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Background Ozonated water (O3 wat) soil drench and/or foliar spray applications were evaluated for their potential to control the root-knot nematode Meloidogyne incognita (RKN) and the airborne pathogen Tomato spotted wilt virus (TSWV) in tomato. We investigated how O3 wat modulates the salicylic acid/jasmonic acid/ethylene (SA/JA/ET) signalling network in the host, locally and systemically, to induce resistance to nematode and virus. Results The application as soil drench was effective in reducing the number of galls and egg masses, but did not reduce the incidence and severity of TSWV infection. Conversely, O3 wat applied by foliar spray decreased TSWV disease incidence and severity (-20%), but was not able to control M. incognita infection. SA-related genes were generally upregulated in both locally treated and systemically reached tissues, showing a positive action of the O3 wat treatment on SA signalling. Neither O3 wat application method significantly altered JA-related gene expression in either direction. ET-related genes were differentially regulated by root or leaf treatments, indicating that O3 wat may have different effects on ET-mediated signalling in different organs. JA/ET/SA related pathways were differentially modulated by O3 wat in the presence of either RKN or TSWV. Conclusion O3 wat had a higher efficacy when applied directly to organs challenged by the pathogens, although it was potentially able to stimulate defence responses through the activation of SA signalling. Owing to its safety and effectiveness in controlling nematode and virus infections, O3 wat can be considered as a possible alternative tool for sustainable disease management practices. © 2019 Society of Chemical Industry.

This research was carried out in order to evaluate the presence and distribution of viral infections causing severe disease in eggplant plants collected from different districts in Bekaa valley, Lebanon. Most infected plants showed virus-like symptoms consisting predominantly of leaf blotch, mottling chlorotic and ring spots, leaf twisting and plant dwarf were also observed in the visited fields. Symptomatic and asymptomatic plants were collected and screened by ELISA test for the presence of several different pathogenic viruses potentially present in the area. Results showed that potato virus Y (PVY) was the most prevalent virus found by ELISA (detected in the 15.3% of the tested plants), followed by eggplant mottled dwarf virus (EMDV, 2.9%) and cucumber mosaic virus (CMV, 1.2%), while tomato spotted wilt virus (TSWV), alfalfa mosaic virus (AMV) and pepper mottle virus (PepMoV) were not detected. Biological indexing of symptomatic ELISA-negative plants, followed by electron microscopy, indicated the presence of virus-like particles of the genus Potexvirus, which was subsequently confirmed as potato virus X (PVX) by RT-PCR and Sanger sequencing. PVX was found in 35.3% of the tested plants, all sampled in the northern Bekaa area. In a phylogenetic analysis, the partial coat protein gene sequence of a selected Lebanese isolate, PVX-AK1, clustered together with other PVX isolates from Asia. Furthermore, the 124-aa sequence of PVX-AK1 shared 100% identity with PVX-UK3, an isolate which is known as avirulent in potato genotypes carrying either Nx or Rx resistance genes. This work revealed a picture of the previously uninvestigated phytosanitary status of eggplant crops in an important horticultural area of Lebanon.

Plants defend themselves from pathogenic virus infections by deploying a complex defense system. Fundamental in the plant antiviral immune system is the RNA silencing (RS) pathway, which consists in a multilayered set of enzymatic components. The RS machinery targets the double-stranded RNA, generating 21–24 nucleotides (nt) small interfering RNAs (siRNAs) that guide the cleavage of viral RNA in a sequence-specific manner. RS signals are then amplified and transmitted cell-to-cell and systemically, in a process that is provided with self-regulatory feedback mechanisms. All the steps of the RS pathway were revealed to be targeted by viral counterattack strategies that allow pathogens to escape host defenses and establish a successful infection. Viral suppressors of RNA silencing (VSRs) are virus-encoded proteins that interfere with RS by blocking or destabilizing the host antiviral functions. This chapter reviews the current knowledge on VSRs mechanisms of action, and describes the latest discoveries in this fascinating evolutionary arms race between plants and viruses.

