Widely targeted analysis of metabolomic changes of Cucumis sativus induced by cucurbit chlorotic yellows virus.

BACKGROUND: Plant metabolites play vital roles in regulating the behavior of herbivore insects. Virus infection can universally alter plant metabolites to manipulate the orientation and feeding behaviors of insect vector, to favor the transmission of virus. Thus, determining the differentially accumulated metabolites of plant upon virus infection could provide insights into understanding how the triple interactions among plant, virus and insect vector happens. Our previous studies have found that vector whitefly Bemisia tabaci (Gennadius, Hemiptera: Aleyrodidae) showed different orientation behavior and performance on CCYV-infected and healthy cucumber plants. Cucurbit chlorotic yellows virus (CCYV) is exclusively transmitted by B. tabaci in a semi-persistent mode. In this study, we take the CCYV, B. tabaci and cucumber as a research system to explore the functions of phyto-metabolites in the triple interactions. RESULTS: A total of 612 metabolites changed upon CCYV infection were monitored. Metabolites mainly enriched in flavonoids, lipids, nucleotides and their derivatives. At 7 days post CCYV inoculation (dpi), the contents of lipids, terpenoids and flavonoids remarkably decreased, while amino acids, nucleotides and their derivatives notably up-accumulated. At 15 dpi, the accumulation of flavonoids were still significantly reduced upon CCYV infection, while lipids, amino acids, nucleotides and derivatives were remarkably enhanced. Most of significantly increased metabolites were lipids (lysophosphatidylethanolamine, LPE; lysophosphatidylcholine, LPC and their isomers). Also, the number of significantly changed metabolites increased with the infection period. However, only a few organic acids and phenolic acids showed difference between CCYV-infected and healthy cucumber plants. CONCLUSIONS: CCYV infection repressed the defensive flavonoids, terpeneoids metabolism but triggered the lipids, amino acids and nucleotides metabolism with the inoculation period. This result suggests that CCYV-infection makes cucumber plants more susceptible for whiteflies attack and CCYV infection. The reduction of defensive comounds and the increase of amino acids may be partially responsible for enhancing feeding preference of whiteflies to CCYV-infected hosts. CCYV may hijacked lipid metabolism for virus replication and assembly.

Plasticity of the lettuce infectious yellows virus minor coat protein (CPm) in mediating the foregut retention and transmission of a chimeric CPm mutant by whitefly vectors.

Transmission of the crinivirus, lettuce infectious yellows virus (LIYV), is determined by a minor coat protein (CPm)-mediated virion retention mechanism located in the foregut of its whitefly vector. To better understand the functions of LIYV CPm, chimeric CPm mutants engineered with different lengths of the LIYV CPm amino acid sequence and that of the crinivirus, lettuce chlorosis virus (LCV), were constructed based on bioinformatics and sequence alignment data. The 485 amino acid-long chimeric CPm of LIYV mutant, CPmP-1, contains 60 % (from position 3 to 294) of LCV CPm amino acids. The chimeric CPm of mutants CPmP-2, CPmP-3 and CPmP-4 contains 46 (position 3 to 208), 51 (position 3 to 238) and 41 % (position 261 to 442) of LCV CPm amino acids, respectively. All four mutants moved systemically, expressed the chimeric CPm and formed virus particles. However, following acquisition feeding of the virus preparations, only CPmP-1 was retained in the foreguts of a significant number of vectors and transmitted. In immuno-gold labelling transmission electron microscopy (IGL-TEM) analysis, CPmP-1 particles were distinctly labelled by antibodies directed against the LCV but not LIYV CPm. In contrast, CPmP-4 particles were not labelled by antibodies directed against the LCV or LIYV CPm, while CPmP-2 and -3 particles were weakly labelled by anti-LIYV CPm but not anti-LCV CPm antibodies. The unique antibody recognition and binding pattern of CPmP-1 was also displayed in the foreguts of whitefly vectors that fed on CPmP-1 virions. These results are consistent with the hypothesis that the chimeric CPm of CPmP-1 is incorporated into functional virions, with the LCV CPm region being potentially exposed on the surface and accessible to anti-LCV CPm antibodies.

Nuances of Whitefly Vector-Crinivirus Interactions Revealed in the Foregut Retention and Transmission of Lettuce Chlorosis Virus by Two Bemisia tabaci Cryptic Species.

