Deciphering Resistance to Root-Knot Nematodes in Prunus for Rootstock Breeding: Sources, Genetics and Characterization of the Ma Locus

: Root-knot nematode (RKN) species are predominant pests of crops, attacking stone fruit crops Prunus spp. under Mediterranean climate conditions worldwide. Natural resistance for rootstock breeding is a control method that is gaining interest as an alternative to the highly toxic nematicides. This review ﬁrst reports an outline of the root-knot nematodes parasitizing stone fruit crops and the Prunus species and rootstocks. It then describes the main sources of resistance detected among the Prunus germplasm and focuses on the major resistance genes identiﬁed and their characteristics (spectrum, durability, histological mechanism, effect of temperature, interaction with other pests and diseases, etc.). In peach, besides the RMia reference gene, the new genes PkMi and Mf , also located on chromosome 2, need to be characterized regarding their spectrum and relationship. The two other Prunus reference genes, Ma from plum (complete spectrum) and RMja from almond (more restricted spectrum), are orthologs that belong to a TIR-NB-LRR (TNL) cluster on chromosome 7. The review ﬁnally summarizes the positional cloning of the Ma gene and the characterization of its unique TNL structure, encompassing a ﬁve-times repeated post-LRR domain. Deciphering how this structure is functionally involved in Ma ’s remarkable biological properties is a real challenge for the future.


Introduction
In many perennials, rootstocks contribute to the control of various abiotic constraints such as drought, salinity and limestone, together with diverse biotic constraints due to soil-borne pests and diseases. Amongst soil pests, the root-knot nematode (RKN) species Meloidogyne spp. are a major problem in stone fruit crops Prunus spp., and breeding programs throughout the world target their control as one of the major traits to improve [1][2][3][4]. These programs may exploit the high variability of the interaction between the pest and the plant species and in particular the natural resistance to the RKN species existing in the Prunus germplasm. RKN resistance is an environmentally friendly alternative to the highly toxic chemical nematicides and is thus of an outstanding interest in rootstock breeding.

Root-Knot Nematodes
Root-knot nematodes (RKNs) (Meloidogyne spp.) are found in Mediterranean, tropical and equatorial areas and to a lesser extent in temperate regions. They have been long considered as the most damaging among nematodes worldwide [5]. The Meloidogyne genus includes the nematode species with the largest agricultural impact, causing losses estimated at tens of billions of euros per year [6] when all crops are confounded. RKNs have long reduced fruit production in several economically important Prunus species. Peach, almond and plum species grown under Mediterranean climate conditions are those most affected [1,7]. Infections by root-knot nematodes induce typical below-ground root galls and lead to the above-ground stunted growth of young trees. Early root infections result in extensive tree death in an orchard. Tree defoliation occurs that ultimately results in yield losses. Sandy soils are the most favorable to RKN attacks and gall development.

Biology and Life Cycle
RKNs are sedentary endoparasitic species and obligate biotrophs that penetrate plant roots and hijack the nutritional resources of the host for their benefit. They are extremely polyphagous and reproduce on thousands of cultivated and wild plants [8]. All Meloidogyne spp. have a similar life cycle. Eggs are deposited in gelatinous masses and survive in the soil or in plant residues. The mobile second-stage juveniles (J2s) hatch from the eggs and move towards the root tips. During compatible interaction between susceptible plants, J2s penetrate the root and migrate downwards, between the cells of the cortex, to the apical meristem region. Then, they move upwards towards the vascular cylinder and fix. They become sedentary and develop into swollen third-and fourth-stage juveniles and finally into adult females. While settling, they induce specialized hypertrophied and multinucleate cells (designated giant cells) that form a feeding site. Females may lay more than 1000 eggs in an egg mass, which is often located on the outer surface of the root. The giant feeding cells and the divisions of the surrounding vascular parenchyma cells give rise to the typical root galls [9].

