# Inducible MdAGG lectins in apple immunity toward Fire Blight: CRISPR/Cas9 validation and their potential for intragenesis approaches

Antoine Bodelot, Nicolas Dousset, Elisa Ravon, Christelle Heintz, Marie-Noëlle Brisset, Alexandre Degrave, Emilie Vergne

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Antoine Bodelot, Nicolas Dousset, Elisa Ravon, Christelle Heintz, Marie-Noëlle Brisset, et al. Inducible MdAGG lectins in apple immunity toward Fire Blight: CRISPR/Cas9 validation and their potential for intragenesis approaches. Horticultural Research (Japan), 2025, uhaf262, 10.1093/hr/uhaf262/8271418. hal-05300148

HAL Id: hal-05300148

https://institut-agro-rennes-angers.hal.science/hal-05300148v1

Submitted on 6 Oct 2025

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**Inducible MdAGG lectins in apple immunity toward Fire Blight: CRISPR/Cas9 validation and their potential for intragenesis approaches.**

**Authors informations**

Antoine Bodelot¹, Nicolas Dousset¹, Elisa Ravon¹, Christelle Heintz¹, Marie-Noelle Brisset¹, Alexandre Degrave¹*, Emilie Vergne¹*

¹ Univ. Angers, Institut Agro, INRAE, IRHS, SFR QUASAV, 49000, Angers, France

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*Alexandre Degrave and Emilie Vergne should be considered the joint senior author

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Corresponding author: Emilie Vergne, Univ. Angers, Institut Agro, INRAE, IRHS, SFR QUASAV, F-49000, Angers, France; Email: [emilie.vergne@inrae.fr](mailto:emilie.vergne@inrae.fr)

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**Others authors email:**

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Antoine Bodelot: antoine.bodelot@inrae.fr

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Nicolat Dousset: nicolas.dousset@inrae.fr

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Elisa Ravon: elisa.ravon@inrae.fr

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Christelle Heintz: christelle.heintz@agrocampus-ouest.fr

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Marie-Noëlle Brisset: marie-noelle.brisset@inrae.fr

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Alexandre Degrave: alexandre.degrave@agrocampus-ouest.fr

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**Running head**

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Induced and effective Fire Blight resistance in apple is conferred by MdAGG lectins.

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© The Author(s) 2025. Published by Oxford University Press. This is an Open Access article distributed under the terms of the Creative Commons Attribution License https://creativecommons.org/licenses/by/4.0/, which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited.

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**Abstract**

Fire blight, caused by the bacterium *Erwinia amylovora*, represents a significant threat to apple (*Malus domestica*) production. Currently, only a limited number of genes effectively involved in resistance to *E. amylovora* have been identified.

Seeking new resistance candidates, we focused on a multigene family encoding amaranthin-like lectins, which are highly upregulated following chemical elicitation by acibenzolar-S-methyl (ASM). These lectins are believed to contribute to downstream defense by promoting bacterial aggregation, which led to their designation as *Malus domestica* agglutinins (MdAGG).

When loss-of-function editions were introduced into MdAGG genes, the plant's ability to mount a fully effective defense response against fire blight upon ASM treatment was compromised, confirming the role of MdAGGs in fire blight resistance. Next, we coupled the pPPO16 promoter, endogenous to apple and known to be rapidly induced during *E. amylovora* infection, with the coding sequence of MdAGG10 to create apple lines with fire blight-inducible MdAGG10 expression. Early MdAGG10 expression in these lines significantly improved resistance to fire blight, and an additional ASM treatment further enhanced this resistance.

In summary, we conclude that MdAGGs act as defense genes whose timely expression can provide effective resistance against *E. amylovora*.

**Key words:** amaranthin-like lectins, CRISPR/Cas9, *Erwinia amylovora*, Intragenesis, *Malus domestica* (apple tree), New Breeding Techniques

**Introduction**

Fire blight is a devastating disease caused by the necrogenic bacteria *Erwinia amylovora* (*Ea*) which affects most members of the *Rosaceae* family, including apple (*Malus domestica*). *Ea* owns two major virulence factors. The first one is its type III secretion system (T3SS) which injects effectors into host cells and these effectors are assumed to be secreted and translocated via this T3SS *ie*. AvrRpt2*Ea*, DspE/A, HopPtoC*Ea*, HrpN, Eop1 and Eop3 (McNally *et al.*, 2015). The second factor is the exopolysaccharide (EPS), external glycans whose production is correlated to *Ea* virulence (Ayers *et al.*, 1979). As a Gram negative bacteria, the external membrane of *Ea* cells also contains lipopolysaccharides (LPS), which are glycolipids protecting the bacterial cells from the oxidative burst launched by the plant during infection
(Berry *et al.*, 2009). EPS and LPS are known bacterial PAMPs (Pathogen-Associated Molecular Patterns), widely conserved components essential for pathogenicity and pathogens survival (Boutrot & Zipfel, 2017).

As the protection practices against fire blight are scarce and often poorly effective (Bonn & Van der Zwet, 2000), the selection of resistant cultivars remains an alternative to be favoured. However, conventional breeding of apple is tedious especially due to a long juvenile phase (Campa *et al.*, 2023). Until now, conventional breeding for fire blight resistance has mostly focused on resistance QTLs (Quantitative Trait Loci), with the strongest ones identified in wild *Malus*: on linkage group (LG) 3 of *Malus × robusta* 5 (Peil *et al.*, 2019), on LG10 of *M. fusca* (Emeriewen *et al.*, 2014) and on LG12 of 'Evereste', *M. floribunda* 821 (Durel *et al.*, 2009) and *M. × arnoldiana* (Emeriewen *et al.*, 2017). Candidate genes under these QTLs have been assigned as either NBS-LRR: *FB_MR5* in *Malus × robusta* 5 (Fahrentrapp *et al.*, 2013); receptor-like kinase: *FB_Mfu10* in *M. fusca* (Emeriewen *et al.*, 2018); and both of them in the QTL on LG12 of 'Evereste' (and *M. floribunda*; Parravicini *et al.*, 2011) and *M. × arnoldiana* (Emeriewen *et al.*, 2021). However, bypasses of the resistance gained from the introgression of QTLs from 'Evereste' (and *M. floribunda*) and *Malus × robusta* (*FB_MR5* gene) into commercial varieties have already been observed in apple, due to the evolution of the bacterial effectors Eop1 and AvrRPT2<sub>Ea</sub>, respectively (Emeriewen *et al.*, 2019).

New breeding technologies, including cisgenesis, intragenesis, RNA interference and genome editing mediated by CRISPR-Cas9 are alternatives to conventional breeding (Campa *et al.*, 2023). These technologies, however, have rarely been used to improve resistance to fire blight, via R recognition mechanisms or via targeting mechanisms considered to be more durable. Cisgenesis has been used to introduce the *FB_MR5* gene from *Malus × robusta* 5 in the susceptible genotype 'Gala Galaxy', leading to resistance levels equivalent to those obtained for classically bred descendants (Kost *et al.*, 2015). Intragenesis has already been employed to improve scab resistance in apple (Joshi *et al.*, 2011), but not yet for fire blight resistance. Successful knockdown of apple susceptibility genes (S-genes; Campa *et al.*, 2019; Pompili *et al.*, 2020) by has been performed by RNA interference on the *HIPM* gene (Campa *et al.*, 2019) or by directed mutagenesis by CRISPR/Cas9 of the *DIPM4* gene (Pompili *et al.*, 2020), both genes encoding proteins that respectively interact with *Ea* effectors HrpN and DspA/E. To the best of our knowledge, no published attempts have been made to improve apple resistance to *Ea* through NBT by targeting defense genes, such as Pathogenesis Related (PR) genes, that are involved in downstream mechanisms of pathogen recognition by R proteins or Pattern
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Recognition Receptors (PRR) and subsequent signal transduction (Pattern-Triggered Immunity, PTI; Jones & Dangl, 2006).

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The candidate family we targeted in the present study was identified in previous works on elicitor-induced resistance in apple against *E. amylovora* and proposed as a new class of PR proteins (Warneys *et al.*, 2018; Chavonet *et al.*, 2022). In particular, the chemical elicitor ASM up-regulates thousands of genes in apple, including a multigenic family partly organized as tandem array genes encoding lectins that have been termed *Malus domestica* agglutinins (MdAGGs, Warneys *et al.*, 2018). Lectins are proteins with at least one non-catalytic site able to reversibly bind mono- or oligosaccharides (Peumans & Van Damme, 1995). Plant PRR harbouring extracellular lectin domains that recognize specific carbohydrates of pathogens and trigger subsequent defense responses have yet received extensive interest (Bellande *et al.*, 2017). In contrast, defense-inducible lectins not related to pathogen recognition and subsequent signalization are less studied, although they can bind carbohydrate patterns and act as key players in plant defense processes through different pathways (De Coninck & Van Damme 2021). According to the classification of Van Damme *et al.* (2008) MdAGG1 to MdAGG18 are defense-inducible merolectins composed of a unique amaranthin-like carbohydrate binding domain. The apple genome also encodes nine other amaranthin-like lectins, MdDiAGG1 to 9, composed of two amaranthin-like domains and an additional aerolisin/ETX pore-forming domain for three of them: MdDiAGG5, 6 and 7 (Warneys *et al.*, 2018). MdDiAGGs are constitutively expressed, unlike MdAGGs, and MdAGG10, which is one of the most ASM-induced genes and aligns best with the MdAGGs consensus sequence (Warneys *et al.*, 2018), has been studied further. Agglutination of *Ea* cells by MdAGG10 has been demonstrated *in vitro*, involving electrostatic interactions with the negatively charged bacterial polysaccharides EPS and LPS. Moreover, *Ea* actively downregulates the expression of MdAGG genes and evades MdAGG10-driven cell aggregation thanks to the secretion of EPS (Chavonet *et al.*, 2022). MdAGGs are therefore assumed to be good candidate defense genes for improving apple PTI against *Ea*. However, there is still no functional evidence supporting the involvement of MdAGGs in apple resistance to *Ea*, as the constitutive overexpression of MdAGG10 in the susceptible 'Gala' genotype did not improve resistance to *Ea* (Bodelot *et al.*, 2023). Moreover, constitutive expression of MdAGG10 is associated with growth and development defaults of apple transgenic lines (Bodelot *et al.*, 2023). Associating MdAGG sequences with an *Ea*-inducible promoter, appears as an alternative to the apparent toxicity of the constitutive accumulation of these proteins, and its possible negative impact on resistance. From this

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perspective, the promoter of the endogenous apple *PPO16* gene, known to be activated by *Ea* infection in a rapid and sustained manner (Gaucher *et al.*, 2022), was considered as a valuable candidate.

