Impacts of Cucurbit Chlorotic Yellows Virus (CCYV) on Biological Characteristics of Its Vector Bemisia tabaci (Hemiptera: Aleyrodidae) MED Species.

Plant viruses can change the phenotypes and defense pathways of the host plants and the performance of their vectors to facilitate their transmission. Cucurbit chlorotic yellows virus (CCYV) (Crinivirus), a newly reported virus occurring on cucurbit plants and many other plant species, is transmitted specifically by Bemisia tabaci MEAM1 (B biotype) and MED (Q biotype) cryptic species in a semipersistent manner. This study evaluated the impacts of CCYV on B. tabaci to better understand the plant-virus-vector interactions. By using CCYV-B. tabaci MED-cucumber as the model, we investigated whether or how a semipersistent plant virus impacts the biology of its whitefly vector. CCYV mRNAs were detectable in nymphs from first to fourth instars and adults of B. tabaci with different titers. Nymph instar durations and adult longevity of female whiteflies greatly extended on CCYV-infected plants, but nymph instar durations and adult longevity of male whiteflies were not significantly influenced. In addition, the body length and oviposition increased in adults feeding on CCYV-infected plants, but the hatching rates of eggs and survival rates of different stages were not affected. Most interestingly, the sex ratio (male:female) significantly reduced to 0.5:1 in whitefly populations on CCYV-infected plants, while the ratio remained about 1:1 on healthy plants. These results indicated that CCYV can significantly impact the biological characteristics of its vector B. tabaci. It is speculated that CCYV and B. tabaci have established a typical mutualist relationship mediated by host plants.

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.

Epidemiology and control of emerging criniviruses in bean.

During the last two decades, new criniviruses emerged in green bean crops in the south-east of Spain. Bean yellow disorder virus (BnYDV) was first detected in 2003 and caused major economic damage in crops grown in greenhouses. It was characterized as the first crinivirus to infect a member species of the Leguminosae family. Symptoms induced during BnYDV infection include interveinal chlorosis and yellowing on leaves, and reduced fruit yield and quality. Similar symptoms, although more severe, were observed in bean crops in the same region during the fall of 2011. From that moment on, BnYDV was not detected anymore in diseased plants, but instead lettuce chlorosis virus (LCV) was associated with the diseased plants. Previously, LCV was detected only in California, USA, infecting lettuce and sugarbeets. The host range and partial genomic sequences lead to the description of the new strain, LCV-SP. The complete sequence of its genome revealed the virus as a recombinant of BnYDV and LCV, in which the latter had lost two ORFs in the RNA1 of the bipartite genome and had acquired two homologue ORFs from BnYDV. Both viruses are transmitted by the whitefly Bemisia tabaci. When compared with other crinivirus pathosystems, the transmission efficiency of BnYDV to its primary host bean, is among the highest, and its persistence in the vector among the longest, up to 9 days. The host range of BnYDV s restricted to several crop species of the Leguminosae: common bean (Phaseolus vulgaris), pea (Pisum sativum), tirabeque (P. sativum subsp. sativum var. macrocarpon), lentil (Lens culinaris) and faba bean (Vicia faba). LCV-SP is also able to infect green bean plants but not lettuce, its original host, probably following its recombinant nature. Symptoms and epidemiology of the bean criniviruses are compared with similar pathosystems that occur in the same region and that involve cucurbit yellow stunting disorder virus and tomato chlorosis virus, infecting cucurbitaceous and solanaceous crops, respectively. Control of the criniviruses in bean crops will depend on efficient control of the vector. Physical control with greenhouses that prevent viruliferous whiteflies from gaining access to crops reduces BnYDV infection in plants and loss of production. Integrated pest management in beans would be preferred and the use of natural enemies to reduce secondary spread within greenhouses must be investigated.

Tomato chlorosis virus, an emergent plant virus still expanding its geographical and host ranges.

