Dilantha Fernando, PhD Professor - Asianpgpr.comasianpgpr.com/4thAsianPGPR-PPT/Dilantha Fernando_4th...
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Dilantha Fernando, PhD Professor 4 th Asian PGPR Conference, Hanoi, Vietnam May 3-6, 2015
Transcript of Dilantha Fernando, PhD Professor - Asianpgpr.comasianpgpr.com/4thAsianPGPR-PPT/Dilantha Fernando_4th...
Dilantha Fernando, PhD Professor
4th Asian PGPR Conference, Hanoi, Vietnam May 3-6, 2015
Red River
Campus of UM
The Crop
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Canada and Canola - 20 million acres
Presenter
Presentation Notes
Canola is grown primarily in regions of western Canada, with some acreage being planted in Ontario and the Pacific northwest. Canola has surpassed wheat as the most valuable crop If you look at the numbers you will notice that total acreage more than doubled in the last ten years. Canadian grown canola contributes $15.4 billion annually to Canada’s economy Canada is the biggest single producer of canola in the world Most of the product is exported, and the biggest client is the united states.
Canada- China: Common interest in Global Canola/Rapeseed Production
EU2736%
India11%
China 21%
Australia3%
US1%
Ukraine3%
Canada20%
Other5%
EU27
India
China
Australia
US
Ukraine
Canada
Other
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Canola - The Health Facts
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The Pathogen: Sclerotinia sclerotiorum
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Sclerotinia Disease cycle
Apothecia germination Windborne ascospores
Lesions spread on stem Sclerotia form in stem Leaf infection
Petal infection
Infection stage
Narrow w indow of protection??
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Inhibition of Sclerotinia by P. chlororaphis PA-23 PA-23 Plant Beneficial Bacterium
PA-23
phenazine
Sclerotinia
Savchuk and Fernando, FEMS Microbiology Ecology 2004
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Pseudomonas chlororaphis PA23
• Gram-negative rod • Rhizobacterium • Biocontrol activity
Laser scanning confocal microscope image of PA23 with blue
autofluorescence thought to be due to phenazine and pyoverdin
(siderophore) production
Presenter
Presentation Notes
- from roots of soy bean plants
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P. chlororaphis (PA23) vs. stem rot of canola
Fernando et al, 2007 Crop Protection
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Investigation of Petals for Ascospore Germination (Savchuk and Fernando, FEMS Microbiology Ecology 2004)
(Fernando et al, 2007, Crop Protection)
S.s. Ascospores & PA23
9h
48h
S.s. Ascospores only
9h
18h
36h
48h
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Field experiments in 2001, thru 2004 Carman, Manitoba
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Fernando et al, 2007, Crop Protection
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Fernando et al, 2007, Crop Protection
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Effect of P. chlororaphis (PA- 23) on canola stem rot under field conditions
PA-23 (9 x108cfu/ml) was sprayed at 30 and 50% bloom followed by ascospore spray (8 x 104 cfu/ml)
PA23* - Double spray (9 x 108 cfu/ml)
IC – Inoculated control (8x104 cfu/ml)
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PA23 PA23* IC
Treatments
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infe
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PPI PSI
PPI - Percent petal infestation; PSI – Percent stem rot incidence
ab ab
b b
a a
Fernando et al. 2007 Crop Protection
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Detection of PCA biosynthetic gene by PCR
1650bp
Phz1/phz2
Neg.ck
2-79 PA
-23 1kb ladder
Positive strain: P. fluorescens 2-79: 1400bp
1000bp
Zhang et al., 2006 Can J Microbiology
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1650bp
Pf-5 PA
-23 Pf-5 PA
-23 Pf-5 PA
-23 1kb ladder
Positive strain: P. fluorescens Pf-5:
1050bp 768bp
719bp 1000bp
Detection of pyrrolnitrin biosynthetic gene by PCR
Zhang et al., 2006 Can J Microbiology
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HPLC bioassay for phenazine production
Zhang et al. Can J M icrobiology 2006
•Phenazines
•Pyrrolnitrin
-Other compounds:
-Volatile antibiotics, e.g. HCN
-Protease, lipase, chitinase
-Siderophores
P. chlororaphis PA23
Zhang, et al, 2006: Can. J M icrobiology Poritsanos et al, 2006, Can J M icrobiology
Presenter
Presentation Notes
Characterization of PA23 revealed it produces an arsenal of potential AF products including: PHZ PRN HCN Degradative enzymes & siderophores
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Global Regulation of Secondary Metabolite Production in P. chlororaphis
Antifungal Activity
Mutant 63 (phzE Tn mutant) – increased AF activity compared to PA-23 wild type
Mutant 314 (gacS Tn mutant) no AF activity
