Engineering of stable infectious cDNA constructs of a fluorescently tagged tomato chlorosis virus.

Tomato chlorosis virus (ToCV) is an emerging pathogen that cause severe yellow leaf disorder syndrome in tomato plants. In this study, we aimed to generate a recombinant ToCV tagged with green fluorescent protein (GFP) to enable real-time monitoring of viral infection in living plants. Transformation of the full-length cDNA construct of ToCV RNA1 into Escherichia coli resulted in instability issues, which were successfully overcome by inserting a plant intron into RNA1. Subsequently, a GFP tag was engineered into a cDNA construct of ToCV RNA2. The resulting recombinant ToCV-GFP could systemically infect Nicotiana benthamiana plants, and GFP expression was observed along the major veins. Utilizing ToCV-GFP, we also showed that ToCV engages in antagonistic relationships with two different tomato-infecting viruses in mixed infections in N. benthamiana. This study demonstrates the potential of ToCV-GFP as a valuable tool for the visual tracking of infection and movement of criniviruses in living plants.

Enhanced Susceptibility to Tomato Chlorosis Virus (ToCV) in Hsp90- and Sgt1-Silenced Plants: Insights from Gene Expression Dynamics.

The emerging whitefly-transmitted crinivirus tomato chlorosis virus (ToCV) causes substantial economic losses by inducing yellow leaf disorder in tomato crops. This study explores potential resistance mechanisms by examining early-stage molecular responses to ToCV. A time-course transcriptome analysis compared naïve, mock, and ToCV-infected plants at 2, 7, and 14 days post-infection (dpi). Gene expression changes were most notable at 2 and 14 dpi, likely corresponding to whitefly feeding and viral infection. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses revealed key genes and pathways associated with ToCV infection, including those related to plant immunity, flavonoid and steroid biosynthesis, photosynthesis, and hormone signaling. Additionally, virus-derived small interfering RNAs (vsRNAs) originating from ToCV predominantly came from RNA2 and were 22 nucleotides in length. Furthermore, two genes involved in plant immunity, Hsp90 (heat shock protein 90) and its co-chaperone Sgt1 (suppressor of the G2 allele of Skp1) were targeted through viral-induced gene silencing (VIGS), showing a potential contribution to basal resistance against viral infections since their reduction correlated with increased ToCV accumulation. This study provides insights into tomato plant responses to ToCV, with potential implications for developing effective disease control strategies.

Emergence of yellowing disease in cucurbitaceous vegetables caused by Crinivirus and Polerovirus in India.

Totally 102 symptomatic samples of cucurbitaceous vegetables showing yellowing were collected from fields of Uttar Pradesh and screened by RT-PCR assay for the presence of Crinivirus and Polerovirus. Among them, Crinivirus (16%) and Polerovirus (23%) were tested positive with the universal primer pairs. Based on the sequence analysis of amplified product, two Crinivirus (Cucurbit chlorotic yellows virus - CCYV and Cucurbit yellow stunting disorder virus - CYSDV) and two Polerovirus (Cucurbit aphid-borne yellows virus - CABYV and Luffa aphid-borne yellows virus - LABYV) species were characterized. Phylogenetic analysis revealed less genetic distance among the Indian isolates of CCYV, CYSDV and LABYV whereas CABYV closely related to Chinese isolates. To the best of our knowledge, this study documents infection of CCYV on cucumber, round melon and muskmelon; CYSDV on satputia and sponge gourd; CABYV on ivy gourd; and LABYV on ridge gourd, satputia and muskmelon for the first time in India.

Interactions of Tomato Chlorosis Virus p27 Protein with Tomato Catalase Are Involved in Viral Infection.