Lettuce infectious yellows virus is the first crinivirus for which the retention of purified virions ingested into the whitefly (Bemisia tabaci New World (NW)) vector's foregut, has been demonstrated to be a requisite for successful virus transmission. This key finding supports the hypothesis that the determinant of foregut retention and transmission is present on the virion itself. However, whether this is also true for other criniviruses has not been established. Here, we provide evidence that lettuce chlorosis virus (LCV) acquired from plants is retained in the foreguts of both the B. tabaci NW and Middle East-Asia Minor 1 (MEAM1) vector species and transmitted upon inoculation feeding. An association between foregut retention and transmission by NW vectors is also observed following the acquisition and inoculation feeding of LCV virions purified using a standard procedure involving 2% or 4% (v/v) Triton™ X-100 (TX-100). However, while virions purified with 2% or 4% TX-100 are also retained in the foreguts of MEAM1 vectors, transmission is observed with the 4% TX-100-purified virions or when more vectors are used for acquisition and inoculation feeding. These results suggest that an intrinsic difference exists between NW and MEAM1 vectors in their interactions with, and transmission of, LCV virions.

In Vitro Identification and In Vivo Confirmation of Inhibitors for Sweet Potato Chlorotic Stunt Virus RNA Silencing Suppressor, a Viral RNase III.

Sweet potato virus disease (SPVD), caused by synergistic infection of Sweet potato chlorotic stunt virus (SPCSV) and Sweet potato feathery mottle virus (SPFMV), is responsible for substantial yield losses all over the world. However, there are currently no approved treatments for this severe disease. The crucial role played by RNase III of SPCSV (CSR3) as an RNA silencing suppressor during the viruses' synergistic interaction in sweetpotato makes it an ideal drug target for developing antiviral treatment. In this study, high-throughput screening (HTS) of small molecular libraries targeting CSR3 was initiated by a virtual screen using Glide docking, allowing the selection of 6,400 compounds out of 136,353. We subsequently developed and carried out kinetic-based HTS using fluorescence resonance energy transfer technology, which isolated 112 compounds. These compounds were validated with dose-response assays including kinetic-based HTS and binding affinity assays using surface plasmon resonance and microscale thermophoresis. Finally, the interference of the selected compounds with viral accumulation was verified in planta In summary, we identified five compounds belonging to two structural classes that inhibited CSR3 activity and reduced viral accumulation in plants. These results provide the foundation for developing antiviral agents targeting CSR3 to provide new strategies for controlling sweetpotato virus diseases.IMPORTANCE We report here a high-throughput inhibitor identification method that targets a severe sweetpotato virus disease caused by coinfection with two viruses (SPCSV and SPFMV). The disease is responsible for up to 90% yield losses. Specifically, we targeted the RNase III enzyme encoded by SPCSV, which plays an important role in suppressing the RNA silencing defense system of sweetpotato plants. Based on virtual screening, laboratory assays, and confirmation in planta, we identified five compounds that could be used to develop antiviral drugs to combat the most severe sweetpotato virus disease.

A temperature-driven model for potato yellow vein virus transmission efficacy by Trialeurodes vaporariorum (Hemiptera: Aleyrodidae).

Management of viral plant diseases can be improved by using models to predict disease spread. Potato yellow vein virus (PYVV) of the genus Crinivirus (Closteroviridae) is transmitted in a semi-persistent manner by the greenhouse whitefly Trialeurodes vaporariorum (Hemiptera: Aleyrodidae). Although several approaches exist for modeling insect population growth, modeling vector-born virus spread remains difficult because fundamental knowledge on the relationship between virus transmission and temperature is lacking for most vector transmitted viruses. To address this challenge, we initially developed a temperature-dependent phenology model for the whitefly vector using the Insect Life Cycle Modeling (ILCYM) software. In the present study, the effect of temperature on the efficiency of virus transmission by the whitefly was determined through controlled laboratory experiments at 8 constant temperatures in the range from 10 to 25 °C. The vector capacity to transmit the virus was highest at 15 °C (about 70 % probability of infection) but decreased radically as temperature deviated from this optimum temperature to <10 % at temperatures of 10 and 20 °C, respectively. The temperature-dependent probability of virus transmission by a single adult whitefly could be described by a nonlinear function, which was validated by transmission frequencies observed at fluctuating temperatures. This function combined with life table parameters calculated from previously established temperature-dependent phenology model for the vector provided a full temperature-responsive model for predicting PYVV spread potential and transmission probabilities. For spatial risk predictions, we devised two virus transmission risk indexes and tested their performance in correctly predicting virus presence/absence with field survey data. The best performing risk index was used to generate risk maps, which reflected well the current (real) occurrence of the virus but also predicted areas at high risk, where the virus has not previously been reported. One of them in western Panama was targeted for surveillance and resulted in identification of the virus in the country, where it was not previously known to occur. Simulated risk maps for the year 2050 revealed that climate change may significantly affect, the risk of distribution, generally reducing in tropical areas of the world, but increasing in the temperate regions.

