Host-plant resistance is an effective, environmentally friendly means of controlling insect pests, including aphids. Here, we consider plant resistance to be a heritable trait, displaying genotype-dependent variability within a plant species. Resistance to aphids has been described in several crops (Dogimont et al., 2010; Smith and Chuang, 2014). This resistance is controlled by one or several genes, which may be recessive or dominant. Resistance deters aphids from the crop, and affects their biotic potential, including their growth, development, and reproduction. So resistance is generally detected through these central aphid life history traits, rather than by a visible plant phenotype. The melon Vat gene is unique among the known resistance genes in that it has a pleiotropic effect as it also confers resistance to the viruses transmitted by aphids.

Melon crops are primarily colonized by only one aphid species, the melon aphid Aphis gossypii, a cosmopolitan aphid species. This aphid causes stunting and severe leaf-curling, and heavy colonization can result in plant death. Aphids also excrete honeydew onto the leaves and fruits. This sticky sweet substance acts as an ideal growth medium for sooty mold, which greatly decreases fruit quality. Moreover, A. gossypii is an efficient vector for viruses, contributing to the spread of diseases.

Resistance to A. gossypii in melon was first observed in the mid-20th century (Ivanoff, 1944). In 1967, an American team of entomologists and plant geneticists began a systematic study of resistance to A. gossypii in melon. They focused on the Indian line PI 371795, later called PI 414723, which suffers only mild attacks in the field (Kishaba et al., 1971; Bohn et al., 1972). In controlled no-choice tests, few aphids survive on this line, and the fecundity of those that do is low (Kishaba et al., 1971). This resistance is a dominant trait in PI 414723, and is controlled by a major gene and several minor genes (Kishaba et al., 1976). A French team of virologists and plant geneticists studied the resistance of the Korean line PI 161375 to Cucumber mosaic virus (CMV) in detail. They discovered an original phenotype of this line: complete resistance to CMV when the aphid A. gossypii inoculated the plant with the virus. Moreover, A. gossypii aphids departed from PI 161375 plants. These two phenotypes cosegregated in PI 161375 and were controlled by a single dominant gene (Pitrat and Lecoq, 1980). Complete resistance to CMV was also observed in PI 414723 when CMV was introduced into the plant by the aphid A. gossypii (Pitrat and Lecoq, 1982). PI 414723 and PI 161375 thus have similar features: resistance to CMV when A. gossypii inoculates the plant with the virus cosegregating with resistance to A. gossypii controlled by a single dominant gene (Pitrat and Lecoq, 1982). In both lines, the resistance to viruses is expressed only if the aphid inoculating the plant with the virus is A. gossypii. PI 161375 and PI 414723 plants are susceptible to viruses when other aphid species, such as A. citricola, A. craccivora, A. fabae, and Myzus persicae, inoculate the plant with viruses, or if viruses are introduced mechanically (Lecoq et al., 1979, 1980; Romanow et al., 1986). The resistance to viruses when A. gossypii inoculated the plant is also fully effective against unrelated viruses (Lecoq et al., 1980). The gene controlling this double phenotype has been named Vat, for ‘virus aphid transmission’ (Pitrat and Lecoq, 1982).

Several hundreds of accessions were tested for their effect on the aphid traits (Pitrat et al., 1996; Fergany et al., 2011). These large screenings have suggested that about 5% of accessions display resistance to colonization by A. gossypii. Among them, only a small number have been tested for the double phenotype characteristic of Vat, resistance to virus and resistance to aphids. Up to now, the double phenotype has been identified in 25 melon lines (Pitrat and Lecoq, 1980; Soria et al., 2000; Thomas et al., 2012b; Boissot et al., 2016). These melon accessions or lines originate from Asia, Africa, America, and Europe.

Two independent breeding programs were conducted early on, to transfer resistance to A. gossypii into cultivars, with the transfer of resistance from PI 161375 into Charentais-type melons and resistance from PI 414723 into Western Shipper–type melons. Consistent with the cosegregation of resistance to melon aphid and resistance to viruses, which were introduced by melon aphids, the inbred lines obtained in both programs also displayed resistance to viruses when the melon aphid inoculated the plant (Kishaba et al., 1992; Boissot et al., 2016). Margot became the first melon cultivar declared resistant to the melon aphid A. gossypii to be listed in the French catalog in 1987. Since then, 110 Charentais-type cultivars declared resistant to this aphid have been released in France (GEVES data). Melons are cultivated in the South East (SE) and South West (SW) of France, and on two islands of the Lesser Antilles (LA). Given the commercial success of some of the resistant cultivars, about 80% of the melon crops cultivated in SE France since 2000 are thought to have carried this resistance (Boissot et al., 2016).

Since these seminal studies were conducted, the molecular structure of the Vat gene has been elucidated, its double phenotype has been investigated at the cellular level, and its effect on the behavior and life-history traits of the aphid has been studied. Its spectrum of activity against the clonal diversity of A. gossypii has been studied in the laboratory, and its efficacy and the durability of these effects have been studied in situ. All these points will be reviewed after a short presentation of the three protagonists: Cucumis melo, A. gossypii and the viruses transmitted by A. gossypii.

