overall hypothesis if n-glycans on prrs are the first to recognize invading pathogens, then...
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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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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.
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Figure 1 Background
• Method: – tDNA insertion mutants– 100nM elf18 or flg22 treatment
GOI - Gene of InterestARM - Antibiotic Resistance Marker
GOI
Ti Plasmid
ARM
Agrobacterium
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Figure 1A
Out of ALL mutants, only stt3a-2 was strongly insensitive to MAMP treatment
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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.
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Results: Supplemental Figure 2
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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.
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Figure 1B•Mutants showed to be more susceptible to bacteria
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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.
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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
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SDS-Page
• Separates proteins according to their size
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Figure 2A
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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.
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Figure 2B
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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.
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Confocal Microscopy
http://www.olympusfluoview.com/theory/index.html
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Figure 2C
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Supplemental Figure 5A & B
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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.
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Figure 3A
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Figure 3B
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Figure 3C
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Figure 3D
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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.
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EFR
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Figure 4A
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Figure 4B
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Figure 4C
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