Incidence and Epidemiology of Citrus Viroids in Greece: Role of Host and Cultivar in Epidemiological Characteristics.

Viroids represent a threat to the citrus industry and also display an intricate matter for citrus tristeza virus (CTV) control as most of the commercial citrus rootstocks that are resistant/tolerant to CTV appear to be highly susceptible to viroid infection. Therefore, a detailed knowledge of the viroid's incidence and distribution, along with the assessment of unexplored epidemiological factors leading to their occurrence, are necessary to further improve control measures. Herein, a large-scale epidemiological study of citrus viroids in five districts, 38 locations and 145 fields in Greece is presented, based on the analysis of 3005 samples collected from 29 cultivars of six citrus species. We monitored the occurrence of citrus exocortis (CEVd), hop stunt (HSVd), citrus dwarfing (CDVd), citrus bark cracking (CBCVd), and citrus bent leaf (CBLVd) viroids, and addressed their epidemiological patterns and factors shaping their population structure. Our results show a high frequency and wide distribution of four viroids in all areas and in almost all hosts, whereas CBLVd occurrence was restricted to Crete. Mixed infections were found in all districts in which a wide spread of viroids was observed. We identified a potential pathogens' different preferences that could be partially explained by the host and cultivar, including the type of infection (single or mixed) and the number of viroids in the mixed infections. Overall, this work provides the first detailed epidemiological study on citrus viroids, enriching our knowledge for the implementation, production, and distribution of certified citrus propagative material, and the development of sustainable control strategies.

Molecular Characterization of the Coat Protein Gene of Greek Apple Stem Pitting Virus Isolates: Evolution through Deletions, Insertions, and Recombination Events.

A RT-PCR assay developed to amplify the full coat protein (CP) gene of apple stem pitting virus (ASPV) was evaluated using 180 Greek apple and pear samples and showed a broad detection range. This method was used to investigate the presence of ASPV in quince in Greece and showed a high incidence of 52%. The sequences of 14 isolates from various hosts with a distinct RFLP profile were determined. ASPV population genetics and the factors driving ASPV evolution were analyzed using the Greek ASPV sequences, novel sequences from Brazilian apple trees and Chinese botanical Pyrus species, and homologous sequences retrieved from GenBank. Fourteen variant types of Greek, Brazilian and botanical isolates, which differ in CP gene length and presence of indels, were identified. In addition, these analyses showed high intra- and inter-group variation among isolates from different countries and hosts, indicating the significant variability present in ASPV. Recombination events were detected in four isolates originating from Greek pear and quince and two from Brazilian apples. In a phylogenetic analysis, there was a tendency for isolates to cluster together based on CP gene length, the isolation host, and the detection method applied. Although there was no strict clustering based on geographical origin, most isolates from a given country tended to regroup in specific clusters. Interestingly, it was found that the phylogeny was correlated to the type, position, and pattern of indels, which represent hallmarks of specific lineages and indicate their possible role in virus diversification, rather than the CP size itself. Evidence of recombination between isolates from botanical and cultivated species and the clustering of isolates from botanical species and isolates from cultivated species suggest the existence of a possible undetermined transmission mechanism allowing the exchange of ASPV isolates between the cultivated and wild/ornamental hosts.

Serological and molecular analysis indicates the presence of distinct viral genotypes of Apple stem pitting virus in India.

Recombination leads to the generation of new viral progeny which remain undetected by routine testing procedures and may be a threat to the infected host. Here, we have characterised the complete genome sequences of two isolates of Apple stem pitting virus from apple cv. Red Chief (Palampur) and cv. Gold Spur (N) with distinct serological reactivities. The viral genomes consisted of 9267 nucleotides for isolate Palampur and 9254 nucleotides for isolate N, excluding the poly (A) tail and contained 5five open reading frames (ORFs). Isolate N shared 80.8% sequence identity with ASPV apple isolate GA2 from China, while isolate Palampur shared 81.4% sequence identity with ASPV apple isolate PB66 from the United Kingdom. The serological difference of isolates N and Palampur along with their low sequence identity indicated the existence of two distinct virus genotypes which was corroborated by evolutionary and genetic differentiation analyses. Recombination events were detected in the RdRp and CP sequences of Palampur isolate thereby suggesting the role of recombination in the evolution of distinct virus genotypes. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s13205-021-02798-5.