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The present study aimed to determine if *MdAGGs* genes are effectively involved in apple resistance against *Ea*. Thanks to CRISPR/Cas9, now widely mastered in apple (Campa *et al.*, 2023) we evaluated the consequences of *MdAGGs* knockdown on ASM-induced resistance to *Ea*. Using the intragenic association between the fire blight inducible *pPPO16* promoter and the coding sequence of *MdAGG10* allowed us to study the effect of *Ea*-driven expression of *MdAGG10* on disease progression. From a methodological perspective, we evaluated the use of *in vitro* apple shoots for preliminary phenotyping of transformed lines, and a qPCR-based method to calculate the overall editing rate of edited lines.

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<table><caption>**Table 1** Information on the gRNA1 and gRNA2 targeted DNA zones on GDDH13 genome and the two phases of 'Gala' genome. gRNAs target all *MdAGG* members of the family. gRNA1 and 2 were designed on *MdAGG10* (MD10G1027210) on GDDH13 genotype. On the two genotypes, gRNA1 targets all *MdAGGs* with more or less mismatches, but also the off-target coding sequence *MdDiAGG4* (MD14G1239550) with two mismatches. On the two genotypes, gRNA2 targets all others *MdAGGs* with more or less mismatches except MdAGG14 (MD10G1027250), MdAGG17 (MD05G1031110) and MdAGG18 (MD05G1031120); it has no predicted off-target. Mismatch tolerance is considered effective ("Yes") for up to two base mismatches (Charrier et al, 2019), occurring between positions 20 and 4 relative to the PAM. Dashes (__) indicate non-off-target sequences.</caption><thead><tr><th rowspan="3">Gene name<sup>a</sup></th><th colspan="10">Information on GDDH13 genotype*</th><th colspan="10">Information on 'Gala'<sup>d</sup></th></tr><tr><th rowspan="2">Gene ID</th><th rowspan="2">Genome localization</th><th rowspan="2">Positions of gRNA1 "offtargets" with 0, 1 or 2 mismatches**</th><th rowspan="2">gRNA 1 mismatches</th><th rowspan="2">gRN A1 edit</th><th colspan="4">Information on 'Gala'<sup>d</sup> phase A</th><th colspan="4">Information on 'Gala'<sup>d</sup> phase B</th><th colspan="4">Information on GDDH13 genotype*</th><th colspan="4">Information on 'Gala'<sup>d</sup> phase A</th><th colspan="4">Information on 'Gala'<sup>d</sup> phase B</th></tr><tr><th>gRNA 1 mismatches</th><th>gRN A1 edit</th><th>gRNA 1 mismatches</th><th>gRN A1 edit</th><th>gRNA 2 mismatches</th><th>gRN A2 edit</th><th>gRNA 1 mismatches</th><th>gRN A1 edit</th><th>gRNA 1 mismatches</th><th>gRN A1 edit</th><th>gRNA 2 mismatches</th><th>gRN A2 edit</th><th>gRNA 1 mismatches</th><th>gRN A1 edit</th><th>gRNA 1 mismatches</th><th>gRN A1 edit</th></tr></thead><tbody><tr><td>MdAG G1</td><td>MD10G1 026610</td><td>Chr10:3293389.. 3293886 (- strand)</td><td>Position: Chr10:329 3855- 3293877:+ Position: Chr10:329 7058- 3297080:+ Position: Chr10:330 7773- 3307795:+ Position: Chr10:332816.. 3323313 (- strand)</td><td>1</td><td>Yes</td><td>1</td><td>Yes</td><td>1</td><td>Yes</td><td>0</td><td>Yes</td><td>0</td><td>Yes</td><td>0</td><td>Yes</td><td>0</td><td>Yes</td><td>0</td><td>Yes</td><td>0</td><td>Yes</td><td>0</td><td>Yes</td></tr><tr><td>MdAG G2</td><td>MD10G1 026620</td><td>Chr10:3296592.. 3297089 (- strand)</td><td>Position: Chr10:329 7058- 3297080:+ Position: Chr10:330 7773- 3307795:+ Position: Chr10:332 3282- 3323304:+ Position: Chr10:332 6446- 3326468:+ Position: Chr10:3339497.. 3339994 (- strand)</td><td>1</td><td>Yes</td><td>1</td><td>Yes</td><td>1</td><td>Yes</td><td>0</td><td>Yes</td><td>0</td><td>Yes</td><td>0</td><td>Yes</td><td>0</td><td>Yes</td><td>0</td><td>Yes</td><td>0</td><td>Yes</td><td>0</td><td>Yes</td></tr><tr><td>ABS1</td><td>Intergenic region</td><td>-</td><td>Chr10:3322816.. 3323313 (- strand)</td><td>1</td><td>Yes</td><td>1</td><td>Yes</td><td>1</td><td>Yes</td><td>0</td><td>Yes</td><td>0</td><td>Yes</td><td>0</td><td>Yes</td><td>0</td><td>Yes</td><td>0</td><td>Yes</td><td>0</td><td>Yes</td><td>0</td><td>Yes</td></tr><tr><td>MdAG G3</td><td>MD10G1 026810</td><td>Chr10:3325980.. 3326477 (- strand)</td><td>Position: Chr10:332 3282- 3323304:+ Position: Chr10:332 6446- 3326468:+ Position: Chr10:3339497.. 3339994 (- strand)</td><td>1</td><td>Yes</td><td>1</td><td>Yes</td><td>1</td><td>Yes</td><td>0</td><td>Yes</td><td>0</td><td>Yes</td><td>0</td><td>Yes</td><td>0</td><td>Yes</td><td>0</td><td>Yes</td><td>0</td><td>Yes</td><td>0</td><td>Yes</td></tr><tr><td>MdAG G4</td><td>MD10G1 026820</td><td>Chr10:3325980.. 3326477 (- strand)</td><td>Position: Chr10:332 3282- 3323304:+ Position: Chr10:332 6446- 3326468:+ Position: Chr10:3339497.. 3339994 (- strand)</td><td>2</td><td>Yes</td><td>2</td><td>Yes</td><td>2</td><td>Yes</td><td>0</td><td>Yes</td><td>0</td><td>Yes</td><td>0</td><td>Yes</td><td>0</td><td>Yes</td><td>0</td><td>Yes</td><td>0</td><td>Yes</td><td>0</td><td>Yes</td></tr><tr><td>MdAG G5</td><td>MD10G1 027010</td><td>Chr10:3349989.. 3350522 (- strand)</td><td>Position: Chr10:335 0491- 3350513:+ Position: Chr10:336 2749- 3362771:+ Position: Chr10:338 4748- 3384770:+ Position: Chr10:338 5523- 3385545:+ Position: Chr10:342 3632- 3423654:+ targeted gene of gRNA1 (position 10 - strand)</td><td>2</td><td>Yes</td><td>2</td><td>Yes</td><td>2</td><td>Yes</td><td>0</td><td>Yes</td><td>0</td><td>Yes</td><td>0</td><td>Yes</td><td>0</td><td>Yes</td><td>0</td><td>Yes</td><td>0</td><td>Yes</td><td>0</td><td>Yes</td></tr><tr><td>MdAG G6</td><td>MD10G1 027020</td><td>Cluster Chr. 10</td><td>Chr10:3362283.. 3362780 (- strand)</td><td>0</td><td>Yes</td><td>0</td><td>Yes</td><td>0</td><td>Yes</td><td>0</td><td>Yes</td><td>0</td><td>Yes</td><td>0</td><td>Yes</td><td>0</td><td>Yes</td><td>0</td><td>Yes</td><td>0</td><td>Yes</td><td>0</td><td>Yes</td></tr><tr><td>MdAG G7</td><td>MD10G1 027025</td><td>250 kb</td><td>Chr10:3384282.. 3384779 (- strand)</td><td>0</td><td>Yes</td><td>0</td><td>Yes</td><td>1</td><td>Yes</td><td>0</td><td>Yes</td><td>0</td><td>Yes</td><td>0</td><td>Yes</td><td>0</td><td>Yes</td><td>0</td><td>Yes</td><td>0</td><td>Yes</td><td>0</td><td>Yes</td></tr><tr><td>MdAG G8</td><td>MD10G1 027030</td><td>0</td><td>Chr10:3384282.. 3384779 (- strand)</td><td>0</td><td>Yes</td><td>0</td><td>Yes</td><td>0</td><td>Yes</td><td>0</td><td>Yes</td><td>0</td><td>Yes</td><td>0</td><td>Yes</td><td>0</td><td>Yes</td><td>0</td><td>Yes</td><td>0</td><td>Yes</td><td>0</td><td>Yes</td></tr><tr><td>ABS2</td><td>Intergenic region</td><td>-</td><td>Chr10:3423166.. 3423663 (- strand)</td><td>2</td><td>Yes</td><td>0</td><td>Yes</td><td>0</td><td>Yes</td><td>0</td><td>Yes</td><td>0</td><td>Yes</td><td>0</td><td>Yes</td><td>0</td><td>Yes</td><td>0</td><td>Yes</td><td>0</td><td>Yes</td><td>0</td><td>Yes</td></tr><tr><td>MdAG G9</td><td>MD10G1 027110</td><td>Chr10:3455323.. 3455820 (- strand)</td><td>Position: Chr10:342 3632- 3423654:+ targeted gene of gRNA1 (position 10 - strand)</td><td>0</td><td>Yes</td><td>0</td><td>Yes</td><td>0</td><td>Yes</td><td>0</td><td>Yes</td><td>0</td><td>Yes</td><td>0</td><td>Yes</td><td>0</td><td>Yes</td><td>0</td><td>Yes</td><td>0</td><td>Yes</td><td>0</td><td>Yes</td></tr><tr><td>MdAG G10</td><td>MD10G1 027210</td><td>Chr10:3455323.. 