After the frst outbreak of tomato brown rugose fruit virus (ToBRFV) in tomato in Jordan (Salem et al. 2016), this virus has been reported from other neighboring countries and worldwide. In early 2020, virus-like symptoms consisting in chlorosis, mosaic and leaves discolorations accompanied with brown stems and fruit deformation were observed on greenhouse-cultivated sweet pepper plants in the coastal regions of Syria (Tartous) and Lebanon (Byblos). A total of 40 symptomatic and 20 asymptomatic plants were sampled in diferent sites in both countries and tested for ToBRFV by real-time RT-PCR using CaTa28/CSP1325 primers and probes (diagnostic protocol PM 7/146, EPPO Bulletin 2021). ToBRFV was found in 44 samples including all symptomatic ones. Furthermore, RT-PCR using specifc primers ToBRFV-F5722/ToBRFV-R6179 amplifying a 458-bp fragment of the coat protein gene (Panno et al. 2019) confrmed the identifcation of ToBRFV. All the samples tested negative for tomato spotted wilt virus, pepino mosaic virus and cucumber mosaic virus in RT-PCR. To confrm the specifcity of the amplifed DNA, one representative RTPCR amplicon from each country was sequenced in both directions. Sequences were deposited in GenBank under the accession numbers OU600529 and OU600530 for the Syrian and Lebanese isolates, respectively. Bioinformatics and BLAST analysis of the sequenced amplicons showed 99.78% similarity between the Syrian and the Lebanese isolates and 99.56% nucleotide identity with TBRFV isolate Ant-Pep (MT002973.1) from Turkey. This is the frst report of ToBRFV infecting sweet pepper plants in Syria and Lebanon.

Potato virus Y (PVY) isolate PVYC-to induces growth reduction and foliar symptoms in tomato, but new vegetation displays symptom recovery at a later stage. In order to investigate the role of micro(mi)RNA and secondary small(s)RNA-regulated mechanisms in tomato defenses against PVY, we performed sRNA sequencing from healthy and PVYC-to infected tomato plants at 21 and 30 days post-inoculation (dpi). A total of 792 miRNA sequences were obtained, among which were 123 canonical miRNA sequences, many isomiR variants, and 30 novel miRNAs. MiRNAs were mostly overexpressed in infected vs. healthy plants, whereas only a few miRNAs were underexpressed. Increased accumulation of isomiRs was correlated with viral infection. Among miRNA targets, enriched functional categories included resistance (R) gene families, transcription and hormone factors, and RNA silencing genes. Several 22-nt miRNAs were shown to target R genes and trigger the production of 21-nt phased sRNAs (phasiRNAs). Next, 500 phasiRNA-generating loci were identified, and were shown to be mostly active in PVY-infected tissues and at 21 dpi. These data demonstrate that sRNA-regulated host responses, encompassing miRNA alteration, diversification within miRNA families, and phasiRNA accumulation, regulate R and disease-responsive genes. The dynamic regulation of miRNAs and secondary sRNAs over time suggests a functional role of sRNA-mediated defenses in the recovery phenotype.

