# Identification and expression of the peach TNL RMia genes for the resistance to the root-knot nematode Meloidogyne incognita

Henri Duval, Laure Heurtevin, Naïma Dlalah, Claire Caravel, Caroline Callot, Cyril van Ghelder

► To cite this version:

Henri Duval, Laure Heurtevin, Naïma Dlalah, Claire Caravel, Caroline Callot, et al. Identification and expression of the peach TNL RMia genes for the resistance to the root-knot nematode Meloidogyne incognita. Scientia Horticulturae, 2025, 343, pp.114081. 10.1016/j.scienta.2025.114081. hal-05036400

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Research Paper

# Identification and expression of the peach TNL *RMia* genes for the resistance to the root-knot nematode *Meloidogyne incognita*

Henri Duval<sup>a, *</sup>, Laure Heurtevin<sup>a</sup>, Naïma Dlahal<sup>a</sup>, Claire Caravel<sup>b</sup>, Caroline Callot<sup>c</sup>, Cyril Van Ghelder<sup>b, *</sup>

<figure><img src="image_2.png" /></figure>

## ARTICLE INFO

**Keywords:**

Peach

Root-Knot Nematodes (RKN)

TIR-NB-LRR (TNL)

NLR

Resistance gene

Long read RNAseq

RMia

## ABSTRACT

Root-Knot Nematodes (RKN) are major pests for world agriculture. As RKN are polyphagous endoparasites, present in various climates and able to multiply rapidly, the quest to discover new natural resistances in plants is essential. In *Prunus* species, several major resistance genes to RKN that belong to the NLR family, have been detected in recent years. In Peach, the *RMia* gene, which confers resistance to the RKN *Meloidogyne incognita*, *M. arenaria*, and *M. ethiopica*, was localized on the chromosome 2. In this study, we used short- and long-read sequencing technologies to identify the *RMia* gene in two independent RKN-resistant peach varieties. We characterized three candidate genes and studied their expression profile upon RKN infection. Our study identified and characterized these candidates that are most likely involved in the RKN resistance. Ultimately, we developed new molecular markers associated to this resistance that can be used in breeding programs.

## 1. Introduction

Among the plant parasitic nematodes, Root-Knot Nematodes (RKN), *Meloidogyne spp.*, are often described as the most important genus in terms of scientific studies and caused damages (Jones et al., 2013). The model species *M. incognita* together with *M. javanica*, *M. arenaria*, *M. hapla* and the emerging species, *M. floridensis* and *M. enterolobii* represent a threat for agriculture due to their host range, distribution, and life traits. Indeed, these telluric pathogens have a worldwide distribution, are able to infect almost all the cultivated plant species, and can complete their life cycle in a few weeks producing a thousand juveniles per female stage in the model species *M. incognita* for instance (Abad et al. 2008). With the progressive removal of the most harmful nematicides, other alternatives to control these pests are required. The combination of innovative cultural methods, the use of biocontrol agents and the reasoned utilization of resistant plant cultivars is the most promising approach to control RKN and minimizing harmful effects on the environment (Djian-Caporalino et al. 2019). Therefore, the identification and characterization of major Resistance genes (R genes) against RKN are crucial in this context. As the risk of R gene circumvention increases in perennial plants, due to longer contact between host and pathogens,

the opportunity to combine several R genes in one cultivar (i.e. gene pyramiding) reinforces resistance's durability. This approach is particularly relevant for the *Prunus* genus that gathers many species of agro-nomical interest, including peach, plum and almond, which are susceptible to RKN. As hybrids and grafts can be carried out between *Prunus* species, investigating the *Prunus* R genes repertoire is particularly promising.

NLR genes or Nucleotide Binding and Leucine Rich-Repeat receptor genes (NB-LRR) represent a major resistant gene family in plants. They are composed of 3 different classes mainly characterized by a conserved structure made of the Nucleotide Binding and the Leucine Rich-Repeat domains, and different N-terminal domains. The CNL (including a N-terminal Coiled-Coil domain), the RNL (RPW8 domain) and the TNL (TIR or Toll-like Interleukin Receptor-1 domain) form this gene super-family. Although NLR numbers are numerous in plant genomes (i.e. often >100 per genomes), the ratio between the three classes greatly varies in plant genomes reflecting various defense strategies (Shao et al. 2014). NLR are intracellular receptors that can detect the presence of pathogens through the direct or indirect recognition of molecules (grouped under the name of effectors) secreted by invaders to feed on plants and escape plant immunity. This detection is often carried out by

<footnote><sup>a</sup> INRAE, UR1052, Unité de Génétique et Amélioration des Fruits et Légumes (GAFL), F-84143 Montfavet, France</footnote>

<footnote><sup>b</sup> INRAE, Université Côte d'Azur, CNRS, Institut Sophia Agrobiotech (ISA), Sophia Antipolis F-06903, Sophia Antipolis, France</footnote>

<footnote><sup>c</sup> CNRGV, Université de Toulouse, INRAE, CNRS, F-31326 Castanet Tolosan, France</footnote>

<footnote>* Corresponding authors.</footnote>

<footnote>E-mail addresses: henri.duval@inrae.fr (H. Duval), cyril.van-ghelder@inrae.fr (C. Van Ghelder).</footnote>
C-terminal domains of NLR sensors which will activate, through polymerization and N-terminal domains, other NLR helpers to trigger a downstream signaling pathway leading to cell death. Thus, NLR act in complex networks to confer plant immunity (Castel et al. 2019). Therefore, NLR are pivotal factors of the so-called Effector-Triggered Immunity (ETI) in plants. Recently, different TNL models have been structurally studied, revealing that TNL C-terminal domains (i.e. Leucine Rich Repeat and Post-LRR C-JID domains) directly bind the corresponding effector. Then, a superstructure (resistosome), made of a tetramerization of the TNL sensor, will lead to cell death through a mechanism that still needs to be more documented (Ma et al. 2020; Martin et al. 2020)

