A natural substitution of a conserved amino acid in eIF4E confers resistance against multiple potyviruses.

Eukaryotic translation initiation factor 4E (eIF4E), which plays a pivotal role in initiating translation in eukaryotic organisms, is often hijacked by the viral genome-linked protein to facilitate the infection of potyviruses. In this study, we found that the naturally occurring amino acid substitution D71G in eIF4E is widely present in potyvirus-resistant watermelon accessions and disrupts the interaction between watermelon eIF4E and viral genome-linked protein of papaya ringspot virus-watermelon strain, zucchini yellow mosaic virus or watermelon mosaic virus. Multiple sequence alignment and protein modelling showed that the amino acid residue D71 located in the cap-binding pocket of eIF4E is strictly conserved in many plant species. The mutation D71G in watermelon eIF4E conferred resistance against papaya ringspot virus-watermelon strain and zucchini yellow mosaic virus, and the equivalent mutation D55G in tobacco eIF4E conferred resistance to potato virus Y. Therefore, our finding provides a potential precise target for breeding plants resistant to multiple potyviruses.

Watermelon crinkle leaf-associated virus 1 and watermelon crinkle leaf-associated virus 2 have a bipartite genome with molecular signatures typical of the members of the genus Coguvirus (family Phenuiviridae).

Watermelon crinkle leaf-associated virus 1 and watermelon crinkle leaf-associated virus 2 (WCLaV-1 and WCLaV-2), two unclassified members of the order Bunyavirales, are phylogenetically related to members of the genus Coguvirus (family Phenuiviridae). The genome of both viruses was reported previously to be composed of three RNA segments. However, the terminal sequences of two genomic RNA segments, namely those encoding the putative movement protein (MP) and the nucleocapsid (NP) protein, remained undetermined. High-throughput sequencing of total RNA and small RNA preparations, combined with reverse transcription PCR amplification followed by sequencing, revealed that the WCLaV-1 and WCLaV-2 possess a bipartite genome consisting of a negative-sense RNA1, encoding the RNA-dependent RNA polymerase, and an ambisense RNA2, encoding the putative movement (MP) and nucleocapsid (NP) proteins. The two open reading frames of RNA2 are in opposite orientations and are separated by a long AU-rich intergenic region (IR) that may assume a hairpin conformation. RNA1 and RNA2 of both viruses share almost identical 5' and 3' termini, which are complementary to each other up to 20 nt. This genome organization is typical of members of the genus Coguvirus, with which WCLaV-1 and WCLaV-2 also share similar terminal 5' and 3' sequences of RNA1 and RNA2. These molecular features, together with phylogenetic reconstructions support the classification of WCLaV-1 and WCLaV2 as members of two new species in the genus Coguvirus.

A tobacco ringspot virus-based vector system for gene and microRNA function studies in cucurbits.

Cucurbits are economically important crops worldwide. The genomic data of many cucurbits are now available. However, functional analyses of cucurbit genes and noncoding RNAs have been impeded because genetic transformation is difficult for many cucurbitaceous plants. Here, we developed a set of tobacco ringspot virus (TRSV)-based vectors for gene and microRNA (miRNA) function studies in cucurbits. A TRSV-based expression vector could simultaneously express GREEN FLUORESCENT PROTEIN (GFP) and heterologous viral suppressors of RNA silencing in TRSV-infected plants, while a TRSV-based gene silencing vector could knock down endogenous genes exemplified by PHYTOENE DESATURASE (PDS) in Cucumis melo, Citrullus lanatus, Cucumis sativus, and Nicotiana benthamiana plants. We also developed a TRSV-based miRNA silencing vector to dissect the functions of endogenous miRNAs. Four representative miRNAs, namely, miR159, miR166, miR172, and miR319, from different cucurbits were inserted into the TRSV vector using a short tandem target mimic strategy and induced characteristic phenotypes in TRSV-miRNA-infected plants. This TRSV-based vector system will facilitate functional genomic studies in cucurbits.

Progression of Watermelon Bud Necrosis Virus Infection in Its Vector, Thrips palmi.

