chromatin genes could inhibit subsequent Agrobacterium-mediated transformation. In particular, blocking expression of genes encoding histone H2A and H3 decreased transformation.…”

mentioning

confidence: 99%

Overexpression of SeveralArabidopsisHistone Genes IncreasesAgrobacterium-Mediated Transformation and Transgene Expression in Plants

et al. 2009

View full text Add to dashboard Cite

The Arabidopsis thaliana histone H2A-1 is important for Agrobacterium tumefaciens-mediated plant transformation. Mutation of HTA1, the gene encoding histone H2A-1, results in decreased T-DNA integration into the genome of Arabidopsis roots, whereas overexpression of HTA1 increases transformation frequency. To understand the mechanism by which HTA1 enhances transformation, we investigated the effects of overexpression of numerous Arabidopsis histones on transformation and transgene expression. Transgenic Arabidopsis containing cDNAs encoding histone H2A (HTA), histone H4 (HFO), and histone H3-11 (HTR11) displayed increased transformation susceptibility, whereas histone H2B (HTB) and most histone H3 (HTR) cDNAs did not increase transformation. A parallel increase in transient gene expression was observed when histone HTA, HFO, or HTR11 overexpression constructs were cotransfected with double-or single-stranded forms of a gusA gene into tobacco (Nicotiana tabacum) protoplasts. However, these cDNAs did not increase expression of a previously integrated transgene. We identified the N-terminal 39 amino acids of H2A-1 as sufficient to increase transient transgene expression in plants. After transfection, transgene DNA accumulates more rapidly in the presence of HTA1 than with a control construction. Our results suggest that certain histones enhance transgene expression, protect incoming transgene DNA during the initial stages of transformation, and subsequently increase the efficiency of Agrobacterium-mediated transformation. INTRODUCTIONAgrobacterium tumefaciens-mediated plant genetic transformation is an important experimental tool for investigation of various aspects of plant biology and for agricultural biotechnology. Development of this transformation technology represents the culmination of many decades of effort to improve tissue culture and plant genetic engineering techniques. Agrobacteriummediated transformation is based on a conjugative transfer-like process, which eventually takes the T-DNA to the host cell nucleus (Gelvin, 2000(Gelvin, , 2003a(Gelvin, , 2009Tzfira and Citovsky, 2003;Citovsky et al., 2007). After attachment of the bacterium to plant cells and induction of Agrobacterium virulence (vir) genes, a single-stranded DNA (the T-strand) is processed from the resident tumor-inducing or root-inducing plasmid and transported from the bacterium to the plant cell. In addition, a number of Virulence effector proteins, including VirD2 (attached to the T-strand), the single-strand DNA binding protein VirE2, plus VirE3, VirD5, and VirF are also transferred to the plant (Otten et al., 1984;Stahl et al., 1998;Vergunst et al., 2000Vergunst et al., , 2003Vergunst et al., , 2005Schrammeijer et al., 2003). These proteins are likely involved in protecting T-DNA from nuclease digestion, directing T-DNA to the nucleus, stripping proteins from the T-strand prior to integration, and integrating T-DNA into the plant genome. T-DNA integration occurs randomly into genomic DNA by the process of illegitimate recombination (...

show abstract

Plants: Genetic Transformation

Siemens

2009

Encyclopedia of Industrial Biotechnology

View full text Add to dashboard Cite

The majority of cultivated plants, including all major cereals, can be transformed either by Agrobacterium ‐mediated transformation or by particle bombardment, which are the two widely used methods for genetic manipulation of plants. Virus‐based vectors can be efficiently used for high transient expression of foreign proteins in transfected plants, providing a low cost alternative to stable transformation. Beside these three major methods, further techniques such as protoplast‐based direct gene transfer, tissue electroporation, silicon carbide fiber‐mediated transformation, sonoporation, and microinjection have yielded transgenic plants, and still find applications.

show abstract

Identification and Characterization of Plant Genes Involved inAgrobacterium-Mediated Plant Transformation by Virus-Induced Gene Silencing

Cited by 78 publications

References 67 publications

Agrobacterium May Delay Plant Nonhomologous End-Joining DNA Repair via XRCC4 to Favor T-DNA Integration

Agrobacterium May Delay Plant Nonhomologous End-Joining DNA Repair via XRCC4 to Favor T-DNA Integration

Overexpression of SeveralArabidopsisHistone Genes IncreasesAgrobacterium-Mediated Transformation and Transgene Expression in Plants

Plants: Genetic Transformation

Contact Info

Product

Resources

About