Tomato chlorosis virus (ToCV) causes an important disease that primarily affects tomato, although it has been found infecting other economically important vegetable crops and a wide range of wild plants. First described in Florida (USA) and associated with a 'yellow leaf disorder' in the mid-1990s, ToCV has been found in 35 countries and territories to date, constituting a paradigmatic example of an emergent plant pathogen. ToCV is transmitted semipersistently by whiteflies (Hemiptera: Aleyrodidae) belonging to the genera Bemisia and Trialeurodes. Whitefly transmission is highly efficient and cases of 100% infection are frequently observed in the field. To date, no resistant or tolerant tomato plants are commercially available and the control of the disease relies primarily on the control of the insect vector. TAXONOMY: Tomato chlorosis virus is one of the 14 accepted species in the genus Crinivirus, one of the four genera in the family Closteroviridae of plant viruses. VIRION AND GENOME PROPERTIES: The genome of ToCV is composed of two molecules of single-stranded positive-sense RNA, named RNA1 and RNA2, separately encapsidated in long, flexuous, rod-like virions. As has been shown for other closterovirids, ToCV virions are believed to have a bipolar structure. RNA1 contains four open reading frames (ORFs) encoding proteins associated with virus replication and suppression of gene silencing, whereas RNA2 contains nine ORFs encoding proteins putatively involved in encapsidation, cell-to-cell movement, gene silencing suppression and whitefly transmission. HOST RANGE: In addition to tomato, ToCV has been found to infect 84 dicot plant species belonging to 25 botanical families, including economically important crops. TRANSMISSION: Like all species within the genus Crinivirus, ToCV is semipersistently transmitted by whiteflies, being one of only two criniviruses transmitted by members of the genera Bemisia and Trialeurodes. DISEASE SYMPTOMS: Tomato 'yellow leaf disorder' syndrome includes interveinal yellowing and thickening of leaves. Symptoms first develop on lower leaves and then advance towards the upper part of the plant. Bronzing and necrosis of the older leaves are accompanied by a decline in vigour and reduction in fruit yield. In other hosts the most common symptoms include interveinal chlorosis and mild yellowing on older leaves. CONTROL: Control of the disease caused by ToCV is based on the use of healthy seedlings for transplanting, limiting accessibility of alternate host plants that can serve as virus reservoirs and the spraying of insecticides for vector control. Although several wild tomato species have been shown to contain genotypes resistant to ToCV, there are no commercially available resistant or tolerant tomato varieties to date.

Accumulation of 24 nucleotide transgene-derived siRNAs is associated with crinivirus immunity in transgenic plants.

RNA silencing is a conserved antiviral defence mechanism that has been used to develop robust resistance against plant virus infections. Previous efforts have been made to develop RNA silencing-mediated resistance to criniviruses, yet none have given immunity. In this study, transgenic Nicotiana benthamiana plants harbouring a hairpin construct of the Lettuce infectious yellows virus (LIYV) RNA-dependent RNA polymerase (RdRp) sequence exhibited immunity to systemic LIYV infection. Deep sequencing analysis was performed to characterize virus-derived small interfering RNAs (vsiRNAs) generated on systemic LIYV infection in non-transgenic N. benthamiana plants as well as transgene-derived siRNAs (t-siRNAs) derived from the immune-transgenic plants before and after LIYV inoculation. Interestingly, a similar sequence distribution pattern was obtained with t-siRNAs and vsiRNAs mapped to the transgene region in both immune and susceptible plants, except for a significant increase in t-siRNAs of 24 nucleotides in length, which was consistent with small RNA northern blot results that showed the abundance of t-siRNAs of 21, 22 and 24 nucleotides in length. The accumulated 24-nucleotide sequences have not yet been reported in transgenic plants partially resistant to criniviruses, and thus may indicate their correlation with crinivirus immunity. To further test this hypothesis, we developed transgenic melon (Cucumis melo) plants immune to systemic infection of another crinivirus, Cucurbit yellow stunting disorder virus (CYSDV). As predicted, the accumulation of 24-nucleotide t-siRNAs was detected in transgenic melon plants by northern blot. Together with our findings and previous studies on crinivirus resistance, we propose that the accumulation of 24-nucleotide t-siRNAs is associated with crinivirus immunity in transgenic plants.

Transcriptome analysis of Cucumis sativus infected by Cucurbit chlorotic yellows virus.

BACKGROUND: Cucurbit chlorotic yellows virus (CCYV) is a recently reported bipartite crinivirus that causes chlorotic leaf spots and yellowing symptoms on the leaves of cucurbit plants. The virus-host interaction of CCYV remains to be elucidated, and the influence of criniviruses on the host gene transcriptome requires analysis. METHODS: We used transcriptome sequencing to analyse the differentially expressed genes (DEGs) caused by CCYV infection. RESULTS: CCYV infection resulted in 865 DEGs. The Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis identified 67 pathways, and the three major enrichment pathways (according to the P-values) were photosynthesis-antenna proteins (KO00196), phenylalanine metabolism (KO00360a), and phenylpropanoid biosynthesis (KO00940). Of the 13 DEGs identified in phenylalanine metabolism, 11 genes encode disease resistance-related phenylalanine ammonia-lyase (PAL) genes. Using quantitative real-time PCR, we validated the differential expression of 12 genes. CONCLUSIONS: Our study based on the CCYV-cucumber interaction provides comprehensive transcriptomic information, and will improve our understanding of host-crinivirus interactions.

