Dr Jonathan Trouvé

Paolo Zardi

Dr Shaymaa Al-Shehimy

Dr Thierry Roisnel

Rafael Gramage-Doria
email: rafael.gramage-doria@univ-rennes1.fr

Enzyme-Like Supramolecular Iridium Catalysis Enabling C-H Bond Borylation of Pyridines with meta-Selectivity

The use of secondary interactions between substrates and catalysts is a promising strategy to discover selective transition metal catalysts for atom-economy C-H bond functionalizations. Unfortunately, the most powerful catalysts are still found via trial-anderror screening due to the low association constants between the substrate and the catalyst in which small stereo-electronic modifications in the catalyst (and/or the substrate) can lead to completely different reactivity patterns. In order to circumvent these limitations and to increase the level of reactivity prediction in this type of important reactions, we report herein a supramolecular catalyst harnessing Zn … N interactions that bind to pyridine-like substrates as tight as it could be found in some enzymes. Furthermore, the distance and spatial geometry between the catalytically active site and the substrate binding site is ideal to target unprecedented meta-selective iridium-catalyzed C-H bond borylations with enzymatic Michaelis-Menten kinetics, besides unique substrate-selectivity and dormant reactivity patterns.

Introduction

In the last three decades, C-H bond functionalizations enabled by transition metal catalysts have shown a remarkable impact in the advancement of chemical synthesis due to high atom-and stepeconomy it represents. [1] Besides substrates with a biased reactivity [2] or controlled by metal-directing groups, [3] the reactivity at inherently unreactive sites is of paramount importance to access new chemical dimensions. To meet this challenge, a standard approach deals with extensive fine-tuning of the catalyst first coordination sphere with different stereo-electronic modifications [4] or to introduce multiple catalytic cycles. [5] Although useful, substantial costs and efforts need to be undertaken to trialand-error studies to search for the optimal catalytic system.

On the other hand, enzymes, Nature's catalysts, exploit alternative approaches to control chemical reactivity. [6] Importantly, they incorporate a number of kinetically reversible interactions in the second coordination sphere of the active site to stabilize key transition states. [7] For chemists, this has been a source of inspiration to mimic in abiological catalysis. [START_REF]Supramolecular Catalysis[END_REF] As such, different strategies have been developed to design transition metal catalysts incorporating remote functionalities to preorganize substrates in a specific geometry to access unprecedented C-H bond functionalizations. [9] In this context, the incorporation of hydrogen bonding, [10] ion-pairing [11] or Lewisadducts formation, [12] respectively, to transition metal catalysts has emerged as promising approaches to control the regioselectivity in iridium-catalyzed C-H bond borylation reactions (Figure 1A). [13] Borylated products are relevant as they can straightforward be engaged in state-of-the-art carbon-carbon and carbon-heteroatom bond forming process. [14] Unfortunately, the predictability of these catalytic systems is still poor rationalized, basically due to the low association constants between the substrate and the catalyst, generally with K < 10 2 M -1 . [10][11][12] Consequently, small modifications in the commonly-used bipyridine-derived ligand and/or the substrate can lead to a complete lack of reactivity or absence of selectivity. [10][11][12] Precisely, we aimed at circumventing the poor reactivity of pyridine derivatives at meta position. [15a] Iridium-catalyzed C-H bond borylations typically occur at the meta position of pyridine derivatives when bulky substituents are present in the ortho site in order to prevent the undesired coordination of the pyridine nitrogen lone pair to the iridium catalyst, which gives unproductive and unselective catalysis (Figure 1B). [12a,15b] To overcome these challenges, we anticipated that a geometrically-constrained iridium catalyst featuring a relatively strong pyridine-to-catalyst interaction may increase the activity and selectivity for C-H bond borylation reactions. Herein, we present a rationally-designed supramolecular iridium catalyst exhibiting strong affinity for pyridine derivatives (K > 10 4 M -1 ) by means of kinetically labile Zn … N coordination (Figure 1C). [START_REF] Bonar-Law | kinetically labile Zn … N interactions for purely organic reactions[END_REF] Due to the perfect geometry and ideal atom-precise distance between the active site and the substrate-recognition site, only metaborylated pyridines were obtained (Figure 1C). In addition, the catalytic system displayed enzyme-like features such as Michaelis-Menten kinetics and substrate selectivity. Applications to five-membered nitrogen-containing heterocycles and dormant reactivity are also disclosed.

Results and Discussion

Semi-empirical molecular modelling (PM3-level) showed that the geometry of ligand L (Scheme 1 and Figure 1C) as well as the distance between the zinc(II)-porphyrin fragment (the substrate binding site) and the iridium N,N-chelating motif (the potentially catalytically active site) in the periphery is suitable to accommodate pyridine substrates with selective activation at the meta-C-H bond site. [START_REF] Boller | The active catalytic species in iridium-catalyzed C-H bond borylation is postulated to be a N,N-chelated iridium trisboryl complex, for seminal works see[END_REF] In addition, the restricted motion between the N,N-chelating motif and the zinc(II)-porphyrin backbone will increase the rigidity of the system by keeping the peripheral iridium active site just above the zinc(II)-porphyrin plane. With this in mind, the targeted supramolecular ligand L was readily synthesized in a four-step reaction sequence starting from commercially available chemicals (see Scheme S1). [18] As expected, ligand L is in keeping with a Cs-symmetrical molecule according to NMR spectroscopy studies. [18] 1 H NMR and UV-vis binding studies established the ability of L to interact with pyridine as the model substrate with an association constant of K1.1 = 5.7 x 10 4 ± 1.5 M -1 at room temperature (Scheme 1, see Figures S1-S3). [18,19] Notably, this substrate-to-catalyst binding is as tight as it can be found in some enzymes. [20] The binding of pyridine to L was also evidenced by 1 H NMR studies at temperatures up to 90 o C and by DOSY experiments that showed that the up-field shifted proton signals belonging to the pyridine diffused together with those corresponding to L (see Figures S4-S7). [18] Scheme 1. Binding of pyridine to L via kinetically labile Zn … N coordination.

Moreover, single crystals suitable for X-ray diffraction studies were obtained from a solution containing L and pyridine in an equimolar ratio (Figure 2, left). [21] As anticipated, the nitrogen atom from pyridine binds to the zinc(II) center of L. Gratifyingly, the coordinating arm of L stands in the same face as the substrate with a volumetric space between both motifs enough to accommodate a catalytically active iridium center. Analogously single crystals with a potential substrate, N-methylimidazole, were also obtained and analysed by X-ray diffraction studies (Figure 2, right). As expected, the non-methylated nitrogen atom (N1) from the substrate was bound to the zinc(II) center from L. The binding of N-methylimidazole to L via Zn … N interaction was also corroborated in solution by 1 H NMR spectroscopy studies (see Figure S13). [18] In parallel, coordination chemistry studies by 1 H NMR and HRMS (m/z = 1121.2599) showed that the reaction between L and 0.5 equivalents of [Ir(COD)(Cl)]2 afforded the corresponding cationic iridium complex L1 (Scheme 2, see Figures S8-S10). [18] Subsequent 1 H NMR studies by treatment of the iridium complex L1 with increasing amounts of pyridine showed a single set of upfield shifted protons belonging to pyridine (Scheme 2) in a similar way as it was observed with the titration studies between L and pyridine (see Figures S11-S12). [18] Scheme 2. Simultaneous coordination of both iridium and pyridine to L.

Having established that L enables the binding of pyridine inside the zinc(II)-porphyrin pocket simultaneously to the coordination of the N,N-chelating unit towards the potentially, catalytically active iridium center, we embarked in the catalytic assessment of L as a supramolecular ligand in iridium-catalyzed C-H bond borylations between unfunctionalized pyridine and bis(pinacolato)diboron (B2pin2) as a model reaction (Table 1). This reaction is well-known to give a mixture of meta-and paraborylated products with classical bipyridine ligands [15a] and it represents a benchmark test for evaluating the activity and the regio-selectivity outcome. [13] Initially, we screened solvents that

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Angewandte Chemie were known to be used in iridium catalyzed C-H bond borylation of (hetero)arenes such as heptane and ethers at temperatures near to their boiling point. [10][11][12][13][14][15]22] Although poor conversions were observed, an exquisite meta selectivity was evidenced as only the products resulting from mono-and bis-borylation 1 and 2 formed without any detectable para or ortho regioisomers 3 or 4 (Table 1, entries 1-4). The reaction in toluene as solvent at 70 o C revealed as the optimal one for obtaining the mono-functionalized product with meta selectivity in an isolated yield of 54% (Table 1, entry 5, GC-yield 90%). [START_REF]A similar conversion and yield was obtained under extensive ligand optimization in dioxane at room temperature[END_REF] This is rather unexpected because nonfunctionalized arenes (i.e. toluene) are typically more reactive than pyridine using bipyridine-or phenantroline-derived ligands for iridium-catalyzed C-H bond borylation reactions. [13,22,24] In our case, we only detected trace amounts of borylated toluene and up to 15% (based on B2pin2) in the case of the reaction performed at higher 80 o C, that again exhibited excellent meta-selectivity for the C-H bond borylation of pyridine (Table 1, entry 6). For instance, with 58 times more toluene than pyridine in the reaction mixture, our supramolecular catalyst exhibits a high preference for the pyridine substrate than for the aromatic toluene one with an overall selectivity S estimated at 380 for the most unfavorable scenario. [25] Decreasing the temperature to 60 o C completely inhibited the catalysis (Table 1,entry 7). This suggests that the substrate-catalyst or product-catalyst binding via Zn … N coordination is strong and it only becomes reversible to enable turnovers in catalysis at higher temperatures. [26] Switching the toluene solvent for a more bulky p-xylene led to no borylation at the solvent and high reactivity towards meta-selectivity in only 12 hours (Table 1,entry 8). Doubling the amounts of both B2pin2 and the iridium catalyst led to the bis-borylated, meta-selective product 2 in an isolated yield of 70% (Table 1,entry 9), which represents the best result so far obtained for bis-functionalization to date. [27] The fact that aromatic apolar solvents such as toluene or p-xylene are crucial for the activity and selectivity of the catalysis, indirectly indicates that the polar ones (i.e. ethers) significantly disturb the binding of the substrate to the ligand L. [START_REF] Kadri | O-containing polar solvents (DMF, DMSO, acetone, THF, etc.) completely cleave the Zn … N interaction between zinc(II)-porphyrinoids and pyridine derivatives, for examples see references: a)[END_REF] A similar reasoning may explain the higher reactivity encountered when using [Ir(COD)(Cl)]2 instead of the typically more reactive [Ir(COD)(OMe)]2 [9][10][11][12][13]15,[START_REF] Boller | The active catalytic species in iridium-catalyzed C-H bond borylation is postulated to be a N,N-chelated iridium trisboryl complex, for seminal works see[END_REF][22][START_REF]A similar conversion and yield was obtained under extensive ligand optimization in dioxane at room temperature[END_REF][24] as the released methoxide anion from the former could bind to the zinc(II) center in L (Table 1, entry 2). The reactions performed in the absence of iridium or in the absence of ligand L, respectively, led to no conversion of pyridine substrate (see Table S1). [18] Table 1. Reaction optimization for the iridium-catalyzed C-H bond borylation of pyridine. [a] Entry Solvent T ( o C) t (h) Conv. (%) [b] (1+2):3:4 [c] 1:2 [d] 1 heptane 80 48 30 100:0:0 90:10