Zucchini yellow mosaic virus (ZYMV) was first reported in Italy, but no Italian isolates have been sequenced so far. A ZYMV isolate, designated Z104, was isolated from a zucchini plant grown in the open field in Piedmont, Italy, and showing severe disease symptoms. We report the full-length genome sequence of Z104 as determined by Sanger sequencing and small (s)RNA deep sequencing. The latter technique was useful to confirm the isolate sequence, that was deposited in the NCBI database with the accession number MK956829 and to investigate the sequence variability of Z104 isolate. The genome of 9611 nucleotides (nt) consisted of an open reading frame of 9243 nt encoding a polyprotein of 3080 amino acids (349.88 kDa), flanked by the 5′- and 3′-untranslated regions, and an overlapping ORF of 74 amino acids, the P3N-PIPO. As in previously sequenced ZYMV isolates, Z104 polyprotein consists of 10 putative functional proteins. Several domains in the HC-Pro and P3 proteins related to the transmissibility by aphids and virus aggressiveness are conserved in Z104. Symptoms induced both in the field and laboratory by Z104 together with its molecular features indicate that it is an aggressive strain. Deep sequencing of sRNAs revealed a particularly high variability in the genome sequence at eight nucleotide positions, three of which resulted in missense mutations in the P3, 6K1, and NIb proteins. This may be indicative of the natural ZYMV genome variability within the host. The phylogenetic analysis of the whole genome or the sole coat protein of several ZYMV isolates did not provide evidence for a close relationship of Z104 with other isolates from European countries. To the best of our knowledge, this is the first-reported complete genome of a ZYMV isolate from Italy.

Ozonated water (OThe application as soil drench was effective in reducing the number of galls and egg masses, but did not reduce the incidence and severity of TSWV infection. Conversely, OO

The powdery mildew disease affects several crop species and is also one of the major threats for pea (Pisum sativum L.) cultivation all over the world. The recessive gene er1, first described over 60 years ago, is well known in pea breeding, as it still maintains its efficiency as a powdery mildew resistance source. Genetic and phytopathological features of er1 resistance are similar to those of barley, Arabidopsis, and tomato mlo powdery mildew resistance, which is caused by the loss of function of specific members of the MLO gene family. Here, we describe the obtainment of a novel er1 resistant line by experimental mutagenesis with the alkylating agent diethyl sulfate. This line was found to carry a single nucleotide polymorphism in the PsMLO1 gene sequence, predicted to result in premature termination of translation and a non-functional protein. A cleaved amplified polymorphic sequence (CAPS) marker was developed on the mutation site and shown to be fully co-segregating with resistance in F(2) individuals. Sequencing of PsMLO1 from three powdery mildew resistant cultivars also revealed the presence of loss-of-function mutations. Taken together, results reported in this study strongly indicate the identity between er1 and mlo resistances and are expected to be of great breeding importance for the development of resistant cultivars via marker-assisted selection.

Mixed infection with the SON41 strain of Potato virus Y (PVY-SON41) in tomato increased accumulation of RNAs of strains Fny and LS of Cucumber mosaic virus (CMV-Fny and CMV-LS, respectively) and enhanced disease symptoms. By contrast, replication of PVY-SON41 was downregulated by CMV-Fny and this was due to the CMV-Fny 2b protein. The CMV-FnyΔ2b mutant was unable to systemically invade the tomato plant because its movement was blocked at the bundle sheath of the phloem. The function needed for invading the phloem was complemented by PVY-SON41 in plants grown at 22°C whereas this complementation was not necessary in plants grown at 15°C. Mutations in the 2b protein coding sequence of CMV-Fny as well as inhibition of translation of the 2a/2b overlapping region of the 2a protein lessened both the accumulation of viral RNAs and the severity of symptoms. Both of these functions were complemented by PVY-SON41. Infection of CMV-Fny supporting replication of the Tfn-satellite RNA reduced the accumulation of CMV RNA and suppressed symptom expression also in plants mixed-infected with PVY-SON41. The interaction between CMV and PVY-SON41 in tomato exhibited different features from that documented in other hosts. The results of this work are relevant from an ecological and epidemiological perspective due to the frequency of natural mixed infection of CMV and PVY in tomato.

