Loquat (Eriobotrya japonica) Is a New Natural Host of Tomato Mosaic Virus and Citrus Exocortis Viroid.

Loquat leaves exhibiting obvious yellowing, blistering, mosaic, leaf upward cupping, crinkle, and leaf narrowing were identified in Panzhihua City, Sichuan Province, China. High-throughput sequencing (HTS) with the ribo-depleted cDNA library was employed to identify the virome in the loquat samples; only tomato mosaic virus (ToMV) and citrus exocortis viroid (CEVd) were identified in the transcriptome data. The complete genome sequence of ToMV and CEVd were obtained from the loquat leaves. The full-length genome of the ToMV-loquat is 6376 nt and comprises four open reading frames (ORFs) encoding 183 kDa protein, RNA-dependent RNA polymerase (RdRp), movement protein (MP), and coat protein (CP), respectively. A pairwise identity analysis showed that the complete sequence of the ToMV-loquat had a nucleotide identity between 98.5 and 99.3% with other ToMV isolates. A phylogenetic analysis indicated that ToMV-loquat was more closely related to ToMV-IFA9 (GenBank No. ON156781). A CEVd sequence with 361 nt in length was amplified based on the HTS contigs, sequence alignment indicated CEVd-loquat had the highest identity with the strain of CEVd-Balad (GenBank No. PP869624), phylogenetic analysis showed that CEVd-loquat was more closely related to CEVd-lettuce (GenBank No. ON993891). This significant discovery marks the first documentation and characterization of ToMV and CEVd infecting loquat plants, shedding light on potential threats to loquat cultivation and providing insights for disease management strategies.

TOM1 family conservation within the plant kingdom for tobacco mosaic virus accumulation.

The susceptibility factor TOBAMOVIRUS MULTIPLICATION 1 (TOM1) is required for efficient multiplication of tobacco mosaic virus (TMV). Although some phylogenetic and functional analyses of the TOM1 family members have been conducted, a comprehensive analysis of the TOM1 homologues based on phylogeny from the most ancient to the youngest representatives within the plant kingdom, analysis of support for tobamovirus accumulation and interaction with other host and viral proteins has not been reported. In this study, using Nicotiana benthamiana and TMV as a model system, we functionally characterized the TOM1 homologues from N. benthamiana and other plant species from different plant lineages. We modified a multiplex genome editing tool and generated a sextuple mutant in which TMV multiplication was dramatically inhibited. We showed that TOM1 homologues from N. benthamiana exhibited variable capacities to support TMV multiplication. Evolutionary analysis revealed that the TOM1 family is restricted to the plant kingdom and probably originated in the Chlorophyta division, suggesting an ancient origin of the TOM1 family. We found that the TOM1 family acquired the ability to promote TMV multiplication after the divergence of moss and spikemoss. Moreover, the capacity of TOM1 orthologues from different plant species to promote TMV multiplication and the interactions between TOM1 and TOM2A and between TOM1 and TMV-encoded replication proteins are highly conserved, suggesting a conserved nature of the TOM2A-TOM1-TMV Hel module in promoting TMV multiplication. Our study not only revealed a conserved nature of a gene module to promote tobamovirus multiplication, but also provides a valuable strategy for TMV-resistant crop development.

Genetic Variability and Molecular Evolution of Tomato Mosaic Virus Populations in Three Northern China Provinces.

RNA viruses tend to mutate during transmission and host infection, which is critical to viral adaptation and evolution. Tomato mosaic virus (ToMV) is a member of the genus Tobamovirus (family Virgaviridae) and an economically important virus with detrimental effects on tomatoes worldwide. Although the ToMV gene sequences have been completed in China, their genetic diversity and population structure remain unclear. We collected 425 tomato samples from tomato-growing areas in three northern Chinese provinces 2016. Reverse transcription PCR results showed that the average incidence of the virus in the field samples was 67.15%, and ToMV was detected in all test areas. The analysis of ToMV single nucleotide polymorphisms in China showed that ToMV was evolutionarily conserved, and the variation in the whole genome was uneven. Pairwise identity analysis showed significant variability in genome sequences among ToMV strains with genomic nucleotide identities of 73.2-99.6%. The ToMV population in the northern Chinese provinces had purification and selection functions, which were beneficial in the evolution of the ToMV population. Although there has been some distribution of ToMV strains in China, the virus was generally stabilized as a uniform strain under the pressure of purification selection. Our findings show how to monitor the prevalent strains of ToMV and their virulence in China and provide useful information for its prevention and control.

