A single substitution in Vacuolar protein sorting 4 is responsible for resistance to Watermelon mosaic virus in melon.

In plants, introgression of genetic resistance is a proven strategy for developing new resistant lines. While host proteins involved in genome replication and cell to cell movement are widely studied, other cell mechanisms responsible for virus infection remain under investigated. Endosomal sorting complexes required for transport (ESCRT) play a key role in membrane trafficking in plants and are involved in the replication of several plant RNA viruses. In this work, we describe the role of the ESCRT protein CmVPS4 as a new susceptibility factor to the Potyvirus Watermelon mosaic virus (WMV) in melon. Using a worldwide collection of melons, we identified three different alleles carrying non-synonymous substitutions in CmVps4. Two of these alleles were shown to be associated with WMV resistance. Using a complementation approach, we demonstrated that resistance is due to a single non-synonymous substitution in the allele CmVps4P30R. This work opens up new avenues of research on a new family of host factors required for virus infection and new targets for resistance.

Specificity of Resistance and Tolerance to Cucumber Vein Yellowing Virus in Melon Accessions and Resistance Breaking with a Single Mutation in VPg.

Cucumber vein yellowing virus (CVYV) is an emerging virus on cucurbits in the Mediterranean Basin, against which few resistance sources are available, particularly in melon. The melon accession PI 164323 displays complete resistance to isolate CVYV-Esp, and accession HSD 2458 presents a tolerance, i.e., very mild symptoms despite virus accumulation in inoculated plants. The resistance is controlled by a dominant allele Cvy-11, while the tolerance is controlled by a recessive allele cvy-2, independent from Cvy-11. Before introducing the resistance or tolerance in commercial cultivars through a long breeding process, it is important to estimate their specificity and durability. Upon inoculation with eight molecularly diverse CVYV isolates, the resistance was found to be isolate-specific because many CVYV isolates induced necrosis on PI 164323, whereas the tolerance presented a broader range. A resistance-breaking isolate inducing severe mosaic on PI 164323 was obtained. This isolate differed from the parental strain by a single amino acid change in the VPg coding region. An infectious CVYV cDNA clone was obtained, and the effect of the mutation in the VPg cistron on resistance to PI 164323 was confirmed by reverse genetics. This represents the first determinant for resistance-breaking in an ipomovirus. Our results indicate that the use of the Cvy-11 allele alone will not provide durable resistance to CVYV and that, if used in the field, it should be combined with other control methods such as cultural practices and pyramiding of resistance genes to achieve long-lasting resistance against CVYV.

Sixty Years After the First Description: Genome Sequence and Biological Characterization of European Wheat Striate Mosaic Virus Infecting Cereal Crops.

High-throughput sequencing technologies were used to identify plant viruses in cereal samples surveyed from 2012 to 2017. Fifteen genome sequences of a tenuivirus infecting wheat, oats, and spelt in Estonia, Norway, and Sweden were identified and characterized by their distances to other tenuivirus sequences. Like most tenuiviruses, the genome of this tenuivirus contains four genomic segments. The isolates found from different countries shared at least 92% nucleotide sequence identity at the genome level. The planthopper Javesella pellucida was identified as a vector of the virus. Laboratory transmission tests using this vector indicated that wheat, oats, barley, rye, and triticale, but none of the tested pasture grass species (Alopecurus pratensis, Dactylis glomerata, Festuca rubra, Lolium multiflorum, Phleum pratense, and Poa pratensis), are susceptible. Taking into account the vector and host range data, the tenuivirus we have found most probably represents European wheat striate mosaic virus first identified about 60 years ago. Interestingly, whereas we were not able to infect any of the tested cereal species mechanically, Nicotiana benthamiana was infected via mechanical inoculation in laboratory conditions, displaying symptoms of yellow spots and vein clearing evolving into necrosis, eventually leading to plant death. Surprisingly, one of the virus genome segments (RNA2) encoding both a putative host systemic movement enhancer protein and a putative vector transmission factor was not detected in N. benthamiana after several passages even though systemic infection was observed, raising fundamental questions about the role of this segment in the systemic spread in several hosts.

