Mutations in CmVPS41 controlling resistance to cucumber mosaic virus display specific subcellular localization.

Resistance to cucumber mosaic virus (CMV) in melon (Cucumis melo L.) has been described in several exotic accessions and is controlled by a recessive resistance gene, cmv1, that encodes a vacuolar protein sorting 41 (CmVPS41). cmv1 prevents systemic infection by restricting the virus to the bundle sheath cells, preventing viral phloem entry. CmVPS41 from different resistant accessions carries two causal mutations, either a G85E change, found in Pat-81 and Freeman's cucumber, or L348R, found in PI161375, cultivar Songwhan Charmi (SC). Here, we analyzed the subcellular localization of CmVPS41 in Nicotiana benthamiana and found differential structures in resistant and susceptible accessions. Susceptible accessions showed nuclear and membrane spots and many transvacuolar strands, whereas the resistant accessions showed many intravacuolar invaginations. These specific structures colocalized with late endosomes. Artificial CmVPS41 carrying individual mutations causing resistance in the genetic background of CmVPS41 from the susceptible variety Piel de Sapo (PS) revealed that the structure most correlated with resistance was the absence of transvacuolar strands. Coexpression of CmVPS41 with viral movement proteins, the determinant of virulence, did not change these localizations; however, infiltration of CmVPS41 from either SC or PS accessions in CMV-infected N. benthamiana leaves showed a localization pattern closer to each other, with up to 30% cells showing some membrane spots in the CmVPS41SC and fewer transvacuolar strands (reduced from a mean of 4 to 1-2) with CmVPS41PS. Our results suggest that the distribution of CmVPS41PS in late endosomes includes transvacuolar strands that facilitate CMV infection and that CmVPS41 re-localizes during viral infection.

CRISPR/Cas9 gene editing uncovers the roles of CONSTITUTIVE TRIPLE RESPONSE 1 and REPRESSOR OF SILENCING 1 in melon fruit ripening and epigenetic regulation.

Melon (Cucumis melo) has emerged as an alternative model to tomato for studying fruit ripening due to the coexistence of climacteric and non-climacteric varieties. Previous characterization of a major quantitative trait locus (QTL), ETHQV8.1, that is able to trigger climacteric ripening in a non-climacteric background resulted in the identification of a negative regulator of ripening CTR1-like (MELO3C024518) and a putative DNA demethylase ROS1 (MELO3C024516) that is the orthologue of DML2, a DNA demethylase that regulates fruit ripening in tomato. To understand the role of these genes in climacteric ripening, in this study we generated homozygous CRISPR knockout mutants of CTR1-like and ROS1 in a climacteric genetic background. The climacteric behavior was altered in both loss-of-function mutants in two growing seasons with an earlier ethylene production profile being observed compared to the climacteric wild type, suggesting a role of both genes in climacteric ripening in melon. Single-cytosine methylome analyses of the ROS1-knockout mutant revealed changes in DNA methylation in the promoter regions of the key ripening genes such as ACS1, ETR1, and ACO1, and in transcription factors associated with ripening including NAC-NOR, RIN, and CNR, suggesting the importance of ROS1-mediated DNA demethylation for triggering fruit ripening in melon.

Quantitative trait loci analysis of melon (Cucumis melo L.) domestication-related traits.

KEY MESSAGE: Loci on LGIV, VI, and VIII of melon genome are involved in the control of fruit domestication-related traits and they are candidate to have played a role in the domestication of the crop. The fruit of wild melons is very small (20-50 g) without edible pulp, contrasting with the large size and high pulp content of cultivated melon fruits. An analysis of quantitative trait loci (QTL) controlling fruit morphology domestication-related traits was carried out using an in vitro maintained F2 population from the cross between the Indian wild melon "Trigonus" and the western elite cultivar 'Piel de Sapo'. Twenty-seven QTL were identified in at least two out of the three field trials. Six of them were also being detected in BC1 and BC3 populations derived from the same cross. Ten of them were related to fruit morphological traits, 12 to fruit size characters, and 5 to pulp content. The Trigonus alleles decreased the value of the characters, except for the QTL at andromonoecious gene at linkage group (LG) II, and the QTL for pulp content at LGV. QTL genotypes accounted for a considerable degree of the total phenotypic variation, reaching up to 46%. Around 66% of the QTL showed additive gene action, 19% exhibited dominance, and 25% consisted of overdominance. The regions on LGIV, VI, and VIII included the QTL with more consistent and strong effects on domestication-related traits. QTLs on those regions were validated in BC2S1, BC2S2, and BC3 families, with "Trigonus" allele decreasing the fruit morphological traits in all cases. The validated QTL could represent loci involved in melon domestication, although further experiments as genomic variation studies across wild and cultivated genotypes would be necessary to confirm this hypothesis.