The Vat locus was mapped to C. melo’s linkage group V using segregating populations from a cross between the susceptible line Védrantais and the resistant accession PI 161375 (Pitrat, 1991; Baudracco-Arnas and Pitrat, 1996). It was localized to a subtelomeric position on a saturated map combining two recombinant inbred populations resulting from crosses between Védrantais and two resistant accessions PI 161375 and PI 414723 (Périn et al., 2002). In early 2000, a map-based strategy was used to isolate the Vat gene. This approach involved the use of 6000 plants from a back-cross population derived from Védrantais and PI 161375. Recombination events within the terminal region of linkage group V were screened and recombinant plants were phenotyped for resistance to aphids. A physical map encompassing the Vat gene was obtained by screening a melon bacterial artificial chromosome (BAC) library constructed from PI 161375, and the genomic sequence spanning the Vat region was annotated. A comparison of molecular data and phenotypic data for resistance to melon aphid and resistance to viruses when the melon aphid inoculated the viruses showed that the Vat gene was a single functional locus conferring both types of resistance (Pauquet et al., 2004). Nine of the 14 of the back-cross progeny displaying recombination in the genomic sequence spanning the Vat region are presented in Figure 1, with their phenotype. The Vat gene is 6-kb long, and consists of five exons and four introns. It encodes a predicted 1467-amino acid protein presumed to be located in the cytoplasm (Dogimont et al., 2014). This protein belongs to the coiled-coil (CC)-nucleotide binding site (NBS)-leucine-rich repeat (LRR) family (Figure 2). Only three other genes conferring resistance to aphids or other hemipterans, Mi-1 in tomato and Bph14 and Bph26 in rice are known to encode proteins from the NLR family (Rossi et al., 1998; Nombela et al., 2003; Casteel et al., 2006; Du et al., 2009; Tamura et al., 2014). For confirmation of the effect of the Vat resistance allele, an 11-kb DNA fragment harboring Vat’s coding region, promoter, and 3′-flanking region, was introduced into two susceptible C. melo lines by Agrobacterium-mediated transformation (Dogimont et al., 2014). Four lines derived from independent transformation events were obtained and all lines displayed high levels of resistance to NM1 melon aphids and complete resistance to viruses when the NM1 aphids inoculate the transgenic plants with CMV, WMV, and ZYMV.

All the bioassays conducted to identify Vat in the melon genome to date have used NM1. This clone has been used since the early studies by the French team and has provided the most clear-cut differentiation between susceptible and resistant accessions for both resistance to melon aphid and resistance to the viruses introduced into the plant by melon aphid (Boissot et al., 2016). In a study investigating whether the Vat allele of PI 161375 had a specific aphid clone effect or a much broader effect, one of the transgenic lines was tested with a set of A. gossypii clones from the cucurbit host-race. The bioassay used assessed the resistance to viruses transmitted by the aphid. In the transgenic line, the resistance to viruses introduced by an aphid was aphid clone-specific. Surprisingly, for some clones used for inoculation purposes, resistance to the virus was expressed in the native line, PI 161375 but not in the transgenic line (Table 2). These remarkable differences reveal that at least one other gene is involved in the resistance elicited by some A. gossypii clones in PI 161375 (Boissot et al., 2016). In accordance with general rules for the naming of genes, it has been suggested that the gene isolated from PI 161375 (Dogimont et al., 2014) should be renamed Vat-1, and the additional gene Vat-2. There may be allelic series for both these loci (Boissot et al., 2016).

It is not clear from the results presented above whether Vat-1 and Vat-2 form a cluster. This point has been investigated indirectly. PI 161375 is a Korean line harboring Vat-1 and Vat-2; it belongs to the Chinensis botanical group (Table 1). Vat-1 was introgressed from this line into a Charentais line (Cantalupensis group). The process of introgression consists in a first crossing between a Charentais line and PI 161375 and after, backcrossing the aphid-resistant progeny with the Charentais line, referred to as the recurrent line. The bioassays, to select plants resistant to aphids from each back-cross progeny, used the aphid clone NM1. Remarkably, the spectrum of resistance to viruses transmitted by aphids in PI 161375 was found to be conserved in the line Margot, which was obtained after seven back-crosses, (Table 2) and therefore Margot carries Vat-2 (Boissot et al., 2016). This suggests that Vat-1 and Vat-2 are probably very tightly linked. We will therefore use the name ‘Vat’ for the region containing Vat-1 and Vat-2.

Several genomics studies have focused on the region containing Vat. Genes conferring resistance to various pathogens are located in the vicinity of Vat: resistance to Podosphaera xanthii (Yuste-Lisbona et al., 2001; Perchepied et al., 2005a), Cucumber vein yellowing virus (Ibn Oaf, 2012), the Fn gene (Pitrat and Lecoq, 1982) triggering plant necrosis in response to some isolates of ZYMV (Risser et al., 1981), and the quantitative trait loci (QTL) FomV-2 conferring partial resistance to Fusarium oxysporum f. sp. melonis (Perchepied et al., 2005b). The density of resistance genes in melon is highest in the region containing Vat (Garcia-Mas et al., 2012); 28 genes of the NLR family have been identified in a 1-Mb region containing Vat (González et al., 2014). Characterization of four C. melo accessions displaying resistance to viruses when different A. gossypii clones inoculated the plants has identified Vat-1-related sequences (protein identity over 80%; Figures 1 and 2).

