Interplay between drought and plant viruses co-infecting melon plants.

Drought affects crops directly, and indirectly by affecting the activity of insect pests and the transmitted pathogens. Here, we established an experiment with well-watered or water-stressed melon plants, later single infected with either cucumber mosaic virus (CMV: non-persistent), or cucurbit aphid-borne yellow virus (CABYV: persistent), or both CMV and CABYV, and mock-inoculated control. We tested whether i) the relation between CMV and CABYV is additive, and ii) the relationship between water stress and virus infection is antagonistic, i.e., water stress primes plants for enhanced tolerance to virus infection. Water stress increased leaf greenness and temperature, and reduced leaf water potential, shoot biomass, stem dimensions, rate of flowering, CABYV symptom severity, and marketable fruit yield. Virus infection reduced leaf water potential transiently in single infected plants and persistently until harvest in double-infected plants. Double-virus infection caused the largest and synergistic reduction of marketable fruit yield. The relationship between water regime and virus treatment was additive in 12 out of 15 traits at harvest, with interactions for leaf water content, leaf:stem ratio, and fruit set. We conclude that both virus-virus relations in double infection and virus-drought relations cannot be generalized because they vary with virus, trait, and plant ontogeny.

P3N-PIPO but not P3 is the avirulence determinant in melon carrying the Wmr resistance against watermelon mosaic virus, although they contain a common genetic determinant.

Viruses employ a series of diverse translational strategies to expand their coding capacity, which produces viral proteins with common domains and entangles virus-host interactions. P3N-PIPO, which is a transcriptional slippage product from the P3 cistron, is a potyviral protein dedicated to intercellular movement. Here, we show that P3N-PIPO from watermelon mosaic virus (WMV) triggers cell death when transiently expressed in Cucumis melo accession PI 414723 carrying the Wmr resistance gene. Surprisingly, expression of the P3N domain, shared by both P3N-PIPO and P3, can alone induce cell death, whereas expression of P3 fails to activate cell death in PI 414723. Confocal microscopy analysis revealed that P3N-PIPO targets plasmodesmata (PD) and P3N associates with PD, while P3 localizes in endoplasmic reticulum in melon cells. We also found that mutations in residues L35, L38, P41, and I43 of the P3N domain individually disrupt the cell death induced by P3N-PIPO, but do not affect the PD localization of P3N-PIPO. Furthermore, WMV mutants with L35A or I43A can systemically infect PI 414723 plants. These key residues guide us to discover some WMV isolates potentially breaking the Wmr resistance. Through searching the NCBI database, we discovered some WMV isolates with variations in these key sites, and one naturally occurring I43V variation enables WMV to systemically infect PI 414723 plants. Taken together, these results demonstrate that P3N-PIPO, but not P3, is the avirulence determinant recognized by Wmr, although the shared N terminal P3N domain can alone trigger cell death.IMPORTANCEThis work reveals a novel viral avirulence (Avr) gene recognized by a resistance (R) gene. This novel viral Avr gene is special because it is a transcriptional slippage product from another virus gene, which means that their encoding proteins share the common N-terminal domain but have distinct C-terminal domains. Amazingly, we found that it is the common N-terminal domain that determines the Avr-R recognition, but only one of the viral proteins can be recognized by the R protein to induce cell death. Next, we found that these two viral proteins target different subcellular compartments. In addition, we discovered some virus isolates with variations in the common N-terminal domain and one naturally occurring variation that enables the virus to overcome the resistance. These results show how viral proteins with common domains interact with a host resistance protein and provide new evidence for the arms race between plants and viruses.

Ability of Non-Hosts and Cucurbitaceous Weeds to Transmit Cucumber Green Mottle Mosaic Virus.

