OGs triggered MAPK phosphorylation, independently of NO production

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Production of pathogenesis-related proteins (PR proteins)

PR proteins are synthesized in response to pathogens in many plant species. They were classified into 17 families on the basis of their biochemical and biological properties (Van Loon et al., 2006). Most of them have antimicrobial properties and act through hydrolytic activities, resulting in degradation of the wall of the pathogen.
For example, -1,3-glucanase (PR-2) and chitinases (PR-3, PR-4, PR-8 and PR-11) break down the cell walls of fungi. The family of PR-7 includes endoproteases. The PR-12 (defensin), PR-13 (thionins) and some lipid transfer proteins (PR-14) have both antimicrobial and antifungal properties. Protein families PR-1 and PR-5 (thaumatin-like protein) seem to act against oomycetes and the family of PR-10 (ribonuclease-like protein) is involved in the defense against viruses. Class 6 protein contains protease inhibitors and their targets are nematodes and herbivorous insects. PR-15 (oxalate oxidase) and PR-16 (oxalate oxidase-like protein) have a superoxide dismutase activity that generates H2O2, which can be toxic or play a role in signaling.
However, some seem rather involved in the development of resistance as PR-9 (anionic peroxidase) which is likely peroxidases involved in strengthening the cell wall of the plant (Van Loon et al., 2006).
The expression of the PR genes is under the control of the phytohormones SA, JA and/or ET (Van Loon et al., 2006). Thus, these PR proteins can serve as a marker of the involvement of one of the three phytohormones. For example, PR-1 protein is used as a marker of SA-dependent defense pathway and for SAR.

Hypersensitive response (HR)

Many authors have described first hypersensitive response (HR) similar to apoptosis in animals (Greenberg and Yao, 2004). The HR is characterized by several cellular events including condensation of cytoplasm and chromatin, the release of cytochrome c from the mitochondria or the involvement of cysteine proteases (Wall et al., 2008).
In plant-pathogen interaction, the HR is defined as a localized cell death at the site of infection by the pathogens (fungi, bacteria and viruses), which causes the appearance of necrotic lesions. In responses to biotrophic or hemibiotrophic pathogens, this local resistance limits the development of the pathogen by reducing access to available nutrients (Dangl et al., 1996; Greenberg and Yao, 2004). In contrast, it was observed that the HR supports the development of necrotrophic pathogens that feed on dead tissue, such as the fungus B. cinerea (Govrin and Levine, 2000). The HR is considered by some authors as the final stage of development of resistance (Mur et al., 2008). It is often associated with resistance to race-specific type and can be triggered by general elicitors (Heath, 2000; Dangl and Jones, 2006). For example, some PAMPs, such as harpin, can induce HR responses (Jones and Dangl, 2006). In contrast polysaccharides (e.g. OGs, laminarin) do not induce HR and necrosis.
The precise molecular mechanisms contributing to the establishment of HR remain controversial. This probably reflects the fact that the events underlying its implementation vary depending on the pathosystem, and even considered the effector (Shapiro and Zhang, 2001). The signal transduction of programmed cell death (PCD) begins with an increase in free [Ca2+] cyt and [Ca2+] of nuclear core, observed during cell death triggered by cryptogein in tobacco cell suspensions (Ma and Berkowitz, 2007; Lecourieux et al., 2006). The link between HR and transporting calcium into the cell was established using A. thaliana mutant dnd1 to avirulentpathogens (Clough et al., 2000).

Systemic acquired resistance (SAR)

SAR is a form of resistance set up after avirulent pathogens attack and spreads in the whole plant through the vascular system (Sticher et al., 1997). It should be noticed that elicitors such as polysaccharides or elicitins could also induce SAR. It allows protecting the plant against a subsequent attack by a broad spectrum of pathogens including viruses, bacteria, oomycetes and fungi and is effective at least for several weeks. Many studies have shown that the establishment of the SAR involved the SA-dependent pathway. It accumulates at the point of infection and in uninfected tissues (Figure 1.10). In addition, an increased expression of genes encoding some PR proteins is observed in the SAR, contributing to the maintenance of the state of plant resistance (Durrant and Dong, 2004). Although the mobile signal for SAR has been the subject of considerable research over years, its identity remained controversial (Liu et al., 2011).

Induced systemic resistance (ISR)

Among the bacteria in the rhizosphere, some rhizobacteria, called PGPR (plant growth- promoting rhizobacteria), are able to stimulate plant growth and improve its strength vis-à-vis many stress (Van Loon et al., 1998). The ISR is also observed in the case of plants colonized by mycorrhiza (Pozo and Azcona-Aguilar, 2007). ISR provides better resistance to the plant during subsequent attacks by pathogens (Pieterse et al., 1996). This resistance is used in different SAR signaling pathways because it is regulated by JA and ET, and is independent of SA (Van der Ent et al., 2009; Figure 1.10). ISR triggered by beneficial microorganisms is associated with priming rather than with direct activation of defence (Conrath et al., 2006; Pozo et al., 2008; Van Wees et al., 2008). ISR induced modulation of gene expression, mainly involved in the defense or the regulation of transcription in roots (Verhagen et al., 2004) against pathogen or insect that are sensitive/or respond to JA and ET (Ton et al., 2002; Van Oosten et al., 2008).