These sequences display polymorphisms within all parts of the gene (Figure 2). In the LRR part of Vat, two types of polymorphism are observed: single-nucleotide polymorphisms (SNP) and length polymorphisms. The length polymorphisms occur in a specific domain, domain D or LRR2 (Figure 2) (Dogimont et al., 2008a, 2014). This domain consists of near-perfect repeats of 65 amino acids. The Vat-1-related sequences contain two to five repeats. The repeats are 83.1–89.2% identical (Dogimont et al., 2008a). PI 161375 has a Vat-1-related sequence with three repeats known as Vat-like. Vat-like is located 17 kb from Vat-1 (Figure 1) and is not involved in the resistance elicited by the NM1 clone (Dogimont et al., 2008b). In PI 414723, a line exhibiting resistance to several clones, four Vat-1-related sequences have been identified. One of these sequences has only a few SNPs relative to Vat-1. Two sequences have five repeats in LRR2, and both are strong candidates for the control of resistance to P. xanthii (Dogimont et al., 2008b). Both these Vat-related sequences have few SNPs relative to the sequence of Vat-1. The fourth Vat-1-related sequence has two repeats in LRR2 and more SNPs relative to Vat-1 than the other Vat-1-related sequences. In 90625, a line exhibiting resistance restricted to only one clone, NM1 (Boissot et al., 2016), only one Vat-1-related sequence has been identified. This sequence contains four repeats in LRR2 and several SNPs. In Védrantais, a Charentais line resistant to viruses when only one aphid clone, C4, inoculate the plant (Boissot et al., 2016), two Vat-1-related sequences with several SNPs relative to the sequence of Vat-1 and two repeats in LRR2 have been identified. Finally, the cadre of Vat-like genes in each accession may not be complete, since no complete genome sequences are available for these accessions. Involvement of these Vat-1-related sequences in the aphid resistance has not been demonstrated. Actually, the melon reference genome was built from a line (DHL92) given as susceptible to A. gossypii (González et al., 2014), but accurate double-phenotypic data, with a set of characterized clones, are missing for that line. This line has a Vat-1 homolog, MELOC004317, shorter (1038 aa) than the reference one (1467 aa), in particular the LRR2 part is fully absent (Figure 2) (González et al., 2014).

The presence of large numbers of NLR genes in close proximity to Vat, including Vat-like genes, makes assembly difficult and even unsatisfactory when sequences are obtained for small fragments. Sequencing studies of a set of accessions are required with longer fragments for an accurate assembly of the area. Comprehensive cross-comparison between molecular and phenotypic data is therefore required to obtain a full understanding of the genetic control of resistance to A. gossypii aphids and the viruses they transmit. The use of transgenic lines, even if difficult to obtain (Chovelon et al., 2011), will clearly help us to decipher the role of each locus in this cluster. As soon as candidates are identified, transformation with these candidates could be used to validate their roles in the resistance spectrum. This approach could provide new opportunities for genomic selection for resistance in melon.

The Vat-1 gene belongs to the NLR gene family. According to the general framework developed for this category of resistance genes, its functioning involves separate recognition and response phases (Figure 3A). In this case, the recognition phase involves perception of an aphid effector by the plant’s VAT protein, which may be direct or indirect. The vast majority of plant-pathogen effectors have been shown to interact indirectly with a NLR protein of their host. To date, modality of the interaction between aphid effectors and NLR proteins of their host is still unknown. This interaction should occur in the cytoplasm of cells (the predicted location of the VAT protein), and the effector molecule must therefore be delivered to the plant cell. Aphid mouthparts enclose two flexible stylets that the insect drives into the plant tissue to puncture the phloem, so that they can feed on plant sap. On their way to the phloem, the aphid stylets bend around the cells, and the salivary channel ejects a salivary gel that forms a sheath around the stylets. Along the way to their destination, the aphid stylets briefly puncture cells, into which the salivary channel ejects a watery saliva (Tjallingii, 2006). The effector is probably injected into the cells during these puncture events. No aphid effector capable of interacting with an NLR protein has yet been identified. A transcriptomic approach has been used to identify candidates involved in the virulence of A. gossypii on Vat melon (Dutartre-Fricaux et al., 2014). Genetically similar virulent and avirulent clones were used, and a head-reference transcriptome of more than 33000 contigs was generated by de novo assembly. This reference transcriptome has been used to search for candidate effector genes based on their differential expression and/or presenting sequence polymorphisms between virulent and avirulent aphid clones.

At the molecular plant level, the response phase is thought to involve the activation of a signaling cascade, leading to the rapid accumulation of reactive oxygen species (ROS) and defense hormones. In a Vat melon line, the levels of miRNAs involved in the post-transcriptional regulation of gene expression change rapidly after puncture by A. gossypii (Sattar et al., 2012b). Within 12 h of the infestation by melon aphids, 23 families of miRNAs display modulations. Their potential targets suggest a physiological function in disease and stress responses (5), phytohormone perception and signaling (11), miRNA biogenesis (2), and plant growth and development (Sattar et al., 2016). Ethylene, jasmonic acid and auxin have been identified as potential defense hormones in Vat plants infested with A. gossypii (Anstead et al., 2010; Sattar et al., 2016). In two Vat melon lines, peroxidase activity was found to increase within 10 min of aphid puncture (Sarria Villada et al., 2009). Callose, a polysaccharide usually laid down at plasmodesmata, is deposited within 20 min of aphid infestation, and lignin, a macromolecule derived from phenyl propanoids essential for cell wall thickening, is deposited 4.5 h after aphid infestation. The plasma membrane is damaged and the cells collapse. Callose and lignin are deposited in the wall of cells adjacent to the stylet sheath. These reactions do not occur in non-Vat plants. These data clearly illustrate the massive transcriptional reprogramming induced by A. gossypii infestation in Vat-resistant melon plants, triggering a wide range of plant defense responses (Dogimont and Boissot, 2016; Sattar et al., 2016).