Resistance Against Melon Chlorotic Mosaic Virus and Tomato Leaf Curl New Delhi Virus in Melon.

Thirty-one melon accessions were screened for resistance to the begomoviruses Melon chlorotic mosaic virus (MeCMV) and Tomato leaf curl New Delhi virus (ToLCNDV). Five accessions presented nearly complete resistance to both viruses. Accession IC-274014, showing the highest level of resistance to both viruses, was crossed with the susceptible cultivar Védrantais. The F1, F2, F3/F4, and both backcross progenies were mechanically inoculated with MeCMV. Plants without symptoms or virus detection by enzyme-linked immunosorbent assay and/or PCR were considered as resistant. The segregations were compatible with two recessive and one dominant independent genes simultaneously required for resistance. Inheritance of resistance to ToLCNDV in the F2 was best explained by one recessive gene and two independent dominant genes simultaneously required. Some F3 and F4 families selected for resistance to MeCMV also were resistant to ToLCNDV, suggesting that common or tightly linked genes were involved in resistance to both viruses. We propose the names begomovirus resistance-1 and Begomovirus resistance-2 for these genes (symbols bgm-1 and Bgm-2). Resistance to MeCMV in IC-274014 was controlled by bgm-1, Bgm-2, and the recessive gene melon chlorotic mosaic virus resistance (mecmv); resistance to ToLCNDV was controlled by bgm-1, Bgm-2, and the dominant gene Tomato leaf curl New Delhi virus resistance (Tolcndv).

Molecular and biological characterization of a new Tomato mild yellow leaf curl Aragua virus strain producing severe symptoms in tomato.

Tomato mild yellow leaf curl Aragua virus (ToMYLCV) is a begomovirus first reported infecting tomato (Solanum lycopersicum) and milkweed (Euphorbia heterophylla) in Venezuela. In this study, a ToMYLCV isolate (Zulia-219) was completely sequenced and its host range was evaluated. The DNA-A and DNA-B components of isolate Zulia-219 showed 93 and 85% nucleotide sequence identity with the respective counterparts of the ToMYLCV type strain. According to current demarcation criteria for begomovirus species, Zulia-219 is a new strain of ToMYLCV. Interestingly, tomato plants inoculated with ToMYLCV Zulia-219 displayed severe symptoms, including severe chlorotic leaf curling, in contrast to mild symptoms associated with the type strain of this begomovirus. These results indicate potential risks associated with this new ToMYLCV strain for tomato production in Venezuela.

Characterization of a new cucurbit-infecting ipomovirus from Sudan.

Two members of the genus Ipomovirus (family Potyviridae) are known to infect cucurbits: cucumber vein yellowing virus (CVYV), which is emerging throughout the Mediterranean Basin, and squash vein yellowing virus (SqVYV), which has been described in America and the Caribbean Basin, and more recently in Israel. In this work, an ipomovirus different from CVYV and SqVYV, tentatively named coccinia mottle virus (CocMoV), was detected in a sample of the cucurbit Coccinia grandis collected in central Sudan in 2012. Sequence identity in nt was 68 % with CVYV, 59-60 % with SqVYV, cassava brown streak virus and Ugandan cassava brown streak virus, and less than 50 % with other members of the family Potyviridae. Preliminary biological and epidemiological studies indicate that CocMoV has a narrow natural host range and a low prevalence.

Cloning of the Arabidopsis rwm1 gene for resistance to Watermelon mosaic virus points to a new function for natural virus resistance genes.

Arabidopsis thaliana represents a valuable and efficient model to understand mechanisms underlying plant susceptibility to viral diseases. Here, we describe the identification and molecular cloning of a new gene responsible for recessive resistance to several isolates of Watermelon mosaic virus (WMV, genus Potyvirus) in the Arabidopsis Cvi-0 accession. rwm1 acts at an early stage of infection by impairing viral accumulation in initially infected leaf tissues. Map-based cloning delimited rwm1 on chromosome 1 in a 114-kb region containing 30 annotated genes. Positional and functional candidate gene analysis suggested that rwm1 encodes cPGK2 (At1g56190), an evolutionary conserved nucleus-encoded chloroplast phosphoglycerate kinase with a key role in cell metabolism. Comparative sequence analysis indicates that a single amino acid substitution (S78G) in the N-terminal domain of cPGK2 is involved in rwm1-mediated resistance. This mutation may have functional consequences because it targets a highly conserved residue, affects a putative phosphorylation site and occurs within a predicted nuclear localization signal. Transgenic complementation in Arabidopsis together with virus-induced gene silencing in Nicotiana benthamiana confirmed that cPGK2 corresponds to rwm1 and that the protein is required for efficient WMV infection. This work uncovers new insight into natural plant resistance mechanisms that may provide interesting opportunities for the genetic control of plant virus diseases.