Basic concepts of NO synthesis in animals

In animals, NO is synthesized from L-arginine and oxygen by nitric oxide synthase (NOS). Three highly homologous mammalian isoforms of NOS have been identified: neuronal NOS (nNOS), endothelial NOS (eNOS) and inducible NOS (iNOS; Wendehenne et al., 2001).
Each NOS is a modular enzyme that consists of a C-terminal reductase domain and an N-terminal oxygenase domain, both domains being separated by a short calmodulin (CaM) binding site (Poulos et al., 1998; Figure 1.11). In addition to the CaM binding site, NOS contains binding sites for NADPH, flavin mononucleotide (FMN), flavin adenine dinucleotide (FAD), tetrahydrobiopterin (BH4) and a heme group (Wendehenne et al., 2003; Figure 1.11). Functional NOSs are active as a homodimer and transfer electron from NADPH to their heme center via FMN and FAD, where L-arginine is oxidized to L-citrulline and NO (Poulos et al., 1998; Wendehenne et al., 2003; Figure 1.12).
The electron transfer between the reductase and oxygenase domains requires CaM binding. nNOS and eNOS are constitutively expressed and their activity are strickly Ca2+/CaM- dependent and therefore transient (over a matter of minutes) (Mayer and Hemmens, 1997; Nathan and Xie, 1994). The context that is currently de rigueur states that NO produced by eNOS and iNOS acts as a signalling compound. iNOS is expressed in response to cytokines and microbial products. Remarkably, iNOS binds CaM in the absence of free Ca2+ (Griffith and Stuehr, 1995) and, consequently, produces large amount of NO for an extended period (hours to days), in accordance with its involvement as a toxic compound in the immune response. It should be specified that the classification constitutive versus inducible NOSs is not absolute as constitutive NOSs and inducible NOS were also shown to be regulated at the transcriptional and post- translational level, respectively. NO is also produced in bacteria. Gram-positive bacteria encode smaller NOS proteins, containing only the oxygenase domain. Bacterial NOS uses non-specific cellular reductases to produce NO (Wang et al., 2007; Gusarov et al., 2008).

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NR-dependent NO production

The nitrite-dependent NO synthesis involved mainly nitrate reductase (NR), a major cytosolic enzyme of nitrogen assimilation. NR catalyzes the reduction of nitrate (NO3 -) into nitrite (NO2 -) using NAD(P)H as electron donor (Crawford, 1995) which then are converted to ammonium by nitrite reductase (NiR) but could also reduce nitrite to NO both in vitro and in vivo (Yamasaki et al., 2000; Figure 1.13). The production occur in specific physiological context in which the cytosolic nitrite concentration reach high concentrations such as hypoxia (in the range of mM). NO produced by NR is involved in various physiological processes such as stomatal closure (Neill et al., 2008), the response to abiotic stress (Sang et al., 2008) or response to elicitors such as chitosan (Srivastava et al., 2009). In A. thaliana, two cytosolic isoforms have been identified (NR1 and NR2). For example, using the NR deficient A. thaliana mutants (nia1 nia2 double mutant), Bright et al., (2006) demonstrate that NR is the main enzymatic source in ABA-induced stomatal closure, highlighting a role for NR in NO-dependent signaling processes. As this NO production was shown to be H2O2 dependent, Neill et al., (2008) proposed a signaling cascade, ABA-H2O2-NO, leading to stomatal closure. Similarly, it was observed that the NR- deficient double mutant, which shows substantially reduced NO level after bacterial or fungal inoculation, showed no HR and was hyper-susceptible to P. syringae and to the necrotrophic fungal pathogen Sclerotinia sclerotiorum (Modolo et al. 2006; Oliveira et al., 2009; Perchepied et al., 2010).

Table of contents :