At the molecular aphid level, changes in gene expression were investigated in A. gossypii feeding on Vat and non-Vat plants. There is an unexpectedly high abundance of 27 nt-long sRNA sequences in aphids feeding on Vat plants (Sattar et al., 2012a). These sRNAs belong to the Piwi-interacting RNA (piRNA) family. This class of sRNAs is absent from plants. Their biogenesis in animals is still poorly understood. They have been shown to be involved in the silencing of transposable elements exclusively in animal gonads (Vodovar and Saleh, 2012), facilitating short-term adaptation. Their role in A. gossypii remains unknown, but may relate to the lifting of maternal effects. Such effects are observed, for example, in aphids collected from cotton, which have low rates of reproduction in the first generation after transfer onto eggplant, but higher rates in subsequent generations (Satar et al., 2013). Eighty-one conserved microRNAs (miRNAs), 12 aphid-specific miRNAs, and nine novel candidate miRNAs have also been identified (Sattar et al., 2012a). These candidate miRNAs have been shown to be differentially regulated between aphids feeding on Vat and non-Vat plants and may affect their reproductive rates as described below.

Aphid feeding is disrupted on Vat plants. Electrical-Penetration graph (EPG), in which the pathway of aphid stylets from epidermis to phloem can be followed, have been conducted on several Vat melon lines, with different genetic backgrounds and on melon aphid clones originating from different geographic areas (Kennedy et al., 1978; Chen et al., 1997; Klingler et al., 1998; Boissot et al., 2000; Garzo et al., 2002). The journey of the stylets through the mesophyll to the phloem takes from 90 to 140 min in non-Vat plants (Kennedy et al., 1978; Chen et al., 1997; Klingler et al., 1998; Boissot et al., 2000; Garzo et al., 2002), but is disrupted in Vat plants. The observed cellular response seems to occur after the aphid stylets have punctured plant cells rather than during the intercellular penetration of plant tissues by the stylets (Sarria Villada et al., 2009), consistent with the hypothesis that recognition occurs after the delivery of the effector to the cell. Cytological studies have shown that there are more stylet sheaths in Vat plants than in non-Vat plants (Kennedy et al., 1978), suggesting that early mesophyll cell puncture by A. gossypii may be more frequent in Vat plants. The stylets take longer to reach the phloem in Vat plants than in non-Vat plants and are less likely to reach their final destination in Vat plants than in non-Vat plants. Prior exposure of Vat plants to A. gossypii feeding does not modify the expression of this resistance (Chen et al., 1997).

Findings on Vat melon suggest that the plant responses elicited by short cell punctures either hinder the passage of the stylets between cells due to the deposition of callose and/or lignin in the cell walls, or deter the aphid from progressing further into the tissues, through an oxidative burst detected by aphid after brief periods of ingestion following the release of saliva into the cell. Moreover, melon aphids reaching the phloem of Vat plants do not remain there to feed (less than 10 min, if at all; Kennedy et al., 1978; Chen et al., 1997; Klingler et al., 1998; Boissot et al., 2000; Garzo et al., 2002). This suggests that feeding may be difficult in these plants, possibly due to phloem clogging, although this plant reaction has not yet been described in Vat melon. Few quantitative differences in phloem sap have been identified between Vat and non-Vat plants that might explain the deterrence of A. gossypii (Chen et al., 1997). None of the studies, EPG or histological studies, investigating these aspects took into account the dual phloem structure of cucurbits and the nature of the phloem in which A. gossypii is able to establish feeding.

Aphids escape from Vat plants. In free-choice tests, winged aphids are less numerous on Vat plants 24 h after being offered a choice of plants (Kennedy and Kishaba, 1977), and wingless aphids are less numerous on Vat leaf disks from 30 min after being offered a choice of leaf disks (Garzo et al., 2002). Without choice, i.e., only one plant accession is available, wingless aphids walk away from the plant in the 2–3 days after their deposition. This behavior has been observed on several Vat melon lines, with aphid clones originating from different geographic areas that probably displayed marked genetic differences (Pitrat and Lecoq, 1982; Garzo et al., 2001; Boissot et al., 2010; Thomas et al., 2012b). Based on the timing of these events, we can conclude that early plant responses, occurring rapidly after cell puncture by the aphid, have an immediate effect on aphid behavior.

Aphids poorly reproduce on Vat plants. When wingless aphids are encaged on Vat plants, they display low rates of reproduction, mostly due to a longer pre-reproductive period and a smaller number of progeny (Klingler et al., 1998; Garzo et al., 2002; Boissot et al., 2010). This lower reproductive rate may be directly due to poor, disrupted feeding on the contents of phloem (Kennedy et al., 1978; Chen et al., 1997; Klingler et al., 1998; Boissot et al., 2000; Garzo et al., 2002). This hypothesis is supported indirectly by the observation that aphids produce far less honeydew when feeding on Vat plants than on non-Vat plants (Klingler et al., 1998). The resistance factor reducing the reproductive rate is not transmitted through grafting (Kennedy and Kishaba, 1977), consistent with the notion that feeding is somehow difficult, rather than with the phloem being toxic. The rate of aphid reproduction is quantitatively affected by Vat and QTLs (Kishaba et al., 1976; Boissot et al., 2010; Thomas et al., 2012b).

