overall hypothesis if n-glycans on prrs are the first to recognize invading pathogens, then...
TRANSCRIPT
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 - Gene of InterestARM - Antibiotic Resistance Marker
GOI
Ti Plasmid
ARM
Agrobacterium
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 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.
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 N-Glycosylation, THEN point mutations to
elimate N-Glycosylation motifs will result in EFR dysfunction.
EFR
Figure 4A
Figure 4B
Figure 4C