Direct evidence for the semipersistent transmission of Cucurbit chlorotic yellows virus by a whitefly vector.

Cucurbit chlorotic yellows virus (CCYV) (genus Crinivirus, family Closteroviridae) is an emerging plant virus, and is now spreading and causing severe economic losses to cucurbit crops in many Asian countries. CCYV is believed to be transmitted specifically by the sweetpotato whitefly, Bemisia tabaci, in a semipersistent manner. In the present study, we provide direct evidence for the semipersistent transmission of CCYV by Mediterranean (MED) cryptic species of B. tabaci complex. We investigated CCYV transmission characteristics, and immunofluorescently labeled and localized the virus retention site within the vector by laser confocal microscopy. Whiteflies required ≥1 h of acquisition access period (AAP) to successfully acquire CCYV, and the proportion of RT-PCR positive whitefly individuals reached to 100% at 48 h of AAP. CCYV virons could be retained within vectors as long as 12 d, but the proportion of RT-PCR positive whiteflies dropped to 55% by 3 d. Groups of thirty whiteflies given a 24 h of inoculation access period (IAP) to inoculate CCYV on cucumber plants showed a transmission efficiency rate of 72.73%. The retention site of CCYV virons was located in the foregut of virion-fed vectors. These results definitely indicated the semipersistent transmission mode of CCYV by B. tabaci MED.

Infectious cDNA clones of the crinivirus Tomato chlorosis virus are competent for systemic plant infection and whitefly-transmission.

Tomato chlorosis virus (ToCV) (genus Crinivirus, family Closteroviridae) causes important emergent diseases in tomato and other solanaceous crops. ToCV is not transmitted mechanically and is naturally transmitted by whiteflies. The ToCV genome consists of two molecules of linear, positive-sense RNA encapsidated into long flexuous virions. We present the construction of full-length cDNA clones of the ToCV genome (RNA1 and RNA2) fused to the SP6 RNA polymerase promoter and under the control of the CaMV 35S promoter. RNA1 replicated in the absence of RNA2 in Nicotiana benthamiana and tomato protoplasts after inoculation with cDNA-derived in vitro transcripts. Agroinfiltration of RNA1 and RNA2 under the 35S promoter resulted in systemic infection in N. benthamiana plants. In addition, tomato plants were infected by grafting with agroinfected N. benthamiana scions, showing the typical ToCV symptoms. The viral progeny generated in tomato was transmissible by the whitefly Bemisia tabaci.

Agroinoculation of the cloned infectious cDNAs of Lettuce chlorosis virus results in systemic plant infection and production of whitefly transmissible virions.

Lettuce chlorosis virus (LCV) is a single stranded, positive strand RNA virus that is solely transmitted by specific whitefly vectors (Bemisia tabaci biotypes A and B) but not by mechanical leaf-rub inoculation. The roles of viral encoded proteins involved in the infection cycle of LCV have not yet been characterized due to the lack of reverse genetic tools. We present here a report of the successful development of an Agrobacterium-mediated inoculation system for the cloned cDNA constructs of LCV. The cDNAs of both LCV RNAs 1 and 2 were engineered into binary vectors in which the expression of LCV RNAs was regulated under a Cauliflower mosaic virus (CaMV) 35S promoter. In addition, by engineering the sequence elements of the Hepatitis delta virus ribozyme and the nopaline synthase 3' untranslated region immediately downstream of the last nucleotide of LCV RNAs 1 and 2 in the binary vector constructs, the in planta produced LCV transcripts were expected to bear authentic 3' termini. Both constructs were transformed into Agrobacterium tumefaciens cells and infiltrated in Nicotiana benthamiana plants. Three to four weeks post-agroinoculation, the N. benthamiana plants developed typical interveinal chlorosis and LCV infection was detected in the systemic leaves by reverse transcription-PCR. Virions purified from the LCV-infected N. benthamiana plants were flexuous rod-shaped and were transmissible by both B. tabaci biotypes A and B following membrane feeding. These results support the conclusion that Agrobacterium-mediated inoculation of LCV binary vectors in N. benthamiana plants results in LCV infection and the production of biologically active, whitefly transmissible virions. This system represents an important tool for use with reverse genetics designed for the study of LCV gene functions.

