WO2019110538A1 - Novel phytopharmaceutical compounds and compositions, preparation and uses thereof - Google Patents

Abstract

The present invention relates to novel phytopharmaceutical compounds, in particular to elicitors of natural plant defenses, to compositions comprising such compounds, and to uses thereof. The invention also relates in particular to a process for producing the novel phytopharmaceutical compounds.

Description

NOVEL PHYTOPHARMACEUTICAL COMPOUNDS AND COMPOSITIONS, PREPARATION AND USES THEREOF

FIELD OF THE INVENTION

The present invention relates to novel phytopharmaceutical compounds, in particular to elicitors of natural plant defenses, to compositions comprising such compounds, and to uses thereof. The invention also relates in particular to a process for producing the novel phytopharmaceutical compounds.

BACKGROUND

Plant pathogens, including pathogenic fimgus and pathogenic bacteria, use a variety of strategies ranging from stealth to brute force to colonize plants and derive nutrients from their hosts. A plant attacked by a pathogen can resist infection by activating its own defense strategy. These defenses are known to be triggered by a wide range of elicitors, either derived from the plant pathogen or from the host plant (Randoux, 2010). An elicitor is a compound that, when it is perceived by the plant, leads to biochemical and/or physiological plant cell reactions such as the synthesis, or increase of the synthesis, of plant defense molecule(s), for example ethylene and/or jasmonic acid, the production of reactive oxygen species (ROS), expression of specific defense-related genes and proteins, for example polygalacturonase inhibitor proteins (PGIP). Then, the activation of signal transduction pathway(s) leads to late defense gene expression and secondary metabolites.

WOOO/17215 describes the use of sulfated fuco-oligosaccharides for plant protection. Simpson et al. (“ Short chain oligogalacturonides induce ethylene production and expression of the gene encoding aminocyclopropane 1 -carboxylic acid oxidase in tomato plants Glycobiology, vol. 8, no. 6, Uune 1998) teaches that, in contrast with many other effects, only oligogalacturonic acids (OGAs) in the size range of DP 4-6 were active both in eliciting ethylene forming enzyme aminocyclopropane- 1 -carboxylic acid oxidase (ACO) and in the production of ethylene in tomato plants. The authors conclude that specific OGA size ranges exert very different biological activities, particularly in the field of plant defense responses. Randoux (2010) shows that a mix of oligogalacturonides (OGs) with different degree of polymerization elicit defenses in wheat against haustoria de Blumeria. According to W02008/065151, oligogalacturonides exert a bioactive effect on plant defenses if they present an“egg box” conformation (such a conformation requires the addition of one or more cations) and a DP above 8. Benedetti et al. (“Plant immunity triggered by engineered in vivo release of oligogalacturonides, damage-associated molecular patterns Proceedings National Academy of Sciences PNAS, vol. 112, no. 17, 13 April 2015) describe the production by transgenic A. thaliana plants of oligogalacturonides with a specific DP range between 6 and 13 functioning as damage-associated molecular patterns (DAMPs) signals to trigger transgenic plant immunity, and teach that in vivo generated OGs can at the same time negatively affect plant growth and development. W02009/090346 relates to heteropolysaccharides elicitors used for stimulating the natural defenses of plants. W001/00025 relates to the use of oligo 1,4 b-D -mannuromans as phytosanitary products for the protection of plants against pathogens and/or fertilizer.

Oligogalacturonides present an important diversity in their structure. OGs are plant cell wall fragments characterized by their degrees of polymerization (DP), methylation (DM) and acetylation (DA). They come from the degradation of specific pectic regions, the homogalacturonans (HG), that are homopolymers of a-l-4-linked GalA residues (Galacturonic acid) potentially acetylated at the C₂ and/or C₃, and methylated at the C₆ (Ridley et al., 2001). HG are secreted in an esterified form and after secretion, they can be deacetylated and/or demethylesterified in muro by pectin acetylesterases and pectin methyl esterases (PME) respectively (Senechal et al., 2014). This leads to the formation of polygalacturonic acid stretches highly susceptible to degradation by polygalacturonases (PG) or pectate lyases (PL) (Senechal et al., 2014). During infection of plants by pectinolytic bacteria and fungi, OGs are produced by pectin degrading enzymes like PME, PG, PL and pectin lyases (PNL) that decompose and convert HG in fungal biomass, releasing a mix of OGs (Van Kan et al., 2006). These OGs trigger plant defense signaling including production of ethylene, jasmonic acid or the expression of specific defense-related genes like polygalacturonase inhibitor proteins (PGIP) (Bishop et al., 1981; Campbell et al., 1991; Hahn MG et al., 1981; Norman et al, 1999; Ridley et al, 2001).

For the first time, the pectin-derived oligosaccharides in Botrytis cinerea infected Arabidopsis leaves have been generated and characterized by inventors. Inventors herein confirm that the in vivo generated OGs are far more complex than the ones used in particular by the scientific community to elicit resistance in plants, and herein identify new advantageous compounds among these OGs. SUMMARY OF THE INVENTION

Novel compounds eliciting natural plant defenses are herein described. A preferred compound is a methylated and acetylated oligogalacturonide with a degree of polymerization (DP) of 4 (also herein identified as“GaUMcAc”).

Also herein described is the use of a methylated and acetylated oligogalacturonide with a degree of polymerization (DP) of 4 as herein described, typically the use of a methylated and acetylated oligogalacturonide with a 4,5 unsaturated galacturonic acid residue at the non reducing end and a degree of polymerization (DP) of 4, for preventing, controlling or treating a plant against infection by a plant pathogen.

A composition, typically a phytopharmaceutical composition, comprising a (typically at least one) methylated and acetylated oligogalacturonide with a degree of polymerization (DP) of 4, typically a methylated and acetylated oligogalacturonide with a 4,5 unsaturated galacturonic acid residue at the non-reducing end and a degree of polymerization (DP) of 4, is also herein described. In a typical aspect, the composition also comprises a phytopharmaceutically acceptable vehicle or support. In a particular aspect, the composition of the invention is an antifungal and/or anti-bacterial adjuvant.

Also herein described is a process for preparing a methylated and acetylated oligogalacturonide with a degree of polymerization (DP) of 4, typically a methylated and acetylated oligogalacturonide with a 4,5 unsaturated galacturonic acid residue at the non reducing end and a degree of polymerization (DP) of 4, as herein described, wherein the method comprises a step of contacting a pectinic substrate with a pectin lyase (PNL), typically in presence of a polygalacturonase inhibitor protein (PGIP), and preferably further comprises a step of purifying the thereby obtained oligogalacturonide. In a particular process herein described, the amino acid sequence of the pectin lyase is selected from SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36 and SEQ ID NO: 37.

The method may also comprise, typically before the purification step, a step of enriching a composition [obtained by contacting a pectinic substrate with a pectin lyase (PNL) in presence of a polygalacturonase inhibitor protein (PGIP) and comprising several oligogalacturonides] in the oligogalacturonide of interest [i.e. in the methylated and acetylated oligogalacturonide with a degree of polymerization (DP) of 4]

Further herein disclosed is the use of a compound or of a phytopharmaceutical composition as herein described wherein the methylated and acetylated oligogalacturonide with a degree of polymerization (DP) of 4 has the formula (I):

wherein Ac is C0CH3 and Me is CH3, or is obtained by carrying out a process comprising an enzymatic hydrolysis step using a pectin lyase (PNL), or by carrying out a process comprising a step of contacting a pectinic substrate with a pectin lyase (PNL) in presence of a polygalacturonase inhibitor protein (PGIP). The process is preferably carried out in a bioreactor.

The present description further relates to a method for obtaining a plant that is resistant to pathogens, wherein the method comprises a step of contacting a plant with at least one methylated and acetylated oligogalacturonide with a degree of polymerization (DP) of 4, typically with an effective amount of at least one methylated and acetylated oligogalacturonide with a degree of polymerization (DP) of 4.

Also provided is a method for eliciting natural plant defenses, for activating plant defense and resistance reactions against plant pathogen and/or for stimulating the production of plant defense molecules against plant pathogens. Such a method comprises a step of contacting a plant with at least one methylated and acetylated oligogalacturonide with a degree of polymerization (DP) of 4, typically with an effective amount of at least one methylated and acetylated oligogalacturonide with a degree of polymerization (DP) of 4 such as a methylated and acetylated oligogalacturonide with a 4,5 unsaturated galacturonic acid residue at the non reducing end and a degree of polymerization (DP) of 4.

Further herein described is a method for selecting a plant resisting to plant pathogens. This method typically comprises a step of infecting a plant with a plant pathogen, in particular with a plant fungal pathogen, and a step of determining the presence or absence of at least one methylated and acetylated oligogalacturonide with a degree of polymerization (DP) of 4, typically a methylated and acetylated oligogalacturonide with a 4,5 unsaturated galacturonic acid residue at the non-reducing end and a degree of polymerization (DP) of 4, in a plant sample and, if the plant sample contains such a methylated and acetylated oligogalacturonide with a degree of polymerization (DP) of 4, a step of selecting the plant as resistant to plant pathogens. A method for the early detection of a plant infected by a plant pathogen is also herein described. This method comprises a step of determining the presence or absence of a GafMcAc and/or of an oxidized Gal A ₂ (GalA₂ox) in a plant sample and, if the plant sample contains a GafMcAc and/or a GalA₂ox, a step of identifying the plant as infected by a plant pathogen.

DETAILED DESCRIPTION OF THE INVENTION

Inventors have characterized and herein disclose the structure of OGs produced during infection of a plant by Botrytis cinerea ( B . cinerea) and herein identify the selected ones that trigger an advantageous plant immune response.

B. cinerea is a necrotrophic fimgal plant pathogen that causes diseases in hundreds of dicots plants (Elad et al, 2004). Up to now, five polygalacturonases (PGs) have been characterized on the 15 putative ones, BcPGl, BcPG2, BcPG3, BcPG4 and BcPG6 (Kars et al, 2005b), and two pectin methyl esterases (PME), PME1 and PME2 (Kars et al, 2005a). Study of corresponding mutant strains showed that BcPGl and BcPG2 are necessary for full virulence on various plant species (ten Have et al, 1998; Kars et al, 2005b) while BcPMEl and BcPME2 are dispensable to infect tomato and grapevine leaves and pear fruit (Kars et al, 2005a). The OGs structure released during infection have been investigated only once by Joo An et al (2005) from B. cinerea- infected tomato fruit tissue. They observed that PG, PME and, surprisingly, PNL were involved in OGs production and that most of them were esterified. They concluded that in vivo generated OGs are more complex than the ones extensively used by the scientific community to elicit resistance in plants. Indeed, up to now, the actual model exclusively relies on the defense activation by in vitro unmethylated polygalacturonic acid (PGA) fragments ranging from DP3 to DP 15 (Ferrari et al, 2007 ; Moscatiello et al, 2006 ; Denoux et al, 2008 ; Moloshok et al, 1992 ; Simpson et al, 1998 ; Thain et al, 1990 ; Davidsson et al, 2017).

