Transcript Jiang Lab Progress - University of California, Berkeley

Jiang Lab Role
• Gene expression profiling of key potato
biology (polyploidy, tuberization, late blight
resistance)
• Provide services to the project for potato
transformation and greenhouse late blight
resistance evaluation
Gene Expression Profiling Progress
– Polyploidy (20 Arrays)
• 12 arrays completed on leaflets
• 8 arrays completed on root tips
• Comparative profiling using cDNA-AFLP
– Tuberization (22 Arrays)
• 10 arrays completed on in vitro tuber development
• 12 arrays completed on in vivo tuber development
• Patatin gene family expression profiling
– Late blight resistance (24 arrays)
• 24 arrays completed on transgenic RB lines
Transformation and Resistance Evaluation
Potato Transformation
Transgenic RB lines in Russet Burbank, Superior, Dark Red Norland
Constructs from the Baker lab (VF36 with GSS-4 and GSS-5)
Five constructs from the Jahn lab
RNAi constructs for RPR and SGT, Two RB-tagging lines
Late Blight Resistance Evaluation
635 clones, 2163 plants, in 29 inoculation experiments
Publications from Jiang Lab
Colton, L.M., Groza, H.I., Wielgus, S.M., and Jiang, J. (2006) Marker-assisted
selection for the broad-spectrum potato late blight resistance conferred by gene RB
derived from a wild potato species. Crop Sci. 46: 589-594.
Stupar, R.M., Beaubien, K.A., Jin, W., Song, J., Lee, M.-K., Wu, C., Zhang, H.-B.,
Han, B. and Jiang, J. (2006) Structural diversity and differential transcription of the
patatin multicopy gene family during potato tuber development. Genetics (February
issue)
Stupar et al. (2006) Phenotypic and gene expression changes associated with
autopolyploidization. To be submitted to Plant Cell within ~2 months.
Ploidy and Gene Expression
Potato Autopolyploidy series
Wild diploid:
AA
(24 chromo.;
Heterozygous)
Anther culture:
A
(12 chromo.)
Leaf disc: AA
AAAA
(24 chromo.) (48 chromo.)
Homozygous Homozygous
1x
2xR3
2xR5
4x
Series P77
1x
2xR3
2xR5
4x
P77 Monomorphism
M
1x 2xR3 2xR5 4x T
B
1x 2xR3 2xR5 4x T
B
M
1x
2xR3
2xR5
4x
Array experimental design
Biological Rep 1
Biological Rep2
Biological Rep3
1x
1x
1x
2xR3
2xR5
4x
2xR3
2xR5
4x
2xR3
2xR5
4x
A
4x-1x; 2x-1x; 4x-2x
10 genes
B
2 genes
1x-4x; 1x-2x; 2x-4x
4x-1x
135 genes
PloidyUpregulated
Log(2) Expression
Profiles
4x-1x; 2x-1x
248 genes
PloidyDownregulated
Log(2) Expression
Profiles
155 genes
1x-4x
127 genes
1x-4x; 1x-2x
4x-1x; 4x-2x
22 genes
23 genes
1x-4x; 2x-4x
Ploidy
Ploidy
Leaf ploidy array summary
• ~10% of all genes showed significant
expression changes over ploidy levels
• ~50% of ribosomal protein genes showed
significant expression changes over ploidy
levels
• ~75% of histone genes showed significant
expression changes over ploidy levels
Real-time PCR on selected genes
C
YFold
AxisChange
Title
3
3
2
2
1
1
0
Ploidy: 1 2 4 1 2 4 1 2 4 1 2 4
-- IR76
JM09 CZ25 CF69
Ribosomal protein genes
124 124
124 124 124
--- IO91 IZ46 -CenH3
EW61 GF62
X Histone
Axis genes
Title
124
DE81
124 124
-CV58
IS17
Cyclin
Downregulated
Conclusions
• Ploidy affects plant growth rates and cell size
• Gene families that change with ploidy
– Ribosomal protein genes, histones, cyclins
• Important for “cellular infrastructure”
• “Ploidy genes” are context-dependent
• Autopolyploid vs. Allopolyploid
– We find much more subtle expression changes in
autopolyploids than in allopolyploids
RB-Associated Gene Expression
10 arrays were completed in 2005 to test the timing of transcriptome
changes after late blight inoculation
24 arrays completed in the last three weeks for final profiling
“Super-resistant” transgenic RB clone
The late blight resistance phenotypes of two transgenic Katahdin lines under an intensive
inoculation conditions. Left Panel: Left: A transgenic Katahdin clone containing a single
copy of the RB gene; Middle: S. bulbocastanum clone PT29; Right: Katahdin control. The
transgenic Katahdin plant, which is resistant to late blight under regular inoculation
condition, shows a susceptible phenotype. Right Panel: Left: A transgenic Katahdin clone
containing multiple copies of the RB gene; Middle: S. bulbocastanum clone PT29; Right:
Katahdin control. In Southern hybridization, DNA from the transgenic plants was cut with
HindIII (left lane) and EcoR1 (right lane) and hybridized with a vector probe
Schematic of RNA pooling and
microarray hybridization: Four identical
plants from each of the three Katahdin plants
(control Katahdin, Katahdin containing one RB
gene, Katahdin containing multiple copies of
RB gene) are sampled under each of the three
time points (2, 5, and 10 hours after
inoculation). Eight RNA samples will be
isolated from these four plants. Challenged
and unchallenged RNA samples from two
plants will be pooled, amplified and labeled.
The challenged aRNA from the first pair of
plants is labeled with Cy5 and the
unchallenged aRNA labeled with Cy3.
Alternately, the challenged aRNA in the
second pair of plants is labeled with Cy3 and
the unchallenged with Cy5. Both labeled
samples are mixed from each pair of plants
and hybridized to the array. This procedure
creates a dye-swap with two biological
replicates.
(A) Filtered data from Preliminary Array Experiment 1 is shown colored by the normalized
intensity ratio (log transformed) along the vertical axis. The horizontal axis represents gene
expression occurring 5, 15 and 25 hours post inoculation. (B) Filtered data from Preliminary
Array Experiment 2 is shown colored by the normalized intensity ratio (log transformed)
along the vertical axis. The horizontal axis represents gene expression occurring 2, 5 and 10
hours post inoculation
Coming soon:
Final results on gene expression profiling on RBmediated late blight resistance pathway
Acknowledgments
Richard Veilleux
Brian Yandell
Robin Buell
Amy Hart
Willem Rensink
National
Science
Foundation