Reconceptualizing transcriptional slippage in plant RNA viruses.

RNA viruses have evolved sophisticated strategies to exploit the limited encoded information within their typically compact genomes. One of them, named transcriptional slippage (TS), is characterized by the appearance of indels in nascent viral RNAs, leading to changes in the open reading frame (ORF). Although members of unrelated viral families express key proteins via TS, the available information about this phenomenon is still limited. In potyvirids (members of the Potyviridae family), TS has been defined by the insertion of an additional A at An motifs (n ≥ 6) in newly synthesized transcripts at a low frequency, modulated by nucleotides flanking the A-rich motif. Here, by using diverse experimental approaches and a collection of plant/virus combinations, we discover cases not following this definition. In summary, we observe (i) a high rate of single-nucleotide deletions at slippage motifs, (ii) overlapping ORFs acceded by slippage at an U8 stretch, and (iii) changes in slippage rates induced by factors not related to cognate viruses. Moreover, a survey of whole-genome sequences from potyvirids shows a widespread occurrence of species-specific An/Un (n ≥ 6) motifs. Even though many of them, but not all, lead to the production of truncated proteins rather than access to overlapping ORFs, these results suggest that slippage motifs appear more frequently than expected and play relevant roles during virus evolution. Considering the potential of this phenomenon to expand the viral proteome by acceding to overlapping ORFs and/or producing truncated proteins, a re-evaluation of TS significance during infections of RNA viruses is required.IMPORTANCETranscriptional slippage (TS) is used by RNA viruses as another strategy to maximize the coding information in their genomes. This phenomenon is based on a peculiar feature of viral replicases: they may produce indels in a small fraction of newly synthesized viral RNAs when transcribing certain motifs and then produce alternative proteins due to a change of the reading frame or truncated products by premature termination. Here, using plant-infecting RNA viruses as models, we discover cases expanding on previously established features of plant virus TS, prompting us to reconsider and redefine this expression strategy. An interesting conclusion from our study is that TS might be more relevant during RNA virus evolution and infection processes than previously assumed.

The melon Fom-1-Prv resistance gene pair: Correlated spatial expression and interaction with a viral protein.

The head-to-head oriented pair of melon resistance genes, Fom-1 and Prv, control resistance to Fusarium oxysporum races 0 and 2 and papaya ringspot virus (PRSV), respectively. They encode, via several RNA splice variants, TIR-NBS-LRR proteins, and Prv has a C-terminal extra domain with a second NBS homologous sequence. In other systems, paired R-proteins were shown to operate by "labor division," with one protein having an extra integrated domain that directly binds the pathogen's Avr factor, and the second protein executing the defense response. We report that the expression of the two genes in two pairs of near-isogenic lines was higher in the resistant isoline and inducible by F. oxysporum race 2 but not by PRSV. The intergenic DNA region separating the coding sequences of the two genes acted as a bi-directional promoter and drove GUS expression in transgenic melon roots and transgenic tobacco plants. Expression of both genes was strong in melon root tips, around the root vascular cylinder, and the phloem and xylem parenchyma of tobacco stems and petioles. The pattern of GUS expression suggests coordinated expression of the two genes. In agreement with the above model, Prv's extra domain was shown to interact with the cylindrical inclusion protein of PRSV both in yeast cells and in planta.

Snake melon asteroid mosaic virus, a Tentative New Member of the Genus Sobemovirus Infecting Cucurbits.