2 [e] heptane 9 [g] p-xylene 80 48 >99 (70) [f] 100:0:0 13:87 To further rationalize the origin of this high meta-selectivity for the iridium-catalyzed C-H bond borylation reaction, a number of control experiments were performed (Scheme 3). First, the catalysis was attempted replacing the supramolecular ligand L for the individual components forming it, that is, zinc(II)tetraphenylporphyrin (ZnTPP) and the N,N-chelating ligand L* (Scheme 3A). Under the standard reaction conditions, the starting material pyridine was fully recovered unreacted, highlighting the relevance of covalently-linking the substrate recognition site to the catalytically active site as it is the case in the supramolecular ligand L. For comparison purposes, the reaction performed using 4,4'-di-tert-butyl-2,2'-dipyridyl (dtbpy) as ligand with or without the presence of ZnTPP led to an almost statistical mixture of meta-and para-borylated products (Scheme 3B). [15a] The relevance of the substrate binding to the zinc(II)porphyrin pocket was further evidenced by the lack of reactivity observed for pyridines having no lone pair available for binding (i.e. pyridinium derivatives, Scheme 3C) as well as for pyridines unable to bind to the zinc atom due to steric shields (i.e. 2methylpyridine, Scheme 3C). [START_REF] Kadri | O-containing polar solvents (DMF, DMSO, acetone, THF, etc.) completely cleave the Zn … N interaction between zinc(II)-porphyrinoids and pyridine derivatives, for examples see references: a)[END_REF][START_REF] Morisue | For selected examples of negligible binding between 2-substituted pyridines and zinc(II)-porphyrinioids, see: a)[END_REF] A last experiment was performed adding to the standard reaction conditions zinc(II)salphen (ZS) as a substrate competitive inhibitor (Scheme 3D) since it is known that zinc(II)-salphen derivatives bind to pyridine derivatives typically two orders of magnitude higher than zinc(II)porphyrins. [28a,30] In this scenario, an almost complete inhibition of 10.1002/ange.202101997

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Angewandte Chemie catalysis took place with one equivalent of ZS. These observations show that the substrate is significantly bound to the zinc(II)-salphen ZS in which no catalysis takes place and that the catalysis do occur only if the substrate binds to the supramolecular ligand L. Next, a substrate scope was evaluated. The supramolecular catalytic system was found compatible with 3-substituted pyridine derivatives giving the respective meta-borylated products in yields up to 99% (Table 2). For instance, alkyl, aryl, ether, ester, and halogen (F, Cl, Br, CF3, except iodide) functionalities were tolerated (5a-5h, Table 2). On the other hand, no reactivity was observed for carboxylic acid or amide groups (Table 2). 5membered ring heterocycles such as N-protected imidazoles delivered the -substituted borylated products in virtually quantitative yields (6a-6c), which appears promising with respect to literature precedents. [START_REF] Smith | Process for Producing Oxazole, Imidazole, Pyrazole Boryl Compounds[END_REF] Additionally, semi-empirical molecular modelling (PM3-level) showed that the supramolecular iridium catalyst fits well also for the -C-H bond selectivity observed for 5-membered ring heterocycles (see Figure S15). Interestingly, the 2-methylimidazole substrate which is known to bind to zinc(II)porphyrinoids derivatives [START_REF] Nappa | For selected examples of binding between 2-methylimidazole and zinc(II)-porphyrinoids, see: a)[END_REF] did afford the corresponding -C-H bond borylated derivative 6c in 97% isolated yield. This strikingly contrast with the lack of reactivity found for 2-methylpyridine (Scheme 3C) and it clearly shows that the geometry of the substrate is a key parameter for the reactivity observed within this supramolecular catalyst. Limitations of the catalytic system appeared when considering 4-substituted pyiridines and pyrimidines which did not react due to steric effects as it was noted elsewhere (Table 2). [33] Table 2. Substrate evaluation for the supramolecular iridium-catalyzed C-H bond meta-borylation controlled by remote Zn … N interactions. [a] [a] Yields estimated by GC analysis and isolated yields shown in brackets, the slight difference in these values is due to loss and/or partial protodeboration of the products during purification.

We then embarked on the kinetic evaluation of this supramolecular catalysis with a model reaction using 3methylpyridine as the substrate. [18] For this purpose, preliminar reaction progress kinetic analysis (RPKA) were carried out at same and different excess concentrations (see Section 5.4 in the Supporting Information). [34] As it was found before, [17b] the reaction features an incubation period that depends on the catalyst loading, being shorter at higher catalyst loadings (Figure 3, left). In addition, we noted that at lower concentrations of B2pin2, its consumption was not directly correlated to the formation of the product as it is the case when considering the 3-methylpyridine substrate consumption (Figure 3, middle). Higher reaction rates have been observed with B2pin2 instead of HBpin for a certain types of substrates. [15b] This suggests, that a second catalytic cycle is operating to some extent involving a degradation sideproduct from B2pin2, probably HBpin because H2 (another sideproduct) [START_REF]conditions led to 79% of borylated product and 15% of unreacted HBpin, indicating that B2pin2 is a better borylating reagent than HBpin under our standard reaction conditions[END_REF][START_REF] Wright | Although we do not have any spectroscopical evidence for the formation of HBpin or any other borylated species derived from B2pin2, previous reports from the literature indicate that HBpin is indeed a borylating reagent with side-formation of H2, but less reactive than B2pin2[END_REF] is detected by 1 H NMR spectroscopy studies. [18] Besides these observations, the RPKA analysis showed that the catalysis is first order in iridium and pseudo-zero-order in B2pin2, as it could be expected. [15b,17a] On the other hand, the order in substrate did not match to zero as shown by Hartwig with a nonsupramolecular catalyst, [15b] but it rather fitted to a pseudo-first order as it has been seen with non-heteroaromatic substrates. [17a] This strongly supports that the resting state in this supramolecular catalysis does not involve an iridium complex coordinated to the N-containing substrate via Ir-N bonding such as Ir-1 (Figure 1B), [15b] but rather a tris-boryl iridium species formed later in the catalytic cycle likely upon substrate binding to the zinc(II)porphyrin site such as A (Scheme 4). Although the overlay was not fully precise by RPKA analysis due to the limitations previously described, unambiguous catalyst deactivation was evidenced that we ascribed to catalyst degradation [START_REF]Attempts to isolate L after the catalysis indicate substantial degradation according to TLC analysis[END_REF] as no

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Angewandte Chemie product inhibition was observed by RPKA analysis. [18] This indicates that the catalyst immediately releases the product after formation in a similar way as enzymes do. We anticipate that the bulkiness of the product might be at the origin for the absence of product inhibition, thus indirectly enhancing substrate binding under catalytic conditions. Consequently, such catalytic system may follow an enzymatic Michaelis-Menten kinetic behavior in which the whole catalytic events occur upon binding of the substrate in the zinc(II)-porphyrin pocket (Figure 3, right). In fact, a decent fitting (R 2 = 0.994) was found for the plot of rate versus concentration of 3-methylpyridine substrate with Vmax = 0.104 (±0.02) M h -1 and Kmm = 0.107 (±0.04) M. Aiming at identifying potential catalytically active species, the meta-C-H bond borylation reaction was followed on time under catalytic conditions using mulitnuclei NMR spectroscopy (Scheme 4). First, the pre-active iridium catalyst Ir-2 involving trisboryl species was identified at catalytically-relevant temperatures (see Section 6 in the Supporting Information). [18] The 1 H NMR studies of Ir-2 showed two set of signals at  = 1.07 and 1.06 ppm corresponding to two different set of Bpin groups in a 1:2 ratio (one Bpin fragment was in axial position and the other two Bpin fragments were in equatorial position; note that they are not equivalent at high temperatures due to the low symmetry of the species). [17a] Similar observations were evidenced by 11 B NMR studies ( = 22 and 23 ppm in a 1:2 ratio). [18] Next, the reaction between 3-methylpyridine and B2pin2 with the in situ formed Ir-2 was followed on time. [18] The 3-methylpyridine substrate was bound to the supramolecular ligand L via Zn … N interaction under catalytic conditions as regards of the up-field shifted pyridine proton signals strongly supporting the formation of species A (Scheme 4). Although the transient iridium-hydride species B (as well as D) was not detected, the product-to-catalyst species C was formed according to the disappearance of the pyridine signals belonging to A and the merger of a set of three new upfield shifted pyridine proton signals (Scheme 4). Further GC analysis and 1 H NMR spectroscopy studies indicated quantitative formation of meta-borylated product, indicating that the product is easily released from the catalyst pocket under the catalytic conditions. The overall combination of above-described results together with previous data from the literature, [START_REF] Mkhalid | [END_REF] enabled us to propose a catalytic cycle in which all the reaction steps occur after substrate binding to the porphyrin pocket of L with final release of the product after selective meta-C-H bond borylation (Scheme 4). To further show the enzyme-like behavior of this supramolecular iridium catalyst, we wondered whether it would be possible to perform substrate-selective catalysis with a reaction comprising a mixture of all three possible regioisomers derived from 3-methylpyridine as the substrates. [39] Indeed, the supramolecular catalyst formed upon combination of the iridium precursor and L gave a selective system towards the exclusive meta-selective borylation of the meta isomer with no reactivity observed at the other substrates even upon 24 hours reaction time (Scheme 5). A single product formed out of the 10 possible ones considering the different regio-and site-selectivities. Thus, the system leads to substrate-selectivity maintaining an exquisite level of regio-selectivity as well.

