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 NorlandConstructs from the Baker lab (VF36 with GSS-4 and GSS-5)Five constructs from the Jahn labRNAi 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

2xR5

2xR3

4x

M 1x 2xR3 2xR5 4x T B 1x 2xR3 2xR5 4x T B M

P77 Monomorphism

1x 2xR3

2xR5 4x

1x

2xR3 2xR5

4x

1x

2xR3 2xR5

4x

1x

2xR3 2xR5

4x

Biological Rep 1 Biological Rep2 Biological Rep3

Array experimental design

Ploidy Ploidy

Ploidy-Downregulated

Log(2) ExpressionProfiles

A B

Ploidy-Upregulated

Log(2) ExpressionProfiles

4x-1x; 2x-1x; 4x-2x10 genes

4x-1x135 genes

4x-1x; 2x-1x248 genes

4x-1x; 4x-2x22 genes

2 genes1x-4x; 1x-2x; 2x-4x

23 genes1x-4x; 2x-4x

127 genes1x-4x; 1x-2x

155 genes1x-4x

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

-- -- -- -- --0

1

2

3

X Axis Title

C

Ploidy: 1 2 4 1 2 4 1 2 4 1 2 4 1 2 4 1 2 4 1 2 4 1 2 4 1 2 4 1 2 4 1 2 4 1 2 4

Fold

Cha

nge

3

2

1

JM09 CZ25 CF69 IR76 CenH3 EW61 GF62 IO91 IZ46 DE81 CV58 IS17

Ribosomal protein genes Histone genes Cyclin Downregulated

Real-time PCR on selected genes

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

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

“Super-resistant” transgenic RB clone

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 RB-mediated late blight resistance pathway

Acknowledgments

Richard Veilleux Robin BuellAmy HartWillem Rensink

Brian Yandell NationalScienceFoundation