A virus isolate (Su-95-67) was obtained from a snake melon (Cucumis melo var. flexuosus) plant presenting severe chlorotic spots, mosaic, stunting, and leaf deformations collected in Eastern Sudan in 1995. Su-95-67 was easily mechanically transmissible and had a host range limited to a few cucurbit species. Isometric virus particles approximately 30 nm in diameter were observed in leaf dip preparations. A cytopathological study did not reveal alterations specific for a virus genus or family. A polyclonal antiserum was obtained and used in double-antibody sandwich enzyme-linked immunosorbent assay (DAS-ELISA). Su-95-67 was transmitted by seed at a low rate, by the red melon beetle (Aulacophora foveicollis), but not by the melon aphid (Aphis gossypii). Because Su-95-67 shared several properties with sobemoviruses, generic Sobemovirus reverse-transcription polymerase chain reaction primers were developed. They allowed amplification of a 384-bp fragment from extracts of plants infected by two sobemoviruses or by Su-95-67 but not from healthy plants extracts. Sequence comparison confirmed that Su-95-67 belongs to a new tentative Sobemovirus species for which we propose the name Snake melon asteroid mosaic virus (SMAMV). DAS-ELISA tests conducted on extracts of virus-infected cucurbit plants collected from 1992 to 2003 revealed the presence of SMAMV in 10.2% of 600 samples originating from different regions of Sudan.

The Bunyavirales: The Plant-Infecting Counterparts.

Negative-strand (-) RNA viruses (NSVs) comprise a large and diverse group of viruses that are generally divided in those with non-segmented and those with segmented genomes. Whereas most NSVs infect animals and humans, the smaller group of the plant-infecting counterparts is expanding, with many causing devastating diseases worldwide, affecting a large number of major bulk and high-value food crops. In 2018, the taxonomy of segmented NSVs faced a major reorganization with the establishment of the order Bunyavirales. This article overviews the major plant viruses that are part of the order, i.e., orthospoviruses (Tospoviridae), tenuiviruses (Phenuiviridae), and emaraviruses (Fimoviridae), and provides updates on the more recent ongoing research. Features shared with the animal-infecting counterparts are mentioned, however, special attention is given to their adaptation to plant hosts and vector transmission, including intra/intercellular trafficking and viral counter defense to antiviral RNAi.

Asymmetrical over-infection as a process of plant virus emergence.

Disentangling the role of epidemiological factors in plant pathogen emergences is a prerequisite to identify the most likely future invaders. An example of emergence was recently observed in France: in 10 years, "classic" (CL) strains of Watermelon mosaic virus (WMV) were displaced at a regional scale by newly introduced "emerging" (EM) strains. Here we analyse a 3 years dataset describing the co-dynamics of CL and EM strains at field scale using state-space models estimating jointly: (i) probabilities of primary and secondary infection and (ii) probabilities of over-infecting with a CL [EM] strain a plant already infected with an EM [CL] strain. Results especially indicate that it is more than 3 times less probable for a CL strain to over-infect an EM infected plant than for an EM strain to over-infect a CL infected plant. To investigate if these asymmetric interactions can explain the CL/EM shift observed at regional scale, an exploratory model describing WMV epidemiology over several years in a landscape composed of a reservoir and a cultivated compartment is introduced. In most simulations a shift is observed and both strains do coexist in the landscape, reaching an equilibrium that depends on the probabilities of over-infection.

A novel natural mutation in HC-Pro responsible for mild symptomatology of Zucchini yellow mosaic virus (ZYMV, Potyvirus) in cucurbits.

A natural mild isolate of Zucchini yellow mosaic virus was found to contain a mutation in the helper component (HC-Pro) within a conserved motif, "CDNQLD", located 12 residues downstream from the "FRNK" motif involved in symptom severity. Introducing the mutation in an infectious cDNA clone of ZYMV resulted in an almost complete absence of symptoms, although viral accumulation was only partially reduced. The FRNK(X)(12)CDNQLD sequence might be part of a larger motif that is conserved in potyviruses and plays a role in symptomatology and/or silencing inhibition.

Host Range Evolution of Potyviruses: A Global Phylogenetic Analysis.