Distinct cavemoviruses interact synergistically with sweet potato chlorotic stunt virus (genus Crinivirus) in cultivated sweet potato.

Two serologically unrelated sweet potato viruses causing symptoms of vein clearing in the indicator plant Ipomoea setosa were isolated and their genomes have been sequenced. They are associated with symptomless infections in sweet potato but distinct vein-clearing symptoms and higher virus titres were observed when these viruses co-infected with sweet potato chlorotic stunt virus (SPCSV), a virus that is distributed worldwide and is a mediator of severe virus diseases in this crop. Molecular characterization and phylogenetic analysis revealed an overall nucleotide identity of 47.6 % and an arrangement of the movement protein and coat protein domains characteristic of members of the genus Cavemovirus, in the family Caulimoviridae. We detected both cavemoviruses in cultivated sweet potato from East Africa, Central America and the Caribbean islands, but not in samples from South America. One of the viruses characterized showed a similar genome organization as, and formed a phylogenetic sublineage with, tobacco vein clearing virus (TVCV), giving further support to the previously suggested separation of TVCV, and related viral sequences, into a new caulimovirid genus. Given their geographical distribution and previous reports of similar but yet unidentified viruses, sweet potato cavemoviruses may co-occur with SPCSV more often than previously thought and they could therefore contribute to the extensive yield losses and cultivar decline caused by mixed viral infections in sweet potato.

Agroinoculation of the Crinivirus, Lettuce infectious yellows virus, for systemic plant infection.

Lettuce infectious yellows virus (LIYV) is phloem-limited, non-mechanically transmissible, and is transmitted to plants only by Bemisia tabaci. Here, we developed agroinoculation to deliver LIYV to plants thereby obviating the need for B. tabaci. Agroinfiltration of RNA 1 containing a green fluorescent protein gene into Nicotiana benthamiana leaves resulted in subliminal infections, as judged by green fluorescence. Agroinfiltration of LIYV wild-type RNA 1 and 2 constructs resulted in systemic infections in N. benthamiana plants and typical LIYV symptoms. In addition, partially purified LIYV virions from agroinoculated N. benthamiana plants were successfully acquired via membrane-feeding and transmitted to lettuce plants by B. tabaci. Agroinoculation coupled with targeted mutagenesis technologies will greatly enhance LIYV reverse genetics studies to characterize LIYV gene functions in planta for processes such as virus replication, recombination, trafficking, symptom elicitation and virus-vector interactions.

Further complexity of the genus Crinivirus revealed by the complete genome sequence of Lettuce chlorosis virus (LCV) and the similar temporal accumulation of LCV genomic RNAs 1 and 2.

The sequence of Lettuce chlorosis virus (LCV) (genus Crinivirus) was determined and found to contain unique open reading frames (ORFs) and ORFs similar to those of other criniviruses, as well as 3' non-coding regions that shared a high degree of identity. Northern blot analysis of RNA extracted from LCV-infected plants identified subgenomic RNAs corresponding to six prominent internal ORFs and detected several novel LCV-single stranded RNA species. Virus replication in tobacco protoplasts was investigated and results indicated that LCV replication proceeded with novel crinivirus RNA accumulation kinetics, wherein viral genomic RNAs exhibited a temporally similar expression pattern early in the infection. This was noticeably distinct from the asynchronous RNA accumulation pattern previously observed for Lettuce infectious yellows virus (LIYV), the type member of the genus, suggesting that replication of the two viruses likely operate via dissimilar mechanisms.

Synergistic interaction between the Potyvirus, Turnip mosaic virus and the Crinivirus, Lettuce infectious yellows virus in plants and protoplasts.

