Next-generation sequencing identification and multiplex RT-PCR detection for viruses infecting cigar and flue-cured tobacco.

BACKGROUND: Early, precise and simultaneous identification of plant viruses is of great significance for preventing virus spread and reducing losses in agricultural yields. METHODS AND RESULTS: In this study, the identification of plant viruses from symptomatic samples collected from a cigar tobacco planting area in Deyang and a flue-cured tobacco planting area in Luzhou city, Sichuan Province, China, was conducted by deep sequencing of small RNAs (sRNAs) through an Illumina sequencing platform, and plant virus-specific contigs were generated based on virus-derived siRNA sequences. Additionally, sequence alignment and phylogenetic analysis were performed to determine the species or strains of these viruses. A total of 27930450, 21537662 and 28194021 clean reads were generated from three pooled samples, with a total of 105 contigs mapped to the closest plant viruses with lengths ranging from 34 ~ 1720 nt. The results indicated that the major viruses were potato virus Y, Chilli veinal mottle virus, tobacco vein banding mosaic virus, tobacco mosaic virus and cucumber mosaic virus. Subsequently, a fast and sensitive multiplex reverse transcription polymerase chain reaction assay was developed for the simultaneous detection of the most frequent RNA viruses infecting cigar and flue-cured tobacco in Sichuan. CONCLUSIONS: These results provide a theoretical basis and convenient methods for the rapid detection and control of viruses in cigar- and flue-cured tobacco.

A calmodulin-binding transcription factor links calcium signaling to antiviral RNAi defense in plants.

RNA interference (RNAi) is an across-kingdom gene regulatory and defense mechanism. However, little is known about how organisms sense initial cues to mobilize RNAi. Here, we show that wounding to Nicotiana benthamiana cells during virus intrusion activates RNAi-related gene expression through calcium signaling. A rapid wound-induced elevation in calcium fluxes triggers calmodulin-dependent activation of calmodulin-binding transcription activator-3 (CAMTA3), which activates RNA-dependent RNA polymerase-6 and Bifunctional nuclease-2 (BN2) transcription. BN2 stabilizes mRNAs encoding key components of RNAi machinery, notably AGONAUTE1/2 and DICER-LIKE1, by degrading their cognate microRNAs. Consequently, multiple RNAi genes are primed for combating virus invasion. Calmodulin-, CAMTA3-, or BN2-knockdown/knockout plants show increased susceptibility to geminivirus, cucumovirus, and potyvirus. Notably, Geminivirus V2 protein can disrupt the calmodulin-CAMTA3 interaction to counteract RNAi defense. These findings link Ca2+ signaling to RNAi and reveal versatility of host antiviral defense and viral counter-defense.

Analysis of the Role of Bradysia impatiens (Diptera: Sciaridae) as a Vector Transmitting Peanut Stunt Virus on the Model Plant Nicotiana benthamiana.

Bradysia species, commonly known as fungus gnats, are ubiquitous in greenhouses, nurseries of horticultural plants, and commercial mushroom houses, causing significant economic losses. Moreover, the insects from the Bradysia genus have a well-documented role in plant pathogenic fungi transmission. Here, a study on the potential of Bradysia impatiens to acquire and transmit the peanut stunt virus (PSV) from plant to plant was undertaken. Four-day-old larvae of B. impatiens were exposed to PSV-P strain by feeding on virus-infected leaves of Nicotiana benthamiana and then transferred to healthy plants in laboratory conditions. Using the reverse transcription-polymerase chain reaction (RT-PCR), real-time PCR (RT-qPCR), and digital droplet PCR (RT-ddPCR), the PSV RNAs in the larva, pupa, and imago of B. impatiens were detected and quantified. The presence of PSV genomic RNA strands as well as viral coat protein in N. benthamiana, on which the viruliferous larvae were feeding, was also confirmed at the molecular level, even though the characteristic symptoms of PSV infection were not observed. The results have shown that larvae of B. impatiens could acquire the virus and transmit it to healthy plants. Moreover, it has been proven that PSV might persist in the insect body transstadially. Although the molecular mechanisms of virion acquisition and retention during insect development need further studies, this is the first report on B. impatiens playing a potential role in plant virus transmission.

Infection of Arabidopsis by cucumber mosaic virus triggers jasmonate-dependent resistance to aphids that relies partly on the pattern-triggered immunity factor BAK1.

