Agrobacterium Delivery Research Articles

A cytometry microparticle platform approach for screening tobacco microRNA changes after agrobacterium delivery

MicroRNAs are a class of non-coding regulatory RNAs that can modulate development as well as alter innate antiviral defenses in plants. In this study we explored changes in Nicotiana benthamiana tobacco microRNA expression as it relates to expression of a recombinant anti-Ebola GP1 antibody. The antibody was delivered to tobacco leaves through a bacterial Agrobacterium tumefaciens “agroinfiltration” expression strategy. A multiplex microparticle-based cytometry assay tracked the expression changes of 53 host tobacco microRNAs. Our results revealed that the most abundant microRNAs in actively growing leaves corresponded to nanoparticle probes specific to nta-mir-6149 and nta-miR-168b. After agroinfiltration, probes specific for nta-mir-398, and nta-mir-482d were significantly altered in their respective expression levels, however changes were partially attributed to the infiltration broth medium used in the antibody delivery process. Confirmation of nta-mir-398 and nta-mir-482d expression changes was also verified through RT-qPCR. To our knowledge this study is the first to profile medium and Agrobacterium injection at the microRNA level through a multiplex microparticle approach.

Maize (Zea mays L.) transformation by Agrobacterium tumefaciens infection of pollinated ovules

A novel transformation system was established for maize using Agrobacterium infection of in vitro cultured ovules. The maize ovules were isolated 24h after pollination and infected with Agrobacterium. The embryos were isolated from the pollinated ovules 2–3 weeks after Agrobacterium infection, regenerated to plantlets and investigated for transgene expression and inheritance. Experimental evaluations were focused on the four main aspects. Firstly, through the introduction of gus gene for monitoring transformation and development of embryo, it was confirmed that transgenic plants can be generated from in vitro cultured maize ovules infected with Agrobacterium. Secondly, in order to standardize the transformation protocol, several important factors that affected transformation efficiency were optimized. They included Agrobacterium delivery approach, surfactant, AS concentration, and cocultivation duration. Thirdly, stable expression and Mendelian inheritance of the introduced genes were analyzed in independent lines over two generations. Fourthly, the pollinated ovule culture-regeneration potential and transformation efficiency of five maize inbred lines were investigated to confirm the genotype independence of this transformation system. We conclude that the transformation system established in this study can be used to generate high-quality transgenic maize plants rapidly and directly.

A High Throughput Barley Stripe Mosaic Virus Vector for Virus Induced Gene Silencing in Monocots and Dicots

Barley stripe mosaic virus (BSMV) is a single-stranded RNA virus with three genome components designated alpha, beta, and gamma. BSMV vectors have previously been shown to be efficient virus induced gene silencing (VIGS) vehicles in barley and wheat and have provided important information about host genes functioning during pathogenesis as well as various aspects of genes functioning in development. To permit more effective use of BSMV VIGS for functional genomics experiments, we have developed an Agrobacterium delivery system for BSMV and have coupled this with a ligation independent cloning (LIC) strategy to mediate efficient cloning of host genes. Infiltrated Nicotiana benthamiana leaves provided excellent sources of virus for secondary BSMV infections and VIGS in cereals. The Agro/LIC BSMV VIGS vectors were able to function in high efficiency down regulation of phytoene desaturase (PDS), magnesium chelatase subunit H (ChlH), and plastid transketolase (TK) gene silencing in N. benthamiana and in the monocots, wheat, barley, and the model grass, Brachypodium distachyon. Suppression of an Arabidopsis orthologue cloned from wheat (TaPMR5) also interfered with wheat powdery mildew (Blumeria graminis f. sp. tritici) infections in a manner similar to that of the A. thaliana PMR5 loss-of-function allele. These results imply that the PMR5 gene has maintained similar functions across monocot and dicot families. Our BSMV VIGS system provides substantial advantages in expense, cloning efficiency, ease of manipulation and ability to apply VIGS for high throughput genomics studies.