Inventors developed and herein describe a new analytical method. They first analyzed OGs produced by purified polygalacturonases and B. cinerea germinating spores from commercial pectins. They showed that the fimgus exclusively releases polygalacturonase products from commercial pectins. They next identified OGs produced during infection of Arabidopsis leaves by B. cinerea. They discovered that the OGs are mainly products of pectin lyase (PNL) activities. They also observed that non-methylated PG products are oxydized in muro probably by a plant enzyme. At last, using B. cinerea mutants affected in OGs production and displaying opposite virulence, inventors identified among the high amount of different OGs produced, a specific PNL product structure that correlated to the defense activation in planta and herein reveal this structure. The present invention relates to novel compounds, typically to novel phytopharmaceutical compounds, in particular to a novel compound structure which is a methylated and acetylated oligogalacturonide (OG) with a degree of polymerization (DP) of 4 (also herein identified as “GaUMeAc”).

This compound presents at least one of the following structural characteristics, typically and preferably 2 or 3 of the following characteristics:

- an unsaturated galacturonic acid residue at the non-reducing end, the non-reducing end galacturonic acid residue being typically a 4,5 unsaturated galacturonic acid residue,

- an acetylester, typically an acetyl ester group, which may be located on any galacturonic acid (GalA) unit, and is preferably located on the GalA unit located at the non-reducing end, on any carbon of the GalA unit, preferably on the C₂ or C₃ of a GalA unit, and

- a methyl, which may be located on any GalA unit, and is preferably located on the second galacturonic acid (starting from the reducing end), on any carbon of the GalA unit, preferably on the C₆ of a GalA unit.

In a particular aspect, the compound of the invention comprises an unsaturated galacturonic acid residue at the non-reducing end and an acetylester.

In another particular aspect, the compound of the invention comprises an unsaturated galacturonic acid residue at the non-reducing end and a methyl.

In a further particular aspect, the compound of the invention comprises an acetylester and a methyl.

In a preferred aspect, the compound of the invention comprises an unsaturated galacturonic acid residue at the non-reducing end, an acetylester and a methyl.

In another preferred aspect, the compound of the invention has the below represented formula

(I):

In formula (I), the oligogalacturonide has a 4, 5 -unsaturated galacturonic acid residue at the non-reducing end,“Ac” designates an acetyl ester group (COCH₃) and“Me” designates a methyl group (CH₃).

In a particular aspect, the compound of the invention, typically the compound as herein above described of formula (I), is produced through an enzymatic reaction of a pectin lyase on a pectinic substrate.

Inventors also herein disclose a method or process for preparing a methylated and acetylated oligogalacturonide with a degree of polymerization (DP) of 4. This process preferably comprises a step of contacting a pectinic substrate with a pectin lyase (PNL), typically in presence of a polygalacturonase inhibitor protein (PGIP).

In a particular aspect of the process, the pectinic substrate is a commercial substrate, vegetable waste or a mixture thereof

A commercial substrate can be for example a citrus pectin, sugar beet pectin, apple pectin, or any mixture or combination thereof

Vegetable waste can be for example algae, sugar beet pulp, sugarcane bagasse or any mixture thereof

In another particular aspect, the process for preparing a methylated and acetylated oligogalacturonide with a degree of polymerization (DP) of 4 comprises a step wherein the pectinic substrate is contacted with cells, in particular with a culture of cells producing a PNL or with a PNL as herein described of SEQ ID NO: 33 (PNL of Aspergillus niger ) or of SEQ ID NO: 34 (see also Figure l9a), SEQ ID NO: 35, SEQ ID NO: 36 or SEQ ID NO: 37, preferably in presence of a PGIP, typically at least one PGIP. Said cells producing PNL are typically bacterial or fimgal cells. In a preferred aspect, the culture of cells comprises or consists in B. cinerea cells, preferably avirulent or low virulent B. cinerea strain cells, more preferably B. cinerea mutant strain cells altered in the polygalacturonase 1 (BcPGl) function, even more preferably the Bcpgl mutant strain cells described in Ten Have et al. (1998).

In another particular aspect, the culture of cells is a cellular system allowing the heterologous expression of a PNL (also herein identified as“heterologous expression system”), preferably of a PNL such as herein described of SEQ ID NO: 33 (PNL of Aspergillus niger ) or of SEQ ID NO: 34 (see also Figure l9a), SEQ ID NO: 35, SEQ ID NO: 36 or SEQ ID NO: 37.

In a particular aspect, the polygalacturonase inhibitor protein (PGIP) is a recombinant PGIP. The PGIP can be produced via a method involving a heterologous expression system. This method optionally comprises a PGIP purification step.

In a preferred aspect, the polygalacturonase inhibitor protein (PGIP) is a plant PGIP capable of inhibiting the OG enzymatic activity, such as a PGIP from A. thaliana, for example a PGIP selected from AtPGIPl, AtPGIP2, and a combination thereof.

“Heterologous expression system” refers to a system wherein the cells producing the protein of interest are from a particular species and the PGIP is from a different species, for example a system wherein one of the A. thaliana PGIP protein, like AtPGIPl or PGIP2, is expressed in a yeast such as Saccharomyces cerevisiae or Pichia pastoris.

In a particular aspect, the process for preparing a methylated and acetylated oligogalacturonide with a degree of polymerization (DP) of 4 as herein described is performed in a bioreactor.

“Bioreactor” herein refers to any manufactured or engineered device or system preferably containing a pectinic substrate, cells producing PNL or a PNL as herein described of SEQ ID NO: 33 (PNL of Aspergillus niger ) or of SEQ ID NO: 34 (see also Figure l9a), SEQ ID NO: 35, SEQ ID NO: 36 or SEQ ID NO: 37, a PGIP and a culture medium, typically in conditions allowing the culture of cells, the production of PNL and the enzymatic reaction of PNL on the pectinic substrate.

The person of ordinary skill in the art will easily modulate internal conditions during the process carried out in a bioreactor, such as agitation intensity, temperature, cell culture density, kinds and levels of nutrients, kind of culture (batch, fed-batch or continuous) depending on the nature of cells and substrates in order to allow, preferably facilitate, the culture of cells, the production of PNL and the enzymatic reaction of PNL on the pectinic substrate. The method may also comprise a step of enriching a composition, obtained by contacting a pectinic substrate with a pectin lyase (PNL) in presence of a polygalacturonase inhibitor protein (PGIP) and comprising several oligogalacturonides, in the oligogalacturonide of interest [i.e. in the methylated and acetylated oligogalacturonide with a degree of polymerization (DP) of 4] Such a step may be performed by desalting the composition using any method known by the skilled person (cf. experimental part for example).

In a particular aspect, any of the herein described process of producing a methylated and acetylated oligogalacturonide with a degree of polymerization (DP) of 4 further comprises a step of purifying the obtained methylated and acetylated oligogalacturonide with a DP of 4. Methods for purifying such an oligogalacturonide are well known by the skilled artisan.

In a particular aspect, the methylated and acetylated oligogalacturonide with a degree of polymerization (DP) of 4 having the formula (I):

wherein the oligogalacturonide has a 4,5-unsaturated galacturonic acid residue at the non reducing end, and wherein Ac is C0CH₃ and Me is CH₃, has been obtained by carrying out a process, as herein above described, comprising an enzymatic hydrolysis step using a pectin lyase (PNL), and typically a step of contacting a pectinic substrate with a pectin lyase (PNL), preferably in presence of a polygalacturonase inhibitor protein (PGIP).

The present invention, more particularly relates to a novel compound, typically a novel phytopharmaceutical compound or“elicitor” capable of eliciting natural plant defenses, of activating plant defense and resistance reactions against plant pathogen, of stimulating the production of plant defense molecules against plant pathogen and/or of preventing, controlling or treating a plant against infection by a plant pathogen. This compound is typically a methylated and acetylated oligogalacturonide (OG) with a degree of polymerization (DP) of 4 as herein described. The present invention thus relates to the use of a methylated and acetylated oligogalacturonide with a degree of polymerization (DP) of 4 as herein described for eliciting natural plant defenses, for activating plant defense and resistance reactions against a plant pathogen, for stimulating the production of plant defense molecules against a plant pathogen, and/or for preventing, controlling or treating a plant against infection by a plant pathogen (also called a phytopathogen).

The herein described novel compound is typically for use for eliciting natural plant defenses, for activating plant defense and resistance reactions against a plant pathogen, for stimulating the production of plant defense molecules against a plant pathogen, and/or for preventing, controlling or treating a plant against infection by a plant pathogen.

"Preventing" means avoiding occurrence of at least one adverse effect or symptom, preferably all adverse effects or symptoms induced by a plant pathogen infection.

"Controlling" means stopping the progression of a plant pathogen infection. More precisely it means preventing the plant pathogen spread across the healthy parts of a plant or of an organ of a plant, or from an infected plant to another plant, typically to a neighboring plant.

"Treating" means ameliorating the symptom(s) of an infection, or completely curing an infection, typically by reducing or completely eliminating a phytopathogen (typically a fungus or bacterium), i.e. by eliminating any viable phytopathogen in the plant or in an, several or each organ(s) of the plant.

Inventors herein describe for the first time the use of a methylated and acetylated oligogalacturonide with a DP of 4, as herein described, for preparing a product, or a composition comprising such a product, typically a phytopharmaceutical product or composition, for eliciting natural plant defenses, for activating plant defense and resistance reactions against a plant pathogen, for stimulating the production of plant defense molecules against a plant pathogen, and/or for preventing, controlling or treating a plant against infection by a plant pathogen.

Inventors also herein provide a method for eliciting natural plant defenses, for activating plant defense and resistance reactions against a plant pathogen, for stimulating the production of plant defense molecules against a plant pathogen, and/or for preventing, controlling or treating a plant against infection by a plant pathogen. This method comprises a step of contacting a plant and/or a specific organ of the plant with an effective amount of a methylated and acetylated oligogalacturonide with a DP of 4 as herein described, or with a composition comprising such a product.

The term“organ” and“organ of a plant” or“plant’s organ” refer to a part of plant or to a plant propagation material. Examples of plant’s organ include, but are not limited to, leaves, stems, fruits, seeds, cuttings, tubers, roots, bulbs, rhizomes and the like.

The contacting step of the methylated and acetylated oligogalacturonide with a DP of 4, or of a composition comprising such a product, with the plant or organ can be performed in various ways, for example by spraying, drenching, soaking, dipping, injection, through soil feeding, and any combination thereof.

Alternatively, the composition can be applied on a plant or organ by supplying a volatile form of the composition in the vicinity of the plant tissue and allowing the composition to diffuse to the plant or organ through the atmosphere. The composition application way will be adapted by the one ordinary skill in the art to the particular use.

In a preferred aspect, the disclosure relates to a composition, typically to a phytopharmaceutical composition, comprising at least one methylated and acetylated oligogalacturonide with a DP of 4 as herein described, as well as to uses thereof.

In a particular aspect, the composition comprises several oligogalacturonides (OGs) and the at least one methylated and acetylated oligogalacturonide with a DP of 4 as herein described represents at least 25% of said OGs (total OGs), preferably at least 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98% or 99% of said OGs. A composition comprising OGs wherein the at least one methylated and acetylated oligogalacturonide with a DP of 4 as herein described, typically the methylated and acetylated oligogalacturonide with a 4,5 unsaturated galacturonic acid residue at the non-reducing end and a degree of polymerization (DP) of 4, represents at least 25% of the total OGs is herein described as a composition“enriched in” methylated and acetylated oligogalacturonides with a DP of 4.