Cucumber green mottle mosaic virus (CGMMV) is a Tobamovirus of economic importance affecting cucurbit crops and Asian cucurbit vegetables. Non-host crops of CGMMV, including capsicum (Capsicum annum), sweetcorn (Zea mays), and okra (Abelmoschus esculentus), were tested for their susceptibility to the virus, with field and glasshouse trials undertaken. After 12 weeks post-sowing, the crops were tested for the presence of CGMMV, and in all cases, no CGMMV was detected. Commonly found within the growing regions of cucurbits and melons worldwide are weeds, such as black nightshade (Solanum nigrum), wild gooseberry (Physalis minima), pigweed (Portulaca oleracea), and Amaranth species. Several weeds/grasses were tested for their ability to become infected with CGMMV by inoculating weeds directly with CGMMV and routinely testing over a period of eight weeks. Amaranthus viridis was found to be susceptible, with 50% of the weeds becoming infected with CGMMV. To further analyse this, six Amaranth samples were used as inoculum on four watermelon seedlings per sample and tested after eight weeks. CGMMV was detected in three of six watermelon bulk samples, indicating that A. viridis is a potential host/reservoir for CGMMV. Further research into the relationship between CGMMV and weed hosts is required. This research also highlights the importance of proper weed management to effectively manage CGMMV.

Genetic Differentiation and Migration Fluxes of Viruses from Melon Crops and Crop Edge Weeds.

Weeds surrounding crops may act as alternative hosts, playing important epidemiological roles as virus reservoirs and impacting virus evolution. We used high-throughput sequencing to identify viruses in Spanish melon crops and plants belonging to three pluriannual weed species, Ecballium elaterium, Malva sylvestris, and Solanum nigrum, sampled at the edges of the crops. Melon and E. elaterium, both belonging to the family Cucurbitaceae, shared three virus species, whereas there was no virus species overlap between melon and the other two weeds. The diversity of cucurbit aphid-borne yellows virus (CABYV) and tomato leaf curl New Delhi virus (ToLCNDV), both in melon and E. elaterium, was further studied by amplicon sequencing. Phylogenetic and population genetics analyses showed that the CABYV population was structured by the host, identifying three sites in the CABYV RNA-dependent RNA polymerase under positive selection, perhaps reflecting host adaptation. The ToLCNDV population was much less diverse than the CABYV one, likely as a consequence of the relatively recent introduction of ToLCNDV in Spain. In spite of its low diversity, we identified geographical but no host differentiation for ToLCNDV. Potential virus migration fluxes between E. elaterium and melon plants were also analyzed. For CABYV, no evidence of migration between the populations of the two hosts was found, whereas important fluxes were identified between geographically distant subpopulations for each host. For ToLCNDV, in contrast, evidence of migration from melon to E. elaterium was found, but not the other way around. IMPORTANCE It has been reported that about half of the emerging diseases affecting plants are caused by viruses. Alternative hosts often play critical roles in virus emergence as virus reservoirs, bridging host species that are otherwise unconnected and/or favoring virus diversification. In spite of this, the viromes of potential alternative hosts remain largely unexplored. In the case of crops, pluriannual weeds at the crop edges may play these roles. Here, we took advantage of the power of high-throughput sequencing to characterize the viromes of three weed species frequently found at the edges of melon crops. We identified three viruses shared by melon and the cucurbit weed, with two of them being epidemiologically relevant for melon crops. Further genetic analyses showed that these two viruses had contrasting patterns of diversification and migration, providing an interesting example on the role that weeds may play in the ecology and evolution of viruses affecting crops.

Editing melon eIF4E associates with virus resistance and male sterility.

The cap-binding protein eIF4E, through its interaction with eIF4G, constitutes the core of the eIF4F complex, which plays a key role in the circularization of mRNAs and their subsequent cap-dependent translation. In addition to its fundamental role in mRNA translation initiation, other functions have been described or suggested for eIF4E, including acting as a proviral factor and participating in sexual development. We used CRISPR/Cas9 genome editing to generate melon eif4e knockout mutant lines. Editing worked efficiently in melon, as we obtained transformed plants with a single-nucleotide deletion in homozygosis in the first eIF4E exon already in a T0 generation. Edited and non-transgenic plants of a segregating F2 generation were inoculated with Moroccan watermelon mosaic virus (MWMV); homozygous mutant plants showed virus resistance, while heterozygous and non-mutant plants were infected, in agreement with our previous results with plants silenced in eIF4E. Interestingly, all homozygous edited plants of the T0 and F2 generations showed a male sterility phenotype, while crossing with wild-type plants restored fertility, displaying a perfect correlation between the segregation of the male sterility phenotype and the segregation of the eif4e mutation. Morphological comparative analysis of melon male flowers along consecutive developmental stages showed postmeiotic abnormal development for both microsporocytes and tapetum, with clear differences in the timing of tapetum degradation in the mutant versus wild-type. An RNA-Seq analysis identified critical genes in pollen development that were down-regulated in flowers of eif4e/eif4e plants, and suggested that eIF4E-specific mRNA translation initiation is a limiting factor for male gametes formation in melon.