In the past two decades, several RKN resistance genes have been detected and identified in *Prunus* species (Saucet et al. 2016). In plum, the dominant TNL *Ma* gene, cloned in 2008, is a sole R gene that confers a full resistance to all *Meloidogyne* species tested including the emerging species *M. enterolobii* and *M. floridensis* (Duval et al. 2019). To date, this gene, displaying a unique structure among TNL, is not circumvented by high and durable inoculum pressure or by high temperature (Claverie et al. 2011). *Meloidogyne* juveniles will be able to penetrate the roots of the Ma-carrier accession, *P. cerasifera* P2175, however they will not induce any feeding site, preventing any nematode development. Following nematode recognition, the *Ma* gene will induce an Hypersensitive-like Reaction around RKN juveniles (Khallouk et al. 2011). Two genes, *Rjap* and *RMja*, which are most likely the orthologs of the *Ma* gene in *P. salicina* and in *P. dulcis*, respectively, display different spectra of resistance. While the *Rjap* gene has the same broad-spectrum of resistance as *Ma*, *RMja* does not confer a resistance to *M. incognita* and *M. floridensis*, making it an interesting model to study (Van Ghelder et al. 2018; Duval et al. 2019). Besides, two other TNL genes (*PsoRPM2* and *PsoRPM3*) isolated from the wild myrobalan plum (*Prunus sogdiana*) conferred a resistance to *M. incognita* when transferred to tobacco (Zhu et al. 2017; Xiao et al. 2021). In peach, the *RMia* gene represents a major source of resistance to RKN as it confers a resistance to *M. incognita*, *M. arenaria* and *M. ethiopica* (Duval et al. 2019). In a previous study, the gene has been localized in a region of approximately 92Kb flanked by 2 SNP markers (*SNP_APP91* and *SNP_APP92*, named hereafter SP91 and SP92 using a high resolution mapping approach based on 790 hybrids involving the *RMia* carrier, *P. persica* var. 'Nemared' (Duval et al. 2014).

Our objectives are to develop a method to pyramid the three sources of resistance *Ma*, *RMja* and *RMia* in a single *Prunus* genotype in order to obtain *Prunus* rootstock that are resistant to all Meloidogyne species. Since molecular markers for screening the *Ma* and *RMja* genes are now available, identification of the *RMia* gene and development of molecular markers are therefore crucial steps for *RMia* pyramiding. To this aim, in this study we obtained the genomic sequence lying between the two SNP markers SP91 and SP92 of two peach varieties carrying the *RMia* gene, 'WFP' and 'Nemared'. We performed an *in silico* structural and functional annotation of the targeted region, based on long-read transcriptomic data, and measured the differential expression of three detected candidate TNL genes, with and without RKN infection. We developed reliable molecular markers that will be of interest for marker-assisted selection of *RMia* genes and tested the reliability of these markers on a range of peach genotypes. By revealing this resistance cluster, our study paves the way for future functional studies in a species of great agronomic interest.

# 2. Materials and methods

## 2.1. Plant materials

All accessions listed in Table 1 with known; (Esmenjaud et al., 1997; Fernandez et al., 1994; Kester and Asay, 1986) or unknown resistance phenotypes were phenotyped for resistance to *M. incognita* and genotyped using new molecular markers. The Weeping Flower Peach (WFP) clone was used for BAC library construction and long-read RNAseq

<table><caption>**Table 1**
List of the Prunus accessions that have been phenotyped either in this study or in previous studies.</caption><thead><tr><th>Accessions</th><th>Prunus specie</th><th>Phenotype<br/>*<br/>M. incognita</th><th>Resistance<br/>origin</th><th>Phenotyping<br/>Reference</th></tr></thead><tbody><tr><td>WFP weeping</td><td>P. persica</td><td>R</td><td>unknown</td><td>this study</td></tr><tr><td>Nemared</td><td>P. persica</td><td>R</td><td>Nemaguard</td><td>Ramming,<br/>1983</td></tr><tr><td>Montclar®</td><td>P. persica</td><td>S</td><td></td><td>Fernandez,<br/>1994</td></tr><tr><td>Rubira</td><td>P. persica</td><td>S</td><td></td><td>this study</td></tr><tr><td>Pamirskii</td><td>P. persica</td><td>S</td><td></td><td>this study</td></tr><tr><td>Hybrid PRMN<br/>Z64P40</td><td>P. persica</td><td>R</td><td>Nemared</td><td>this study</td></tr><tr><td>GF305</td><td>P. persica</td><td>S</td><td></td><td>Esmenjaud,<br/>1997</td></tr><tr><td>Summergrand</td><td>P. persica</td><td>R</td><td>unknown</td><td>Esmenjaud,<br/>1997</td></tr><tr><td>Missour 61-3</td><td>P. persica</td><td>R</td><td>unknown</td><td>this study</td></tr><tr><td>Nemaguard</td><td>P. persica</td><td>R</td><td>unknown</td><td>Esmenjaud,<br/>1997</td></tr><tr><td>(Nemaguard<br/>xP1908)5</td><td>P. persica x P.<br/>daviana</td><td>R</td><td>Nemaguard</td><td>this study</td></tr><tr><td>Hansen 536</td><td>P. persica x P.<br/>dulcis</td><td>R</td><td>Okinawa<br/>peach</td><td>Kester, 1986</td></tr><tr><td>GF557</td><td>P. dulcis x P.<br/>persica</td><td>R</td><td>Shalil peach</td><td>Esmenjaud,<br/>1997</td></tr><tr><td>Garnem (GN15)</td><td>P. dulcis x P.<br/>persica</td><td>R</td><td>Nemared</td><td>Esmenjaud,<br/>1997</td></tr></tbody></table>