A particular liquid composition comprises from about lpg/mL to about 100 mg/mL of at least one methylated and acetylated oligogalacturonide with a DP of 4 as herein described, typically the methylated and acetylated oligogalacturonide with a 4,5 unsaturated galacturonic acid residue at the non-reducing end and a degree of polymerization (DP) of 4, preferably of GalA₄MeAc-H₂0, most preferably from about lpg/mL to about lmg/mL of at least one methylated and acetylated oligogalacturonide with a DP of 4 as herein described, for example 50 Lig/m 1 of at least one methylated and acetylated oligogalacturonide with a DP of 4 as herein described.

In another typical aspect, the composition also comprises a phytopharmaceutically acceptable vehicle or support. In another aspect, the composition further comprises at least one additional component.

The expression“Phytopharmaceutically acceptable vehicle or support” refers to a vehicle or support that does not produce any adverse effect when applied on a plant or on an organ of the plant. Examples of phytopharmaceutically acceptable vehicle or support include, but are not limited to, water, solvent, and a solid carrier. A preferred vehicle or support is water.

Examples of solvents include, but are not limited to, aromatic hydrocarbons, such as, for example, xylene mixtures or substituted naphthalenes; phthalates, such as, for example, dibutyl phthalate or dioctyl phthalate; aliphatic hydrocarbons, such as, for example, cyclohexane or paraffins; alcohols and glycols and their ethers and esters, such as, for example, ethanol, ethylene glycol, ethylene glycol mono methyl or monoethyl ether; ketones, such as, for example, cyclohexanone; strongly polar solvents, such as, for example, N-methyl- 2-pyrrolidone, dimethyl sulfoxide or dimethylformamide; vegetable oils or epoxidised vegetable oils, such as, for example, epoxidised coconut oil or soybean oil; and water. In a particular aspect, the solvent is a volatile solvent, such as methanol and ethanol.

Examples of solid carriers include, but are not limited to, natural mineral fillers, such as, for example, calcite, talcum, kaolin, montmorillonite or attapulgite; highly dispersed silicic acid or highly dispersed absorbent polymers; pumice, broken brick, sepiolite or bentonite; calcite or sand; dolomite or pulverized plant residues.

The expression “additional component” refers to phytopharmaceutically acceptable component known to be useful for applying materials on growing plants such as a cuticle solubilizing molecule or composition, a buffer material for pH control, a natural or regenerated mineral substance, a dispersant, a surfactant, a wetting agent, a tackifier, a thickener, a binder and any combination thereof.

In a particular aspect, the composition has a liquid, a gel or a volatile form. Examples of liquid forms are, without being limited to, a suspension, a solution, or an emulsion, such as for example, an oil-in-water emulsion or a water-in-oil emulsion. An example of gel form is, without being limited to, a gelified aqueous solution. An example of a volatile form is, without being limited to, a composition comprising a volatile organic solvent.

In another particular aspect, the composition has a solid form. Examples of solid form are, without being limited to, powder, granules, pellets, water dispersible powder, water dispersible granules or water dispersible pellets.

In another particular aspect, the composition is formulated as a concentrate to be diluted before use, such as, for example, a soluble concentrate, an emulsifiable concentrate, a liquid concentrate and the like.

The use of a methylated and acetylated oligogalacturonide with a DP of 4 or of a composition as herein described, for eliciting natural plant defenses, for activating plant defense and resistance reactions against a plant pathogen, for stimulating the production of plant defense molecules against a plant pathogen, and/or for preventing, controlling or treating a plant against infection by a plant pathogen is more particularly herein described.

In a preferred embodiment, the composition of the invention is an antifungal and/or an antibacterial adjuvant. In the context of the present invention, an antifungal and/or an antibacterial adjuvant is a product that assists in the prevention or treatment of a plant disease typically caused by fungi or bacteria.

In the present invention, the plant typically designates a plant infected by or presenting a susceptibility to infection by a plant pathogen, typically at least one pectino lytic phytopathogen.

For example, the plant belongs to the clad of Angiosperm.

In one aspect, the plant belongs to the clade of dicots. Examples of plants from the dicots clade include, but are not limited to, the Solanaceae family, comprising Solanum lycopersicum (tomato), Solanum tuberosum (potatoes), Solanum melongena (eggplant), Capsicum genus (pepper) and Nicotiana tabacum (tobacco); the Vitaceae family comprising the Vitis genus (grapevines); the Brassicaceae family, comprising Brassica rapa (turnip and chines cabbage), mustard species and Arabidospis thaliana, and the Rosacceae family, comprising Malus pumila (apple) and Pyrus species (pear). In a preferred aspect, the dicot plant is selected from A. thaliana, tomato, grapevine, apple and pear. In a particularly preferred aspect, the dicot plant is selected from A. thaliana, tomato, grapevine and apple or from A. thaliana, tomato and grapevine. In another aspect, the plant belongs to the clade of monocots. An example of plants from the monocot clade includes, but is not limited to, the Poaceae family. A preferred example of plant from the monocots clade, belonging to the Poaceae family is Zea mays (maize).

In the context of the present invention, the expression“plant pathogen” typically designates a pectinolytic pathogen. This pectinolytic pathogen is typically a bacterium or a fungus.

In a particular aspect, the plant pathogen is a fungus, typically a phytopathogenic fungus.

The expression“phytopathogen fungi” refers to fungi pathogens for plant that infect organs. Examples of phytopathogenic fungi include, but are not limited to, fungi belonging to the Ascomycetes and Basidiomycetes classes, such as, for example, fungi of the order of Helotiales (such as, for example, family Sclerotiniaceae, Botrytis/ Botryotinia); fungi of the order of Hypocreales (such as, for example, family Nectriaceae, genus Fusarium); fungi of the order of Uredinales (such as, for example, family Pucciniaceae, genus Puccinia); fungi of the order of Ustilaginales (such as, for example, family Ustilaginaceae, genus Ustilago); fungi of the order of Sordariomycetes (such as, for example, family Glomerellaceae, genus Colletotrichum).

In a particular aspect the phytopathogenic fungus is selected from a necrotrophic fimgus, a hemibiotrophic fungus and a biotrophic fungus.

During the colonization of plant hosts, most fimgal pathogens exhibit one of two modes of nutrition: biotrophy, in which nutrients are obtained from living host cells, and necrotrophy, in which nutrients are obtained from host cells which have been previously killed by the fungus. A third fimgus mode of nutrition is hemibiotrophy. In this context, the fimgus has an initial period of biotrophy followed by a period of necrotrophy.

Phytopathogenic fungi can then be distinguished depending on their mode of nutrition: necrotrophic, biothrophic or hemibiotrophic.

In a particular aspect herein described, targeted plant pathogens are necrotrophic fungi, for example B. cinerea.

In another particular aspect, targeted plant pathogens are biotrophic fungi. A biotrophic fimgus is for example Ustilago maydis, a pathogen that cause com smut disease. This disease is characterized by tumors (galls) on leaves, stems, tassels, and ears of the maize.

In another particular aspect, targeted plant pathogens are hemibiotrophic fungi, such as Colletotrichum higginsianum. Colletotrichum species are notorious plant pathogens with a later necrotrophic phase associated with severe symptoms. Colletotrichum higginsianum was reported to cause anthracnose lesions blights on dicot and monocot crop plants in temperate, tropical and subtropical regions, for example on the leaves, petioles, and stems of turnip, mustard, and Chinese cabbage.

In another particular aspect herein described, the plant pathogen is a pectinolytic bacterium. Pectino lytic bacterium designates bacterium phytopathogen having a pectinolytic activity against the cell wall of a plant. Examples of pectinolytic bacterium are bacterium of the genus Pectobaterium such as Pectobacterium atrosepticum or Pectobacterium carotovorum, and bacterium of the genus Ralstonia such as Ralstonia solanacearum.

In a particular aspect, the targeted plant pathogen is the bacteria Ralstonia solanacearum, which is an aerobic non spore-forming, Gram-negative, plant pathogenic bacterium. R. solanacearum has a very broad range of hosts. It infects hundreds of species in many plant families. The majority of hosts are dicots, for example of the Solanaceae family such as tobacco, pepper, eggplant and Irish potato.

In the present invention, the plant infection by plant pathogen typically designates a plant infection by at least one phytopathogen. This infection can occur on any organ of the plant.

In a particular aspect, the plant infection is for example a B. cinerea infection, for example a B. cinerea infection of A. thaliana, tomato, grapevines, or apple; a C. higginsianum infection, for example C. higginsianum infection of turnip, Chinese cabbage, mustard, A. thaliana, or apple; a U. maydis infection, for example a U. maydis infection of maize; a R. solanacearum infection, for example a R. solanacearum infection of tomato, potatoes, eggplant, pepper, and tobacco; or any combination thereof, such as a B. cinerea and/or C. higginsianum of A. thaliana, an apple infection by B. cinerea and/or C. higginsianum or a tomato infection by B. cinerea and/or R. solanacearum.

The present description further relates to a method for obtaining a plant that is resistant to pathogens and to a method for activating, or eliciting, plant defense and resistance reactions against plant pathogens. Each of these methods comprises a step of contacting a plant or an organ of a plant with at least one methylated and acetylated oligogalacturonide with a DP of 4, or with a composition comprising such least one methylated and acetylated oligogalacturonide with a DP of 4, as herein described, typically with an effective amount thereof. In a preferred embodiment, the acetylated oligogalacturonide with a DP of 4 has the formula (I). In a particular aspect, anyone of the herein described methods comprises a step of contacting, a plant or an organ of a plant with an effective amount of a composition, typically a phytopharmaceutical composition, as herein described.

The contacting step may be performed once or several times (for example regularly or periodically, for example on the appropriate season or at the appropriate plant development stage).

As used herein,“an effective amount” refers to an amount of the (active) compound of the invention which induces or elicits plant natural defense, activate plant defense and resistance reaction against plant pathogen, stimulates the production of plant defenses molecules against plant pathogen resulting in obtaining a plant that is resistant to pathogen(s).

In a particular example, the effective amount of at least one methylated and acetylated oligogalacturonide with a DP of 4 as herein described for obtaining a plant that is resistant to pathogens, for controlling the plant infection by a phytopathogen, or for treating a plant infection by a phytopathogen, in a liquid composition comprising water as phytopharmaceutically acceptable vehicle, is from about lpg/mL to about lOOmg/mL, most preferably from about 1 lig/rnL to about lmg/mL.

The effective amount is understood to be variable, as it may be affected by many factors, including but not limited to the type of plant treated, treatment dosages and application rates, method of contacting, weather and seasonal conditions experienced during the plant growing cycle, pathogen susceptibility, etc. Such variables are commonly encountered and understood by, the one of ordinary skill in the art, who may adjust the prophylactic or treatment regimen, e.g., application rate, application timings and/or frequencies, and application way.

In another particular aspect of the method, the organ is at least one, for example two, three, or each of the following organs: leaves, roots, stems and fruits. Preferably, the organ is selected from leaves, roots and/or fruits.