Tomato chlorosis virus (ToCV) severely threatens tomato production worldwide. P27 is known to be involved in virion assembly, but its other roles in ToCV infection are unclear. In this study, we found that removal of p27 reduced systemic infection, while ectopic expression of p27 promoted systemic infection of potato virus X in Nicotiana benthamiana. We determined that Solanum lycopersicum catalases (SlCAT) can interact with p27 in vitro and in vivo and that amino acids 73 to 77 of the N-terminus of SlCAT represent the critical region for their interaction. p27 is distributed in the cytoplasm and nucleus, and its coexpression with SlCAT1 or SlCAT2 changes its distribution in the nucleus. Furthermore, we found that silencing of SlCAT1 and SlCAT2 can promote ToCV infection. In conclusion, p27 can promote viral infection by binding directly to inhibit anti-ToCV processes mediated by SlCAT1 or SlCAT2.

Small RNA Profiling of Cucurbit Yellow Stunting Disorder Virus from Susceptible and Tolerant Squash (Cucurbita pepo) Lines.

RNA silencing is a crucial mechanism of the antiviral immunity system in plants. Small RNAs guide Argonaut proteins to target viral RNA or DNA, preventing virus accumulation. Small RNA profiles in Cucurbita pepo line PI 420328 with tolerance to cucurbit yellow stunting disorder virus (CYSDV) were compared with those in Gold Star, a susceptible cultivar. The lower CYSDV symptom severity in PI 420328 correlated with lower virus titers and fewer sRNAs derived from CYSDV (vsRNA) compared to Gold Star. Elevated levels of 21- and 22-nucleotide (nt) size class vsRNAs were observed in PI 420328, indicating more robust and efficient RNA silencing in PI 420328. The distribution of vsRNA hotspots along the CYSDV genome was similar in both PI 420328 and Gold Star. However, the 3' UTRs, CPm, and p26 were targeted at a higher frequency in PI 420328.

Tomato chlorosis virus p22 interacts with NbBAG5 to inhibit autophagy and regulate virus infection.

Tomato chlorosis virus (ToCV) is a member of the genus Crinivirus in the family Closteroviridae. It has a wide host range and wide distribution, causing serious harm to the vegetable industry. The autophagy pathway plays an important role in plant resistance to virus infection. Viruses and plant hosts coevolve in defence and antidefence processes around autophagy. In this study, the interaction between ToCV p22 and Nicotiana benthamiana B-cell lymphoma2-associated athanogenes5 Nicotiana benthamiana (NbBAG5) was examined. Through overexpression and down-regulation of NbBAG5, results showed that NbBAG5 could negatively regulate ToCV infection. NbBAG5 was found to be localized in mitochondria and can change the original localization of ToCV p22, which is colocalized in mitochondria. NbBAG5 inhibited the expression of mitophagy-related genes and the number of autophagosomes, thereby regulating viral infection by affecting mitophagy. In summary, this study demonstrated that ToCV p22 affects autophagy by interacting with NbBAG5, established the association between viral infection, BAG proteins family, and the autophagy pathway, and explained the molecular mechanism by which ToCV p22 interacts with NbBAG5 to inhibit autophagy to regulate viral infection.

Crinivirus Tomato Chlorosis Virus Compromises the Control of Tomato Yellow Leaf Curl Virus in Tomato Plants by the Ty-1 Gene.