analysis. This 'WFP' clone and the 'PRMN Z64P40' hybrid were used for qPCR gene expression analysis. Short-read resequencing was carried out for the five peach accessions: 'WFP', 'Nemared', 'Montclar', 'Rubira' and 'Pamirskii'. These accessions are maintained in the INRAE collection in 32-litre pots under insect-proof tunnels. Clone S2678 (WFP) was introduced into the INRAE collection from Clemson University. This clone has double red flowers, a weeping habit and is resistant to the aphids Myzus persicae and Myzus variant (Duval et al. 2022). 'Nemared' was registered by the USDA Fresno, California, and originates from F3 seedlings of 'Nemaguard' x 'a red-leaf seedling' (Ramming and Tanner, 1983). The 'PRMN Z64P40' hybrid was obtained by self-fertilization of a nematode-resistant hybrid from a cross between three susceptible peach varieties and 'Nemared': [(Pamirskii x Rubira) x (Montclar x Nemared)]. The plants used for the RKN infestation tests were produced by cuttings, with the exception of the 'PRMN Z64P40' hybrid which was tested from self-pollinated seedlings.

## 2.2. Root-knot nematodes inoculation

For phenotypic resistance testing, 5 two-month-old plants per accession were inoculated at the end of March and grown for 75 days. For gene expression analysis with RNA extraction, we also inoculated 5 cuttings of the 'WFP' accession and 5 two-month-old plants of the 'PRMN Z64P40' hybrid. The RKN, *M. incognita*, was propagated using susceptible tomato plants, *Solanum lycopersicum* cv. 'St Pierre', in a greenhouse (temperature between 25 and 30 °C). The nematode identity was verified by esterase profiling (Esbenshade and Triantaphyllou 1985). After six weeks, tomato roots were harvested and washed. The root system, full of galls, was placed on a sieve inside a bucket, and the whole system was placed in a mist chamber for 2-3 days. Freshly hatched stage 2 (J2) juveniles were collected in tap water and counted. Inoculation was carried out in a greenhouse under controlled conditions. Around 10,000 *M. incognita* J2 were used to inoculate each plant grown in a 1-liter pot, and were distributed in four holes around the base of the plant to ensure homogeneous inoculation. *RMia* resistance to *M. incognita* is not quantitative, so assessment for resistance is a simple

<footnote>* R: Resistant, S: Susceptible.</footnote>
notation of presence/absence of galls in the root systems. In June, plants were assessed for the presence or absence of galls. Varieties were classified as resistant (R) if the entire root system was free of galls for all five replicates, and susceptible (S) if one or more galls were detected.

## 2.3. Gene expression (qRT-PCR)

10 days after inoculation, for both 'WFP' and 'PRMN Z64P40' accessions, root apexes from the 5 nematode-inoculated and 5 control plants were retrieved and flash frozen for storage. Total RNA was extracted from frozen samples using the NucleoSpin®RNA Plant Kit (Macherey-Nagel, Dueren, Germany) according to the manufacturer's instructions. RNA yield and purity were assessed using a Nanodrop® by spectrophotometric absorbance and agarose gel electrophoresis. In total, 500 µg of cDNAs were synthesized using the Affinity Script Multiple Temperature cDNA Synthesis Kit (Agilent), following the instructions provided. Expression analysis was performed by quantitative real-time PCR (qRT-PCR) using a Stratagene® Mx3005P® qPCR System Instrument and Brilliant III SYBR Green qPCR Master Mix with Low ROX (Agilent Technologies). The reaction was prepared with each primer (0.5 µL, 10 µM), 2 µL of 1:10 diluted cDNA, RNase-free water (5 µL), and Brilliant III SYBR Green qPCR Master Mix with High ROX (7 µL) in a total volume of 15 µL. PCR conditions were 95 °C for 10 min, followed by 40 cycles of 95 °C for 30 s and 60 °C for 1 min. All qPCR reactions were normalized using the Ct value corresponding to the EiF4γ peach gene (PpEif4γ) and the 60 S L13 ribosomal protein gene (PpRPL13). Each of the 5 infected and control plants was measured with three technical replicates. Expression analyses were performed according to MIQE standards (Bustin et al. 2009). An adapted R script (Hilliou and Tran 2013) was used to calculate the normalized expression of the genes studied. An analysis of variance (ANOVA) was applied to transcript variations. The list of the designed primers for the qPCR is in Table S1.

## 2.4. Nucleic acid extraction, illumina sequencing and genotyping

Two resistant peach trees, 'Nemared' and 'WFP', and tree susceptible peach trees, 'Montclar', 'Pamirskii' and 'Rubira' were re-sequenced. The genomic DNA was extracted using 100 mg of frozen young leaves. Each sample was ground with a mixer mill in 330 µL of extraction buffer (sorbitol 0.35 M, Tris 0.1 M, EDTA 5 mM, 4 mg of sodium metabisulfite), 330 µL of lysis buffer (Tris 0.2 M, EDTA 50 mM, NaCl 2 M, CTAB 2%), and 130 µL of sarkosyl 5%. After a chloroform-isoamyl alcohol (24:1) procedure, precipitation with isopropanol and three ethanol washes, the DNA content was eluted in water and treated with RNAses. DNA libraries were prepared according to Illumina Nextera's instructions and quantified using an Agilent high-sensitivity chip (Duval et al. 2022). 125 paired-end sequencing was performed using an Illumina Hiseq 2500, and data pre-processing and sample quality checks were carried out. Library preparations and sequencing were carried out by the MGX-Montpellier platform. DNA for genotyping with molecular markers, was extracted from leaves according to a rapid genomic DNA extraction protocol for rosaceous species (Antanaviciute et al. 2015). PCRs were carried out using the GoTaq® G2 Flexi DNA Polymerase following the manufacturer's instructions.