Poritsanos et al., 2006 CJM
Pyrrolnitrin? Wt PA23
phzE: Phenazine deficient
Pyrrolnitrin - deficient
Phenazine and Pyrrolnitrin deficient
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4 2
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Selin et al. 2010. FEMS Microbiology Ecology
Presenter
Presentation Notes
We next went on to look at whether PRN was this critical compound. No Tn mutants in the prnABCD biosynthetic locus were identified with our original screen Thus, a prn knock out mutant was created (by allelic exchange) We also generated a PRN/PHZ double mutant Analysis of these strains in AF assays showed that the prn-deficient strain was markedly reduced in its zoi; the double mutant had a total loss of AF activity Thus, PRN plays a more critical role in controlling S.s.; PHZ also plays a role but a more minor one
Plant studies - canola
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Disease control
PA23 PA23-63 (phz-)
PA23-1 (prn-)
PA23-63-1 (prn/phz-)
Healthy control
Dis
ease
sev
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(0-7
sca
le)
a a
b b b
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Selin et al. 2010. FEMS Microbiology Ecology
Presenter
Presentation Notes
Taking this analysis to the greenhouse, we observed similar findings – explain results
Figure Biosynthesis of PRN and PHZ from shikimic acid. ADIC, 2-amino-4 deoxychorismic acid; PCA, phenazine-1-carboxylic acid; PRN, pyrrolnitrin.
• Why does the PHZ mutant exhibit ↑ AF activity in vitro?
• It produces 2x as much PRN as the WT strain (HPLC analysis)
• no change in prn gene transcription
• PHZ and PRN share a common biosynthetic pathway
• increased PRN levels in PHZ mutant likely due to the an increase of available chorismic acid
Selin et al 2010 FEMS Microbiology Ecology
Presenter
Presentation Notes
One of the perplexing questions was why did we observed increased AF activity in vitro? Analysis of culture extracts showed that the PHZ mutant was producing 2x as much PRN as the wt Analysis of a prnA-lacZ transcriptional (TS) fusion showed no change in gene expression When we looked at the biosynthetic pathways for the two antibiotics we noticed that they have some common steps Specifically, they both use chorismic acid. Thus, we believe that the phzE mutation leads to increased available chorismic acid which can then be used for PRN biosyntheis
Conclusion Pyrrolnitrin production is sufficient for PA23 biocontrol of S. sclerotiorum
Selin et al 2010 FEMS Microbiology Ecology
Presenter
Presentation Notes
This finding is important, because when we look at what factors affect antibiotic expression, we can focus our attention on PRN (and not worry about PHZ) for control of S.s. Many of the conditions that favor expression of one antibiotic are different from those needed for maximal expression of another – this holds true for PRN and PHZ. Again, we can concentration of maximizing PRN production.
Stringent Response
• Global regulatory mechanism that allows bacteria to survive nutrient deprivation
• Shift activities from those involved in growth to those necessary for survival
• Highly relevant to bacteria (e.g. PA23) in environment where nutrients are scarce (phyllosphere??)
• How does the SR affect PA23 biocontrol?
(Manuel et al. 2012, M icrobiology)
• Stress response induced upon nutrient deprivation. – (p)ppGpp accumulation = global regulator
• In Gram-negative bacteria: – RelA: synthetase – SpoT: hydrolase/synthetase
Stringent Response
GTP/GDP
ppGpp
ATP RelA
SpoT
Pi (Manuel et al. 2012, M icrobiology)
Presenter
Presentation Notes
To provide some background on the SR and how it affects bacterial physiology – this is a stress response that becomes activated upon nutrient starvation These conditions lead to accumulation of a small molecule called pppGpp – sometimes referred to as an alarmone Accumulation of this molecule leads to a global shift in gene expression Cells turn off gene needed for rapid growth, protein synthesis, cell division, etc. Turn on genes required for survival – amino acid biosynthesis, carbon sequestration, etc. Once nutrients become available, metabolism shifts back to normal Accumulation of pppGpp is brought about by two enzymes: RelA synthesizes it SpoT can act as either a synthetase or hydrolase (i.e. breaks it down) depending on the conditions
Nutrient Stress
RelA SpoT
(p)ppGpp
Stress Response Genes
Direct Indirect
(Manuel et al. 2012, M icrobiology)
Presenter
Presentation Notes
So, when cells undergo nutrient deprivation, RelA and SpoT get stimulated to generate pppGpp, This then affects the RNA polymerase and shifts gene expression towards those involved in stress response and survival This is also linked to the RpoS (stress response sigma factor) regulon
• Does the SR (nutrient deprivation) affect the biocontrol ability of PA23?
