Investigation of the anti-virC2 transcript in Agrobacterium tumefaciens

Agrobacterium tumefaciens has long been an important tool for plant genetic transformation. Many important crop plants such as maize, soybean, wheat, and rice, as well as numerous other dicot plant species are able to be transformed using A. tumefaciens. While many potential regulatory RNA have been previously identified in A. tumefaciens, very few have been verified with a functional investigation. With the growing understanding of regulatory RNA, and its importance in gene regulation, it is essential to further classify regulatory RNA in the transformative tool A. tumefaciens. It is possible that regulatory RNA could be involved in virulence and their expression altered to increase transformation efficiency. In this work, a particular candidate regulatory RNA, anti-virC2, was selected and investigated. While anti-virC2 was identified previous to this research, the function of anti-virC2 has never before been examined. The location of anti-virC2 is on the Ti-plasmid antisense to the virulence gene virC2 and it was initially hypothesized that anti-virC2 acted to regulate the virC2 gene in cis. Through deletion of the promoter region, expression of the anti-virC2 transcript was reduced by ~39% in mutant strain C58 ∆Pavc2. Using this strain, changes in virC2 transcript and protein abundance were able to be observed and compared to wild type. In addition, other possible changes in virulence due to the drop in anti-virC2 expression were examined through Kalanchoe daigremontiana tumorigenesis assays and Arabidopsis thaliana infection experiments. This work shows that anti-virC2 does not act to regulate the cis encoded virC2 gene but may have some impact on virulence. Additional anti-virC2 targets were also predicted and may be investigated in future studies. As well as the findings made regarding anti-virC2, this study provides an example for the functional study of additional regulatory RNA and may serve as a model for the investigation of other potential regulatory RNAs

Genetic engineering possibilities for CELSS: A bibliography and summary of techniques

A bibliography of the most useful techniques employed in genetic engineering of higher plants, bacteria associated with plants, and plant cell cultures is provided. A resume of state-of-the-art genetic engineering of plants and bacteria is presented. The potential application of plant bacterial genetic engineering to CELSS (Controlled Ecological Life Support System) program and future research needs are discussed

Silencing the Agrobacterium tumefaciens oncogenes

Crown gall disease is an agricultural problem caused by the soil-borne bacterium, Agrobacterium tumefaciens. A. tumefaciens oncogenes cause transformed plant cells to overproduce the hormones, auxin and cytokinin. High hormone levels cause unorganized plant cell growth resulting in a gall. Control of crown gall disease is difficult because after plant cell transformation has occurred, the bacterium is no longer required for the disease to progress. Apple trees engineered to express double-stranded RNA of two A. tumefaciens oncogenes, ipt and iaaM, silenced the expression of the wild-type oncogenes and prevented crown gall disease. Only the iaaM oncogene was targeted for posttranscriptional gene silencing (PTGS) as measured by biological assays and by quantitative reverse-transcription polymerase chain reaction (q-RTPCR) on transgenic tissue. However, if the translation initiation sequence of the iaaM construction was eliminated, gall formation was not prevented, indicating that translatable RNA initiates silencing whereas untranslatable RNA does not. Other data indicate that the Arabidopsis thaliana micro-RNA pathway gene is involved in A. tumefaciens-mediated tumorigenesis. A. thaliana plants with a mutation in HEN1, a gene required for micro-RNA maturation, demonstrated a tenfold reduction in tumorigenesis upon A. tumefaciens infection compared to wild-type. The same plant line showed no difference in T-DNA transfer and nuclear uptake

Protein encoded by oncogene 6b from Agrobacterium tumefaciens has a reprogramming potential and histone chaperone-like activity

Crown gall tumors are formed mainly by actions of a group of genes in the T-DNA that is transferred from Agrobacterium tumefaciens and integrated into the nuclear DNA of host plants. These genes encode enzymes for biosynthesis of auxin and cytokinin in plant cells. Gene 6b in the T-DNA affects tumor morphology and this gene alone is able to induce small tumors on certain plant species. In addition, unorganized calli are induced from leaf discs of tobacco that are incubated on phytohormone-free media; shooty teratomas and morphologically abnormal plants, which might be due to enhanced competence of cell division and meristematic states, are regenerated from the calli. Thus, the 6b gene appears to stimulate a reprogramming process in plants. To uncover mechanisms behind this process, various approaches including the yeast-two-hybrid system have been exploited and histone H3 was identified as one of the proteins that interact with 6b. It has been also demonstrated that 6b acts as a histone H3 chaperon in vitro and affects the expression of various genes related to cell division competence and the maintenance of meristematic states. We discuss current views on a role of 6b protein in tumorigenesis and reprogramming in plants