Thrips are important pests of agricultural, horticultural, and forest crops worldwide. In addition to direct damages caused by feeding, several thrips species can transmit diverse tospoviruses. The present understanding of thrips-tospovirus relationships is largely based on studies of tomato spotted wilt virus (TSWV) and Western flower thrips (Frankliniella occidentalis). Little is known about other predominant tospoviruses and their thrips vectors. In this study, we report the progression of watermelon bud necrosis virus (WBNV) infection in its vector, melon thrips (Thrips palmi). Virus infection was visualized in different life stages of thrips using WBNV-nucleocapsid protein antibodies detected with FITC-conjugated secondary antibodies. The anterior midgut was the first to be infected with WBNV in the first instar larvae. The midgut of T. palmi was connected to the principal salivary glands (PSG) via ligaments and the tubular salivary glands (TSG). The infection progressed to the PSG primarily through the connecting ligaments during early larval instars. The TSG may also have an ancillary role in disseminating WBNV from the midgut to PSG in older instars of T. palmi. Infection of WBNV was also spread to the Malpighian tubules, hindgut, and posterior portion of the foregut during the adult stage. Maximum virus-specific fluorescence in the anterior midgut and PSG indicated the primary sites for WBNV replication. These findings will help to better understand the thrips-tospovirus molecular relationships and identify novel potential targets for their management. To our knowledge, this is the first report of the WBNV dissemination path in its vector, T. palmi.

Transmission mode of watermelon silver mottle virus by Thrips palmi.

Thrips and thrips-transmitted tospoviruses cause significant losses in crop yields worldwide. The melon thrips (Thrips palmi) is not only a pest of cucurbit crops, but also a vector that transmits tospoviruses, such as the watermelon silver mottle virus (WSMoV). Vector transmission of tospoviruses has been well studied in the tomato spotted wilt virus (TSWV)-Frankliniella occidentalis model system; however, until now the transmission mode of WSMoV by T. palmi has not been sufficiently examined. The results of the transmission assays suggest that T. palmi transmits WSMoV in a persistent manner, and that the virus is mainly transmitted by adults, having been ingested at the first-instar larval stage. Complementary RNAs corresponding to the NSm and NSs genes of WSMoV were detected in viruliferous thrips by reverse transcription-polymerase chain reaction; NSs protein was also detected in viruliferous thrips by western blotting, verifying the replication of WSMoV in T. palmi. Furthermore, we demonstrated that in thrips infected with WSMoV at the first-instar larval stage, the virus eventually infected various tissues of the adult thrips, including the primary salivary glands. Taken together, these results suggest that T. palmi transmits WSMoV in a persistent-propagative mode. The results of this study make a significant contribution to the understanding of the transmission biology of tospoviruses in general.

Effectiveness of disinfectants against the spread of tobamoviruses: Tomato brown rugose fruit virus and Cucumber green mottle mosaic virus.

BACKGROUND: Tobamoviruses, including tomato brown rugose fruit virus (ToBRFV) on tomato and pepper, and cucumber green mottle mosaic virus (CGMMV) on cucumber and watermelon, have caused many disease outbreaks around the world in recent years. With seed-borne, mechanical transmission and resistant breaking traits, tobamoviruses pose serious threat to vegetable production worldwide. With the absence of a commercial resistant cultivar, growers are encouraged to take preventative measures to manage those highly contagious viral diseases. However, there is no information available on which disinfectants are effective to deactivate the virus infectivity on contaminated hands, tools and equipment for these emerging tobamoviruses. The purpose of this study was to evaluate a collection of 16 chemical disinfectants for their effectiveness against mechanical transmission of two emerging tobamoviruses, ToBRFV and CGMMV. METHODS: Bioassay was used to evaluate the efficacy of each disinfectant based on virus infectivity remaining in a prepared virus inoculum after three short exposure times (10 s, 30 s and 60 s) to the disinfectant and inoculated mechanically on three respective test plants (ToBRFV on tomato and CGMMV on watermelon). Percent infection of plants was measured through symptom observation on the test plants and the presence of the virus was confirmed through an enzyme-linked immunosorbent assay with appropriate antibodies. Statistical analysis was performed using one-way ANOVA based on data collected from three independent experiments. RESULTS: Through comparative analysis of percent infection of test plants, a similar trend of efficacy among 16 disinfectants was observed between the two pathosystems. Four common disinfectants with broad spectrum activities against two different tobamoviruses were identified. Those effective disinfectants with 90-100% efficacy against both tobamoviruses were 0.5% Lactoferrin, 2% Virocid, and 10% Clorox, plus 2% Virkon against CGMMV and 3% Virkon against ToBRFV. In addition, SP2700 generated a significant effect against CGMMV, but poorly against ToBRFV. CONCLUSION: Identification of common disinfectants against ToBRFV and CGMMV, two emerging tobamoviruses in two different pathosystems suggest their potential broader effects against other tobamoviruses or even other viruses.

