Perception of B. phytofirmans-derived flg22 induces weaker defense responses in grapevine than do X. campestris- or P. aeruginosa-derived flg22

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MAMP responsiveness in cells and in vitro plantlets

Cell culture equilibration for early signaling bioassays

To measure early signaling events, such as ROS production, variations in cytosolic Ca2+ concentrations ([Ca2+]cyt) and MAPK phosphorylation, cells were collected and washed three times with M10 buffer (175 mM mannitol, 0.5 mM CaCl2, 0.5 mM K2SO4, 10 mM MES, pH 5.3) and
suspended at 0.1 g fresh weight of cells (FWC).ml-1 in M10 buffer. For measurement of H2O2 production or MAPK phosphorylation, cells were equilibrated 1h (130 rpm, 25°C) before elicitor treatments. For [Ca2+]cyt variations, washed cells were processed as described elsewhere (§ 2.1.3).
Arabidopsis Col-0 cells were washed with M10 pH 6.2, equilibrated 1h (130 rpm, 25°C) and used for measurements of H2O2 production.

Luminol-based oxidative burst analysis

After equilibration, cells were treated with elicitors or a control treatment. At a given time point post treatment, 250-μL cell aliquots were mixed with 300 μL of H50 buffer (50 mM HEPES, 175 mM mannitol, 10 mM CaCl2, 0.5 mM K2SO4, pH 8.5) and 50μL of 0.3 mM luminol.
Luminescence, expressed in relative luminescence units (RLU), was integrated over 10s by a luminometer (Lumat LB9507, Berthold Technologies) and was converted into nmol H2O2.g-1 FWC, using a standard calibration curve obtained by addition of H2O2 in grapevine cell suspension aliquots. For dose response, oxidative burst was measured at 15 min post treatment or at maximal response. Oxidative burst in Arabidopsis cell suspensions was monitored using the same protocol as for grapevine.
The ROS production in Arabidopsis leaf discs was measured in two discs per plant from at least 6 plants. Leaf discs (4 mm diameter) were cut and floated on 100 μl ultrapure water in a 96-well plate overnight in darkness at room temperature. Then 16 h later, water was replaced with 100 μl of the reaction/elicitation mixture (60 μM luminol, 1 U horse radish peroxidase, elicitor) and the luminescence (RLU) was recorded every 90 s (integration over 1 s) and until 60 min, using a microplate luminescence reader (Mithras LB 940, Berthold Technologies).
For grapevine, at least 12 discs (4 mm diameter) from 3 leaves of 2 plants were vacuuminfiltrated with water and floated on 100 μl water in a 96-well plate overnight in darkness at room temperature. Then 16 h later, water was replaced with 100 μl of the reaction mix (60 μM luminol and 10 U horse radish peroxidase in H50 buffer, pH 8.5) and luminescence was counted as described for Arabidopsis leaf discs. Once ROS levels decreased to the basal level ≤ 80 RLU (~5 min), elicitor or water was added in each well and the luminescence was recorded every 90s and during a 60 min period.

Analysis of free cytosolic calcium concentration variation

Apoaequorin-expressing grapevine cells were suspended in M10 buffer (§2.1.1) and further incubated for 4h with 3 μM coelenterazine (130 rpm, 25°C, at dark) to perform the in vivo aequorin reconstitution before the elicitor treatments. Then, 250 μl cell aliquots were treated with elicitors and the emitted bioluminescence was recorded as RLU s-1 for 30 min using a luminometer. Remaining aequorin was discharged by automatic injection of 300 μl of lysis buffer containing an excess of Ca2+ (2M CaCl2 in 20% ethanol (v/v)) and luminescence was recorded for another 5 min until values were within 1% of the highest discharge value. RLU values were converted into Ca2+ concentrations using the calibration equation p([Ca2+]cyt) = 0.332588 (-log k) + 5.5593, described in detail by Rentel and Knight (2004), where k is the luminescence counts per second/total luminescence counts remaining (Ranf et al., 2008).

MAPK bioassay

Grapevine cells were first equilibrated (§2.1.1), then treated with elicitors or a control treatment. Aliquots of 1.5 ml were harvested at 0, 5, 10, 15, 30 and 60 min post treatment by filtration on GF/A filters, frozen in liquid N2 and kept at -80°C prior to perform the protein extraction (§2.2.1) and MAPK detection (§2.2.4).
Leaves of in vitro grapevine plantlets were first vacuum-infiltrated with water then floated on water (lower leaf surface facing the solution) during 2 h before adding elicitor solutions.
Treated leaves were collected into liquid N2 at 15 min post treatment and kept at -80°C prior to perform the protein extraction (§2.2.2) and MAPK detection (§2.2.4).