Design, Synthesis, and Anti-ToCV Activity of Novel 4(3H)-Quinazolinone Derivatives Bearing Dithioacetal Moiety.

Tomato chlorosis virus (ToCV) has caused great harm to the production of tomato worldwide. To develop efficient anti-ToCV agents, some novel 4(3H)-quinazolinone derivatives containing dithioacetal were designed and synthesized, and their anti-ToCV activities were evaluated by microscale thermophoresis (MST) using ToCV coat protein (ToCV-CP) as a new target. The results showed that some compounds had a strong binding capacity to ToCV-CP. In particular, compounds C5 and C22 have an excellent binding capacity to ToCV-CP, with binding constant values of 0.24 and 0.25 µM, respectively. Additionally, reduced ToCV-CP gene expression levels of 81.05 and 87.59% could be achieved when tomato was treated with compounds C5 and C22, respectively, which were obviously higher than the levels after ningnanmycin (NNM) treatment (43.88%) and lead compound Xiangcaoliusuobingmi (XCLSBM) treatment (63.56%). Therefore, this work indicates that 4(3H)-quinazolinone derivatives containing dithioacetal moiety can be used as novel anti-ToCV agents.

Silencing of Odorant-Binding Protein Gene OBP3 Using RNA Interference Reduced Virus Transmission of Tomato Chlorosis Virus.

Tomato chlorosis virus (ToCV) is widespread, seriously impacting tomato production throughout the world. ToCV is semi-persistently transmitted by Bemisia tabaci (Gennadius) (Hemiptera: Aleyrodidae). Currently, insect olfaction is being studied to develop novel pest control technologies to effectively control B. tabaci and whitefly-borne virus diseases. Despite current research efforts, no report has been published on the role of odorant-binding proteins (OBPs) in insect preference under the influence of plant virus. Our previous research showed that viruliferous B. tabaci preferred healthy plants at 48 h after virus acquisition. In this study, we determined the effect of OBPs on the host preference interactions of ToCV and whiteflies. Our results show that with the increase in acquisition time, the OBP gene expressions changed differently, and the OBP3 gene expression showed a trend of first rising and then falling, and reached the maximum at 48 h. These results indicate that OBP3 may participate in the host preference of viruliferous whiteflies to healthy plants. When the expression of the OBP3 gene was knocked down by an RNA interference (RNAi) technique, viruliferous Mediterranean (MED) showed no preference and the ToCV transmission rate was reduced by 83.3%. We conclude that OBP3 is involved in the detection of plant volatiles by viruliferous MED. Our results provide a theoretical basis and technical support for clarifying the transmission mechanism of ToCV by B. tabaci and could provide new avenues for controlling this plant virus and its vectors.

Differences in gene expression in whitefly associated with CYSDV-infected and virus-free melon, and comparison with expression in whiteflies fed on ToCV- and TYLCV-infected tomato.