Vat plants display particularly high levels of resistance to viruses when aphids inoculated unrelated viruses transmitted in the non-persistent mode: 100% of the non-Vat plants displayed symptoms, whereas only 0 to a few per cent of Vat plants had symptoms, with this small number of plants displaying full symptoms (Lecoq et al., 1979; Kishaba et al., 1992; Boissot et al., 2016). When Vat resistance was first discovered, it was thought to block virus transmission, and Pitrat and Lecoq therefore named the gene responsible Vat, for ‘virus aphid transmission’ (Pitrat and Lecoq, 1982). Nevertheless, A. gossypii was subsequently shown to acquire the virus from Vat plants and to transmit it to non-Vat plants (Romanow et al., 1986), calling this hypothesis into question. According to the resistance to transmission hypothesis, a plant factor blocks the virus in the stylet (a molecule or a particular pH). The aphid must, therefore, first ingest material from the plant, before it egests saliva into the cells and delivers the viruses. All EPG studies have shown that, after puncturing cells, the aphid first salivates and then ingests the contents of the cell (Martin et al., 1997). Further studies have failed to demonstrate resistance to transmission due to retention of the virus in the stylets.

Vat resistance to viruses has to be considered in the general framework described for NLR resistance (Boualem et al., 2016). The VAT protein of non-Vat plants (i.e., carrying a ‘susceptible’ Vat allele) cannot recognize (directly or otherwise) the aphid effectors and viruses delivered to the cell by the aphid. This lack of recognition leads to systemic viral infection. In Vat plants, the resistant isoform of VAT recognizes an effector molecule from the aphid. This recognition induces resistance mechanisms limiting the replication and movement of the virus. The micro-oxidative burst triggered by aphid puncture in Vat plants (Sarria Villada et al., 2009) is thought to block the viruses in the inoculated cell or in neighboring cells. Callose deposit at plasmodesmata may help to contain virus particles in the inoculated or neighboring cells. The response is local: when a Vat plant is first inoculated with CMV by A. gossypii, CMV superinoculation with M. persicae on the same leaf leads to systemic infection (Mistral and Boissot, 2016). In the absence of the aphid effector, the recognition phase does not occur when the Vat plant is infected with viruses. In this case, the viruses replicate and move around the plant, establishing a systemic virus infection.

Virus aphid transmission (the initial name from which the Vat acronym is derived) does not provide an accurate picture of the action of the Vat gene product according to recent data. It would, therefore, be more appropriate to consider Vat to stand for ‘virus aphid triggered.’

In Vat plants, no resistance to viruses transmitted in the persistent mode has ever been reported. As CABYV is restricted to phloem cells, the aphid must reach the phloem cells and feed for long enough to acquire virus particles. Mechanical inoculation is not possible for this virus, suggesting that effective inoculation is dependent on the delivery of the virus directly into the phloem. As A. gossypii rarely reaches the phloem of Vat plants (Kennedy et al., 1978; Chen et al., 1997; Klingler et al., 1998; Boissot et al., 2000; Garzo et al., 2002), CABYV acquisition and inoculation should be disrupted in Vat plants, but this point needs to be investigated.

The LRR domain of the Vat-1 gene is subject to diversifying selection (Dogimont et al., 2014). This selection responds to the diversifying selection acting on the avirulence gene, as frequently reported for other avirulence genes in plant pathogens (Rouxel and Balescent, 2010). This model describes the general framework for the molecular arms race between plants and pathogens. An aphid clone adapted to a given Vat allele would either not deliver an ‘avirulent’ effector to the plant (deletion) or would deliver a ‘virulent’ effector that is not recognized by the VAT protein. In both cases, the expected phenotype would be colonization of Vat plant by the clone, and susceptibility to viruses introduced into the plant by that aphid clone (Figure 3B).

This model has been challenged by testing transgenic lines containing the Vat-1 gene, for resistance to both A. gossypii and CMV introduced into the plant by six A. gossypii clones (C6, C9, CUC1, GWD, GWD2, and NM1) (Boissot et al., 2016). The phenotypes for five of the six clones were consistent with the general model (Figures 3A,B): the clones were either unable to fully colonize the Vat-1 transgenic line and triggered resistance to CMV (NM1, C9, Figure 3A), or they fully colonized the Vat-1 transgenic line and did not trigger resistance to CMV (C6, CUC1, GWD, Figure 3B). These five clones belonged to the three clusters corresponding to the cucurbit host-race (Table 1). The phenotypes observed with one clone (GWD2) were not consistent with the general model, with the clone triggering resistance to viruses but nevertheless being able to colonize the transgenic plants (Figure 3C).

Unlinking of resistance to viruses triggered by the aphid and resistance to aphids was confirmed by testing eleven Vat lines identified from the natural range of diversity in melon with nine clones, the six previously described and CUC6, CUC3, and C4 (Boissot et al., 2016). Only 52 of the 117 interactions characterized, considering results on transgenic lines and natural Vat lines, were consistent with the general model (Figures 3A,B). It has been hypothesized that the decoupling of the resistances to aphids and viruses (Figure 3C) results from aphid adaptation, making it possible for the aphid to colonize the plant even if plant defenses are elicited. The Vat phenotype proved to be a highly powerful tool for investigating a phenomenon never before studied for plant/pathogen interactions. This new model for adaptation to NLR resistance is revealed by the double phenotype, which can be used to follow the resistance process at two levels: recognition, and the efficacy of the plant defenses triggered.