Defense gene induction assay

For defense gene expression kinetics on grapevine cell suspensions, the cell culture density was adjusted to 0.1 g FWC ml-1 with sterile Nitsch-Nitsch medium 16 h prior to experiment.
Otherwise, they were maintained under their culture conditions (25°C, continuous light, 120 rpm). Cells were then treated with elicitors or a control treatment and sampled under sterile conditions.
Aliquots of 1 ml were harvested at indicated time points into liquid N2.
Leaves of in vitro grapevine plantlets were floated on elicitor solutions with the lower leaf surface facing treatment in the growth climatic chamber (25°C). After 6 h of treatment, leaves were harvested into liquid N2. For the basal level of VvPRR transcripts, leaves from 2-month old plantlets were frozen.
All harvested tissues were kept at -80°C prior to RNA extraction (§2.2.7) and qPCR (§2.2.8).

Table of contents :

I. General introduction
1 Socio-economical context
2 Plant immunity
2.1 MAMP-triggered immunity
2.1.1 MAMPs
2.1.2 PRRs, receptors to MAMPs and DAMPs
2.1.2.1 Leucine rich repeat (LRR) receptors
2.1.2.2 Lysin motif (LysM) receptors
2.1.3 PRR-mediated signaling and defense
2.1.4 PRR-mediated disease resistance
2.2 Effector-triggered immunity
2.2.1 Effectors target MTI
2.2.2 ETI responses
3 Grapevine: its biotic interactions and immunity
3.1 Biotic interactions
3.1.1 Fungi
3.1.2 Bacteria
3.2 Grapevine MAMP-triggered immunity
II. Flagellin-triggered immunity
1 Bacterial flagellum and flagellin
2 Flagellin: a general elicitor of MTI
2.1 flg22/FLS2 perception system in plants
2.1.1 Ligand binding
2.1.2 Downstream signaling
2.1.3 FLS2/flg22 signaling regulation
2.1.4 FLS2 protein structure
2.1.5 Crosstalk with brassinosteroid signaling and other MAMP signaling pathways
2.1.6 FLS2 polymorphism: ligand specificities
2.2 Extra-flg22 flagellin recognition
2.2.1 flgII-28: a novel flagellin epitope for plants?
2.2.2 Flagellin glycosylation
2.3 Flagellin/TLR5 perception in animals
3 The flagellin perception upon plant-bacteria interaction
3.1 Evasion of flagellin-mediated immunity
3.2 The role of FLS2-mediated sensing
III. Chitin-triggered immunity
1 Microbial GlcNAc-containing ligands
2 Chitin, a structural component of fungal cell walls
3 Chitin: elicitor of MTI in plants
3.1 OsCEBiP/OsCERK1 perception system in rice
3.2 CERK1: perception system in Arabidopsis
3.2.1 AtLYM2-mediated chitin perception independently of AtCERK1
3.3 Tight regulation of chitin and PGN perception
3.4 Role of other LysM-RLKs (LYKs) in Arabidopis
3.5 Chitosan perception
3.6 Chitin perception by animals
4 Role of chitin perception in plant immunity
AIMS OF WORK
MATERIALS AND METHODS
1 Materials
1.1 Grapevine materials
1.1.1 Cell suspensions
1.1.2 In vitro plantlets
1.1.3 Plants
1.2 Arabidopsis materials
1.2.1 Cell suspensions
1.2.2 Plants
1.3 Microorganisms
1.3.1 Botrytis cinerea
1.3.2 Plasmopara viticola
1.3.3 Burkholderia phytofirmans
1.4 Elicitors
1.4.1 Peptides
1.4.2 Oligosaccharides
1.4.3 Elicitor doses
2 Methods
2.1 MAMP responsiveness in cells and in vitro plantlets
2.1.1 Cell culture equilibration for early signaling bioassays
2.1.2 Luminol-based oxidative burst analysis
2.1.3 Analysis of free cytosolic calcium concentration variation
2.1.4 MAPK bioassay
2.1.5 Defense gene induction assay
2.1.6 Cell death quantification
2.2 Biochemistry, molecular biology and bioinformatics
2.2.1 Total protein extraction from grapevine cells and Arabidopsis
2.2.2 Total protein extraction from in vitro grapevine plantlets
2.2.3 Protein extraction enriched in membrane fraction
2.2.4 Detection of phosphorylated MAPK by Western blotting