The RKNs Affecting Prunus Species
The three predominant RKNs affecting the Prunus species are M. arenaria, M. incognita and M. javanica, which are all extremely polyphagous, have a worldwide tropical and Mediterranean distribution and reproduce asexually via mitotic parthenogenesis. Another widely distributed species is the northern RKN M. hapla, present under temperate climates, which reproduces by meiotic parthenogenesis or amphimixis but develops poorly on Prunus spp. [10]. Additionally, several species with a more restricted distribution have been reported. Among them, one species, reported on peach in Florida in the 1960s as overcoming the resistance of 'Nemaguard' and 'Okinawa' rootstocks [11], has been more recently described as the peach RKN M. floridensis [12,13]. This new species from the USA has been detected in Florida, neighboring southeastern states [14][15][16][17][18] and California [19]. Towards the RKNs M. arenaria, M. incognita, M. javanica and M. floridensis, the behavior of predominant rootstocks belonging to peach, almond-peach or plum has been reported [3].

Diversity of Prunus Species and Rootstocks
The genus Prunus, containing the stone fruit crops, is divided into five main subgenera that comprise more than 400 species distributed worldwide [28]. Among them, a majority of fruit-producing and rootstock species belong to two subgenera: 1.
The genus Prunus might have appeared 61 My ago (Eocene) in Oriental Asia. Eurasia harbors the highest diversity, in agreement with its putative native area [29]. Dispersion and speciation events would explain the current distribution from this area. Amygdalus species moved from oriental to occidental Asia 48 My ago, preceding a diversification and an evolution in which the Himalayan chain separated the almonds (Middle East) and the peaches (Oriental Asia). European plums (P. cerasifera, P. spinosa and P. domestica) migrated from Oriental Asia more recently (31 My) [29]. Nowadays, the distribution area of Myrobalan plum (P. cerasifera) ranges from Central Asia to Southern Europe with the highest diversity being found around the Caspian Sea [30].
Stone fruit crops are grown on fruit cultivars carrying selected nutritional and gustative characteristics grafted onto rootstocks providing vigor and adaptation to soil abiotic and biotic conditions. Fruit breeders have to face autosterility within the allogamous species, almond, plums or cherries, and cope with the low genetic diversity of the mainly autogamous species, peaches and apricots. As members of the Prunus species can be crossed with each other, rootstock breeders can exploit the high interspecific variability of the different parental species to select and combine their desirable features in hybrid material. In order to obtain polyvalent rootstocks, for example, the Myrobalan plum can be easily crossed with peach, almond or apricot [31].

Control of RKNs in Prunus: The Resistance Strategy
Interaction between Prunus and RKN species is either compatible or incompatible. Compatibility involves a nematode behavior that is similar, at the cellular level as well as in the expression of symptoms, in perennial crops such as Prunus and in annual crops such as tomato [32,33]. In Prunus, galls may reach sizes that are impressively large despite the lignification of the root tissues.
Incompatibility is a natural resistance phenomenon. Breeding naturally resistant rootstocks is the most economical and environmentally sound method for managing rootknot nematodes in the Prunus species and for avoiding their dissemination. Rootstock resistance has been investigated and exploited in several countries. For example, the resistant rootstock 'Nemaguard' has been widely used in California because of the high RKN frequency in almond orchards [3].