• Mutants were generated in genes encoding
RelA and SpoT
• No longer able to produce pppGpp and undergo SR
• Analyzed for affect on biocontrol
(Manuel et al. 2012, M icrobiology)
Presenter
Presentation Notes
Thus, we were interested to learn whether the expression of biocontrol factors was affected by the SR (i.e. nutrient deprivation) Mutants of PA23 were generated in the genes encoding RelA and SpoT such that they could no longer make pppGpp and undergo the SR These were then characterized to see how they were impacted
PA23 Antifungal Activity Strain Zone of Inhibition (cm)
PA23 (pUCP22) 1.8 (0.06) PA23relA (pUCP22) 2.3 (0.15) PA23relAspoT (pUCP22) 2.4 (0.23)
PA23relA (pUCPrelA) 1.8 (0.21) PA23relAspoT (pUCPrelA) 2.0 (0.06)
(p)ppGpp Biocontrol Activity represses
Selin et al, 2014 Biological Control
Presenter
Presentation Notes
The first thing that was examined was AF activity Analysis of the SR mutants revealed increase activity Addition of relA in trans (pUCPrelA) restored/reduced AF activity back to wt RelA is the primary enzyme that generate pppGpp (we didn’t add back spoT because it acts as both hydrolase and synthetase) Thus, the SR has a repressive effect on PA23 fungal antagonism
Antibiotic Production
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Activ
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iller
Uni
ts)
Time (hours)
• The stringent response: • negatively impacts pyrrolnitrin production • does not impact phenazine and HCN expression
Strains Pyrrolnitrin Production
(µg/ml) PA23 (pUCP22) 0.0080 (0.005)
PA23relA (pUCP22) 0.0183 (0.001)
PA23relAspoT (pUCP22)
0.0190 (0.002)
PA23relA (pUCPrelA) 0.0087 (0.003)
PA23relAspoT (pUCPrelA)
0.0096 (0.006)
PA23
∆relA
∆relAspoT
prnA-lacZ activity
Presenter
Presentation Notes
Analysis of Antibiotic production revealed that the SR mutants had elevated production of PRN (Table) No change in either PHZ or HCN Gene expression studies revealed that prn gene transcription was elevated in the SR mutants
SR & production of biocontrol factors
(p)ppGpp Pyrrolnitrin
Protease Lipase
Phenazines, HCN
Hypothesis: When cells are facing starvation conditions, they shift activities from non-essential (i.e. biocontrol) to those required to cope with starvation
Presenter
Presentation Notes
Thus, the SR has no effect on production of PHZ and HCN It represses production of PRN, protease and lipase (because the knock out mutants showed elevated levels of these compounds)
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Biofilms
Surfaced-attached community
Extra-cellular polymeric matrix = “slime”
Single/mixed species
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Advantages to “Gated Community” Living
Tolerance to antimicrobials
Shelter from environment:
UV, desiccation, rainfall, temp. variations, wind, humidity, etc.
Share metabolic byproducts
Horizontal gene transfer
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CELL CLUSTERING/
MICROCOLONY FORMATION
PRODUCTION OF MUSHROOM-
LIKE STRUCTURES
DETACHMENT
PLANKTONIC CELLS
ATTACHMENT &
PROLIFERATION
OF CELLS
Bacterial Biofilm Development
Flagella Motility
Pili
Type I/IV Pili Biosurfactants
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Biofilms and PA23
Does PA23 have PGPR activity?
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Biocontrol agents “prime” plant defences
• Faster/stronger induction of basal resistance
• Systemic acquired resistance (SAR)
• Induced systemic
resistance (ISR)
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Pieterse et al. Nature Chemical Biology 5, 308 - 316 (2009) doi:10.1038/nchembio.164
Presenter
Presentation Notes
Plants have an innate immune system! -> can be ‘primed’: faster/stronger induction of basal resistance Priming: Activation of a latent defence mechanisms that are expressed upon subsequent challenge. Interconnected signalling pathways in which hormones play a role SAR: SA-inducible defences (hostile organisms)�ISR: JA-inducible defences (plant beneficial organisms (bacteria/fungi))
Changes that occur in “primed” plants
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V. Pastor et al. Environmental and Experimental Botany 94 (2013) 46– 56
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PA23
• ACO: ethylene biosynthesis (ISR) • AOS: jasmonic acid biosynthesis (ISR) • PR-1: pathogenesis-related protein (SAR) • PDF1.2: ISR and SAR
How does PA23 affect the expression of genes involved in priming?
