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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:
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.
Figure 1 Background
• Method:
– tDNA insertion mutants
– 100nM elf18 or flg22 treatment
GOI
Ti Plasmid
Agrobacterium
GOI - Gene of Interest
ARM - Antibiotic Resistance Marker
Figure 1A
Out of ALL mutants, only stt3a-2 was strongly
insensitive to MAMP treatment
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.
Results: Supplemental Figure 2
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.
Figure 1B
•Mutants showed to
be more susceptible
to bacteria
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.
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
SDS-Page
• Separates proteins according to their size
Figure 2A
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.
Figure 2B
Results: Figure 2
• Localization of PRRs in selected Nglycosylation 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.
Confocal Microscopy
http://www.olympusfluoview.com/theory/index.html
Figure 2C
Supplemental Figure 5A & B
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.
Figure 3A
Figure 3B
Figure 3C
Figure 3D
Results Figure 4
• Hypothesis for Figure 4A
IF EFR function is solely based on NGlycosylation, THEN point mutations to
elimate N-Glycosylation motifs will result in
EFR dysfunction.
EFR
Figure 4A
Figure 4B
Figure 4C