The present description also relates to a method for selecting a plant resisting to plant pathogens, wherein the method comprises a step of infecting a plant with a plant pathogen, in particular with a plant fungal pathogen, and a step of determining the presence or absence of at least one methylated and acetylated oligogalacturonide with a degree of polymerization (DP) of 4, typically a methylated and acetylated oligogalacturonide with a 4,5 unsaturated galacturonic acid residue at the non-reducing end and a degree of polymerization (DP) of 4, in a plant sample and, if the plant sample contains such a methylated and acetylated oligogalacturonide with a degree of polymerization (DP) of 4, a step of selecting the plant as resistant to plant pathogens.

In a preferred embodiment, said acetylated oligogalacturonide with a DP of 4 has the formula

CO-

In a particular aspect, the step of infecting the plant is by contacting the plant, typically an organ of a plant, with a plant pathogen suspension or solid culture (such as a plant pathogen agar culture). The step of infecting is for example carried out by contacting plant’s aerial parts, such as leaves, stems and fruits with a plant pathogen suspension, such as a suspension or fungal spores’ or a bacterial suspension. In another example, the step of infecting a plant is by contacting the plant’s roots or rhizomes, with a plant pathogen suspension, such as a suspension of fungal spores or a bacterial suspension. The parameters of the infecting step, such as the pathogen suspension concentration or the nature of organ(s) of the plant to be infected, depend on the plant species and/or on the pathogen species. Those parameters will be easily modulated by the one of ordinary skill in the art.

In another particular aspect, the step of determining the presence or absence of at least one methylated and acetylated oligogalacturonide with a DP of 4 in a plant sample is by the identification of OG products from plant cell wall degradation by the pathogen in an infected plant sample, typically by carrying out a HSEC-MS method such as described in the experimental part, a HILIC (hydrophilic interaction liquid chromatography) or an LCMSMS (liquid chromatography coupled to a tandem mass spectrometry). Examples of plant samples are, without being limited to, any sample of an infected plant’s organ, or an extract of such a sample.

The present description further relates to a method for the early detection of a plant infected by a plant pathogen, wherein the method comprises a step of determining the presence or absence of at least one OG selected from a methylated and acetylated oligogalacturonide with a degree of polymerization (DP) of 4 (GaUMeAc), typically a methylated and acetylated oligogalacturonide with a 4,5 unsaturated galacturonic acid residue at the non-reducing end and a degree of polymerization (DP) of 4, and an oxidized GalA₂ (GalA₂ox), typically the presence or absence of both, in a plant sample and, if the plant sample contains such at least one OG or both OGs, a step of identifying the plant as infected by a plant pathogen.

Inventors herein provide new markers for early detection of a plant infected by a plant pathogen: GafMcAc and GalA₂ox. They observed that some PG products, such as short non- methylated OG, typically GalA₂, are oxidized in muro by a urinate oxidase, a plant enzyme secreted by the plant when exposed to a pathogen. GalA₂ox is the product of a plant’s urinate oxidase on GalA₂ (produced by the action of a pathogenic PG on the plant cell wall).

In a particular aspect, the early detection can be performed as soon as 20h following plant infection, more preferably as soon as 18h following plant infection.

In another particular aspect, the step of determining the presence or absence of an OG, for example of GafMcAc and/or of an oxidized GalA₂ (GalA₂ox), in a plant sample is carried out via a method, such as a HSEC-MS, a HILIC or a LCMSMS method, allowing the detection, and optionally characterization and quantification, of OG products in a plant sample.

Examples of plant samples are, without being limited to, any samples of any infected plant’s organ, or extract of such a sample.

LEGENDS TO THE FIGURES

Figure 1. Elution profile of various oligogalacturonides produced by Aspergillus aculeatus polygalacturonase from citrus pectins using MS-detection.

(a) OG separation on a BEH-HILIC column (b) OG separation on a HP-SEC column. Oligogalacturonides (OGs) are named GalA_xMe_y. Subscript numbers indicate the degree of polymerization and the number of methylester groups respectively. GalA: galacturonic acid; Me: methylester group; Intens. : signal intensity.

Figure 2. A sensitive and high-performance size-exclusion chromatography method for separating complex mixes of oligogalacturonides.

(a) Base peak chromatogram obtained by LC-MS analysis in negative ionization mode of various methylesterified OGs obtained from citrus pectins digested by commercial polygalacturonase (PG) from Aspergillus aculeatus. (b) Impact of the presence of methyl- and acetyl- esters on the retention time (RT) of OGs. OGs were obtained from sugar beet pectins digested by the commercial A. aculeatus PG. (c) Comparison of the structures of methylesterified OGs with a DP from 3 to 4 released by A. aculeatus PG and their respective RT in min. Oligogalacturonides (OGs) are named GalA_xMe_yAc_z. Subscript numbers indicate the degree of polymerization and the number of methyl- and acetyl- ester groups respectively. DP: degree of polymerization; GalA: galacturonic acid; Ac: acetylester group; Me: methylester group; Intens.: signal intensity. Figure 3. Example of oligogalacturonides MS² fragmentation pattern.

MS² fragmentation pattern of GalA_<5Me₂ oligomer (m/z 550.118) produced by Aspergillus aculeatus polygalacturonase from sugar beet pectins. Subscript numbers indicate the degree of polymerization and the number of methylester groups respectively. GalA: galacturonic acid; Me: methylester group; Intens. : signal intensity.

Figure 4. MS² fragmentation patterns of various short oligogalacturonides produced by Aspergillus aculeatus polygalacturonase from sugar beet pectins.

(a) GalAsMe, (b) GalA₄Me, (c) GalA₄Me₂.

Figure 5. Kinetics of oligogalacturonides produced overtime by the pectinolytic activities of purified Botrytis cinerea enzymes from commercial citrus peel pectins.

(a) OGs released by BcPG2 over time (b) Impact of BcPMEl on OGs released by BcPG2 over time (c) OGs released by BcPG3 over time (d) Impact of BcPMEl on OGs released by BcPG3 over time. Oligogalacturonides (OGs) are named GalA_xMe_yAc_z. Subscript numbers indicate the degree of polymerization and the number of methyl and acetyl-ester groups. GalA: galacturonic acid; Ac: acetylester group; Me: methylester group.

Figure 6. Oligogalacturonomic (OGome) analysis to characterize pectinolytic activities of Botrytis cinerea on commercial citrus peel pectins.

(a) Characterization of OGs released by BcPG2 and BcPG3 in presence or absence of BcPMEl after 20 h of incubation with citrus pectins. Data are means ± SD; n = 3. (b) Kinetics of OGs produced by WT B. cinerea strain over time from citrus pectins and evolution of the OG methylesterification rate over time. Data are means ± SD; n = 3. Oligogalacturonides (OGs) are named GalA_xMe_y. Subscript numbers indicate the degree of polymerization and the number of methylester groups. GalA: galacturonic acid; Me: methylester group.

Figure 7. OGome of Arabidopsis thaliana - Botrytis cinerea interaction.

(a) Proportion of pectin lyase products among total OGs produced after 20 h infection of A. thaliana leaves by B. cinerea. Data are means ± SD; n = 3. (b, c) Fine characterization of OGs produced after 20h infection by HP-SEC-MS. (b) Pectin lyase products, (c) Polygalacturonase products. Data are means ± SD; n = 3. (d) MS² fragmentation pattern of oxidized GalA₂ oligomer (m/z 385.0683) eluting at 7.8 min in Fig. la using HP-SEC with online MS. (e) Kinetics of GalA₃, GalA₂ and oxidized GalA₂ upon infection. Data are means ± SD; n = 3. Oligogalacturonides (OGs) are named GalA_xMe_yAc_z. Subscript numbers indicate the degree of polymerization and the number of methyl and acetyl- ester groups respectively. DP: degree of polymerization; GalA: galacturonic acid; Ac: acetylester group; Me: methylester group; Intens.: signal intensity. Figure 8. Botrytis cinerea BcPGl to BcPG6 and BcPMEl and BcPME2 gene expression measured by RT-qPCR after 6 and 16 hours of incubation with citrus pectins.

Figure 9. Bcpmel/2 and Bcpgl to Bcpg6 strains are defective in citrus pectin digestion and Bcpgl, Bcpg2 and Bcpmel/2 are impaired in oligogalacturonides production.

(a) Light scatter detection of size-exclusion chromatographic profile of non-digested and digested citrus pectins by WT B. cinerea and mutant strains after 6 h of incubation coupled to light scatter detection (b) OGs production of Bcpmel/2 and Bcpgl to Bcpg6 mutants compared to WT strain after 16 h ad 24 h of citrus pectin digestion. Oligogalacturonides (OGs) are named GalA_xMe_y. Subscript numbers indicate the degree of polymerization and the number of methylester groups respectively. GalA: galacturonic acid; Me: methylester group. Figure 10. Size-exclusion chromatographic (SEC) profiles of non-digested and digested citrus pectins by WT Botrytis cinerea strain, Bcpmel/2 and Bcpgl to Bcpg6 mutants after 6 h of incubation.

(a) Refractive index detection of SEC profiles (b) Viscosity detection of SEC profiles (c) Pectin size after 6 h of incubation assessed by SEC-MALLS analysis. n= 2.

Figure 11. Principal component analysis (PC A) of oligogalacturonides production by WT Botrytis cinerea and mutant strains from citrus pectins.

Unit variance scaling is applied to rows; SVD with imputation is used to calculate principal components. X and Y axis show principal component 1 (PC1) and principal component 2 (PC2) respectively (a) PCA assuming genotypes as variables after 16 h of incubation. PC1 and PC2 explain 36.1 % and 32.7 % of the total variance respectively (b) PCA assuming genotypes as variables after 24 h of incubation. PC1 and PC2 explain 47.7 % and 21.7 % of the total variance respectively. Oligogalacturonides (OGs) are named GalA_xMe_y. Subscript numbers indicate the degree of polymerization and the number of methylester groups. GalA: galacturonic acid; Me: methylester group.

Figure 12. Bcpmel/2 and Bcpgl display opposite virulence correlated to different plant defense activation during infection of Arabidopsis thaliana leaves.

(a) Extracted ion chromatograms obtained by HP-SEC-MS analysis in negative ionization mode of oligogalacturonides endogenously produced during infection of A. thaliana leaves infected by WT B. cinerea strain and Bcpgl, Bcpmel/2 mutants over time (b) Infection assays of WT B. cinerea and mutants strains on rosette leaves of A. thaliana. Statistical data of lesion size 72 hours post infection. Values are means ± SEM (n = 15); **P < 0.01 by Student test (c) B. cinerea co-infection assay on rosette leaves of A. thaliana. Leaves were infected with WT B. cinerea strain or Bcpmel/2 followed 24 h later by Bcpgl or a mock control (PDB medium). Lesion size of the infected leaves was measured 72 h later. Values are means ± SEM (n = 25); * P < 0.05 by Student test. DP: degree of polymerization.

Figure 13. Principal component analysis (PCA) of oligogalacturonides production by WT Botrytis cinerea strain and Bcpmel/2 20 hours post infection of Arabidopsis thaliana leaves.