BACKGROUND: Cucurbit yellow stunting disorder virus (CYSDV; genus Crinivirus, Closteroviridae) is transmitted in a semipersistent manner by the whitefly, Bemisia tabaci, and is efficiently transmitted by the widely prevalent B. tabaci cryptic species, MEAM1. In this study, we compared transcriptome profiles of B. tabaci MEAM1, after 24 h, 72 h and 7 days of acquisition feeding on melon plants infected with CYSDV (CYSDV-whiteflies) with those fed on virus-free melon, using RNA-Seq technology. We also compared transcriptome profiles with whiteflies fed on tomato plants separately infected with Tomato chlorosis virus (ToCV), a crinivirus closely related to CYSDV, and Tomato yellow leaf curl virus (TYLCV), a member of the genus Begomovirus, which has a distinctly different mode of transmission and their respective virus-free controls, to find common gene expression changes among viruliferous whiteflies feeding on different host plants infected with distinct (TYLCV) and related (CYSDV and ToCV) viruses. RESULTS: A total of 275 differentially expressed genes (DEGs) were identified in CYSDV-whiteflies, with 3 DEGs at 24 h, 221 DEGs at 72 h, and 51 DEGs at 7 days of virus acquisition. Changes in genes encoding orphan genes (54 genes), phosphatidylethanolamine-binding proteins (PEBP) (20 genes), and AAA-ATPase domain containing proteins (10 genes) were associated with the 72 h time point. Several more orphan genes (20 genes) were differentially expressed at 7 days. A total of 59 common DEGs were found between CYSDV-whiteflies and ToCV-whiteflies, which included 20 orphan genes and 6 lysosomal genes. A comparison of DEGs across the three different virus-host systems revealed 14 common DEGs, among which, eight showed similar and significant up-regulation in CYSDV-whiteflies at 72 h and TYLCV-whiteflies at 24 h, while down-regulation of the same genes was observed in ToCV-whiteflies at 72 h. CONCLUSIONS: Dynamic gene expression changes occurred in CYSDV-whiteflies after 72 h feeding, with decreased gene expression changes associated with 7 days of CYSDV acquisition. Similarities in gene expression changes among CYSDV-whiteflies, ToCV-whiteflies and TYLCV-whiteflies suggest the possible involvement of common genes or pathways for virus acquisition and transmission by whiteflies, even for viruses with distinctly different modes of transmission.

Expanding Knowledge of the Host Range of Tomato chlorosis virus and Host Plant Preference of Bemisia tabaci MEAM1.

The crinivirus Tomato chlorosis virus (ToCV) is often found infecting tomato crops in Brazil, with variable incidence, but associated with prevalence of its primary vector, Bemisia tabaci MEAM1. ToCV control is difficult because there are no resistant commercial tomato varieties or hybrids available and chemical spray for control of the whitefly vector has not been effective. The present study evaluated the partial host range of a Brazilian isolate of ToCV and the preference of B. tabaci MEAM1 for oviposition on those species identified as susceptible to the virus. Subsequently, transmission tests were performed using plants of each ToCV host species as sources of inoculum to elucidate the epidemiological importance of nontomato sources of inoculum for infection of tomato. Among 80 species experimentally inoculated, 25 were susceptible, including 6 previously not known to be hosts (Jaltomata procumbens, Physalis pruinosa, Solanum aculeatissimum, S. viarum, Beta vulgaris var. cicla, and Chenopodium quinoa). Preference of whitefly for oviposition and infection by ToCV under free-choice transmission tests varied among the susceptible species. When ToCV-infected tomato, eggplant, and C. quinoa were used separately as sources of inoculum for virus transmission to tomato plants, mean percentages of infected plants were 76.6, 3, and 0%, respectively. Average oviposition of Bemisia tabaci on these three hosts were 2.7, 10.6, and 0.0 eggs/cm2, respectively. Additional studies will be necessary to evaluate the importance of ToCV host plants under field conditions and their efficiency as sources of inoculum for virus acquisition and transmission to tomato crops.

Infection by the semi-persistently transmitted Tomato chlorosis virus alters the biology and behaviour of Bemisia tabaci on two potato clones.

Insect-borne plant viruses usually alter the interactions between host plant and insect vector in ways conducive to their transmission ('host manipulation hypothesis'). Most studies have tested this hypothesis with persistently and non-persistently transmitted viruses, while few have examined semi-persistently transmitted viruses. The crinivirus Tomato chlorosis virus (ToCV) is semi-persistently transmitted virus by whiteflies, and has been recently reported infecting potato plants in Brazil, where Bemisia tabaci Middle East Asia Minor 1 (MEAM1) is a competent vector. We investigated how ToCV infection modifies the interaction between potato plants and B. tabaci in ways that increase the likelihood of ToCV transmission, in two clones, one susceptible ('Agata') and the other moderately resistant (Bach-4) to B. tabaci. Whiteflies alighted and laid more eggs on ToCV-infected plants than mock-inoculated plants of Bach-4. When non-viruliferous whiteflies were released on ToCV-infected plants near mock-inoculated plants, adults moved more intensely towards non-infected plants than in the reverse condition for both clones. Feeding on ToCV-infected plants reduced egg-incubation period in both clones, but the egg-adult cycle was similar for whiteflies fed on ToCV-infected and mock-inoculated plants. Our results demonstrated that ToCV infection in potato plants alters B. tabaci behaviour and development in distinct ways depending on the host clone, with potential implications for ToCV spread.

