Hop Latent Viroid: A Hidden Threat to the Cannabis Industry.

Hop latent viroid (HLVd) is the biggest concern for cannabis and hop growers worldwide. Although most HLVd-infected plants remain asymptomatic, research on hops has demonstrated a decrease in both the α-bitter acid and terpene content of hop cones, which affects their economic value. The HLVd-associated "dudding" or "duds" disease of cannabis was first reported in 2019 in California. Since then, the disease has become widespread in cannabis-growing facilities across North America. Although severe yield loss associated with duds disease has been recorded, little scientific information is available to growers in order to contain HLVd. Consequently, this review aims to summarise all of the scientific information available on HLVd so as to be able to understand the effect of HLVd on yield loss, cannabinoid content, terpene profile, disease management and inform crop protection strategies.

Characterization of the virome of shallots affected by the shallot mild yellow stripe disease in France.

To elucidate the etiology of a new disease of shallot in France, double-stranded RNAs from asymptomatic and symptomatic shallot plants were analyzed using high-throughput sequencing (HTS). Annotation of contigs, molecular characterization and phylogenetic analyses revealed the presence in symptomatic plants of a virus complex consisting of shallot virus X (ShVX, Allexivirus), shallot latent virus (SLV, Carlavirus) and two novel viruses belonging to the genera Carlavirus and Potyvirus, for which the names of shallot virus S (ShVS) and shallot mild yellow stripe associated virus (SMYSaV), are proposed. Complete or near complete genomic sequences were obtained for all these agents, revealing divergent isolates of ShVX and SLV. Trials to fulfill Koch's postulates were pursued but failed to reproduce the symptoms on inoculated shallots, even though the plants were proved to be infected by the four viruses detected by HTS. Replanting of bulbs from SMYSaV-inoculated shallot plants resulted in infected plants, showing that the virus can perpetuate the infection over seasons. A survey analyzing 351 shallot samples over a four years period strongly suggests an association of SMYSaV with the disease symptoms. An analysis of SMYSaV diversity indicates the existence of two clusters of isolates, one of which is largely predominant in the field over years.

N-terminal region of cysteine-rich protein (CRP) in carlaviruses is involved in the determination of symptom types.

Plant viruses in the genus Carlavirus include more than 65 members. Plants infected with carlaviruses exhibit various symptoms, including leaf malformation and plant stunting. Cysteine-rich protein (CRP) encoded by carlaviruses has been reported to be a pathogenicity determinant. Carlavirus CRPs contain two motifs in their central part: a nuclear localization signal (NLS) and a zinc finger motif (ZF). In addition to these two conserved motifs, carlavirus CRPs possess highly divergent, N-terminal, 34 amino acid residues with unknown function. In this study, to analyse the role of these distinct domains, we tested six carlavirus CRPs for their RNA silencing suppressor activity, ability to enhance the pathogenicity of a heterologous virus and effects on virus accumulation levels. Although all six tested carlavirus CRPs showed RNA silencing suppressor activity at similar levels, symptoms induced by the Potato virus X (PVX) heterogeneous system exhibited two different patterns: leaf malformation and whole-plant stunting. The expression of each carlavirus CRP enhanced PVX accumulation levels, which were not correlated with symptom patterns. PVX-expressing CRP with mutations in either NLS or ZF did not induce symptoms, suggesting that both motifs play critical roles in symptom expression. Further analysis using chimeric CRPs, in which the N-terminal region was replaced with the corresponding region of another CRP, suggested that the N-terminal region of carlavirus CRPs determined the exhibited symptom types. The up-regulation of a plant gene upp-L, which has been reported in a previous study, was also observed in this study; however, the expression level was not responsible for symptom types.

An RNA virus-encoded zinc-finger protein acts as a plant transcription factor and induces a regulator of cell size and proliferation in two tobacco species.

Plant viruses cause a variety of diseases in susceptible hosts. The disease symptoms often include leaf malformations and other developmental abnormalities, suggesting that viruses can affect plant development. However, little is known about the mechanisms underlying virus interference with plant morphogenesis. Here, we show that a C-4 type zinc-finger (ZF) protein, p12, encoded by a carlavirus (chrysanthemum virus B) can induce cell proliferation, which results in hyperplasia and severe leaf malformation. We demonstrate that the p12 protein activates expression of a regulator of cell size and proliferation, designated upp-L (upregulated by p12), which encodes a transcription factor of the basic/helix-loop-helix family sufficient to cause hyperplasia. The induction of upp-L requires translocation of the p12 protein into the nucleus and ZF-dependent specific interaction with the conserved regulatory region in the upp-L promoter. Our results establish the role of the p12 protein in modulation of host cell morphogenesis. It is likely that other members of the conserved C-4 type ZF family of viral proteins instigate reprogramming of plant development by mimicking eukaryotic transcriptional activators.