A rapid fluorescence-based real-time isothermal assay for the detection of Cucurbit yellow stunting disorder virus in squash and watermelon plants.

Cucurbit yellow stunting disorder virus (CYSDV) is a single-stranded positive-sense RNA virus that produces devastating disease in watermelon and squash. Foliar symptoms of CYSDV consist of interveinal yellowing, brittleness, and thickening of older leaves leading to reduced plant vigor. A rapid diagnostic method for CYSDV would facilitate early detection and implementation of best viral-based management practices. We developed a rapid isothermal reverse transcription-recombination polymerase amplification (exo RT-RPA) assay for the detection of CYSDV. The primers and a 6-fluorescein amidite (6-FAM) probe were developed to target the nucleocapsid gene. The real-time assay detected CYSDV at 2.5 pg purified total RNA extracted from CYSDV-infected leaf tissue and corresponded to 10 copies of the target molecule. The assay was specific and did not cross-react with other common cucurbit viruses found in Florida and Georgia. The performance of the exo RT-RPA was evaluated using crude extract from 21 cucurbit field samples and demonstrated that the exo RT-RPA is a rapid procedure, thus providing a promising novel alternative approach for the detection of CYSDV.

Genome-wide identification of long non-coding RNAs and circular RNAs reveal their ceRNA networks in response to cucumber green mottle mosaic virus infection in watermelon.

Long non-coding RNAs (lncRNAs) and circular RNAs (circRNAs) play vital roles in plant defense responses against viral infections. However, there is no systematic understanding of lncRNAs and circRNAs and their competing endogenous RNA (ceRNA) networks in watermelon under cucumber green mottle mosaic virus (CGMMV) stress. Here, we present the characterization and expression profiles of lncRNAs and circRNAs in watermelon leaves 48-h post-inoculation (48 hpi) with CGMMV, with mock inoculation as a control. Deep sequencing analysis revealed 2373 lncRNAs and 606 circRNAs in the two libraries. Among them, 67 lncRNAs (40 upregulated and 27 downregulated) and 548 circRNAs (277 upregulated and 271 downregulated) were differentially expressed (DE) in the 48 hpi library compared with the control library. Furthermore, 263 cis-acting matched lncRNA-mRNA pairs were detected for 49 of the DE-lncRNAs. KEGG pathway analysis of the cis target genes of the DE-lncRNAs revealed significant associations with phenylalanine metabolism, the citrate cycle (TCA cycle), and endocytosis. Additionally, 30 DE-lncRNAs were identified as putative target mimics of 33 microRNAs (miRNAs), and 153 DE-circRNAs were identified as putative target mimics of 88 miRNAs. Furthermore, ceRNA networks of lncRNA/circRNA-miRNA-mRNA in response to CGMMV infection are described, with 12 DE-lncRNAs and 65 DE-circRNAs combining with 22 miRNAs and competing for the miRNA binding sites on 29 mRNAs. The qRT-PCR validation of selected lncRNAs and circRNAs showed a general correlation with the high-throughput sequencing results. This study provides a valuable resource of lncRNAs and circRNAs involved in the response to CGMMV infection in watermelon.

iTRAQ-Based Proteomic Analysis of Watermelon Fruits in Response to Cucumber green mottle mosaic virus Infection.