Chapter 1 INTRODUCTION
1. Plant responses against microbial pathogens
1.1. Plant immunity
1.1.1. PAMP-triggered immunity (PTI)
1.1.2. Effectors-triggered immunity (ETI)
1.2. Signal transduction
1.2.1. Ion Fluxes
1.2.2. Oxidative brust
1.2.3. Activation of MAPKs
1.2.3.1. Mitogen-activated protein kinases (MAPKs)
1.2.3.2. Calcium-dependent protein kinases (CDPKs)….
1.2.4. Nitric oxide
1.2.5. Role of plant hormones
1.3. Plant defense response
1.3.1. Strengthening of cell wall
1.3.2. Synthesis of antimicrobial compounds
1.3.3. Production of pathogenesis related proteins (PR proteins)
1.3.4. Hypersensitive response
1.3.5. Systemic resistance
1.3.5.1. Systemic acquired resistance..
1.3.5.2. Induced systemic resistance
2. Nitric oxide (NO), a key player in plant defence signaling
2.1. NO synthesis
2.1.1. Basic concepts of NO synthesis in animals
2.1.2. Biosynthesis of NO in plants
2.1.2.1. Oxidative pathways
2.1.2.1.1. Arginine dependent NO production
2.1.2.1.2. PAOX pathway
2.1.2.1.3. Hydroxylamine pathway
2.1.2.2. Reductive pathways
2.1.2.2.1. NR-dependent NO production
2.1.2.2.2. NI-NOR pathway
2.1.2.2.3. Mitochondrial pathway
2.1.2.2.4. Xanthine oxidoreductase (XOR) pathway
2.1.2.3. Non enzymatic pathways
2.2. Mechanism of action of NO signaling
2.3. Role of NO in plant defense
2.3.1. NO production in different plant pathogen context
2.3.2. NO production mediates Plant defense
3. The oligogalacturonides/Arabidopsis thaliana model
3.1. The oligogalacturonides (OGs)
3.2. OGs and plant defense responses
3.3. OGs and induced resistance
Chapter 2 Basic concepts of NO signaling in animals Metal Nitrosylation
S-Nitrosylation
Tyrosine nitration
Interplays between NO and Ca2+
NO signaling in plants
Interplays between NO and Ca2+
NO act as a Ca2+ mobilizing messenger
Underlying mechanisms
Impacts of the NO/Ca2+ pathways
Interplays between NO and protein kinases
NO modulates MAPK activities
NO and Ca2+-dependent protein kinases
NO and SnRKs
Interplays between NO and ROS
Impacts of the NO/ROS balance in HR
Candidate sites of interaction between NO and ROS during the HR
A protective molecule?
Conclusion
Chapter 3
1. Material and Methods
1. Biological materials
1.1. Plant material
1.2. Elicitor
1.3. Fungal pathogens
2. Methods
2.1. Genotyping
2.2. NO detection by spectrofluorometry
2.2.1. DAF-2DA detection method
2.2.2. CuFL detection method
2.3. In vivo assay of Nitrate Reductase
2.4. Total RNA islation
2.5. cDNA synthesis
2.6. Analysis of transcript accumulation by Real time qPCR
2.7. PCR fragment cloning
2.8. Immunodetection of phosporylated mitogen activated protein kinases
2.9. In gel kinases assay
2.10. Reactive oxygen species measurement in Arabidopsis thaliana
2.10.1. DAB staining
2.10.2. Detection by chemiluminescent assay
2.11. Transcriptome analysis
2.11.1. RNA extraction, cDNA synthesis and labeling
2.11.2. Array hybridization and scanning
2.11.3. Microarray data analysis
Chapter 4 (Part 1)
Chapter 4 (Part 1)
INTRODUCTION
Material and methods
Plant material and growth conditions
NO measurements
Gene expression analysis by quantitative Real Time-PCR
In vivo Nitrate Reductase activity
H2O2 measurement
Botrytis cinerea culture and infection method
Results
OGs induced NO production in Arabidopsis thaliana
OGs induced NO production is L-NAME sensitive
NR is involved in OGs induced NO production
Ca+2 influx modulates NO production in response to OGs
NO modulates OGs triggered ROS production in Arabidopsis thaliana
NO regulation of OGs responsive genes
NO participate to basal resistance to fungel pathogen Botrytis cinerea
Discussion
Enzymatic sources of OGs induced NO production
OGs induced NO production depends on Ca+2 influx
NO production modulates AtRBOHD-mediated oxidative burst
OGs induced NO dependent genes are involved in A. thaliana basal resistance to
B.cinerea
Chapter 4 (part 2) Relationship between MAPK/CDPK activities and OGs –induced NO production
Results
OGs triggered MAPK phosphorylation, independently of NO production
CDPK are regulated by OGs, independently of NO production
Role of CDPKs in OGs-induced NO production
Role of CDPKs A. thaliana / B. cinerea interaction
Role of CDPKs in the target genes of NO
Discussion
MAPK activation is independent of NO production
CDPK control NO production, NO-mediated response to OGs and resistance
to B. cinerea
Chapter 5 INTRODUCTION
RESULTS
1. Transcriptomic response of A. thaliana leaves to OGs treatment
1.1. In silico functional categorization of OGs-induced genes
1.2. Comparative analysis of the A. thaliana transcriptomic response to OGs
2. Nitric oxide-regulated transcriptomic response to OGs
2.1. Identification of NO-responsive genes
2.2. In silico functional annotation of NO-responsive genes
2.3. Identification of transcription factor binding site (TFBS)
2.4. Functional characterization of NO-responsive genes
2.4.1. Validation of microarray data by RT- qPCR
2.4.2. Genotyping of T-DNA insertion mutant lines
2.4.3. Role of candidate genes in Arabidopsis thaliana / Botrytis cinerea interaction
3. DISCUSSION
Transcriptomic response to oligogalacturonides
Categorization of NO-responsive genes
Functional characterization of NO-responsive genes in the B. cinerea/A. thaliana
interaction
Functional analysis of transcription factors
Functional analysis of disease related proteins
CONCLUSIONS AND PERSPECTIVES
REFERENCES……

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