RICERCA ITALIANA     

THE APOPLAST AND THE INTEGRATION OF PROCESSES REGULATING DEVELOPMENT AND INNATE IMMUNITY OF PLANTS

Università degli Studi Roma Tre

Abstract

ABSTRACT
The main goal of our research project is to investigate the apoplast-specific events that orchestrate processes related to growth and development and the mechanisms that allow a plant to resist to pathogen infection. Recent findings indicate that the apoplastic perception systems associated to both hormonal or pathogen-derived stimuli are often associated and orchestrate coordinated signal trasduction pathways. In the absence of an adaptive immune system, plants base their defence strategies on an innate immune system, analogous to the one found in animals, that includes kinase receptors with leucine-rich repeat (LRR) domains that are implicated in the recognition of “elicitors”, i.e. pathogen-derived molecules. These include the so-called pathogen-associated molecular patterns (PAMPs) such as fungal chitin and bacterial flagellin. The oligogalacturonides (OGs) are instead host-associated molecular patterns (HAMPs). They are released from the plant cell wall homogalacturonan by the action of pathogen-derived polygalacturonases whose enzymatic activity is modulated by specific apoplastic polygalacturonase inhibiting proteins (PGIPs). The OGs regulate some important developmental processes and share with a number of PAMPs common signal transduction pathways for the activation of defence responses. The LRR-RLK mediated immunity is a multi-component defence strategy that is associated to drastic changes in the apoplast redox state. In particular the production of reactive oxygen species (ROS) represents a common response to all defence mechanisms (both PAMPs and HAMPs induce an oxidative burst). An important role in these events is played by apoplastic amine oxidases, a family of enzymes that oxidize polyamines with the production of hydrogen peroxide, and by a high potential cytochrome, Air12, implicated in regulating the apoplastic redox homeostasis. Secretory events and endocytosis are also important for the assembly and the remodelling of the plant cell wall both during the activation of innate immunity and in growth and development.
Five research units, that have developed unique tools and experience in the study of apoplast modifications and of the plant-pathogen interactions, and that have a consolidated experience in complementary research areas, will be integrated in this project to reach the following objectives:
A) The identification of the OG receptor using both reverse genetics and biochemical/proteomics approaches, with the use of tagged OGs. The production and characterization of chimeric LRR-RLK proteins, as a tool for studying the OG receptor. The characterization of the structural requirements for the PG-PGIP interaction and of the cellular dynamics of FLS2, a LRR-RLK protein that recognizes flagellin and flg22, a flagellin-derived peptide. It is also our plan to identify key elements in the OG-mediated signal transduction and the molecular basis of OG/auxin antagonism.
B) The determination of the role of apoplastic or plasmalemma proteins implicated in the regulation of the apoplastic redox state both in the context of defence and development. In particular to structurally characterize the apoplastic copper amine oxidases and flavin-dependent polyamine oxidases, that play a crucial role in the production of hydrogen peroxide necessary to both innate immunity and development, and of Air12, a high potential apoplastic cytochrome b. The analysis of their role in regulating the apoplast redox state through the characterization of insertional mutants. To clarify the functional relationships among amino oxidases, Air12 and proteins implicated in the brassinosteroids and OG signalling. This will be achieved through the characterization of insertional mutants and of specific crossings during their development and in response to abiotic and biotic stress.
C) To dissect the secretion and endocytosis pathways associated to innate immunity and development through i) the preparation of fluorescent protein markers to identify the sorting of proteins implicated in the wall synthesis and remodelling and in the transmission of PAMP-derived signals, ii) the analysis of the apoplastic sorting and putative endocytosis/exocytosis of Air12, amino oxidases and PAMP and OG receptors, following a specific stimulus or not, iii) the characterization of the functional interactions of a number of syntaxins and Rab in the regulation of secretion and endocytosis of these receptors, iv) the analysis of the role played by N-glycosylation and GPI anchors in the secretion and endocytosis processes of proteins induced by PAMPS and OGs, and those regulating apoplast redox state. <<<