Plant DNA Repair and Agrobacterium T−DNA Integration

Agrobacterium species transfer DNA (T−DNA) to plant cells where it may integrate into plant chromosomes. The process of integration is thought to involve invasion and ligation of T-DNA, or its copying, into nicks or breaks in the host genome. Integrated T−DNA often contains, at its junctions with plant DNA, deletions of T−DNA or plant DNA, filler DNA, and/or microhomology between T-DNA and plant DNA pre-integration sites. T−DNA integration is also often associated with major plant genome rearrangements, including inversions and translocations. These characteristics are similar to those often found after repair of DNA breaks, and thus DNA repair mechanisms have frequently been invoked to explain the mechanism of T−DNA integration. However, the involvement of specific plant DNA repair proteins and Agrobacterium proteins in integration remains controversial, with numerous contradictory results reported in the literature. In this review I discuss this literature and comment on many of these studies. I conclude that either multiple known DNA repair pathways can be used for integration, or that some yet unknown pathway must exist to facilitate T−DNA integration into the plant genome

KYRT1, a Disarmed Version of a Highly Tumorigenic \u3cem\u3eAgrobacterium Tumefaciens\u3c/em\u3e Strain Identified as Chry5

Disarmed A. tumefaciencs strain KYRT1, derived from a highly tumorigenic strain identified as A. tumefaciens strain Chry5. Disarming is accomplished by inactivation of plasmid pTiChry5 T-DNA sequences by, for example, deletion of sequences comprising the T-DNA right border. Methods of making transgenic plants using the novel A. tumefaciens strains are also provided

New Approaches to Agrobacterium tumefaciens-Mediated Gene Transfer to Plants

Agrobacterium tumefaciens, a plant pathogen, is commonly used as a vector for the introduction of foreign genes into plants and consequent regeneration of transgenic plants. A. tumefaciens naturally infects the wound sites in dicotyledonous plants and induces diseases known as crown gall. The bacterium has a large plasmid that induces tumor induction, and for this reason, it was named tumor-inducing (Ti) plasmid. The expression of T-DNA genes of Ti-plasmid in plant cells causes the formation of tumors at the infection site. The molecular basis of Agrobacterium-mediated transformation is the stable integration of a DNA sequence (T-DNA) from Ti (tumor-inducing) plasmid of A. tumefaciens into the plant genome. A. tumefaciens-mediated transformation has some advantages compared with direct gene transfer methods such as integration of low copy number of T-DNA into plant genome, stable gene expression, and transformation of large size DNA segments. That is why manipulations of the plant, bacteria and physical conditions have been applied to increase the virulence of bacteria and to increase the transformation efficiency. Preculturing explants before inoculation, modification of temperature and medium pH, addition chemicals to inoculation medium such as acetosyringone, changing bacterial density, and co-cultivation period, and vacuum infiltration have been reported to increase transformation. In this chapter, four new transformation protocols that can be used to increase the transformation efficiency via A. tumefaciens in most plant species are described

Epigenetics of Host-Pathogen Interactions: The effect of acetosyringone on Ti Plasmid methylation patterns in Agrobacterium tumefaciens C58

The plant pathogen Agrobacterium tumefaciens C58 can transfer a portion of its tumor-inducing (Ti) plasmid to plant hosts in response to the plant wound signals, including Acetosyringone. The portion transferred is aptly titled Transfer DNA (T-DNA) which encode genes involved in tumor production and biosynthesis of a unique bacterial food source called opines that provide an advantage to the inducing Agrobacterium. The Ti plasmid contains a number of genes, including the virulence region that enables T-DNA transfer. Epigenetics investigates how chemical modifications to DNA that don’t alter sequence are used to control gene expression (for example, genes involved in pathogen virulence). Epigenetic modifications can be detected by a next generation sequencing technology called Single Molecule Real Time (SMRT) sequencing. SMRT detects these modifications by tracking kinetic shifts during DNA synthesis. Given the unique inter-kingdom DNA transfer, and the importance of epigenetic regulation in other bacteria and plant species, a comparative exploration of methylation patterns of the Ti plasmid under conditions that induce virulence was undertaken. Over a dozen genes with a variety of purposes (virulence regulation, ion transport, DNA replication, etc.) lost methylation following exposure to the virulence inducing molecule acetosyringone, suggesting increased transcription. Three genes, VirD5, TraM and a phosphate/sodium symporter gained methylation throughout the gene, suggesting down regulation. The patterns discovered, while intriguing, sre limited by possible methodological flaws in SMRT sequencing due to incongruities between reported findings and those described in the literature. A further examination of the expression profiles of these genes is warranted given these findings