Investigations on annual spreading of viruses infecting cucurbit crops in Uttar Pradesh State, India.

During 2018 an intensive study was conducted to determine the viruses associated with cucurbitaceous crops in nine agroclimatic zones of the state of Uttar Pradesh, India. Total of 563 samples collected and analysed across 14 different cucurbitaceous crops. The results showed the dominance of Begomovirus (93%) followed by Potyvirus (46%), cucumber green mottle mosaic virus (CGMMV-39%), Polerovirus (9%), cucumber mosaic virus (CMV-2%) and Orthotospovirus (2%). Nearly 65% of samples were co-infected with more than one virus. Additionally, host range expansion of CMV, CGMMV and polerovirus was also observed on cucurbit crops. A new potyvirus species, zucchini tigre mosaic virus, earlier not documented from India has also been identified on five crops during the study. Risk map generated using ArcGIS for virus disease incidence predicted the virus severity in unexplored areas. The distribution pattern of different cucurbit viruses throughout Uttar Pradesh will help identify the hot spots for viruses and will facilitate to devise efficient and eco-friendly integrated management strategies for the mitigation of viruses infecting cucurbit crops. Molecular diversity and evolutionary relationship of the virus isolates infecting cucurbits in Uttar Pradesh with previously reported strains were understood from the phylogenetic analysis. Diverse virus infections observed in the Eastern Plain zone, Central zone and North-Eastern Plain zone indicate an alarming situation for the cultivation of cucurbits in the foreseeable future.

The mitochondrial and chloroplast dual targeting of a multifunctional plant viral protein modulates chloroplast-to-nucleus communication, RNA silencing suppressor activity, encapsidation, pathogenesis and tissue tropism.

Plant defense against melon necrotic spot virus (MNSV) is triggered by the viral auxiliary replicase p29 that is targeted to mitochondrial membranes causing morphological alterations, oxidative burst and necrosis. Here we show that MNSV coat protein (CP) was also targeted to mitochondria and mitochondrial-derived replication complexes [viral replication factories or complex (VRC)], in close association with p29, in addition to chloroplasts. CP import resulted in the cleavage of the R/arm domain previously implicated in genome binding during encapsidation and RNA silencing suppression (RSS). We also show that CP organelle import inhibition enhanced RSS activity, CP accumulation and VRC biogenesis but resulted in inhibition of systemic spreading, indicating that MNSV whole-plant infection requires CP organelle import. We hypothesize that to alleviate the p29 impact on host physiology, MNSV could moderate its replication and p29 accumulation by regulating CP RSS activity through organelle targeting and, consequently, eluding early-triggered antiviral response. Cellular and molecular events also suggested that S/P domains, which correspond to processed CP in chloroplast stroma or mitochondrion matrix, could mitigate host response inhibiting p29-induced necrosis. S/P deletion mainly resulted in a precarious balance between defense and counter-defense responses, generating either cytopathic alterations and MNSV cell-to-cell movement restriction or some degree of local movement. In addition, local necrosis and defense responses were dampened when RSS activity but not S/P organelle targeting was affected. Based on a robust biochemical and cellular analysis, we established that the mitochondrial and chloroplast dual targeting of MNSV CP profoundly impacts the viral infection cycle.

Molecular analysis of Greek isolates of cucumber mosaic virus from vegetables shows a low prevalence of satellite RNAs and suggests the presence of host-associated virus strains.

