In silico analysis of fungal small RNA accumulation reveals putative plant mRNA targets in the symbiosis between an arbuscular mycorrhizal fungus and its host plant.

BACKGROUND: Small RNAs (sRNAs) are short non-coding RNA molecules (20-30 nt) that regulate gene expression at transcriptional or post-transcriptional levels in many eukaryotic organisms, through a mechanism known as RNA interference (RNAi). Recent studies have highlighted that they are also involved in cross-kingdom communication: sRNAs can move across the contact surfaces from "donor" to "receiver" organisms and, once in the host cells of the receiver, they can target specific mRNAs, leading to a modulation of host metabolic pathways and defense responses. Very little is known about RNAi mechanism and sRNAs occurrence in Arbuscular Mycorrhizal Fungi (AMF), an important component of the plant root microbiota that provide several benefits to host plants, such as improved mineral uptake and tolerance to biotic and abiotic stress. RESULTS: Taking advantage of the available genomic resources for the AMF Rhizophagus irregularis we described its putative RNAi machinery, which is characterized by a single Dicer-like (DCL) gene and an unusual expansion of Argonaute-like (AGO-like) and RNA-dependent RNA polymerase (RdRp) gene families. In silico investigations of previously published transcriptomic data and experimental assays carried out in this work provided evidence of gene expression for most of the identified sequences. Focusing on the symbiosis between R. irregularis and the model plant Medicago truncatula, we characterized the fungal sRNA population, highlighting the occurrence of an active sRNA-generating pathway and the presence of microRNA-like sequences. In silico analyses, supported by host plant degradome data, revealed that several fungal sRNAs have the potential to target M. truncatula transcripts, including some specific mRNA already shown to be modulated in roots upon AMF colonization. CONCLUSIONS: The identification of RNAi-related genes, together with the characterization of the sRNAs population, suggest that R. irregularis is equipped with a functional sRNA-generating pathway. Moreover, the in silico analysis predicted 237 plant transcripts as putative targets of specific fungal sRNAs suggesting that cross-kingdom post-transcriptional gene silencing may occur during AMF colonization.

Nondestructive Raman Spectroscopy as a Tool for Early Detection and Discrimination of the Infection of Tomato Plants by Two Economically Important Viruses.

Global population forecasts dictate a rapid adoption of multifaceted approaches to fulfill increasing food requirements, ameliorate food dietary value and security using sustainable and economically feasible agricultural processes. Plant pathogens induce up to 25% losses in vegetable crops and their early detection would contribute to limit their spread and economic impact. As an alternative to time-consuming, destructive, and expensive diagnostic procedures, such as immunological assays and nucleic acid-based techniques, Raman spectroscopy (RS) is a nondestructive rapid technique that generates a chemical fingerprinting of a sample, at low operating costs. Here, we assessed the suitability of RS combined to chemometric analysis to monitor the infection of an important vegetable crop plant, tomato, by two dangerous and peculiarly different viral pathogens, Tomato yellow leaf curl Sardinia virus (TYLCSV) and Tomato spotted wilt virus (TSWV). Experimentally inoculated plants were monitored over 28 days for symptom occurrence and subjected to RS analysis, alongside with measuring the virus amount by quantitative real-time PCR. RS allowed to discriminate mock inoculated (healthy) from virus-infected specimens, reaching an accuracy of >70% after only 14 days after inoculation for TYLCSV and >85% only after 8 days for TSWV, demonstrating its suitability for early detection of virus infection. Importantly, RS also highlighted spectral differences induced by the two viruses, providing specific information on the infecting agent.

From root to fruit: RNA-Seq analysis shows that arbuscular mycorrhizal symbiosis may affect tomato fruit metabolism.