3455820 (- strand)</td><td>Position: Chr10:342 3632- 3423654:+ targeted gene of gRNA1 (position 10 - strand)</td><td>0</td><td>Yes</td><td>0</td><td>Yes</td><td>0</td><td>Yes</td><td>0</td><td>Yes</td><td>0</td><td>Yes</td><td>0</td><td>Yes</td><td>0</td><td>Yes</td><td>0</td><td>Yes</td><td>0</td><td>Yes</td><td>0</td><td>Yes</td></tr><tr><td>MdAG G11</td><td>MD10G1 027220</td><td>Chr10:3483442.. 3483939 (- strand)</td><td>Position: Chr10:342 3632- 3423654:+ targeted gene of gRNA1 (position 10 - strand)</td><td>0</td><td>Yes</td><td>0</td><td>Yes</td><td>0</td><td>Yes</td><td>0</td><td>Yes</td><td>0</td><td>Yes</td><td>0</td><td>Yes</td><td>0</td><td>Yes</td><td>0</td><td>Yes</td><td>0</td><td>Yes</td><td>0</td><td>Yes</td></tr><tr><td>MdAG G12</td><td>MD10G1 027230</td><td>3509813 (- strand)</td><td>Chr10:3509319.. 3509813 (- strand)</td><td>0</td><td>Yes</td><td>0</td><td>Yes</td><td>0</td><td>Yes</td><td>0</td><td>Yes</td><td>0</td><td>Yes</td><td>0</td><td>Yes</td><td>0</td><td>Yes</td><td>0</td><td>Yes</td><td>0</td><td>Yes</td><td>0</td><td>Yes</td></tr></tbody></table>
<table><tbody><tr><td>MdAG<br>G13</td><td>MD10G1<br>027240</td><td>Chr10:3524494..<br>3524991 (-<br>strand)</td><td>Position:<br>Chr10:352<br>4960-<br>3524982:+<br>Position:<br>Chr10:353<br>7561-<br>3537583:+<br>Position:<br>Chr10:355<br>3744-<br>3553766:+</td><td>0</td><td>Yes</td><td>0</td><td>Yes</td><td>0</td><td>Yes</td><td>Position:<br>Chr10:352<br>4736-<br>3524758:-<br>Position:<br>Chr10:353<br>7337-<br>3537359:+<br>Position:<br>Chr10:355<br>3520-<br>3553542:-</td><td>1</td><td>Yes</td><td>1</td><td>Yes</td><td>1</td><td>Yes</td></tr><tr><td>MdAG<br>G14</td><td>MD10G1<br>027250</td><td>Chr10:3537095..<br>3537592 (-<br>strand)</td><td></td><td>1</td><td>Yes</td><td>1</td><td>Yes</td><td>1</td><td>Yes</td><td></td><td>1</td><td>No</td><td>1</td><td>No</td><td>1</td><td>No</td></tr><tr><td>MdAG<br>G15</td><td>MD10G1<br>027260</td><td>Chr10:3553278..<br>3553775 (-<br>strand)</td><td></td><td>0</td><td>Yes</td><td>0</td><td>Yes</td><td>0</td><td>Yes</td><td></td><td>0</td><td>Yes</td><td>0</td><td>Yes</td><td>0</td><td>Yes</td></tr><tr><td>MdAG<br>G16</td><td>MD06G1<br>232610</td><td>Chr06:36368957<br>..36369454 (-<br>strand)</td><td>Position:<br>Chr06:363<br>69423-<br>36369445:<br>+</td><td>2</td><td>Yes</td><td>-</td><td>-</td><td>2</td><td>Yes</td><td>Position:<br>Chr06:363<br>69199-<br>36369221:<br>-</td><td>1</td><td>Yes</td><td>-</td><td>-</td><td>1</td><td>Yes</td></tr><tr><td>MdAG<br>G17</td><td>MD05G1<br>031110</td><td>Chr05:5050912..<br>5051415 (+<br>strand)</td><td>Position:<br>Chr05:505<br>0921-<br>5050943:-<br>Position:<br>Chr05:507<br>2629-<br>5072651:-</td><td>2</td><td>Yes</td><td>2</td><td>Yes</td><td>2</td><td>Yes</td><td>Position:<br>Chr05:505<br>1145-<br>5051167:+<br>Position:<br>Chr05:507<br>2853-<br>5072875:+</td><td>1</td><td>No</td><td>1</td><td>No</td><td>1</td><td>No</td></tr><tr><td>MdAG<br>G18</td><td>MD05G1<br>031120</td><td>Chr05:5072620..<br>5073123 (+<br>strand)</td><td></td><td>2</td><td>Yes</td><td>2</td><td>Yes</td><td>2</td><td>Yes</td><td></td><td>1</td><td>No</td><td>1</td><td>No</td><td>1</td><td>No</td></tr><tr><td>MdDiA<br>GG1</td><td>MD06G1<br>232620</td><td>Chr06:36378219<br>..36379187 (-<br>strand)</td><td></td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td></td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td></tr><tr><td>MdDiA<br>GG2</td><td>MD06G1<br>232630</td><td>Chr06:36397896<br>..36398933 (-<br>strand)</td><td></td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td></td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td></tr><tr><td>MdDiA<br>GG3</td><td>MD06G1<br>233110</td><td>Chr06:36438595<br>..36439497 (+<br>strand)</td><td></td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td></td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td></tr><tr><td>MdDiA<br>GG4</td><td>MD14G1<br>239550</td><td>Chr14:31867464<br>..31868432 (-<br>strand)</td><td>Position:<br>Chr14:318<br>67930-<br>31867952:<br>+</td><td>2</td><td>Yes</td><td>-</td><td>-</td><td>2</td><td>Yes</td><td></td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td></tr><tr><td>MdDiA<br>GG5</td><td>MD07G1<br>171100</td><td>Chr07:24677707<br>..24679209 (+<br>strand)</td><td></td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td></td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td></tr><tr><td>MdDiA<br>GG6</td><td>MD01G1<br>104300</td><td>Chr01:21733076<br>..21734572 (+<br>strand)</td><td></td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td></td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td></tr><tr><td>MdDiA<br>GG7</td><td>MD02G1<br>240000</td><td>Chr02:28852956<br>..28854545 (+<br>strand)</td><td></td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td></td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td></tr><tr><td>MdDiA<br>GG8</td><td>MD03G1<br>201500</td><td>Chr03:27573652<br>..27575032 (+<br>strand)</td><td></td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td></td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td></tr><tr><td>MdDiA<br>GG9</td><td>MD14G1<br>241050</td><td>Chr14:31980059<br>..31981000 (-<br>strand)</td><td></td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td></td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td></tr></tbody></table>

§(Warneys et al., 2018), ABS1 and 2 are intergenic regions not encoding MdAGGs or

MdDiAGGs

*on https://iris.angers.inra.fr/gddh13

**positions from http://crispor.gi.ucsc.edu/

‡blast on sequences from genomes "Malus_domestica_Gala.altA" (phase A) and "Malus_domestica_Gala.altB" (phase B) (Sun et al., 2020)

## Results

### Generation of transgenic lines

A total of 419 leaf explants were inoculated with *Agrobacterium tumefaciens* containing the binary vector with the CRISPR-MdAGGAll construct (T-DNA in Fig. S1a) targeting supposedly all of *MdAGG* family members in 'Gala' (Table 1). The five kanamycin resistant-
regenerants which survived over 6 months of micropropagation were analyzed by PCR to confirm transformation and absence of *A. tumefaciens* contamination (Fig. S2a). Amplification using primers I (Table S1) designed for *EF1α* gene was used as a positive PCR control, plasmid DNA extracted from *A. tumefaciens* strain containing the CRISPR-MdAGGAll construct as a positive control of T-DNA presence and genomic DNA extracted from a non-transgenic 'Gala' as a negative control of T-DNA-presence. K primers (Table S1) amplifying 23S ribosomal RNA from *A. tumefaciens* showed that all tested lines were free from *A. tumefaciens* contamination. Amplification using J primers (Table S1), which target the *nptII* selection gene, confirmed that all five lines were true transgenics containing the selectable marker. Finally, amplification using primers B and L (Table S1), which respectively target gRNAs sequences and the Cas9 coding sequence, demonstrated that all lines had integrated the full construct. Considering the initial number of explants (419), the transformation rate was therefore 1.19%. The five lines were called *mdagg-1* to *mdagg-5* and further analyzed.