Principal Investigator

Riccardo Angelini Università degli Studi ROMA TRE

Research Objectives

The main goal of this integrated research project is to investigate the apoplast-specific events that orchestrate processes related to growth and development and the mechanisms that allow a plant to resist to pathogen infection. The evidence that the brassinosteroid co-receptor BAK1 is also necessary for the function of innate immunity receptors, implicated in the recognition at the apoplastic level of pathogen-associated molecular patterns (PAMP), and that this interaction is at the origin of an integrated regulation of development and defence responses, sheds a new light on these wide research areas. The dualism defence/development applies also to plant-derived elicitors (HAMP: Host-Associated Molecular Patterns) such as the oligogalacturonides (OG). The OG regulate some important developmental processes and share with a number of PAMPs the same signal transduction pathways for the activation of defence responses. They are released from the homogalacturonan of the plant cell wall by pathogen-derived polygalacturonases (PG) whose activity is modulated by specific plant inhibitors, localized at the apoplast [PG Inhibiting Proteins (PGIP)]. The production of reactive oxygen species (ROS) is also a response that is typically associated both to the activation of defence mechanisms (PAMP and HAMP induce an oxidative burst) and to the differentiation processes associated to development, which are also intimately related with secretion processes and endocytosis. The structural and functional analysis of this network is fundamental for the development of breeding programmes to increase plant resistance to disease and environmental stresses.
SPECIFIC OBJECTIVES
A) IDENTIFICATION OF THE OG RECEPTOR AND SIGNAL TRASDUCTION PATHWAY, STRUCTURAL CHARACTERIZATION OF THE PG-PGIP INTERACTION AND OF PROTEINS THAT MEDIATE PAMP AND HAMP RECOGNITION. The OG receptor, in analogy with the majority of the plant innate immunity receptors and receptors of ialuronan fragments in animals, might be characterized by an extracellular leucine-rich repeat (LRR) domain and belong to the LRR-RLK (receptor-like kinases) subfamily, that includes 235 members in Arabidopsis. A reverse genetic approach will be pursued; moreover, since it is possible that OG are perceived by non-LRR receptors, a biochemical/proteomics approach will also be undertaken with the use of tagged OG. It is also our goal to identify key elements in the signal transduction pathway activated by OG and to investigate the biological significance of the well-known OG/auxin antagonism. Moreover we also mean i) to analyze the structural determinants of the interaction between FLS2, a LRR-RLK protein that recognizes flagellin, and flg22, a flagellin-derived peptide, and the cellular dynamics of this class of receptors, ii) to characterize chimeric LRR-RLK as a tool for the study of the OG receptor and iii) to identify and characterize the PGIP “hot spots” for the interaction with PG.
B) STRUCTURAL CHARACTERIZATION AND DETERMINATION OF THE ROLE PLAYED BY APOPLASTIC OR PLASMA MEMBRANE PROTEINS INVOLVED IN REDOX REGULATION OF THE APOPLAST DURING DEVELOPMENT AND DEFENSE
In order to understand how ROS production can be regulated in relation with defense responses and development, single systems need to be characterized and functional interactions need to be clarified. A major goal of the project consists in the structural and functional characterization of apoplastic copper amine oxidase and flavin polyamine oxidase, which play a crucial role in the production of hydrogen peroxide. H2O2 is required for both defense mechanisms and formation of cross-links, fundamental for the architecture of the apoplast during growth and differentiation. Moreover, the role of Air12 in the regulation of the redox state of the apoplast. will be determined. Air12 is an apoplastic, high potential cytochrome b which may be involved in the regeneration of reduced ascorbate in the cell wall, although partner proteins interacting with Air12 are required to sustain a transmembrane redox transfer. The potential interactors of Air12 will be searched, possible candidates are the flavoprotein FQR1, the copper oxidase SKU5 and a transmembrane protein which contains a cytochrome b561 domain fused with the DOMON domain of Air12 (CY-DOM). We will study whether Air12, plasma membrane NADPH-oxidase and amino oxidases are involved in the oxidative burst elicited by OG and if they contribute to the transduction pathway activated by these elicitors. A strategic goal will be to clarify the functional relationships between amine oxidases, Air12 and proteins involved in OG and brassinosteroid signalling. This will achieved through the functional characterization of insertional mutants and specific crosses, both during development and under biotic and abiotic stresses.
C) DISSECTION OF EXOCYTOSIS AND ENDOCYTOSIS ASSOCIATED TO DEFENSE MECHANISMS AND DEVELOPMENT PROCESSES.
Secretory and endocytotic events play an important role in cell wall assembling and remodelling during growth and differentiation associated to the development, as shown in the apoplast formation in polarised cells or secondary differentiation, and activation of innate immunity. The differential sorting toward specific plasma membrane sub-domains through Trans Golgi Network, as well as the endocytosis, requires the formation of distinct vesicle populations with specific target and the involvement of SNARE and RabGTPases. To dissect this complex network of events during growth and differentiation associated to development and innate immunity, the following objectives will be realised: i) to prepare fluorescent marker proteins to identify the protein sorting involved in cell wall formation and remodelling and in the signal transmission of PAMP, ii) to determine the sorting to the apoplast and the eventual endocytosis of Air 12, amine oxidase, PAMP and OG receptors after either specific stimulation or not, iii) to characterize the functional interactions of some SNARE and Rab in the events that regulate secretion and the endocytosis of such receptors, iv) to clarify the role of N-glycosylation and GPI anchor in the secretion and endocytotic processes of the proteins induced by PAMP and OG.
To overcome the many conceptual and methodological problems that hamper progress in this field, five research units that in the past years have developed unique tools and expertise in the study of the apoplastic milieu and/or plant-pathogen interactions will integrate and complement their actions to achieve the specific goals described in this proposal. The units have complementary expertise in biochemistry, proteomics, molecular biology, molecular genetics, in vivo imaging, structural biology and bioinformatics. Many of the tasks will be developed in collaboration, undertaking complementary activities and sharing protocols, equipment, plasmid constructs, fluorescent probes and biological materials. Established methodologies, such as heterologous expression in eukaryotic cells, confocal microscopy, proteomics, Real-time PCR, surface plasmon resonance for protein-protein interaction studies combined to mass spectrometry (BIA-MS), FRET, and MALS-QELS will be shared This joint activity also will provide the ground for the advanced training of the numerous young Italian researchers that participate to this project. <<<