BACKGROUND: Tomato (Solanum lycopersicum) establishes a beneficial symbiosis with arbuscular mycorrhizal (AM) fungi. The formation of the mycorrhizal association in the roots leads to plant-wide modulation of gene expression. To understand the systemic effect of the fungal symbiosis on the tomato fruit, we used RNA-Seq to perform global transcriptome profiling on Moneymaker tomato fruits at the turning ripening stage. RESULTS: Fruits were collected at 55 days after flowering, from plants colonized with Funneliformis mosseae and from control plants, which were fertilized to avoid responses related to nutrient deficiency. Transcriptome analysis identified 712 genes that are differentially expressed in fruits from mycorrhizal and control plants. Gene Ontology (GO) enrichment analysis of these genes showed 81 overrepresented functional GO classes. Up-regulated GO classes include photosynthesis, stress response, transport, amino acid synthesis and carbohydrate metabolism functions, suggesting a general impact of fungal symbiosis on primary metabolisms and, particularly, on mineral nutrition. Down-regulated GO classes include cell wall, metabolism and ethylene response pathways. Quantitative RT-PCR validated the RNA-Seq results for 12 genes out of 14 when tested at three fruit ripening stages, mature green, breaker and turning. Quantification of fruit nutraceutical and mineral contents produced values consistent with the expression changes observed by RNA-Seq analysis. CONCLUSIONS: This RNA-Seq profiling produced a novel data set that explores the intersection of mycorrhization and fruit development. We found that the fruits of mycorrhizal plants show two transcriptomic "signatures": genes characteristic of a climacteric fleshy fruit, and genes characteristic of mycorrhizal status, like phosphate and sulphate transporters. Moreover, mycorrhizal plants under low nutrient conditions produce fruits with a nutrient content similar to those from non-mycorrhizal plants under high nutrient conditions, indicating that AM fungi can help replace exogenous fertilizer for fruit crops.

The arbuscular mycorrhizal symbiosis attenuates symptom severity and reduces virus concentration in tomato infected by Tomato yellow leaf curl Sardinia virus (TYLCSV).

The arbuscular mycorrhizal (AM) symbiosis is considered a natural instrument to improve plant health and productivity since mycorrhizal plants often show higher tolerance to abiotic and biotic stresses. However, the impact of the AM symbiosis on infection by viral pathogens is still largely uncertain and little explored. In the present study, tomato plants were grown under controlled conditions and inoculated with the AM fungus Funneliformis mosseae. Once the mycorrhizal colonization had developed, plants were inoculated with the Tomato yellow leaf curl Sardinia virus (TYLCSV), a geminivirus causing one of the most serious viral diseases of tomatoes in Mediterranean areas. Biological conditions consisted of control plants (C), TYLCSV-infected plants (V), mycorrhizal plants (M), and TYLCSV-infected mycorrhizal plants (MV). At the time of analysis, the level of mycorrhiza development and the expression profiles of mycorrhiza-responsive selected genes were not significantly modified by virus infection, thus indicating that the AM symbiosis was unaffected by the presence and spread of the virus. Viral symptoms were milder, and both shoot and root concentrations of viral DNA were lower in MV plants than in V plants. Overall F. mosseae colonization appears to exert a beneficial effect on tomato plants in attenuating the disease caused by TYLCSV.

Identification of DNA Viruses in Ancient DNA from Herbarium Samples.

Herbaria encompass millions of plant specimens, mostly collected in the nineteenth and twentieth centuries that can represent a key resource for investigating the history and evolution of phytopathogens. In the last years, the application of high-throughput sequencing technologies for the analysis of ancient nucleic acids has revolutionized the study of ancient pathogens including viruses, allowing the reconstruction of historical genomic viral sequences, improving phylogenetic based molecular dating, and providing essential insight into plant virus ecology. In this chapter, we describe a protocol to reconstruct ancient plant and soil viral sequences starting from highly fragmented ancient DNA extracted from herbarium plants and their associated rhizospheric soil. Following Illumina high-throughput sequencing, sequence data are de novo assembled, and DNA viral sequences are selected, according to their similarity with known viruses.

Exogenous Application of dsRNA for Protection against Tomato Leaf Curl New Delhi Virus.

Tomato leaf curl New Delhi virus (ToLCNDV) is an emerging plant pathogen, fast spreading in Asian and Mediterranean regions, and is considered the most harmful geminivirus of cucurbits in the Mediterranean. ToLCNDV infects several plant and crop species from a range of families, including Solanaceae, Cucurbitaceae, Fabaceae, Malvaceae and Euphorbiaceae. Up to now, protection from ToLCNDV infection has been achieved mainly by RNAi-mediated transgenic resistance, and non-transgenic fast-developing approaches are an urgent need. Plant protection by the delivery of dsRNAs homologous to a pathogen target sequence is an RNA interference-based biotechnological approach that avoids cultivating transgenic plants and has been already shown effective against RNA viruses and viroids. However, the efficacy of this approach against DNA viruses, particularly Geminiviridae family, is still under study. Here, the protection induced by exogenous application of a chimeric dsRNA targeting all the coding regions of the ToLCNDV DNA-A was evaluated in zucchini, an important crop strongly affected by this virus. A reduction in the number of infected plants and a delay in symptoms appearance, associated with a tendency of reduction in the viral titer, was observed in the plants treated with the chimeric dsRNA, indicating that the treatment is effective against geminiviruses but requires further optimization. Limits of RNAi-based vaccinations against geminiviruses and possible causes are discussed.