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11.25
15.00
0 HRS 3 HRS 6 HRS 12 HRS 24 HRS 48 HRS
Fold
cha
nge
ACO PR1
Presenter
Presentation Notes
qPCR: PA23 done 3x (average of the averages used); others done 1x. ct values range: 19-34. PA23: Also checked AOS, PDF1.2 All primers from U.Sask. paper: PR1: too large region amplified (640bp) PR2: too large; 26 copies in B. napus PR5: too large; 2 copies AAO1: (auxin) region aplified is downstream of one of the copies; did not work at all. 8 copies expected. ACO: 2 copies; both correspond to primers used CCR: 4 copies. Different genes targeted by thes primers: “CCR-related” OPCL: 3 copies. None amplified - R primer doesn’t match OPR2: 2 copies aplified. SAM3: 4 copies. 1 copy amplifies.
Why is RNAseq more suitable?
Frequency of sequences in a library population Advantages:
• No transcript-specific probes required
• Highly sensitive • High dynamic range
Wang et al., Nat Rev Genet. 2009 Jan; 10(1): 57–63. 43
Presenter
Presentation Notes
detect novel transcripts, gene fusions, single nucleotide variants, indels (small insertions and deletions), and other previously unknown changes that arrays cannot detect. RNA-seq is better at detecting differential gene expression at low expression levels than is microarrays Most current sequencing platforms are capable of providing only relatively short sequence reads (~40-400bp depending upon the platform). Therefore, most protocols incorporate a fragmentation step to improve sequence coverage over the transcriptome.
Establishing large-scale datasets of the Sclerotinia-canola pathosystem = extending to a Tripartite system
Infected leaves Tissues collected with laser microdissection
Next Generation Sequencing
Laser Microdissection (LMD), has the unique ability of isolating individual cells and tissues without contamination for further genetic analyses: • Genomics, transcriptomics, proteomics,
metabolomics
Tissue is embedded in paraffin, cut into thin sections (10 microns), and placed on a membrane
A high powered laser is then used to excise desired tissues
What is laser microdissection?
Presenter
Presentation Notes
Insert light microscopy here… video…
How do the plants respond to stress at the RNA Level?
Infect leaf tissue with petal inoculation
Excise lesion & RNA isolation with βME
Illumina RNA-sequencing: 100bp PE reads
Data analysis and visualization
1 2
3 4
RNA quality assessment, cDNA synthesis & library
preparation
Presenter
Presentation Notes
Consider describing exactly how much infected leaf tissue you used; indicate that the library prep & other associated steps were all done in China
Differential expression analysis gives scope of transcriptional reprogramming in infected leaves
What are some of the ways we can extract biological meaning from these numbers? • Enrichment of AT homologues for
identification of over-represented GO terms
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f DE
tran
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Fold change analysis
WestarZY821
Up regulated
1561 3834 2059
Westar ZY821
Down regulated
2377 4151 1916
Westar ZY821
Presenter
Presentation Notes
Differential gene expression analysis identifies transcripts involved in the infection process. Genes are considered DE if they have a minimum log2 fold change of 2 AND a p-value < 0.01 How can I use this data to get biological relevant information, can do it by using arabidopsis homologs,
Enriched GO terms
Description Shared down Shares up ZY821
down ZY821 up ZY821
specific down
ZY821 specific up
Westar down Westar up
Westar specific down
Westar specific up
apoplast 0
-3.8846260
23
-1.2665151
29
-4.3497779
41
-2.0459046
01
-1.9406180
13 0
-15.706970
83 0
-14.608833
27
cell wall -
2.88764798
-5.1600970
63
-2.2094771
88
-3.2490074
31
-1.0931890
12
-1.1442521
02
-1.2877167
81
-15.583810
6 0
-13.584511
62
plasma membrane -
3.581352013
-7.4006396
41
-3.5693116
74
-6.7940657
78
-1.7570709
37
-2.4538094
56
-1.8309040
96
-11.864536
27 0
-6.5699864
9
chloroplast stroma -
2.18894376
-2.6511988
81
-3.8549434
93
-1.8408045
1
-3.0375758
27 0 0
-10.264407
35 0
-10.210898
45
response to chitin 0
-2.0932730
23 0
-5.0171648
71
-0.8784448
09
-3.3848603
14 0
-6.6785711
44 0
-8.1606609
15
response to wounding 0
-0.9196706
31 0
-4.7521935
26 0
-4.8653423
26 0