A total of 1150 leaf explants were inoculated with *A. tumefaciens* containing the binary vector with the *pPPO16::MdAGG10* construct (T-DNA in Fig. S1b) assumed to induce MdAGG10 expression upon *Ea* infection (Gaucher *et al.*, 2022). The nine kanamycin resistant-regenerants which survived over 6 months of micropropagation were analyzed by PCR, with similar controls as describe above for CRISPR lines, to confirm transformation and absence of *A. tumefaciens* contamination (Fig. S2b). Amplification with primers H (Table S1) for the *pPPO16::MdAGG10* association presence showed that all lines had integrated the full construct. Considering the initial number of explants (1150), the transformation rate was therefore 0.78%. Six of the nine lines, called *pPPO16::MdAGG10-1* to *pPPO16::MdAGG10-6* were further analyzed.
<figure><img src="image_3.png" /><figcaption>**Fig. 1** Dual guide-RNA gene edition strategy leads to *MdAGG* genes knockdown in apple. Four cloned sequences from each of the 5 independent *mdagg* edited apple mutants were analyzed. Each box represents one *MdAGG* sequence. Unedited *MdAGG* sequences are represented by a darkgrey box, short indels leading to frameshift mutations protein sequence (respectively early insertion of stop codon) are represented by darkgrey boxes with vertical lines (respectively with horizontal lines), larger deletions occurring within a single *MdAGG* sequence and leading to frameshift mutations are represented with a smaller box with grey horizontal lines, deletions occurring between different *MdAGG* sequences and leading to large genomic DNA excisions are represented with a darkgrey and black box, sequences for which no information is available are represented by lightgrey boxes. « * » corresponds to a specific case observed in clone 4 of *mdagg-4* where a 200 nucleotides portion of *MdAGG15* was invertedly inserted into the targeted site of *MdAGG3*. gRNAs efficiencies are represented in (i) blue if the edits occurred in the gRNA1 targeted sequence, in (ii) red if edits occurred in gRNA2 targeted sequence and in (iii) green if edits occurred in both gRNAs targeted sequences. The targeted phase of ‘Gala’</figcaption></figure>
216 genome was specified when possible: orange for phase A, light blue for phase B and black for phase A or B.
217

218

219 Dual guide-RNA edition of *MdAGG* genes leads to multiple loss-of-function mutations

220

221 For each of the five lines, named *mdagg-1* to *mdagg-5*, four bacterial clones (referred to as 'clones') were sequenced as a check of the occurrence of editing events (Fig. 1). The two target sites proved effective, and the five lines proved edited, displaying various types of edits: (i) indel or substitution of several nucleotides in one *MdAGG* sequence (called after type A edit), (ii) deletion of the sequence between the two target sites, of around two hundred nucleotides, in one *MdAGG* sequence (called after type B edit) and (iii) deletion of several thousands of nucleotides between two different *MdAGGs* sequences (called after type C edit). This characterization also reported the insertion of one *MdAGG* sequence between the two targeted sites of another *MdAGG* sequence (called after Type D edit). In *mdagg-1* line, we observed four type A edits. In *mdagg-2* line, we observed two type B edits (clone 1 and 2), one type A edit (clone 3) and one type C edit (clone 4) displaying a 126 kb deletion. In *mdagg-3* line, we observed three unedited *MdAGG* sequences (clones 2, 3 and 4) and one type A edit (clone 1). For *mdagg-4* line, clones 1 and 2 displayed type A edits, clone 3 displayed a type C edit with a 212 kb deletion. Ultimately, clone 4 displayed a type D edit, where 200 nucleotides of *MdAGG15* were invertedly inserted between the two targeted sites of *MdAGG3*. For *mdagg-5* line, we observed three type B edits (clones 1, 2 and 3), as well as one type A edit (clone 4). All these edits on *MdAGGs* coding sequence led to the early insertion of a stop codon resulting in a truncated protein, except for one *MdAGG10* allele (clone 3) of *mdagg-1* line, where edits led to substitution or indels of amino acids (Fig. 1). Altogether, this analysis showed that both gRNAs were effective and that they led to multiple loss-of-function mutations in the *MdAGG* genes.

242
<figure><img src="image_4.png" /><figcaption>**Fig. 2** *In vitro* culture shoots of MdAGGs edited apple mutants except *mdagg-4* are still susceptible to *Erwinia amylovora* despite ASM treatment. Fire blight disease incidence (± standard deviation) and AUDPC scoring over a 8-day period after *Ea* infection. Each point represents the AUDPC value for individual shoots (28<n< 32), diamonds represent the mean AUDPC for each condition. Significance was evaluated with a Kruskal-Wallis test followed by a Dunn test, different letters indicate different statistical classes (p<0.05).</figcaption></figure>

**MdAGG loss-of-function mutants are impaired in their ASM-induced resistance response toward *E. amylovora***

To assess the effect of loss-of-function mutations of *MdAGGs* in inducible-resistance, *in vitro*
shoot cultures of lines *mdagg-1* to 5 were treated with ASM and further inoculated with *Ea*.
Susceptible control (untransformed susceptible variety ‘Gala’ (WT), water-treated), displayed
significantly higher disease incidence and severity scores than resistant one (ASM-treated
WT). Compared to the resistant control, fire blight incidence was significantly higher in all
*mdagg* mutant lines treated with ASM, except for line *mdagg-4* (Fig. 2). Regarding fire blight
severity, the AUDPC of ASM-treated *mdagg-4* mutants was equivalent to that of the resistant
control whereas the AUDPC of the four other ASM-treated *mdagg* mutants was equivalent to
that of the susceptible control (Fig. 2). Altogether, these results show that except for line
*mdagg-4*, *in vitro* shoots of *mdagg* mutant lines were not able to mount a fully-effective
resistance response toward *Ea* upon ASM treatment. Mutant lines *mdagg-1* and *mdagg-2* were
further analyzed.

<figure><img src="image_5.png" /><figcaption>**Fig. 3** Greenhouse-grown *MdAGG* edited apple mutants do not upregulate *MdAGG* upon ASM treatment and are more susceptible to *Erwinia amylovora*. (a) Quantification of editing rates at the gRNA1 target sequences in *mdagg-1* and *-2* lines. The editing rate (e) was calculated as followed: e = 1/x where x corresponds to the relative quantity of unedited DNA sequences of</figcaption></figure>
272 *MdAGGs* (lightgrey bars) or *MdDiAGG4* (darkgrey bars) measured by qPCR. The values 273 shown are the average measured in 4 to 6 samples from two independent biological repeats. 274 The data are compared with the ‘Gala’ non transformed genotype, as an unedited reference. 275 Significance of the Wilcoxon rank test: *p<0.05; ns not significant. (b) Relative expression 276 (log₂ ratio) of *MdAGGs* in the non-inoculated leaves just before bacterial infection. Means, 277 medians, and interquartile range (IQR) are indicated by diamonds, lines, boxes, respectively. 278 Whiskers indicate the most extreme values between the nearest quartile and 1.5 IQR. The mean 279 value from samples of water-pretreated controls (WT) was used for the calculation of the log₂ 280 ratio. Each dot represents one biological replicate, *i.e* a pool of two leaves of independent plants 281 (7<n<9). (c) MdAGGs protein detection by western-blot (upper panel) in the non-inoculated 282 leaves just before bacterial infection. Homogeneity of loaded proteins (15 µg per lane) was 283 verified by Coomasie brilliant blue staining (lower panel). (d) Fire blight disease incidence (± 284 standard deviation) 21 days after *E. amylovora* infection and phenotypic assessment by 285 AUDPC calculation over these 21 days. Each point represents the AUDPC for individual plants 286 (52<n<70 from two independent biological repeats). Diamonds represent the mean AUDPC for 287 each condition. For all quantified parameters, significance was evaluated with a Kruskal-Wallis 288 test followed by a Dunn test, different letters indicating significant differences (p<0.05). 289

290 We completed the quantitative characterization of the edits in *mdagg-1* and *mdagg-2* using a 291 qPCR-based method we developed to measure the editing rates at the gRNA1 targets, because 292 this guide seemed to be very effective for MdAGGs edits (Fig. 1). The targets of gRNA1 293 consisted of MdAGGs target sites and the MdDiAGG4 off-target site (Table 1). The forward 294 primers for both the MdAGGs targets and MdDiAGG4 off-target were designed to end just 295 after the Cas9 cut site (4 nucleotides from the PAM) of the gRNA1 target. This design allows 296 amplification only in wild-type alleles, where no cutting or modification has occurred, and in 297 rare cases where synonymous repairs may have taken place. Estimated global editing rates were 298 then calculated as inverses of these quantities of unmodified sequences. The results show that 299 both mutant lines *mdagg-1 and -2* were significantly edited at the *MdAGGs* target sites (Fig. 3a, lightgrey bars), while neither displayed significant edits at the off-target site *MdDiAGG4* (Fig. 3a, darkgrey bars). 300 301