Tomato yellow leaf curl disease (TYLCD) causes severe damage to tomato crops in warm regions of the world, and is associated with infections of several whitefly (Bemisia tabaci)-transmitted single-stranded (ss)DNA begomoviruses (genus Begomovirus, family Geminiviridae). The most widespread begomovirus isolates associated with TYLCD are those of the type strain of the Tomato yellow leaf curl virus species, known as Israel (TYLCV-IL). The Ty-1 gene is widely used in commercial tomato cultivars to control TYLCV-IL damage, providing resistance to the virus by restricting viral accumulation and tolerance to TYLCD by inhibiting disease symptoms. However, several reports suggest that TYLCV-IL-like isolates are adapting to the Ty-1 gene and are causes of concern for possibly overcoming the provided control. This is the case with TYLCV-IL IS76-like recombinants that have a small genome fragment acquired by genetic exchange from an isolate of Tomato yellow leaf curl Sardinia virus, another begomovirus species associated with TYLCD. Here we show that TYLCV-IL IS76-like isolates partially break down the TYLCD-tolerance provided by the Ty-1 gene and that virulence differences might exist between isolates. Interestingly, we demonstrate that mixed infections with an isolate of the crinivirus (genus Crinivirus, family Closteroviridae) species Tomato chlorosis virus (ToCV), an ssRNA virus also transmitted by B. tabaci and emerging worldwide in tomato crops, boosts the breakdown of the TYLCD-tolerance provided by the Ty-1 gene either with TYLCV-IL IS76-like or canonical TYLCV-IL isolates. Moreover, we demonstrate the incorporation of the Ty-2 gene in Ty-1-commercial tomatoes to restrict (no virus or virus traces, no symptoms) systemic infections of recombinant TYLCV-IL IS76-like and canonical TYLCV-IL isolates, even in the presence of ToCV infections, which provides more robust and durable control of TYLCD.

Whole genome sequence of mulberry crinivirus, a new member of the genus Crinivirus.

The whole genome sequence of mulberry crinivirus (MuCV), a novel member of the genus Crinivirus (family Closteroviridae) identified in mulberry (Morus alba L), was determined. The virus possesses a bipartite genome. RNA1 contains 8571 nucleotides (nt) with four open reading frames (ORFs). ORF1a encodes a putative polyprotein with papain-like protease, methyltransferase, and RNA helicase domains. ORF1b putatively encodes an RNA-dependent RNA polymerase (RdRp), which is probably expressed via a + 1 ribosomal frameshift. RNA2 consists of 8082 nt, containing eight ORFs that are similar in size and position to orthologous genes of other criniviruses. Phylogenetic analysis based on RdRp amino acid sequences of criniviruses placed MuCV in group 1.

Effects of insulin-like peptide 7 in Bemisia tabaci MED on tomato chlorosis virus transmission.

BACKGROUND: Tomato chlorosis virus (ToCV) is a semi-persistent plant virus that is primarily transmitted by the whitefly Bemisia tabaci (Hemiptera: Aleyrodidae). It causes a serious disease that lowers tomato yield. Insulin-like peptide (ILP), an insulin homolog, regulates trehalose metabolism in a variety of insects. In a previous study, we discovered that trehalose metabolism is required for whiteflies to transmit ToCV effectively. Furthermore, transcriptome sequencing revealed that the BtILP7 gene was highly expressed in B. tabaci infected with ToCV. Therefore, the whitefly ILP7 gene may facilitate the transmission of ToCV and be an attractive target for the control of whiteflies and subsequently ToCV. RESULTS: The ToCV content in B. tabaci MED was found to be correlated with BtILP7 gene expression. Subsequent RNA interference (RNAi) of the BtILP7 gene had a significant impact on B. tabaci MED's trehalose metabolism and reproductive capacity, as well as ability to transmit ToCV. CONCLUSIONS: These results indicate that the BtILP7 gene was closely related to ToCV transmission by regulating trehalose metabolism and reproduction behavior, thus providing a secure and environmentally friendly management strategy for the control of whiteflies and ToCV-caused disease. © 2022 Society of Chemical Industry.

A plant virus enhances odorant-binding protein 5 (OBP5) in the vector whitefly for more actively olfactory orientation to the host plant.