-5.9997665
89
-1.1264087
65
-7.8440262
3
chloroplast -
1.717308848
-3.1407776
81
-1.6199643
32
-1.6907848
64 0 0 0
-6.6391495
71 0
-5.0140324
69
transcription factor activity 0 0 0 0 0 0
-1.9533688
52 0
-5.4124550
4
-1.6016833
13
defense response -
1.829165449
-2.3452950
56
-1.4042061
19
-5.3297870
3 0
-4.9388291
42
-1.4704664
92
-2.2467401
58
-1.3450970
62
-2.2386033
29
defense response to
-1 6137422
-0 8562982
-1 7133587
-0 9846055
-0 7827133
-4 2253335
-3 8827561
Presenter
Presentation Notes
Gene ontology, or GO, is a major bioinformatics initiative to unify the representation of gene and gene product attributes across all species.[1] More specifically, the project aims to: Maintain and develop its controlled vocabulary of gene and gene product attributes; Annotate genes and gene products, and assimilate and disseminate annotation data; Provide tools for easy access to all aspects of the data provided by the project, and to enable functional interpretation of experimental data using the GO, for example via enrichment analysis. GO is part of a larger classification effort, the Open Biomedical Ontologies (OBO).[2]
Enriched GO term: Apoplast
Westar 48 hpi ZY821 48 hpi
Cell degradation is more pronounced in the susceptible line Westar
Description Shared down
Shares up
ZY821 down ZY821 up
ZY821 specific down
ZY821 specific
up
Westar down
Westar up
Westar specific down
Westar specific
up
Apoplast 0
-3.884626
023
-1.266515
129
-4.349777
941
-2.045904
601
-1.940618
013 0
-15.70697
083 0
-14.60883
327
Presenter
Presentation Notes
Apotosis go term
Enriched GO term: JA signaling pathway
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control 12HPI 24HPI 48HPI
AOC
Westar ZY821
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80
100
120
140
control 12HPI 24HPI 48HPI
AOS
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20
30
40
50
60
70
control 12HPI 24HPI 48HPI
PDF1.2
Rela
tive
mRN
A le
vel
D
Description Shared down Shares up ZY821
down ZY821 up ZY821
specific down
ZY821 specific
up
Westar down
Westar up
Westar specific down
Westar specific
up
jasmonic acid mediated signaling pathway
-0.697864
622
-1.429203
603 0
-3.519697
826 0
-2.911604
873
-0.705267
39
-2.066786
018 0
-1.788475
829
Presenter
Presentation Notes
GOSEQ: Gene ontology analysis for RNA-seq GOSEQ, a new module to MeV 4.7, is a technique for identifying differentially expressed sets of genes, such as GO terms while accounting for the biases inherent to sequencing data.
0%
25%
50%
75%
100%
Ss Water
Infe
ctio
n ra
te
Petals Leaves
Ss
PA23/Ss
PA23
Water PA23 prevents infection of canola with S.
sclerotiorum
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Does PA23 prevent S. sclerotiorum growth on canola?
52
What is the role of ROS in plant “priming”?
• ROS: normal products of plant cellular metabolism • Excessive amounts: cell death • O2˙¯ and H2O2: strengthen cell wall • H2O2: diffusible signalling molecule • Arabidopsis: Some priming dependent on H2O2
Sharma et al., 2012. doi:10.1155/2012/217037
Presenter
Presentation Notes
H2O2 has no unpaired electrons so it can easily cross membranes
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ROS: What do we expect?
• Moderate ROS production: biocontrol agent (PA23)
• Excessive ROS production: pathogen (S.s.)
• Biocontrol agent + pathogen: somewhere in the middle
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Hydrogen peroxide production
Water PA23 + S.s. PA23 S.s.
PA23 induces moderate H2O2 production
Staining with 3,3-diaminobenzidine (DAB) -> brown pigment
Presenter
Presentation Notes
Staining with 3,3-diaminobenzidine (DAB)
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Superoxide production
PA23 PA23 + S.s. Water S.s.
PA23 induces moderate superoxide production
Staining with nitrotetrazolium blue chloride (NBT) -> blue pigment
Presenter
Presentation Notes
Staining with nitrotetrazolium blue chloride (NBT) -> blue pigment
Acknowledgements • Collaborators:
– Teri de Kievit, Mark Belmonte
• Biocontrol work: Transcriptome work: – Carrie Selin (Ph.D.) – Jerrylynn Manuel (Ph.D.) Sherry Mao – Rahim Habibian (former Post Doc/RA) Kelly Duke – Yilan Zhang (MSc) – Nicole Poritsanos (MSc) – S. Nakkeeran (Post Doc)
• Funding: • NSERC Discovery, CIHR-IPM, ARDI, and Canola
Council of Canada
Thank you!