Cucumber mosaic virus (CMV) is a generalist pathogen that infects many economically important crops in Greece. The present study was designed to evaluate the genetic variability of Greek CMV isolates in combination with their satellite RNAs (satRNAs). To achieve this goal, 77 CMV isolates were collected from symptomatic Greek vegetables, mainly tomatoes and cucurbits, alongside their neighboring crops, during a four-year period from 2015 to 2018. Phylogenetic analysis of a partial coat protein (CP) gene segment revealed that all of the isolates belong to CMV subgroups IA and IB and that they are closely related to previously reported Greek isolates. It should be noted, however, that the latter mainly included tomato isolates. Network analysis of the evolutionary relationships among the CP sequences of the Greek isolates in comparison to the corresponding sequences obtained from the GenBank database indicated two predominant common ancestors and at least three differentiated peripherals, and possibly host-associated (tomatoes, legumes, cucurbits) haplogroups (strain groups). More specifically, host-adaptive evolution can be postulated regarding the tomato isolates in subgroup IB. Necrogenic or non-necrogenic satRNAs were detected in four samples from tomato and melon, and this is the first report of non-necrogenic satRNAs in CMV in Greece.

Complete sequence of an isolate of snake melon asteroid mosaic virus confirms that it is a member of a distinct sobemovirus species.

A virus tentatively named "snake melon asteroid mosaic virus" (SMAMV) was found in Sudan in cucurbit crops (10% of 600 samples) between 1992 and 2003. Biological and cytological properties as well as sequence data on a 345-nt fragment suggested that SMAMV was a member of the genus Sobemovirus. However, no complete sequence had been obtained, and the relationship between SMAMV and the acknowledged sobemoviruses had not been ascertained. In this work, we obtained the full-length sequence of an SMAMV isolate. The sequence was 4225 nt long, with a typical sobemovirus genetic organization. Sequence identity to other sobemoviruses was below 50%, both for the full-length genome and for individual proteins. These data confirm that SMAMV belongs to a novel sobemovirus species.

Detection and complete genome sequence of a divergent isolate of cucumber fruit mottle mosaic virus on Coccinia grandis in Sudan.

Cucurbit-infecting tobamoviruses known so far belong to six acknowledged or tentative species. Except for cucumber green mottle mosaic virus (CGMMV), which is present worldwide, they are geographically restricted, mostly to Asia, and have not been observed in Africa so far. A tobamovirus isolate infecting a wild Coccinia grandis plant was collected in central Sudan in 2012. Its host range appeared to be mostly limited to cucurbits. Its full-length genome sequence was determined and found to be 85% identical to those of isolates of cucumber fruit mottle mosaic virus (CFMMV) described in Israel and Korea, whereas the aa sequence identity to CFMMV isolates was 92 to 95%, depending on the protein. Based on its biological and molecular properties, we suggest that the Sudanese isolate should be considered a divergent isolate of CFMMV. This is the first description of CFMMV in Africa. Its high divergence from isolates from Israel and Korea suggests a lack of recent exchanges between CFMMV from Sudan and the other known populations.

Melon Genome Regions Associated with TGR-1551-Derived Resistance to Cucurbit yellow stunting disorder virus.

Cucurbit yellow stunting disorder virus (CYSDV) is one of the main limiting factors of melon cultivation worldwide. To date, no commercial melon cultivars resistant to CYSDV are available. The African accession TGR-1551 is resistant to CYSDV. Two major quantitative trait loci (QTLs) have been previously reported, both located near each other in chromosome 5. With the objective of further mapping the gene or genes responsible of the resistance, a recombinant inbred line (RIL) population derived from the cross between TGR-1551 and the susceptible cultivar 'Bola de Oro' was evaluated for resistance to CYSDV in five different assays and genotyped in a genotyping by sequencing (GBS) analysis. The major effect of one of the two QTLs located on chromosome 5 was confirmed in the multienvironment RIL assay and additionally verified through the analysis of three segregating BC1S1 populations derived from three resistant RILs. Furthermore, progeny test using the offspring of selected BC3 plants allowed the narrowing of the candidate interval to a 700 kb region. The SNP markers identified in this work will be useful in marker-assisted selection in the context of introgression of CYSDV resistance in elite cultivars.

Can Winged Aphid Abundance Be a Predictor of Cucurbit Aphid-Borne Yellows Virus Epidemics in Melon Crop?