Resistance Sources
Resistance sources have been investigated and characterized mainly in peaches, almonds and plums.
Peach is self-fertile and thus has a low level of genetic variability [34]. Wild relatives, including P. davidiana and P. kansuensis from China [35,36], are used as sources of resistance to pests and diseases for fruit varieties and rootstocks. Peach was the first Prunus species for which RKN resistance (R) factors were reported [37]. The first RKN-resistant peaches, Shalil, Bokhara and Yunnan, originated from northwest India, the Central Asian republics and China, respectively [11], and were resistant to both M. arenaria and M. incognita. New peach varieties, such as Nemaguard, Nemared and Okinawa, were then selected in the USA, from the 1960s onwards, for their higher resistance towards M. javanica [35]. Since the 1990s, several rootstocks for peach have been derived from these accessions, including the complex almond-peach hybrid cv. Flordaguard, the almond-peach cv. Garnem and the peach × P. davidiana cv. Cadaman [3].
The almond originates from Central Asia and has been cultivated in the Middle East and Mediterranean areas since Antiquity for its sweet seeds, with bitter-seeded varieties being used as rootstocks. Resistance to M. javanica has been detected in bitter seeds of Alnem seedlings in Israel, and these seedlings were later shown to resist M. arenaria, M. enterolobii and M. ethiopica, but they did not show resistance to either M. incognita or M. floridensis [38][39][40].
Plum species are diverse and adapted to a broad range of climatic and edaphic conditions. They were domesticated independently in Europe (P. domestica, P. insititia), China (P. salicina) and North America (P. hortulana) [41][42][43]. Most fruit varieties of plums belong to the European species P. domestica and P. insititia, or to P. salicina. Resistance has been identified in a number of plum rootstocks, including the St Julien (P. insititia), Damas (P. spinosa × P. domestica) and Myrobalan plums (P. cerasifera) [44]. The Myrobalan plum, a near-wild allogamous species found in temperate and Mediterranean climates, originated in the Caucasus and Crimean regions [30]. Myrobalan accessions P. 2175, P. 1079 and P. 2980 express complete resistance to more than 30 RKN isolates from M. arenaria, M. incognita, M. javanica, M. hispanica and M. hapla [10,45]. Recent evaluations showed that this resistance also controls the species M. ethiopica and M. enterolobii [40].

Genetics and Mapping of RKN Resistance
A major dominant reference R gene has been identified in each of the plum, almond and peach species (Table 1).
In plum, the resistance carried out by the Myrobalan plum has been shown to be conferred by a single major dominant gene called Ma. The Ma gene controls resistance to all tested RKN species and to the predominant RKN species M. arenaria, M. incognita and M. javanica [46,47], in particular. Ma also controls the peach RKN M. floridensis [47] and the highly aggressive species M. enterolobii (=M. mayaguensis), which is uncontrolled by the Mi1-2 R gene from tomato [48]. More recently Ma has also been shown to confer resistance to M. ethiopica [40] (Table 1), a species widely present in stone fruits and grape in South America [25]. Ma, as the RKN reference gene for plum, is located on the linkage group (LG) 7 of the Prunus map [49] (chromosome 7 of the peach reference genome [50]). The steps of the genetics and mapping studies in the Myrobalan plum are summarized in the first part of Figure 1.
In almond, bitter cultivars of the 'Alnem' series carry resistance to M. javanica conferred by a single gene identified as soon as 1975 [51] and later designated as RMja [52]. This almond RKN reference gene also controls M. arenaria, M. ethiopica and M. enterolobii [40] but neither M. incognita nor M. floridensis ( Table 1). The RMja gene, first shown as closely linked to the Ma gene [52], has recently been characterized as an orthologue of this plum gene [53].
In peach, the 'Nemaguard' and 'Nemared' rootstocks carry the RMia gene for resistance to M. arenaria and M. incognita [49]. This gene, most likely having a populationdependent activity towards M. javanica, controls neither M. enterolobii nor M. ethiopica [40] ( Table 1). RMia, the RKN reference gene for peach, is independent from Ma and RMja and is located on LG2 in the Prunus map [49] (chromosome 2 of the peach reference genome [50]).
Additionally, a Chinese wild peach (P. kansuensis) has been shown to carry an R gene to M. incognita named PkMi [54,55], which is also located on LG2, but its relationship with RMia needs to be clarified. An accession of P. kansuensis has also been proposed by other authors as carrying a single-locus dominant (Mf )/recessive (mf ) model for the inheritance of resistance to M. floridensis [56], as the cognate Mf locus is also located on LG2 [57]. It will be important to determine the spectrum of this resistance towards other RKN species and to shed light on the relationship between the RMia, PkMi and Mf genes from peach resistance sources.
Other R genes have also been detected in other plum species. The Rjap gene, identified in the Japanese plum P. salicina and located on LG7, carries a wide resistance spectrum that might be the same as that of Ma [49]. Even though no fine mapping has been performed for Rjap, it is highly plausible that it is an Ma orthologous gene. More recently a gene for resistance to M. incognita [58], named PsoRPM2, was reported in the wild Chinese plum species P. sogdiana, which is close to the Myrobalan plum.

