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Transgenic Plants

Methods of Gene Transfer

Prof. (Dr.) Shiburaj S

Gene Transfer methods in Plants

There are mainly two species of Agrobacterium :

i. A. tumefaciens that induces crown gall disease.

Virulence through Ti Plasmid (pTi)

Vector Mediated Gene Transfer ii. A. rhizogenes ( Rhizobium rhizogenes ) that induces hairy root disease. Virulence through Ri Plasmid (pRi)

Agrobacterium tumefaciens – pathogen and useful genetic tool Casimiro, I., Marchant, A., Bhalerao, R.P., Beeckman, T., Dhooge, S., Swarup, R., Graham, N., Inzé, D., Sandberg, G., Casero, P.J. and Bennett, M. (2001). Auxin Transport Promotes Arabidopsis Lateral Root Initiation. Plant Cell. 13: 843 - 852. Herb Pilcher Cherry Agrobacterium tumefaciens is a plant pathogen that induces tumors on about 60% of dicotyledonous angiosperms and gymnosperms Its tumor-inducing property also makes it a valuable tool for introducing genes into plants for research and agricultural purposes

Crown gall disease The first written record of crown gall disease, on grape, dates from 1853 Fridiano Cavara (1897) found that a bacterium causes crown gall in grape Crown gall induces growths at wound sites and severely limits crop yields and growth vigor

gall gall They isolated a bacterium from galls on daisy. When inoculated onto other plants, galls were produced

The story begins about 100 years ago when two USDA

plant pathologists, Erwin Smith and Charles Townsend

published a paper in Science ( 1907 ) reporting that a

bacterium that they named Bacterium

tumefaciens caused plant tumors or crown galls on a

variety of plants.

A few days after inoculation, tumors become independent of bacteria Braun, A.C. (1943) Studies on tumor inception in the crown-gall disease. Am. J. Bot. 30: 674 - 677 Periwinkle ( Catharanthus roseus ) stems were inoculated with Agrobacterium tumefaciens , and then incubated at room temperature for various times, followed by 5 days at 47*C to kill the bacteria When the tissue was incubated at room temperature for four days before heat-killing the bacteria, many tumors were formed When the tissue was incubated at room temperature for one day before heat- killing the bacteria, no tumors were formed Viable bacteria are no longer necessary beyond two days post-inoculation. After this period, tumors become independent of the bacteria, because the bacteria have altered the host cells, by transferring some factors into them.

© 2014 American Society of Plant Biologists TIP- tumor inducing principle"

• Braun developed the concept that has guided so much subsequent research on Agrobacterium namely, a "tumor inducing principle" or TIP is responsible for tumor development.
• Using temperature shift experiments, he demonstrated that the TIP must be elaborated during the first 10 hours after plant inoculation.
• Heating inoculated plants to 32°C before this time, a temperature which does not kill either the bacteria or the plant, prevented tumor formation.
• After 10 hours, heating did not affect tumor formation.
• Braun also discovered that Agrobacterium transforms plant cells permanently in that tumor cells can grow in vitro indefinitely on medium that does not support the growth of normal plant tissue.
• In a remarkably prescient statement made in 1947, Braun suggested that DNA was a possible candidate for TIP.

Some DNA from the Ti plasmid is transferred into the plant cells (1977) Reprinted from Chilton, M.-D., Drummond, M.H., Merlo, D.J., Sciaky, D., Montoya, A.L., Gordon, M.P. and Nester, E.W. (1977). Stable incorporation of plasmid DNA into higher plant cells: the molecular basis of crown gall tumorigenesis. Cell. 11: 263 - 271. with permission from Elsevier. See also Yadav, N.S., Postle, K., Saiki, R.K., Thomashow, M.F. and Chilton, M.D. (1980). T-DNA of a crown gall teratoma is covalently joined to host plant DNA. Nature. 287: 458 - 461. Ti plasmid Restriction enzyme digestion “Our results suggest that the tumor- inducing principle first proposed by Braun (1947) is indeed DNA, as many investigators have long suspected.” Pos. controls (Ti plasmid) Neg. control (untransformed plant DNA) DNA from crown gall Renaturation kinetics of labeled plasmid DNA fragments with various unlabeled DNA samples Increasing amounts of labeled Ti plasmid DNA The key finding was that Ti plasmid DNA anneals with DNA isolated from the crown gall, meaning that the gall contains Ti DNA

Structure and function analysis of the Ti plasmid T-DNA pTi The virulence ( vir ) genes are required for T-DNA movement into the plant cell Transfer DNA (T-DNA) moves into the plant cell nucleus. It is flanked by two direct 25 bp repeat border sequences, shown as yellow triangles Tumour inducing (Ti) plasmid is a plasmid found in pathogenic species of Agrobacterium , including A. tumefaciens , A. rhizogenes , A. rubi and A. vitis. The organization of Ti plasmids varies between isolates, but all carry one or more T-DNA region and one vir region. Ti plasmid that is transferred via conjugation into host plant cells after an injury site is sensed by the bacteria.

The T-DNA region: tumor-inducing genes and opine synthesis genes T-DNA pTi Auxin synthesis Cytokinin synthesis Opine synthesis to “feed” Agrobacterium Autonomous growth Plant cell T4SS T4SS = Type IV Secretion System

OPINES – The unusual compounds Octopine Octopine- utilizing strain Nopaline- Nopaline utilizing strain The type of opine is determined by the bacterium, not the plant Opines are low molecular weight compounds found in plant crown gall tumors or hairy root tumors produced by pathogenic bacteria of the genus Agrobacterium and Rhizobium There are some 30 different opines described so far. Opine biosynthesis is catalyzed by specific enzymes encoded by genes contained in a small segment of DNA (known as the T-DNA, for 'transfer DNA’), which is part of the Ti plasmid or Ri plasmid. The opines are used by the bacterium as an important energy, carbon and nitrogen source. Chemically, opines fall into two major structural classes:

1. Amine derivatives (most opines belongs to this)
2. Sugar-phosphodiesters (e.g. Agrocinopines) Opines out leak into intercellular spaces Bacteria growing in intercellular spaces of tumor make enzyme allowing them to take up and metabolize opines The name opine comes from octopine, the first opine discovered in 1927, not in crown galls, but in octopus muscle. Nopaline (N2-(D-1,3-dicarboxypropyl)-L-arginine) was first isolated from crown gall tumors.