302
303 Next, to determine if *MdAGGs* are involved in the ability of greenhouse-grown apple to mount
304 an effective ASM-induced resistance response toward *Ea*, mutant lines *mdagg-1* and *mdagg-2*
were acclimatized in greenhouse, treated with ASM and further inoculated with *Ea*. WT plants, either treated with water or ASM, were included as susceptible and resistant controls, respectively. Targeted gene expression and protein production were also analyzed in the remaining leaf tissues cut out during the inoculation procedure. *MdAGGs* expression was monitored by RT-qPCR with primers O (Table S1) designed to allow amplification of transcripts only in absence of edition (Fig. S1c). As expected, the results show that *MdAGGs* expression was significantly upregulated by the ASM treatment compared with the water treatment in untransformed control plants. Interestingly, no significant difference in *MdAGGs* transcript accumulation was observed between the susceptible control and the ASM-treated mutant lines (Fig. 3b), suggesting that among the *MdAGGs* successfully disrupted in these mutant lines are those primarily responsible for the ASM-induced expression observed in the resistant control. Protein accumulation analysis by western-blot confirmed loss-of-function for *mdagg-1* and *mdagg-2* mutants in sofar MdAGGs proteins were only detected in the resistant control (Fig. 3c). After inoculation with *Ea*, fire blight disease symptoms were scored every 3 to 4 days during 3 weeks and resistance was evaluated by determining disease incidence at the end of the experiment or disease severity by AUDPC calculation (Fig. 3d, Table S2). As expected, the ASM treatment reduced by more than 50 % disease incidence and severity for the WT. In contrast, although disease incidence and severity in ASM-treated *mdagg-1* and 2 lines were both significantly lower than for the susceptible control, they were significantly higher than for the resistant control (Fig. 3d). This shows that despite ASM elicitation, *mdagg-1* and *mdagg-2* greenhouse-grown mutants are less resistant to fire blight than the resistant control.

327

Overall, these results demonstrate that the expression and accumulation of functional MdAGGs are crucial for apple to mount a fully effective resistance response induced by ASM.

330
<figure><img src="image_6.png" /><figcaption>**Fig. 4** Fire blight inducible *PPO16* promoter upregulates *MdAGGs* expression upon *Erwinia amylovora* infection on *in vitro* culture shoots of *pPPO16::MdAGG10* transgenic lines. Light (respectively dark) grey boxplots represent *MdAGGs* expressions 24 (respectively 40) hours after *E. amylovora* infection by infiltration, versus water infiltration (mock). Each point represents the *MdAGGs* expressions (log~2~ ratio) in individual inoculated samples relative to the mean value of *MdAGGs* expressions in the water-infiltrated biological repeats at the same day of sampling. Means, medians, interquartile range (IQR) are indicated by diamonds, lines, boxes, respectively. Each biological replicate is a pool of all leaves sampled from two independent plants (6<n<8). Significance was evaluated with a Kruskal-Wallis test followed by a Dunn test, different letters indicate different significance (p<0.05). Boxes with same letter and same colour represent medians that are not significantly different (p<0.05).</figcaption></figure>

**Molecular and phenotyping characterization of pPPO16::MdAGG10 inducible lines under *E. amylovora* inoculation challenge**

Six of the nine lines obtained after *A. tumefaciens* transformation, called *pPPO16::MdAGG10-1* to *pPPO16::MdAGG10-6*, were analyzed to determine if bacterial infection with *Ea*, known to activate *pPPO16* promoter, effectively led to overexpression of *MdAGG10*. Transcript accumulation of *MdAGG10* was assessed by RT-qPCR at 24 and 40 hours post infiltration (hpi) in mock- or *Ea*-infiltrated leaves of *in vitro* shoot cultures. The untransformed ‘Gala’ variety
(WT) was used as a control. Differential gene expression analysis shows that untransformed plants did not upregulate *MdAGG10* in response to *Ea* infection whereas the transformed lines did, with a log₂ ratio (compared to the WT line) varying from 1 to 5 depending on the lines and the day of sampling (24 or 40 hpi - Fig. 4).

<figure><img src="image_7.png" /><figcaption>**Fig. 5** *In vitro* culture shoots of *pPPO16::MdAGG10* lines are less susceptible to *Erwinia amylovora*. Fire blight disease incidence (± standard deviation) 12 days after *E. amylovora* infection and phenotypic assessment by AUDPC calculation over these 12 days. Each dot represents the AUDPC of one shoot (29<n<35). Each diamond represents the mean AUDPC of each condition. Significance was evaluated with a Kruskal-Wallis test followed by a Dunn test, different letters indicate different statistical classes (p<0.05).</figcaption></figure>

To determine if conditional upregulation of *MdAGG10* enhances apple resistance toward *Ea*, *in vitro* shoot cultures of *pPPO16::MdAGG10-1* to 6 lines were inoculated with *Ea* and disease symptoms were scored daily over 12 days following inoculation. Resistance was evaluated by determining disease incidence at the end of the experiment or disease severity by AUDPC calculation. The WT plants, either treated with water or ASM, were included as a respectively susceptible and resistant controls. Water-treated WT plants displayed significantly higher
disease incidence and severity scores than ASM-treated WT plants. Fire blight incidence was significantly higher than in the susceptible control in four of the six lines (pPPO16::MdAGG10-2 to -4 and 5), and intermediate to those of resistant and susceptible controls in pPPO16::MdAGG10-1 and 6, although more similar to incidence measured for the susceptible control (Fig. 5). In four of the six lines (pPPO16::MdAGG10-1 to -3 and -6), disease severity was intermediate to those of resistant and susceptible controls, whereas in pPPO16::MdAGG10-4 and -5, it was equivalent to that of the susceptible control (Fig. 5). In brief, except line pPPO16::MdAGG10-4 and -5, most of pPPO16::MdAGG10 lines in vitro shoots were less severely affected by fire blight than the susceptible control. Lines pPPO16::MdAGG10-1 and pPPO16::MdAGG10-6 were further analyzed in greenhouse.

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<figure><img src="image_8.png" /><figcaption>**Fig. 6** Greenhouse-grown *pPPO16::MdAGG10* apple lines are less susceptible to *Erwinia amylovora* (a) Representative picture of water-treated apple lines inoculated with *E. amylovora*, 19 days post-inoculation (b) Fire blight disease incidence (± standard deviation) 19 days after inoculation and phenotypic assessment by AUDPC calculation over these 19 days. Each dot represents the AUDPC of one plant (29<n<35). Each diamond represents the mean AUDPC of each condition. Significance was evaluated with a Kruskal-Wallis test followed by a Dunn test, different letters indicate different statistical classes (p<0.05).</figcaption></figure>

We next investigated whether infection-inducible *MdAGG10* expression provides effective resistance toward *Ea* lines *pPPO16::MdAGG10-1* and 6 grown in greenhouse with no apparent
development problems (Table S3), and if ASM further enhances this resistance, knowing that the *pPPO16* promoter had little reaction to ASM, in comparison to its reaction to *Ea* infection (Fig. S3, adapted from Chavonet *et al.*, 2022). Plants were therefore treated with ASM or water and then inoculated with *Ea*. The untransformed susceptible variety 'Gala', either treated with water or ASM, was included as a respectively susceptible and resistant control. Nineteen days after inoculation, plants of water-treated *pPPO16::MdAGG10-1* and 6 lines were obviously less affected by fire blight compared to the susceptible control. Disease incidence reached almost 94% in the susceptible control, which results in the stunted phenotype of plants systemically affected by *Ea* (Fig. 6a). In contrast, significantly lower disease incidence of the water-treated *pPPO16::MdAGG10-1* and 6 plants is evidenced by actively growing plants for both of these transgenic lines (Fig. 6a, b). Regarding disease severity, water-treated *pPPO16::MdAGG10-1* and 6 lines were intermediate to those of resistant and susceptible controls. Plants of both transgenic lines that were previously treated with ASM displayed lower disease incidence and severity than the resistant control (Fig. 6b, Table S4).

Altogether, these results show that infection-induced *MdAGG10* expression alone provides effective resistance to *Ea* in greenhouse-grown plants, with ASM treatment offering additional protection, reducing disease symptoms by approximately 90%.

## Discussion

Genetic improvement by NBT of apple varieties susceptible to fire blight have already been performed by transferring QTLs genes from wild relatives, or knocking-out of S-genes. Here, we provide evidence that intragenic association of a disease-induced promoter with a downstream defense gene constitutes a supplementary strategy for the biotechnological toolbox aiming at enhancing and studying fire blight disease resistance in apple.