In tomato, resistance to Tomato spotted wilt virus (TSWV) is conferred by the dominant gene, designated Sw-5. Virulent Sw-5 resistance breaking (SRB) mutants of TSWV have been reported on Sw-5 tomato cultivars. Two different PCR-based allelic discrimination techniques, namely Custom TaqMan(TM) SNP Genotyping and high-resolution melting (HRM) assays, were developed and compared for their ability to distinguish between avirulent (Sw-5 non-infecting, SNI) and SRB biotypes. TaqMan assays proved to be more sensitive (threshold of detection in a range of 50-70 TSWV RNA copies) and more reliable than HRM, assigning 25 TSWV isolates to their correct genotype with an accuracy of 100%. Moreover, the TaqMan SNP assays were further improved developing a rapid and simple protocol that included crude leaf extraction for RNA template preparations. On the other hand, HRM assays showed higher levels of sensitivity than TaqMan when used to co-detect both biotypes in different artificial mixtures. These diagnostic assays contributed to gain preliminary information on the epidemiology of TSWV isolates in open field conditions. In fact, the presented data suggest that SRB isolates are present as stable populations established year round, persisting on both winter (globe artichoke) and summer (tomato) crops, in the same cultivated areas of Southern Italy.

In diverse eukaryotic organisms, Dicer-processed, virus-derived small interfering RNAs direct antiviral immunity by RNA silencing or RNA interference. Here we show that in addition to core dicing and slicing components of RNAi, the RNAi-mediated viral immunity in Arabidopsis thaliana requires host RNA-directed RNA polymerase ( RDR ) 1 or RDR6 to produce viral secondary siRNAs following viral RNA replication-triggered biogenesis of primary siRNAs. We found that the two antiviral RDRs exhibited specificity in targeting the tripartite positive-strand RNA genome of cucumber mosaic virus (CMV). RDR1 preferentially amplified the 5′-terminal siRNAs of each of the three viral genomic RNAs, whereas an increased production of siRNAs targeting the 3′ half of RNA3 detected in rdr1 mutant plants appeared to be RDR6-dependent. However, siRNAs derived from a single-stranded 336-nucleotide satellite RNA of CMV were not amplified by either antiviral RDR, suggesting avoidance of the potent RDR-dependent silencing as a strategy for the molecular parasite of CMV to achieve preferential replication. Our work thus identifies a distinct mechanism for the amplification of immunity effectors, which together with the requirement for the biogenesis of endogenous siRNAs, may play a role in the emergence and expansion of eukaryotic RDRs.

Plant viruses are obligate parasites that exploit host components for replication and spread inside the host. Transport of the viral genome is enabled by movement proteins (MPs) targeting the cell periphery to mediate passage throughout plasmodesmata (PD). Pectin methylesterase (PME) is one of the critical host factors facilitating MPs in PD gating, and a direct interaction of PME with Tobacco mosaic virus (TMV) MP is required for viral movement and in turn for virus viability. PME is a critical enzyme for host development and defence, acting via complex mechanisms involving multigenic and tissue specific isoforms and endogenous inhibitors. This composite activity of PME suggests that level and timing of protein accumulation, with respect to virus inoculation and MP expression, can be critical for the functional outcome of the PME-MP interaction and in turn for the success of a viral infection. Based on this notion, we tested different experimental conditions to evaluate the beneficial effect of the downregulation of PME gene expression on the development of TMV-induced disease and on plant protection. We used virus induced gene silencing technology (VIGS) to downregulate PME gene expression, which resulted in a 30–45 % reduction of TMV symptom severity and, correspondingly, to a 60 % reduction of TMV RNA accumulation in systemic leaves. VIGS proved to be a rapid and effective technology for PME gene silencing in functional assays and for plant defence from viral infection. Our findings indicate that N. benthamiana plants with hindered expression of PME survive a TMV infection, which kills non-silenced plants within a week.