Recombination profiles between Tomato yellow leaf curl virus and Tomato yellow leaf curl Sardinia virus in laboratory and field conditions: evolutionary and taxonomic implications.

Tomato yellow leaf curl Sardinia virus and Tomato yellow leaf curl virus have co-existed in Italian tomato crops since 2002 and have reached equilibrium, with plants hosting molecules of both species plus their recombinants being the most frequent case. Recombination events are studied in field samples, as well as in experimental co-infections, when recombinants were detected as early as 45 days following inoculation. In both conditions, recombination breakpoints were essentially absent in regions corresponding to ORFs V2, CP and C4, whereas density was highest in the 3'-terminal portion of ORF C3, next to the region where the two transcription units co-terminate. The vast majority of breakpoints were mapped at antisense ORFs, supporting speculation that the rolling-circle replication mechanism, and the existence of sense and antisense ORFs on the circular genome, may result in clashes between replication and transcription complexes.

Identification of Known and Novel Arundo donax L. MicroRNAs and Their Targets Using High-Throughput Sequencing and Degradome Analysis.

MicroRNAs (miRNAs) are a class of non-coding molecules involved in the regulation of a variety of biological processes. They have been identified and characterized in several plant species, but only limited data are available for Arundo donax L., one of the most promising bioenergy crops. Here we identified, for the first time, A. donax conserved and novel miRNAs together with their targets, through a combined analysis of high-throughput sequencing of small RNAs, transcriptome and degradome data. A total of 134 conserved miRNAs, belonging to 45 families, and 27 novel miRNA candidates were identified, along with the corresponding primary and precursor miRNA sequences. A total of 96 targets, 69 for known miRNAs and 27 for novel miRNA candidates, were also identified by degradome analysis and selected slice sites were validated by 5'-RACE. The identified set of conserved and novel candidate miRNAs, together with their targets, extends our knowledge about miRNAs in monocots and pave the way to further investigations on miRNAs-mediated regulatory processes in A. donax, Poaceae and other bioenergy crops.

Women in the European Virus Bioinformatics Center.

Viruses are the cause of a considerable burden to human, animal and plant health, while on the other hand playing an important role in regulating entire ecosystems. The power of new sequencing technologies combined with new tools for processing "Big Data" offers unprecedented opportunities to answer fundamental questions in virology. Virologists have an urgent need for virus-specific bioinformatics tools. These developments have led to the formation of the European Virus Bioinformatics Center, a network of experts in virology and bioinformatics who are joining forces to enable extensive exchange and collaboration between these research areas. The EVBC strives to provide talented researchers with a supportive environment free of gender bias, but the gender gap in science, especially in math-intensive fields such as computer science, persists. To bring more talented women into research and keep them there, we need to highlight role models to spark their interest, and we need to ensure that female scientists are not kept at lower levels but are given the opportunity to lead the field. Here we showcase the work of the EVBC and highlight the achievements of some outstanding women experts in virology and viral bioinformatics.

Identification of grapevine microRNAs and their targets using high-throughput sequencing and degradome analysis.

In plants, microRNAs (miRNAs) comprise one of three classes of small RNAs regulating gene expression at the post-transcriptional level. Many plant miRNAs are conserved, and play a role in development, abiotic stress responses or pathogen responses. However, some miRNAs have only been found in certain species. Here, we use deep-sequencing, computational and molecular methods to identify, profile, and describe conserved and non-conserved miRNAs in four grapevine (Vitis vinifera) tissues. A total of 24 conserved miRNA families were identified in all four tissues, and 26 known but non-conserved miRNAs were also found. In addition to known miRNAs, we also found 21 new grapevine-specific miRNAs together with their star strands. We have also shown that almost all of them originated from single genes. Furthermore, 21 other plausible miRNA candidates have been described. We have found that many known and new miRNAs showed tissue-specific expression. Finally, 112 target mRNAs of known and 44 target mRNAs of new grapevine-specific miRNAs were identified by genomic-scale high-throughput sequencing of miRNA cleaved mRNAs.