Finally, we studied the ability of this supramolecular iridium catalyst to display dormant reactivity in a way that the borylation only occurs when an external stimuli is applied to the system. More precisely, and considering the lack of reactivity of pyridinium salts (vide supra), we decided to introduce after 3 hours of reaction time one equivalent of N,N-diisopropylethylamine base (DIPEA) as an external chemical stimuli with the hope that an in situ deprotonation of the substrate will bring the pyridine to bind the zinc pocket of the supramolecular ligand L following iridiumcatalyzed remote borylation. Indeed, we were pleased to detect 90% conversion of pyridine after 16 h according to the above described reaction design (Figure 4).

Conclusion

In summary, we have reported a rationally-designed supramolecular borylating iridium catalyst displaying C-H bond meta-selectivity for challenging pyridine-like derivatives via reversible and dynamic Zn … N binding. [START_REF] Bonar-Law | kinetically labile Zn … N interactions for purely organic reactions[END_REF] The predictability of the selectivity was made possible considering the relatively strong association constant between the substrates and the catalyst and the precise distance between the active site and the recognition site supported by a straightforward combination of experimental and theoretical (semi-empirical) studies. The catalytic system displayed unique enzymatic features as regards of the kinetics, mechanism and substrate-selectivity herein presented. The incorporation of substrate-recognition sites based on zinc(II)porphyrin scaffolds in the second coordination sphere of a priori inactive or unselective catalytic moieties clearly constitutes an alternative to the more classical fine-tuning at the first coordination sphere. In addition, we showed that N,N-chelating ligands beyond bipyridine or phenantroline derivatives are suitable for iridium-catalyzed C-H bond borylations. [40] Because this presented approach is readily tunable at the active site and the substrate-recognition site, other types of transition metal-

Accepted Manuscript

Angewandte Chemie catalyzed transformations towards unbiased substrates could be envisioned as well as exploiting them for temporal control of reactivity considering the preliminary dormant reactivity studies.

Figure 1 .

 1 Figure 1. Overview of regio-selective iridium-catalyzed C-H bond borylation reactions guided by secondary interactions and issues encountered in pyridine meta-C-H bond reactivity (state-of-the art vs present work). B = (pinacolato)boron. B2pin2 = bis(pinacolato)diboron. COD = 1,5-cyclooctadiene.

Figure 2 .

 2 Figure 2. X-ray structures of the supramolecular assemblies between ligand L and pyridine (left) as well as N-methylimidazole (right) [capped sticks representation except the zinc atom that is ball representation, hydrogen atoms have been omitted except those from the nitrogen-containing substrates]. Color code: carbon (brown), nitrogen (blue), hydrogen (white). Selected distances [Å]: Zn … N1 2.14, H … N2 3.54, H … N3 3.92 (left) and Zn … N1 2.09, H … N2 3.79, H … N3 3.95 (right).

  [a] Reaction conditions: pyridine (0.162 mmol), B2pin2 (0.162 mmol), [Ir(COD)(Cl)]2 (1.5 mol%), L (3 mol%), solvent (1 mL).[b] Conversion determined as pyridine consumption.[c] Ratio meta/para/ortho functionalization of pyridine determined by 1 H NMR and GC using n-dodecane as internal standard.[d] Ratio mono/bis-functionalization of the meta-borylated product determined by 1 H NMR and GC-MS using n-dodecane as internal standard.[d] Reaction performed with [Ir(COD)(OMe)]2 instead of [Ir(COD)(Cl)]2.[f] Isolated yield of the main product displayed in brackets.[g] Reaction performed with 3 equivalents of B2pin2, 3 mol% of [Ir(COD)(Cl)]2 and 6 mol% of L.

Scheme 3 .

 3 Scheme 3. Control experiments.

  COD)Cl] 2 (1.5 mol%) L (3 mol%), ZS (1 equiv.) B 2 pin 2 (1 equiv.) p-xylene, 80 o C

Figure 3 .

 3 Figure 3. Graphical representation of the kinetic profiles under standard (stoichiometric) reaction conditions (left, [3-methylpyridine] = 0.162 M, [B2pin2] = 0.162 M) and under half concentration of B2pin2 with respect to the substrate (middle; [3-methylpyridine] = 0.130 M, [B2pin2] = 0.065 M); and graphical representation of the reaction rate versus substrate concentration plots together with the fitting to the Michaelis-Menten rate equation (right).

  Scheme 4. Postulated reaction mechanism for the supramolecular iridiumcatalyzed C-H bond meta-borylation of pyridines using L. B = (pinacolato)boron. The ppm values refer to the proton signals from the pyridine C-H bonds pointed out by the gray arrows that were determined by 1 H NMR spectroscopy at 80 o C under catalytic conditions.

Scheme 5 .

 5 Scheme 5. Substrate-selectivity targeted using the supramolecular iridium catalyst featuring secondary Zn … N interactions.

Figure 4 .

 4 Figure 4. Dormant reactivity featured by the supramolecular iridium catalyst using a base (DIPEA) as an external chemical stimuli.

Acknowledgements

The research leading to these results has received funding from the People Programme (Marie Curie Actions) of the European Union's Seventh Framework Programme (FP7/2007-2013) under REA grant agreement n. PCOFUND-GA-2013-609102, through the PRESTIGE programme coordinated by Campus France (P.Z.). Financial support from ANR-JCJC (PhD grant to J.T.), Fondation Rennes 1 (MSc grant to S.A.-S.), CNRS, Universite de Rennes 1, Rennes Metropole, and Region Bretagne (Strategie d'Attractivite Durable and Boost'Europe) is acknowledged. Keywords: iridium • pyridine • porphyrinoids • homogeneous catalysis • supramolecular chemistry

RESEARCH ARTICLE

Entry for the Table of Contents

Insert graphic for Table of Contents here.

An iridium-based supramolecular catalyst (see figure) has been designed to selectively recognize pyridine-lilke derivative via secondary Zn … N interactions. Owing to the distance between the substrate binding site and the active site as well as the geometrically constraints, meta-selective borylation reactions were accomplished displaying catalytic behaviors typically encountered in enzymes.

Institute and/or researcher Twitter usernames: @INC_CNRS @UnivRennes1 @Rafa_gramage ((max. width: 5.5 cm; max. height: 5.0 cm))


domain: sde sdv

text:
Alexandre A Degrave

Marine Wagner

Pierre George

Laurent Coudard

Xavier Pinochet

Magali Ermel

Elise J Gay

Isabelle Fudal

Onesimo Moreno-Rico

Thierry Rouxel

| Marine Wagner

| Xavier Pinochet

| Onesimo Moreno-Rico

Marie-Hélène Balesdent
email: marie-helene.balesdent@inrae.fr

Alexandre Deg

A new avirulence gene of Leptosphaeria maculans , AvrLm14 , identifies a resistance source in American broccoli ( Brassica oleracea ) genotypes

Keywords: Brassica napus, gene-for-gene, resistance introgression, stem canker

niveau recherche, publiés ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

| INTRODUC TI ON

Crops of agronomic importance are submitted to multiple pathogen challenges, threatening global food security. Whenever enough genetic variation is available, genetic improvement of crops is the most reliable, least expensive and most environment-friendly disease management strategy. However, the use of major gene resistance obeying the gene-for-gene postulate often exerts a strong selection pressure on pathogen populations whenever the corresponding avirulence gene is prevalent in populations, leading to possible "breakdown" of the new resistance source [START_REF] Lo Presti | Fungal effectors and plant susceptibility[END_REF][START_REF] Rouxel | Life, death and rebirth of avirulence effectors in a fungal pathogen of Brassica crops, Leptosphaeria maculans[END_REF]. Moreover, it is now thought that a single mutation in one avirulence gene may allow the pathogen to defeat several resistance genes simultaneously, and some complex genefor-gene systems rely on recognition specificities that can be masked by other avirulence genes [START_REF] Plissonneau | Unusual evolutionary mechanisms to escape effectortriggered-immunity in the fungal phytopathogen Leptosphaeria maculans[END_REF]. This indicates that one strategy to hamper resistance gene breakdown is a temporal or geographic alternating of resistance sources exerting sequential nonredundant pressure on populations. The counterpart of the deployment of such strategies is the need for a given crop to have a diversified portfolio of major R genes to develop informed strategies to use in a perspective of durability [START_REF] Brown | Durable resistance of crops to disease: a Darwinian perspective[END_REF].