Cucumber green mottle mosaic virus (CGMMV) is an important viral pathogen on cucurbit plants worldwide, which can cause severe fruit decay symptoms on infected watermelon (usually called "watermelon blood flesh"). However, the molecular mechanism of this disease has not been well understood. In this study, we employed the isobaric tags for relative and absolute quantitation (iTRAQ) technique to analyze the proteomic profiles of watermelon fruits in response to CGMMV infection. A total of 595 differentially accumulated proteins (DAPs) were identified, of which 404 were upregulated and 191 were downregulated. Functional annotation analysis showed that these DAPs were mainly involved in photosynthesis, carbohydrate metabolism, secondary metabolite biosynthesis, plant-pathogen interaction, and protein synthesis and turnover. The accumulation levels of several proteins related to chlorophyll metabolism, pyruvate metabolism, TCA cycle, heat shock proteins, thioredoxins, ribosomal proteins, translation initiation factors, and elongation factors were strongly affected by CGMMV infection. Furthermore, a correlation analysis was performed between CGMMV-responsive proteome and transcriptome data of watermelon fruits obtained in our previous study, which could contribute to comprehensively elucidating the molecular mechanism of "watermelon blood flesh". To confirm the iTRAQ-based proteome data, the corresponding transcripts of ten DAPs were validated by determining their abundance via quantitative reverse transcriptase-polymerase chain reaction (qRT-PCR). These results could provide a scientific basis for in-depth understanding of the pathogenic mechanisms underlying CGMMV-induced "watermelon blood flesh", and lay the foundation for further functional exploration and verification of related genes and proteins.

Assessing the Temporal Effects of Squash vein yellowing virus Infection on Settling and Feeding Behavior of Bemisia tabaci (MEAM1) (Hemiptera: Aleyrodidae).

Insect vector behavior and biology can be affected by pathogen-induced changes in the physiology and morphology of the host plant. Herein, we examined the temporal effects of Squash vein yellowing virus (family Potyviridae, genus Ipomovirus) infection on the settling, oviposition preference, and feeding behavior of its whitefly vector, Bemisia tabaci (Gennadius) Middle East-Asia Minor 1 (MEAM1), formerly known as B. tabaci biotype B. Settling and oviposition behavioral choice assays were conducted on pairs of infected and mock-inoculated watermelon (Citrullus lanatus (Thunb) Matsum and Nakai) (Cucurbitales: Cucurbitaceae) at 5-6 days post inoculation (DPI) and 10-12 DPI. Electropenetrography, or electrical penetration graph (both abbreviated EPG), was used to assess differences in feeding behaviors of whitefly on mock-inoculated, 5-6 and 10-12 DPI infected watermelon plants. Whiteflies showed no preference in settling or oviposition on the infected and mock-inoculated plants at 5-6 DPI. However, at 10-12 DPI, whiteflies initially settled on infected plants but then preference of settling shifted to mock-inoculated plants after 8 h. Only at 10-12 DPI, females laid significantly more eggs on mock-inoculated plants than infected plants. EPG revealed no differences in whitefly feeding behaviors among mock-inoculated, 5-6 DPI infected and 10-12 DPI infected plants. The results highlighted the need to examine plant disease progression and its effect on vector behavior and performance, which could play a crucial role in Squash vein yellowing virus spread.

DNA Methylation Analysis of the Citrullus lanatus Response to Cucumber GreenMottle Mosaic Virus Infection by Whole-Genome Bisulfite Sequencing.

DNA methylation is an important epigenetic mark associated with plant immunity, butlittle is known about its roles in viral infection of watermelon. We carried out whole-genomebisulfite sequencing of watermelon leaves at 0 h (ck), 48 h, and 25 days post-inoculation withCucumber green mottle mosaic virus (CGMMV). The number of differentially methylated regions(DMRs) increased during CGMMV infection and 2788 DMR-associated genes (DMGs) werescreened out among three libraries. Most DMRs and DMGs were obtained under the CHH context.These DMGs were significantly enriched in the Kyoto Encyclopedia of Genes and Genomes (KEGG)pathways of secondary biosynthesis and metabolism, plant-pathogen interactions, Toll-likereceptor signaling, and ABC transporters. Additionally, DMGs encoding PR1a, CaMs, calciumbindingprotein, RIN4, BAK1, WRKYs, RBOHs, STKs, and RLPs/RLKs were involved in thewatermelon-CGMMV interaction and signaling. The association between DNA methylation andgene expression was analyzed by RNA-seq and no clear relationship was detected. Moreover,downregulation of genes in the RdDM pathway suggested the reduced RdDM-directed CHHmethylation plays an important role in antiviral defense in watermelon. Our findings providegenome-wide DNA methylation profiles of watermelon and will aid in revealing the molecularmechanism in response to CGMMV infection at the methylation level.