Histological Mechanisms of RKN Resistance
Histological resistance mechanisms have been studied in plum and peach. Maresistant plums have no galls, and no swollen larval nematode stages are observed, regardless of the RKN species considered. In those plums, the early penetration of M. arenaria J2s was shown to be similar in resistant and susceptible (S) (=lacking the Ma resistance gene) accessions [33]. In both the R and S accessions studied, J2s enter the elongation zone above the root cap, migrate downwards to the apical meristematic region and move then upwards in the direction of the stele [32]. In susceptible tissues, J2s complete their life cycle, whereas in resistant tissues, a hypersensitive-like response (HLR) of the root cells blocks the nematode development in its filiform state, either in the apical meristematic region or in the stele. Severe damage due to J2s in the apical meristem induces the development of new subterminal rootlets [32,33], which is an active reaction to the necroses of terminal root cells.
In peach, studies on the histological mechanisms of resistance in the rootstocks Nemaguard and Nemared have been conducted. In both rootstocks, which are considered resistant to certain M. javanica populations despite the formation of small galls, a protective 'walling off' process isolating young giant cells from neighboring cells has been observed. The giant cells then became necrotic and collapsed, thereby preventing the maturation of the females, and this nematode restriction was accompanied by callose deposition within the compact cell layer around the infection site [59][60][61].

Other Features of Resistance in Prunus: Durability, Stability at High Temperature and Relationship with Other Pests and Pathogens
An important feature for breeding RKN-resistant Prunus cultivars is the level of durability conferred by the R genes. This is a key trait since the perennial nature of trees increases the risk of resistance breakdown. Durable resistance, as defined by Johnson [62], is resistance that remains effective during extensive use in agriculture, over a long period, in an environment conducive to the disease. In interspecific Prunus hybrids, the resistance conferred by Ma alone, RMia alone or by a combination of Ma and RMia, has not been overcome when challenged, under greenhouse conditions, by continuous M. incognita inoculum pressure for up to four years. By contrast the resistance conferred by Mi-1 in tomato broke down after only one to two years [63]. Additionally, the effect of temperature on resistance expression has also been assessed. The Ma heterozygous-resistant accession P. 2175 (Ma/ma)and the Ma homozygous-resistant accession P. 1079 (Ma/Ma) were not affected by a continuous temperature of 32 • C, which showed that Ma resistance is stable at high temperatures [64].
The relationship between resistance to RKNs and other pests and pathogens of the Prunus spp. has been studied towards the soil bacterium Agrobacterium. tumefaciens and towards the root endoparasitic nematode Pratylenchus vulnus. Agrobacterium tumefaciens is responsible for a very common disease in the roots of stone fruit crops, the crown gall disease. Meloidogyne spp., when present, facilitate the expression of crown gall symptoms in RKN-susceptible rootstocks. The Ma gene has been shown to have a protective effect against the expression of bacterial symptoms in the presence of RKN infections, as no nematode or bacterial symptoms have been observed in Ma-resistant materials [65]. This effect of Ma and presumably of other RKN R genes against Meloidogyne-transmitted crown gall is an additional argument for gene introgression into Prunus rootstocks.
The root-lesion nematode (RLN) P. vulnus is an endoparasitic species that differs from Meloidogyne spp. with its migratory behaviour at all its developmental stages. A study by Stalin et al. [66] illustrated that the Ma gene does not confer resistance to the RLN P. vulnus, which could be expected given the completely different mode of parasitism of the two nematode genera.