Arbuscular mycorrhizal symbiosis limits foliar transcriptional responses to viral infection and favors long-term virus accumulation.

Tomato (Solanum lycopersicum) can establish symbiotic interactions with arbuscular mycorrhizal (AM) fungi, and can be infected by several pathogenic viruses. Here, we investigated the impact of mycorrhization by the fungus Glomus mosseae on the Tomato spotted wilt virus (TSWV) infection of tomato plants by transcriptomic and hormones level analyses. In TSWV-infected mycorrhizal plants, the AM fungus root colonization limited virus-induced changes in gene expression in the aerial parts. The virus-responsive upregulated genes, no longer induced in infected mycorrhizal plants, were mainly involved in defense responses and hormone signaling, while the virus-responsive downregulated genes, no longer repressed in mycorrhizal plants, were involved in primary metabolism. The presence of the AM fungus limits, in a salicylic acid-independent manner, the accumulation of abscissic acid observed in response to viral infection. At the time of the molecular analysis, no differences in virus concentration or symptom severity were detected between mycorrhizal and nonmycorrhizal plants. However, in a longer period, increase in virus titer and delay in the appearance of recovery were observed in mycorrhizal plants, thus indicating that the plant's reaction to TSWV infection is attenuated by mycorrhization.

No Evidence for Seed Transmission of Tomato Yellow Leaf Curl Sardinia Virus in Tomato.

Seed transmission is an important factor in the epidemiology of plant pathogens. Geminiviruses are serious pests spread in tropical and subtropical regions. They are transmitted by hemipteran insects, but a few cases of transmission through seeds were recently reported. Here, we investigated the tomato seed transmissibility of the begomovirus tomato yellow leaf curl Sardinia virus (TYLCSV), one of the agents inducing the tomato yellow leaf curl disease, heavily affecting tomato crops in the Mediterranean area. None of the 180 seedlings originating from TYLCSV-infected plants showed any phenotypic alteration typical of virus infection. Moreover, whole viral genomic molecules could not be detected in their cotyledons and true leaves, neither by membrane hybridization nor by rolling-circle amplification followed by PCR, indicating that TYLCSV is not a seed-transmissible pathogen for tomato. Examining the localization of TYLCSV DNA in progenitor plants, we detected the virus genome by PCR in all vegetative and reproductive tissues, but viral genomic and replicative forms were found only in leaves, flowers and fruit flesh, not in seeds and embryos. Closer investigations allowed us to discover for the first time that these embryos were superficially contaminated by TYLCSV DNA but whole genomic molecules were not detectable. Therefore, the inability of TYLCSV genomic molecules to colonize tomato embryos during infection justifies the lack of seed transmissibility observed in this host.

A Primer on the Analysis of High-Throughput Sequencing Data for Detection of Plant Viruses.

High-throughput sequencing (HTS) technologies have become indispensable tools assisting plant virus diagnostics and research thanks to their ability to detect any plant virus in a sample without prior knowledge. As HTS technologies are heavily relying on bioinformatics analysis of the huge amount of generated sequences, it is of utmost importance that researchers can rely on efficient and reliable bioinformatic tools and can understand the principles, advantages, and disadvantages of the tools used. Here, we present a critical overview of the steps involved in HTS as employed for plant virus detection and virome characterization. We start from sample preparation and nucleic acid extraction as appropriate to the chosen HTS strategy, which is followed by basic data analysis requirements, an extensive overview of the in-depth data processing options, and taxonomic classification of viral sequences detected. By presenting the bioinformatic tools and a detailed overview of the consecutive steps that can be used to implement a well-structured HTS data analysis in an easy and accessible way, this paper is targeted at both beginners and expert scientists engaging in HTS plant virome projects.

ORTom: a multi-species approach based on conserved co-expression to identify putative functional relationships among genes in tomato.