Virus host range, i.e., the number and diversity of host species of viruses, is an important determinant of disease emergence and of the efficiency of disease control strategies. However, for plant viruses, little is known about the genetic or ecological factors involved in the evolution of host range. Using available genome sequences and host range data, we performed a phylogenetic analysis of host range evolution in the genus Potyvirus, a large group of plant RNA viruses that has undergone a radiative evolution circa 7000 years ago, contemporaneously with agriculture intensification in mid Holocene. Maximum likelihood inference based on a set of 59 potyviruses and 38 plant species showed frequent host range changes during potyvirus evolution, with 4.6 changes per plant species on average, including 3.1 host gains and 1.5 host loss. These changes were quite recent, 74% of them being inferred on the terminal branches of the potyvirus tree. The most striking result was the high frequency of correlated host gains occurring repeatedly in different branches of the potyvirus tree, which raises the question of the dependence of the molecular and/or ecological mechanisms involved in adaptation to different plant species.

Distribution and evolution of the major viruses infecting cucurbitaceous and solanaceous crops in the French Mediterranean area.

Plant viral diseases represent a significant burden to plant health, and their highest impact in Mediterranean agriculture is on vegetables grown under intensive horticultural practices. In order to understand better virus evolution and emergence, the most prevalent viruses were mapped in the main cucurbitaceous (melon, squashes) and solanaceous (tomato, pepper) crops and in some wild hosts in the French Mediterranean area, and virus diversity, evolution and population structure were studied through molecular epidemiology approaches. Surveys were performed in summer 2016 and 2017, representing a total of 1530 crop samples and 280 weed samples. The plant samples were analysed using serological and molecular approaches, including high-throughput sequencing (HTS). The viral species and their frequency in crops were quite similar to those of surveys conducted ten years before in the same areas. Contrary to other Mediterranean countries, aphid-transmitted viruses remain the most prevalent in France whereas whitefly-transmitted ones have not yet emerged. However, HTS analysis of viral evolution revealed the appearance of undescribed viral variants, especially for watermelon mosaic virus (WMV) in cucurbits, or variants not present in France before, as for cucumber mosaic virus (CMV) in solanaceous crops. Deep sequencing also revealed complex virus populations within individual plants with frequent recombination or reassortment. The spatial genetic structure of cucurbit aphid-borne yellows virus (CABYV) was related to the landscape structure, whereas in the case of WMV, the recurrence of introduction events and probable human exchanges of plant material resulted in complex spatial pattern of genetic variation.

Impact of Vat resistance in melon on viral epidemics and genetic structure of virus populations.

Cultivar choice is at the heart of cropping systems and resistant cultivars should be at the heart of disease management strategies whenever available. They are the easiest, most efficient and environmentally friendly way of combating viral diseases at the farm level. Among the melon genetic resources, Vat is a unique gene conferring resistance to both the melon aphid Aphis gossypii and the viruses it carries. The 'virus side' of this pleiotropic phenotype is seldom regarded as an asset for virus control. Indeed, the effect of Vat on virus epidemics in the field is expected to vary according to the composition of aphid populations in the environment and long-term studies are needed to draw a correct trend. Therefore, the first objective of the study was to re-evaluate the potential of Vat to reduce viral diseases in melon crops. The second objective was to investigate the potential of Vat to exert a selection pressure on virus populations. We monitored the epidemics of Cucurbit aphid-borne yellows virus (CABYV), Cucumber mosaic virus (CMV), Watermelon mosaic virus (WMV) and Zucchini yellow mosaic virus (ZYMV) in two melon lines having a common genetic background, a resistant line (R) and a susceptible line (S), in eight field trials conducted in southeastern France between 2011 and 2015. Vat had limited impact if any on WMV epidemics probably because A. gossypii is not the main vector of WMV in the field, but a favorable impact on CMV, yet of variable intensity probably related to the importance of A. gossypii in the total aphid population. Vat had a significant impact on CABYV epidemics with mean incidence reduction exceeding 50% in some trials. There was no effect of Vat on the structure of virus populations, both for the non-persistent WMV transmitted by numerous aphid species and for the persistent CABYV transmitted predominantly by A. gossypii.

Potential Involvement of Melon Fruit in the Long Distance Dissemination of Cucurbit Potyviruses.