From a methodological point of view, being able to use apple shoots cultivated *in vitro* for pre-screening of transformed lines, before their acclimatization in a greenhouse, can save valuable time and money. In this work, although the observed effects were weak, they nonetheless allowed a pre-screening, which results were then confirmed and reinforced in greenhouse for the two selected lines, as already observed for plum resistance to sharka (Garcia-Almodovar *et al.*, 2015), but only partially for pear resistance to fire blight, presumably due to a higher inoculum concentration in greenhouse than *in vitro* (Malnoy *et al.*, 2005).
Still from a methodological point of view, the two gRNAs designed for targeting all MdAGG members of the family, and the tandem array organization of MdAGG1 to MdAGG15 on chromosome 10 (Warneys et al., 2018) allowed large DNA deletions of several kb eliminating numerous MdAGG sequences in their entirety. CRISPR/Cas9 is a powerful and precise method to induce targeted mutagenesis, but it is known to create editing chimerism in plants whose transformation involves somatic organogenesis potentially involving several cells at the origin of the regenerated line, which is the case for apple (Charrier et al., 2019). Rounds of adventitious regeneration step can efficiently reduce chimerism (Malabarba et al., 2020) but stay time-consuming. As already demonstrated in Pompili et al. (2020), HTS (High throughput sequencing) targeted resequencing methods can be used to capture this edition diversity in first-generation edited apple lines, both in qualitative and quantitative terms. As an initial strategy, we opted here for a partial qualitative sequencing analysis of the edits by examining a few alleles of the targeted sequences. This strategy was intended to verify that editing events had occurred, but it could not provide the complete editing profile of each line. This approach nevertheless provided a solid overview of the different types of edits involved, especially the large deletions. In the mutant lines *mdagg-1* and -2, selected for further study, the gRNA1 targets consisting of *MdAGGs* target sites and the *MdDiAGG4* off-target site, were then analyzed quantitatively using an easy-to-use and effective qPCR-based method, confirming that these lines were suitable for robust greenhouse experiments.

We first demonstrated *in vitro* that four of the five *mdagg* lines were more susceptible to fire blight than the WT plants despite the application of ASM. Mutant *mdagg-4* was the only line whose ASM-induced resistance was comparable to that of WT plants, although it displayed editions affecting MdAGG sequences. It is unclear whether cell types that are at first line in the early steps of the foliar infection process, such as those surrounding the vascular tissue (Billing, 2011), remained unedited for this mutant, or if certain edits—such as the 212 kb deletion observed in one clone—enhance resistance by eliminating unidentified susceptibility factors. An in-depth analysis of this mutant line remains to be performed to explain its inconsistent phenotype. For the mutants selected for greenhouse assays, the decrease in resistance to *Ea* correlated with the absence of MdAGGs accumulation at the transcriptional and protein levels. To the best of our knowledge, it is the first time that amaranthin-like lectins are demonstrated to be involved in plant resistance against a bacteria by loss-of-function in the native species, rather than through ectopic expression (Cabrales-Orona et al., 2022). Here, MdAGGs are lectins containing a unique amaranthin-like domain, able to agglutinate bacterial cells *in vitro*
but devoid of proven bactericidal effect (Bodelot *et al.*, 2023). Whether MdAGGs act by physically slowing bacterial progression within apple tissues thanks to their agglutination properties or if this agglutination also facilitates the antimicrobial activity of other plant-derived compounds remains to be investigated.

Once demonstrated that MdAGGs are necessary to mount an effective ASM-triggered resistance response to fire blight, we wanted to determine if gain-of-function by inducible-expression of MdAGG10 improves resistance to *Ea*. Because of its high expression after ASM treatment and its closest proximity to the consensus sequence of the other 17 members of the family (Warneys *et al.*, 2018), we choose *MdAGG10* as the best candidate to select, to promote fire blight resistance. A previous attempt performed by constitutive overexpression of *MdAGG10* did not meet our expectations in so far overexpressing lines were as susceptible to *Ea* as wildtype ones (Bodelot *et al.*, 2023). We wondered whether plants engineered to conditionally express *MdAGG10* following *Ea* infection could display partial resistance toward fire blight. The *Ea*-inducible promoter *pPPO16* (Gaucher *et al.*, 2022) was therefore used to produce transgenic *pPPO16::MdAGG10* apple lines. Here, our results showed that *MdAGG10* exhibited strong expression in the early stages of *Ea* infection, thanks to its association with *pPPO16*, and this strong expression was associated with enhanced resistant to the disease. Moreover, combining inducible expression of *MdAGG10* and treatment with ASM led to a drastic reduction of the disease. The *pPPO16* promoter being much less responsive to ASM treatment than to *Ea* infection, it is rather unlikely that this additional disease reduction is due to the small additional induction of the promoter by ASM. The mechanisms responsible for the important disease reduction evidenced on ASM-treated *pPPO16::MdAGG10* lines remain to be determined. These plants could either have benefited from the addition of *Ea*-induced *MdAGG10* cisgene expression with that of the ASM-induced *MdAGGs* endogenes, but they could also have been protected thanks to the induction of hundreds of others defense-related genes known to be upregulated by ASM (Warneys *et al.* 2018). These results, when compared to those from the previous study involving constitutive overexpression of the same *MdAGG10* gene (Bodelot *et al.*, 2023), underscore the advantage of prioritizing inducible expression over constitutive expression when the aim is to enhance the activity of genes of interest. Inducible and transient expression of *MdAGG10* had here no negative impact on plant development and conferred partial resistance, whereas its constitutive CaMV p35S-driven overexpression led to pleiotropic effects on plant growth without enhancing resistance (Bodelot *et al.*, 2023). Such pleiotropic effects have already been documented in studies dedicated to constitutive
overexpression of lectin-encoding genes (Cabrales-Orona *et al.*, 2022). The past studies which
demonstrated that the ectopic expression of the amaranthin encoding gene from *Amaranthus
caudatus* protects recipient plants from aphids without pleiotropic effects were performed using
phloem-specific promoters (Wu *et al.*, 2006). It seems therefore that amaranthin-like encoding
genes can be engineered to promote plant resistance but their regulation in space and time has
to be considered cautiously to avoid advert effects on plant fitness. Using native pathogen- or
pest-inducible promoters like *pPPO16* to drive amaranthin-like lectin encoding genes, as we
did here, could be an interesting option to test in other systems.

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Until now, breeding apple resistance has focused on resistance QTLs (Emeriewen *et al.*, 2021).
However, the ongoing plant-pest arms race often results in the selection of mutations in the
pest's genome. These mutations alter the structure of its avirulence proteins, allowing it to
bypass the plant's R genes and evade recognition. Bypasses of resistance QTLs to *Ea* have
already been observed in apple (Emeriewen *et al.*, 2018). Here, we demonstrate that it is
possible to create a resistance in apple by focusing on a downstream defense gene, *MdAGG10*,
which interacts with *Ea*'s PAMPs LPS and EPS, possibly through electrostatic interactions
(Chavonet *et al.*, 2022). Indeed, the most important feature seems to be the acidic nature (*ie.*
negatively charged) of the polysaccharide (Chavonet *et al.*, 2022). Mutants of *Ea* engineered
to produce uncharged amylovoran (the principal component of *Ea*'s EPS) were shown to be
non-pathogenic on apple, phenocopying bacterial mutants that do not produce amylovoran at
all (Wang *et al.*, 2012). Altogether, these studies suggest that the downstream defense protein
*MdAGG10* targets bacterial features under strong evolutionary constraints, making it possibly
difficult for *Ea* to bypass the provided resistance. These considerations lead us to believe that
the intragenic *pPPO16-MdAGG10* association is a good candidate for breeding durable
resistance.

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Conclusion
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To conclude, in this work, we have validated MdAGGs as important compounds of apple's
defense toolbox against *Ea* and created a partial resistance to fire blight thanks to the intragenic
association of the disease-inducible *pPPO16* promotor and the coding sequence of *MdAGG10*.
But *pPPO16::MdAGG10* plants analyzed here do not meet the standards of intragenic plants:
1) the terminator of this construct is of viral origin and 2) the corresponding T-DNA does not
have a recombination system such as in the PMF1-GFP-dest plasmid (Schaart *et al.*, 2010),
529 which allows the elimination of other elements (selection gene, recombination system itself)
530 than the construct of interest after transformation. Functional intragenic apple lines could be
531 obtained using the Rubisco terminator, which has already been employed in apple (Joshi et al.,
532 2011). Combined with the overmentioned recombination system, such intragenic lines
533 allowing MdAGG10 expression upon Ea infection, could be upgraded toward higher resistance
534 by pyramiding more genes involved in partial resistance. Further combined with other control
535 methods, such as treatments with environment-friendly non-chemical plant resistance inducers,
536 unlike ASM, will contribute to generate robust and high-level resistance enabling better control
537 of fire blight.
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539

## Material and Methods

540

541

1. Bacterial strains, culture and suspensions preparation

542 *A. tumefaciens* strain EHA105 containing plasmids of interest was grown in Luria Broth (LB)
543 media (Duchefa Biochemie, DH Haarlem, Netherland) at 28°C. Suspensions of *A. tumefaciens*
544 were prepared in a medium consisting of 1.95 g/L MES medium (Duchefa Biochemie), 2 g/L
545 MgCL2, 20 g/L sucrose and 30 mg/L acetosyringone, with pH adjusted to 5.6. *Ea* strain
546 CFBP1430 was grown on King's B media (King *et al.*, 1954) at 26°C. Suspensions of *Ea* were
547 prepared in sterile water from solid exponential growth phase culture.
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2. Construction of vectors and generation of transgenic lines

551 Binary vector CRISPR-MdAGGAll used in this study (T-DNA in Fig. S1a) derived from pDE-
552 CAS9Kr vector (Charrier *et al.*, 2019). To maximize the probability to target the 18 MdAGG
553 genes, this construct contained two guide-RNAs with a different promoter (gRNA1 with MdU3
554 and gRNA2 with MdU6), that were designed so that either one or both are complementary to
555 each MdAGG gene, while minimizing off-target sequences (Table 1) MdDiAGG4 was the only
556 coding sequence potentially off-targeted by gRNA1 and ABS1 was not considered because
557 non-coding and with no evidence of transcription factor binding sites within it (checked on
558 PlantPAN https://plantpan.itps.ncku.edu.tw/plantpan4/TF_TFBS_search.php with
559 Arabidopsis and *Malus domestica* genomes, Chow *et al.*, 2024). We speculated that this
560 strategy would lead to (i) punctual mutations at either gRNA site, (ii) deletions of several
561 hundreds of nucleotides between both gRNA sites within MdAGG genes or (iii) deletions of
562 several thousands of nucleotides between gRNA sites of different MdAGG genes organized in
tandem array. Each gRNA cassette flanked with attB gateway sites was synthesized