BACKGROUND: The whitefly Bemisia tabaci (Hemiptera: Aleyrodidae) is a notorious agricultural pest and the effective vector of many plant viruses worldwide. Cucurbit chlorotic yellows virus (CCYV), exclusively transmitted by B. tabaci in a semipersistent manner, is a serious causal agent in cucurbit crops in many countries. Plant viruses can manipulate the behaviors of insect vectors to promote the spread of themselves, but underlying mechanisms are remaining unclear. RESULTS: In this study, our observations indicated that B. tabaci, when carrying CCYV, oriented more actively to the host plant cucumber. Transcriptome analysis and quantitative polymerase chain reaction with reverse transcription analysis showed that the odorant-binding protein 5 (OBP5) was upregulated with viral acquisition. Sequence and phylogenetic analysis showed that BtabOBP5 was highly homologous with nine OBPs from other hemipteran insects. In addition, OBP5-silenced whiteflies significantly altered their orientation behavior towards cucumber plants and towards some typical volatile organic compounds released from cucumbers. CONCLUSION: This study described a novel mechanism by which the olfactory system of vector insects could be regulated by a semipersistent plant virus, thereby affecting insect olfactory behavior and relationship with host plants. These results provided a basis for developing potential olfaction-based pest management strategies in the future. © 2022 The Authors. Pest Management Science published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

Development of a RT-LAMP assay for real-time detection of criniviruses infecting tomato.

Yellowing symptoms caused by tomato chlorosis virus (ToCV) and tomato infectious chlorosis virus (TICV), both assigned to the genus Crinivirus, resemble nutrient deficiencies. Therefore, early diagnosis of infections will prevent crop damage and the spread of the viruses. In this study, we established a rapid detection method for ToCV and TICV by reverse transcription-loop-mediated isothermal amplification (RT-LAMP). We first designed primer sets for RT-LAMP specific for ToCV and TICV. Next, by selecting the optimum primer set and determining the optimum conditions for the RT-LAMP reaction, each virus was detected within 50 min by piercing the diseased area of a tomato leaf with a toothpick, immersing the toothpick in the reaction solution, and conducting the RT-LAMP reaction. To verify the accuracy of the procedure, 61 tomato leaf samples showing disease symptoms were collected from five regions of Indonesia, and the RT-LAMP results for the samples were identical to those obtained with the commonly used reverse transcription-polymerase chain reaction.

Discovery of novel whitefly vector proteins that interact with a virus capsid component mediating virion retention and transmission.

Specificity and efficiency of plant virus transmission depend largely on protein-protein interactions of vectors and viruses. Cucurbit chlorotic yellows virus (CCYV), transmitted specifically by tobacco whitefly, Bemisia tabaci, in a semi-persistent manner, has caused serious damage on cucurbit and vegetable crops around the world. However, the molecular mechanism of interaction during CCYV retention and transmission are still lacking. CCYV was proven to bind particularly to the whitefly foregut, and here, we confirmed that the minor coat protein (CPm) of CCYV is participated in the interaction with the vector. In order to identify proteins of B. tabaci that interact directly with CPm of CCYV, the immunoprecipitation (IP) assay and DUALmembrane cDNA library screening technology were applied. The cytochrome c oxidase subunit 5A (COX), tubulin beta chain (TUB) and keratin, type I cytoskeletal 9-like (KRT) of B. tabaci shown strong interactions with CPm and are closely associated with the retention within the vector and transmission of CCYV. These findings on whitefly protein-CCYV CPm interactions are crucial for a much better understanding the mechanism of semi-persistent plant virus transmission by insect vectors, as well as for implement new strategies for effective management of plant viruses and their vector insects.

Silencing of the Prophenoloxidase Gene BtPPO1 Increased the Ability of Acquisition and Retention of Tomato chlorosis virus by Bemisia tabaci.