Molecular dissection of distinct symptoms induced by tomato chlorosis virus and tomato yellow leaf curl virus based on comparative transcriptome analysis.

The viral infection of plants may cause various physiological symptoms associated with the reprogramming of plant gene expression. However, the molecular mechanisms and associated genes underlying disease symptom development in plants infected with viruses are largely unknown. In this study, we employed RNA sequencing for in-depth molecular characterization of the transcriptional changes associated with the development of distinct symptoms induced by tomato chlorosis virus (ToCV) and tomato yellow leaf curl virus (TYLCV) in tomato. Comparative analysis of differentially expressed genes revealed that ToCV and TYLCV induced distinct transcriptional changes in tomato and resulted in the identification of important genes responsible for the development of symptoms of ToCV (i.e., chlorosis and anthocyanin accumulation) and TYLCV (i.e., yellowing, stunted growth, and leaf curl). Our comprehensive transcriptome analysis can provide molecular strategies to reduce the severity of disease symptoms as well as new insights for the development of virus-resistant crops.

Detection and epidemic dynamic of ToCV and CCYV with Bemisia tabaci and weed in Hainan of China.

BACKGROUND: In recent years, two of the crinivirus, Tomato chlorosis virus (ToCV) and Cucurbit chlorotic yellows virus (CCYV) have gained increasing attention due to their rapid spread and devastating impacts on vegetable production worldwide. Both of these viruses are transmitted by the sweet potato whitefly, Bemisia tabaci (Gennadius), in a semi-persistent manner. Up to now, there is still lack of report in Hainan, the south of China. METHODS: We used observational and experimental methods to explore the prevalence and incidence dynamic of CCYV and ToCV transmitted by whiteflies in Hainan of China. RESULTS: In 2016, the chlorosis symptom was observed in the tomato and cucumber plants with a large number of B. tabaci on the infected leaves in Hainan, China, with the incidence rate of 69.8% and 62.6% on tomato and cucumber, respectively. Based on molecular identification, Q biotype was determined with a viruliferous rate of 65.0% and 55.0% on the tomato and cucumber plants, respectively. The weed, Alternanthera philoxeroides near the tomato and cucumber was co-infected by the two viruses. Furthermore, incidence dynamic of ToCV and CCYV showed a close relationship with the weed, Alternanthera philoxeroides, which is widely distributed in Hainan. CONCLUSION: Our results firstly reveal that the weed, A. philoxeroides is infected by both ToCV and CCYV. Besides, whiteflies showed a high viruliferous rate of ToCV and CCYV. Hainan is an extremely important vegetable production and seed breeding center in China. If the whitefly can carry these two viruses concurrently, co-infection in their mutual host plants can lead to devastating losses in the near future.

Whitefly feeding behavior and retention of a foregut-borne crinivirus exposed to artificial diets with different pH values.

Transmission of plant viruses by phytophagous hemipteran insects encompasses complex interactions underlying a continuum of processes involved in virus acquisition, retention and inoculation combined with vector feeding behavior. Here, we investigated the effects of dietary pH on whitefly (Bemisia tabaci) feeding behavior and release of Lettuce infectious yellows virus (LIYV) virions retained in the vector's foregut. Electrical penetration graph analysis revealed that variables associated with whitefly probing and ingestion did not differ significantly in pH (4, 7.4, and 9) adjusted artificial diets. To investigate virus retention and release, whiteflies allowed to acquire LIYV virions in a pH 7.4 artificial diet were fed pH 4, 7.4, or 9 virion-free artificial (clearing) diets. Immunofluorescent localization analyses indicated that virions remained bound to the foreguts of approximately 20%-24% of vectors after they fed on each of the 3 pH-adjusted clearing diets. When RNA preparations from the clearing diets were analyzed by reverse transcription (RT) nested-PCR and, in some cases, real-time qPCR, successful amplification of LIYV-specific sequence was infrequent but consistently repeatable for the pH 7.4 diet but never observed for the pH 4 and 9 diets, suggesting a weak pH-dependent effect for virion release. Viruliferous vectors that fed on each of the 3 pH-adjusted clearing diets transmitted LIYV to virus-free plants. These results suggest that changes in pH values alone in artificial diet do not result in observable changes in whitefly feeding behaviors, an observation that marks a first in the feeding of artificial diet by whitefly vectors; and that there is a potential causal and contingent relationship between the pH in artificial diet and the release/inoculation of foregut bound virions.