2.2.5 Detection of GFP and VvFLS2 by Western blotting
2.2.6 Generation of VvFLS2 antibody and dot-blot specificity test
2.2.7 Isolation of total RNA
2.2.8 cDNA synthesis and quantitative real-time PCR (qPCR)
2.2.9 Bioinformatics
2.2.10 General cloning technics
2.2.11 Cloning of GFP-tagged or antisense VvPRRs by Gateway® technology
2.3 Plant transformation
2.3.1 Grapevine transformation and plantlet generation via somatic embryogenesis
2.3.2 Arabidopsis transformation and mutant screening
2.4 Histochemical GUS detection in Arabidopsis pPR1::GUS seedlings
2.5 Flg22- triggered growth inhibition assays on Arabidopsis and grapevine
2.6 Protection assays on grapevine leaf discs
2.7 Grapevine infection with B. phytofirmans
2.8 Confocal microscopy
RESULTS AND DISCUSSION
I. Screening of MAMP responsiveness in grapevine
II. Flagellin perception system in grapevine
Results
1 Flg22 induces immune responses and resistance against Botrytis cinerea in grapevine
2 In silico characterization of the predicted grapevine FLAGELLIN SENSING 2 receptor: VvFLS2
3 VvFLS2 functionally complements the Arabidopsis fls2 mutant and is localized at the plasma membrane
4 Recognition specificities of flagellin perception in grapevine
4.1 Perception of B. phytofirmans-derived flg22 induces weaker defense responses in grapevine than do X. campestris- or P. aeruginosa-derived flg22
4.2 AtFLS2 and VvFLS2 have different recognition specificities
4.3 B. phytofirmans overcomes Xc flg22-induced MTI to colonize grapevine plants
5 Silencing of VvFLS2 in grapevine induced defects in flg22 immune signaling
5.1 Generation of antisense VvFLS2 lines
5.2 Screening of asVvFLS2 lines for VvFLS2 transcript amounts and flg22 responsiveness
5.3 The line #2-22 is affected in flg22 signaling
5.4 VvFLS2 protein detection
6 FLS2-like gene in grapevine
Discussion
1 The FLS2/flg22 perception system is conserved in grapevine and triggers a typical MTI
2 The reduction in VvFLS2 transcript levels affects the flg22 signaling in grapevine
3 Weak eliciting activity of Bp flg22 in grapevine
4 AtFLS2 and VvFLS2 have different recognition specificities
5 B. phytofirmans overcomes MTI in Arabidopsis and grapevine to colonize plants
6 VvFLS2-like gene in grapevine
Perspectives
III. Chitin perception system in grapevine
Results
1 Chitin and chitosan induce defense responses in grapevine
2 LysM-RLKs (LYKs) in grapevine and identification of putative AtCERK1 orthologs in grapevine
2.1 In silico characterization of the predicted grapevine CHITIN ELICITOR RECEPTOR KINASE 1
orthologs: VvCERK1, 2 and 3
2.2 Functional complementation of the Arabidopsis cerk1-2 mutant with grapevine VvCERKs
2.2.1 Constitutive overexpression of VvCERK1 does not complement cerk1-2
2.2.2 Constitutive overexpression of VvCERK2 or VvCERK3 leads to cell death
2.2.3 Inducible expression of VvCERKs in cerk1-2 background
2.3 Silencing of VvCERKs in grapevine
3 LysM-RLPs (LYPs) family and identification of putative OsCEBiP ortholog in grapevine (VvCEBiP)
3.1 In silico characterization of the predicted grapevine CHITIN ELICITOR BINDING PROTEIN
orthologs
3.2 Silencing of VvCEBiP1 in grapevine
Discussion
1 Chitin is a weak elicitor in grapevine
2 Role of VvCERK1
3 Role of VvCERK2
4 VvCERK3 can partly complement the chitin-induced ROS burst in Atcerk1-2
5 A partial loss of VvCERK3 in grapevine does not attenuate chitin responses
6 VvCERK-associated cell death phenotype
7 Lethality of antisense VvCERK1 and VvCERK2 calli
8 The role of the closest grapevine ortholog of OsCEBiP in chitin perception

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