First Results

EXPECTED RESULTS

OBJECTIVE A. STRUCTURAL CHARACTERIZATION OF THE PG-PGIP INTERACTION AND OF PROTEINS THAT MEDIATE PAMP AND HAMP RECOGNITION. STUDIES ON THE OG-MEDIATED SIGNAL TRASDUCTION PATHWAY

A. 1.IDENTIFICATION AND CHARACTERIZATION OF PGIP “HOT SPOTS” FOR PG RECOGNITION
-We expect to identify the minimal PGIP structural requirements for the recognition of each different PGs. These data will be used to rationally design PGIP2 variants able to recognize fungal PGs that wild-type PGIP2 does not inhibit.

A. 2.CRYSTALLIZATION OF THE PG-PGIP COMPLEX, A CHIMERIC APPROACH
-The minimal expectation is to obtain diffracting crystals of the PG-PGIP chimera and, possibly, to determine its structure within the project time span.

A. 3. STRUCTURAL ANALYSIS OF THE FLS2-FLG22 INTERACTION
-We expect to have obtained by the end of the program a system for the heterologous expression of FLS2-LRR, to have developed a purification protocol to obtain the protein in milligram quantities and to have performed a complete crystallization screen of the protein alone and in complex with flg22. Crystals obtained will be subjected to the crystallographic analysis.
- Availability of constructs for in planta expression of two FLS2/EFR chimeric receptors, also fused with GFP, and of transgenic Arabidopsis ecotype Wassiliewskija (Ws) plants, which normally do not respond to fgl22, expressing these receptors, as well as the fusion FLS2-GFP.
- Availability of transgenic plants obtained from the transformation of the Arabidopsis efr mutant with a construct for the expression of the fusion EFR-GFP.
- Information on which chimeric receptor is able to activate, upon recognition of flg22, the signal transduction cascade typical of EFR.
- Information in the structural requirement for the construction of functional chimeric receptors
- Availability of a method for the preparation of OG-containing IWFs and of at least an assays for the determination of OG levels in IWF.

A. 4.CHARACTERIZATION OF SIGNAL TRANSDUCTION PATHWAY REGULATED BY OG
- Information about the involvement of Et, SA, JA, auxin, RbohD, Air12, AtCuAO1, AtCuAO2 and AtPAO1 in OG-mediated induction of the expression of marker genes and protection against B. cinerea.
- Information on the features of the OG/auxin antagonism in both IAA-induced root formation in Arabidopsis leaf explants and the protection against B. cinerea (active concentrations of OGs, degree of polymerization of active OGs, temporal kinetics of the action of OGs and auxin).
- Information about the regulation of the expression of miRNA393 and auxin receptor transcripts in response to OGs
- Information about the resposne to OGs and B. cinerea of plants overexpressing miR393, mutants defective in miRNA formation, auxin receptor mutants or other auxin mutants.