During the 20th century, breeding for resistance in nearly every crop species resulted in deployment of major disease-resistance genes. Interspecific and intergeneric hybridizations are performed between cultivated species and between cultivated species and their wild relatives, allowing introgression of resistance traits from sexually compatible wild relatives [START_REF] Katche | Interspecific hybridization for Brassica crop improvement[END_REF][START_REF] Stam | When resistance gene pyramids are not durable -the role of pathogen diversity[END_REF]. Each parent species can harbour different resistance genes and resynthesizing interspecific hybrids is thus a way to diversify resistance sources. Agricultural crops often are allopolyploids resulting from interspecific crosses between related species (e.g., wheat, oilseed rape, brown mustard, tobacco, triticale) [START_REF] Mason | Creating new interspecific hybrid and polyploid crops[END_REF]. Brassica napus (oilseed rape, OSR, canola) is a natural interspecific hybrid between two diploid Brassica species, B. rapa (turnip, Chinese cabbage, etc.) and B. oleracea (cabbages) (U, 1935).

Being of recent origin, B. napus is known to have a narrow genetic basis and low diversity of resistance genes, while both parents show a remarkable diversity of morphotypes and genetic diversity [START_REF] Girke | Extending the rapeseed genepool with resynthesized Brassica napus L. I: Genetic diversity[END_REF]. In Brassicaceae crop breeding programmes, the resistance gene diversity in parent diploid species can be exploited to develop new cultivars with biotic and abiotic stress resistance [START_REF] Katche | Interspecific hybridization for Brassica crop improvement[END_REF].

One of the main diseases of brassica crops is stem canker (blackleg), caused by the ascomycete Leptosphaeria maculans (Rouxel & Balesdent, 2005). The fungus develops typical gene-for-gene interaction with its host and most sources of resistance available (Rlm genes) originate from B. rapa or from other related brassicas such as brown mustard (B. juncea) or black mustard (B. nigra). In contrast, B. oleracea is known to be highly susceptible to the disease, with few resistance sources from this gene pool identified to date. For example, an analysis of 392 accessions of B. oleracea from diverse geographic origins and with different morphotypes showed most of these were extremely susceptible to four L. maculans isolates [START_REF] Dilmaghani | Migration patterns and changes in population biology associated with the worldwide spread of the oilseed rape pathogen Leptosphaeria maculans[END_REF]. Only six accessions showed moderate resistance against two or three of the isolates. More recently, a survey of 32 Korean cabbage germplasms identified two resistant lines against two L. maculans isolates without investigating the genetic control of resistance [START_REF] Robin | Korean Brassica oleracea germplasm offers a novel source of qualitative resistance to blackleg disease[END_REF].

Here we analyse a small group of genotypes of B. oleracea grown in Mexico and identify a novel resistance source both in controlled conditions when inoculated with isolates obtained from B. napus in Europe and when the cultivars are grown in the field. Genetic analyses on the fungal side show avirulence is due to a single gene genetically independent from all currently identified AvrLm genes and termed AvrLm14. Candidate-based and map-based cloning of AvrLm14 point to a typical avirulence effector gene of L. maculans in terms of gene structure, regulation, and genome environment. This first identification and characterization of an avirulence gene towards B. oleracea in L. maculans may be indicative of the occurrence of a single-gene matching major resistance in broccoli and shows that B. oleracea diversity can also be a resource to consider to diversify the resistance genes portfolio used in B. napus.

| RE SULTS

| A new resistance found in American broccoli genotypes

Six American broccoli cultivars, Domador, Ironman, Marathon, Monaco, Neptuno, and Tlaloc, were found in a preliminary screening to show resistance responses when inoculated with two isolates of L. maculans obtained from oilseed rape, v23.1.2 and PHW1223 (data not shown; see Table 1 for a detailed description of the isolates).

All these cultivars were previously shown to be highly susceptible to Mexican isolates of L. maculans, typically belonging to race Av1-2-4-5-6-7-(8), suggesting these cultivars are devoid of the cognate resistance genes Rlm1/ LepR3, Rlm2, Rlm4, Rlm5, Rlm6, and Rlm7 (Dilmaghani et al., 2012). To refine these data by including identification of Rlm3 and Rlm9, an additional round of screening was done with a set of eight differential isolates developed at INRAE (Table 1).

The expected differential responses were expressed when using control oilseed rape genotypes (e.g., expression of the Rlm4 resistance towards isolate v23.2.1 only by cv. Jet Neuf) (Figure 1), while, with the main exception of cv. Ironman, all broccoli cvs expressed a moderate but nondifferential resistance response to all eight isolates (Figure 1). This was expressed as a larger than usual symptom with patches of dark necrotic responses and no or sparse sporulation (scoring around "3" in Figure 1) similar to what was described for the AvrLm1-Rlm1 interaction [START_REF] Ansan-Melayah | Genetic characterization of AvrLm1, the first avirulence gene of Leptosphaeria maculans[END_REF] but differing from the <1 mm hypersensitive response (HR) observed in typical gene-for-gene interactions such as the AvrLm4-Rlm4 or the AvrLm7-Rlm7 interactions (typical scoring "1" in Figure 1). The highest level of resistance was observed for cv. Monaco, while it was much less effective for cv. Ironman, showing complete susceptibility to isolate v34.18.12 and moderate but variable susceptibility to four other isolates (Figure 1). Lastly, focusing only on cvs Monaco and Ironman we investigated whether this new resistance could operate against an 87-isolate field population collected in France in the growing season 2003/2004[START_REF] Huang | Fitness cost associated with loss of the AvrLm4 avirulence function in Leptosphaeria maculans (phoma stem canker of oilseed rape)[END_REF]. Using the cotyledon-inoculation test, control isolates behaved as expected: European or Australian isolates v23.1.2 (JN2), IBCN18, and WT50 (Table 1) were avirulent while the Mexican isolate obtained from B. oleracea, OMR13, was highly virulent towards the two broccoli genotypes (Figure S1). Four other isolates, IBCN14, IBCN80 and the oldest available isolates in collections, BBA62908 and IBCN74 (both Av1-2-4-5-6-7-8), were also fully or moderately virulent towards the two broccoli genotypes (Figure S1). In the field population, nearly all isolates were avirulent towards the two broccoli genotypes and mostly induced HRs on cv.

Monaco. However, two field isolates were moderately or highly virulent towards broccolis, with one showing virulence similar to that

IBCN18 Australia F 1988 OSR M1 Av1-2-4-7-(8) Figure S1, Table 4 [1] IBCN56* Canada F 1992 OSR BJ-121 Av1-2-3-5-6-9 Figure 1 [1] IBCN80 Canada F 1985 OSR Leroy Av1-2-3-5-6-9 Figure S1 [1] WT50 Australia F 2004 OSR Av5-7-(8) Figure S1 [4] OMR13 Mexico F 2008 Cabbage Av1-2-4-(5)-6-7-(8) Figure S1 [3] OMR19 Mexico F 2008 Cabbage Av1-2-4-5-6-7-(8) Figure 2, Table 4 [3] OMR21 Mexico F 2008 Cabbage Av1-2-4-(5)-6-7-(8) Table 3, Table 4 [3]
19.4.24* L Av3-5-6-8 Figure 1 [5]

19.2.1 L Av3-5-6-8 Table 4 [5] v23.2.1* L Av4-5-6-7-8 Figure 1 [6] v23.1.2* L JN2 Av5-6-7-8
Figure 1, Figure 2, Figure S1, Table 3,

| The new resistance is effective in the field in France

To assess the effectiveness of this new resistance source under 2). Mostly, typical greyish leaf symptoms were recovered from Goéland, leading to the isolation of a vast majority of L. maculans isolates. In contrast, fewer and less typical symptoms were recovered from leaves of the FU-Eurol-Rlm10, Ironman, and Monaco genotypes, leading to the isolation of a majority of isolates of another Leptosphaeria species, L. biglobosa, unaffected by Rlm genes from Brassica species (Table 2). Moreover, the ratio of L. biglobosa:L. maculans was much higher when isolated from the broccoli genotypes compared to FU-Eurol-Rlm10 (Table 2), substantiating the scarcity of typical symptoms that can be obtained from broccoli genotypes. While more than half of the L. maculans isolates recovered from FU-Eurol-Rlm10 were virulent towards the never used before Rlm10 resistance, only one isolate fully virulent towards the novel B. oleracea resistance could be recovered (Table 2).

Overall, these data indicate that none of the known resistance genes (Rlm1-Rlm9) are present in American genotypes of broccoli included in this study and that a new resistance source is present in these genotypes. This resistance is effective when faced with French field populations of L. maculans but virulence is present in a small ratio of field isolates. The fact that isolates virulent towards the new resistance source are already present in French populations isolated from OSR could be indicative of the occurrence of a typical gene-for-gene interaction and thus that the B. oleracea genotypes harbour a single-gene major resistance, tentatively named Rlm14. The high number of recombinant genotypes in the progeny (Table 3) indicated genetic independence between AvrLm14 and all three other avirulence genes. Cross 67, involving the reference sequenced isolate v23.1.3, was then used for genetic mapping of the new avirulence locus.