Unit variance scaling is applied to rows; SVD with imputation is used to calculate principal components. X and Y axis show principal component 1 (PC1) and principal component 2 (PC2). (a) PCA assuming genotypes as variables. PC1 and PC2 explain 42.5 % and 18.3 % of the total variance, respectively (b) PCA assuming OGs as variables. PC1 and PC2 explain 51.3 % and 26.4 % of the total variance, respectively. Oligogalacturonides (OGs) are named GalA_xMe_yAc_z. Subscript numbers indicate the degree of polymerization and the number of methyl and acetyl-ester groups. GalA: galacturonic acid; Ac: acetylester group; Me: methylester group.

Figure 14. Identification of a novel oligogalacturonide elicitor, GaLAiMeAc-IhO.

(a) Heatmap of all the OGs (OG; 71 rows) for 12 samples (columns). Annotation labels refer to OG structure. Rows are centered; unit variance scaling is applied to rows. Both rows and columns are clustered using McQuitty distance and maximum linkage (b) Extracted ion chromatograms for GalA₄MeAc-H₂0 produced by Bcpgl, Bcpmel/2 and WT B. cinerea strains over time during infection of A. thaliana leaves (c) GUS activity quantified by fluorescence in leaves from transgenic A. thaliana plants expressing the GUS reporter gene under the control of AtPGIPl promoter. Leaves were infiltrated either by GaLAtMeAc-ELO or with a control consisting of a mix of methylesterified OGs from DP4 to DP 10. Values are means ± SEM (n = 6); * P < 0.05 by Mann- Whitney test (d) Fragmentation pattern of GaLAiMeAc-EbO. Oligogalacturonides (OGs) are named GalA_xMe_yAc_z. Subscript numbers indicate the degree of polymerization and the number of methyl- and acetyl- ester groups respectively. GalA: galacturonic acid; Ac: acetylester group; Me: methylester group; Intens.: signal intensity.

Figure 15. Principal component analysis (PCA) of oligogalacturonides production by WT Botrytis cinerea strain and Bcpmel/2 20 hours post infection of Arabidopsis thaliana leaves after the exclusion of GalA₂ox.

Unit variance scaling is applied to rows; SVD with imputation is used to calculate principal components. X and Y axis show principal component 1 and principal component 2 respectively (a) PCA assuming OGs as variables. PC1 and PC2 explain 40.9 and 33.1 % respectively (b) PCA loadings show that Bcpmel/2 and the WT strains are separated according to PC2. Oligogalacturonides (OGs) are named GalA_xMe_yAc_z. Subscript numbers indicate the degree of polymerization and the number of methyl- acetyl- ester groups. GalA: galacturonic acid ; Ac: acetylester group; Me: methylester group.

Figure 16. Relative abundance of oligogalacturonides present in semi-purified extracts used to infiltrate A. thaliana leaves.

(a) Mix of OGs containing low levels of GalA₄MeAc-H₂0. (b) Mix of OGs enriched in GaLMMeAc-FhO, i.e. a mix wherein GahMMeAc-FhO represents at least 25%, typically at least 50%, of the OGs. Oligogalacturonides (OGs) are named GalA_xMe_yAc_z. Subscript numbers indicate the degree of polymerization and the number of methyl- and acetyl- ester groups. OGs were purified by HP-Sec from leaves infected by bcpmel/2 (a) or by WT (b). DP: degree of polymerization, GalA: galacturonic acid; Ac: acetylester group; Me: methylester group.

Figure 17. BcPNLl is active on highly methylesterified pectins and at alkaline pH.

(a) Pectin lyase activity was measured at 235 nm at different pH, with glycine-NaOH and Tris-HCl buffers. Data represent the mean ± SE of three replicates (b) Pectin lyase activity was measured at 235 nm using substrates with various degrees of methylesterification (DM). Data represent the mean ± SE of three replicates (c) MS identification of OGs from DP4 to DP5 released by BcPNLl from Arabidopsis thaliana leaf cell wall. Boxed OGs are also detected in OGome produced during the Arabidopsis thaliana - Botrytis cinerea interaction. Figure 18. JA-signaling inhibition is modulated by the DP4/DP5 ratio produced during infection.

(a) Quantification of transcripts from JA and defense-related genes in non-infected leaves (mock) and leaves infected by WT Botrytis cinerea and Bcpmel/2 strains (b) Composition of semi-purified OGs from leaves infected either by WT Botrytis cinerea or Bcpmel/2 strains (c) Expression of AtPAD3 and ATPR1 and AtJOX3/AtJRG21 in leaves incubated either by semi-purified OGs from leaves infected either by WT Botrytis cinerea or Bcpmel/2 strains. Values are means ± SEM (n = 4); * P < 0.05 by Mann- Whitney test. Subscript numbers indicate the degree of polymerization and the number of methyl- and acetyl- ester groups respectively. GalA: galacturonic acid; Ac: acetylester group; Me: methylester group; Intens.: signal intensity.

Figure 19. Sequences alignment and expression of BcPNLl.

(a) Sequence alignment of BcPNLl and pectin lyase, family 1 (PelA) of Aspergillus niger. The residues underlined are involved in the enzyme-substrate interaction. The arginine residues, boxed in grey, are predicted to be N-glycosylated. (b) Western blot verification of the expression of BcPNLl by using anti-his antibodies and chromogenic detection. L: molecular weight markers.

Figure 20. MS identification of OGs from DP6 to DP9 released by BcPNLl from Arabidopsis thaliana leaf cell wall.

Boxed oligogalacturonides (OG) are also detected in OGome produced during the Arabidopsis thaliana - Botrytis cinerea interaction. OGs are named GalA_xMe_yAc_z. Subscript numbers indicate the degree of polymerization and the number of methyl and acetyl- ester groups respectively. GalA: galacturonic acid; Ac: acetylester group; Me: methylester group; Intens.: signal intensity.

Figure 21. Spectra of semi-purified OG preparations from WT- (a) and Bcpmel/2- (b) infected leaves.

Further aspects and advantages of the present invention will be disclosed in the following experimental section and figures which shall be considered as illustrative only.

EXPERIMENTAL SECTION

Materials and Methods

Plant Growth

A. thaliana wild-type Wassilewskija (WS) plants were grown in soil in a growth chamber at 22 °C, 70% humidity, under irradiance of 100 pmol-nf^s^-1 with a photoperiod of 8h light/ 16h dark.

Fungal strains and growth

The wild-type B. cinerea B05.10 strain and Bcpgl-6 and Bcpmel/2 mutants (Ten Have et al., 2001; Kars et al, 2005a) were grown on potato dextrose agar at 23 °C under continuous light. After 10 days, each strain produced a dense carpet of conidia.

Fungal growth for OG analysis

The spores were washed from the surface of the plate using Gamborg’s B5 basal medium, 2% (w/v) fructose and 10 mM phosphate buffer pH 6.4. Fungal hyphae were removed from the suspension by filtering. The concentration of spores was determined using a Malassez cell and adjusted. To analyze OGs released from citrus pectins (Sigma, P9135), a 0,8% pectic solution in Gamborg medium was mixed v/v with spore suspensions at 6 x 10⁵ spores/ml and incubated on a rotary shaker at 100 rpm at 23 °C during 6, 16 or 24 h. To analyze OGs released during infection, isolated A. thaliana leaves of 5-week-old plants were directly immersed in a B. cinerea suspension (6 leaves for 10 ml of suspension at 3 x 10⁵ spores/ml) and incubated on a rotary shaker at 100 rpm at 23 °C during 12, 15, 18 or 20 h. For all the experiments, the liquid media was then collected and an equal volume of 96% ethanol was added to precipitate the largest molecules. After centrifugation (5000 g during 10 min), the supernatant was collected and dried in a speed vacuum concentrator at room temperature. The obtained pellet was then diluted. For OGs produced from pectins, 2 ml were dried and diluted in 200 mΐ. For OGs released during infection, the equivalent of the digestate of 3 Arabidopsis leaves of 5-week-old A. thaliana plants was dried and diluted in 200 mΐ. 10 mΐ were injected for MS analysis.

Source of enzymes and pectins

Endopolygalacturonase M2 from Aspergillus aculeatus (Megazyme) was used as reference. Pichia pastoris lines expressing B. cinerea BcPG2, BcPG3 and BcPMEl, were obtained from Jan A. L. Van Kan and grown for 3 days at 30 °C in Yeast Extract Peptone Dextrose (YEPD) solid medium (Kars et al, 2005b). The methods to purify BcPMEl, BcPG2 and BcPG3 have been described previously (Kars et al, 2005b; L’Enfant et al., 2015). Enzymatic activities were tested using commercial pectic substrates (Citrus peel pectins with a degree of methylesterification (DM) of 70% (Sigma), sugar beet pectins).

OG characterization and quantification

Hydrophilic interaction liquid chromatography (HILIC). Pectin digests, diluted to 1 mg/ml in 50% (v/v) acetonitrile, were analyzed using an UltiMate™ 3000 RSLCnano System system (Thermo Scientific, Waltham, MA, USA) coupled to an Impact II UHR-QqTOF (Bruker). Chromatographic separation was performed on an Acquity UPLC BEH HILIC column (1.7 pm, 2.1 mm x 150 mm, Waters Corporation, Milford, MA, USA). Elution was performed at a flow rate of 500 m 1/m in and a column oven temperature of 40 °C. The injection volume was set to 1 mΐ.

The composition of the two mobile phase lines were (A) 99: l(v/v) water/acetonitrile 15 mM with (water/ ACN) 0.1% formic acid, (B) 90% (v/v) ACN ammonium formate 15 mM/ formic acid 0.1%. The following elution profile was used: 0-1 min, isocratic 100% B; 1-30 min, linear from 100% to 60% B; followed by column re-equilibration; 35-45 isocratic 100 % B. MS-detection was performed in negative mode with the end plate offset set voltage to 500 V, capillary voltage to 2500 V, Nebulizer 50 psi, dry gas 10 Emin and dry temperature 200 °C. Mass spectra were acquired over the scan range m z 150-2000. Compass 1.8 software, (Bruker Daltonics) was used to acquire and process the data.

High-performance size- exclusion chromatography (HP-SEC). Samples were diluted at 1 mg/ml in ammonium formate 50 mM, formic acid 0.1%. Chromatographic separation was performed on an ACQUITY UPLC Protein BEH SEC Column (125 A, 1.7 pm, 4.6 mm X 300 mm, Waters Corporation, Milford, MA, USA). Elution was performed in 50 mM ammonium formate, formic acid 0.1% at a flow rate of 400 m 1/m in and a column oven temperature of 40 °C. The injection volume was set to 10 mΐ. MS-detection was performed in negative mode with the end plate offset set voltage to 500 V, capillary voltage to 4000 V, Nebulizer 40 psi, dry gas 8 Emin and dry temperature 180 °C.

Data analysis

Major peaks were annotated following accurate mass annotation, isotopic pattern and MS/MS analysis (Fig. 14). The MS fragmentation pattern is indicated according to the nomenclature of Domon and Costello (1988). The fragments are designated as X for cross-ring cleavages and Y, Z for glycosidic bond cleavages when charge is retained at the reducing end and A (cross ring cleavages) and B, C (glycosidic bond cleavages) when charge is retained at the non-reducing end. Sugars, indicated as subscript number, are numbered from the reducing end for X, Y and Z ions and from the non-reducing end for the others. For cross-ring cleavages, the cleaved bonds are indicated by superscript numbers. At last, ions produced as a result of more than one cleavage are designated with a slash between cleavage sites (e.g. ^0,2A₄/^1,5X2). Inventors took in account that Z- and C-type were found to be dominant over Y- and B-type ions in negative ion mode (Komer et al., 1999; Quemener et ah, 2003a; Quemener et al., 2003b).