563
independently by Integrated DNA Technology, Inc. (San Jose, CA, USA) and subsequently
cloned in pDONR207 vector via BP cloning (Gateway system; Thermo Fisher Scientific,
Waltham, MA, USA). The vector pDONR207-U3gRNA1-U6gRNA2 was constructed by
placing the 'U6gRNA2' cassette after the 'U3gRNA1' cassette using restriction/ligation at
XhoI/PstI sites in the donor vector followed by SalI/PstI cloning in the destination vector. It
was then used to create vector CRISPR-MdAGGAll using Gateway LR cloning with the vector
pDE-CAS9Kr. Primers used to verify each cloning step (primers A: BP cloning and gRNAs
addition, primers B: LR cloning) are indicated in Table S1. BLAST analysis with AtU3 and
AtU6 sequences (respectively, found upstream X52629 and X52528; Marshallsay et al., 1990)
confirmed that MdU3 and MdU6 promoters are respectively placed upstream MD10G1073100
and MD07G1138500 genes (https://iris.angers.inra.fr/gddh13/). Sequences are given in
Charrier et al. (2019). The CRISPOR software (http://crispor.tefor.net/; Haeussler et al., 2016)
was used to choose the target sequences of gRNAs based on the Version 1.1 of the *Malus domestica* INRAE GGDH13 genome (https://iris.angers.inra.fr/gddh13/; Daccord et al., 2017).
Binary vector pPPO16::MdAGG10 used in this study (T-DNA in Fig. S1b) derived from
pKGWFS7 (Karimi et al., 2002) and was constructed as followed. The promotor *pPPO16* from
'MM106' (MK873007, about two kilobases - kb) was amplified with Q5 High Fidelity DNA
Polymerase (New England Biolabs, Ipswich, MA, USA) according to the manufacturer
instructions and with primers C adding an *EcoRI* restriction site in 3'. Amplified fragment was
then cloned into p-ENTR/D TOPO (Invitrogen, Carlsbad, CA, USA) and then transferred into
the destination vector pKGWFS7 by Gateway recombination to create pKGWFS7-pPPO16.
*MdAGG10* CDS (MD10G1027210, 498 base pairs) was also amplified with the Q5 high
fidelity DNA polymerase according to the manufacturer instructions and with primers F adding
*EcoRI* and *NcoI* restriction sites in 5' and 3' respectively, then cloned into pGEM-T easy
(Promega, Madison, WI, USA) according to the manufacturer instructions. The *EcoRI*-*MdAGG10-NcoI* fragment was then inserted in pKGWFS7-pPPO16 by restriction/ligation to
create pPPO16::MdAGG10. Primers used to clone or to verify each cloning step (primers C to
H) are indicated in Table S1.

Apple transgenic lines were generated as previously described in Malabarba *et al.* (2020).
Briefly, transformation was performed on the youngest leaves of 4-week-old *in vitro* apple
shoots. The leaves were vacuum-infiltrated at -0.9 bar for 1 min in a suspension of
*Agrobacterium tumefaciens* (EHA105) at 1 × 10⁸ bacteria/mL and Silwet L-77R (Lehle Seeds,
Round Rock, TX, USA) at 0.002%. Leaves were then wounded transversely with a scalpel and
transferred to apple regeneration medium containing Murashige and Skoog salts (MS)
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(Murashige and Skoog, 1962), 30g/L saccharose, 5 mg/L thidiazuron, 0.5 mg/L naphthalene acetic acid and 100 mM acetosyringone, solidified with Phytagel TM (Sigma-Aldrich, Saint-Louis, MO, USA) at 3 g/L. Leaves were co-incubated for two days in the dark at 22-24 °C with *A. tumefaciens*. Leaves were then placed on their respective regeneration medium containing 150 mg/L timentin, 100 mg/L kanamycin and 300 mg/L cefotaxime. Explants were kept in dark conditions for a total of 6 months, transferred monthly to fresh medium and monitored for emergence of adventitious buds.

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### 3. Plant growth conditions and ASM treatment

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*In vitro* shoots of non-transgenic 'Gala' were micropropagated as in Malabarba *et al.* (2020), on MS salts (Murashige and Skoog, 1962) supplemented with 30g/L saccharose, 0.1 mg/L 3-indolebutyric acid (IBA) and 0.5 mg/L 6-benzyladenine and solidified with Agar (Condalab, Madrid, Spain) at 7.5 g/L. Regenerated buds from transformation, and subsequent *in vitro* shoots of transgenic lines were micropropagated in the same medium, with the addition of 150 mg/L timentin, 100 mg/L kanamycin and 300 mg/L cefotaxime. Cultures were transferred monthly to fresh medium. All the plants were cultivated at 22-24 °C in a growth chamber with a 16:8 h light:dark photoperiod (cool white fluorescent tubes, 40-60 mmol.m⁻².s⁻¹).

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The rooting conditions were as followed: *in vitro* shoots of non-transgenic 'Gala', and of transgenic lines, were transferred for one week, in the dark and at 22-24 °C, on an induction medium composed of MS salts (Murashige and Skoog, 1962) supplemented with 20g/L saccharose and 3 mg/L IBA and solidified with Agar (Condalab, Madrid, Spain) at 7.5 g/L. *In vitro* shoots were then transferred for 3 weeks, at 22-24 °C with a 16:8 h light:dark photoperiod, on an expression medium composed of MS salts (Murashige and Skoog, 1962) supplemented with 20g/L saccharose and solidified with Agar (Condalab) at 7.5 g/L. The rooted shoots were then acclimatized into greenhouse and grown under 22°C, humidity rate of 80% and shading of 500 W.m⁻² during 8 weeks after acclimatation. These 8-week-old acclimatized plants are referred to as 'greenhouse grown plants' throughout the manuscript, whether they are non-transgenic 'Gala' control plants or transgenic lines.

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*In vitro* shoots and greenhouse grown plants (non-transgenic control and transgenic lines) are treated with a 0.2 g.L⁻¹ ASM (Bion® 50WG, Syngenta, Basel, Switzerland) solution 3 days before inoculation, with either an Ecospray sprayer (Seidden, Madrid, Spain) for *in vitro* shoots or a 750 mL hand sprayer for greenhouse grown plants. The ASM solution was filter-sterilized at 0.22 µm (Pall Corporation, Port Washington, NY, USA) before spraying the *in vitro* shoots.

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631 4. Plant inoculation, phenotyping and sampling

632 For phenotyping analyses (Fig. 2, 3d, 5 and 6b), wild-type or transgenic *in vitro* shoots and
633 greenhouse grown plants were inoculated with a 10⁷ colony forming unit.mL⁻¹ *Ea* suspension
634 prepared in sterile water. Plants were inoculated with scissors previously dipped in *Ea*
635 suspension, by cutting the lower third of one of the apical leaf for *in vitro* shoots, or of the last
636 fully developed leaf for greenhouse grown plants. In the experiments performed on wild-type
637 or *mdagg-1* and -2 lines grown in greenhouse (Fig. 3b and c), the remaining leaf tissues cut
638 out for inoculation were then pooled by pair and frozen at -80°C for subsequent analysis of
639 *MdAGGs* expression (7<n<9) or MdAGGs protein detection (n = 1)). Fire blight incidence was
640 measured by checking the presence of necrotized cells beyond the inoculated leaf petiole. Fire
641 blight severity corresponded to the Area Under Disease Progression Curve (AUDPC - Shaner
642 and Finney, 1977) calculated with fire blight symptoms measured differently on *in vitro* shoots
643 or greenhouse grown plants. On greenhouse grown plants, they were measured 4, 7, 11, 14 and
644 19 day post inoculation (dpi) by measuring the length of necrotized shoot whereas on *in vitro*
645 shoots, a score has been assigned at 4, 5, 6, 7, 8 dpi for *MdAGGs* edited lines test (Fig. 2) and
646 at 4, 5, 6, 7, 8 10 and 12 dpi for *pPPO16::MdAGG10* lines test (Fig. 5), according to the
647 proportion of necrotized tissues observed on each plant. These scores are summarized in Table
648 S5. For expression analyses on *in vitro* shoots of non-transgenic 'Gala' control and
649 *pPPO16::MdAGG10* transgenic lines 1 to 6 (Fig. 4), inoculation was done similarly as in
650 Gaucher *et al.* (2022). Bacterial inocula was prepared in sterile water at a concentration of 10⁷
651 colony forming unit.mL⁻¹, supplemented with 0.01 % (v/v) of wetting agent Silwet (L-77, De
652 Sangosse Ltd, Cambridge, UK). The mock solution corresponded to sterile water supplemented
653 with the wetting agent Silwet. Shoots were totally submerged in mock or inoculum and vacuum
654 infiltrated at -0.09 MPa for 2 min. Infiltrated shoots were dried on sterile filter paper and placed
655 on micropropagation medium for 3 days before sampling. Twenty-four and 40 hpi, mock- or
656 *Ea*-infiltrated shoot leaves were sampled, immediately frozen in liquid nitrogen and kept at -
657 80°C until analysis. Each biological replicate is a pool of all leaves sampled from two
658 independent shoots (6<n<8).