Aphid-borne viruses are frequent yield-limiting pathogens in open field vegetable crops. In the absence of curative methods, virus control relies exclusively on measures limiting virus introduction and spread. The efficiency of control measures may greatly benefit from an accurate knowledge of epidemic drivers, in particular those linked with aphid vectors. Field experiments were conducted in southeastern France between 2010 and 2019 to investigate the relationship between the epidemics of cucurbit aphid-borne yellows virus (CABYV) and aphid vector abundance. Winged aphids visiting melon crops were sampled daily to assess the abundance of CABYV vectors (Aphis gossypii, Macrosiphum euphorbiae and Myzus persicae) and CABYV was monitored weekly by DAS-ELISA. Epidemic temporal progress curves were successfully described by logistic models. A systematic search for correlations was undertaken between virus variables including parameters µ (inflection point of the logistic curve) and γ (maximum incidence) and aphid variables computed by aggregating abundances on periods relative either to the planting date, or to the epidemic peak. The abundance of A. gossypii during the first two weeks after planting was found to be a good predictor of CABYV dynamics, suggesting that an early control of this aphid species could mitigate the onset and progress of CABYV epidemics in melon crops.

Development of a Virus-Induced Gene Silencing System for Dioecious Coccinia grandis.

Coccinia grandis is an interesting model system to understand dioecy in Cucurbitaceae family. Recent transcriptomics and proteomics studies carried out to understand the sex expression in C. grandis have resulted in identification of many candidate sex-biased genes. In absence of an efficient genetic transformation protocol for C. grandis, virus-induced gene silencing (VIGS) would be a powerful tool to enable gene functional analysis. In current study, we explored the apple latent spherical virus (ALSV) for gene knockdown in C. grandis. The viral infection was achieved through mechanical inoculation of ALSV-infected Chenopodium quinoa leaf extract onto the cotyledons of C. grandis. ALSV-VIGS mediated knockdown of CgPDS gene was successfully achieved in C. grandis by mechanical inoculation method resulting in characteristic photobleaching. Subsequently, we developed agroinfiltration compatible vectors for direct infection of C. grandis and shortened the time-frame by skipping viral propagation in C. quinoa. Typical yellow-leaf phenotype was observed in C. grandis plants agroinfiltrated with ALSV-CgSU constructs, indicating robust silencing of CgSU gene. In addition, we improved the infection efficiency of ALSV by co-infiltration of P19 viral silencing suppressor. These results suggest that ALSV-VIGS is suitable for characterization of gene function in dioecious C. grandis and it can help us understand the mechanism of sex expression.

Priming Melon Defenses with Acibenzolar-S-methyl Attenuates Infections by Phylogenetically Distinct Viruses and Diminishes Vector Preferences for Infected Hosts.

Plant virus management is mostly achieved through control of insect vectors using insecticides. However, insecticides are only marginally effective for preventing virus transmission. Furthermore, it is well established that symptoms of virus infections often encourage vector visitation to infected hosts, which exacerbates secondary spread. Plant defense elicitors, phytohormone analogs that prime the plant immune system against attack, may be a viable approach for virus control that complements insecticide use by disrupting pathologies that attract vectors. To explore this, we tested the effect of a commercial plant elicitor, acibenzolar-S-methyl (ASM), on infection rates, virus titers, and symptom development in melon plants inoculated with one of two virus species, Cucumber mosaic virus (CMV) and Cucurbit yellow stunting disorder virus (CYSDV). We also conducted behavioral assays to assess the effect of ASM treatment and virus inoculation on vector behavior. For both pathogens, ASM treatment reduced symptom severity and delayed disease progression. For CYSDV, this resulted in the attenuation of symptoms that encourage vector visitation and virion uptake. We did observe slight trade-offs in growth vs. defense following ASM treatment, but these effects did not translate into reduced yields or plant performance in the field. Our results suggest that immunity priming may be a valuable tool for improving management of insect-transmitted plant viruses.

Development of Loop-Mediated Isothermal Amplification Assay for Rapid Detection of Cucurbit Leaf Crumple Virus.