| A single-gene control and genetic independence from known avirulence genes

| Identification and characterization of AvrLm14 points to a typical AvrLm gene

To identify the candidate effector gene encoding AvrLm14, we firstly undertook an a priori strategy involving systematic PCR amplification of 43 of the genes encoding putative effectors whose repertoire has been established in [START_REF] Rouxel | Effector diversification within compartments of the Leptosphaeria maculans genome affected by Repeat-Induced Point mutations[END_REF] on a series of isolates showing avirulence or virulence towards broccoli genotypes. Only five of the genes of the repertoire showed complete lack of amplification in the two parental isolates virulent towards broccolis, OMR19 and OMR21 (Table 4). Of these, four genes were also lacking in isolate IBCN74, found here to be virulent towards Monaco and Ironman (Table 4 and Figure S1). In addition, one of the candidates, Lema_T200070, was not predicted in the new version of the genome annotation [START_REF] Dutreux | De novo assembly and annotation of three Leptosphaeria genomes using Oxford Nanopore MinION sequencing[END_REF] due to lack of transcriptomic support. Among the three remaining candidates, only the Symptoms were scored at 13 days postinoculation on a 1-6 scale [START_REF] Balesdent | Analysis of Leptosphaeria maculans race structure in a worldwide collection of isolates[END_REF], with scores 1-3 and 4-6 corresponding to avirulent and virulent phenotypes, respectively. Each bar represents the average (+ SE) of 14-22 inoculation points per interaction and data with different letters are significantly different (Kruskal-Wallis test: p < .0001) S1) that covered 547 kb and encompassed 54 predicted genes. This region mostly corresponded to a 399 kb AT-rich isochore (Figure S2). Six genes predicted as encoding putative secreted proteins were identified among the 54 genes, of which only one, the candidate gene Lmb_JN3_05547, showed a 0.984 probability to be an effector according to the EffectorP prediction tool [START_REF] Sperschneider | Improved prediction of fungal effector proteins from secretomes with EffectorP 2.0[END_REF], and was located as a solo-gene in the middle of the AT-rich isochore. Exploitation of RNA-Seq data [START_REF] Dutreux | De novo assembly and annotation of three Leptosphaeria genomes using Oxford Nanopore MinION sequencing[END_REF] pointed to expression kinetics typical of known avirulence genes for this candidate, with a low level of expression in axenic conditions and a strong overexpression upon cotyledon infection 5-9 days postinoculation (dpi) followed by a drastically reduced expression level at later time points (Figure 3). In addition to its location within a large transposable element (TE)-rich genome region, Complementation of an avrLm14 isolate with Lmb_JN3_05547 indicated that 13 out of the 15 complemented isolates had a restored AvrLm14 phenotype and that the two isolates that were still virulent had no expression of the transgene (data not shown).

F I G U R E 2
As a complementary approach, 14 transformants were recovered following RNAi silencing of the candidate AvrLm14 in the reference isolate v23.1.3. All of these transformants remained fully virulent on the control oilseed rape cv. Westar (data not shown). On Monaco,

isolates showing a low level of silencing of Lmb_JN3_05547 (residual expression of 60% or higher compared to the avirulent control) still displayed an avirulent phenotype (more than 90% of the inoculation sites), while transformants with a residual expression of Lmb_ JN3_05547 lower than 52% of that of the control displayed virulent phenotypes on a majority of inoculation sites (Figure 4a). Moreover, the two field isolates found to be virulent on Ironman and Monaco (Figure S1) displayed no expression of Lmb_JN3_05547, although they contained the corresponding gene (data not shown). All these data agree to confirm that Lmb_JN3_05547 is AvrLm14.

| Fitness analyses indicate AvrLm14 restricts expansion of lesion size on cotyledons

Based on the residual expression level of AvrLm14 in RNAi transformants and their pathogenicity on Monaco, these transformants were classified into two groups, that is, silenced or not (Figure 4). Germinating conidia in axenic medium were used as a reference areas were measured at 12 dpi. Analysis of variance (ANOVA) of mean size of cotyledon lesion indicated no effect of the cultivar on the lesion size (p = .802), while the silencing had a significant effect (p = .015); the inactivation of AvrLm14 not only suppressed the avirulence on Monaco (Figure 4a), but led to an increase of cotyledon lesion size on susceptible cultivars (Figure 4b). In addition, this effect functioned in a quantitative manner because the cotyledon lesion size was negatively correlated to the level of residual expression of AvrLm14 (Spearman test, p = .017; Figure 4b).

| D ISCUSS I ON

Control of the stem canker disease of Brassica strongly depends on the use of resistance genes in the host, at least in part of continental Europe, where no fungicides are used against L. maculans.

Due to limited genetic resources in B. napus, this led in the past to a series of boom-and-bust cycles during which one new effective resistance gene was introduced and widely used on large acreages, exerting a very high selection pressure on fungal populations and eventually leading to a shift from avirulence to virulence in populations and ineffectiveness of the resistance [START_REF] Rouxel | Life, death and rebirth of avirulence effectors in a fungal pathogen of Brassica crops, Leptosphaeria maculans[END_REF]. Here, we identified a novel resistance source in B. oleracea and cloned the corresponding avirulence gene, AvrLm14. Without having performed genetic analyses on the plant side, the identification of a single avirulence gene in the fungus strongly suggests the plant-pathogen interaction obeys a canonical gene-for-gene relationship involving one resistance gene in the plant, responsible for resistance to avirulent populations of the fungus. Similar postulates have been done multiple times ever since we cloned the first avirulence gene in L. maculans and have always proven to be the rule (e.g., [START_REF] Ansan-Melayah | Genes for race-specific resistance against blackleg disease in Brassica napus L[END_REF]. In this respect the relatively easy cloning of fungal avirulence genes provides us with an efficient tool to identify corresponding single-gene resistance sources in the host.

The Rlm14 resistance speculated here seems to be effective against a large proportion of L. maculans French field populations both when using a collection of isolates in controlled conditions and in field settings under diverse pedoclimatic conditions. However, virulent isolates preexist at a low level in collections (one fully virulent isolate out of 87 field isolate collections) and in field populations (one fully virulent isolate obtained from leaf symptoms). Preexistence of virulent isolates towards a new resistance source has been observed

for nearly all Rlm genes introduced in France when data were available on population structure prior to the use of the resistance gene (e.g., estimated frequencies of avrLm7 isolates ranging from 0.006% to 1.3% at Versailles prior to the use of Rlm7, or 3.2% of virulent field isolates towards the never used Rlm11; [START_REF] Balesdent | The dispensable chromosome of Leptosphaeria maculans shelters an effector gene conferring avirulence towards Brassica rapa[END_REF][START_REF] Daverdin | Genome structure and reproductive behaviour influence the evolutionary potential of a fungal phytopathogen[END_REF]. This is attributable to the conjunction of large The cloning of the 10th avirulence gene (AvrLm14) in L. maculans illustrates a remarkable identity of traits among all currently cloned AvrLm genes, even though they share no sequence identity and are scattered throughout the genome, often on distinct chromosomes.

As noticed when having cloned the first avirulence gene of L. maculans more than a decade ago [START_REF] Gout | Lost in the middle of nowhere: the AvrLm1 avirulence gene of the Dothideomycete Leptosphaeria maculans[END_REF], all of them are "lost in the middle of nowhere" genes isolated in large AT-rich blocks of degenerated transposable elements (for a review, see [START_REF] Rouxel | Life, death and rebirth of avirulence effectors in a fungal pathogen of Brassica crops, Leptosphaeria maculans[END_REF]. In relation to this genome location we also found recently that all AvrLm genes are enriched in H3K9me3 (trimethylation of lysine 9 of histone H3) marks, usually regarded as characteristic of constitutive heterochromatin in other organisms [START_REF] Soyer | Genome-wide mapping of histone modifications in two species of Leptosphaeria maculans showing contrasted genomic organization and different host specialization[END_REF]. Being associated with this repressive mark, none of the AvrLm genes are expressed during vegetative growth [START_REF] Soyer | Genome-wide mapping of histone modifications in two species of Leptosphaeria maculans showing contrasted genomic organization and different host specialization[END_REF], but all of them show the same kinetics of expression when the fungus is inoculated onto cotyledons of B. napus, with a strong peak of overexpression 5-9 days postinoculation before symptoms are expressed (Figure 3) [START_REF] Gay | Large-scale transcriptomics to dissect two years of the life of a fungal phytopathogen interacting with its host plant[END_REF]. Additionally, all of them encode small secreted proteins, often but not always enriched in cysteine residues, and showing no or few matches in databases, no recognizable domain, and no postulated function. The 10 known AvrLm genes are expressed together with tens of other putative effectors [START_REF] Gay | Large-scale transcriptomics to dissect two years of the life of a fungal phytopathogen interacting with its host plant[END_REF][START_REF] Gervais | Different waves of effector genes with contrasted genomic location are expressed by Leptosphaeria maculans during cotyledon and stem colonization of oilseed rape[END_REF][START_REF] Haddadi | Genome-wide transcriptome analyses provides insights into the lifestyle transition and effector repertoire of Leptosphaeria maculans during colonization of B. napus seedlings[END_REF]. Generally, the quantity of effectors produced at the same time in the same place raises the question of why so many effectors are produced together.

This led to the postulate of functional redundancy substantiating the dispensability of avirulence effectors under resistance gene selection [START_REF] Lo Presti | Fungal effectors and plant susceptibility[END_REF]. The data obtained on AvrLm14, along with those previously obtained for AvrLm1, AvrLm3, AvrLm4-7, or

AvrLm10A, showed that functional inactivation or silencing of AvrLm genes often results in an observable phenotype, suggesting additive effects rather than redundancy. In many cases, and irrespective of the approach used, L. maculans AvrLm genes were first postulated to promote development of leaf lesions. This was shown when comparing near-isogenic lines of the fungus for AvrLm1 and AvrLm4-7, by functional complementation for AvrLm4-7, and following RNAi silencing for AvrLm3 [START_REF] Huang | Fitness cost associated with loss of the AvrLm4 avirulence function in Leptosphaeria maculans (phoma stem canker of oilseed rape)[END_REF][START_REF] Huang | Fitness cost of virulence differs between the AvrLm1 and AvrLm4 loci in Leptosphaeria maculans (phoma stem canker of oilseed rape)[END_REF]Novakova et al., 2016;[START_REF] Plissonneau | Unusual evolutionary mechanisms to escape effectortriggered-immunity in the fungal phytopathogen Leptosphaeria maculans[END_REF]. In contrast to AvrLm1, AvrLm3, or AvrLm4-7, two other AvrLm genes, AvrLm10A [START_REF] Petit-Houdenot | A two genes-for-one gene interaction between Leptosphaeria maculans and Brassica napus[END_REF] and AvrLm14 (this study) have been found, following RNAi silencing experiments, to limit the expansion of the lesions. This suggests the existence of a trade-off between a series of effectors that are produced at the same time with antagonistic effects on lesion size to eventually control the expansion of the leaf lesion. Such fine tuning is likely to be important for infection of oilseed rape, because it is known that leaf lesions supporting asexual sporulation play a minor role in the fungal life cycle due to the low infectivity of pynidiospores and the low importance of secondary infection compared to continuous infection by ascospores under European field conditions (Rouxel & Balesdent, 2005). As postulated by Petit-Houdenot et al.