Transcriptome analysis of the whitefly, Bemisia tabaci MEAM1 during feeding on tomato infected with the crinivirus, Tomato chlorosis virus, identifies a temporal shift in gene expression and differential regulation of novel orphan genes.

BACKGROUND: Whiteflies threaten agricultural crop production worldwide, are polyphagous in nature, and transmit hundreds of plant viruses. Little is known how whitefly gene expression is altered due to feeding on plants infected with a semipersistently transmitted virus. Tomato chlorosis virus (ToCV; genus Crinivirus, family Closteroviridae) is transmitted by the whitefly (Bemisia tabaci) in a semipersistent manner and infects several globally important agricultural and ornamental crops, including tomato. RESULTS: To determine changes in global gene regulation in whiteflies after feeding on tomato plants infected with a crinivirus (ToCV), comparative transcriptomic analysis was performed using RNA-Seq on whitefly (Bemisia tabaci MEAM1) populations after 24, 48, and 72 h acquisition access periods on either ToCV-infected or uninfected tomatoes. Significant differences in gene expression were detected between whiteflies fed on ToCV-infected tomato and those fed on uninfected tomato among the three feeding time periods: 447 up-regulated and 542 down-regulated at 24 h, 4 up-regulated and 7 down-regulated at 48 h, and 50 up-regulated and 160 down-regulated at 72 h. Analysis revealed differential regulation of genes associated with metabolic pathways, signal transduction, transport and catabolism, receptors, glucose transporters, α-glucosidases, and the uric acid pathway in whiteflies fed on ToCV-infected tomatoes, as well as an abundance of differentially regulated novel orphan genes. Results demonstrate for the first time, a specific and temporally regulated response by the whitefly to feeding on a host plant infected with a semipersistently transmitted virus, and advance the understanding of the whitefly vector-virus interactions that facilitate virus transmission. CONCLUSION: Whitefly transmission of semipersistent viruses is believed to require specific interactions between the virus and its vector that allow binding of virus particles to factors within whitefly mouthparts. Results provide a broader understanding of the potential mechanism of crinivirus transmission by whitefly, aid in discerning genes or loci in whitefly that influence virus interactions or transmission, and subsequently facilitate development of novel, genetics-based control methods against whitefly and whitefly-transmitted viruses.

Cucurbit chlorotic yellows virus: Insights Into Its Natural Host Range, Genetic Variability, and Transmission Parameters.

Cucurbit chlorotic yellows virus (CCYV) (genus Crinivirus, family Closteroviridae) is implicated in cucurbit yellows disease (CYV), causing typical interveinal yellowing symptoms in leaves, and is transmitted by Bemisia tabaci Mediterranean (MED) and Middle East-Asia Minor 1 (MEAM1). Due to its recent report in cucurbit crops in Greece, field surveys were conducted during 2011-2016 to determine the presence of the virus in symptomatic cucurbits and alternative hosts among arable weed species. Results indicated the restricted spread of the virus and identified 13 weed species as CCYV hosts for the first time. Sequence analysis of the RNA-dependent RNA polymerase (RNA1) coat and minor coat proteins (RNA2) revealed very low genetic diversity (<0.1%) among the Greek isolates. Transmission experiments were also conducted using B. tabaci MED with retention determined at four days, whereas transmission efficiency was positively correlated with the number of adults used, features linked to the virus semipersistent mode of transmission.

A 3'-end structure in RNA2 of a crinivirus is essential for viral RNA synthesis and contributes to replication-associated translation activity.

The terminal ends in the genome of RNA viruses contain features that regulate viral replication and/or translation. We have identified a Y-shaped structure (YSS) in the 3' terminal regions of the bipartite genome of Lettuce chlorosis virus (LCV), a member in the genus Crinivirus (family Closteroviridae). The YSS is the first in this family of viruses to be determined using Selective 2'-Hydroxyl Acylation Analyzed by Primer Extension (SHAPE). Using luciferase constructs/replicons, in vivo and in vitro assays showed that the 5' and YSS-containing 3' terminal regions of LCV RNA1 supported translation activity. In contrast, similar regions from LCV RNA2, including those upstream of the YSS, did not. LCV RNA2 mutants with nucleotide deletions or replacements that affected the YSS were replication deficient. In addition, the YSS of LCV RNA1 and RNA2 were interchangeable without affecting viral RNA synthesis. Translation and significant replication were observed for specific LCV RNA2 replicons only in the presence of LCV RNA1, but both processes were impaired when the YSS and/or its upstream region were incomplete or altered. These results are evidence that the YSS is essential to the viral replication machinery, and contributes to replication enhancement and replication-associated translation activity in the RNA2 replicons.