High-Resolution Mapping and Cloning of the Ma Locus
The Ma gene, the first RKN R gene characterized in a perennial, has remarkable biological properties in comparison with the Mi1-2 tomato R gene. As reported earlier the resistance that Ma confers is high and durable, whatever the temperature, and controls all the RKN species attacking the Prunus spp. These remarkable features have justified a positional cloning strategy in order to reveal its structure and function. Within stone fruit crops, it is also the first R gene to be cloned [67,68]. Successive steps to reach this objective are summarized hereafter and in the second part of Figure 1. Molecular mapping of the Ma locus began with the detection of two SCAR (sequence characterized amplified region) markers, SCAL19 and SCAFLP2, tightly linked to Ma [69,70] using a BSA (bulked segregant analysis) approach. Ma was then mapped on the chromosome 7 of the reference European Prunus map in a 2.7 cM interval between the SSR (simple sequence repeat) pchgms6 and SCAL19 markers [71]. A total of 1332 individuals revealed 31 individuals recombining between SCAL19 and pchgms6 in this interval [71]. The recombinant individuals allowed a finer location of the gene, as Ma co-segregated with the SCAFLP2 marker and was separated from SCAFLP3 (proximal) and SCAFLP4 (distal) by a single and 11 recombination events, respectively.
The Myrobalan accession P. 2175, heterozygous for Ma, was used for the construction of a DNA BAC library comprising 30,720 clones with a mean insert size of 145 kb and a 14-15x Prunus haploid genome coverage. A few BAC clones from the library allowed to land on the Ma spanning interval and to construct the resistant and susceptible physical contigs using PCR screening with codominant markers. Additional microsatellite markers were designed from BAC subcloning or BAC end sequencing. Finally, in the resistant contig, a single 287-kb BAC clone (BAC 76H19) was shown to carry the Ma gene (=R dominant allele); this BAC clone contained two flanking markers on each side of the gene as well as two cosegregating markers [71]. BAC 40K9 (170 kb) was shown to carry the S recessive allele (ma).

Evidence of the TNL Cluster Containing Ma
Complete sequencing of BAC 76H19 allowed new SCAR and SSR markers to be placed on the sequence and the determination of a physical interval of 174 kb containing Ma [72]. The new markers SSR2 and SCAFLP4, manually designed within this interval, reduced the region containing the gene to 70 kb with four candidate genes: one lectin protein kinase (LecPK) and three TIR-NB-LRRs (TNLs). In order to locate Ma more precisely, a final round of mapping using 1780 additional segregating individuals was undertaken. Among these, some carried the RMia gene from the Nemared peach and were separated from those carrying Ma by resistance assays using M. floridensis. A total of 19 new recombinants were then genotyped for nine SCAR or SSR markers designed within the 70-kb candidate interval. The phenotype of five recombinant genotypes located within this interval and evaluated for M. floridensis reduced the final candidate region to the 32-kb sequence encompassing a cluster of three TNL genes (TNL1 to TNL3).
The sequence of the susceptible BAC 40K9, 170 kb long, encompassing the S allele of the Ma candidate region [71], allowed for the identification of the three TNLs (tnl1 to tnl3) orthologous to the resistant haplotype (TNL1 to TNL3). A prediction of intron/exon structures showed that TNL2 was a pseudogene (deletion of a single base leading to a frameshift mutation) and that TNL3 was truncated in its LRR repeat motif and was much shorter. Thus, TNL1 appeared to be the best candidate gene.

Validation of the Final Candidate TNL1 and Evidence of the Remarkable Structure of the Ma Gene
The complete cDNA sequence of this best candidate revealed a gene size of 6147 bp (without UTRs) and a predicted protein of 2048 aa [72]. A TNL1 sequence of 15 kb containing the 10 kb genomic sequence and a 5 kb native promotor sequence was cloned. Validation of the TNL1 candidate gene was then performed using hairy roots (A. rhizogenes), and several transformation events of a susceptible clone [73] were resistant to all RKNs tested, which unequivocally proved that TNL1 was the Ma gene. Thus, the Ma gene is a TNL consisting of nine exons separated by introns of variable sizes. The first four exons code, respectively, for the protein domains TIR, NB, NLL and LRR, the so-called 'NLL' exon (NBS-LRR Linker), encoding a sequence linking the very different structures of NB and LRR domains. The last five exons, designated as post-LRR (PL) domains because of their location in C-terminal position in the gene, encode five modules, separated by introns containing microsatellites [72]. The similarities noted between these PL domains indicate that they are repeated exons of approximately 200-250 amino acids.
The addition of these five PL exons to the gene structure makes Ma a TNL twice as large as the so-called classical TNLs. A further study showed that TNL genes with multiple PL domains are rare in peach and the other plants screened. Moreover, the five-PL domain pattern is probably unique to Ma and its orthologues within Prunus and closely related genera from the Rosaceae and was probably inherited from the common ancestor of these plants in the subfamily Spiraeoideae. To date, Ma is the longest TNL R gene cloned in plants [74].