## 2.5. Bacterial artificial chromosome (BAC)

A BAC library was constructed for the resistant peach 'WFP' using the approach described in (Duval et al. 2022) to obtain the region encompassing the *RMia* gene. The 'WFP' BAC library was screened for the SP91 and SP92 region using 3 labeled probes, which were designed using the Primer 3 software (Table S1). Selected clones were sequenced using the PacBio RS II sequencer. After a demultiplexing step, the sequence assembly was performed following the HGAP PacBio workflow (Chin et al. 2013)

## 2.6. PacBio technology-based full-length cDNA library construction, preparation and sequencing

Total RNA was extracted from two nematode-infested root samples using the NucleoSpin® RNA Plant kit (Macherey Nagel). The quality of total RNA was evaluated using an Agilent 2100 Bioanalyzer (Agilent Technologies, CA, USA). Only samples with RNA integrity number (RIN) ≥ 8 were used for deep sequencing. To build the library for PacBio sequencing, RNA qualified was reverse transcribed using the SMARbell® Express Template Prep Kit 2.0. PacBio Sequel® Systems was used to sequence transcriptomes. The Sub-reads shorter than 50 bp, the poor-quality reads and specific adaptor sequences were removed (CTCAGTCTGTGTGT for reverse primer of RNA roots). We used the Iso-Seq 3 bioinformatics workflow in SMRT Link Version 7.0.1.66975 to produce full-length transcript sequences and splice variants of good quality, with no assembly required. The Iso-Seq analysis workflow starts with the generation of high-fidelity reads using the circular consensus sequencing (CCS) method and the required trimmed poly(A) tail. The minimum accepted subread length was 50 bp, and we added the isoseq-mode option. We used the Iso-Seq 3 bioinformatics workflow in SMRT Link Version 7.0.1.66975 to produce full-length transcript sequences and splice variants of good quality, with no assembly required. The Iso-Seq analysis workflow starts with the generation of high-fidelity reads using the circular consensus sequencing (CCS) method and the required trimmed poly(A) tail. The minimum accepted subread length was 50 bp, and we added the isoseq-mode option.

## 2.7. Sequence assembly and analysis

The sequence mapping on the peach genome V2 was obtained using BWA (v 0.7.12-r1039) with the BWA-MEM algorithm for mapping. The read alignment and index files (BAM and BAI files), genomic feature files (GFF3) on the reference Peach V2, and *de novo* sequences were visualized with the Integrative Genomics Viewer (Robinson et al. 2011). We used the QIAGEN CLC Genomics Workbench (https://digitalinsights.qiagen.com) for the sequence trimmings, gene annotations, and motif research. The software used for the gene prediction was FGENESH (Solovyev et al. 2006), with *P. persica* as organism-specific gene-finding parameters. Protein analysis and domain research were carried out using Interproscan (Paysan-Lafosse et al. 2023). Synteny was visualized using Kablammo software (Wintersinger and Wasmuth 2015)

# 3. Results

## 3.1. Genomic region encompassing the *RMia* locus in the resistant varieties 'WFP' and 'Nemared'

A fine mapping study localized the *RMia* locus in the peach resistant variety 'Nemared', in the chromosome 2 between two KASP™ markers, SP92 and SP91 (Duval et al. 2014). In the first version of the peach reference genome (Lovell; v1.0) (Verde et al. 2013), four TNL genes were annotated in this interval, *ppa023253m*, *ppa018595m*, *ppa000501m* and *ppa023503m* (Fig. 1a). In the next version of the reference genome (v2.0) (Verde et al. 2017), the region delineated by the same markers, contains 2 complete TNL genes (*Prupe.2G055600* and *Prupe.2G055700*), 1 truncated TNL (*Prupe.2G055500*) (Fig. 1b) and 3 other genes coding for, a trafficking protein particle complex, a short transmembrane protein and an unknown protein. Amplification with specific primers of these three TNL by PCR using 'Nemared' DNA failed, suggesting that some polymorphism exists in the region. We therefore carried out NGS sequencing of both resistant accessions 'Nemared' and 'WFP' and the susceptible rootstocks 'Montclar' and 'Pamirskii'. While mapping NGS short reads of both resistant genotypes on the SP92-SP91 region of the peach reference sequence (v2.0), we observed that a large part of this region was not covered by reads, in line with the presence of a large indel in this region (Fig. S1ab). Conversely, the same experiment
<figure><img src="image_3.png" /><figcaption>**Fig. 1.** Schematic representation of the genomic interval ‘SP92-SP91’ from the chromosome 2. (a) TNL positions in the peach reference genome (*P. persica* cv. *Lovell*) V1. (b) TNL positions in the V2. (c) Position of predicted TNL genes in the resistant varieties ‘Nemared’ and ‘WFP’. (d) Expressed genes in the SP92-SP91 interval.</figcaption></figure>

using the reads of the susceptible rootstocks ‘Montclar’ and ‘Pamirskii’ led to a complete mapping onto the reference sequence meaning that no such large indels exist in both susceptible varieties. Consequently, we postulated that this targeted sequence was distinct between, on one hand, the susceptible rootstocks Lovell, ‘Montclar’ and ‘Pamirskii’, and on the other hand, the resistant accessions ‘WFP’ and ‘Nemared’. A BAC library was constructed to obtain the sequence of the SP92-SP91 region in the resistant genotype ‘WFP’. We designed specific probes inside this region to screen the BAC clones (Table S1). After aligning the BAC ends of five screened BACs on the Peach V2, we selected one BAC (43N01) and sequenced it using PacBio long-read sequencing technology. We confirmed the accuracy of the BAC sequence by mapping the ‘WFP’ NGS short-reads on this *de novo* BAC sequence. In addition, we also mapped the NGS short-reads of ‘Nemared’ on the BAC sequence and we observed that the SP92-SP91 regions are similar in ‘Nemared’ and ‘WFP’ (Fig. S1c). This sequence, named hereafter ‘R_SP92SP91’, encompassing the *RMia* locus is 145,198 kb long. Therefore, the R_SP92SP91 region is 71.7 kb larger than the V2_SP92-SP91 region in the peach reference genome (v2.0). The visualization of the syntenic region between both sequences indicated that this extended sequence is mainly the result of a large insertion in the central part of the region (Fig. S2).