The p22 RNA Silencing Suppressor of the Crinivirus Tomato chlorosis virus is Dispensable for Local Viral Replication but Important for Counteracting an Antiviral RDR6-Mediated Response during Systemic Infection.

Among the components of the RNA silencing pathway in plants, RNA-dependent RNA polymerases (RDRs) play fundamental roles in antiviral defence. Here, we demonstrate that the Nicotiana benthamiana RDR6 is involved in defence against the bipartite crinivirus (genus Crinivirus, family Closteroviridae) Tomato chlorosis virus (ToCV). Additionally, by producing a p22-deficient ToCV infectious mutant clone (ToCVΔp22), we studied the role of this viral suppressor of RNA silencing in viral infection in both wild-type and RDR6-silenced N. benthamiana (NbRDR6i) plants. We demonstrate that p22 is dispensable for the replication of ToCV, where RDR6 appears not to have any effect. Furthermore, the finding that ToCV∆p22 systemic accumulation was impaired in wild-type N. benthamiana but not in NbRDR6i plants suggests a role for p22 in counteracting an RDR6-mediated antiviral response of the plant during systemic infection.

Infectious clones of the crinivirus cucurbit chlorotic yellows virus are competent for plant systemic infection and vector transmission.

Cucurbit chlorotic yellows virus (CCYV), a recently identified bipartite crinivirus, causes economic losses in cucurbit plants. CCYV is naturally transmitted only by whitefly Bemisia tabaci. Here we constructed full-length cDNA clones of CCYV (RNA1 and RNA2) fused to the T7 RNA polymerase promoter and the cauliflower mosaic virus 35S promoter. CCYV replicated and accumulated efficiently in Cucumis sativus protoplasts transfected with in vitro transcripts. Without RNA2, RNA1 replicated efficiently in C. sativus protoplasts. Agroinoculation with the infectious cDNA clones of CCYV resulted in systemic infection in the host plants of C. sativus and Nicotiana benthamiana. Virus derived from the infectious clones could be transmitted between cucumber plants by vector whiteflies. This system will greatly enhance the reverse genetic studies of CCYV gene functions.

The p22 RNA silencing suppressor of the crinivirus Tomato chlorosis virus preferentially binds long dsRNAs preventing them from cleavage.

Viruses encode silencing suppressor proteins to counteract RNA silencing. Because dsRNA plays a key role in silencing, a general silencing suppressor strategy is dsRNA binding. The p22 suppressor of the plant virus Tomato chlorosis virus (ToCV; genus Crinivirus, family Closteroviridae) has been described as having one of the longest lasting local suppressor activities. However, the mechanism of action of p22 has not been characterized. Here, we show that ToCV p22 binds long dsRNAs in vitro, thus interfering with their processing into small RNAs (sRNAs) by an RNase III-type Dicer homolog enzyme. Additionally, we have studied whether a putative zinc finger motif found in p22 has a role in dsRNA binding and suppressor function. The efficient ability of p22 to suppress RNA silencing, triggered by hairpin transcripts transiently expressed in planta, supports the relationship between its ability to bind dsRNA in vitro and its ability to inhibit RNA silencing in vivo.

Genetic diversity and silencing suppression activity of the p22 protein of Tomato chlorosis virus isolates from tomato and sweet pepper.

As for other bipartite criniviruses (genus Crinivirus, family Closteroviridae), the genome of Tomato chlorosis virus encodes an RNA silencing suppressor, the protein p22, in the 3'-proximal region of RNA1. This protein has been reported as having one of the longest lasting local suppressor activities when transiently expressed in Nicotiana benthamiana. Here, we examined the genetic diversity of the p22 gene in ToCV isolates from tomato and sweet pepper. The p22 gene sequences clearly grouped into two separated clades. However, functional analysis of both types of p22 proteins indicated no evident differences in suppressor activity. Our findings provide experimental evidence that the presence of a "strong" silencing suppressor is a conserved feature of ToCV isolates.