(2019), it seems to make sense to restrict leaf lesion development to prevent early leaf senescence that would compromise the ability of L. maculans to reach the leaf vascular tissue in time and eventually the stem, where sexual reproduction takes place. Interestingly, the ability of AvrLm14 to contribute to restriction of lesion expansion is consistent with its absence in isolates obtained from B. oleracea, either in Mexico or in the oldest isolates of the collection, because the pathogenic cycle of L. maculans on cabbages is different from that on OSR and mostly relies on asexual sporulation on leaf lesions with little evidence of systemic colonization of stems to cause stem canker [START_REF] Dilmaghani | Clonal populations of Leptosphaeria maculans contaminating cabbage in Mexico[END_REF]. Thus it appears that the production of a mix of effectors promoting or restricting lesion size development provides the fungus with a highly flexible template to adapt to different host species and different pathogenic strategies depending on whether it relies on asexual sporulation for short-cycle cultivation practices such as those used for B. oleracea in Mexico or canola in western Canada [START_REF] Dilmaghani | Migration patterns and changes in population biology associated with the worldwide spread of the oilseed rape pathogen Leptosphaeria maculans[END_REF] or rather on sexual reproduction necessitating a lengthy colonization of stem tissues for longcycle hosts such as winter OSR in Europe.

| E XPERIMENTAL PROCEDURE S

| Fungal material

Isolates used in this study are described in whose genome sequence is the reference genome sequence for L. maculans [START_REF] Dutreux | De novo assembly and annotation of three Leptosphaeria genomes using Oxford Nanopore MinION sequencing[END_REF] and its sister isolate, v23.1.2 (JN2). Fungal cultures were maintained and conidia were produced as previously described [START_REF] Ansan-Melayah | Genetic characterization of AvrLm1, the first avirulence gene of Leptosphaeria maculans[END_REF]. To examine genetic linkage between AvrLm1, AvrLm2, AvrLm4-7, and AvrLm14, cross #66 was performed between isolate OMR21 and isolate v23.1.2. To map AvrLm14, a segregating population was built following an in vitro cross (#67) between isolates OMR19 and the reference v23.1.3. In vitro crosses and recovery of random ascospore progeny were done as previously established [START_REF] Plissonneau | A game of hide and seek between avirulence genes AvrLm4-7 and AvrLm3 in Leptosphaeria maculans[END_REF]. et al., 2005), Aviso (Rlm9), ES Astrid (Rlm9), and FU-Eurol-74 (Rlm10) [START_REF] Petit-Houdenot | A two genes-for-one gene interaction between Leptosphaeria maculans and Brassica napus[END_REF]. American broccoli cultivars used were Domador, Ironman, Marathon, Monaco, Neptuno, and Tlaloc.

| Plant genotypes and inoculation tests

All isolates were phenotyped for their virulence spectrum using a cotyledon-inoculation test. Cotyledons of 10-to 12-day-old seedlings were puncture-inoculated with 10 µl of inoculum (10 7 pycnidiospores/ml [START_REF] Balesdent | Genetic control and host range of avirulence toward Brassica napus cultivars Quinta and Jet Neuf in Leptosphaeria maculans[END_REF]). Spore suspensions of each isolate were inoculated on 10-12 plants of each of the B. napus differentials and B. oleracea cultivars. Symptoms were scored 12-18 dpi using a semiquantitative 1 (avirulent) to 6 (virulent) rating scale in which scores 1-3 represent different levels of resistance (from typical HR to delayed resistance) and 4-6 represent different levels of susceptibility (mainly evaluated by the intensity of sporulation on lesions; [START_REF] Balesdent | Analysis of Leptosphaeria maculans race structure in a worldwide collection of isolates[END_REF].

For RNA-Seq analyses, inoculations were done on cv. Darmor-bzh as described previously in [START_REF] Dutreux | De novo assembly and annotation of three Leptosphaeria genomes using Oxford Nanopore MinION sequencing[END_REF] Development of symptoms was assessed weekly and symptomatic leaves were collected to obtain single pycnidium isolates from leaf spots as described in [START_REF] Balesdent | A largescale survey of races of Leptosphaeria maculans occurring on oilseed rape in France[END_REF].

| Field experiments

| Genetic mapping

Generation of minisatellite markers and primers for mapping and PCR amplification were done as reported in [START_REF] Bally | FONZIE: an optimized pipeline for minisatellite marker discovery and primer design from large sequence data sets[END_REF]. On cross #67, linkage analysis among loci was performed using MapMaker/

| DNA and RNA manipulations

For PCR, genomic DNA was extracted from conidia with the DNeasy 96 Plant Kit (Qiagen) as already described [START_REF] Daverdin | Genome structure and reproductive behaviour influence the evolutionary potential of a fungal phytopathogen[END_REF].

Standard PCRs were performed in an Mastercycler EP gradient thermocycler (Eppendorf) as described in [START_REF] Daverdin | Genome structure and reproductive behaviour influence the evolutionary potential of a fungal phytopathogen[END_REF].

Sequencing was performed using a CEQ 8000 automated sequencer (Beckman Coulter) according to the manufacturer's instructions.

Total RNA was extracted from infected cotyledons of B. napus or B. oleracea genotypes using TRIzol reagent (Invitrogen) according to manufacturer's protocol. Total RNA was treated with RNase-free DNase I (New England Biolabs).

| Gene annotation

Gene annotation was firstly based on the RNA-Seq data from [START_REF] Dutreux | De novo assembly and annotation of three Leptosphaeria genomes using Oxford Nanopore MinION sequencing[END_REF]. Annotation of 5′ and 3′ UTRs, transcription start and stop sites, and intron positions were then confirmed following PCR amplification and sequencing of 3′-and 5′-ends of cDNA using the SMARTer RACE cDNA Amplification kit (ClonTech) according to the manufacturer's recommendation and using primers AvrLm14-F, AvrLm14-R, and AvrLm14-int1F (Table S1).

| Expression analyses using RNA-Seq and RT-qPCR

For RNA-Seq, protocols are described in [START_REF] Dutreux | De novo assembly and annotation of three Leptosphaeria genomes using Oxford Nanopore MinION sequencing[END_REF]. For the RT-qPCR experiments, all RNA samples were adjusted to 3 μg of RNA and single-strand cDNA was generated using oligo(dT)-primed reverse transcription with PrimerScript reverse transcriptase (TaKaRa) according to the manufacturer's protocol. Samples were analysed from a 10-fold dilution of the original RT products as described by [START_REF] Fudal | Heterochromatin-like regions as ecological niches for avirulence genes in the Leptosphaeria maculans genome: map-based cloning of AvrLm6[END_REF]. Quantitative PCR was performed using a 7900 fast real-time PCR system (Applied Biosystems) and ABsolute SYBR Green ROX dUTP mix (ThermoScientific). To evaluate the expression profile of AvrLm14 in complemented isolates, C t values were analysed according to the method described by [START_REF] Muller | Processing of gene expression data generated by quantitative real-time RTPCR[END_REF]. In silencing assays, the residual expression of AvrLm14, compared to those of the wild-type isolate, were analysed according to the 2 -ΔΔCt method [START_REF] Livak | Analysis of relative gene expression data using real-time quantitative PCR and the 2 -ΔΔCt method[END_REF]. Fungal

Actin or β-tubulin was used as a constitutive reference gene and L. maculans EF1α relative expression to Actin was used as a supplementary control to verify that there was no variation in the level of expression of Actin between conditions and between biological replicates. For each value measured, two to three technical replicates from two biological replicates were performed. Water and uninfected plants were used as negative controls. The RT-qPCR primers used are indicated in Table S1.

| Functional validation

For functional complementation experiments, a fragment was amplified using primers AvrLm14-XhoIF, which contains a XhoI site,

FI G U R E 1

 1 Box plot representation of interaction phenotypes of a set of differential isolates with known avirulence genes with a series of oilseed rape (OSR) cultivars and five Mexican broccoli genotypes. The differential OSR set consists of Westar (susceptible control, no Rlm gene) (a), Jet Neuf (Rlm4) (b), Bristol (Rlm2 & Rlm9) (c), Goéland (Rlm9) (d), Columbus (Rlm1 & Rlm3) (e), and 01.23.2.1 (Rlm7) (f). The five Mexican cultivars of B. oleracea are Neptuno (g), Tlaloc (h), Marathon (i), Monaco (j), and Ironman (k). Spore suspensions were deposited on wounded cotyledons, and symptoms were scored at 14 days postinoculation (dpi) and 18 dpi. Symptom expression at 18 dpi is displayed (except for 01.23.2.1, 14 dpi) using a semiquantitative 1 (avirulent) to 6 (virulent) rating scale in which scores 1-3 represent different levels of resistance (from typical hypersensitive response [HR] to delayed resistance) and 4-6 represent different levels of susceptibility. For each box, the red cross indicates the average scoring (10-12 plants per inoculation point, repeated twice) while the black horizontal line in the rectangle indicates the median scoring; the rectangles comprise 75% of the rating scores of the most virulent isolates available in the collection and isolated from B. oleracea, OMR13 and IBCN74 (Figure S1).