Tomato chlorosis virus (ToCV) has seriously impacted tomato production around the world. ToCV is semi-persistently transmitted by the whitefly, Bemisia tabaci, which is a serious agricultural pest in the world. However, the interaction mechanism between ToCV and its whitefly vector is still poorly understood. Our previous transcriptome analysis demonstrated that the expression level of an immune-related gene, prophenoloxidase (PPO), in B. tabaci increased after ToCV acquisition, which indicates that the PPO may be involved in the interaction mechanism between the ToCV and its vector. To determine the role of the PPO in the acquisition and retention of ToCV by B. tabaci, we cloned the complete Open Reading Frames (ORF) of the BtPPOs (BtPPO1 and BtPPO2), and then structure and phylogenetic analyses were performed. BtPPOs were closely related to the PPO genes of Hemiptera insects. Spatial-temporal expression detection was qualified by using reverse transcription quantitative PCR (RT-qPCR), and this revealed that BtPPOs were expressed in all tissues and developmental stages. We found that only BtPPO1 was significantly upregulated after B. tabaci acquired ToCV for 12 and 24 h. According to the paraffin-fluorescence probe-fluorescence in situ hybridization (FISH) experiment, we verified that ToCV and BtPPO1 were co-located in the thorax of B. tabaci, which further revealed the location of their interaction. Finally, the effects of the BtPPOs on ToCV acquisition and retention by B. tabaci were determined using RNA interference (RNAi). The results showed that the RNAi of the responsive gene (BtPPO1) significantly increased the titer of ToCV in B. tabaci. These results demonstrate that BtPPO1 participates in ToCV acquisition and retention by B. tabaci.

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.

Wild Radish (Raphanus raphanistrum L.) Is a Potential Reservoir Host of Cucurbit Chlorotic Yellows Virus.

Cucurbit chlorotic yellows virus (CCYV) belongs to the genus Crinivirus and is part of a complex of whitefly-transmitted viruses that cause yellowing disease in cucurbits. In the southeastern USA, heavy incidences of CCYV have been observed on all cucurbits grown in the fall. CCYV was detected from wild radish (Raphanus raphanistrum L.), a common weed that grows in the southeastern USA by high-throughput sequencing as well as RT-PCR. CCYV sequence from wild radish was 99.90% and 99.95%, identical to RNA 1 and RNA 2 of cucurbit isolates of CCYV from the region. Transmission assays using whiteflies demonstrated that wild radish is a good host for CCYV. Whiteflies were also able to acquire CCYV from wild radish and transmit the virus to cucurbit hosts, which developed typical symptoms associated with CCYV. Using quantitative PCR, the titer of CCYV in wild radish was also estimated to be on par with that of cucurbit hosts of the virus. Whitefly bioassays revealed that wild radish is an acceptable feeding and reproductive host plant. These results indicate that wild radish could serve as a reservoir host for CCYV in the USA and other parts of the world where similar conditions exist.

Coinfection of Tomato Plants with Tomato yellow leaf curl virus and Tomato chlorosis virus Affects the Interaction with Host and Whiteflies.

Susceptible plants infected by single or multiple viruses can differ in symptoms and other alterations influencing virus dissemination. Furthermore, behavior of viruliferous vectors may be altered in certain cases to favor acquisition and inoculation processes conductive to virus transmission. We explored single and mixed infections frequently occurring in tomato crops, caused by two viruses transmitted by the whitefly Bemisia tabaci: Tomato yellow leaf curl virus (TYLCV, Begomovirus, Geminiviridae) and Tomato chlorosis virus (ToCV, Crinivirus, Closteroviridae). Coinfection of both viruses in tomato plants showed more severe symptoms at late stages compared with single infections, although at earlier stages the interaction began with attenuation. This asymmetric synergism correlated with the dynamics of ToCV accumulation and expression of the salicylic acid responsive gene PR-P6. Visual and olfactory cues in whitefly preference were evaluated under controlled conditions in choice assays, testing viruliferous and nonviruliferous adult whiteflies. In experiments allowing both visual and olfactory cues, whiteflies preferred symptomatic leaflets from plants infected either with TYLCV alone or with TYLCV and ToCV, over those infected with ToCV alone or noninfected leaflets, suggesting that TYLCV drove host selection. Odor cues tested in Y-tube olfactometer assays showed neutral effects on whiteflies' preference, and bioassays comparing the attractiveness of colored sticky cards confirmed preference for sectors colored to mimic TYLCV symptomatic leaves compared with asymptomatic leaves. Our results show that the presence of coinfecting viruses affect the host and could alter the behavior of insect vectors.