## 3.2. Structural and functional annotation of the R_SP92SP91 region

The analysis of the R_SP92SP91 sequence revealed that three full-length TNL genes (hereafter named as RMiaGC1, RMiaGC2 and RMiaGC3) were predicted along with 35 other putative genes including a large majority of genes coding for small peptides with no functional annotation and several transposable elements (Table S2). RMiaGC2 and RMiaGC3 are arranged only 5 kb apart from each other in a head-to-head organization while RMiaGC1 is located 67 kb upstream (Fig. 1c). We did a long-read RNAseq analysis from RNA of nematode-infected roots to assess (i) the predicted structural annotation and (ii) the expression of the predicted genes in the region. Only the three TNL genes were expressed in the region R_SP92SP91 (Fig. 1d). The structure of the expressed genes, including the number and location of exons, introns, and UTR, was ascertained by mapping the full-length transcripts on the *de novo* BAC sequence. While RMiaGC1 exhibits only one transcript (Fig. S1c), RMiaGC2 and RMiaGC3 display 5 and 3 isoforms, respectively. As isoforms mainly differ in the 3' and 5' UTR length, it may affect

the expression regulation of these candidates but unlikely their protein sequences. Only one isoform of the RMiaGC2 displays a slight variation in the LRR domain. RMiaCG1, RMiaGC2 and RMiaGC3 code for 3 predicted proteins of 1178 aa, 1154 aa, and 1095 aa, respectively (Fig. 2). The percentage identity between these 3 genes ranged between 74.56% to 80.17% (Table S3). A BlastP search using the three candidate genes as queries against the proteome of Lovell, detects the highest identity for RMiaGC1 and RMiaGC2, (79% and 71%, respectively) with a TNL (Prupe.2G018300), located outside the SP92SP91 region, 4.88 Mb away on the chromosome 2 (Table S3). The percent identity between these 3 genes and the 3 TNL present in the SP92-SP91 region of the peach reference sequence (i.e. Prupe.2G055500, Prupe.2G055600 and Prupe.2G055700) ranged from 59.55% to 83.72% (Table S3). The functional annotation revealed the canonical organization of TNL proteins (i.e. the following chain of domains from the N-terminus to the C-terminus end: TIR, NB-ARC, NLL, LRR and PL-CJID) (Fig. 3b). As expected, the 3 first domains exhibit a low polymorphism with all the canonical/functional motifs described in the literature. The LRR domain is also relatively conserved between the 3 candidates, except a short deletion of 10 aa located in RMiaGC2 (Fig. S3). Conversely, PL-CJID domains show a higher polymorphism, including larger indels. Beyond these discrepancies, the 3 PL-CJID domains, harbor the classic motifs of the domain and atypical acidic regions made of repeat of aspartic and glutamic acids (D and E) (Figs. 3 and S3). While polymorphic regions between motifs 1 and 2 and in the C-terminal end involve regions rich in acidic amino acids, polymorphic regions between motifs 2 and 4 are enriched in polar amino acids.

## 3.3. Quantitative expression analysis of *RMia* candidate genes

We analyzed the relative normalized expression profiles of RMiaCG1, RMiaGC2 and RMiaGC3 from ‘WFP’ seedlings infested with nematodes and from seedlings free of nematodes, using specific primers for each candidate gene (Table S1). The RMiaGC1 gene was statistically strongly up-regulated 10 days after nematode infection (Fig. 4). RMiaGC2 and RMiaGC3 were also statistically up-regulated upon nematode infection but in a clear restrained manner. RMiaGC2 and RMiaGC3 harbor a similar intensity in response to RKN infection. We repeated the experiment using a resistant peach hybrid, ‘PRMN Z64P40’, obtained from *P. persica* (Pamirskii × Rubira) × (Montclar ×
<figure><img src="image_4.png" /><figcaption>**Fig. 2.** Structural annotation of the three *RMia* gene candidates. (a) Structure of the three genes RMiaCG1, RMiaGC2 and RMiaGC3. Size of exons and introns are in bp. Exons are represented in boxes, introns by black lanes. (b) Predicted domains of the three *RMia* candidate proteins in amino acids.</figcaption></figure>

<figure><img src="image_5.png" /><figcaption>**Fig. 3.** Detailed view of the PL-CJID domain of the three *RMia* gene candidates. The picture highlights the polymorphic regions between the three *RMia* candidates and the existing knowledge from other TNL studies for RPP1 (Ma et al. 2020), Roq1 (Martin et al., 2020) and WRR4A (Castel et al. 2019).</figcaption></figure>

Nemared). In this crossing, the resistance trait to RKN is exclusively carried by 'Nemared', which contains the *RMia* gene, whereas the three other parents are fully susceptible to RKN. Interestingly, the expression pattern of the 3 candidate genes follows the same trend, thus confirming that a similar regulation occurs within this cluster of TNL in response to RKN.