Delivery into plants of T-DNAs containing promoter, terminator, or coding sequences generated small interfering RNAs (siRNAs) specific to each type of sequence. When both promoter and transcribed sequences were simultaneously present in the T-DNA, accumulation of siRNAs to transcribed sequences was favored over accumulation of siRNAs to the nontranscribed upstream promoter sequences. The generation of specific siRNA sequences occurred even in the absence of T-DNA homology to sequences in the plant. Delivery of T-DNA, with homology to the transgene limited to the nontranscribed cauliflower mosaic virus 35S promoter (35SP) and the transcribed nopaline synthase transcription termination (NosT)signal sequences, into transgenic plants expressing the green fluorescent protein (GFP), generated siRNAs in infiltrated tissues to both 35SP (35SsiRNAs) and NosT (NosTsiRNAs), but not to the GFP sequence (GFPsiRNAs). In infiltrated tissues, the 35SsiRNAs failed to trigger the transcriptional silencing of the transgene, accumulation of 35SsiRNAs could be prevented by the potyviral HC-Pro, and the NosTsiRNAs required an initial amplification to trigger efficient transgene silencing, which is mediated by transcripts from the exogenous T-DNA, and not from the transgene. In upper leaves, silencing correlated with the presence of GFPsiRNAs and the absence of 35SsiRNAs, confirming that its spread was posttranscriptionally mediated by the transgene mRNA.

Transgenic resistance to plant viruses is an important technology for control of plant virus infection, which has been demonstrated for many model systems, as well as for the most important plant viruses, in terms of the costs of crop losses to disease, and also for many other plant viruses infecting various fruits and vegetables. Different approaches have been used over the last 28 years to confer resistance, to ascertain whether particular genes or RNAs are more efficient at generating resistance, and to take advantage of advances in the biology of RNA interference to generate more efficient and environmentally safer, novel "resistance genes." The approaches used have been based on expression of various viral proteins (mostly capsid protein but also replicase proteins, movement proteins, and to a much lesser extent, other viral proteins), RNAs [sense RNAs (translatable or not), antisense RNAs, satellite RNAs, defective-interfering RNAs, hairpin RNAs, and artificial microRNAs], nonviral genes (nucleases, antiviral inhibitors, and plantibodies), and host-derived resistance genes (dominant resistance genes and recessive resistance genes), and various factors involved in host defense responses. This review examines the above range of approaches used, the viruses that were tested, and the host species that have been examined for resistance, in many cases describing differences in results that were obtained for various systems developed in the last 20 years. We hope this compilation of experiences will aid those who are seeking to use this technology to provide resistance in yet other crops, where nature has not provided such.

Transgenic resistance to plant viruses is an important technology for control of plant virus infection, which has been demonstrated for many model systems, as well as for the most important plant viruses, in terms of the costs of crop losses to disease, and also for many other plant viruses infecting various fruits and vegetables. Different approaches have been used over the last 28 years to confer resistance, to ascertain whether particular genes or RNAs are more efficient at generating resistance, and to take advantage of advances in the biology of RNA interference to generate more efficient and environmentally safer, novel “resistance genes.” The approaches used have been based on expression of various viral proteins (mostly capsid protein but also replicase proteins, movement proteins, and to a much lesser extent, other viral proteins), RNAs [sense RNAs (translatable or not), antisense RNAs, satellite RNAs, defective-interfering RNAs, hairpin RNAs, and artificial microRNAs], nonviral genes (nucleases, antiviral inhibitors, and plantibodies), and host-derived resistance genes (dominant resistance genes and recessive resistance genes), and various factors involved in host defense responses. This review examines the above range of approaches used, the viruses that were tested, and the host species that have been examined for resistance, in many cases describing differences in results that were obtained for various systems developed in the last 20 years. We hope this compilation of experiences will aid those who are seeking to use this technology to provide resistance in yet other crops, where nature has not provided such.

