overall hypothesis

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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.

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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. Results: Figure 1. Figure 1A & 1B Hypothesis: - PowerPoint PPT Presentation

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Page 1: Overall Hypothesis

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.

Page 2: Overall Hypothesis

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.

Page 3: Overall Hypothesis

Figure 1 Background

• Method: – tDNA insertion mutants– 100nM elf18 or flg22 treatment

GOI - Gene of InterestARM - Antibiotic Resistance Marker

GOI

Ti Plasmid

ARM

Agrobacterium

Page 4: Overall Hypothesis

Figure 1A

Out of ALL mutants, only stt3a-2 was strongly insensitive to MAMP treatment

Page 5: Overall Hypothesis

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.

Page 6: Overall Hypothesis

Results: Supplemental Figure 2

Page 7: Overall Hypothesis

Figure 1B Background

• Method: – Col-O and mutants treated with 0.5x108 cfu/ml of

Pseudomonas psyringae pv. 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.

Page 8: Overall Hypothesis

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

Page 9: Overall Hypothesis

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.

Page 10: Overall Hypothesis

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

Page 11: Overall Hypothesis

SDS-Page

• Separates proteins according to their size

Page 12: Overall Hypothesis

Figure 2A

Page 13: Overall Hypothesis

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.

Page 14: Overall Hypothesis

Figure 2B

Page 15: Overall Hypothesis

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.

Page 16: Overall Hypothesis

Confocal Microscopy

http://www.olympusfluoview.com/theory/index.html

Page 17: Overall Hypothesis

Figure 2C

Page 18: Overall Hypothesis

Supplemental Figure 5A & B

Page 19: Overall Hypothesis

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.

Page 20: Overall Hypothesis

Figure 3A

Page 21: Overall Hypothesis

Figure 3B

Page 22: Overall Hypothesis

Figure 3C

Page 23: Overall Hypothesis

Figure 3D

Page 24: Overall Hypothesis
Page 25: Overall Hypothesis

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.

Page 26: Overall Hypothesis

EFR

Page 27: Overall Hypothesis

Figure 4A

Page 28: Overall Hypothesis

Figure 4B

Page 29: Overall Hypothesis

Figure 4C

Page 30: Overall Hypothesis