IDENTIFICATION AND CHARACTERIZATION OF DNA REPAIR COMPONENTS INVOLVED IN AGROBACTERIUMMEDIATED PLANT

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IDENTIFICATION AND CHARACTERIZATION OF DNA REPAIR
COMPONENTS INVOLVED IN AGROBACTERIUM-MEDIATED
PLANT TRANSFORMATION

• Feng Zhang
• Summer Noble Scholar 06
• Mysore Lab

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Crown Gall Disease
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(No Transcript)
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T-DNA Integration(whats known)

• Agrobacterium T-DNA is thought to integrate into
  plant chromosome randomly via a non-homologous
  recombination (NHR) pathway.
• Several repair and recombination proteins
  required for T-DNA integration in yeast (via NHR
  pathway) are reported in literature.
• T-DNA knockouts of Arabidopsis DNA repair gene
  KU80 have been shown to be defective in T-DNA
  integration (Li et al., PNAS 10219231).

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Objectives of the Project

• To identify plant genes that encode proteins
  responsible for
• Conversion of the single stranded T-DNA to a
  double-stranded intermediate.
• The integration of double stranded T-DNA into the
  plant genome
• Ligation of double stranded T-DNA into
  chromosomal DNA.

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Virus-Induced Gene Silencing (VIGS)
Dicer
Plant cell
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Tobacco Rattle Virus (TRV) -- VIGS Vector
RNA1
Rz
Replicase
MP
16K
2X35S
NOSt
RNA2
40K
CP
32.8K
VIGS-RNA2
Dr. Dinesh-Kumar S.P., Yale University
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VIGS Approach Agro- inoculation
TRV-RNA2-Target gene
TRV-RNA1
Inoculation by Infiltration
Dr. Dinesh-Kumar S.P., Yale University
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Materials and Methods

• Candidate genes were chosen from literature and
  from annotated N. benthamiana database at TIGR.
• Primer sequences based on tomato and N.
  benthamiana sequences were designed with GATEWAY
  cloning sites.
• 10 candidate gene fragments were amplified,
  cloned into pTRV2 vector for VIGS and sequenced.
• TRVGFP (control) and 10 DNA Repair (DR) gene
  fragments, cloned in pTRV2 (TRVDR), were
  infiltrated into 3 week old N. benthamiana
  plants.
• Samples were collected 3 weeks later for leaf
  disk transformation assays.
• Plants were inoculated for in planta
  tumorigenesis assays.

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Methods Materials (Contd)

• Leaf disks were inoculated with tumorigenic
  strain of Agrobacterium tumefaciens to assay
  tumor formation potential.
• Another stable transformation assay with
  non-tumorigenic strain of Agrobacterium
  containing a binary vector was performed.
• In vitro GUS assays were performed to check for
  transient and stable GUS expression.
• Non-selective callus inducing medium was used to
  confirm normal cell division and proliferation of
  VIGS samples.
• Semi-quantitative RT-PCR was performed to check
  for down-regulation of transcripts.
• THREE INEPENDENT EXPERIMENTS WERE CONDUCTED.

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Experimental Pipeline for Identification of
Candidate Genes Involved in Agrobacterium-Mediated
Transformation
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Amplification of DR templates from N. benthamiana
cDNA
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In Planta Tumorigenesis Assay
Control
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Semi-Quantitative RT-PCR to Check for Transcript
Reduction in Leaves of Silenced Plants
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In vitro Cell Division Assay
TRVGFP
TRVDR8
TRVDR9
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In Vitro GUS Expression Assay
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Quantitation of in vitro ßGlucuronidase Activity
Using Fluorescent Substrates

Each value is the mean of 12 readings
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Leaf Disc Tumorigenesis Assay
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Leaf Disc Transformation Assay
TRVGFP
TRVDR8
TRVDR9
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Summary

• DR8 and DR9 silenced plants show severely stunted
  phenotype while others appeared normal.
• Cell division profiles appear normal in all the
  silenced plants including the stunted plants.
• DR8 and DR9 silenced plants showed marked
  decrease in transient GUS expression and also in
  stable GUS expression.
• In agreement with the above results, leaf discs
  derived from DR8 and DR9 silenced plants also
  showed reduced tumorigenesis.
• DR3, DR8 and DR9 silenced plants showed reduction
  in fresh/dry weight of calli in the leaf disc
  transformation assay reinforcing the conclusion,
  that these plants are blocked in stable
  transformation.

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Future Directions

• Repeat stable tranformation assay with lower
  titre of Agrobacterium strain.
• Use qRT-PCR to assess amount of integrated T-DNA
  copies in control and VIGS samples.
• Obtain Arabidopsis knockouts for interesting
  candidates to validate results via root
  tumorigenesis assays and floral dip
  transformation assays.
• Check sensitivity of VIGS plants to DNA damage
  agents and UV radiation.

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Acknowledgements

• Dr. Zarir Vaghchhipawala
• Dr. Kiran Mysore
• Mysore Lab
• Steve Rhines and Emily Edwards
• Noble Foundation Summer Scholar Program
• Workshop Coordinators
• Summer Research Scholars Kirsti Burr, Emily
  Combs, Trilby Hillenbrand
• NSF and Noble Foundation for funding this project