Motivations. RNA silencing, or post-transcriptional gene silencing (PTGS), is a conserved mechanism in a broad range of eukaryotes. In plants, PTGS acts as an antiviral system and a successful virus infection requires suppression or evasion of the induced silencing response. Small interfering RNAs (siRNAs) accumulate in plants infected with RNA and DNA viruses and provide specificity to this RNA-mediated immune system. High-throughput sequencing has contributed to expanding our previously knowledge of siRNA populations better describing their abundance, complexity and diversity in infected tissues. It is now known that siRNAs from virus-infected plants are extraordinarily abundant and diverse, and are widespread in near saturation at any region of either positive and negative genomic RNAs. However, certain regions of viral genomes ("hot spots") are usually more represented than others in sequenced siRNA populations. A gene involved in chlorophyll biosynthesis has been shown targeted by a siRNA derived from viral satellite RNA, revealing PTGS mechanism at basis of the symptom. Potato virus Y (PVY) is the type species of Potyvirus, a genus of agricultural importance belonging to the largest plant virus family, Potyviridae. The potyviral genome is a single-stranded, positive-sense RNA of ca. 10 kilobases (kb). PVYc-to and PVY-SON41 are two isolates of PVY that infect solanaceous hosts. While PVYc-to induces severe leaf distortion in different cultivars of tomato (Solanum lycopersicum), PVY-SON41 produces in the same host only a mild mosaic, followed by recovery. In order to elucidate the molecular mechanism underlying the different symptoms induced by PVYc-to and PVY-SON41 infections on tomato, we set up an in silico approach, mining genomic regions of PVY isolates and looking at possible RNA-based mechanisms where siRNA putatively generated from the PVY genome could target and suppress accumulation of host messenger RNA (mRNA), leading to dysfunctional biological processes that could explain different disease phenotypes. Methods. A Perl script was used to extract the complete datasets of 21-mers from isolates PVYc-to and PVY-SON41 complete genomes, leading the scanning of the complete sequence, shifted by one base at the time. MySQL was used for identification of the 21-mers shared and unique between the two viral genomes, highlighting sequence differences that could be at the base of diverse induced symptoms. The data obtained in the previous step were used to build the genomic map of identical regions, by fancyGene. A BLAST analysis was conducted with the identified 21-mer dataset, considering only the dissimilar sequences between the two isolates (blastn-short identity > 94%, max 2 mismatch or gap, alignment length 21 bp). The 21-mer were used as query and the tomato mRNA database (Solgenomics, release ITAG2.3) was used as target. The results were further used as input in a gene ontology analysis, through Blast2GO. Results. Despite the high identity (> 91%) shared by the two viral genomes, the 21-mer dataset produced a high degree of variation, as the analyses showed 1750 identical and 9671 and 9680 dissimilar sets of 21-mer, for PVYc-to and PVY-SON41, respectively. Data showed that 315 and 381 tomato mRNA were complementary to, and thus possible targets of, the in silico-generated siRNAs deduced from of PVYc-to and PVY-SON41 sequences, respectively. The GO analysis showed different putative gene targets, involved in several metabolic pathways indicating specific targets for each isolate. Of particular interest, for instance, are putative target mRNAs of transcription factors with a known role concerning leaf development and symmetry. These genes, if differentially modulated during the infections of the two different PVY isolates, could cause the leaf malformations observed as major differential symptoms between the two PVY isolates. To validate the bioinformatic approach, target genes with highly significant complementarity have been selected from the in silico data for experimental validation in tomato plants mechanically infected by virus isolates.

The biological and molecular characterization is reported of a Watermelon mosaic virus isolate, denoted WMV-Le, associated with a necrotic phenotype of watermelon plants grown in the Provinces of Lecce and Taranto (Apulia, southern Italy). The fully sequenced WMV-Le genome consists of 10,045 nucleotides and is 99.1% similar to that of WMV-C05-270, a French isolate from melon of the WMV molecular group 3. Using recombination detection program RDP3, putative recombination breakpoints were identified close to nucleotide positions 42 to 1892, covering the 5′UTR/P1/HC-Pro region. The event represents the insertion of a sequence fragment of an isolate similar to WMV-FBR04-37 in the background of an isolate similar to WMV-FMF00-LL1. The field symptomatology was reproduced in watermelon plants grown in an experimental greenhouse but the virus induced severe symptoms also in Cucumis sativus, C. melo, Cucurbita maxima and C. pepo .

