Overall Hypothesis

1. Overall Hypothesis IF N-glycans on PRRs are the first to recognize invading pathogens, THEN mutations in genes that encode for N-glycosylation enzymes will cause a decreased or non-existent immune response.

2. Results: Figure 1 • Figure 1A & 1B Hypothesis: IF N-glycans recognize invading pathogens and stimulate a seedling growth arrest immune response, THEN mutations in genes that encode for N-glycosylation enzymes will leave growth unaffected.

3. Figure 1 Background • Method: • tDNA insertion mutants • 100nM elf18 or flg22 treatment GOI ARM Ti Plasmid Agrobacterium GOI - Gene of Interest ARM - Antibiotic Resistance Marker

4. Figure 1A Out of ALL mutants, only stt3a-2 was strongly insensitive to MAMP treatment

5. Results: Figure 1 • Hypothesis Supplemental Figure 2 IF N-glycans recognize invading pathogens and stimulate an “oxidative burst” immune response, THEN mutations in genes that encode for N-glycosylation enzymes will decrease the “oxidative burst” immune response.

6. Results: Supplemental Figure 2

7. Figure 1B Background • Method: • Col-O and mutants treated with 0.5x108cfu/ml of Pseudomonas psyringaepv. tomato DC3000 bacteria. • Hypothesis Figure 1B IF an immune response decreases bacterial viability, THEN mutations in N-glycosylation that decrease immune response will have no effect on bacterial viability.

8. Figure 1B • Mutants showed to be more susceptible to bacteria

9. Figure 2 Background • Method: • Cross-linking • SDS-PAGE • Hypothesis Figure 2A: IF peptide shape is essential to pathogen recognition, THEN cross-linked peptides will result in a loss of function for N-glycosylation mutants.

10. Cross-linking • Radioactivity-labeled elf26 and flg22 peptides(MAMP variants) • in vitro • Bind to receptors EFR and FLS2 • If receptor is still present we will see a band at 150kDa (EFR) or 175kDa (FLS2) • Shows ligand binding and response

11. SDS-Page • Separates proteins according to their size

12. Figure 2A

13. Results: Figure 2 • Hypothesis Figure 2B IF N-glycosylation is responsible for protein folding, then mutation in the N-glycosylation pathway will result in decreased PRR accumulation.

14. Figure 2B

15. Results: Figure 2 • Localization of PRRs in selected N-glycosylation mutants IF EFR and FLS2 are truly membrane-bound proteins, THEN a fluorescent tag on these PRRs will result in localization at the plasma membrane.

16. Confocal Microscopy http://www.olympusfluoview.com/theory/index.html

17. Figure 2C

18. Supplemental Figure 5A & B

19. Results: Figure 3 • Hypothesis for Figure 3 IF tunicamycin causes N-glycan degradation, THEN a gel will reveal band shift proportional to N-glycans present on wildtype PRRs.

20. Figure 3A

21. Figure 3B

22. Figure 3C

23. Figure 3D

25. Results Figure 4 • Hypothesis for Figure 4A IF EFR function is solely based on N-Glycosylation, THEN point mutations to elimate N-Glycosylation motifs will result in EFR dysfunction.

26. EFR

27. Figure 4A

28. Figure 4B

29. Figure 4C