Combined use of genetic and genomics resources to understand virus resistance and fruit quality traits in melon.

The availability of the genome sequence of many crop species during the past few years has opened a new era in plant biology, allowing for the performance of massive genomic studies in plant species other than the classical models Arabidopsis and rice. One of these crop species is melon (Cucumis melo), a cucurbit of high economic value that has become an interesting model for the study of biological processes such as fruit ripening, sex determination and phloem transport. The recent availability of the melon genome sequence, together with a number of genetic and genomic resources, provides powerful tools that can be used to assist in the main melon breeding targets, namely disease resistance and fruit quality. In this review, we will describe recent data obtained combining the use of a melon near isogenic line (NIL) population and genomic resources to gain insight into agronomically important traits as fruit ripening, resistance to Cucumber Mosaic virus (CMV) and the accumulation of sugars in fruits.

Selective silencing of 2Cys and type-IIB Peroxiredoxins discloses their roles in cell redox state and stress signaling.

Peroxiredoxins (Prx) catalyse the reduction of hydrogen peroxide (H2O2) and, in association with catalases and other peroxidases, may participate in signal transduction by regulating intercellular H2O2 concentration that in turn can control gene transcription and cell signaling. Using virus-induced-gene-silencing (VIGS), 2-Cys Peroxiredoxin (2CysPrx) family and type-II Peroxiredoxin B (PrxIIB) gene were silenced in Nicotiana benthamiana, to study the impact that the loss of function of each Prx would have in the antioxidant system under control (22 °C) and severe heat stress conditions (48 °C). The results showed that both Prxs, although in different organelles, influence the regeneration of ascorbate to a significant extent, but with different purposes. 2CysPrx affects abscisic acid (ABA) biosynthesis through ascorbate, while PrxIIB does it probably through the xanthophyll cycle. Moreover, 2CysPrx is key in H2O2 scavenging and in consequence in the regulation of ABA signaling downstream of reactive oxygen species and PrxIIB provides an important assistance for H2O2 peroxisome scavenges.

cmv1-Mediated Resistance to CMV in Melon Can Be Overcome by Mixed Infections with Potyviruses.

Resistance to cucumber mosaic virus (CMV) strain LS in melon is controlled by the gene cmv1, which restricts phloem entry. In nature, CMV is commonly found in mixed infections, particularly with potyviruses, where a synergistic effect is frequently produced. We have explored the possibility that this synergism could help CMV-LS to overcome cmv1-mediated resistance. We demonstrate that during mixed infection with a potyvirus, CMV-LS is able to overcome cmv1-controlled resistance and develop a systemic infection and that this ability does not depend on an increased accumulation of CMV-LS in mechanically inoculated cotyledons. Likewise, during a mixed infection initiated by aphids, the natural vector of both cucumoviruses and potyviruses that can very efficiently inoculate plants with a low number of virions, CMV-LS also overcomes cmv1-controlled resistance. This indicates that in the presence of a potyvirus, even a very low amount of inoculum, can be sufficient to surpass the resistance and initiate the infection. These results indicate that there is an important risk for this resistance to be broken in nature as a consequence of mixed infections, and therefore, its deployment in elite cultivars would not be enough to ensure a long-lasting resistance.

Knock-Out of CmNAC-NOR Affects Melon Climacteric Fruit Ripening.

Fruit ripening is an important process that affects fruit quality. A QTL in melon, ETHQV6.3, involved in climacteric ripening regulation, has been found to be encoded by CmNAC-NOR, a homologue of the tomato NOR gene. To further investigate CmNAC-NOR function, we obtained two CRISPR/Cas9-mediated mutants (nor-3 and nor-1) in the climacteric Védrantais background. nor-3, containing a 3-bp deletion altering the NAC domain A, resulted in ~8 days delay in ripening without affecting fruit quality. In contrast, the 1-bp deletion in nor-1 resulted in a fully disrupted NAC domain, which completely blocked climacteric ripening. The nor-1 fruits did not produce ethylene, no abscission layer was formed and there was no external color change. Additionally, volatile components were dramatically altered, seeds were not well developed and flesh firmness was also altered. There was a delay in fruit ripening with the nor-1 allele in heterozygosis of ~20 days. Our results provide new information regarding the function of CmNAC-NOR in melon fruit ripening, suggesting that it is a potential target for modulating shelf life in commercial climacteric melon varieties.

Modulating climacteric intensity in melon through QTL stacking.