A loop-mediated isothermal amplification (LAMP) assay was developed for simple, rapid and efficient detection of Cucurbit leaf crumple virus (CuLCrV), one of the most important begomoviruses that infects cucurbits worldwide. A set of six specific primers targeting a total 240 nt sequence regions in the DNA A of CuLCrV were designed and synthesized for detection of CuLCrV from infected leaf tissues using real-time LAMP amplification with the Genie® III system, which was further confirmed by gel electrophoresis and SYBR™ Green I DNA staining for visual observation. The optimum reaction temperature and time were determined, and no cross-reactivity was seen with other begomoviruses. The LAMP assay could amplify CuLCrV from a mixed virus assay. The sensitivity assay demonstrated that the LAMP reaction was more sensitive than conventional PCR, but less sensitive than qPCR. However, it was simpler and faster than the other assays evaluated. The LAMP assay also amplified CuLCrV-infected symptomatic and asymptomatic samples more efficiently than PCR. Successful LAMP amplification was observed in mixed virus-infected field samples. This simple, rapid, and sensitive method has the capacity to detect CuLCrV in samples collected in the field and is therefore suitable for early detection of the disease to reduce the risk of epidemics.

A single amino acid substitution in the movement protein enables the mechanical transmission of a geminivirus.

Begomoviruses of the Geminiviridae are usually transmitted by whiteflies and rarely by mechanical inoculation. We used tomato leaf curl New Delhi virus (ToLCNDV), a bipartite begomovirus, to address this issue. Most ToLCNDV isolates are not mechanically transmissible to their natural hosts. The ToLCNDV-OM isolate, originally identified from a diseased oriental melon plant, is mechanically transmissible, while the ToLCNDV-CB isolate, from a diseased cucumber plant, is not. Genetic swapping and pathological tests were performed to identify the molecular determinants involved in mechanical transmission. Various viral infectious clones were constructed and successfully introduced into Nicotiana benthamiana, oriental melon, and cucumber plants by Agrobacterium-mediated inoculation. Mechanical transmissibility was assessed via direct rub inoculation with sap prepared from infected N. benthamiana. The presence or absence of viral DNA in plants was validated by PCR, Southern blotting, and in situ hybridization. The results reveal that mechanical transmissibility is associated with the movement protein (MP) of viral DNA-B in ToLCNDV-OM. However, the nuclear shuttle protein of DNA-B plays no role in mechanical transmission. Analyses of infectious clones carrying a single amino acid substitution reveal that the glutamate at amino acid position 19 of MP in ToLCNDV-OM is critical for mechanical transmissibility. The substitution of glutamate with glycine at this position in the MP of ToLCNDV-OM abolishes mechanical transmissibility. In contrast, the substitution of glycine with glutamate at the 19th amino acid position in the MP of ToLCNDV-CB enables mechanical transmission. This is the first time that a specific geminiviral movement protein has been identified as a determinant of mechanical transmissibility.

Assessing the Impact on Virus Transmission and Insect Vector Behavior of a Viral Mixed Infection in Melon.

Mixed viral infections in plants are common, and can result in synergistic or antagonistic interactions. Except in complex diseases with severe symptoms, mixed infections frequently remain unnoticed, and their impact on insect vector transmission is largely unknown. In this study, we considered mixed infections of two unrelated viruses commonly found in melon plants, the crinivirus cucurbit yellow stunting disorder virus (CYSDV) and the potyvirus watermelon mosaic virus (WMV), and evaluated their vector transmission by whiteflies and aphids, respectively. Their dynamics of accumulation was analyzed until 60 days postinoculation (dpi) in mixed-infected plants, documenting reduced titers of WMV and much higher titers of CYSDV compared with single infections. At 24 dpi, corresponding to the peak of CYSDV accumulation, similar whitefly transmission rates were obtained when comparing either individual or mixed-infected plants as CYSDV sources, although its secondary dissemination was slightly biased toward plants previously infected with WMV, regardless of the source plant. However, at later time points, mixed-infected plants partially recovered from the initially severe symptoms, and CYSDV transmission became significantly higher. Interestingly, aphid transmission rates both at early and late time points were unaltered when WMV was acquired from mixed-infected plants despite its reduced accumulation. This lack of correlation between WMV accumulation and transmission could result from compensatory effects observed in the analysis of the aphid feeding behavior by electrical penetration graphs. Thus, our results showed that mixed-infected plants could provide advantages for both viruses, directly favoring CYSDV dissemination while maintaining WMV transmission.