Cucurbit chlorotic yellows virus p22 is a suppressor of local RNA silencing.

RNA silencing is a major antiviral mechanism in plants, which is counteracted by virus-encoded proteins with silencing suppression activity. ORFs encoding putative silencing suppressor proteins that share no structural or sequence homology have been identified in the genomes of four criniviruses. In this study, we investigated the RNA silencing suppression activity of several proteins encoded by the RNA1 (RdRp, p22) and RNA2 (CP, CPm and p26) of cucurbit chlorotic yellows virus (CCYV) using co-agroinfiltration assays on Nicotiana benthamiana plants. Our results indicate that p22 is a suppressor of local RNA silencing that does not interfere with cell-to-cell movement of the RNA silencing signal or with systemic silencing. Furthermore, comparisons of the suppression activity of CCYV p22 with that of two other well-known crinivirus suppressors (CYSDV p25 and ToCV p22) revealed that CCYV p22 is a weaker suppressor of local RNA silencing than the other two proteins. Finally, a comparative sequence analysis of the p22 genes of seven Greek CCYV isolates was performed, revealing a high level of conservation. Taken together, our research advances our knowledge about plant-virus interactions of criniviruses, an emergent group of pathogens that threatens global agriculture.

A Thioredoxin Domain-Containing Protein Interacts with Pepino mosaic virus Triple Gene Block Protein 1.

Pepino mosaic virus (PepMV) is a mechanically-transmitted tomato pathogen of importance worldwide. Interactions between the PepMV coat protein and triple gene block protein (TGBp1) with the host heat shock cognate protein 70 and catalase 1 (CAT1), respectively, have been previously reported by our lab. In this study, a novel tomato interactor (SlTXND9) was shown to bind the PepMV TGBp1 in yeast-two-hybrid screening, in vitro pull-down and bimolecular fluorescent complementation (BiFC) assays. SlTXND9 possesses part of the conserved thioredoxin (TRX) active site sequence (W__PC vs. WCXPC), and TXND9 orthologues cluster within the TRX phylogenetic superfamily closest to phosducin-like protein-3. In PepMV-infected and healthy Nicotiana benthamiana plants, NbTXND9 mRNA levels were comparable, and expression levels remained stable in both local and systemic leaves for 10 days post inoculation (dpi), as was also the case for catalase 1 (CAT1). To localize the TXND9 in plant cells, a polyclonal antiserum was produced. Purified α-SlTXND9 immunoglobulin (IgG) consistently detected a set of three protein bands in the range of 27⁻35 kDa, in the 1000 and 30,000 g pellets, and the soluble fraction of extracts of healthy and PepMV-infected N. benthamiana leaves, but not in the cell wall. These bands likely consist of the homologous protein NbTXND9 and its post-translationally modified derivatives. On electron microscopy, immuno-gold labelling of ultrathin sections of PepMV-infected N. benthamiana leaves using α-SlTXND9 IgG revealed particle accumulation close to plasmodesmata, suggesting a role in virus movement. Taken together, this study highlights a novel tomato-PepMV protein interaction and provides data on its localization in planta. Currently, studies focusing on the biological function of this interaction during PepMV infection are in progress.

Multifaceted capsid proteins: multiple interactions suggest multiple roles for Pepino mosaic virus capsid protein.

Pepino mosaic virus (PepMV) (family Alphaflexiviridae, genus Potexvirus) is a mechanically transmitted tomato pathogen that, over the last decade, has evolved from emerging to endemic worldwide. Here, two heat-shock cognate (Hsc70) isoforms were identified as part of the coat protein (CP)/Hsc70 complex in vivo, following full-length PepMV and CP agroinoculation. PepMV accumulation was severely reduced in Hsp70 virus-induced gene silenced and in quercetin-treated Nicotiana benthamiana plants. Similarly, in vitro-transcribed as well as virion RNA input levels were reduced in quercetin-treated protoplasts, suggesting an essential role for Hsp70 in PepMV replication. As for Potato virus X, the PepMV CP and triple gene-block protein 1 (TGBp1) self-associate and interact with each other in vitro but, unlike in the prototype, both PepMV proteins represent suppressors of transgene-induced RNA silencing with different modes of action; CP is a more efficient suppressor of RNA silencing, sequesters the silencing signal by preventing its spread to neighboring cells and its systemic movement. Here, we provide evidence for additional roles of the PepMV CP and host-encoded Hsp70 in viral infection, the first as a truly multifunctional protein able to specifically bind to a host chaperone and to counterattack an RNA-based defense mechanism, and the latter as an essential factor for PepMV infection.