The quantification of messenger RNA expression levels by real-time reverse-transcription polymerase chain reaction requires the availability of reference genes that are stably expressed regardless of the experimental conditions under study. We examined the expression variations of a set of eight candidate reference genes in tomato leaf and root tissues subjected to the infection of five taxonomically and molecularly different plant viruses and a viroid, inducing diverse pathogenic effects on inoculated plants. Parallel analyses by three commonly used dedicated algorithms, geNorm, NormFinder and BestKeeper, showed that different viral infections and tissues of origin influenced, to some extent, the expression levels of these genes. However, all algorithms showed high levels of stability for glyceraldehyde 3-phosphate dehydrogenase and ubiquitin, indicated as the most suitable endogenous transcripts for normalization in both tissue types. Actin and uridylate kinase were also stably expressed throughout the infected tissues, whereas cyclophilin showed tissue-specific expression stability only in root samples. By contrast, two widely employed reference genes, 18S ribosomal RNA and elongation factor 1α, demonstrated highly variable expression levels that should discourage their use for normalization. In addition, expression level analysis of ascorbate peroxidase and superoxide dismutase showed the modulation of the two genes in virus-infected tomato leaves and roots. The relative quantification of the two genes varied according to the reference genes selected, thus highlighting the importance of the choice of the correct normalization method in such experiments.

The differential response of 29 genotypes of tomato and wild tomato relatives (Solanum section Lycopersicon species) to cucumber mosaic virus strain Fny (CMV-Fny), alone or in combination with three different satellite RNA (satRNA) variants, allowed the identification of four disease phenotype patterns, each including plants that developed very severe symptoms (leaf malformations, top stunting and lethal necrosis) and plants that remained asymptomatic. No resistance or tolerance to CMV-Fny was observed, whilst individual host genotypes displayed latent infection upon inoculation with one (CMV-Fny/Tfn-satRNA, phenotype patterns 1 and 4), two (CMV-Fny/Tfn-satRNA and CMV-Fny/TTS-satRNA, phenotype pattern 2) or all three (the former two plus CMV-Fny/77-satRNA, phenotype pattern 3) CMV/satRNA combinations. RNA gel-blot analyses showed that latent infection generally correlated with a strong downregulation of CMV RNA accumulation levels. Introgression lines derived from a cross between Solanum habrochaites LA1777, which displayed disease phenotype pattern 2, and Solanum lycopersicum were screened for tolerance to the stunting phenotype induced by CMV-Fny/TTS-satRNA, and only one line, carrying an introgression on chromosome 6, was identified as being partially tolerant. Solanum chilense LA1932×S. lycopersicum back-cross introgression lines were screened for tolerance to lethal necrosis induced by CMV-Fny/77-satRNA (phenotype pattern 3); the tolerant phenotype was observed in 33 % of plants of the BC1F2 progeny and 1F3 progeny. Thus, potentially useful sources of tolerance to CMV/satRNA-induced diseases were identified, although the tolerant phenotypes appeared to be controlled by complex quantitative trait loci.

Mixed infection with the SON41 strain of Potato virus Y (PVY-SON41) in tomato increased accumulation of RNAs of strains Fny and LS of Cucumber mosaic virus (CMV-Fny and CMV-LS, respectively) and enhanced disease symptoms. By contrast, replication of PVY-SON41 was downregulated by CMV-Fny and this was due to the CMV-Fny 2b protein. The CMV-Fny?2b mutant was unable to systemically invade the tomato plant because its movement was blocked at the bundle sheath of the phloem. The function needed for invading the phloem was complemented by PVY-SON41 in plants grown at 22°C whereas this complementation was not necessary in plants grown at 15°C. Mutations in the 2b protein coding sequence of CMV-Fny as well as inhibition of translation of the 2a/2b overlapping region of the 2a protein lessened both the accumulation of viral RNAs and the severity of symptoms. Both of these functions were complemented by PVY-SON41. Infection of CMV-Fny supporting replication of the Tfn-satellite RNA reduced the accumulation of CMV RNA and suppressed symptom expression also in plants mixed-infected with PVY-SON41. The interaction between CMV and PVYSON41 in tomato exhibited different features from that documented in other hosts. The results of this work are relevant from an ecological and epidemiological perspective due to the frequency of natural mixed infection of CMV and PVY in tomato.