Tobamovirus infection aggravates gray mold disease caused by Botrytis cinerea by manipulating the salicylic acid pathway in tomato.

Botrytis cinerea is the causative agent of gray mold disease, and infects more than 1400 plant species, including important crop plants. In tomato, B. cinerea causes severe damage in greenhouses and post-harvest storage and transport. Plant viruses of the Tobamovirus genus cause significant damage to various crop species. In recent years, the tobamovirus tomato brown rugose fruit virus (ToBRFV) has significantly affected the global tomato industry. Most studies of plant-microbe interactions focus on the interaction between the plant host and a single pathogen, however, in agricultural or natural environments, plants are routinely exposed to multiple pathogens. Here, we examined how preceding tobamovirus infection affects the response of tomato to subsequent infection by B. cinerea. We found that infection with the tobamoviruses tomato mosaic virus (ToMV) or ToBRFV resulted in increased susceptibility to B. cinerea. Analysis of the immune response of tobamovirus-infected plants revealed hyper-accumulation of endogenous salicylic acid (SA), upregulation of SA-responsive transcripts, and activation of SA-mediated immunity. Deficiency in SA biosynthesis decreased tobamovirus-mediated susceptibility to B. cinerea, while exogenous application of SA enhanced B. cinerea symptoms. These results suggest that tobamovirus-mediated accumulation of SA increases the plants' susceptibility to B. cinerea, and provide evidence for a new risk caused by tobamovirus infection in agriculture.

Elicitation of Hyoscyamine Production in Datura stramonium L. Plants Using Tobamoviruses.

Datura stramonium L. produces tropane alkaloids, and the hyoscyamine is dominant among them. Hyoscyamine is produced by hairy root cultures in vitro derived from native plants or plants with the genetically modified biosynthetic pathway for hyoscyamine. A common procedure is extraction from cultivated plants. Elicitors for increased production can be used in both cases. Live viruses are not well known for use as elicitors, therefore, D. stramonium plants grown in soil were artificially infected with the tobamoviruses Pepper mild mottle virus (PMMoV), Tomato mosaic virus (ToMV), and Tobacco mosaic virus (TMV). Differences in the content of hyoscyamine were between capsules and roots of infected and non-infected plants. Elicitation increased content of hyoscyamine in capsules 1.23-2.34 times, compared to the control. The most effective viruses were PMMoV and ToMV (isolate PV143), which increased content to above 19 mg/g of fresh weight of a capsule. The effect of each virus elicitor was expressed also in hyoscyamine content in roots. Elicited plants contained 5.41-16.54 times more hyoscyamine in roots compared to non-elicited plants. The most effective elicitor was ToMV SL-1, which raised production above 20 mg/g fresh weight of roots. It has been shown that tobamoviruses can be used as biotic elicitors.

Knockout of SlTOM1 and SlTOM3 results in differential resistance to tobamovirus in tomato.

During tobamovirus-host coevolution, tobamoviruses developed numerous interactions with host susceptibility factors and exploited these interactions for replication and movement. The plant-encoded TOBAMOVIRUS MULTIPLICATION (TOM) susceptibility proteins interact with the tobamovirus replicase proteins and allow the formation of the viral replication complex. Here CRISPR/Cas9-mediated mutagenesis allowed the exploration of the roles of SlTOM1a, SlTOM1b, and SlTOM3 in systemic tobamovirus infection of tomato. Knockouts of both SlTOM1a and SlTOM3 in sltom1a/sltom3 plants resulted in an asymptomatic response to the infection with recently emerged tomato brown rugose fruit virus (ToBRFV). In addition, an accumulation of ToBRFV RNA and coat protein (CP) in sltom1a/sltom3 mutant plants was 516- and 25-fold lower, respectively, than in wild-type (WT) plants at 12 days postinoculation. In marked contrast, sltom1a/sltom3 plants were susceptible to previously known tomato viruses, tobacco mosaic virus (TMV) and tomato mosaic virus (ToMV), indicating that SlTOM1a and SlTOM3 are not essential for systemic infection of TMV and ToMV in tomato plants. Knockout of SlTOM1b alone did not contribute to ToBRFV and ToMV resistance. However, in triple mutants sltom1a/sltom3/sltom1b, ToMV accumulation was three-fold lower than in WT plants, with no reduction in symptoms. These results indicate that SlTOM1a and SlTOM3 are essential for the replication of ToBRFV, but not for ToMV and TMV, which are associated with additional susceptibility proteins. Additionally, we showed that SlTOM1a and SlTOM3 positively regulate the tobamovirus susceptibility gene SlARL8a3. Moreover, we found that the SlTOM family is involved in the regulation of plant development.