Fruit ripening is one of the main processes affecting fruit quality and shelf life. In melon there are both climacteric and non-climacteric genotypes, making it a suitable species to study fruit ripening. In the current study, in order to fine tune ripening, we have pyramided three climacteric QTLs in the non-climacteric genotype "Piel de Sapo": ETHQB3.5, ETHQV6.3 and ETHQV8.1. The results showed that the three QTLs interact epistatically, affecting ethylene production and ripening-related traits such as aroma profile. Each individual QTL has a specific role in the ethylene production profile. ETHQB3.5 accelerates the ethylene peak, ETHQV6.3 advances the ethylene production and ETHQV8.1 enhances the effect of the other two QTLs. Regarding aroma, the three QTLs independently activated the production of esters changing the aroma profile of the fruits, with no significant effects in fruit firmness, soluble solid content and fruit size. Understanding the interaction and the effect of different ripening QTLs offers a powerful knowledge for candidate gene identification as well as for melon breeding programs, where fruit ripening is one of the main objectives.

Fruit Morphology and Ripening-Related QTLs in a Newly Developed Introgression Line Collection of the Elite Varieties 'Védrantais' and 'Piel de Sapo'.

Melon is an economically important crop with widely diverse fruit morphology and ripening characteristics. Its diploid sequenced genome and multiple genomic tools make this species suitable to study the genetic architecture of fruit traits. With the development of this introgression line population of the elite varieties 'Piel de Sapo' and 'Védrantais', we present a powerful tool to study fruit morphology and ripening traits that can also facilitate characterization or pyramidation of QTLs in inodorous melon types. The population consists of 36 lines covering almost 98% of the melon genome, with an average of three introgressions per chromosome and segregating for multiple fruit traits: morphology, ripening and quality. High variability in fruit morphology was found within the population, with 24 QTLs affecting six different traits, confirming previously reported QTLs and two newly detected QTLs, FLQW5.1 and FWQW7.1. We detected 20 QTLs affecting fruit ripening traits, six of them reported for the first time, two affecting the timing of yellowing of the rind (EYELLQW1.1 and EYELLQW8.1) and four at the end of chromosome 8 affecting aroma, abscission and harvest date (EAROQW8.3, EALFQW8.3, ABSQW8.3 and HARQW8.3). We also confirmed the location of several QTLs, such as fruit-quality-related QTLs affecting rind and flesh appearance and flesh firmness.

A novel introgression line collection to unravel the genetics of climacteric ripening and fruit quality in melon.

Introgression lines are valuable germplasm for scientists and breeders, since they ease genetic studies such as QTL interactions and positional cloning as well as the introduction of favorable alleles into elite varieties. We developed a novel introgression line collection in melon using two commercial European varieties with different ripening behavior, the climacteric cantalupensis 'Védrantais' as recurrent parent and the non-climacteric inodorus 'Piel de Sapo' as donor parent. The collection contains 34 introgression lines, covering 99% of the donor genome. The mean introgression size is 18.16 Mb and ~ 3 lines were obtained per chromosome, on average. The high segregation of these lines for multiple fruit quality traits allowed us to identify 27 QTLs that modified sugar content, altered fruit morphology or were involved in climacteric ripening. In addition, we confirmed the genomic location of five major genes previously described, which control mainly fruit appearance, such as mottled rind and external color. Most of the QTLs had been reported before in other populations sharing parental lines, while three QTLs (EAROQP11.3, ECDQP11.2 and FIRQP4.1) were newly detected in our work. These introgression lines would be useful to perform additional genetic studies, as fine mapping and gene pyramiding, especially for important complex traits such as fruit weight and climacteric ripening.

Shaping melons: agronomic and genetic characterization of QTLs that modify melon fruit morphology.

The consistency of quantitative trait locus (QTL) effects among genetic backgrounds is a key factor for introgressing QTLs from initial mapping experiments into applied breeding programs. We have selected four QTLs (fs6.4, fw4.3, fw4.4 and fw8.1) involved in melon fruit morphology that had previously been detected in a collection of introgression lines derived from the cross between a Spanish cultivar, "Piel de Sapo," and the Korean accession PI161375 (Songwan Charmi). Introgression lines harboring these QTLs were crossed with an array of melon inbred lines representative of the most important cultivar types. Hybrids of the introgression and inbred lines, with the appropriate controls, were evaluated in replicated agronomic trials. The effects of the QTLs were consistent among the different genetic backgrounds, demonstrating the utility of these QTLs for applied breeding programs in modifying melon fruit morphology. Three QTLs, fw4.4, fs6.4 and fs12.1 were subjected to further study in order to map them more accurately by substitution mapping using a new set of introgression lines with recombination events within the QTL chromosome region. The position of the QTLs was narrowed down to 36-5 cM, depending on the QTL. The results presented in the current study set the basis for the use of these QTLs in applied breeding programs and for the molecular characterization of the genes underlying them.