Reverse transcription recombinase polymerase amplification assay for rapid detection of the cucurbit chlorotic yellows virus.

The cucurbit chlorotic yellows virus (CCYV) causes severe economic losses in cucurbit plants. Although it has been widely known in various countries for several years, CCYV is rarely recognized due to the lack of rapid and effective detection methods in the early stage of the disease. Recombinase polymerase amplification (RPA) is a new, efficient, and simple technology for nucleic acid detection. In the present study, reverse transcription (RT)-RPA and quantitative RT-RPA were developed and utilized for fast detection of CCYV in field-collected melon samples. The analysis was performed under constant temperature conditions without the necessity for a thermal cycler in just 20 min. Moreover, the detection limit of RT-RPA for CCYV was determined at 10 pg. In the study, 58 field-collected samples were employed to evaluate the performance of the two assays. The positive rates were established at 72.4 % (42/58) and 75.9 % (44/58) by RT-RPA and qRT-RPA, respectively, and were consistent with the RT-PCR results. The successful application of RPA for the detection of CCYV in field-collected melon samples indicated its potential applicability. Thus, the developed RPA assays provide an alternative for fast, efficient, sensitive, and reliable detection of CCYV in diagnostic laboratories, which lack the precise instrumentation, and fields without appropriate equipment.

A simplified RT-PCR assay for the simultaneous detection of tomato chlorosis virus and tomato yellow leaf curl virus in tomato.

Tomato chlorosis virus (ToCV), a species of single-stranded RNA virus belonging to the Crinivirus genus, and Tomato yellow leaf curl virus (TYLCV), a species of single-stranded circular DNA virus belonging to the Begomovirus genus, are two major emerging viruses transmitted by whiteflies and are causing huge losses to tomato production worldwide. To facilitate the simultaneous detection of both viruses in co-infected plants for disease control, a duplex reverse-transcription PCR assay was developed. The assay used three primers, a degenerate reverse primer targeting a conserved region of TYLCV and the RNA2 of ToCV, and two virus-specific forward primers targeting the minor coat protein gene of ToCV and the C3 gene of TYLCV, respectively, to amplify a 762-bp and a 338-bp fragment from ToCV and TYLCV, respectively, in a single reaction. The concentration of the primers, annealing temperature and amplification cycles used in the assay were optimized, and the sensitivity of the assay was assessed. Using this assay, 150 tomato leaf samples collected from the field during 2018 were tested. The results showed that both viruses could be detected simultaneously in co-infected field samples. The assay should benefit the rapid detection of these two viruses in tomato crops and would facilitate early warning of infections for the control of the two virus diseases.

A multiplex RT-PCR assay combined with co-extraction of DNA and RNA for simultaneous detection of TYLCV and ToCV in whitefly.

Tomato yellow leaf curl virus (TYLCV) and tomato chlorosis virus (ToCV) were transmitted by the sweet potato whitefly Bemisia tabaci (Gennadius) and cause serious yield losses on tomato around the world. To understand the actual situation of co-infection of TYLCV and ToCV of individual whiteflies, we developed multiplex RT-PCR combined with co-extraction of DNA and RNA from a single whitefly. First, a nucleic acid extraction method previously reported was modified and adopted to obtain the RNA-DNA mixture including TYLCV and ToCV in a simple form without manual homogenization. Second, primers were newly designed in actin gene of B. tabaci for the confirmation of extraction and PCR success, and multiplex RT-PCR method was developed using specific primer sets for TYLCV, ToCV and B. tabaci. This method enables the detection of TYLCV and ToCV from a single insect and efficient use of field samples obtained using sticky traps. The method will be useful to monitor infection status of TYLCV and ToCV in the field while reducing labor and cost.