Rapid detection of Cucumber green mottle mosaic virus in watermelon through a recombinase polymerase amplification assay.

Cucumber green mottle mosaic virus (CGMMV), a member of the genus Tobamovirus, is an important quarantine plant virus worldwide, and often causes seriously damages to productions of watermelon, melon, cucumber and other cucurbit crops. In this study, we developed a novel isothermal recombinase polymerase amplification (RPA) technique for detection of CGMMV in watermelon samples. A pair of CGMMV specific RPA primers was prepared based on the conserved CGMMV coat protein gene sequences. The result showed that this RPA detection method can be performed at 38 °C and completed in about 30 min, and there was no cross-reactivity with other common cucurbit viruses. Sensitivity assay showed that this RPA method was more sensitive compared with the regular RT-PCR. Using field-collected watermelon tissue samples, we have demonstrated that this newly developed method is rapid, easy to use and reliable for CGMMV detection, especially in resource-limited laboratories or on-site facilities.

T4 DNA polymerase-assisted upgrade of a nicking/polymerization amplification strategy for ultrasensitive electrochemical detection of Watermelon mosaic virus.

An upgraded nicking/polymerization strategy for ultrasensitive electrochemical detection of Watermelon mosaic virus (WMV) is proposed on the basis of the exonuclease and polymerase activity of T4 DNA polymerase and Mg2+-dependent DNAzyme-assisted and hemin/G-quadruplex DNAzyme-assisted cascade amplification strategies. Briefly, the hybridized DNA of the target WMV sequence, HP1, and P1 was recognized and nicked by nicking endonuclease Nb.BbvCI, and two DNA segments (P1-25 and P1-6) were produced. P1-25 was digested in the 3'→5' direction, and digestion was halted at the 3'-terminal G locus with the exonuclease activity of T4 DNA polymerase. When dNTP solution mix was added to the mixture, an intact enzymatic sequence of Mg2+-dependent DNAzyme was synthesized by T4 DNA polymerase, which hybridized with its substrate sequence in the loop segment of HP2 immobilized on a gold electrode and initiated the cleavage round. The caged G-quadruplex sequence was released and formed hemin/G-quadruplex-based DNAzyme, resulting in sharply increased electrochemical signals. A correlation between the differential pulse voltammetry signal and the concentration of target WMV sequence was obtained in the range from 50 fM to 1 nM, with 50 fM detection limit. Because the nicking and polymerization reactions are irreversible and share the same buffer, the cascade amplification strategy is an ultrasensitive and high-efficiency strategy, indicating potential for viral detection. Graphical abstract An upgrade nicking/polymerization strategy for ultrasensitive electrochemical detection of Watermelon mosaic virus (WMV) was proposed based on DNAzyme-assistant cascade amplification strategies.

Expression and Localization Patterns of a Small Heat Shock Protein that Interacts with the Helicase Domain of Cucumber Green Mottle Mosaic Virus.

Cucumber green mottle mosaic virus (CGMMV), a member of the genus Tobamovirus (family Virgaviridae), is an economically important virus that has detrimental effects on cucurbit crops worldwide. Understanding the interaction between host factors and CGMMV viral proteins will facilitate the design of new strategies for disease control. In this study, a yeast two-hybrid assay revealed that the CGMMV helicase (HEL) domain interacts with a Citrullus lanatus small heat shock protein (sHSP), and we verified this observation by performing in vitro GST pull-down and in vivo coimmunoprecipitation assays. Measurement of the levels of accumulated sHSP transcript revealed that sHSP is upregulated on initial CGMMV infection in both Nicotiana benthamiana and C. lanatus plants, although not in the systemically infected leaves. We also found that the subcellular localization of the sHSP was altered after CGMMV infection. To further validate the role of sHSP in CGMMV infection, we produced and assayed N. benthamiana transgenic plants with up- and down-regulated sHSP expression. Overexpression of sHSP inhibited viral RNA accumulation and retarded disease development, whereas sHSP silencing had no marked effect on CGMMV infection. Therefore, we postulate that the identified sHSP may be one of the factors modulating host defense mechanisms in response to CGMMV infection and that the HEL domain interaction may inhibit this sHSP function to promote viral infection.