Differential Detection of the Tobamoviruses Tomato Mosaic Virus (ToMV) and Tomato Brown Rugose Fruit Virus (ToBRFV) Using CRISPR-Cas12a.

CRISPR/Cas12a-based detection is a novel approach for the efficient, sequence-specific identification of viruses. Here we adopt the use of CRISPR/Cas12a to identify the tomato brown rugose fruit virus (ToBRFV), a new and emerging tobamovirus which is causing substantial damage to the global tomato industry. Specific CRISPR RNAs (crRNAs) were designed to detect either ToBRFV or the closely related tomato mosaic virus (ToMV). This technology enabled the differential detection of ToBRFV and ToMV. Sensitivity assays revealed that viruses can be detected from 15-30 ng of RT-PCR product, and that specific detection could be achieved from a mix of ToMV and ToBRFV. In addition, we show that this method can enable the identification of ToBRFV in samples collected from commercial greenhouses. These results demonstrate a new method for species-specific detection of tobamoviruses. A future combination of this approach with isothermal amplification could provide a platform for efficient and user-friendly ways to distinguish between closely related strains and resistance-breaking pathogens.

Ameliorating the Adverse Effects of Tomato mosaic tobamovirus Infecting Tomato Plants in Egypt by Boosting Immunity in Tomato Plants Using Zinc Oxide Nanoparticles.

Tomato mosaic virus (ToMV) is one of the economically damageable Tobamovirus infecting the tomato in Egypt that has caused significant losses. It is therefore of great interest to trigger systemic resistance to ToMV. In this endeavor, we aimed to explore the capacity of ZnO-NPs (zinc oxide nanoparticles) to trigger tomato plant resistance against ToMV. Effects of ZnO-NPs on tomato (Solanum lycopersicum L.) growth indices and antioxidant defense system activity under ToMV stress were investigated. Noticeably that treatment with ZnO-NPs showed remarkably increased growth indices, photosynthetic attributes, and enzymatic and non-enzymatic antioxidants compared to the challenge control. Interestingly, oxidative damage caused by ToMV was reduced by reducing malondialdehyde, H2O2, and O2 levels. Overall, ZnO-NPs offer a safe and economic antiviral agent against ToMV.

Production of ORF8 protein from SARS-CoV-2 using an inducible virus-mediated expression system in suspension-cultured tobacco BY-2 cells.

KEY MESSAGE: Production of ORF8 protein from SARS-CoV-2 in tobacco BY-2 cells.

Identification and Mapping of Tomato Genome Loci Controlling Tolerance and Resistance to Tomato Brown Rugose Fruit Virus.

Tomato brown rugose fruit virus (ToBRFV) was identified in Israel during October 2014 in tomato plants (Solanum lycopersicum). These plants, carrying the durable resistance gene against tomato mosaic virus, Tm-22 , displayed severe disease symptoms and losses to fruit yield and quality. These plants were found infected with a tobamovirus similar to that discovered earlier in Jordan. This study was designed to screen and identify tomato genotypes resistant or tolerant to ToBRFV. The identified resistance and tolerance traits were further characterized virologically and genetically. Finally, DNA markers linked to genes controlling these traits were developed as tools to expedite resistance breeding. To achieve these objectives, 160 genotypes were screened, resulting in the identification of an unexpectedly high number of tolerant genotypes and a single genotype resistant to the virus. A selected tolerant genotype and the resistant genotype were further analyzed. Analysis of genetic inheritance revealed that a single recessive gene controls tolerance whereas at least two genes control resistance. Allelic test between the tolerant and the resistant genotype revealed that these two genotypes share a locus controlling tolerance, mapped to chromosome 11. This locus displayed a strong association with the tolerance trait, explaining nearly 91% of its variation in segregating populations. This same locus displayed a statistically significant association with symptom levels in segregating populations based on the resistant genotype. However, in these populations, the locus was able to explain only ~41% of the variation in symptom levels, confirming that additional loci are involved in the genetic control of the resistance trait in this genotype. A locus on chromosome 2, at the region of the Tm-1 gene, was finally found to interact with the locus discovered on chromosome 11 to control resistance.