For the targeted analysis of 72 specific oligosaccharides, the theoretical exact masses were used with 4 significant figures with a scan width of 5 ppm. The resulting extracted ion chromatograms were integrated and the area under the curve was used for relative quantitation. Data were analyzed using principle component analysis (PCA). The heatmap presented in Fig. 14a was generated using ClustVis (“http://biit.cs.ut.ee/clustvis”).

Semi-purification of OGs and GUS quantification

OG-containing samples corresponding to 16 leaves infected either by WT strain or Bcpmel/2 were desalted using Pierce Graphite spin column (Thermo Scientific, Waltham, MA, USA). A sample containing a high relative amount of GaLMMeAc-EbO was selected. As a control, Bcpmel/2 samples containing the highest relative amount of OG DP4 was used. After desalting, both samples were dried. GaLMMeAc-EbO-enriched samples (i.e. samples wherein GalA₄MeAc-H₂0 represents at least 25% of the OGs) were resuspended at a final concentration of 50 Lig/m 1. The GaLViMeAc-EEO OGs were next infiltrated in leaves of 5- week-old seedlings expressing the defense reporter construct pAtPGIPlr. GUS. GUS activity analyses were performed on the aerial part of 5-week-old seedlings as described by Elmayan and Vaucheret (1996) with some modifications: the GUS buffer does not contain any b- mercaptoethanol and the measures were performed with a fluoroskan ascent (Thermo Scientific, Waltham, MA, USA). Three pools of two leaves of two different replicates were analyzed.

Size-exclusion chromatography and multi-angle laser-scattering

Pectins (1 mg/ml in 0.1 M L1NO3) were injected on an on-line size-exclusion chromatography (SEC) column coupled with multi-angle laser-light scattering (MALLS), a differential refractive index (dRI) detector and a viscometer (Viscostar, Wyatt Technology Inc., Santa Barbara, USA). Experiments were performed as described in Rihouey et al. (2017).

Quantitative polymerase chain reaction

After incubation for 6 and 16 h, total RNA was extracted from harvested biomass using Trizol reagent (Invitrogen, Carsbad, CA, U.S.A.). Reverse transcription was performed using an oligo-dT20 for a primer and Superscript II RnaseH-reverse transcriptase (Invitrogen). Real time quantitative PCR analysis was performed using Bio-rad Cfx Connect. A 1 :5 dilution of cDNA (2.5 mΐ) was amplified in a 7.5 mΐ reaction mix containing Power Syber green PCR master mix (Applied Biosystems) and 10 mM of each primer (Table 1). Gene expression values were normalized to expression of B. cinerea gene actin.

Table 1. List of primers used for RT q-PCR experiments.

The BcPNLl cloning

The coding sequence of BcPNLl (Bo fuT4_P032630) (Leroch et al, 2013) including the native peptide signal part, was amplified by PCR using Phusion®Taq polymerase (Thermofisher scientific) from B. cinerea gDNA with two specific primers (Table 1). The expression vector pPICZaB (Invitrogen, Cat. No. VI 9520) was digested by Rv/BI and Vo/I, and the insert was ligated into the vector. After transformation in E. coli TOP 10 (Invitrogen, Cat. No. C404003), the insert was verified by sequencing, the linearized construct was used to transform Pichia pastoris X-33 strain as described in the instruction manual P. pastoris expression kit (Invitrogen, Cat. No. K1710-01). Transformants were selected on Zeocin. Protein extraction and purification, enzymatic activities

The P. pastoris line expressing BcPNLl were grown in baffled flasks in 10 mL of buffered glycerol-complex medium, overnight at 30°C using the appropriate antibiotic. Cells were then collected by centrifugation and resuspended to an OD600 of 1.0 in 100 mL of buffered methanol complex medium. A final concentration of 0.5 % (v/v) methanol was added every 24h to maintain induction. After 72h of induction, the culture was centrifuged at 1 500 g for 10 min. The supernatant was loaded onto a lml HisTrap excel column (GE Healthcare) to affinity purification. The eluate fractions were concentrated using centrifugal filter units (Amicon® Ultra-4, Millipore). 6 pg of eluate were loaded into a 10% SDS-PAGE with Coomassie blue staining. The protein concentrations were determined using the Bradford assay with bovine serum albumin as a standard. To identify the recombinant protein by Western blot, SDS-PAGE was transferred from resolving gel to PVDF blotting membrane using the appropriate cathode and anode buffers and a Trans-Blot TURBO Transfer System (Bio-Rad, Cat. No. 170-4155) at 0.1 A for 30 min. TBS-T (0.5% Tween 20 in TBS) was used as washing buffer and 4% non-fat dried milk in TBS-T was used as blocking reagent. Transferred proteins were incubated for 1 h at room temperature under shaking with 1 :4000 dilution of anti-his antibody coupled with peroxidase (Sigma, Cat. No. A7058). After washes, the reagent DAB substrate (ThermoFisher Scientific, Cat. No. 34002) was used to detect the protein of interest according to the supplier’s instructions. Substrate specificities of pectin lyase were tested on following substrates: polygalacturonic acid (Sigma, Cat. No. 81325); Citrus pectin, degree of methylesterification (DM) 20-34% (Sigma, Cat. No. P9311); Citrus pectin, DM 55-70% (Sigma, Cat. No. P9436); Citrus pectin, DM >85% (Sigma, Cat. No. P9561); apple pectin DM 70-75% (Sigma, Cat. No. 76282); sugar beet pectin, DM 42%, degree of acetylation 31% (CPKelco). Pectin lyase activity from purified BcPNLl was determined using a protocol adapted from Albersheim (1966). 25 mΐ of purified BcPNLl (17.2 ng) was added into 100 mΐ of pre-heated 0.5 % (w/v) substrate in 50 mM Tris-HCl buffer (pH7.8) and incubated at 40 °C. Pectin lyase activity of forming unsaturated products was determined by measuring a linear increase in absorbance at 235 nm for 20 min.

The optimal pH of BcPNLl was assayed in 50 mM with glycine-NaOH buffer (pH 7.7-10.0) and Tris-HCl buffer (pH 6.8-8.2) at 40°C, according to the same conditions as previously described and using high DM Citrus pectin as substrate.

One enzyme unit is defined as the formation of 1 pmo 1 unsaturated pectin per min, with a molar extinction coefficient of 5500 M ¹ cmf¹. Each enzymatic measurement was performed in triplicate. Quantitative RT-PCR

After incubation for 3h with semi-purified OGs (50 pg/mL), leaves were quick-frozen in liquid nitrogen and stored at -80°C. RNA was extracted using Qiagen RNAeasy kit (Qiagen, Paris, France) lpg of RNA was treated with RNA- free DNAse and used for RT-PCR using the RevertAid H using ingredients from ThermoFisher (Villebon-sur-Yvette, France). 10 ng of RNA was used for each q-PCR. 3 technical repeats were performed for each primer pair (Table 1) and 3 biological repeats for each sample. Transcript levels were normalized using the reference gene UBI4 according to the formula E*2^A(Cq target - Cq ref). Gene transcript levels were expressed relative to the mock (treatment without OG), i.e. the calibrator for which expression level was set to 1.

Affymetrix Microarray analysis

24h after infection with Bcpme or WT strains, leaves were collected and RNA was extracted. Total RNA extraction was carried out from three biological replicates of infected leaves using the Nucleospin RNA XS purification kit according to the manufacturer’s instructions (Macherey Nagel, Germany), including the removal of genomic DNA. RNA samples were quantified using a Nanodrop spectrophotometer (Thermo Scientific, USA) and quality control (RIN>8) was assessed by a 2100 Bioanalyzer (Agilent Technologies, Santa Clara, USA). cRNAs, prepared from 100 ng of total RNA according to the GeneChip Whole Transcript (WT) PLUS protocol (Affymetrix, Thermo Fisher Scientific, USA), were used to generate single- stranded DNAs, which were fragmented and biotinylated according to the manufacturer’s instructions. The labelled single-stranded DNAs were hybridized for 18 hours at 48°C on Affymetrix four-arrays strips (Arabidopsis Gene 1.1 ST Array strip) in Affymetrix GeneAtlas hybridization station. After hybridization, strips were washed (Affymetrix GeneAtlas Fluidics Station) and imaged (GeneAtlas imaging station).

Data were normalized using the Expression Console software (Affymetrix) using default RMA-sketch normalization. Normalized files were then analyzed by Transcriptome Analysis Console (TAC) 4.0 software (Affymetrix) including Limma differential expression analysis with eBayes correction of ANOVA variance using default settings. Normalized expression values were filtered for statistical relevance of differential expression using FDR F-Test p- value<0,0l.

Statistical analyses

For testing the normality of distribution, the Shapiro-Wilk test was performed. For normally distributed data, a Bartlett’s test was performed to compare the variance of samples. Unpaired t-tests were used. For smaller set of data, inventors performed a Mann- Whitney test.

Results Development of a sensitive separative method for OG characterization

In a first experiment, OGs were analyzed in the hydrolysis products of commercial citrus pectins (degree of methylation (DM): 70% (Luzio et al, 2013)) digested for 20 h with Aspergillus aculeatus PG. OGs were separated using hydrophilic interaction liquid chromatography (HILIC) (Remoroza et al, 2012; Leijdekkers et al, 2011; Remoroza et al., 2014), and characterized using high-resolution mass spectrometry (HR-MS) in negative mode. Although a number of methylated and non-methylesterified GalA oligomers originating from the HG backbone could be distinguished in the HILIC elution pattern (Fig. 1), non- or poorly methylesterified OGs were insufficiently separated in broad peaks resulting in low MS sensitivity (Fig. 1). To improve the separation power, inventors developed a high- performance size-exclusion chromatography (HP-SEC)-MS-based method, which yielded sharper peaks (a few seconds) resulting in a higher signal to noise ratio relative to the HILIC separation (Fig. 2a). This method allowed the DP and methylation pattern of each OG to be determined based upon its retention time, accurate mass annotation, isotopic pattern and MS/MS analysis (Fig. 3 and Table 2).

Table 2. List of oligogalacturonides produced by the commercial Aspergillus aculeatus polygalacturonase from citrus and sugar beet pectins.

The retention time was surprisingly not only determined by the DP but also by the methylesterification status of the OG (Fig. 2a and Table 2), methylesterified OGs eluting later (corresponding to a smaller hydrodynamic volume) than their unmethylesterified counterparts. In contrast, the presence of the, more bulky, acetylester groups did not affect the retention time as shown by the analysis of OGs produced from highly acetylesterified sugar beet pectins (Fig. 2b and Table 2). This indicates that CV, methylesterification, unlike C2/C3 acetylesterification, can reduce the hydrodynamic volume of the OGs. The structural basis for this effect was revealed by the analysis of OGs that did not follow this rule (Fig. 2c, Fig. 4 and Table 2), such as GalA₃Me2, which co-eluted with, not after, GalA₃Me (GalA-GalAMe- GalA) and GalA₄Me₃, which co-eluted with GalA₄Me2 (GalA-GalAMe-GalAMe-GalA). Comparison of the structures (Fig. 2c) showed that OGs with two adjacent unmethylesterified GalAs systematically eluted earlier than OGs of the same size lacking this feature. The increased hydrodynamic volume therefore can be attributed to the electrostatic repulsion between two adjacent carboxylic functions, making the OG more extended (Fig. 2c).

