Jenna Kausner PBIO 427 April15, 2011

1. Expression of insect (Microderapuntipennisdzungarica) antifreeze protein MpAFP149 confers the cold tolerance to transgenic tobacco Jenna Kausner PBIO 427 April15, 2011

2. Background • Antifree proteins (AFPs) have been isolated from several organisms. • Fish, insects, plants, bacteria • Bind to ice crystals, inhibit growth, lower freezing point and not melting point.

3. Background • Thermal hysteresis activity (THA)- difference between melting point and nonequilibrium freezing point. • Used as an indicator of AFPs activity, so AFPs often referred to as thermal hysteresis proteins (THPs). • Varies among species: Insects (3-6 ºC), Fish (0.7-1.5 ºC), Plants (0.2-0.5 ºC)

4. Background • AFP-producing insects: goal is to avoid freezing, cannot survive if body fluids actually freeze. • AFPs lower freezing point of hemolymph and gut fluid to prevent freezing from the external ice across the body surface.

5. Background • Can achieve higher crop yields by improving freezing tolerance of plants. • Therefore, want to express AFPs in frost-susceptible crops to increase their cold tolerance.

6. Purpose • Some of the most effective ATPs found in insects. • Want to test the MpAFP149 gene, isolated from the beetle Microderapuntipennisdzungarica,in its ability to increase cold-tolerance of transgenic tobacco plants and protect them from freezing damage.

7. MpAFP149 gene • 363 bp with a signal peptide sequence • Transcript encoding 98 amino acids of mature peptide is 68.37% homologous with published AFP from Tenebriomolitor (Tm) • Tm expressed successfully in E. coli; high activity at protecting bacteria at low temperature, linearly correlated with AFP concentration.

8. Transient expression of MpAFP149 in onion cells • Confirm expression of MpAFP149 in plants and visualize sub-cellular localization • MpAFP149 gene with 35S promoter fused with green fluorescent protein (GFP) in plasmid pCAMBIA1302-GFP • Introduced into onion epidermal cells via particle bombardment

9. Plasmid constuction • MpAFP149 gene with signal peptide sequence obtained by PCR. • Gene construct CaMV35S-MpAFP149-Nos inserted into plasmid pCAMBIA1302 with HindIII and EcoRI to form expression vector pCAMBIA1302-MpAFP149

10. Gene construct

11. Plant transformation • Expression vector pCAMBIA1302-MpAFP149 transferred into competent Agrobacterium cells (EHA105 strain) • DNA extracted from kanamycin-resistant surviving Agrobacterium colonies • PCR to confirm presence of MpAFP149 transgene

12. Plant transformation • 1-2 in. young tobacco leaf discs infected with EHA105 Agrobacterium containing pCAMBIA1301-MpAFP149. • Cultivated in the dark at 28 ºC for 2 days. • Leaf discs transferred to generation medium supplemented with hygromycin. • T0 plants allowed to grow and flower and set seeds in a growth chamber.

13. Germination and growth • Allowed to grow 15 weeks in green house before harvesting seed capsules • T0 and Wild-type seeds sterilized by soaking in 1:9 (v/v) 30% bleach:ethanol • Rinsed 5 times with ethanol and left overnight to volatize ethanol

14. Germination and growth • T0 seeds germinated on plates with hygromycin to select for seedlings carrying HPTII gene • Transplanted into pots to full growth at 25 ºC

15. Identification of transgenic plants • Extracted genomic DNA and performed PCR to identify MpAFP149 gene • RNA isolated from plant leaves and reverse transcription carried out • RT-PCR products run through agarose gel electrophoresis to check MpAFP140 transcription

16. Immunolocalization of AFP • Wild-type leaves and transgenic tobacco leaves • Polyclonal antibody raised in mouse against MpAFP149 protein • Immunogold labeling (Antibodies conjugated to gold particles)

17. Western blot • Extracted apoplastic proteins from leaves and separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) • Western blot with antibody against MpAFP149 protein

18. Cold-resistance analysis • After growing for one month, three transgenic and three wild-type plants of similar growth states and no visible phenotypical differences were chosen to undergo cold treatment, measure electrolyte leakage, and Malondialdehyde (MDA) content.

19. Cold-resistance analysis • Set temperature in freezing chamber to -1 ºC for 0, 24, 48, and 72 hours and observed phentypes. • In addition, leaf samples from each group were washed with deionized water and then immersed in deionized water. • After vacuum infiltration, the electric conductivity of supernatant was detected.

20. MDA content • MDA- natural occuring reactive species that is a marker for oxidative stress • The level of MDA at -1 ºC was determined to analyze the comparative rate of lipid peroxidation.

21. Results • The localization of MpAFP149 was determined by expressing MpAFP149:GFP construct plasmid in onion epidermal cells. • For the control, fluorescence was seen throughout the entire cell and for the transformed cells it was solely in the apoplast(see Figure next slide).

22. Localization of MpAFP149:GFP fusion protein

23. Results • 35S-MpAFP149-NOS vector transformed into tobacco using Agrobacterium-mediated gene transfer. • Screened by hygromycin and tested for the presence of the vector by PCR. • Two samples, T0-5 and T0-39 showed higher transcript level by RT-PCR. • These two lines were chosen for detailed analysis.

24. RT-PCR on T1 transgenic tobacco plants

25. Immunolocalization of AFP • Immunogold labeling approach used to determine if MpAFP149 protein was expressed and where it localized in transgenic tobacco. • Showed that MpAFP149 protein accumulated in outer layers of cell wall in transgenic plant, but absent in control tobacco plant

26. Immunolocalization of AFP

27. Western Blot • Western blot for apoplastic proteins showed expected protein band of 10.2 kDa, indicating that mature peptide protein MpAFP149 synthesized in transgenic tobacco.

28. Western Blot

29. Cold resistance analysis • When exposed for 1 day, both transgenic and wild-type tobacco plants only exhibited moderate dehydration. • When exposed for 2 and 3 days, most leaves of wild-type were frozen but transgenic tobacco only exhibited dehydration of a few older leaves near the plant base.

30. Cold resistance analysis • After returning to room temperature, MpAFP149 plants overcame dehydration and recovered completely. • Wild-type displayed permanent damage. • Transgenic line displayed improved cold tolerance and enhanced recovery

32. Ion Leakage • Low temperatures disrupted semi-permeability of tobacco cytomembranes • Effusion of electrolytes resulted in increased electrical activity of tissues • Over time, ion leakage difference increased between control and transgenic tobacco.

33. MDA content • Increase of MDA parallels the increase in conductivity/ion leakage, one does not cause the other. • Wild-type plants suffered higher oxidative lipid injury than transgenic plants; correlated to increases in ion leakage and MDA content.

35. Conclusion • Transgenic tobacco plants expressing MpAFP149 protein with the signal peptide showed improved tolerance to cold and an enhanced recovery. • MpAFP149 may be used as a candiate for the improvement of frost-resistant crops.

36. References • Wang, Y., Qiu, L., Dai, C., Wang, J., Luo, J., Zhang, F., & Ma, J. (2008) Expression of insect (Microderapuntipennisdzungarica) antifreeze protein MpAFP149 confers the cold tolerance to transgenic tobacco. Plant Cell Rep 27: 1349-1358.