Full-Length Infectious Clones of Two New Isolates of Tomato Mosaic Virus Induce Distinct Symptoms Associated with Two Differential Amino Acid Residues in 128-kDa Protein.

In 2017, two new tomato mosaic virus (ToMV) isolates were collected from greenhouses in Buyeo, Chungcheongnam-do, South Korea. Full-length cDNAs of the new ToMV isolates were cloned into dual cauliflower mosaic virus 35S and T7 promoter-driven vectors, sequenced and their pathogenicities investigated. The nucleotide sequences of isolates GW1 (MH507165) and GW2 (MH507166) were 99% identical, resulting in only two amino acid differences in nonconserved region II and the helicase domain, Ile668Thr and Val834Ile. The two isolates were most closely related to a ToMV isolate from Taiwan (KJ207374). Isolate GW1 (Ile668, Val834) induced a systemic hypersensitive response in Nicotiana benthamiana compared with the isolate GW2, which a single residue substitution showed was due to Val834.

Highly Sensitive and Practical Detection of Plant Viruses via Electrical Impedance of Droplets on Textured Silicon-Based Devices.

Early diagnosis of plant virus infections before the disease symptoms appearance may represent a significant benefit in limiting disease spread by a prompt application of appropriate containment steps. We propose a label-free procedure applied on a device structure where the electrical signal transduction is evaluated via impedance spectroscopy techniques. The device consists of a droplet suspension embedding two representative purified plant viruses i.e., Tomato mosaic virus and Turnip yellow mosaic virus, put in contact with a highly hydrophobic plasma textured silicon surface. Results show a high sensitivity of the system towards the virus particles with an interestingly low detection limit, from tens to hundreds of attomolar corresponding to pg/mL of sap, which refers, in the infection time-scale, to a concentration of virus particles in still-symptomless plants. Such a threshold limit, together with an envisaged engineering of an easily manageable device, compared to more sophisticated apparatuses, may contribute in simplifying the in-field plant virus diagnostics.

Salicylic acid and gentisic acid induce RNA silencing-related genes and plant resistance to RNA pathogens.

We have observed that treatments with salicylic acid (SA) or gentisic acid (GA) induced resistance to RNA pathogens such as ToMV and CEVd in tomato and Gynura auriantiaca, respectively. Accumulation of SA and GA has been found to occur in plants infected by these pathogens, thus pointing out a possible defence role of both molecules. To study the molecular basis of the observed induced resistance to RNA pathogens the induction of silencing-related genes by SA and GA was considered. For that purpose, we searched for tomato genes which were orthologous to those described in Arabidopsis thaliana, such as AtDCL1, AtDCL2, AtDCL4, AtRDR1, AtRDR2 and AtRDR6, and we tracked their induction in tomato along virus and viroid infections. We observed that CEVd significantly induced all these genes in tomato, with the exception of ToRDR6, being the induction of ToDCL4 the most outstanding. Regarding the ToMV asymptomatic infection, with the exception of ToRDR2, we observed a significant induction of all the indicated silencing-related genes, being ToDCL2 the most induced gene. Subsequently, we analyzed their transcriptional activation by SA and at the time when ToMV was inoculated on plants. ToDCL2, ToRDR1 and ToRDR2 were significantly induced by both SA and GA, whereas ToDCL1 was only induced by SA. Such an induction resulted more effective by SA treatment, which is in agreement with the stronger SA-induced resistance observed. Our results suggest that the observed delay in the RNA pathogen accumulation could be due to the pre-induction of RNA silencing-related genes by SA or GA.