A. 5. IDENTIFICATION OF OG RECEPTOR
- Availability of Arabidopsis homozygous KO lines for LysM- and LRR-RLK, and BAK1.
- Information about the expression of marker genes in the KO lines.
- Possibly, availability of at least one KO line for an RLK showing a defective response to OGs.
- Availability of OGs labelled with biotin or fluorophors.
- Information about the ability of the labelled OGs to induce the expression of marker genes.
- Identification of proteins binding OGs at high affinity.
- Availability of double or triple KO mutants for RLKs obtained by crossing

OBJECTIVE B. STRUCTURAL CHARACTERIZATION AND DETERMINATION OF THE PHYSIOLOGICAL ROLE OF APOPLASTIC AND PLASMAMEMBRANE PROTEINS INVOLVED IN THE REGULATION OF THE REDOX STATE OF THE APOPLAST

B. 1. BIOCHEMICAL CHARACTERIZATION, EXPRESSION PROFILE AND STUDIES ON INSERTIONAL MUTANTS FOR APOPLASTIC COPPER AMINE OXIDASE (AtCuAO) AND POLIAMINE OXIDASE (AtPAO) in Arabidopsis thaliana
-Heterologous expression and biochemical characterization of the two secretory Arabidopsis CuAO (AtCuAO1 and AtCuAO2 )
-Informations on the regulation of AtCuAO1 and AtCuAO2 during plant development, treatment with a panel of plant hormones and after wounding or biotic stress
-Morpholigical and phenotypical characteristion, lignin, suberin and hydrogen peroxide level determination in Arabidopsis insertional mutants for the two AtCuAO under study, in normal growth conditions or after wounding or B. cinerea infection.
-Determination of AtPAO 1 and AtPAO5 subcellular localisation
- Characterisation of Arabidopsis insertional mutants for AtPAO 1 and AtPAO5
-Morphological and phenotypical characterization, lignin, suberin and hydrogen peroxide level determination in Arabidopsis insertional mutants for AtPAO 1 and AtPAO5 after wounding and fungal or bacterial infection
- Determination of expression pattern of AtPAO1 and AtPAO5.
- Expression of AtPAO5 in heterologous systems
- We expect to determine the structure of AtPAO1 alone and in complex with different highly specific inhibitors within the time span of the project. As for AtCuAO1 and AtCuAO2, we expect to have verified the possibility to express them in milligram quantities and if this prerequisite is fulfilled, to have explored the complete crystallization “space” for at least one of them.

B. 2. STUDIES OF FUNCTIONAL RELATIONSHIP BETWEEN AMINE OXIDASES, LRR-RLK, BAK1, RbohD AND Air12
-Information of the involvement of RbohD, Air 12, BAK1 and other LRR-RLK mutants in hydrogen peroxide production after biotic and abiotic stress and in the presence of CuAO or PAO specific inhibitors
-Characterization of crossing between AtCuAO or AtPAO and RbohD mutants as concerns their contribute to hydrogen peroxide production in the apoplast.
-Characterization of crossing between AtCuAO or AtPAO mutants with BAK1, or other LRR-RLK mutants, to verify if hydrogen peroxide production deriving from specific amine oxidases during development or after stress is under control of signalling pathway different from those regulated from these receptors.

B. 3. POST-TRANSLATIONAL MODIFICATIONS OF MAIZE PAO (ZmPAO): IMPLICATIONS IN REGULATION AND SECRETION OF THE ENZYME
-Identification of phosphorylated isoforms of purified ZmPAO. Analysis of ZmPAO enzyme activity regulation in vivo caused by protein phosphorylation–dephosphorylation.
-Identification of potential S-nitrosylation sites on ZmPAO and of the S-nitrosylation effects on the enzyme activity levels.
-Identification of phosphorylated ZmPAO isoforms in vivo.
-Analysis of the effect of disulphide bridge on ZmPAO enzyme activity.
-Analysis of the role played by carboxyterminal cysteines and Asn105 N-glycosylation events on apoplastic ZmPAO transport.