We speculate that individuals of some clones adapted to Vat defenses they trigger (e.g., GWD2). If these aphids infest a Vat plant successfully, they must accept the Vat plant and reproduce at a high rate on it. EPG has revealed that Vat affects the exploratory behavior of the aphid on the plant, but this effect is quantitative, with some aphids reaching the phloem of Vat plants. Individuals of a clone adapted to Vat defenses probably reach the phloem more often than those of a non-adapted clone. A ‘classical avenue’ of research will involve comparison of the transcriptomes of adapted and non-adapted clones puncturing Vat plants, to track the aphid genes involved in this adaptation. We propose to explore a new avenue of research: does the dual phloem structure of cucurbits, and of melon in particular, play a role in this adaptation?

It is possible a priori that viruses transmitted in the non-persistent mode can overcome Vat-mediated resistance. In this scenario, viral variants may multiply and leave the punctured cells before the defense mechanisms are fully effective, resulting in the development of systemic infections. The expected phenotype would be ‘susceptibility to viruses introduced by an aphid clone that is incapable of colonizing Vat plants’ (Figure 3D). To date, this double phenotype has never been observed in a transgenic line carrying the Vat-1 gene, suggesting that viral adaptation may not occur (Boissot et al., 2016). Consistent with these findings, experimental evolution experiments with viruses on Vat-plants have been unsuccessful. Sequential virus transmissions from infected Vat melon plants to healthy Vat melon plants were established with two aphid clones and three viruses, CMV, ZYMV, and WMV. None of these viruses evolved in response to the resistance triggered by these two clones, even when four sequential virus transmissions could be done (Boissot et al., 2016). These results strongly suggest that viruses transmitted in the non-persistent mode do not readily adapt to the Vat resistance triggered by A. gossypii.

Aphid density is lower on Vat plants than on non-Vat plants (Thomas et al., 2016). With the aim of quantifying the effect of Vat at crop level, we compiled bibliographic data for a density index for Vat and non-Vat plants grown in field experiments conducted at the same location (Schoeny et al., 2014; Thomas et al., 2016). The aphid density index was 44% lower on Vat plants (Figure 4). This index is related to aphid density per m² over the entire cropping period, by an exponential relationship (y = 1463e^0.1088x with r² = 0.72, n = 304). Therefore, for non-Vat plant indexes of 90, 50, and 30, aphid density is reduced by factors of 50, 11, and 4, respectively, on Vat plants.

There are four key phases in the dynamics of crop infestation by aphids: visiting by winged aphids, infestation with the wingless nymphs they generate, development into colonies, and production of winged individuals for dispersal. Melon crops are visited by spring migrant aphids of numerous species. The proportion of A. gossypii among the visiting aphids and the genetic composition of the A. gossypii spring migrant population depend on geographic area (Thomas et al., 2016). Only some of the A. gossypii spring migrants generate progeny (Figure 5), mostly specializing on cucurbits (i.e., belonging to the Cucurbits I, II, and III genetic clusters). This selection leads to a significant decrease in clonal richness between the spring migrant and wingless populations on melon plants (Thomas et al., 2012a), and this decrease continues during subsequent steps, reflecting differences in fitness or competition between clones on melons (Thomas et al., 2016) (Figure 5).

The effect of Vat on the first step of infestation has never been reported at field level. However, Vat plants were found to be less attractive for winged aphids than non-Vat plants in greenhouse experiments based on artificial infestation (Kennedy and Kishaba, 1977). The effect of Vat on subsequent phases of infestation has been characterized in open-field melon crops under natural infestation conditions, but only in French melon production areas and in the Lesser Antilles (Thomas et al., 2016). The wingless populations on Vat plants have a genetic composition different from that of the populations on non-Vat plants, as clonal richness and clonal diversity decreased during infestation. Aphids from group III of the cucurbit host-race are eliminated in favor of aphids from group I. In French production areas, the third step, colony development, is erratic on melon crops, and, in the presence of Vat, this step is very rarely observed, generally in only a few aphid genotypes from group I or II. The fourth phase, the production of winged individuals for dispersal, is density dependent (Dixon, 1985), and therefore very rare on Vat plants. The populations of A. gossypii occurring in French melon crops contain a large proportion of aphids from genetic group III, and the decrease in density and diversity on Vat plants probably reflects selection rather than competition. Consistent with this hypothesis, a laboratory study showed that Vat-mediated resistance affected the population growth of 90% of group III clones, but only 40% of those from group I or II (Lombaert et al., 2009). Only group I aphids are present in the Lesser Antilles, where the third and fourth steps are regularly reached, even on Vat plants. The effect of Vat on aphid clonal richness is illustrated in Figure 5. Only four clones, CUC1, CUC6, GWD, and C6, have regularly been identified in colonies on Vat plants in field experiments (Thomas et al., 2016) and studied in laboratory experiments (Boissot et al., 2016). C6 does not trigger resistance to CMV and probably has a virulent effector not recognized by Vat, whereas CUC1, CUC6, and GWD trigger resistance to CMV and are adapted to plant response.