A dual strategy for the suppression of host antiviral silencing: two distinct suppressors for viral replication and viral movement encoded by potato virus M.

Viruses encode RNA silencing suppressors to counteract host antiviral silencing. In this study, we analyzed the suppressors encoded by potato virus M (PVM), a member of the genus Carlavirus. In the conventional green fluorescent protein transient coexpression assay, the cysteine-rich protein (CRP) of PVM inhibited both local and systemic silencing, whereas the triple gene block protein 1 (TGBp1) showed suppressor activity only on systemic silencing. Furthermore, to elucidate the roles of these two suppressors during an active viral infection, we performed PVX vector-based assays and viral movement complementation assays. CRP increased the accumulation of viral RNA at the single-cell level and also enhanced viral cell-to-cell movement by inhibiting RNA silencing. However, TGBp1 facilitated viral movement but did not affect viral accumulation in protoplasts. These data suggest that CRP inhibits RNA silencing primarily at the viral replication step, whereas TGBp1 is a suppressor that acts at the viral movement step. Thus, our findings demonstrate a sophisticated viral infection strategy that suppresses host antiviral silencing at two different steps via two mechanistically distinct suppressors. This study is also the first report of the RNA silencing suppressor in the genus Carlavirus.

Complementation analysis of triple gene block of Potato virus S (PVS) revealed its capability to support systemic infection and aphid transmissibility of recombinant Potato virus X.

Triple gene block (TGB) sequences derived from isolates of ordinary Potato virus S (PVS-O) and Chenopodium-systemic (PVS-CS) were analyzed. Although the TGB sequences did not reveal any specific difference within the 7K protein, some specific differences within the 25K and 12K ORFs were found. In order to investigate a possible functional divergence of PVS-O and PVS-CS TGB variants, these genes were propagated in chimeric Potato virus X (PVX). Both PVS TGB variants partly complemented PVX TGB in Nicotiana benthamiana. The recombinant viruses multiplied to lower titer than the wild-type PVX in N. benthamiana showed attenuated symptoms. Whereas the recombinant PVX variants were also propagated systemically in Nicotiana glutinosa, Celosia argentea, Nicotiana occidentalis and chimeric PVX bearing TGB from PVS-O in Solanum lycopersicum, neither were propagated systemically in Chenopodium quinoa nor in Nicotiana tabacum cv. Samsun nn and the PVX-resistant Solanum tuberosum cv. Szignal. The potential for recombinant viruses to be transmitted by the aphid Myzus persicae was investigated. Aphid transmission in the recombinant virus was obtained by replacing PVX TGB by TGB from the PVS-CS isolate. These results show the potential function of Carlavirus TGB in aphid transmissibility and underlines the possible biological risks from certain recombinant virus variants.

Role of the zinc-finger and basic motifs of chrysanthemum virus B p12 protein in nucleic acid binding, protein localization and induction of a hypersensitive response upon expression from a viral vector.

The genomes of carlaviruses encode cysteine-rich proteins (CRPs) of unknown function. The 12 kDa CRP of chrysanthemum virus B (CVB), p12, has been shown previously to induce a hypersensitive response (HR) when expressed from potato virus X (PVX). This study demonstrated that a p12-induced HR was preceded by induction of a number of genes related to pathogenesis, stress and systemic acquired resistance. p12 localized predominantly to the nucleus. Interestingly, it was found that p12 bound both RNA and DNA in vitro, but notably exhibited a preference for DNA in the presence of Zn(2+) ions. Mutational analysis of the p12 conserved sequence motifs demonstrated that the basic motif is required for p12 translocation to the nucleus, thus representing part of the protein nuclear localization signal, whereas the predicted zinc finger motif is needed for both Zn(2+)-dependent DNA binding and eliciting an HR in PVX-infected leaves. Collectively, these results link, for the first time, nuclear localization of the protein encoded by a cytoplasmically replicating virus and its DNA-binding capacity with HR induction. Furthermore, these data suggest that p12 may mediate induction of the host genes by binding to the plant genomic DNA, and emphasize that CVB p12 is functionally distinct from other known nuclear-localized proteins encoded by the plant positive-stranded RNA viruses.