Pepino mosaic virus triple gene block protein 1 (TGBp1) interacts with and increases tomato catalase 1 activity to enhance virus accumulation.

Various plant factors are co-opted by virus elements (RNA, proteins) and have been shown to act in pathways affecting virus accumulation and plant defence. Here, an interaction between Pepino mosaic virus (PepMV) triple gene block protein 1 (TGBp1; p26) and tomato catalase 1 (CAT1), a crucial enzyme in the decomposition of toxic hydrogen peroxide (H2O2), was identified using the yeast two-hybrid assay, and confirmed via an in vitro pull-down assay and bimolecular fluorescent complementation (BiFC) in planta. Each protein was independently localized within loci in the cytoplasm and nuclei, sites at which their interaction had been visualized by BiFC. Following PepMV inoculation, CAT mRNA and protein levels in leaves were unaltered at 0, 3 and 6 days (locally) and 8 days (systemically) post-inoculation; however, leaf extracts from the last two time points contained increased CAT activity and lower H2O2 evels. Overexpression of PepMV p26 in vitro and in planta conferred the same effect, suggesting an additional involvement of TGBp1 in potexvirus pathogenesis. The accumulation of PepMV genomic and subgenomic RNAs and the expression of viral coat protein in noninoculated (systemic) leaves were reduced significantly in CAT-silenced plants. It is postulated that, during PepMV infection, a p26-CAT1 interaction increases H2O2 cavenging, thus acting as a negative regulator of plant defence mechanisms to promote PepMV infections.

Cydonia japonica, Pyrus calleryana and P. amygdaliformis: three new ornamental or wild hosts of Apple stem pitting virus.

Japanese quince, ornamental and wild pear symptomless samples were infected with Apple stem pitting virus (ASPV). Identification of ASPV was achieved by different PCR assays that amplified either the RNA polymerase or coat protein gene regions. For further confirmation, 312 bp amplicons within the polymerase gene were sequenced and compared with previously published ASPV sequences and additional sequences of isolates from ancient Italian cultivars. Comparison of the partial sequences isolated from wild/ornamental hosts and from cultivated species revealed significant divergence levels. Among the wild/ornamental isolates, the PCT88 isolate from Pyrus calleryana was the most divergent, having an amino acid deletion and incorporating a unique stretch of amino acids not present in any other isolate. Further to this preliminary partial sequence data, statistical analysis demonstrated that the isolates from wild or ornamental hosts were not more closely related to each other than to isolates from cultivated hosts. These results represent the first report of natural ASPV infection in these novel ornamental and wild Rosaceae hosts.

Pepino mosaic virus capsid protein interacts with a tomato heat shock protein cognate 70.

Plant viral capsid proteins (CP) can be involved in virus movement, replication and symptom development as a result of their interaction with host factors. The identification of such interactions may thus provide information about viral pathogenesis. In this study, Pepino mosaic virus (PepMV) CP was used as bait to screen a tomato (Solanum lycopersicum) cDNA library for potential interactors in yeast. Of seven independent interacting clones, six were predicted to encode the C-termini of the heat shock cognate 70 (Hsc70) proteins. Three full length tomato Hsc70s (named Hsc70.1, .2, .3) were used to confirm the interaction in the yeast two hybrid assay and bimolecular fluorescent complementation (BiFC) in planta. The PepMV CP-Hsc70 interaction was confirmed only in the case of Hsc70.3 for both assays. In BiFC, the interaction was visualized in the cytoplasm and nucleus of agroinfiltrated Nicotiana benthamiana epidermal cells. During PepMV infection, Hsc70.3 mRNA levels were induced and protein accumulation increased at 48 and 72 h post inoculation. In transmission electron microscopy using immunogold labelling techniques, Hsc70 was detected to co-localize with virions in the phloem of PepMV-infected tomato leaves. These observations, together with the co-purification of Hsc70 with PepMV virions further support the notion of a PepMV CP/Hsc70 interaction during virus infection.