B. 4. PRODUCTION OF RECOMBINANT AIR12, BIOCHEMICAL AND STRUCTURAL CHARACTERIZATION AND IN PLANTA LOCALIZATION
- Production of purified recombinant Air12 in its native form and anti-Air12 antibodies.
-Description of the main functional properties of soybean Air12 in vitro. Definition of the main properties of this cytochrome family, on the basis of Air12 characteristics, as well as on those of other known proteins containing the DOMON domain.
-Soybean Air12 structure determination. If the protein can be expressed in heterologous systems such Pichia pastoris, we expect to purify it in quantities amenable for a complete screen of crystallization conditions with the help of the nanodrop crystallization robot
-Cloning of Air12 promoter of Arabidopsis and production of transgenic Arabidopsis plants expressing the GUS reporter gene under control of the Air12 endogenous promoter.
-Definition of conditions, tissues and developmental stages in which Air12 is mainly expressed.
- Information on the regulation of Air12 during auxin-induced root formation in leaf explants.
- Information on the regulation of Air12 in response to OGs and B. cinerea
- Information on the involvement of Et, SA, JA, auxin, RhohD, BAK1 and other LRR-RLKs in the OG- and B. cinerea-mediated induction of Air12 expression

B. 5. PHENOTYPIC CHARACTERIZATION OF ARABIDOPSIS T-DNA LINES FOR AIR12 AND POTENTIAL INTERACTORS
-Isolation of homozygous T-DNA lines for Air12, FQR1 and SKU5 genes
-Phenotypic characterization of homozygous KO lines
- Information on hydrogen peroxide production in response to OGs, flg22 and B. cinerea
- Information about the ability to form adventitious roots in leaf explants in response to auxin
-Wild type phenotype rescue of homozygous T-DNA lines for Air12, FQR1 and SKU5 through reinsertion of the respective genes.
-Production of multiple mutants by crossing.

B. 6. SEARCH OF AIR12 PARTNERS
-Setting of a protocol for the purification of lipid rafts from soybean roots.
-Identification of possible Air12 interactors
-Production of soybean transgenic lines expressing Air12-HA
-Expression and purification of recombinant forms of Air12 interactors
-Complexes reconstruction, stoechiometry and functional analysis of redox activity

OBJECTIVE C DISSECTION OF SECRETION AND ENDOCYTOTIC MECHANISMS ASSOCIATED TO DEFENSE MECHANISMS AND GROWTH EVENTS

C. 1. PREPARATION OF NEW FLUORESCENT MARKERS TO STUDY CELL WALL REMODELLING AND THE ESO/ENDOCYTOTIC EVENTS OF THE PAMP AND HAMP RECEPTORS
- Availability of constructs for the expression of the fusion proteins CslAO2-GFP, CesA6-GFP, Air12-GFP and PAO-GFP
- Information on their localization and dynamics in tobacco protoplasts and agroinfiltrated leaves.
- Information on the ligand-dependent internalization and the dynamics in response to OGs of the fusion proteins FLS2-GFP, EFR-GFP, FLS2/EFR-GFP in protoplasts, agroinfiltrated leaves and Arabidopsis transgenic seedlings and plants.
- Information on the possibility to obtain a differential block of the trafficking of FLS2-GFP, EFR-GFP, FLS2/EFR-GFP in the presence of tyrphostin A23 e BFA and their possible accumulation in prevacuolar or endosomal compartments.
- Information about whether overexpression of CslAO2-GFP in tobacco protoplasts leads to an increase of the mannan synthase activity.
- Information about the possible internalization of PGIP2-GFP in response to OG and its differential inhibition in the presence of tyrphostin A23 and BFA


C. 2. ROLE OF THE GLYCOSYLPHOSPHATIDYLINOSITOL ANCHOR AND N-GLYCOSYLATION IN TARGETING, SECRETION AND ENDOCYTOSIS
- Information on the sorting to ER or Golgi, the plasma membrane and/or the apoplast of PGIP2-GFP, CslAO2-GFP, CesA6-GFP, Air12-GFP, PAO-GFP, FLS2-GFP, EFR-GFP, FLS2/EFR-GFP in the presence of an inhibitor of the synthesis of the GPI anchor.
- Information on the sorting to ER or Golgi, the plasma membrane and/or the apoplast of PGIP2-GFP, CslAO2-GFP, CesA6-GFP, Air12-GFP, PAO-GFP, FLS2-GFP, EFR-GFP, FLS2/EFR-GFP when glycosylation is inhibited.
- Information of the secretion pattern of ZmPAO Asn105Ser.
-Information on the possible internalization of fluorescent OGs

C. 3. DISSECTION OF SECRETION AND ENDOCYTOTIC MECHANISMS THROUGH THE USE OF DOMINANT NEGATIVE (DN) MUTANTS
-Setting an in vivo assay to characterise the functional role of SYP121 and SYP122 in PGIP2-GFP, CslAO2-GFP, CesA6-GFP, Air12-GFP, PAO-GFP sorting.
-Availability of RabGTPases (Rab11a, RabA2, RabA3, RabA5 e RabF2) DN mutants with differential inhibitory effect on exocytosis, endocytosis and endosomial trafficking of the new markers developed in the project.
- To obtain PGIP2 and ZmPAO fragments in fusion to GFP, that reach the apoplast to identify specific cell wall target signals.