An ecological genetics analyze of melon-aphid dynamics has been applied in three different agricultural systems over the last decade, to predict the durability of Vat resistance to A. gossypii (Thomas et al., 2016). It appears that A. gossypii is evolving at a regional level in response to the deployment of Vat melon crops. For example, two different bottlenecks affect the dynamics of adapted clones in melon-producing areas, due to (i) the low levels of dispersal morph production on Vat melon and (ii) the winter extinction of clones. The low levels of dispersal morph production result from the containment of populations at levels of crowding below that required to induce the production of winged morphs. Winter extinction occurs due to the absence of other cucurbit crops to serve as hosts between two melon crop cycles, limiting the maintenance of Vat-adapted clones. In melon-producing areas without bottlenecks (such as the Lesser Antilles), resistance is predicted to be not durable. In areas in which both types of bottlenecks occur (such as South-West France), resistance is predicted to be durable. In South-East France, only one of the two bottlenecks occurs, and cucurbits are cultivated almost year-round. Moreover, in South-East France Vat melons have been cultivated at a large scale since 2000, and Vat resistance is now jeopardized by the emergence of adapted clones. These findings suggest that, for a cosmopolitan pest, such as A. gossypii, decisions concerning resistance deployment should take into account the genetic structure of the pest population at regional scale, the availability of winter host plants for adapted biotypes between crop cycles (Thomas et al., 2016) and the allele composition of the Vat cluster.

The manipulation of agricultural systems to increase the durability of Vat resistance through winter extinction does not appear to be feasible. However, it may be possible to increase durability by preventing the production of dispersal morphs from adapted clones. Different ways of achieving this aim have been investigated. The use of strips of flowering plants sown close to Vat melon crops to attract natural enemies has been investigated (Schoeny et al., 2014), but the efficacy of this approach will need to be confirmed over several years. Alternatively, QTLs decreasing the production of dispersal morphs on Vat plants could be sought. The identification of such QTLs is probably feasible in melon accessions displaying resistance to aphids sensu stricto (no elicitation of resistance to virus by A. gossypii). This phenotype, like classical phenotypes of resistance to aphids described in other crops, has already been observed in the natural range of melon diversity (Boissot et al., 2016). QTLs controlling this phenotype could be combined with Vat resistance in a melon breeding program.

The effect of Vat on virus epidemics is poorly documented. Field experiments were conducted in France in the late 1970s to compare the development of CMV in Vat accession PI 161375 and a non-Vat melon cultivar (Lecoq and Pitrat, 1983). CMV progression curves had the same general “S” shape, with a steep slope, but disease onset was always earlier in the susceptible plots, with symptoms observed 12–24 days later in Vat plots than in non-Vat plots. This evaluation continued into the 1980s, with the resistant cultivar Virgos (Lecoq and Pitrat, 1989). In accordance with previous results, resistance delayed the CMV epidemic development and greatly decreased the rate of disease increase. It should be borne in mind that PI 161375 and Virgos carry composite resistance to CMV: Vat and the oligogenic and recessive resistance to ‘common’ CMV strains (Guiu-Aragones et al., 2014). It is, therefore, difficult to determine the actual contribution of Vat to the control of CMV epidemics. These experiments were more informative for WMV, because ‘resistance to common CMV strains’ is not effective against WMV. In Vat melon plots, WMV epidemics were delayed slightly (by about 5 days), with no significant reduction of the rate of disease increase (Lecoq and Pitrat, 1989).

Recent studies in South-East France compared virus epidemic development in melon lines differing only by the presence/absence of Vat (Schoeny et al., 2014; Boissot et al., 2015). In most field trials, Vat had a significant effect on CMV epidemics, mostly by reducing the rate of disease increase. It had no effect on WMV epidemics, probably because A. gossypii is not the principal aphid vector of this virus.

The partial effect of Vat on CMV epidemics is consistent with Vat resistance being elicited by only a proportion of the viruliferous aphids visiting melon crops. Indeed, more than 80 aphid species are able to transmit CMV, therefore viruliferous aphids belonging to these species trigger epidemics when they visit Vat and non-Vat melon crops. Moreover within A. gossypii, it remains unclear whether A. gossypii not belonging to the Cucurbit host-race can elicit resistance to CMV resistance. Nevertheless, the partial effect of Vat on CMV epidemics remains significant, probably because A. gossypii is one of the most efficient vectors of CMV based on laboratory experiments.

Finally, the use of Vat to control the spread of non-persistent viruses in melon crops is dependent on the importance of viruliferous A. gossypii relative to other vector species in the spread of the virus in the crop. The effect of Vat is not sufficient for the full control of virus epidemics in crops, but the broad spectrum of this effect and the inability of viruses to adapt to it (Boissot et al., 2016) have made this type of resistance much of a ‘holy grail’ for plant breeders. In the postgenomic era, it may be possible to edit this resistance gene to make it possible for any aphid species to trigger resistance or for resistance to occur without the need for aphid triggering.

Concerning persistent viruses, Vat steadily and significantly decreases CABYV epidemics, mostly by delaying them (Schoeny et al., 2014; Boissot et al., 2015). A. gossypii is the principal vector of CABYV so, even though this aspect has not yet been investigated in the laboratory, it appears likely that Vat affects CABYV transmission by decreasing both acquisition and inoculation rates. The effect of Vat on CABYV population genetic diversity has not yet been documented.