French

  field conditions, both Monaco and Ironman were included in a field experimental setting alongside a susceptible OSR control, Goéland and new putative sources of resistance introgressed in OSR, such as Rlm10 originating from black mustard, B. nigra (Petit-Houdenot et al., 2019). Field experiments were established for two consecutive years in up to eight locations in France and leaves with symptoms were collected in autumn for isolation and analysis of fungal isolates (Table

  Genetic control of resistance can easily be approximated by characterization of the genetic control of avirulence on the fungal side. Two crosses were undertaken between Mexican virulent isolates (OMR19, OMR21) and European isolates showing avirulence towards broccoli genotypes (Figure2), cross 66 (OMR21 × v23.1.2; 87 progeny) and cross 67 (OMR19 × v23.1.3; 79 progeny). Cross 66 involved two isolates differing at loci AvrLm1, AvrLm2, and AvrLm4, and was primarily used to evaluate genetic linkage between these loci and the putative TA B L E 2 Characterization of Leptosphaeria maculans isolates obtained from field experiments containing new resistant material (Rlm10 from Brassica nigra in a B. napus background and the new resistance source from B. oleracea described in this study) The isolates recovered from leaf symptoms were classified as Leptosphaeria maculans (Lm) or L. biglobosa (Lb) following PCR amplification of the rDNA internal transcribed spacer (ITS) and Mat loci; nd, the isolate did not belong to any of the species using this criterion or was a mix of both species. b Number (%) of L maculans isolates showing virulence when inoculated on cotyledons of genotypes with novel resistance sources (Rlm10 or the resistant B oleracea). c The isolate showed an intermediate behaviour when inoculated on cotyledons of Monaco. avirulence gene (AvrLm14) towards broccolis. The progeny was thus assessed on control differential OSR genotypes (Westar: no R gene, Columbus: Rlm1, Bristol: Rlm2, and Jet Neuf: Rlm4) and on broccoli cv. Monaco. The progeny was assessed for segregation of avirulence and recombination of avirulence phenotypes between pairs of avirulence genes. All four avirulence genes, including the putative AvrLm14, showed a segregation ratio consistent with a single-gene control [AvrLm1: 45:42 A:V, p(χ 2 ) = .748; AvrLm2: 40:47 A:V, p(χ 2 ) = .453; AvrLm4: 41:45 A:V, p(χ 2 ) = .670; avirulence towards Monaco: 50:37 A:V, p(χ 2 ) = .163; p(χ 2 ) < .001 if considering a two-gene control].

  Interaction phenotypes of parent Leptosphaeria maculans isolates OMR19 (black bars) and JN2 (grey bars) on cotyledons of control oilseed rape genotypes (Westar [no Rlm gene] and Bristol [Rlm2, Rlm9]), and three broccoli cultivars, Monaco, Domador, and Ironman.

  lack of amplification of Lm_JN3_05547 (formerly Lema_P004720) fully cosegregated with the virulence phenotype on Monaco in cross 67. Additional genetic mapping based on minisatellite markers generated from the genomic sequence of the supercontig hosting the candidate gene (Bally et al., 2010) delineated a 11.3 cM genetic interval bordered by markers SSR209 and mpb0.69 (Table

  examination of data generated by[START_REF] Soyer | Genome-wide mapping of histone modifications in two species of Leptosphaeria maculans showing contrasted genomic organization and different host specialization[END_REF] also showed that Lmb_JN3_05547 was strongly enriched in the heterochromatin mark H3K9me3. RACE-PCR was used to confirm the improved gene annotation obtained in Dutreux et al. (2018) and refine it. It showed that Lmb_JN3_05547 is a mono-exonic 405-bp gene, with a 131-bp 5′ untranslated region (UTR) encompassing a 50 bp intron and a 30 or 107-bp 3′ UTR. The gene encoded a 134 amino acid protein encompassing a 17 amino acid signal peptide. The mature protein contained four cysteine residues (Figure S2). The predicted protein had no Blast hit with sequences available in public databases, including in closely related species such as L. biglobosa or other closely related Dothideomycetes.

  Each of these 14 RNAi transformant was inoculated onto two susceptible B. napus varieties, Aviso and ES Astrid, and cotyledon lesion F I G U R E 3 Kinetics of expression of the candidate AvrLm14, Lmb_JN3_05547 (red line) following inoculation of cotyledons of a susceptible oilseed rape cultivar, Darmor-Bzh, at a range of days postinoculation (dpi). This includes the RNA-Seq data described in[START_REF] Dutreux | De novo assembly and annotation of three Leptosphaeria genomes using Oxford Nanopore MinION sequencing[END_REF] and[START_REF] Gay | Large-scale transcriptomics to dissect two years of the life of a fungal phytopathogen interacting with its host plant[END_REF], and encompasses the kinetics of three other avirulence genes, AvrLm2 (green), AvrLm4-7 (black), and AvrLm6 (blue) for comparison. Data are log 2 (RPKM +1) transformed expression data, scaled to expression of all genes in the genome.

  effective pathogen population size, hosting of AvrLm genes in dispensable regions of the genome along with the importance of sexual reproduction in the fungal life cycle that maximizes the ability to continuously and locally generate nonconventional inactivating TA B L E 4 PCR amplification of the repertoire of effector genes (encoding small secreted proteins, SSPs) identifies five candidates with lack of amplification in the two isolates virulent towards Brassica oleracea genotypes OMR19 and OMR 21 according to[START_REF] Rouxel | Effector diversification within compartments of the Leptosphaeria maculans genome affected by Repeat-Induced Point mutations[END_REF] has been moved to v2 following Nanopore resequencing and RNA-Seq-based annotation of isolate JN3[START_REF] Dutreux | De novo assembly and annotation of three Leptosphaeria genomes using Oxford Nanopore MinION sequencing[END_REF].b Descriptions of isolates are in

For

  evaluation of the effectiveness of the resistance source found in broccoli genotypes, field experiments were set up for two growing seasons in eight locations with contrasting agroclimatic environments: En Crambade and Mondonville (south France), Rennes (Brittany, west France), Versailles, Grignon and Verneuil l'Etang (Ilede-France region), Rozières and Blois (central France, a region with high acreages of OSR). In France, OSR is usually sown late August to spend winter under a rosette stage. Ascospore infection mostly takes place in autumn (October-December) and leaf symptoms are mostly expressed during this period. To synchronize growth stages between OSR and broccoli genotypes, and to maximize the chances of submission to a wide ascospore shower at the time when plants are the most susceptible to the disease (i.e., before the 6-leaf stage), seeds were presown in individual pots in the greenhouse on 15 September and 15 October, and then transplanted in the field at the 2-leaf stage on 15 October and 15 November, respectively.

  EXP v. 3.0b software (available online; Whitehead/MIT Center for Genome Research, Cambridge, MA, USA) with a logarithm of odds (LOD) score of 3.0 and a maximum recombination frequency of 20 cM.

and

  AvrLm14-EcoRIR, which contains an EcoRI site. The resulting PCR product was inserted into pGEM-T Easy vector (Promega), generating vector pGEM-AvrLm14. The 1.3 kb XhoI/EcoRI fragment was inserted into the binary vector pPZPnat1 linearized with the corresponding restriction enzymes, yielding plasmid pPZPnat1-AvrLm14. The silencing vectors were constructed by inserting two inversely oriented fragments into pJK11. These fragments were PCR amplified with primers SilEcoRIAvrLm14GF1/SilSacIAvrLm14GR1 and primers SilBamHIAvrLm14pF2/SilSacIAvrLm14pR2 using as a template cDNA from inoculated cotyledons 7 dpi. The resulting PCR products were digested by EcoRI/SacI and BamHI/SacI, respectively, and ligated into the EcoRI/BamHI-linearized pJK11 vector. The resulting plasmids place the hairpin-inverted repeat fragments under the control of the promoter of Glomerella cingulata pgdA and the terminator of Aspergillus nidulans trpC. These expression cassettes were excised by XhoI/SpeI and subcloned into the corresponding sites of pPZPnat1. The different constructs were then introduced into Agrobacterium tumefaciens C58 (pGV2260) by electroporation at 1.5 kV, 200 Ω, and 25 µF. Transformants were selected on minimal medium supplemented with 50 µg/ml nourseothricin (Werner, BioAgents), purified by single conidium isolation and maintained on selective medium. ACK N OWLED G EM ENTS The authors wish to thank Bertrand Auclair (INRAE BIOGER) for plant management, Pascal Bally (INRAE, Bioger) for design of markers for genetic mapping, Hortense Brun (INRAE, IGEPP) for field experiments, and Régine Delourme (INRAE, IGEPP) for B. napus Darmor-bzh seeds. Seeds of FU-Eurol 74 were kindly provided by Anne-Marie Chèvre (IGEPP, Rennes). Alexandre Degrave and the project were funded by the French National Research Agency project AvirLep (ANR GPLA07-024C). The "Effectors and Pathogenesis of L. maculans" group benefits from the support of Saclay Plant Sciences-SPS (ANR-17-EUR-0007). AUTH O R CO NTR I B UTI O N S M.H.B. designed the research and acquired the funding. A.D., M.W., P.G., L.C., I.F., and M.E. performed the experiments. A.D., M.W., E.J.G., M.H.B., and T.R. analysed the data. X.P., O.M.R., T.R., and M.H.B. supervised the project. T.R. and M.H.B. wrote the draft manuscript. All authors revised the manuscript.

ID Country of origin Origin a Year of isolation Host b Other name Race c Interaction phenotype in Table/Figure Reference d

  List and characteristics of Leptosphaeria maculans field or laboratory isolates used in this study

	Isolate BBA62908	Germany	?	1966	Beta vulgaris?		Av1-2-4-5-6-7-8	Figure S1	[1]
	IBCN74	France	F	1974 ?	Cabbage	PHW1245 Av1-2-4-5-6-7-8	Figure S1, Table 4	[1]
	a.2	France	F	1990	OSR		Av1-5-6-7-8	Table 4	[2]
	H5	France	F	1990	OSR		Av5-6-7-8	Table 4	[1]
	Nz-T4* e	New Zealand	F	?	Swede	T	Av5-6-8	Table 4	[1]
	PHW1223*	Australia	F	?	OSR		Av5-6-8-9	Figure 1, Table 4	[1]
	IBCN14	Australia	F	1988	OSR	MD2	Av5-6	Figure S1, Table 4	[1]
	IBCN17	Australia	F	1988	OSR	C13	Av4-5-6-7-8	Table 4	[1]

TA B L E 1

  Host from which the isolate was obtained; swede, Brassica napus rapifera; cabbage, Brassica oleracea; OSR, oilseed rape (Brassica napus oleifera).