High-Resolution Mapping of the Almond RMja Gene, a Ma Orthologue
An F2 almond progeny of approximately 1500 individuals from a cross between P. dulcis cv. Alnem 1, a homozygous carrier of the RMja gene (RMja/RMja), and P. dulcis cv. Lauranne, which is susceptible to M. javanica (rmja/rmja), was used. Simultaneously, SSR and SNP (single nucleotide polymorphism) markers flanking the Ma gene locus were generated from the peach genome and were used to genotype all individuals. Two flanking markers with a single recombinant and thus closely linked to RMja were identified. These data show that, in the peach genome [50], the RMja gene was located in an interval of less than 100 kb. This window spans a cluster of four TNLs in peach that contains the orthologue of the Ma gene. In order to obtain a reliable sequence of the region containing RMja in almond, the BAC libraries of the Alnem 1 and Lauranne parental accessions were generated. Two BAC clones flanking the gene in Alnem 1 were identified, sequenced and used to generate an assembled sequence of 336 kb containing the RMja gene. The location of markers from the peach genome on the Alnem 1 sequence revealed the presence of only three genes in the RMja candidate interval, two of which have an altered structure.
A complementary bioinformatic analysis associated with expression data allows for the conclusion that the RMja gene is the ortholog of the Ma gene in almond. The RMja gene has an architecture which is highly similar to Ma. Nevertheless, the polymorphism, located mainly in the PLs domains, might explain the difference in the resistance spectrum of the two genes. More generally, the comparison of the TNL cluster sequences in almond (from BAC sequences [53]), in peach (from the peach genome [50] and in plum (from the BAC sequences [71,72]) allowed for the proposed hypotheses on the genetic mechanisms that led to the shaping and evolution of the particular structures in this cluster.

Conclusions and Perspectives
Research on resistance to root-knot nematodes in stone fruit crops began early in the USA, and data obtained there was completed later by diverse studies conducted in other Mediterranean countries (Israel, France, Spain, etc.). Beyond the three RKN species identified before the 1980s, new species have been described that have a more restricted geographical distribution but can be destructive when present. In each of the Prunus species most severely affected by these pests, i.e., the peach, the almond and the plum, at least one resistance source has been characterized and the cognate major genes identified and mapped. Studies on the key features of the genes have been conducted; in particular, the resistance spectrum of Ma, RMia and RMja now considers the array of six predominant RKN species attacking stone fruit crops. Such information will facilitate the creation of complete-spectrum resistance in Prunus rootstocks. It also opens the way for breeders to pyramid several resistance layers to M. arenaria and M. ethiopica (Ma + RMia + RMja), M. javanica and M. enterolobii (Ma + RMja), and M. incognita (Ma + RMia), while M. floridensis is the sole species controlled by Ma alone ( Table 1).
The high-resolution mapping of the Ma locus revealed the unprecedented structure of the gene and of one of its alleles (RMja). The fact that Ma controls all Prunus RKN species might underpin an original function of the gene. This function presumably renders it able to recognize, directly or indirectly, either a wide range of effector molecules differing between each of the diverse RKN species controlled, or a single or a few effector molecules that are common to all of them [13]. Another important point is that three Ma allelic (= orthologous) sequences with different resistance spectra are now available. In line with these data, research will have to fully consider comparative genomics within the Ma locus to identify the resistance determinants (sequences and/or motifs) and in particular, those involved in the resistance specificity. Methods and model studies published recently open the way in this objective [75][76][77]. The noteworthy C-terminal part of the gene with a huge post-LRR region (=5 repeated PL domains) makes it the longest R gene ever cloned. A recent work has shown that integrity of the single PL domain often carried by TNL genes such as RPS4 (Arabidopsis) or N (tobacco) is required for function [78], even though its role is still ignored. Consequently, deciphering whether and, in the presumed positive case, how this 5-PL region is involved in the remarkable biological properties of Ma remains a real challenge for the future.
Funding: Not applicable.