The replication-associated proteins encoded by Cucumber mosaic virus (CMV), the 1a and 2a proteins, were detected by immunogold labeling in two host species of this virus, tobacco ( Nicotiana tabacum ) and cucumber ( Cucumis sativus ). In both hosts, the 1a and 2a proteins colocalized predominantly to the vacuolar membranes, the tonoplast. While plus-strand CMV RNAs were found distributed throughout the cytoplasm by in situ hybridization, minus-strand CMV RNAs were barely detectable but were found associated with the tonoplast. In both cucumber and tobacco, 2a protein was detected at higher densities than 1a protein. The 1a and 2a proteins also showed quantitative differences with regard to tissue distributions in tobacco and cucumber. About three times as much 2a protein was detected in CMV-infected cucumber tissues as in CMV-infected tobacco tissues. In tobacco, high densities of these proteins were observed only in vascular bundle cells of minor veins. In contrast, in cucumber, high densities of 1a and 2a proteins were observed in mesophyll cells, followed by epidermis cells, with only low levels being observed in vascular bundle cells. Differences were also observed in the distributions of 2a protein and capsid protein in vascular bundle cells of the two host species. These observations may represent differences in the relative rates of tissue infection in different hosts or differences in the extent of virus replication in vascular tissues of different hosts.

Part of the notoriety of cucumber mosaic cucumovirus (CMV) lies in its ability to support replication of a small (332–405 nucleotide) satellite RNA (satRNA) which can sometimes affect CMV replication, pathogenesis and symptom expression. Variants with attenuating effects (benign satRNAs) have been extensively studied as potential or actual biocontrol agents for the prevention of CMV-induced diseases. This approach has become the basis for experimental and practical field tests where satRNA-mediated protection has been achieved successfully by either preinoculation of seedlings (cross-protection or “vaccination”) or by transgenosis, and apparently no adverse effects have been detected.

Transgenic tomato (Lycopersicon esculentum Mill. cv. UC82) plants expressing a benign variant of Cucumber mosaic virus satellite RNA (CMV Tfn-satRNA) were generated. The transformed plants did not produce symptoms when challenged with a satRNA-free strain of CMV (CMV-FL). The same plant lines initially were susceptible to necrosis elicited by a CMV strain supporting a necrogenic variant of satRNA (CMV-77), but a phenotype of total recovery from the necrosis was observed in the newly developing leaves. The features of the observed resistance were analyzed and are consistent with two different mechanisms of resistance. In transgenic plants inoculated with CMV-FL strain, the symptomless phenotype was correlated to the down-regulation of CMV by Tfn-satRNA, amplified from the transgene transcripts, as the first resistance mechanism. On the other hand, the delayed resistance to CMV-77 in transgenic tomato lines was mediated by a degradation process that targets satRNAs in a sequence-specific manner. Evidence is provided for a correlation between a reduced accumulation level of transgenic messenger Tfn-satRNA, the accumulation of small (approximately 23 nucleotides) RNAs with sequence homology to satRNAs, the progressively reduced accumulation of 77-satRNA in infected tissues, and the transition in infected plants from diseased to healthy. Thus, events leading to the degradation of satRNA sequences indicate a role for RNA silencing as the second mechanism determining resistance of transgenic tomato lines.