### 3.4. Marker development for marker assisted selection

The acquisition of the full sequence of the resistant locus was an essential prerequisite to develop reliable molecular markers. We designed specific intragenic molecular markers in the genes RMiaGC1 and RMiaGC2 to cover the region (Table S1). The position of the markers used is represented in the Fig. 5a. For the gene RMiaGC1, the two markers are G13F1R1 designed for the qPCR expression and another one in the 5'UTR region, G13QP211. For RMiaGC2, we designed a reliable marker, G13F5R5. The Fig. 5b shows the results of the PCR

amplifications for two resistant varieties (WFP and 'Nemared') with one band for the three markers and no amplification for three susceptible varieties ('Montclar', 'Rubira' and 'Pamirskii'). A single signal was visible in the resistant varieties for the markers G13F1R1, G13QP211 and G13F5R5 at 437 bp, 150 bp and 205 bp, respectively. We tested these markers in other peach rootstocks for which RKN-related phenotypic status had been established. In all accessions, the genotype corresponded to the phenotype when using the molecular markers SP92, G13F1R1, G13QP211 and G31F5R5. Conversely, the KASP™ marker SP91 gave some discrepancies between genotypes and phenotypes for 'Nemaguard', the hybrid (Nemaguard x P1908), the peach-almond rootstocks 'Hansen 536', 'GF557' and the peach'Missour 61-3', meaning that this marker was not tightly linked to the *RMia* resistance source (Table 2).
<figure><img src="image_6.png" /><figcaption>**Fig. 4.** Expression pattern of the three genes RM1aCG1, RMiaGC2 and RMiaGC3 in the resistant 'WFP' infected with *M incognita* nematodes (WFP_Inf) and 'WFP' not infected (WFP_No-Inf), and in the PRMN seedlings infected with *M incognita* nematodes (PRMN_Inf) and the PRMN seedlings not infected (PRMN_NoInf). Data represented the mean of five biological replicates with three technical replicates each. *** Significative differences between infected and not-infected plants (_Inf vs. Noinf_). Differences in relative gene expression were statistically assessed using a Kruskal-Wallis test (***P < 0.001, **P < 0.01).</figcaption></figure>

<figure><img src="image_7.png" /><figcaption>**Fig. 5.** Genotyping using molecular markers designed in the *RMia* region for resistant and susceptible peaches. (a) Position of the framing markers SNP092 and SP091 and specific markers b) DNA amplifications for the RMiaGC1 and RMiaGC2 genes with the primers G13F1R1, G13QP211 and G31F5R5, respectively.</figcaption></figure>

# 4. Discussion

## 4.1. *Structural variation in the SP92SP91 region*

In 2014, we identified the genomic location of the *RMia* gene in a region of the chromosome 2 marked out by two SNPs (SP92 and SP91), where four TNL genes were annotated. We observed that almost all susceptible varieties contained a retrotransposon inserted between the two truncated TNL genes, *ppa023253* and *ppa018595* (Duval et al. 2014). Considering their structure, we hypothesized that both genes initially formed a single functional gene, *RMia*, that might be inactivated due to the transposon's insertion. However, we were unable to amplify the whole gene from the resistant peaches 'Nemared' and 'WFP' with specific primers flanking both genes *ppa023253* and *ppa018595m*. In addition, we found other susceptible peach varieties such as 'Pamirskii', that did not include the transposable element. The results of the present study clearly indicate that other genes are present in 'WFP' and

'Nemared' compared to the peach reference genome. The origin of these genes could be either an introgression in this region or could originate from duplication followed by rearrangements in the region as we also found the highest percent identity between the first candidate gene, RMiaGC1, and *Prupe.2G018300*, a gene located upstream the SNP SP92 in the peach reference genome. However, considering the rather weak percentage identity between the 3 *RMia* candidates and the NLRome from the peach reference genome, the *RMia* gene likely originated from introgression coming from a source that still needs to be discovered. As 'Nemared' and 'WFP' are two varieties that are not directly genetically related, the introgression might have occurred some time ago in a common ancestor. We could confirm that this introgression is also present in the resistant 'Okinawa' peach, by mapping the sequence (SRX2914754; Cao et al. 2022) on the SP92SP91 WFP sequence (Fig. S4).
<caption>**Table 2**</caption>

Phenotypes (R: Resistant, no galls; S: Susceptible, presence of galls) and Genotypes of peach rootstocks using the KASP™ markers SP92 (SNP R:C and S:T), SP91 (SNP R: A and S:G) and the molecular size markers G13F1R1, G13QP2111 and G31F5R5 designed in this study (R: one band of 450, 150 and 205 bp respectively; S: no band). For these markers, resistant homozygote (RR) and heterozygote (RS) cannot be distinguished and are noted R.

<table><thead><tr><th rowspan="2">Rootstocks</th><th rowspan="2">Phenotype<br>vs. M. incognita</th><th colspan="5">Markers</th></tr><tr><th>SP92</th><th>G13F1R1</th><th>G13QP211</th><th>G31F5R5</th><th>SP91</th></tr></thead><tbody><tr><td>WFP weeping</td><td>R</td><td>RR</td><td>R</td><td>R</td><td>R</td><td>RR</td></tr><tr><td>Nemared</td><td>R</td><td>RR</td><td>R</td><td>R</td><td>R</td><td>RR</td></tr><tr><td>Montclar®</td><td>S</td><td>SS</td><td>SS</td><td>SS</td><td>SS</td><td>SS</td></tr><tr><td>Rubira</td><td>S</td><td>SS</td><td>SS</td><td>SS</td><td>SS</td><td>SS</td></tr><tr><td>Pamirskii</td><td>S</td><td>SS</td><td>SS</td><td>SS</td><td>SS</td><td>SS</td></tr><tr><td>Hybrid PRMN Z64P40</td><td>R</td><td>RR</td><td>R</td><td>R</td><td>R</td><td>RR</td></tr><tr><td>GF305</td><td>S</td><td>SS</td><td>SS</td><td>SS</td><td>SS</td><td>SS</td></tr><tr><td>Summergrand</td><td>R</td><td>RR</td><td>R</td><td>R</td><td>R</td><td>SS</td></tr><tr><td>Missour 61-3</td><td>R</td><td>RR</td><td>R</td><td>R</td><td>R</td><td>SS</td></tr><tr><td>Nemaguard (Nemaguard xP1908)5</td><td>R</td><td>RS</td><td>R</td><td>R</td><td>R</td><td>SS</td></tr><tr><td>Hansen 536</td><td>R</td><td>RS</td><td>R</td><td>R</td><td>R</td><td>SS</td></tr><tr><td>GF557</td><td>R</td><td>RS</td><td>R*</td><td>R</td><td>R</td><td>RR</td></tr><tr><td>Garnem (GN15)</td><td>R</td><td>RS</td><td>R</td><td>R</td><td>R</td><td>RR</td></tr></tbody></table>

