Agrobacterium rhizogenes GALLS Gene Encodes Two Secreted Proteins Required for Genetic Transformation of Plants Larry D. Hodges, Lan-Ying Lee, Henry McNett, Stanton B. Gelvin, Walt Ream

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Agrobacterium rhizogenes GALLS Gene Encodes Two
Secreted Proteins Required for Genetic
Transformation of PlantsLarry D. Hodges,
Lan-Ying Lee, Henry McNett, Stanton B. Gelvin,
Walt Ream

• Presented by Catherine Crosby

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A. tumefaciens wild type system

• Key Features for this paper
• VirE1/VirE2 facilitate transfer and localization
  of T-strand
• VirE2 binds T-strand inside the plant cell
• Vir E2/E1 are necessary for pathogenicity
• Key features of the A. tumefaciens Ti plasmid
• Transfers DNA between the border sequences
• T-strand induces tumor formation
• Carries all necessary factors

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A. tumefaciens wild type system
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A. rhizogenes system

• Key differences
• Does not have VirE1/E2
• Uses the GALLS genes instead
• Differences in Ri plasmid
• Lacks VirE1/E2
• Contains Galls complex
• Similar structure and sequence of the Vir operon

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Experimental design

• Knock-out VirE1/E2 on the Ti plasmid
• Use the Ri plasmid from A. rhisogenes
• GALLS gene complex can compliment
• Plasmid constructed to carry the Ti T-strand,
  with the Ri GALLS genes replacing Vir E1/E2
• Trace GALLS proteins using molecular techniques
• His taggin, YFP, probes, blotting, protein
  interactions

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Results GALLS Proteins

• Two protein products
• GALLS Full length (FL)
• GALLS C-Terminus (CT)
• GALLS-FL will compliment Vir E1/E2 activity
• GALLS-CT interacts with the T-strand and GALLS-FL
• GALLS-FL localizes to the nucleus
• GALLS-CL is cytoplasmic unless associated with
  GALLS-FL

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Protein interactions
FIG. 2. Immunoblot analysis of proteins encoded
by the GALLS gene. Blots were probed with
GALLS-specific antibodies. FL, GALLSFL His-FL,
His6-tagged GALLS-FL CT, GALLS-CT. Numbered bars
indicate the size (in thousands) of molecular
weight standards. (A) Soluble proteins from A.
tumefaciens containing vector plasmid (left lane)
or the wild-type GALLS gene (right lane). (B)
Soluble proteins were extracted from A.
tumefaciens cells harboring the His6- GALLS-FL
gene and fractionated on Ni-nitrilotriacetic acid
resin bound proteins were eluted with imidazole.
Proteins from the flowthrough (flow), wash, and
first and second eluates are shown. (C) Soluble
proteins from A. tumefaciens containing the
vector plasmid (lane 1), wild-type GALLS gene
(lane 2), the 1 frameshift mutation in codon 803
of the GALLS gene (lane 3, 1 FS), or the
GALLS gene with the Met808Ile Met815Val mutations
(lane 4, MIMV). (D) GALLS proteins expressed in
A. tumefaciens cells containing the GALLS gene
fused to the Trc promoter and 5 UTR from pTrc99
in the absence () or presence () of IPTG
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Take Home from Fig. 2

• GALLS-FL is less abundant than GALLS-CT
• GALLS-FL and GALLS-CT interact

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GALLS Proteins

• Both proteins come from the same mRNA
• They share a promoter!
• Disparity in concentrations is a translation
  effect
• Rarely used Leucine codons
• Anti-sense mRNA
• Little evidence for either
• GALLS-FL is required for complimentation
• GALLS-CT plays a roll in some systems for
  complimentation

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Virulence of GALLS carriers and GALLS mutants
FIG. 3. Virulence of strains expressing wild-type
or mutant GALLS genes. (A) K. daigremontiana
leaves and the basal surfaces of carrot root
slices were inoculated with the A. tumefaciens
mx358 virE2 mutant strain harboring vector
plasmid (top row), the wild-type GALLS gene
(second row), or the mutated GALLS gene (M808I
M815V MIMV) inoculated alone (third row) or
coinoculated with a virE2 mutant harboring the
nonpathogenic GALLShelicase III mutant (16)
(carrots only). (B) A. thaliana root explants
were inoculated with wild-type A. tumefaciens
A348 (black bars) or the A. tumefaciens mx358
virE2 mutant strain expressing both wild-type
GALLS-FL and GALLS-CT (wt FL CT) or only
GALLS-FL (FL-MIMV). Inoculum size is indicated as
CFU/ml.
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Take Home for Fig 3

• Wild Type GALLS restores pathogenicity
• GALLS-FL alone restores pathogenicity for some
  hosts, but not all
• GALLS-CT mutants can be complimented by a
  GALLS-FL mutant

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GALLS Localization
FIG. 4. Localization of GALLS proteins expressed
in plant cells. (A) Tobacco protoplasts were
electroporated with plasmids expressing GALLS-FL
YFP or GALLS-FLNLSYFP from a CaMV35S promoter.
A plasmid expressing RFP was included to mark
transformed cells although RFP is distributed
throughout transformed cells, higher amounts
concentrate in the nucleus. Nuclei were stained
with Hoechst dye (left). Cells were examined by
epifluorescence microscopy using filters for YFP
or RFP, as indicated. Column 3 shows Hoechst
(Nuclei) and YFP signals superimposed. (B)
Epifluorescence and confocal microscopy of GALLS
protein interactions. Tobacco cells were
electroporated with plasmids expressing proteins
fused to complementary nYFP and cYFP. BiFC was
detected by epifluorescence microscopy (top row)
and confocal microscopy with the focal plane
through the nucleus (bottom row). YFP
fluorescence detected by epifluorescence
microscopy (EPI) is superimposed on bright-field
images. Confocal images of YFP fluorescence are
superimposed on differential interphase contrast
(DIC) images.
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Take Home for Fig. 4

• The NLS is critical for GALLS-FL function
• GALLS-CT only enters the nucleus when interacting
  with proteins with a NLS
• GALLS-CT is cytoplasmic
• GALLS proteins also interact with VirD2

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GALLS Conclusions

• A. rhizogenes GALLS genes compliment A.
  tumefaciens VirE1/E2 genes
• GALLS-FL binds the T-strand and carries it to the
  nucleus of the plant host.
• GALLS-FL is a helicase with DNA binding motifs
  and NLS sequence.
• GALLS-CT plays an important roll.

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Questions? Thoughts?

• Two different systems, GALLS and VirE1/E2,
  developed in two related pathogens.
• How do the two systems differ?
• Protein function?
• Motifs/ protein families?