IMPORTANCE OF EXPECTED RESULTS FOR THE ADVANCE OF BIOLOGICAL KNOWLEDGE AND APPLICATIVE REPERCUSSIONS

Understanding molecular mechanisms underlying the integration in the apoplast of processes regulating innate immunity and development of plant organisms has a very important repercussions either in the progress of biological knowledge or in the more applicative field of the genetic improvement of resistance to diseases of species of agronomic interest. Moreover, industrial applications and advanced research training may represent a further aspect creating new employment possibility. We delineate here some of these repercussions:

A) To provide a knowledge base for improvement of crop plants more resistant to pathogens and abiotic stress with a consequent environmental benefit for a lower necessity of chemicals to control diseases or environmental stresses.
Pre- and post- harvest losses due to diseases or other stress may account for up to 40% of some economically important crops. To limit these losses in agriculture, substantial amounts of pesticides or other chemical compounds are used with negative effects on the ecosystems. Development of novel crops with improved resistance traits, may reduce the pre- and post-harvest use of fungicides and other chemicals with corresponding environmental benefits. Although enhanced resistance has been successfully introduced into crops by breeding programs, effective genetic resistance against several pathogens is still lacking, and there is an urgent need to increase knowledge base in this field also as concerns relationship between disease, developmental effects and crop loss. Participants of RUs have made major contributions to recent breakthroughs in the understanding of the role of the apoplast, the first line of defence, in plant-pathogen interactions, in structural characterization of plant cell wall and plasma-membrane proteins, apoplast redox state regulation, exocytic and endocytic processes leading to formation and remodelling of plant cell wall during development and disease. Development of model plants mutated in PAMP and HAMP receptors, in key components of the apoplastic and plasma-membrane redox machinery, exocytic and endocytic system, combined with functional characterization of genes involved in PAMP and HAMP signaling will provide an important knowledge base for development of broad-spectrum and durable resistance in key crops.

B) To implement the research of enterprises
A significant number of SMEs are developing in the EU to exploit the new opportunities arising from plant genetics, physiology, molecular biology, biochemistry of plant cell wall and plant innate immunity (agro-food companies, raw materials, bio-energy). These enterprises and SMEs are key potential users of advanced methodologies of the project for their own research.

C) To implement use of plant crops for industrial use thus creating new employment possibility.
Increased awareness among Italian and European companies of the project objectives, of progress and research results could potentially have a positive effect of employment. Indeed, industrial users can foresee new developments and employment possibilities emerging from increasing market demands for environment-friendly products and plant biomass for many applications.

D) Advanced research training
Advanced research training of young researchers has important repercussions in both competitiveness and employment possibility. <<<

Timescale

24 months

National and international background

SCIENTIFIC BACKGROUND
The plant cell apoplast, mainly represented by the cell wall and the wall/plasma membrane interface, is the site where crucial events of perception and signalling occur, involving both endogenous stimuli that regulate differentiation and development, and exogenous signals associated to biotic and abiotic stresses. Recent evidence indicates that the same perception/transduction elements may mediate the response to these two classes of stimuli and orchestrate the activation of signalling pathways for a coordinated regulation of developmental and defence processes.