Cross protection against the watermelon strain of Papaya ringspot virus through modification of viral RNA silencing suppressor.

Papaya ringspot virus watermelon strain (PRSV-W) causes huge economic losses to cucurbits production. Here, we constructed an infectious clone of PRSV-W, pCamPRSV-W, which can induce similar symptoms and accumulate to same levels as wild type virus in plants of Cucurbita pepo, Cucumis melo, Citrullus lanatus and Cucumis sativus. The green fluorescence protein gene gfp was cloned into pCamPRSV-W to produce pCamPRSV-W-GFP, which produced strong green fluorescence in systemic leaves of inoculated Cucurbita pepo, Cucumis melo, Citrullus lanatus and Cucumis sativus plants, indicating that pCamPRSV-W can be used to express foreign genes. Ten mutants of PRSV-W, obtained by site-directed mutagenesis in the RNA silencing suppressor helper-component proteinase encoding region, produced dramatically attenuated symptoms in plants of Cucumis melo. The Cucumis melo plants pre-infected with mutants K125D and G317 K showed effective protection against the challenge inoculation of wild type PRSV-W. The attenuated mutants generated in this study will be helpful for the eco-friendly control of PRSV-W.

Transcriptome Analysis of Watermelon Leaves Reveals Candidate Genes Responsive to Cucumber green mottle mosaic virus Infection.

Cucumber green mottle mosaic virus (CGMMV) is a member of the genus Tobamovirus, which cause diseases in cucurbits, especially watermelon. In watermelon, symptoms develop on the whole plant, including leaves, stems, peduncles, and fruit. To better understand the molecular mechanisms of watermelon early responses to CGMMV infection, a comparative transcriptome analysis of 24 h CGMMV-infected and mock-inoculated watermelon leaves was performed. A total of 1641 differently expressed genes (DEGs) were identified, with 886 DEGs upregulated and 755 DEGs downregulated after CGMMV infection. A functional analysis indicated that the DEGs were involved in photosynthesis, plant⁻pathogen interactions, secondary metabolism, and plant hormone signal transduction. In addition, a few transcription factor families, including WRKY, MYB, HLH, bZIP and NAC, were responsive to the CGMMV-induced stress. To confirm the high-throughput sequencing results, 15 DEGs were validated by qRT-PCR analysis. The results provide insights into the identification of candidate genes or pathways involved in the responses of watermelon leaves to CGMMV infection.

Host-associated selection of a P3 mutant of zucchini yellow mosaic virus affects viral infectivity in watermelon.

In this study, we found that the infectivity of zucchini yellow mosaic virus (ZYMV) in watermelon lines H1 and K6 changed from partial to complete after propagation in the susceptible watermelon line ZXG637. When using cucumber infected with strain ZYMV-CH87 as an inoculum (named ZYMV-CH87C), the mean incidences of infection in lines H1 and K6 were 6% and 11%, respectively. However, when these lines were inoculated with ZXG637 infected with ZYMV-CH87C (named ZYMV-637), 100% of the plants became infected. Sequencing of ZYMV from these different inoculums revealed two nucleotide changes in the P3 cistron in ZYMV-637, which resulted in changes in the amino acids at positions 768 and 857 of the P3 protein, compared with the original strain ZYMV-CH87. We named this variant the M768I857-variant. The M768I857-variant was detected at low levels (3.9%) in ZYMV-CH87C. When ZYMV-CH87C was passaged with ZXG637, the M768I857-variant was selected by the host, and the original sequence was replaced entirely after two passages. These results may be explained by host-associated selection due to an unknown host-encoded factor. Using the M768I857-variant as an inoculum, 100% of the H1 and K6 plants showed systemic symptoms. These results suggest that (1) changing the individual amino acids at the end of the P3 N-terminus induces resistance-breaking, and (2) the P3 N-terminus may be involved in host recognition.

Exogenously applied dsRNA molecules deriving from the Zucchini yellow mosaic virus (ZYMV) genome move systemically and protect cucurbits against ZYMV.