Detection and some properties of cowpea mild mottle virus isolated from soybean in Iran.

During 2006-2007 growing seasons, survey were carried to identify a virus disease causing mosaic of soybean in the field in Southern region (Khozestan Province) of Iran. To detect the viral infection, diseased leaf samples showing mild mosaic and leaf malformation were collected from soybean fields in Dezful, located in Khozestan Province. Infected samples were carried to the lab in a proper condition on ice packages. TPIA and DAS-ELISA serological tests were applied to identify the viral agent. To investigate the host-range, several indicator plants were mechanically inoculated under green-house condition. Seed transmission of CPMMV was examined using the seeds obtained from infected plants. The virus isolate was not found to be seed-borne in Clark variety of soybean. Different steps of ultracentrifugation including sucrose density gradient (10-40%) were carried out in order to obtain partial purified virus. On the basis of biological, serological and EM results, CPMMV-Carla virus was identified in the infected soybean samples. This is the first report of CPMMV infection of soybean in Iran.

[Interaction of cysteine-rich protein of Carlavirus with plant defense system].

Viruses of genus Carlavirus encode a small cysteine-rich protein (CRP) of unknown function. To investigate the role of CRP of carlavirus chrysanthemum virus B (CVB), a recombinant potato virus X (PVX) genome was constructed, which carried the CVB CRP gene. Expression of CVB CRP in the PVX genetic background drastically changed the PVX symptom phenotype in N. benthamiana. Instead of symptomless infection and mild mosaic, which are characteristic of PVX in this plant host, the recombinant virus expressing CVB CRP induced formation of necrotic local lesions on inoculated leaves and necrosis of the apical leaves. In N. tabacum, the infection pattern depended on the host genotype: the recombinant PVX was able to spread systemically only in N gene-carrying plants. In agroinfiltration-mediated transient expression assay, CVB CRP did not exhibit the properties of avirulence factor in N. benthamiana and was unable to suppress post-transcriptional gene silencing. Thus, CVB CRP is the viral pathogenicity determinant controlling the virus interaction with plant hosts in a manner which depends on plant defense mediated by resistance genes such as the N gene.

Autocatalytic processing of the 223-kDa protein of blueberry scorch carlavirus by a papain-like proteinase.

The first open reading frame of the blueberry scorch carlavirus (BBScV) genome encodes a putative replication-associated protein of 223 kDa (p223). A pulse-chase analysis of viral RNA translated in vitro in rabbit reticulocyte lysate revealed that p223 was proteolytically processed. Using a full-length ORF 1 cDNA clone in a coupled in vitro transcription/translation reaction, we confirmed that the ORF 1 gene product of BBScV processes autocatalytically. From sequence alignments with phylogenetically related viruses, including tymoviruses, we predicted that p223 contained a papain-like proteinase domain with a putative catalytic cysteine994 and histidine1075. A second possible proteinase domain, which contained cysteine895 and histidine984 residues with similar spacing but was otherwise less similar to the viral papain-like proteinases, was identified immediately upstream of the predicted catalytic site. The cleavage site of the proteinase was predicted to be between the putative helicase and the polymerase domains, possibly between or close to glycine1472 and alanine1473. Supporting these predictions, deletion of the 2091 nucleotides encoding the C-terminal region of p223, which contained the putative RNA polymerase domain and the putative cleavage site of the polyprotein, abolished autoproteolysis. Deletion of the 2061 nucleotides encoding the N-terminal region, which contained the putative methyltransferase domain, did not affect autoproteolysis. Alteration of cysteine994, histidine1075, or glycine1472 abolished autoproteolysis in vitro, supporting the involvement of these residues at the catalytic site and cleavage site. Alteration of the upstream cysteine895 and histidine984 residues did not affect processing in vitro. Capped BBScV full-length transcripts containing mutations in the codons for either cysteine994 or histidine1075 were not infectious in the systemic host plants Chenopodium quinoa and C. amaranticolor, whereas alteration of glycine1472 signficantly decreased but did not abolish infectivity. Transcripts containing mutations in the codons for either cysteine895 or histidine984 also were infectious, but resulted in delayed symptom expression in plants.