A Single Amino Acid Substitution in the Intervening Region of 129K Protein of Cucumber Green Mottle Mosaic Virus Resulted in Attenuated Symptoms.

Cucumber green mottle mosaic virus (CGMMV), a member of the genus Tobamovirus, is a major threat to economically important cucurbit crops worldwide. An attenuated strain (SH33b) derived from a severe strain (SH) of CGMMV caused a reduction in the viral RNA accumulation and the attenuation of symptoms, and it has been successfully used to protect muskmelon plants against severe strains in Japan. In this study, we compared GFP-induced silencing suppression by the 129K protein and the methyltransferase domain plus intervening region (MTIR) of the 129K protein between the SH and SH33b strains, respectively. As a result, silencing suppression activity (SSA) in the GFP-silenced plants was inhibited efficiently by the MTIR and 129K protein of SH strain, and it coincided with drastically reduced accumulation of GFP-specific small interfering RNAs (siRNAs) but not by that of SH33b strain. Furthermore, analyses of siRNA binding capability (SBC) by the MTIR of 129K protein and 129K protein using electrophoretic mobility shift assay revealed that SBC was found with the MTIR and 129K protein of SH but not with that of SH33b, suggesting that a single amino acid mutation (E to G) in the MTIR is responsible for impaired SSA and SBC of SH33b. These data suggest that a single amino acid substitution in the intervening region of 129K protein of CGMMV resulted in attenuated symptoms by affecting RNA silencing suppression.

Construction and biological characterization of an Agrobacterium-mediated infectious cDNA of squash mosaic virus.

Squash mosaic virus (SqMV), a member of the species Squash mosaic virus in the genus Comovirus (family Comoviridae), is an important seed-borne virus that causes serious economic losses in cucurbit crops. Here, we constructed infectious cDNA clones of SqMV genomic RNAs (RNA1 and RNA2) under the control of the cauliflower mosaic virus (CaMV) 35S promoter by Gibson assembly. The infectious cDNA clones of SqMV could infect zucchini squash (Cucurbita pepo) plants systemically by agrobacterium-mediated inoculation. The virus progeny from the infectious clones showed no difference from the wild type in terms of pathogenicity and symptom induction. It could be mechanically transmitted to zucchini squash (Cucurbita pepo), pumpkin (Cucurbita moschata), cucumber (Cucumis sativus), and muskmelon (Cucumis melo) but not watermelon (Citrullus lanatus) or Nicotiana benthamiana. This is the first report of construction of a SqMV infection clone and will facilitate the investigation of viral pathogenesis and host interactions.

Thladiantha Dubia Mosaic Virus: A Novel Potyvirus Infecting Manchurian Tubergourd (Thladiantha dubia) in Northeast China.

A new virus with flexuous, filamentous particles approximately 650 nm long was discovered in Manchurian tubergourd (Thladiantha dubia Bunge) leaves exhibiting severe mosaic symptoms. The whole genome sequence of the virus was determined by reverse transcription-polymerase chain reaction (RT-PCR) and rapid amplification of cDNA ends (RACE). The entire genome consisted of 10,112 nucleotides (nt) excluding the poly (A) tail, which shared the highest nucleotide sequence identity (73.8%) with that of papaya leaf distortion mosaic virus Hainan-DF isolate (PLDMV-Hainan-DF). A phylogenetic analysis showed that this virus clustered with PLDMV isolates in a subbranch within the potyviral clade. Of the 23 species of indicator plants tested, only potato and its original host were systemically infected by the virus tested upon mechanical inoculation. A field survey showed that the virus was widely distributed on T. dubia and potatoes in Northeast China. Moreover, this virus displayed a high degree of genetic variation as evaluated by the sequences of the coat protein (CP) gene. Based on these results, the name Thladiantha dubia mosaic virus (ThDMV) is proposed for this new potyvirus.