Oligogalacturonomic (OGome) analysis to characterize pectinolytic activities of B. cinerea

The B. cinerea genome encodes a large array of putative pectinolytic enzymes: 13 PGs, 5 PMEs, 2 PLs and 5 PNLs. Knock-out mutants were obtained for 6 PGs (Ten Have et al., 2001) (BcPGl, 2, 3, 4, 5 and 6) and two BcPMEs (Kars et al, 2005a) (PME1 and 2). Their analysis showed that BcPGl and BcPG2 are necessary for full virulence on various plant species (Ten Have et al., 1998; Kars et al., 2005b), while BcPMEl and 2 are dispensable for normal virulence, at least on tomato and grapevine leaves or pear fruit. The activities of the 5 PGs (BcPGl -4,6) were previously characterized on polygalacturonic acid (PGA) (Kars et al., 2005b). Here, inventors used oligogalacturonomic analysis (OGome) on a more complex methylesterified substrate (citrus pectins, 70% DM) to study in more details the activity of two PGs, BcPG2 and BcPG3, representatives of single attack (non-processive) and multiple attack (processive) enzymes respectively. The former cleave only once after formation of the enzyme-substrate complex and release OGs of varying DPs, whereas the latter attack the substrate multiple times and release GalA and short OGs already from the start of the reaction (Fig. 5).

Inventors first purified both PGs from recombinant Pichia pastoris liquid cultures and incubated the enzymes with citrus pectins. After 20 h of digestion, both PGs produced mainly GalA, non-methylesterified dimers (GahM) and trimers (GalA₃) (Fig. 6a) as well as small amounts of methylesterified OGs. As expected, the non-processive enzyme BcPG2 generated mainly GahM and GalA₃, whereas the processive BcPG3 produced primarily GalA and GalA2. Such processive PG activity requires fully de-methylesterified HG stretches, which are generated by (processive) plant PMEs. However, the presence of methylesterified OGs (up to three methylester groups in GalA Mcs or GalA₆Mc₃) indicates that both PGs can also cleave partially demethylesterified stretches. Inventors next studied the combined activity of BcPMEl and BcPG2 or BcPG3. BcPMEl enhanced almost 5 times the production of GalA, GalA₂ and GalA₃ by BcPG2 and completely suppressed the accumulation of methylesterified OGs (Fig. 5 and Fig. 6a). In contrast, BcPMEl had a much more limited impact on the overall BcPG3 activity but promoted a shift from GalA + GalA₂ towards GalA₂ + GalA₃ for the most abundant OGs. Interestingly, some higher DP OGs, including GalA₆Me₃, resisted further digestion (Fig. 5 and Fig. 6a), suggesting that this OG is a poor substrate for both BcPMEl and BcPG3. Together, these results confirm that BcPG2 requires, and BcPG3 has a preference for, substrates of at least DP4 (Kars et al, 2005b), but also show that their substrates can be partially methylesterified. In addition, they show that non-processive BcPMEl greatly enhances the number of cleavage sites for non-processive PG activities but, as expected, does not enhance the processive PG activity of BcPG3. Finally, they show that BcPMEl demethylesterifies both the HG polymer and OGs, even the trimers, except for GalA₆Mc₃, which, for an unknown reason, is more recalcitrant to the enzyme.

Inventors next used OGome analysis to study the global B. cinerea pectino lytic activity. To this end, citrus pectins were incubated with B. cinerea spores and the OGome was analyzed over time. After 6 h of incubation, only a very small amount of OGs was detected (Fig. 6b). After 16 h, primarily GalA₂ and GalA₃ but also methylesterified OGs from DP4 to DP7 had accumulated. Since purified BcPG2 and BcPG3 did not produce such methylesterified oligomers in large amounts, the production of these OGs most likely involved other pectinolytic enzymes. At 24h, the relative amount of GalA₂ had increased and that of methylesterified OGs from DP4 to DP7 had decreased. This confirms that B. cinerea PMEs and PGs can use OGs as a substrate.

OGome of B. cinerea - A. thaliana interaction

So far, inventors showed the activity of B. cinerea and three of its purified enzymes on citrus pectins, which is a complex but inert substrate. Inventors next investigated how the interaction with living plant cells affects the pectinolytic activity of the fungus. Previous studies have shown that plants produce defensive PG-inhibiting proteins (PGIPs) that attenuate the activity of fungal PGs (De Lorenzo et al., 2002). This is thought to promote the accumulation of large non-methylesterified OGs with elicitor activity (Benedetti et al., 2015). However, very little is known on the OGs that are actually produced for example during the interaction of B. cinerea with its host. Inventors studied the OGome of A. thaliana leaves incubated with B. cinerea spores in liquid culture. Under these conditions, the first plant symptoms can be observed after 10 hours post infection (hpi). They chose 20 hpi to collect the medium, when significant maceration of the tissue could be detected. After concentration, the medium was analyzed by HP-SEC. Given the complexity of the mix analyzed, they used in vitro released OGs as standards. Surprisingly, the most abundant OGs differed by 18 mass units (H₂0) from OGs released from citrus or sugar beet pectins (Table 3). This difference is due to the loss of a water molecule between C₄ and C₅ of the non-reducing end galacturonic acid residue during the B- elimination reaction catalyzed by pectin lyases (PNL) (Linhardt et al, 1987). These OGs are named GalA_xMe_yAc_{z -} H₂0. They conclude that during infection, only 20% of the accumulated OGs are actually produced by PGs, the remaining 80% being the product of PNLs (Fig. 7a). This is in striking contrast with the OGome of B. cinerea grown on citrus pectins, in which no PNL products were found (Fig. 6b). Table 3. List of the oligogalacturonides produced upon infection of Arabidopsis thaliana by Botrytis cinerea.

Among the PNL products, twenty different OGs of DPs ranging from 3 to 10 were identified (Fig. 7b). The main products displayed a DP of 4 and 5, the GalA₄MeAc - H₂0 being the most abundant, representing 25% of the OGs. It is also worth noting that all the PNL- generated OGs contained at least one GalAMe for two GalAs, some of them also being acetylesterified.

The PG-derived OGs were mainly of DP2 and 3. Surprisingly, DP3 OGs were all methylesterified and the bulk of the DP2 OGs was not GalA₂ but an ion at m/z 385, differing by 16 mass units (oxygen) from GalA₂ (369) (Fig. 7c and Table 3). The MS/MS fragmentation pattern of this ion showed two major fragments corresponding to an uronic acid linked to a galactaric acid, which is a Ci-oxidized uronic acid (Fig. 7d). To determine the origin of this oxidized GalA₂ (GalA₂ox), inventors performed a kinetic study of GalA₃, GalA₂ and GalA₂ox from 12 hpi to 18 hpi. Whereas the amount of all three OGs increased from 12 to 15 hpi, GalA₃ and GalA₂ progressively disappeared in favor of GalA₂ox from 15 to 18 hpi. Given the absence of GalA₂ox in the absence of living plant cells, inventors assume that GalA₃ is immediately converted into GalA₂ and oxidized by a plant oxidase.

Relative contribution of the main PG and PME activities in pectin degradation during B. cinerea growth

To gain better insight into OG production during A. thaliana leaf infection and to understand the role of these OGs in plant defense activation, inventors took advantage of B. cinerea mutants altered for each of the six PGs and the double mutant Bcpmel/2. RT-qPCR analysis showed that all the genes were expressed in WT B. cinerea grown in the presence of citrus pectins (Fig. 8) after 6 and 16 hours of incubation. They next analyzed the size of citrus pectins, using size-exclusion chromatography, after 6 h incubation with the WT strain or each of the pg and pine mutants. Interestingly, multiple-angle light scattering (Fig. 9a), refractive index detection and viscosimetry analyses (Fig. 10) showed a higher pectin size for all the mutants relative to the WT strain (Fig. 9a and Fig. 10), indicating that all these enzymes contributed to pectin digestion.

They also analyzed the OGs released after l6h and 24h of B. cinerea growth on citrus pectins. A principal component analysis (PCA) showed that after 16 h of growth, Bcpgl and Bcpmel/2 samples were separated from others (Fig. 11a) and that at 24 hpi, Bcpmel/2 samples formed an independent group (Fig. lib). They scored the results for log2 fold change compared to the WT strain (Fig. 9b). After l6h, Bcpgl, Bcpmel/2 and to a lesser extent Bcpg2 were the most affected in OG production while no differences were observed in the other mutants. Interestingly, Bcpmel/2 was the only mutant affected in GalA₂ production and showed higher levels of methylesterified OGs of DP 6 and DP 7. After 24 h, Bcpmel/2 accumulated, relative to the WT strain, even more methylesterified OGs of DP 5 to 7 and less GalA₃ and GalA₄Me. In addition, all the mutants, except Bcpgl, accumulated less GalA₄Me than the WT strain, suggesting the involvement of all these enzymes in the production of this OG (Fig. 9b). Interestingly, the strong of the OGome of Bcpgl relative to that of the WT at l6h, had completely disappeared at 24h. This indicates that BcPGl is critical for the rate of OG accumulation, not for OG accumulation per se.

Bcpgl and Bcpmel/2 are affected in OG accumulation during infection

Inventors next investigated whether the perturbation of OG accumulation had any impact on the virulence of the mutant fungi. They focused on Bcpgl and Bcpmel/2 since these mutants showed, in addition to compromised degradation of citrus pectins, the most strongly perturbed OG profiles. A. thaliana leaves were infected with mutant and WT B. cinerea strains and the in vivo OGome and virulence were analyzed. HP-SEC-MS/MS analysis confirmed that OG production was affected in the two B. cinerea mutants also during infection of A. thaliana leaves (Fig. 12a). Indeed, for the WT strain, the relative OG composition evolved from 12 hpi to 18 hpi. At 12 hpi, the OG size profile showed two peaks corresponding respectively to OGs having a DP> 7 and to OGs having a DP between 3 and 5 (3<DP<5). The amounts of OGs having a DP> 7 did not change during the time course, the amount of OGs having a DP between 3 and 5, however, dramatically increased between 12 and 15 hpi and leveled off at 18 hpi. At this stage, a new peak corresponding to DP2 OGs (GalA₂ox) had appeared (Fig. 12a). In the mutants, the OG profile was strongly altered. Firstly, whereas, like for the WT strain, the total amount of OGs increased over time, the relative OG composition did not evolve, suggesting that the encoded enzymes were required for a specific step in the conversion of the OGs. For Bcpmel/2, the OGs having a DP> 7 accumulated relative to the WT strain, as observed on citrus pectins. In addition, DP4 levels were strongly reduced and no DP2 accumulated at 18 hpi. BcPME activity therefore appears to be critical for the turnover of the OGs having a DP> 7, the production of DP4 OGs and the conversion of OG into GalA₂ox. For Bcpgl, hardly any OGs having a DP> 7 were detected, again as observed on citrus pectins, and the majority of the OGs was of DP4, with very minor amounts of DP2 or DP3. BcPGl activity therefore appears to contribute to the generation of the OGs having a DP> 7 but also to the turnover of DP4 OGs. B. cinerea infection assays showed low virulence for Bcpgl (Ten Have et ah, 1998/ and comparable (after 72 hpi) virulence for Bcpmel/2 (Kars et al., 2005a/, relative to the WT (Fig. 12b). Given the fact that both mutants are compromised in pectin turnover, the results indicate that the virulence of the fungus does not only reflect its capacity to degrade pectin (which, on first sight, might explain the reduced virulence of Bcpgl ) but also its ability to escape detection by the defense system of the plant, which may be the case for Bcpmel/2. To test the latter possibility, inventors infected leaves with Bcpmel/2 followed, 24 h later, by Bcpgl or a mock treatment (Fig. 12c). Interestingly, the presence of Bcpgl reduced the virulence, relative to the mock control. No such effect was observed when Bcpmel/2 was replaced by the WT strain in the experiment. These results show that the increased virulence of Bcpmel/2, despite its impaired pectinolytic capacity, reflects the absence of a presumably OG elicitor, which is present in leaves infected with Bcpgl or WT B. cinerea strains.