Co-expressed genes are often expected to be functionally related and many bioinformatics approaches based on co-expression have been developed to infer their biological role. However, such annotations may be unreliable, whereas the evolutionary conservation of gene co-expression among species may form a basis for more confident predictions. The huge amount of expression data (microarrays, SAGE, ESTs) has already allowed functional studies based on conserved co-expression in animals. Up to now, the implementation of analogous tools for plants has been strongly limited probably by the paucity and heterogeneity of data. Here we present ORTom, a tomato-centred EST data-mining approach based on conserved co-expression in the Solanaceae family. ORTom can be used to predict functional relationships among genes and to prioritize candidate genes for targeted studies. The method consists in ranking ESTs co-expressed with a gene of interest according to the level of expression pattern conservation in phylogenetically-related plants (potato, tobacco and pepper) to obtain lists of putative functionally-related genes. The lists are then analyzed for Gene Ontology keyword enrichment. The web server ORTom has been implemented to make the results publicly-available and searchable. Few biological examples on how the tool can be used are presented.

The complete nucleotide sequence of an isolate of Tomato yellow leaf curl Sardinia virus found in Sicily.

Partial sequences of Tomato yellow leaf curl Sardinia virus (TYLCSV) derived from tomato samples collected in Sicily in 1999, 2002 and 2004 indicated the presence of a TYLCSV different from the one previously described as the Sic strain. Here, we report a complete DNA sequence that is classified as belonging to the TYLCSV type strain (Sar strain), confirming the co-existence in Sicily of virus populations of both strains. Moreover, comparisons between this new sequence and those of the two recombinants recently described in Sicily revealed unequivocally (99% identity) that their TYLCSV-derived portion originated from the Sar strain.

Different Genetic Sources Contribute to the Small RNA Population in the Arbuscular Mycorrhizal Fungus Gigaspora margarita.

RNA interference (RNAi) is a key regulatory pathway of gene expression in almost all eukaryotes. This mechanism relies on short non-coding RNA molecules (sRNAs) to recognize in a sequence-specific manner DNA or RNA targets leading to transcriptional or post-transcriptional gene silencing. To date, the fundamental role of sRNAs in the regulation of development, stress responses, defense against viruses and mobile elements, and cross-kingdom interactions has been extensively studied in a number of biological systems. However, the knowledge of the "RNAi world" in arbuscular mycorrhizal fungi (AMF) is still limited. AMF are obligate mutualistic endosymbionts of plants, able to provide several benefits to their partners, from improved mineral nutrition to stress tolerance. Here we described the RNAi-related genes of the AMF Gigaspora margarita and characterized, through sRNA sequencing, its complex small RNAome, considering the possible genetic sources and targets of the sRNAs. G. margarita indeed is a mosaic of different genomes since it hosts endobacteria, RNA viruses, and non-integrated DNA fragments corresponding to mitovirus sequences. Our findings show that G. margarita is equipped with a complete set of RNAi-related genes characterized by the expansion of the Argonaute-like (AGO-like) gene family that seems a common trait of AMF. With regards to sRNAs, we detected populations of sRNA reads mapping to nuclear, mitochondrial, and viral genomes that share similar features (25-nt long and 5'-end uracil read enrichments), and that clearly differ from sRNAs of endobacterial origin. Furthermore, the annotation of nuclear loci producing sRNAs suggests the occurrence of different sRNA-generating processes. In silico analyses indicate that the most abundant G. margarita sRNAs, including those of viral origin, could target transcripts in the host plant, through a hypothetical cross-kingdom RNAi.

The Induction of an Effective dsRNA-Mediated Resistance Against Tomato Spotted Wilt Virus by Exogenous Application of Double-Stranded RNA Largely Depends on the Selection of the Viral RNA Target Region.

Tomato spotted wilt virus (TSWV) is a devastating plant pathogen, causing huge crop losses worldwide. Unfortunately, due to its wide host range and emergence of resistance breaking strains, its management is challenging. Up to now, resistance to TSWV infection based on RNA interference (RNAi) has been achieved only in transgenic plants expressing parts of the viral genome or artificial microRNAs targeting it. Exogenous application of double-stranded RNAs (dsRNAs) for inducing virus resistance in plants, namely RNAi-based vaccination, represents an attractive and promising alternative, already shown to be effective against different positive-sense RNA viruses and viroids. In the present study, the protection efficacy of exogenous application of dsRNAs targeting the nucleocapsid (N) or the movement protein (NSm) coding genes of the negative-sense RNA virus TSWV was evaluated in Nicotiana benthamiana as model plant and in tomato as economically important crop. Most of the plants treated with N-targeting dsRNAs, but not with NSm-targeting dsRNAs, remained asymptomatic until 40 (N. benthamiana) and 63 (tomato) dpi, while the remaining ones showed a significant delay in systemic symptoms appearance. The different efficacy of N- and NSm-targeting dsRNAs in protecting plants is discussed in the light of their processing, mobility and biological role. These results indicate that the RNAi-based vaccination is effective also against negative-sense RNA viruses but emphasize that the choice of the target viral sequence in designing RNAi-based vaccines is crucial for its success.