## 4.2. Regulation of RMia candidate genes following RKN infection

The long-read RNAseq analysis obtained from 'WFP' indicated that the three TNL genes, RMiaGC1, RMiaGC2 and RMiaGC3 were expressed in roots. The qPCR analysis showed that the RMiaGC1 gene is the most upregulated gene in the presence of nematodes. The overexpression of RMiaGC1 was confirmed by the same qPCR analysis in the other resistant genotype (PRMN140) including the resistant gene from 'Nemared'. By contrast, the other two candidate genes (RMiaGC2 and RMiaGC3) studied in the same analysis are also overexpressed but at a lower level. Interestingly, both genes display a similar regulation when infected by nematodes that may be explained by shared regulatory elements, likely in the 5kb region between them. Similarly to other NLR genes (Borrelli et al. 2018), the three candidate genes are constitutively expressed at a low level and are upregulated, at different levels of expression, following pathogen's infection. If pathogens usually dampen the immune system in susceptible plants leading to infection, in resistant plants, NLR are often upregulated directly or as a secondary consequence (Castel et al. 2019). The paradigm for NLR has recently evolved. Intricate networks between ETI and PTI components work in concert from the invader's detection to activation of multiple NLR and formation of "resistosome" structures leading to cell death and resistance (Huang et al. 2023). Although sensor NLR are not systematically upregulated, the HR process and resistance come with accumulation of transcripts and proteins of activator/helper NLR (Borrelli et al. 2018). On one hand, the transcription of RMiaGC1 is strongly responsive to RKN infection. On the other hand RMiaGC2 and RMiaGC3 are localized in close proximity to each other and organized in head-to-head configuration that reminds the genetic and functional paired NLR (e.g. RGA4/RGA5 and RPS4/RRS1) (Narusaka et al. 2009; Cesari et al. 2014; Xi et al. 2022). In our study, the RMia resistance might be mediated by one of the candidate genes or the result of the cooperation of 2 or even the 3 candidate genes. One hypothesis may be that RMiaGC2 and RMiaGC3 act similarly to paired TNL (sensors/executors). In addition, this paired TNL complex may induce a strong expression of RMiaGC1 leading to the resistance. Alternatively, the RMiaGC1 gene may be the sole RMia gene and would be able to induce the plant's immune response to nematodes by the large accumulation of its transcripts. A similar cluster of TNL, involved in resistance to the aphid *Myzus persicae*, has been recently identified in the same variety (WFP) (Duval et al. 2022). In such cases, high-resolution mapping studies would hardly settle these intricate models due to the considerable number of hybrids that should be produced to reveal discriminating recombinants. On the other side, transformation of susceptible peaches, with the different candidates to reveal the RMia gene, would be possible in theory. However, and despite the fact that

production of *Prunus* hairy roots using *Agrobacterium rhizogenes* has recently made progress (Xu et al. 2020), the functional validation of the different constructions (seven different possible combinations), which are necessary to elucidate the *RMia* model, would be indubitably challenging in this plant model. The heterologous expression in a model plant such as *Arabidopsis* or *Tobacco* (Xiao et al. 2021) would be an alternative, but with no guarantee of success as other components, required for upstream or downstream signalization, might be absent in such plants. Ultimately, the use of 'new genomic techniques' (NGTs) may be the answer to edit such candidates making their functional characterization easier in the near future (Nerva et al. 2023).

## 4.3. RMia among resistance genes to RKN in Prunus

In recent years, different sources of resistance to RKN have been identified among perennial plants (Saucet et al. 2016). Among woody plants, *Prunus* species hosts several resistance genes to RKN with different spectra of resistance to the most common species of the *Meloidogyne* genus. Apart from the RMia gene in *P. persica*, a succession of TNL genes have been identified. Cao and al (2022) have identified three genes in *P. kansuensis* (CV "HongGenGanSuTao") that are overexpressed in infected plants. They found an indel in the promoter of *Prupe.2G053600*, which would explain the resistance. This deletion is not present in 'Nemared' and 'WFP' (Fig. S5a) and this gene was not expressed in our long-read RNAseq 'WFP' experiment. Their other expressed gene *Prupe.2G055000* with 77.9% identity with RMiaGC1, is not present in 'WFP' and 'Nemared'. By attempting to align HongGenGanSuTao" reads to the RMiaGC1 locus (Fig. S6b), we observed that RMiaGC1 is not present in "HongGenGanSuTao". In a more distant prunus species *P. sogdiana*, the wild myrobalan plum, TNL genes that confer a resistance to *M. incognita* have been identified (Zhu et al. 2017; Xiao et al. 2021). In addition, the widely studied *Ma* gene in *P. cerasifera* confers a full resistance to all *Meloidogyne* species tested (Claverie et al. 2011; Duval et al. 2019). Besides, the *Ma* ortholog, *RMja*, in Almond does not control some *Meloidogyne* species such *M. incognita* or *M. floridensis* but it presents the same unique structure as the *Ma* gene (Van Ghelder et al. 2018; Duval et al. 2019). As *RMia* confers resistance to *M. incognita* but not to *M. javanica*, it is very complementary with the other RKN resistance genes. Considering the gene structure, *Ma* and *RMja* have an atypical repetition of 5 PL-CJID domains, a structure suspected to be involved in the RKN recognition (Van Ghelder et al. 2018). Our study revealed that *RMia* gene candidates have a more conventional structure including the canonical domains of TNL. Recently, the PL-CJID domain, which is present in the large majority of TNL (Van Ghelder and Esmenjaud 2016), has been shown to be directly