CmVPS41 Is a General Gatekeeper for Resistance to Cucumber Mosaic Virus Phloem Entry in Melon.

Melon production is often compromised by viral diseases, which cannot be treated with chemicals. Therefore, the use of genetic resistances is the main strategy for generating crops resistant to viruses. Resistance to Cucumber mosaic virus (CMV) in melon is scarcely described in few accessions. Until recently, the only known resistant accessions were Freeman's Cucumber and PI 161375, cultivar Songwhan Charmi (SC). Resistance to CMV in melon is recessive and generally oligogenic and quantitative. However, in SC, the resistance to CMV strains of subgroup II is monogenic, depending only on one gene, cmv1, which is able to stop CMV movement by restricting the virus to the bundle sheath cells and preventing a systemic infection. This restriction depends on the viral movement protein (MP). Chimeric viruses carrying the MP of subgroup II strains, like the strain LS (CMV-LS), are restricted in the bundle sheath cells, whereas those carrying MP from subgroup I, like the strain FNY (CMV-FNY), are able to overcome this restriction. cmv1 encodes a vacuolar protein sorting 41 (CmVPS41), a protein involved in the transport of cargo proteins from the Golgi to the vacuole through late endosomes. We have analyzed the variability of the gene CmVPS41 in a set of 52 melon accessions belonging to 15 melon groups, both from the spp melo and the spp agrestis. We have identified 16 different haplotypes, encoding 12 different CmVPS41 protein variants. Challenging members of all haplotypes with CMV-LS, we have identified nine new resistant accessions. The resistance correlates with the presence of two mutations, either L348R, previously found in the accession SC and present in other three melon genotypes, or G85E, present in Freeman's Cucumber and found also in four additional melon genotypes. Moreover, the new resistant accessions belong to three different melon horticultural groups, Conomon, Makuwa, and Dudaim. In the new resistant accessions, the virus was able to replicate and move cell to cell, but was not able to reach the phloem. Therefore, resistance to phloem entry seems to be a general strategy in melon controlled by CmVPS41. Finally, the newly reported resistant accessions broaden the possibilities for the use of genetic resistances in new melon breeding strategies.

A mutation in the melon Vacuolar Protein Sorting 41prevents systemic infection of Cucumber mosaic virus.

In the melon exotic accession PI 161375, the gene cmv1, confers recessive resistance to Cucumber mosaic virus (CMV) strains of subgroup II. cmv1 prevents the systemic infection by restricting the virus to the bundle sheath cells and impeding viral loading to the phloem. Here we report the fine mapping and cloning of cmv1. Screening of an F2 population reduced the cmv1 region to a 132 Kb interval that includes a Vacuolar Protein Sorting 41 gene. CmVPS41 is conserved among plants, animals and yeast and is required for post-Golgi vesicle trafficking towards the vacuole. We have validated CmVPS41 as the gene responsible for the resistance, both by generating CMV susceptible transgenic melon plants, expressing the susceptible allele in the resistant cultivar and by characterizing CmVPS41 TILLING mutants with reduced susceptibility to CMV. Finally, a core collection of 52 melon accessions allowed us to identify a single amino acid substitution (L348R) as the only polymorphism associated with the resistant phenotype. CmVPS41 is the first natural recessive resistance gene found to be involved in viral transport and its cellular function suggests that CMV might use CmVPS41 for its own transport towards the phloem.

cmv1 is a gate for Cucumber mosaic virus transport from bundle sheath cells to phloem in melon.

Cucumber mosaic virus (CMV) has the broadest host range among plant viruses, causing enormous losses in agriculture. In melon, strains of subgroup II are unable to establish a systemic infection in the near-isogenic line SC12-1-99, which carries the recessive resistance gene cmv1 from the accession PI 161375, cultivar 'Songwhan Charmi'. Strains of subgroup I overcome cmv1 resistance in a manner dependent on the movement protein. We characterized the resistance conferred by cmv1 and established that CMV-LS (subgroup II) can move from cell to cell up to the veins in the inoculated leaf, but cannot enter the phloem. Immunogold labelling at transmission electron microscopy level showed that CMV-LS remains restricted to the bundle sheath (BS) cells in the resistant line, and does not invade vascular parenchyma or intermediary cells, whereas, in the susceptible line 'Piel de Sapo' (PS), the virus invades all vein cell types. These observations indicate that the resistant allele of cmv1 restricts systemic infection in a virus strain- and cell type-specific manner by acting as an important gatekeeper for virus progression from BS cells to phloem cells. Graft inoculation experiments showed that CMV-LS cannot move from the infected PS stock into the resistant cmv1 scion, thus suggesting an additional role for cmv1 related to CMV transport within or exit from the phloem. The characterization of this new form of recessive resistance, based on a restriction of virus systemic movement, opens up the possibility to design alternative approaches for breeding strategies in melon.