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660 5. Nucleic acids extraction and reverse transcription

661 Genomic DNA (gDNA) was extracted as described in Fulton *et al.* (1995). For RNA extraction,
662 frozen leaf samples were ground into powder using a tissue lyser (Retsch, Hann, Germany)
663 during 20 seconds at 25 Hz twice. Total RNA was extracted with a NucleoSpin RNA Plant Kit
664 (Macherey-Nagel, Düren, Germany) following manufacturer's recommendation and RNA
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concentration was measured with a Nanodrop spectrophotometer (Thermo Fisher Scientific).

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Two µg of RNA were reverse-transcribed into cDNA using M-MLV Reverse Transcriptase (Promega) following manufacturer's instructions. Primers I (Table S1), designed from either side of an intron, were used to confirm absence of genomic DNA by polymerase chain reaction (PCR).

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## 6. Genotyping of transgenic lines by PCR and targeted DNA sequencing

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T-DNA insertion verifications with primers B, H, I, J, K, L (Table S1), were performed by PCR amplifications with GoTaq G2 Flexi DNA polymerase (Promega) according to manufacturer's instructions and by using following PCR reaction conditions: 95 °C for 5 minutes ; 35 cycles at 95 °C for 30 seconds, 58 °C for 45 seconds and 72 °C for 60 seconds, followed by a final extension step at 72 °C for 5 minutes. PCR products were separated on a 2% agarose gel.

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For the qualitative characterization of MdAGGs edits, we used primers M (Table S1) flanking the sequence containing the target zones of the two gRNAs. Amplifications were performed with Q5® High Fidelity 2X MasterMix according to manufacturer's recommendations. The PCR reaction was performed with an initial step at 98°C during 30 seconds followed by 40 cycles at 98°C during 10 seconds, the appropriate melting temperature during 30 seconds and 72°C for 60 seconds with a final extension at 72°C during 120 seconds. Amplicons were cloned with a CloneJET PCR kit (Thermo Fisher Scientific) according to manufacturer's recommendations, and bacterial transformation was performed with Top10 competent *E.coli* cells subsequently spread on LB medium complemented with 100 mg.mL⁻¹ ampicilin for 24 hours at 37°C. Bacterial clones, each containing one allele of the potentially edited *MdAGG* target sequence were selected by on-colony PCR using N primers (Table S1) present on the plasmid and framing the insertion zone, with GoTaq G2 Flexi DNA polymerase according to manufacturer's recommendations. Selection was on the basis of the expected size of the amplicons inserted in the event of slight modifications of the gRNAs targeted sequences (indels or substitutions) or of elimination of the portion of sequence between the two gRNAs targeted sequences. For each transgenic line, 4 bacterial clones containing putative edited MdAGGs sequences were sent for sequencing with the reverse N primer (Table S1) to Azenta Life Science company (Burlington, MA, USA). The resulting sequences were aligned with the CDS of *MdAGG1* to *MdAGG18* from the two phases of 'Gala' genome (Sun *et al.*, 2020) using the MultAlin software (Corpet, 1988).

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699 7. qPCR analysis

700 *MdAGG* expressions and quantifications of *MdAGGs* and *MdDiAGG4* edition rates in *mdagg-1* and *mdagg-2* lines were measured by qPCR. Briefly, cDNAs (expressions) or genomic DNA (edition rates) were mixed with MESA Blue 2X PCR MasterMix for SYBR Green Assays with fluorescein (Eurogentec, Liege, Belgium) and primers (O for expressions, P and Q for edition rates; Table S1) at the appropriate concentrations. Reactions were performed on a CFX Connect Real-Time System (Bio-Rad Laboratories, Hercules, CA, USA) for the qPCR and melt curves were done to check the absence of non-specific amplifications and primer-dimers products. Relative expressions and edition rates quantifications were calculated using the 2-ΔΔCt method. The normalization factor was calculated using 3 housekeeping genes (*Actin*, *GAPDH* and *TuA*; primers R, S, T respectively in Table S1) as recommended by Vandesompele *et al.* (2002).

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712 8. Protein extraction, separation and immunodetection

713 Frozen leaves were ground into powder in liquid nitrogen with mortar and pestle. Phenol
714 Protein phenol extraction and subsequent Bradford quantification were performed as described
715 in Chavonet et al. (2022). Extracted proteins were separated on Mini PROTEAN TGX Precast
716 Gel (Bio-Rad Laboratories Inc, Hercules, CA, USA) for 35 minutes at 200 V (Mini-
717 PROTEAN® Tetra Cell, 4-Gel System, Bio-Rad laboratories Inc, Hercules, CA, USA) and
718 electroblotted onto 0.45 µm polyvinylidene difluoride (PVDF) membranes (immobilon-P,
719 Milli- pore Corp., Bedford, MA, United States). The membrane was subsequently blocked with
720 EveryBlot Blocking Buffer (Bio-Rad Industries Inc, Hercules, CA, USA) and incubated
721 overnight at 4 °C in EveryBlot Blocking Buffer containing 1:1000 of two anti-MdAGGs
722 antibodies described in Warneys *et al.*, 2018. These antibodies are able to recognize all
723 possible MdAGG proteins since they were obtained through rabbit immunization programs
724 (Eurogentec, Liege, Belgium) with synthetic peptides (NKYLRYQLDAESDLN and
725 TEKPNNEDYADKNYV) corresponding to conserved amino acids in MdAGGs. The
726 membrane was then washed 5 times for 5 minutes in a TBSt solution (Tris 20 mM; NaCl 150
727 mM; Tween20 0.005%; v/v; pH 7.6), incubated for 1 hour in EveryBlot Blocking Buffer,
728 containing 1:5000 of HRP conjugated goat anti-rabbit antibodies (Merck KGaA, Darmstadt,
729 Germany) and revealed with the ClarityTM Western ECL Substrates Kit (Bio-Rad Laboratories
730 Inc, Hercules, CA, USA) according to manufacturer's protocol. Membranes were revealed
731 using a ChemiDocTM MP Imaging System (Bio-Rad Laboratories Inc, Hercules, CA, USA).
Before blotting, the gels were activated under UV light in the same imaging system, which allowed to reveal total proteins on blotted membranes by fluorescence emission.

## 9. Data analysis

Data analysis was performed using Rstudio software (R Core Team, 2023) and the graphical representations were generated using the packages "ggplot2" (Wickam, 2016) in association with "ggpubr" (Kassambara, 2023a). To perform statistical analysis using standard deviations on the single values of fire blight incidence (one value per genotype x condition), we carried out bootstrap analysis with the package "boot" (Canty and Ripley, 2024). Tukey's parametric test (one-step multiple pairwise comparison) was used to assess whether or not the mean sizes of plants *pPPO16::MdAGG10-1* and *6* were different from those obtained for the non-transgenic control 'Gala' (Table S3), using the package "rstatix" (Kassambara, 2023b). A non-parametric test (Kruskal-Wallis) followed by a post-hoc Dunn test were used for all other experiments, using packages "FSA" (Ogle *et al.*, 2023) in association with "rcompanion" (Mangiafico, 2024).

## Acknowledgment

The authors are grateful for the technical support provided by the ANAN shared platform of the SFR QUASAV and the PHENOTIC platform (greenhouse facilities). The authors thank their funders: these researches were funded by the Agg-FREDI project (INRAE BAP Department 2020-2021) and the French Priority Research Programme "Cultiver et Protéger Autrement" (PPR-CPA) managed by the Agence Nationale de la Recherche (project ANR-20-PCPA-0003 CapZeroPhyto). A PhD grant was also awarded to AB by INRAE-BAP Department and the Pays-de-la-Loire Region.

## Author contributions

The design of the research was performed by EV and AD. Performance of the research: apple transgenic lines were obtained by AB, ND, ER and EV. Plant inoculation and phenotyping were performed by AB, ND, ER, CH, EV and AD. Genotyping, qPCR analysis, immunodetection were completed by AB, AD and EV. Data analyses were completed by AB, AD and EV. Interpretation and writing of the manuscript: the manuscript was drafted by AB,
765

completed by EV, AD and MNB and reviewed by all the authors. EV, AD and MNB supervised
the research project.

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**Data availability**

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All data supporting the findings of this study are available in the article and in its supplemental
information files.

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**Competing interests**

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The authors declare no competing interests.

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**Supplementary Material**

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Supplementary Material may be found online.

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**Fig. S1** T-DNA constructs and positions of gRNAs targets, quantitative PCR primers, and
western-blot probes, on *MdAGG* sequences.

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**Fig. S2** Genotyping of apple transgenic lines.

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**Fig. S3** MdAGGs and MdPPOs expression modulation by *Ea T3SS* in the susceptible MM106 genotype compared with modulation by ASM in apple seedlings (Adapted from Chavonet et al., 2022).

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**Table S1** Primer used in this work.

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**Table S2** Fire blight lesion length on *mdagg* lines 21 days after *Erwinia amylovora* infection.

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**Table S3** Size of *pPO16::MdAGG10* lines and non-transgenic ‘Gala’ control eight weeks after greenhouse acclimatation.

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**Table S4** Fire blight lesion length on *pPPO16::MdAGG10* lines 19 days after *Erwinia amylovora* infection.

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799 **Table S5** Disease score assigned for AUDPC calculation on *in vitro* culture shoots inoculated
800 by *Erwinia amylovora*.

801

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