Zucchini yellow mosaic virus (ZYMV) causes serious damage in a large number of cucurbits, and control measures are necessary. Transgenic cucurbits expressing parts of the ZYMV genome have been shown to be resistant to the cognate virus. A non-transgenic approach involving the exogenous application of double-stranded RNA (dsRNA) has also been shown to induce resistance in tobacco against Cucumber mosaic virus (CMV) and Tobacco mosaic virus (TMV). In the present study, dsRNA molecules derived from the helper component-proteinase (HC-Pro) and coat protein (CP) genes of the ZYMV_DE_2014 isolate were produced in vitro. On exogenous dsRNA application in cucumber, watermelon and squash plants, dsRNA HC-Pro conferred resistance of 82%, 50% and 18%, and dsRNA CP molecules of 70%, 43% and 16%, respectively. On deep sequencing analysis of ZYMV-infected watermelon, hot-spot regions for viral small interfering RNAs (vsiRNAs) in the genome of ZYMV were identified. Stem-loop reverse transcription-polymerase chain reaction (RT-PCR) detection of selected 21-nucleotide-long vsiRNAs in plants that received only dsRNA molecules suggested that the dsRNAs exogenously applied onto plants were successfully diced, thus initiating RNA silencing. dsRNA molecules were found to be progressively degraded in planta, and strongly detected by semi-quantitative RT-PCR for at least 9 days after exogenous application. Moreover, dsRNA molecules were detected in systemic tissue of watermelon and squash, showing that dsRNA is transported long distances in these plants.

Transcriptome analysis of watermelon (Citrullus lanatus) fruits in response to Cucumber green mottle mosaic virus (CGMMV) infection.

Cucumber green mottle mosaic virus (CGMMV) belongs to the Tobamovirus genus and is a major global plant virus on cucurbit plants. It causes severe disease symptoms on infected watermelon plants (Citrullus lanatus), particularly inducing fruit decay. However, little is known about the molecular mechanism of CGMMV-induced watermelon fruit decay. For this study, comparative analysis of transcriptome profiles of CGMMV-inoculated and mock-inoculated watermelon fruits were conducted via RNA-Seq. A total of 1,621 differently expressed genes (DEGs) were identified in CGMMV-inoculated watermelon, among which 1,052 were up-regulated and 569 were down-regulated. Functional annotation analysis showed that several DEGs were involved in carbohydrate metabolism, hormone biosynthesis and signaling transduction, secondary metabolites biosynthesis, and plant-pathogen interactions. We furthermore found that some DEGs were related to cell wall components and photosynthesis, which may directly be involve in the development of the symptoms associated with diseased watermelons. To confirm the RNA-Seq data, 15 DEGs were selected for gene expression analysis by qRT-PCR. The results showed a strong correlation between these two sets of data. Our study identified many candidate genes for further functional studies during CGMMV-watermelon interactions, and will furthermore help to clarify the understanding of pathogenic mechanism underlying CGMMV infection in cucurbit plants.

Deep Sequencing Data and Infectivity Assays Indicate that Chickpea Chlorotic Dwarf Virus is the Etiological Agent of the "Hard Fruit Syndrome" of Watermelon.

Chickpea chlorotic dwarf virus (CpCDV), a polyphagous mastrevirus, family Geminiviridae, has been recently linked to the onset of the "hard fruit syndrome" of watermelon, first described in Tunisia, that makes fruits unmarketable due to the presence of white hard portions in the flesh, chlorotic mottling on the rind, and an unpleasant taste. To investigate the etiological agent of this disease, total RNA extracted from symptomatic watermelon fruits was subjected to small RNA sequencing through next generation sequencing (NGS) techniques. Data obtained showed the presence of CpCDV and two other viral species. However, following validation through polymerase chain reaction (PCR), CpCDV was the only viral species consistently detected in all samples. Watermelon seedlings were then challenged by an agroinfectious CpCDV clone; several plants proved to be CpCDV-infected, and were able to produce fruits. CpCDV infected and replicated in watermelon fruits and leaves, leading to abnormality in fruits and in seed production, similar to those described in field. These results indicate that CpCDV is the etiological agent of the "hard fruit syndrome" of watermelon.