<footnote>* Presence of a second band (1300 bp).</footnote>
involved in the pathogen recognition in different TNL models. Indeed, structural studies demonstrated that effectors interacted with polymorphic regions between conserved motifs of the PL-CJID domains of RPP1 (Ma et al. 2020) and Roq1 (Martin et al. 2020). Later, the extreme C-terminal end of WRR4A was shown to be essential for the effector recognition of the oomycete *Albugo candida* (Castel et al. 2021). In our study, we shed light on the polymorphism that exists in these specific regions of the PL-CJID domain between RMiaGC1, RMiaGC2, and RMiaGC3. These differences are made of indels and repeats of specific amino acids. Similarly to the PL-CJID domains of WRR4A, the RMiaGC2 C-terminal end is enriched in aspartic and glutamic acids. The three candidate genes also contain repeats of acidic amino acids between motif 1 and 2. Besides, the region between motif 2 and 4 is rich in polar residues. Such type of amino acids has been shown to be involved in effector recognition in RPP1 (amino acids N, S, Q, and T) and Roq1 (NR loop). These regions in the *RMia* gene candidates might be the seat of the RKN effector interaction and recognition. The functional validation of this hypothesis would require the knowledge of interacting effectors, however very rare models involving a R gene together with its cognate nematode effector are described in the literature.

## 5. Conclusion

INRAE's *Prunus* rootstock breeding programs aim to combine the three main dominant resistance genes, *Ma* (plum), *RMja* (almond) and *RMia* (peach) into new rootstocks to increase the durability of nematode resistance. The nematode species, *M. javanica*, *M. enterolobii*, and *M. floridensis* recently described in Florida (Handoo et al. 2004), are able to infest rootstocks carrying the *RMia* gene. More recently, Qiu and colleagues tested two isolates of *M. arenaria* that produce galls on the peach rootstock Flandaguard, whose source of resistance is Okinawa peach, in which the *RMia* gene is present (Qiu et al. 2021). Even though both *Ma* and *RMja* genes have not been circumvented, this recent study shows that some RKN may be able to overcome resistance genes in *Prunus*. It is therefore important to pyramid several sources of resistance to optimize their durability. Marker-Assisted Selection (MAS) for the *Ma* gene is possible using intragenic SCAR markers, CT3-4N and NSCAFLP2 (Claverie et al. 2011). For the *RMja* gene, MAS selection can be carried out using a specific SNP on chromosome 7 at position 9,019,871 in the almond reference genome (*P. dulcis* cv 'Texas' V2), using either the KASP™ markers SP903 and SP904 (Duval et al. 2018), or SNPid 52,023 from the Affyx almond array chip (Duval et al. 2023). Regarding *RMia*, we designed the novel markers G13F1R1, G13QP211, and G31F5R5 and used the KASP™ marker SP92 (Duval et al. 2014) usable in MAS. We developed two robust intragenic markers, G13F1R1 and GQP211, within the RMiaGC1 gene. Besides, the KASP™ marker SP92 is the closest intergenic marker to RMiaGC1 (37.8 Kb) and was shown to be a good indicator as well. For all genotypes tested, including varieties and hybrids, the genotyping using SP92, G13F1R1, G13QP211, and G31F5R5 markers strictly follows the phenotypes, which make them linked to the *RMia* resistance, unlike the KASP™ marker SP91 for which phenotypic predictions were not always correlated to the genotype (Table 2). In conclusion, *Prunus* rootstock breeders have effective markers at their disposal for pyramiding the three genes *Ma*, *RMja* and *RMia*, in a three-way rootstock either with size markers or SNP markers.

### CRediT authorship contribution statement

**Henri Duval:** Writing – review & editing, Writing – original draft, Validation, Methodology, Funding acquisition, Formal analysis, Conceptualization.
**Laure Heurtevin:** Writing – original draft, Data curation, Formal analysis, Software, Data analysis.
**Naïma Dialah:** Writing – review & editing, Formal analysis, Data curation.
**Claire Caravel:** Data curation.
**Caroline Callot:** Software, Data curation.
**Cyril Van Ghelder:** Writing – review & editing, Writing – original draft, Validation, Methodology, Conceptualization.

### Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

### Acknowledgments

The authors thank the genotoul bioinformatics platform Toulouse Occitanie (Bioinfo Genotoul, https://doi.org/10.15454/1.5572369328961167E12) (accessed on 1 January 2018) for providing computing, storage resources, the MGX Platform—Montpellier GenomiX for the NGS sequencing, the INRAE Gentyane platform for the long-read RNAseq sequencing and Jacques Lagnel for advice in the bioinformatic analysis. This study was supported by the INRAE Plant Biology and Breeding Division and the Plant – Nematode Interaction (IPN) team.

### Supplementary materials

Supplementary material associated with this article can be found, in the online version, at doi:10.1016/j.scienta.2025.114081.

### Data availability

Raw data related to *P. persica* 'WFP' can be retrieved in the Bio-Project number **PRJNA854365** including Illumina WGS of 'WFP' (**SRR20115452**) and long read RNAseq of 'WFP' (**SRR20225451**). The R_SP92SP91 sequence containing the three *RMia* gene candidates from 'WFP' can be retrieved from the GeneBank accession number **PQ432884**. Raw data related to the WGS of *P. persica* 'Pamirskii' (**SAMN43405486**), 'Montclar' (**SAMN43416978**) and 'Nemared' (**SAMN43416965**) are publicly available in BioProjects number **PRJNA1154452** and **PRJNA854381**.

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