Four sequence positions of the movement protein of Cucumber mosaic virus determine the virulence against cmv1-mediated resistance in melon.

The resistance to a set of strains of Cucumber mosaic virus (CMV) in the melon accession PI 161375, cultivar 'Songwhan Charmi', is dependent on one recessive gene, cmv1, which confers total resistance, whereas a second set of strains is able to overcome it. We tested 11 strains of CMV subgroups I and II in the melon line SC12-1-99, which carries the gene cmv1, and showed that this gene confers resistance to strains of subgroup II only and that restriction is not related to either viral replication or cell-to-cell movement. This is the first time that a resistant trait has been correlated with CMV subgroups. Using infectious clones of the CMV strains LS (subgroup II) and FNY (subgroup I), we generated rearrangements and viral chimaeras between both strains and established that the determinant of virulence against the gene cmv1 resides in the first 209 amino acids of the movement protein, as this region from FNY is sufficient to confer virulence to the LS clone in the line SC12-1-99. A comparison of the sequences of the strains of both subgroups in this region shows that there are five main positions shared by all strains of subgroup II, which are different from those of subgroup I. Site-directed mutagenesis of the CMV-LS clone to substitute these residues for those of CMV-FNY revealed that a combination of four of these changes [the group 64-68 (SNNLL to HGRIA), and the point mutations R81C, G171T and A195I] was required for a complete gain of function of the LS MP in the resistant melon plant.

RNA silencing suppression by a second copy of the P1 serine protease of Cucumber vein yellowing ipomovirus, a member of the family Potyviridae that lacks the cysteine protease HCPro.

The P1 protein of viruses of the family Potyviridae is a serine proteinase, which is highly variable in length and sequence, and its role in the virus infection cycle is not clear. One of the proposed activities of P1 is to assist HCPro, the product that viruses of the genus Potyvirus use to counteract antiviral defense mediated by RNA silencing. Indeed, an HCPro-coding region is present in all the genomes of members of the genera Potyvirus, Rymovirus, and Tritimovirus that have been sequenced. However, it was recently reported that a sequence coding for HCPro is lacking in the genome of Cucumber vein yellowing virus (CVYV), a member of the genus Ipomovirus, the fourth monopartite genus of the family. In this study, we provide further evidence that P1 enhances the activity of HCPro in members of the genus Potyvirus and show that it is duplicated in the ipomovirus CVYV. The two CVYV P1 copies are arranged in tandem, and the second copy (P1b) has RNA silencing suppression activity. CVYV P1b suppressed RNA silencing induced either by sense green fluorescent protein (GFP) mRNA or by a GFP inverted repeat RNA, indicating that CVYV P1b acts downstream of the formation of double-stranded RNA. CVYV P1b also suppressed local silencing in agroinfiltrated patches of transgenic Nicotiana benthamiana line 16c and delayed its propagation to the neighboring cells. However, neither the short-distance nor long-distance systemic spread of silencing of the GFP transgene was completely blocked by CVYV P1b. CVYV P1b and P1-HCPro from the potyvirus Plum pox virus showed very similar behaviors in all the assays carried out, suggesting that evolution has found a way to counteract RNA silencing by similar mechanisms using very different proteins in viruses of the same family.

Virus-induced gene silencing in plants.

Virus-induced gene silencing (VIGS) is a technology that exploits an RNA-mediated antiviral defense mechanism. In plants infected with unmodified viruses the mechanism is specifically targeted against the viral genome. However, with virus vectors carrying inserts derived from host genes the process can be additionally targeted against the corresponding mRNAs. VIGS has been used widely in plants for analysis of gene function and has been adapted for high-throughput functional genomics. Until now most applications of VIGS have been in Nicotiana benthamiana. However, new vector systems and methods are being developed that could be used in other plants, including Arabidopsis. Here we discuss practical and theoretical issues that are specific to VIGS rather than other gene "knock down" or "knockout" approaches to gene function. We also describe currently used protocols that have allowed us to apply VIGS to the identification of genes required for disease resistance in plants. These methods and the underlying general principles also apply when VIGS is used in the analysis of other aspects of plant biology.