Transformation of Potato (Solanum tuberosumcv.Savalan) by Chitinase and β-1,3-Glucanase Genes of Myco-Parasitic Fungi Towards Improving Resistance to Rhizoctonia solani AG-3

Potato (Solanum tuberosum L.) an agro-economically important food crop in the world, is sensitive to many fungal pathogens including Rhizoctonia solani (AG-3), the causal agent of stem and root rot diseases. Chitinase and glucanase are cell wall degrading enzymes which have been shown to have high antifungal activity against a wide range of phytopathogenic fungi. In the present study, plasmid pBIKE3 harboring a double-gene cassette containing the chitinase (chit42) and β-1,3-glucanase (bgn13.1) genes was constructed. In this construct, the chit42 gene is located between the CaMV 35S promoter and nos terminator derived from pBI121, while the bgn13.1 gene is downstream of a modified CaMV 35S promoter, followed by the nos terminator both of which were derived from the pRTL plasmid. Micro-tubers of potato plants (the Savalan cultivar) were transformed with the pBIKE3 construct via the Agrobacterium delivery system. Integration of these two genes into the potato genome and their expression at the transcriptional level was confirmed by polymerase chain reaction (PCR) and reverse transcription-PCR (RT-PCR). The radial diffusion assay showed that the heterologous expressed chitinase and glucanase enzymes demonstrated antifungal activity on R. solani (AG-3).

Targeted transgene integration in plant cells using designed zinc finger nucleases

Targeted transgene integration in plants remains a significant technical challenge for both basic and applied research. Here it is reported that designed zinc finger nucleases (ZFNs) can drive site-directed DNA integration into transgenic and native gene loci. A dimer of designed 4-finger ZFNs enabled intra-chromosomal reconstitution of a disabled gfp reporter gene and site-specific transgene integration into chromosomal reporter loci following co-transformation of tobacco cell cultures with a donor construct comprised of sequences necessary to complement a non-functional pat herbicide resistance gene. In addition, a yeast-based assay was used to identify ZFNs capable of cleaving a native endochitinase gene. Agrobacterium delivery of a Ti plasmid harboring both the ZFNs and a donor DNA construct comprising a pat herbicide resistance gene cassette flanked by short stretches of homology to the endochitinase locus yielded up to 10% targeted, homology-directed transgene integration precisely into the ZFN cleavage site. Given that ZFNs can be designed to recognize a wide range of target sequences, these data point toward a novel approach for targeted gene addition, replacement and trait stacking in plants.

Use of Potato Virus X (PVX)–Based Vectors for Gene Expression and Virus‐Induced Gene Silencing (VIGS)

Potato virus X-based vectors are a well established system for rapid in planta studies. The vectors can be used for expression of proteins in plants and to down-regulate genes through virus-induced gene silencing. The development of binary-based vectors for Agrobacterium delivery makes this system well suited to high-throughput studies. Protocols are given for establishing infections to achieve expression and VIGS through in vitro transcription and Agrobacterium delivery to glasshouse and in vitro-grown plant material.

Transgenic potato plants expressing soybean beta-1,3-endoglucanase gene exhibit an increased resistance to Phytophthora infestans.

Soybean beta-1,3-endoglucanase represents a model system for studies on early plant responses to infection by fungal pathogens, and it has been implicated in the release of elicitors from fungal cell walls. In the present study, potato plants were transformed with the soybean beta-1,3-endoglucanase cDNA via Agrobacterium delivery system. The transfer of the gene into potato genome was confirmed by (i) PCR amplification, (ii) Northern blot analyses, and (iii) an increase in the activity of beta-1,3-endoglucanase in transgenic plants. The transformation resulted in an increased resistance of selected transgenic plants to infection by Phytophthora infestans, an important pathogen.

Agrobacterium-mediated inoculation of plants with tomato golden mosaic virus DNAs

We have adapted the "agroinfection" procedure of Grimsley and co-workers [4,5] to develop a simple, efficient, reproducible infectivity assay for the insect-transmitted, split-genome geminivirus, tomato golden mosaic virus (TGMV). Agrobacterium T-DNA vectors provide efficient delivery of both components of TGMV when used in mixed inoculation of wild-type host plants. A greater increase in infection efficiency can be obtained by Agrobacterium delivery of the TGMV A component to "permissive" transgenic plants. These "permissive" plants contain multiple tandem copies of the B component integrated into the host genome. An inoculum containing as few as 2000 Agrobacterium cells can produce 100% infection under these conditions. Further, our results show that there is a marked effect of the configuration of the TGMV A components within the T-DNA vector on time of symptom development. We have also found that transgenic plants carrying tandem copies of the A component do not complement the B component. Possible mechanisms to explain these results and the potential use of this system to further study the functions of the geminivirus components in infection are discussed.