A new OG elicitor

To identify such an elicitor, inventors compared at 20 hpi the OGome generated by Bcpmel/2 on A. thaliana leaves with that generated by the WT strain. PCA results revealed clustering of replicate samples and a clear separation between Bcpmel/2 and the WT strain despite the higher variability in Bcpmel/2- infected samples relative to the WT strain (Fig. 13a). PCA analysis on 71 OGs showed a clear separation of GalA₂ox and GalA₄MeAc-H₂0 from the other OGs (Fig. 13b). They observed a high variability of GalA₂ox amount between samples that was not related to fungus genotypes analyzed. Therefore, it was excluded for further analysis. Hierarchical clustering was next performed to classify the remaining 70 OGs according to their relative abundance in the different samples (Fig. 14a and Fig. 15). This showed that GalA₄MeAc-H₂0 was most strongly overrepresented for the WT strain relative to Bcpmel/2. This OG was also the most abundant OG at 20 hpi and clustered with GalA^Mc. Bcpmel/2 instead, showed higher levels of GalA₅Me₂Ac-H₂0 relative to the WT.

To further investigate the correlation between the presence of GalA₄MeAc-H₂0 and reduced virulence, they quantified this OG in WT-, Bcpgl- and Bcpine 1 /2- i nfcctcd leaves at 12, 15 and 18 hpi (Fig. 14b). Interestingly, Bcpgl indeed produced this OG and, despite the reduced overall OG content of the mutant, it released this OG earlier and accumulated higher levels of it relative to the WT. This OG appears therefore as a good candidate for a defense response elicitor. To confirm its elicitor activity, the molecule was compared to a mix of methylesterified OGs produced by Bcpmel/2 as control (see Fig. 16 for OG composition). They next infiltrated leaves of transgenic A. thaliana plants containing the GUS reporter gene under the control of AtPGIPl promoter (pAtPGIPl ::GUS [b-glucuronidase]) which is known to be induced by trimers and OGs having a DP from DP 10 to 15 and whose expression correlates with the mechanisms of plant defense (Davidsson et al, 2017 ; Ferrari et al, 2003). Their results showed that, at 24 hours post infiltration, AtPGIPl was twice more activated by GalA₄MeAc-H₂0 than by OGs produced by Bcpmel/2 (Fig. 14c).

To determine the precise position of the acetyl- and methyl- ester groups on the GalA₄MeAc- H₂0, they analyzed the fragmentation pattern (See Fig. 14d and Methods). The acetyl ester was only found on the A- and C- fragments, corresponding to the non-reducing end, but never on the Z-fragments, which corresponds to the reducing end. Moreover, the ring cleavage product ^0,2 Ai at 157.0051 m/z proves that the acetylesterification occurs on the C₃ of the GalA at the non-reducing end.

A fungal PNL contributes to OG production during infection.

Inventors assumed that the PNL activity detected came from B. cinerea, since so far, only pectate lyase, no pectin lyase activity has been observed in plants (Senechal et al., 2014). They therefore selected, from published transcriptome profiles (Leroch et al., 2013), the most abundant transcript, encoding a putative pectin lyase in germinating B. cinerea conidia, ( BcPNLl ) to express the enzyme in Pichia pastoris. The enzyme, purified from the culture medium (Figures 19a and 19b), showed PNL activity on various commercial substrates with a sharp optimum at pH 8 (Fig. 17a) and a preference for highly methylesterified pectins (Fig. 17b). To assess the potential role of this PNL in the production of unsaturated OGs during infection, inventors analyzed the OGs produced by this enzyme from A. thaliana leaf cell walls by HPSEC-MS. They detected OGs from DP4, all methylesterified and some also acetylesterified (Fig. 17c and Fig. 20). Among these OGs, 15 were also found in B. cinerea- infected A. thaliana leaves (Fig. 17c and Fig. 20), while the other OGs were more methylesterified. Together, these results are consistent with the involvement of BcPNLl, in particular in the initial steps of OG production during B. cinerea infection.

GaLAiMeAc-FhO enhances its JA response at least in part by restricting the accumulation of JOX3 transcripts

The negative correlation between GalA₄MeAc-H₂0 levels and fungal virulence might indicate a specific activity for this OG as opposed to other OGs. Alternatively, all three genotypes may produce OGs with elicitor activity but the difference in virulence of the mutants may reflect differences in the timing of the accumulation of GalA₄MeAc-H₂0 and perhaps other OGs. To clarify the role of GalA4MeAc-H20, inventors compared the transcriptomes of leaves infected for 24 h with WT or Bcpmel/2, when symptoms were already visible. Comparison of fungus- (WT or Bcpmel/2 ) with mock-infected leaves revealed a total of 7395 differentially expressed A. thaliana genes, many of which were annotated as“defense related”. Among those, the pool of transcripts encoding negative regulators of JA signaling fJOX proteins hydroxylate and inactivate JA (Caarls et al., 2017; Smirnova et al., 2017) and JAZ proteins are transcriptional repressors of JA-regulated genes (Zhang et al. 2015)] was slightly upregulated in leaves infected with Bcpmel/2-, relative to the WT strain. This did not simply reflect a more advanced infection stage of Bcpmel/2, since transcript levels for other defense-related genes were comparable, if not slightly lower, in Bcpmel/2 vs WT-infected leaves. This suggests that, among the immune responses elicited by the fungus, JA-dependent defenses are induced more efficiently by the WT strain than by Bcpmel/2 and that GalA₄MeAc-H₂0 is responsible, at least in part, for these differences.

Inventors next produced semi-purified OG preparations from WT- and Bcpmel/2- infected leaves, enriched or depleted for GalA₄MeAc-H₂0 respectively (Fig. 18b and Fig. 21) and which completely lacked the previously identified demethylesterified OG elicitors OG10-15 and OG3. GalA₄MeAc-H₂0 represents 48 % of total semi-purified OGs from WT-infected leaves. Incubation of A. thaliana leaves with either preparation induced, within 3 h, the expression of defense genes PAD 3 and PR1 (Fig. 18c), indicating the presence of OG elicitors in both fractions. .10X3 transcript levels, instead, were slightly higher in leaves treated with the Bcpmel/2- relative to the WT-derived OG fraction. This result supports the hypothesis that the plant may, upon recognition of GalA₄MeAc-H₂0, enhance its JA response, at least in part by restricting the accumulation of JOX3 transcripts (Fig. 18c).

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Claims

1. Use of a methylated and acetylated oligogalacturonide with a 4,5 unsaturated galacturonic acid residue at the non-reducing end and a degree of polymerization (DP) of 4 for preventing, controlling or treating a plant against infection by a plant pathogen.

2. A phytopharmaceutical composition comprising a methylated and acetylated oligogalacturonide with a 4,5 unsaturated galacturonic acid residue at the non-reducing end and a degree of polymerization (DP) of 4, and a phytopharmaceutically acceptable vehicle or support.

3. The phytopharmaceutical composition according to claim 2, wherein the composition is an anti-fimgal and/or an anti-bacterial adjuvant.

4. The use according to claim 1, wherein the methylated and acetylated oligogalacturonide with a degree of polymerization (DP) of 4 has the formula (I):

(I),

wherein Ac is COCH3 and Me is CH3.

5. A process for preparing a methylated and acetylated oligogalacturonide with a degree of polymerization (DP) of 4, wherein the method comprises a step of contacting a pectinic substrate with a pectin lyase (PNL) in presence of a polygalacturonase inhibitor protein

(PGIP).

6. The process according to claim 5, wherein the amino acid sequence of the pectin lyase is selected from SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36 and SEQ ID NO: 37.

7. The process according to claim 5 or 6, wherein the process is performed in a bioreactor.

8. The use according to claim 1 or 4, wherein the methylated and acetylated oligogalacturonide with a 4,5 unsaturated galacturonic acid residue at the non-reducing end and a degree of polymerization (DP) of 4 is obtained by carrying out the process of claim 5, 6 or 7.

9. The composition according to claim 2 or 3, wherein the methylated and acetylated oligogalacturonide with a 4,5 unsaturated galacturonic acid residue at the non-reducing end and a degree of polymerization (DP) of 4 is obtained by carrying out the process of claim 5, 6 or 7.

10. The use according to anyone of claim 1, 4 or 8, wherein the plant is a dicot plant, preferably selected from Arabidopsis thaliana, tomato, grapevine and apple.

11. The use according to anyone of claim 1, 4 or 8, wherein the plant is a monocot plant, preferably maize.

12. The use according to anyone of claims 1, 4, 8, 10 or 11, wherein the plant pathogen is a pectinolytic pathogen selected from a necrotrophic fungus, a hemibiotrophic fungus, a biotrophic fungus and a pectinolytic bacterium.

13. A method for activating plant defense and resistance reactions against plant pathogens, wherein the method comprises a step of contacting a plant, in particular an organ of a plant with an effective amount of at least one methylated and acetylated oligogalactoturonide with a degree of polymerization (DP) of 4.

14. The method of claim 13, wherein contacting of the plant is carried out via a plant’s organ selected from leaves, roots and/or fruits.

15. A method for selecting a plant resisting to plant pathogens, wherein the method comprises a step of infecting a plant with a plant fimgal pathogen and a step of determining the presence or absence of at least one methylated and acetylated oligogalactoturonide with a degree of polymerization (DP) of 4 in a plant sample and, if the plant sample contains a methylated and acetylated oligogalactoturonide with a degree of polymerization (DP) of 4, a step of selecting the plant as resistant to plant pathogens.

16. A method for the early detection of a plant infected by a plant pathogen, wherein the method comprises a step of determining the presence or absence of a methylated and acetylated oligogalacturonide with a degree of polymerization (DP) of 4 and of an oxidized GalA₂ (GalA₂ox) in a plant sample and, if the plant sample contains a methylated and acetylated oligogalacturonide with a degree of polymerization (DP) of 4 and a GalA₂ox, a step of identifying the plant as infected by a plant pathogen.