PLANT INNATE IMMUNITY
In the absence of an adaptive immune system, plants rely for defence against pathogens on a so-called innate immunity system analogous to that found in animals (36). Main elements of this system are germ line-encoded pattern receptors (PRR) for recognition of pathogen-derived molecules indicated as elicitors (generic elicitors or plant-genotype specific elicitors). Generic elicitors are indicated as pathogen-associated molecular patterns (PAMP) and include fungal chitin and bacterial flagellin. Oligogalacturonides (OG) are plant endogenous elicitors (HAMP -Host-Associated Molecular Patterns) that are released from the plant cell wall homogalacturonan (HGA) by pathogen-secreted polygalacturonase (PG), which are also ranked among PAMP (42). OG accumulation is favored by the interaction with specific inhibitors of PGs (PGIP: polygalacturonase-inhibiting proteins) that are located in the plant cell wall (14, 17). PGIPs are leucine-rich repeat (LRR) proteins (16) that bind also OGs with an array of regularly spaced positive charges (47). Treatment with OGs induces protection against the necrotrophic fungus Botrytis cinerea via a still uncharacterized signal transduction pathway that functions independently of the known pathways involving salicylic acid (SA), jasmonate (JA), and ethylene (Et) (18). During a previous PRIN programme, global transcript profiling of Arabidopsis seedlings showed that early (1 h) responses regulated by OG and flg22, a peptide derived from a conserved region of flagellin and a typical PAMP, are highly overlapping (&gt;98%), whereas late (3 and 6 h) responses are divergent, with more than 4000 genes regulated only by the treatment with flg22. Nothing is known about the perception and transduction of OG; in particular, the nature of the OG receptor is still unknown. It is conceivable that it belongs to the LRR-RLK receptor subfamily, which comprises as many as 235 members (36).

THE INNATE IMMUNITY RECEPTORS INTERACT WITH RECEPTORS IMPLICATED IN THE REGULATION OF DEVELOPMENTAL PROCESSES
LRR domains are found in receptors of Drosophila (Toll) and mammals (Toll-like receptors) implicated both in innate immunity (37) and in the regulation of developmental processes (44). The same happens in the case of plant PAMP receptors since it has been recently demonstrated that there is a common regulatory junction between defence responses and developmental processes (11). The plant pathogen perception system is exemplified by the recognition of flagellin by FLS2, a kinase receptor characterized by an extracellular domain composed by the tandem repetition of 28 LRR modules and an intracellular kinase domain (LRR-RLK, 22). FLS2 associates with BAK1, the brassinosteroid (BR) kinase co-receptor in the early stages of bacterial infection and this interaction stimulates defence responses (11). BAK1 in turn also heterodimerizes with the kinase BR receptor BRI1. BRI1 is implicated in the BR-associated development regulation, in the BR-independent control of programmed cell death (25) and in the perception of wound damage, since it is also capable of binding systemin, a peptidic HAMP associated with the systemic signalling of wound (53). It is not known whether the same interactions are also operating in the case of HAMP, even though this appears to be probable since OGs are capable of regulating both defence responses and developmental processes through their inhibition of auxin-induced responses (7, 32). Recently it was demonstrated that flg22, a peptide derived from flagellin, intereferes with the signalling triggered by auxin, by suppressing the expression of auxin receptors through miRNA393 (34). The cooperation between HAMP and PAMP receptors and receptor of hormones probably constitutes an evolutionary strategy whose purpose is to orchestrate defence and development through coordinated perception and signal transduction mechanisms.