Lettuce infectious yellows virus (LIYV), the type member of the genus Crinivirus in the family Closteroviridae, is specifically transmitted by the sweet potato whitefly (Bemisia tabaci) in a semipersistent manner. LIYV infections result in a low virus titer in plants and protoplasts, impeding reverse genetic efforts to analyze LIYV gene/protein functions. We found that synergistic interactions occurred in mixed infections of LIYV and Turnip mosaic virus (TuMV) in Nicotiana benthamiana plants, and these resulted in enhanced accumulation of LIYV. Furthermore, we examined the ability of transgenic plants and protoplasts expressing only the TuMV P1/HC-Pro sequence to enhance the accumulation of LIYV. LIYV RNA and protein titers increased by as much as 8-fold in these plants and protoplasts relative to control plants. LIYV infections remained phloem-limited in P1/HC-Pro transgenic plants, suggesting that enhanced accumulation of LIYV in these plants was due primarily to increased replication efficiency, not to greater spread.

Complete nucleotide sequence of the RNA2 of the crinivirus tomato chlorosis virus.

The complete sequence of genomic RNA2 of Tomato chlorosis virus (ToCV; genus Crinivirus, family Closteroviridae), isolate AT80/99 from Spain, was determined and compared with those from the other members of the genus sequenced to date. RNA2 is 8244 nucleotides (nt) long and putatively encodes nine ORFs that encompass the hallmark gene array of the family Closteroviridae, which includes a heat shock protein 70 family homologue, a 59 kDa protein, the coat protein, and a diverged coat protein. Phylogenetic analysis confirmed assignment of ToCV in the genus Crinivirus, being most similar to sweet potato chlorotic stunt virus and cucurbit yellow stunting disorder virus.

Viral class 1 RNase III involved in suppression of RNA silencing.

Double-stranded RNA (dsRNA)-specific endonucleases belonging to RNase III classes 3 and 2 process dsRNA precursors to small interfering RNA (siRNA) or microRNA, respectively, thereby initiating and amplifying RNA silencing-based antiviral defense and gene regulation in eukaryotic cells. However, we now provide evidence that a class 1 RNase III is involved in suppression of RNA silencing. The single-stranded RNA genome of sweet potato chlorotic stunt virus (SPCSV) encodes an RNase III (RNase3) homologous to putative class 1 RNase IIIs of unknown function in rice and Arabidopsis. We show that RNase3 has dsRNA-specific endonuclease activity that enhances the RNA-silencing suppression activity of another protein (p22) encoded by SPCSV. RNase3 and p22 coexpression reduced siRNA accumulation more efficiently than p22 alone in Nicotiana benthamiana leaves expressing a strong silencing inducer (i.e., dsRNA). RNase3 did not cause intracellular silencing suppression or reduce accumulation of siRNA in the absence of p22 or enhance silencing suppression activity of a protein encoded by a heterologous virus. No other known RNA virus encodes an RNase III or uses two independent proteins cooperatively for RNA silencing suppression.

The Lettuce infectious yellows virus (LIYV)-encoded P26 is associated with plasmalemma deposits within LIYV-infected cells.

Cytological, immunological, and mutagenesis approaches were used to identify the viral factors associated with the formation of plasmalemma deposits (PLDs) in whole plants and protoplasts infected by Lettuce infectious yellows virus (LIYV). Transmission electron microscopy and immunogold labeling using polyclonal antibodies to four of the five LIYV RNA 2-encoded large proteins, capsid protein (CP), minor capsid protein (CPm), HSP70 homolog (HSP70h), and P59, showed specific labeling of LIYV virions or virion aggregates around the vesiculated membranous inclusions, but not PLDs in LIYV-infected Nicotiana benthamiana, Nicotiana clevelandii, Lactuca sativa, and Chenopodium murale plants, and Nicotiana tabacum protoplasts. In contrast, antibodies to the RNA 2-encoded P26 showed specific labeling of PLDs but not virions in both LIYV-infected plants and protoplasts. Virion-like particles (VLPs) were seen in protoplasts infected by all LIYV RNA 2 mutants except for the CP (major capsid protein) mutant. PLDs were more difficult to find in protoplasts, but were seen in protoplasts infected by the CP and CPm mutants, but not in protoplasts infected by the P26, HSP70h, or P59 mutants. Interestingly, although the CPm mutant showed VLPs and PLDs, the PLDs did not show associated virions/virion-like particles as was always observed for PLDs seen in protoplasts infected by wild-type LIYV. Immunoblot analyses performed on purified LIYV virions showed that P26 was not detected with purified virions, but was detected in the cell wall, 1000 g and 30,000 g pellet fractions of LIYV-infected plants. These data suggest that P26 is associated with the LIYV-induced PLDs, and in contrast to the other RNA 2-encoded large proteins, P26 is not a virion protein.