Arbuscular Mycorrhizal Symbiosis Primes Tolerance to Cucumber Mosaic Virus in Tomato.

Tomato plants can establish symbiotic interactions with arbuscular mycorrhizal fungi (AMF) able to promote plant nutrition and prime systemic plant defenses against pathogens attack; the mechanism involved is known as mycorrhiza-induced resistance (MIR). However, studies on the effect of AMF on viral infection, still limited and not conclusive, indicate that AMF colonization may have a detrimental effect on plant defenses against viruses, so that the term "mycorrhiza-induced susceptibility" (MIS) has been proposed for these cases. To expand the case studies to a not yet tested viral family, that is, Bromoviridae, we investigated the effect of the colonization by the AMF Funneliformis mosseae on cucumber mosaic virus (CMV) infection in tomato by phenotypic, physiological, biochemical, and transcriptional analyses. Our results showed that the establishment of a functional AM symbiosis is able to limit symptoms development. Physiological and transcriptomic data highlighted that AMF mitigates the drastic downregulation of photosynthesis-related genes and the reduction of photosynthetic CO2 assimilation rate caused by CMV infection. In parallel, an increase of salicylic acid level and a modulation of reactive oxygen species (ROS)-related genes, toward a limitation of ROS accumulation, was specifically observed in CMV-infected mycorrhizal plants. Overall, our data indicate that the AM symbiosis influences the development of CMV infection in tomato plants and exerts a priming effect able to enhance tolerance to viral infection.

Comparative analysis of expression profiles in shoots and roots of tomato systemically infected by Tomato spotted wilt virus reveals organ-specific transcriptional responses.

Tomato (Solanum lycopersicon), a model species for the family Solanaceae, is severely affected by Tomato spotted wilt virus (TSWV) worldwide. To elucidate the systemic transcriptional response of plants to TSWV infection, microarray experiments were performed on tomato. Parallel analysis of both shoots and roots revealed organ-specific responses, although the virus was present in similar concentration. In the shoots, genes related to defense and to signal transduction were induced, while there was general repression of genes related to primary and secondary metabolism as well as to amino acid metabolism. In roots, expression of genes involved in primary metabolism and signal transduction appear unaffected by TSWV infection, while those related to the response to biotic stimuli were induced and those associated to the response to abiotic stress were generally repressed or unaltered. Genes related to amino acid metabolism were unaffected, except for those involved in synthesis of secondary compounds, where induction was evident. Differential expression of genes involved in metabolism and response to ethylene and abscisic acid was observed in the two organs. Our results provide new insight into the biology of the economically important interaction between tomato and TSWV.

Two new natural begomovirus recombinants associated with the tomato yellow leaf curl disease co-exist with parental viruses in tomato epidemics in Italy.

Two tomato geminivirus species co-exist in protected crops in Sicily, Tomato yellow leaf curl Sardinia virus (TYLCSV, found in 1989) and Tomato yellow leaf curl virus (TYLCV, found in 2002), and mixed infections have been detected. In a field survey conducted in 2004, the viral intergenic region (IR) was amplified from infected plants, and molecules apparently hybrid between the two species were found, but only in plants where one or both parental species were also present. Two of these hybrids, named 2/2 and 2/5, were isolated and infectious clones were obtained. They were both readily whitefly-transmitted to tomato plants; clone 2/5 produced symptoms typical of TYLCSV and TYLCV, while clone 2/2 produced more severe symptoms, with leaves showing downward curling and rugosity. Sequence analysis showed that both 2/2 and 2/5 are newly generated hybrids, with two recombination sites each. One site, common to both hybrids, is in the stem-loop of the IR. The other is close to the 3'-end of the CP ORF in 2/5 and within the Rep ORF in 2/2. Thus, the 2/2 hybrid virus has a hybrid Rep protein, with the 202 amino-terminal aa from TYLCV and the remaining 155 aa from TYLCSV. Replication assays in leaf disc indicated a lower replicative capacity with respect to parental viruses, a fact that might help to explain why plants infected only by a recombinant have not been found so far.