Papaya ringspot virus (PRSV) and Zucchini yellow mosaic virus(ZYMV) are potyviruses frequently reported in cucurbits in Mediterranean, subtropical, and tropical regions. Occasionally, epidemics are also observed in more temperate regions, but the ways these viruses are introduced into new areas are not yet fully determined. We investigated the possibility that infected imported melon fruit could be a route for the introduction of PRSV and ZYMV. Imported melon fruits of the yellow canary type infected by ZYMV and PRSV were exposed in the fields next to healthy melon or squash bait plants. During this period, aphids were observed landing and probing on the fruits. In four independent experiments using different fruits, 3.1 to 25% of bait plants were infected by ZYMV and/or PRSV. PRSV was more frequently transmitted to bait plants than ZYMV. Comparison of partial sequences of the isolates from fruits and from bait plants showed a very high, if not complete, identity within each experiment, confirming that a natural transmission did occur from the fruit to the bait plants. These results suggest that globalization of melon production and international trade may be a factor in the spread of cucurbit potyviruses between countries or continents.

Biological and Molecular Characterization of Moroccan watermelon mosaic virus and a Potyvirus Isolate from Eastern Sudan.

A potyvirus (Su-94-54) was isolated from a naturally infected snake cucumber (Cucumis melo var. flexuosus) plant with severe mosaic and leaf deformation symptoms collected in Eastern Sudan. This isolate has a host range limited to cucurbits and is serologically distantly related to Moroccan watermelon mosaic virus (MWMV) and to Papaya ringspot virus (PRSV). Coat protein sequence analysis of Su-94-54 and MWMV and comparison with other potyviruses indicate that Su-94-54 is more closely related to MWMV than to any other potyvirus. Based on the amino acid sequence identity in the core part of the coat protein with MWMV (86%), this isolate could be regarded as a distinct species. However, because of biological, cytological, and serological affinities with MWMV, we propose that this isolate be considered as a strain of MWMV, possibly an evolutionary intermediate between MWMV and PRSV, until more is known on the structure of the PRSV subgroup within the genus Potyvirus.

A short motif in the N-terminal part of the coat protein is a host-specific determinant of systemic infectivity for two potyviruses.

Although the biological variability of Watermelon mosaic virus is limited, isolates from the three main molecular groups differ in their ability to infect systemically Chenopodium quinoa. Mutations were introduced in a motif of three or five amino acids located in the N-terminal part of the coat protein, and differing in isolates from group 1 (motif: lysine-glutamic acid-alanine (Lys-Glu-Ala) or KEA, systemic on C. quinoa), group 2 (Lys-Glu-Thr or KET, not systemic on C. quinoa) and group 3 (KEKET, not systemic on C. quinoa). Mutagenesis of KEKET in an isolate from group 3 to KEA or KEKEA was sufficient to make the virus systemic on C. quinoa, whereas mutagenesis to KET had no effect. Introduction of a KEA motif in Zucchini yellow mosaic virus coat protein also resulted in systemic infection on C. quinoa. These mutations had no obvious effect on the disorder profile or potential post-translational modifications of the coat protein as determined in silico.

Viruses of cucurbit crops in the Mediterranean region: an ever-changing picture.

Cucurbit crops may be affected by at least 28 different viruses in the Mediterranean basin. Some of these viruses are widely distributed and cause severe yield losses while others are restricted to limited areas or specific crops, and have only a negligible economic impact. A striking feature of cucurbit viruses in the Mediterranean basin is their always increasing diversity. Indeed, new viruses are regularly isolated and over the past 35 years one "new" cucurbit virus has been reported on average every 2 years. Among these "new" viruses some were already reported in other parts of the world, but others such as Zucchini yellow mosaic virus (ZYMV), one of the most severe cucurbit viruses and Cucurbit aphid-borne yellows virus (CABYV), one of the most prevalent cucurbit viruses, were first described in the Mediterranean area. Why this region may be a potential "hot-spot" for cucurbit virus diversity is not fully known. This could be related to the diversity of cropping practices, of cultivar types but also to the important commercial exchanges that always prevailed in this part of the world. This chapter describes the major cucurbit viruses occurring in the Mediterranean basin, discusses factors involved in their emergence and presents options for developing sustainable control strategies.