Vat resistance has only a partial effect on virus epidemics in melon and is not used in that aim by growers. As a matter of fact resistance to viruses sensu stricto needs to be integrated in cultivars to control virus epidemics in crops. This type of resistance to viruses has been identified in C. melo species, but has generally been little used in plant breeding programs (Pitrat, 2016). If deployed at a large scale in melon crops, such resistance would exert a selection pressure on viruses, placing the durability of the resistance at risk. The utility of combining Vat with resistance to viruses sensu stricto has been investigated for CMV and CABYV (Boissot et al., 2015). The epidemic data obtained for Vat and non-Vat melon crops in South-East France have been integrated into a mathematical model of the evolutionary and epidemiological processes shaping the dynamics of a virus population in a landscape composed of a seasonal cultivated compartment and a reservoir compartment containing virus throughout the year (Fabre et al., 2015). Various agro-ecological systems were considered, mimicking the situation of melon crops in South-East France. The deployment of resistance to viruses sensu stricto combined with Vat would probably be beneficial for CABYV control, but the potential benefit remains uncertain (although certainly not negative) for the long-term control of CMV. Another modeling study has suggested that the maintenance of low-population aphid populations could prevent the emergence of highly virulent CMV+N-satRNA isolates (Betancourt et al., 2016).

Finally, since the description of the ‘aphid side’ of the pleiotropic phenotype of Vat in the late 1960s, each decade has contributed to improvements in our knowledge and use of this amazing gene. The ‘virus side’ of the pleiotropic phenotype was elucidated in the late 1970s, with the breeding and deployment of the first Vat cultivars in the 1980s, and mapping in the 1990s. The assignment of this gene to the NLR gene family in the first decade of the 21st century provided clues to its mode of action, which is now at least partially understood. A succession of new technologies over this period provided new insight into the pleiotropic phenotype of Vat. Our knowledge of the genetic diversity of A. gossypii has also been refined over time. A. gossypii genetic diversity presents a major challenge to Vat resistance in the field, but also provides us with opportunities to extend our knowledge of the mechanisms underlying Vat resistance.

The A. gossypii/melon interaction can be investigated within the broader A. gossypii/cucurbit interaction, for at least two points. First, the double phenotype conferred by Vat makes it possible to investigate this interaction over the subgroup of A. gossypii constituting the Cucurbit host-race. How diverse are the A. gossypii strains able to elicit resistance to viruses? The VAT protein probably interacts with ligands present in the A. gossypii species or in the A. gossypii group. Once the avirulence factor interacting with the VAT protein has been identified, it will be possible to perform genetic diversity studies on that factor. Second, the particular structure of the phloem in cucurbits may play a key role in the specialization of A. gossypii, an insect feeding on plant sap, on cucurbits and in adaptation to Vat resistance, which decreases the access of A. gossypii to the phloem.

The double phenotype can also be used as a tool for ‘reading’ the recognition phase independently of the response phase, whether this response is considered in terms of the response of the plant, or that of the aphid. Promising preliminary results have been obtained with this approach, and the double phenotype could be more extensively used for such studies. The observation that some A. gossypii clones trigger resistance responses in Vat plants and are adapted to this response provides new insight into the capacity of pests and pathogens to adapt to NLR-mediated resistance in plants. The general framework for resistance mediated by such genes is that, within a pest/pathogen species, a clone/isolate is considered to have adapted if it does not trigger NLR-mediated resistance (i.e., there is no recognition phase). The models proposed for Vat/A. gossypii interaction suggest that aphid clones are adapted to Vat plants either because their avirulence factors do not trigger resistance or because they can colonize the plants even if plant defenses are triggered. Does this second mechanism exist in other NLR plant resistance/pest or pathogen interactions? If so, it would complicate the identification of avirulence factors, because adapted and non-adapted pests/pathogens could have identical avirulence effectors interacting (directly or indirectly) with the protein encoded by the resistance gene. It would also call into question the validity of durability modeling approaches based exclusively on gene-for-gene interaction.

The second way in which A. gossypii clones can adapt, such that the A. gossypii clones can colonize Vat plants whilst eliciting resistance to viruses, appears to be the most common mechanism in A. gossypii populations developing colonies on Vat crops, raising questions about the evolutionary advantages of such a mode of adaptation. This type of adaptation increases the chances of A. gossypii being able to colonize a melon crop free of viruses (because the viruses transmitted by this aphid are blocked in Vat plants), probably leading to the production of a larger number of progeny. Further studies are required to assess the advantages of these two types of adaptation.

Many different studies of Vat resistance have been carried out, revealing the considerable utility of this gene for addressing research questions and the limitations of the use of resistance genes in agriculture. One key question remains: is this gene really unique among the genes conferring resistance to aphids? No other aphid resistance gene has been reported to confer resistance to viruses transmitted in the non-persistent mode. However, it is not clear how many of the many known aphid resistance genes have been tested for effects on viruses.

NB conceived the review and built the figures and tables. AS contributed to the introduction’, the presentation of viruses transmitted by A. gossypii to melon crops, to molecular features to the effect of Vat at the whole-plant level, Vat effect on viruses populations and conclusion. She revised the entire manuscript. FV-M contributed to the presentation of A. gossypii and the part on Vat effect on A. gossypii populations. She revised the entire manuscript.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

The reviewer IK and handling Editor declared their shared affiliation, and the handling Editor states that the process nevertheless met the standards of a fair and objective review.