								[5, 6]
							Table 4
	v23.1.3		L		JN3	Av1-4-5-6-7-8	Table 4	[5, 6]
	v23.1.11		L		JN11	Av1-4-5-6-7-8	Table 4	[5, 6]
	v23.11.9*		L			Av1-5-6-7-8	Figure 1	[5,6]
	V34.18.12*		L			Av2-(5)-7-(8)	Figure 1	[5]
	m1-401 -m3-409	France	F	2003	OSR	Single-conidium	Figure S1	[7]
					(Mohican)	field population (9
						isolates)
	m2-6xx -m10-6xx	France	F	2004	OSR	Single-ascospore	Figure S1	[7]
					(Mohican)	field population
						(78 isolates)
	a F, Field single-ascospore isolate (single conidium isolates for the m1-401-m3-409 population); L, laboratory isolate originating from in vitro crosses.
	c Races are defined as the combination of avirulent alleles at the AvrLm1 to AvrLm9 loci. Avx-y-z indicates avirulence towards resistance genes Rlmx,
	Rlmy, and Rlmz. Figures in parentheses indicate missing data at a given avirulence locus.
	d [1] Balesdent et al. (2005); [2] Gall et al. (1994); [3] Dilmaghani et al. (2012); [4] Dilmaghani et al. (2009); [5] Balesdent et al. (2002); [6] Balesdent
	et al. (2001); [7] Huang et al. (2006).
	e Isolates marked with an asterisk are those used in Figure 1 to search for known resistance gene in broccoli genotypes.

b

  Segregation analysis of interaction phenotypes in progeny of cross #66 shows genetic independence between AvrLm14 and AvrLm1, AvrLm2, or AvrLm4

					Recombinant
		Parental phenotype 1 c	Parental phenotype 2 a	Recombinant phenotype 1	phenotype 2	p (χ 2 ) b
	AvrLm14 vs.	21 (AvrLm14-avrLm1) c	18 (avrLm14-AvrLm1)	28 (AvrLm14-AvrLm1)	20 (avrLm14-avrLm1)	.456 ns
	AvrLm1
	AvrLm14 vs.	21 (AvrLm14-avrLm2)	19 (avrLm14-AvrLm2)	26 (AvrLm14-AvrLm2)	21 (avrLm14-avrLm2)	.746 ns
	AvrLm2
	AvrLm14 vs.	28 (AvrLm14-avrLm4)	18 (avrLm14-AvrLm4)	22 (AvrLm14-AvrLm4)	18 (avrLm14-avrLm4)	.374 ns
	AvrLm4

TA B L E 3

a Isolate v23.1.2, showing virulence at loci AvrLm1, AvrLm2, and AvrLm4, and avirulence at locus AvrLm14.

b Isolate OMR21, showing avirulence at loci AvrLm1, AvrLm2, and AvrLm4, and virulence at locus AvrLm14.

c Probability associated to the χ 2 test for independent segregation (25% of the progeny in each phenotypic class); ns, not significant.

d AvrLm, avirulent phenotype; avrLm, virulent phenotype.

Table 1 .

 1 Only isolates OMR19, OMR21, and IBCN74 are virulent towards B. oleracea genotypes Ironman and Monaco.

	suggested that domestication was accompanied by impoverishment
	in resistance gene analogs (RGAs) in B. oleracea compared to the an-
	cestral species B. macrocarpa, which displays resistance to pathogens
	such as Sclerotinia sclerotiorum lost in domesticated Brassica species.
	One last speculation could be linked to diversification of genes in
	the A and C genomes of B. napus compared to those of its progen-
	itors. Fu et al. (2019) showed a high 87.1% level of conservation of
	RGAs of the nucleotide binding site-leucine rich repeat (NBS-LRR)
	class in the A genome between B. napus and B. rapa while the ratio
	was much lower (66.4%) for the C genome between B. napus and
	B. oleracea due to expansion of genes of this family in the C genome

c +, presence of the expected PCR product; -, lack of PCR amplification. to difficulties in correctly interpreting incompatibility phenotypes in the C versus the A genome; actually, many of the common Rlm genes induce HR-type, easy to score resistance responses, while intermediate responses are found for B. oleracea genotypes. In this respect, the moderately resistant lines of B. oleracea identified by Dilmaghani et al. (2012) have not been investigated further. Bayer et al. (2019) of B. napus. One important way to increase the gene pool of diversity in terms of resistance sources is to exploit the diversity of related mustard species B. nigra, B. juncea, and B. carinata, all sharing the B genome, absent from B. napus, and being famed to harbour complete resistance or nonhost resistance to L. maculans. Roy (1984) introgressed resistance gene Rlm6 from B. juncea into B. napus. Similarly, Chevre et al. (1996) generated a resistant B. napus line harbouring one chromosome of B. nigra that was later shown to contain the new Rlm10 gene (Petit-Houdenot et al., 2019). More recently, similar works were performed to introduce one chromosome of B. carinata into B. napus, allowing the generation of plants resistant to L. maculans

[START_REF] Navabi | Introgression of B-genome chromosomes in a doubled haploid population of Brassica napus × B. carinata[END_REF]

. To date, none of these works resulted in commercial B. napus varieties harbouring the new effective resistance. This illustrates that, while the mustards are promising sources of new resistance to exploit, there still are difficulties in performing interspecific hybridization between B. napus and the different mustard species to make this the easiest way to generate B. napus varieties with improved resistance

[START_REF] Mason | Creating new interspecific hybrid and polyploid crops[END_REF]

. In contrast, multiple successes were found to resynthesize allopolyploid B. napus by crossing B. rapa and B. oleracea to increase the pool of available diversity

[START_REF] Katche | Interspecific hybridization for Brassica crop improvement[END_REF]

. Our finding that not only B. rapa, but also B. oleracea may harbour diversified sources of resistance justifies a continuous genebank screening effort to identify and exploit both B. rapa and B. oleracea resources. This approach via resynthesized B. napus should provide new B. napus resources with new Rlm

Table 1

 1

	. Briefly, a se-

Grant/ Award Number: ANR GPLA07-024C and ANR-17-EUR-0007

DATA AVA I L A B I L I T Y S TAT E M E N T All genome and transcriptome data are described in Rouxel et al. (2011), Dutreux et al. (2018), and Gay et al. (2020). All other relevant data are provided in the main text or supporting information.

mutations in AvrLm genes [START_REF] Daverdin | Genome structure and reproductive behaviour influence the evolutionary potential of a fungal phytopathogen[END_REF][START_REF] Rouxel | Life, death and rebirth of avirulence effectors in a fungal pathogen of Brassica crops, Leptosphaeria maculans[END_REF].

In our collections, one of the two oldest isolates was obtained in Europe from cabbage while the other was obtained from an uncertain host species. Both have the same AvrLm gene complement as modern Mexican isolates obtained from B. oleracea and are virulent towards Rlm14. We previously postulated a North American origin of L. maculans, partly because the first recorded damaging epidemics of the disease at the beginning of the 20th century were on cabbage in North America [START_REF] Dilmaghani | Migration patterns and changes in population biology associated with the worldwide spread of the oilseed rape pathogen Leptosphaeria maculans[END_REF] B. napus is a species with very low diversity due to its recent origin (7,500 years or less) and to a bottleneck effect associated with intensive selection for low erucic acid and low glucosinolate content traits [START_REF] Katche | Interspecific hybridization for Brassica crop improvement[END_REF]. In addition, there is no natural source of variation because B. napus is not found in nature as a wild type. All resistance genes of B. napus currently used in commercial varieties (Rlm1, Rlm2, Rlm3, Rlm4, Rlm7, Rlm9) are located on the A genome of B. napus [START_REF] Lv | An update on the arsenal: mining resistance genes for disease management of Brassica crops in the genomic era[END_REF] or have been introgressed from B. rapa into B. napus (LepR1, LepR2, LepR3, [START_REF] Yu | Identification of two novel genes for blackleg resistance in Brassica napus[END_REF][START_REF] Yu | Identification and mapping of a third blackleg resistance locus in Brassica napus derived from B. rapa subsp. sylvestris[END_REF]Rlm11, Balesdent et al., 2013;RlmS, Neik et al., 2020). Most of these, except Rlm3 to date, have also been found in genotypes of B. rapa [START_REF] Leflon | Detection, introgression and localization of genes conferring specific resistance to Leptosphaeria maculans from Brassica rapa into B. napus[END_REF], as well as other resistance genes, Rlm8 [START_REF] Balesdent | New avirulence genes in the phytopathognic fungus Leptosphaeria maculans[END_REF] and LepR1 [START_REF] Yu | Identification of two novel genes for blackleg resistance in Brassica napus[END_REF]. This suggests that B. rapa was the main provider of resistance genes in B. napus. In contrast, and while the B. oleracea genome (CC) shares a high ancestral synteny with the A genome of B. rapa [START_REF] Chalhoub | Early allopolyploid evolution in the post-Neolithic Brassica napus oilseed genome[END_REF], suggesting that functional homologues of A genome resistance genes could be found in the C genome, no currently known resistance gene against L. maculans is located on the C genome of B. napus [START_REF] Lv | An update on the arsenal: mining resistance genes for disease management of Brassica crops in the genomic era[END_REF]. In this respect, the few studies having screened B. oleracea genetic resources, and having identified lines showing moderate to high resistance did not further investigate the genetic control of the resistance to conclude on whether major genes for resistance may be present in B. oleracea (Dimaghani et al., 2012;[START_REF] Robin | Korean Brassica oleracea germplasm offers a novel source of qualitative resistance to blackleg disease[END_REF]. Without additional information at this stage, we can only speculate on why the A genome harbours so many resistances to L. maculans compared to the C genome. The first reason could be a biased interpretation due