THE ALTERATION OF APOPLAST REDOX STATE IS A COMMON ELEMENT IN INNATE IMMUNITY AND GROWTH AND DIFFERENTIATION PROCESSES ASSOCIATED TO DEVELOPMENT
The activation of the plant innate immunity system through PRR triggers a multi-component defence response which is often associated to important changes in the redox state of the apoplast. In particular, the production of the reactive oxygen species (ROS; 3) represents a common response during the activation of the defence mechanisms (PAMP and HAMP induce an oxidative burst) and the differentiation processes associated to the development (10, 19, 20, 38, 51). The production of specific ROS in the apoplast is finely regulated and has important functions, such as the regulation of cell–wall loosening or stiffening with important effects on plant growth, the regulation of the cell-wall differentiation during development and the activation of the defence mechanisms against pathogens, including the hypersensitive response and the activation of wound-repairing mechanisms. The ROS production in the apoplast depends on multiply systems [membrane-linked NADPH oxidase, peroxidase, oxalate oxidase, copper amine oxidase (CuAO; 26), FAD-depedent polyamine oxidase (PAO) (1, 3)]. In particular, recent data have evidenced that extracellular amine oxidases have an important role in the production of the hydrogen peroxide necessary for formation of cross-links fundamental to the architecture of the apoplastic compartment during growth and differentiation processes associated to development and defense mechanism (2, 13, 31, 40, 46, 52). For example, it has been shown that treatment of tobacco cells with alpha-difluoromethyl-ornithine, a specific inhibitor of polyamine biosynthesis, blocks the production of hydrogen peroxide induced by cryptogein, a bacterial elicitor which provokes oxidative burst and PCD (54). Furthermore, cryptogein treatment of tobacco cells transformed with a RNAi construct able to suppress the expression of an apoplastic PAO, does not induce hydrogen peroxide production and PCD (55). Ten CuAO-encoding genes are present in Arabidospis thaliana. Two of these, At4g14940 e At1g62810, encode for apoplastic proteins (AtCuAO1 and AtCuAO2). AtCuAO1 is expressed in the early phases of xylem development (33). Microarray data indicate that AtAO1 expression is induced by BR and jasmonic acid. AtCuAO2 gene expression is induced by BR, pathogens (Pytophtora infestant e Botrytis cinerea (unpublished results of RU II). A search of the Arabidopsis database with the amino acid sequence of MPAO has revealed the presence of five cDNAs encoding for PAO (AtPAO1-5) (50). AtPAO1 has very high sequence identity with an apoplastic PAO from tobacco (NtPAO) which has been shown to be implicated in hydrogen peroxide production during TMV- (30) or cryptogein-induced hypersensitive response (55). AtPAO2-4 are localized in the peroxisome while any specific localisation signal is evident for AtPAO5. Ascorbic acid plays a main role in the regulation of apoplastic redox state and therefore in the regulation of the described processes. It is not known whether enzymatic systems for the regeneration of reduced ascorbate exist at the cell wall. B-type cytochromes of the plasma membrane (5, 39) could transfer electrons from intracellular donors to the apoplastic dehydroascorbate. Air12 is the only cytochrome of this kind to be individuated so far (RU IV unpublished data). It is a soluble protein bound to the external side of the membrane through a GPI-anchor (9). This cytochrome interacts with ascorbate, but it needs other proteins to accomplish a trans-membrane redox transport. Redox proteins containing the cytochrome domain of Air12 (named DOMON; 4) fused to other redox domains exist in different organism and indicate that CY-DOM cytochromes (comprising a cytochrome b561 and a DOMON domain) could be among Air12 interactors (15, 30). Cytochrome b561 are trans-membrane proteins (22, 39), whose domain is capable of transferring electrons from ascorbate to dehydroascorbate or other acceptors through the lipid bi-layer (48). Other possible interactors are the flavoprotein FQR1 (27) and the copper oxidase SKU5, which have been both found in association with Air12 in lipid rafts preparations. The possible functional association of Air12, FQR1 and SKU5 is further supported by their being induced by auxin in the same way and time within developing lateral roots (27, 28, 35). Air12 gene expression is induced during lateral root formation stimulated by auxin (28, 35); it is also induced by OG and B.cinerea, as indicated by transcriptomic analyses run by RU II.

SECRETION SPECIFIC OF MACROMOLECULES TO THE APOPLAST AND ENDOCYTOSIS ARE INTEGRATED PROCESSES IN DIFFERENTATION EVENTS ASSOCIATED TO THE DEVELOPMENT AND INNATE IMMUNITY.
The high dynamism of the cell wall depends on the endomembrane system functionality, since cell wall proteins and matrix polysaccharides are synthesised in ER and Golgi apparatus, respectively, and secreted by vesicles to the apoplast (24). Secretory and endocytotic events (45) are important for cell wall assembly and remodelling during growth and differentiation, as demonstrated in the apoplast formation in polarized cells or during secondary differentiation and in the innate immunity activation. The pathogen attack represents a situation in which plant cells require a rapid secretion of large amount of apoplastic materials. Transcriptomic analyses show that in Arabidopsis several genes encoding for secreted and plasma membrane proteins are induced by OG and flg22, as well as genes involved in the regulation of secretion. On the other hand, it has been demonstrated that both the FLS2/BAK1 complex and the BRI1/BAK1 heterodimer are internalized in endocytotic vesicles after stimulation with flg22 and BR respectively (43). The differential sorting toward lytic or storage vacuoles, or toward specific plasma membrane sub-domains through the TGN requires the formation of distinct vesicle populations with specific targets and the involvement of SNARE and RabGTPase proteins. It is widely accepted the idea that plasma membrane and, consequently, the cell wall are the “default” destination for many proteins (6, 24). Probably, these events are more complex than it is thought. Recently, it has been reported that secretion marker proteins (secGFP) and cell-wall polysaccharides move through different secretory pathways (29). Preparation of specific secretion and endocytotic markers, the study of their dynamism as response to PAMP and HAMP, the comprehension of N-glycosylation role and the glycosylphosphatidylinositol (GPI) anchor of specific proteins involved in the cell wall formation and defence response, are fundamental objective to understand the molecular mechanisms that control the apoplast remodelling during growth and biotic stress conditions. <<<