Many aphid-vectored viruses are transmitted nonpersistently via transient attachment of virus particles to aphid mouthparts and are most effectively acquired or transmitted during brief stylet punctures of epidermal cells. In Arabidopsis thaliana, the aphid-transmitted virus cucumber mosaic virus (CMV) induces feeding deterrence against the polyphagous aphid Myzus persicae. This form of resistance inhibits prolonged phloem feeding but promotes virus acquisition by aphids because it encourages probing of plant epidermal cells. When aphids are confined on CMV-infected plants, feeding deterrence reduces their growth and reproduction. We found that CMV-induced inhibition of growth as well as CMV-induced inhibition of reproduction of M. persicae are dependent upon jasmonate-mediated signalling. BRASSINOSTEROID INSENSITIVE1-ASSOCIATED KINASE1 (BAK1) is a co-receptor enabling detection of microbe-associated molecular patterns and induction of pattern-triggered immunity (PTI). In plants carrying the mutant bak1-5 allele, CMV induced inhibition of M. persicae reproduction but not inhibition of aphid growth. We conclude that in wildtype plants CMV induces two mechanisms that diminish performance of M. persicae: a jasmonate-dependent and PTI-dependent mechanism that inhibits aphid growth, and a jasmonate-dependent, PTI-independent mechanism that inhibits reproduction. The growth of two crucifer specialist aphids, Lipaphis erysimi and Brevicoryne brassicae, was not affected when confined on CMV-infected A. thaliana. However, B. brassicae reproduction was inhibited on CMV-infected plants. This suggests that in A. thaliana CMV-induced resistance to aphids, which is thought to incentivize virus vectoring, has greater effects on polyphagous than on crucifer specialist aphids.

Multiple cellular compartments engagement in Nicotiana benthamiana-peanut stunt virus-satRNA interactions revealed by systems biology approach.

KEY MESSAGE: PSV infection changed the abundance of host plant's transcripts and proteins associated with various cellular compartments, including ribosomes, chloroplasts, mitochondria, the nucleus and cytosol, affecting photosynthesis, translation, transcription, and splicing. Virus infection is a process resulting in numerous molecular, cellular, and physiological changes, a wide range of which can be analyzed due to development of many high-throughput techniques. Plant RNA viruses are known to replicate in the cytoplasm; however, the roles of chloroplasts and other cellular structures in the viral replication cycle and in plant antiviral defense have been recently emphasized. Therefore, the aim of this study was to analyze the small RNAs, transcripts, proteins, and phosphoproteins affected during peanut stunt virus strain P (PSV-P)-Nicotiana benthamiana interactions with or without satellite RNA (satRNA) in the context of their cellular localization or functional connections with particular cellular compartments to elucidate the compartments most affected during pathogenesis at the early stages of infection. Moreover, the processes associated with particular cell compartments were determined. The 'omic' results were subjected to comparative data analyses. Transcriptomic and small RNA (sRNA)-seq data were obtained to provide new insights into PSV-P-satRNA-plant interactions, whereas previously obtained proteomic and phosphoproteomic data were used to broaden the analysis to terms associated with cellular compartments affected by virus infection. Based on the collected results, infection with PSV-P contributed to changes in the abundance of transcripts and proteins associated with various cellular compartments, including ribosomes, chloroplasts, mitochondria, the nucleus and the cytosol, and the most affected processes were photosynthesis, translation, transcription, and mRNA splicing. Furthermore, sRNA-seq and phosphoproteomic analyses indicated that kinase regulation resulted in decreases in phosphorylation levels. The kinases were associated with the membrane, cytoplasm, and nucleus components.

Improving Regulation of Enzymatic and Non-Enzymatic Antioxidants and Stress-Related Gene Stimulation in Cucumber mosaic cucumovirus-Infected Cucumber Plants Treated with Glycine Betaine, Chitosan and Combination.

Cucumber mosaic cucumovirus (CMV) is a deadly plant virus that results in crop-yield losses with serious economic consequences. In recent years, environmentally friendly components have been developed to manage crop diseases as alternatives to chemical pesticides, including the use of natural compounds such as glycine betaine (GB) and chitosan (CHT), either alone or in combination. In the present study, the leaves of the cucumber plants were foliar-sprayed with GB and CHT-either alone or in combination-to evaluate their ability to induce resistance against CMV. The results showed a significant reduction in disease severity and CMV accumulation in plants treated with GB and CHT, either alone or in combination, compared to untreated plants (challenge control). In every treatment, growth indices, leaf chlorophylls content, phytohormones (i.e., indole acetic acid, gibberellic acid, salicylic acid and jasmonic acid), endogenous osmoprotectants (i.e., proline, soluble sugars and glycine betaine), non-enzymatic antioxidants (i.e., ascorbic acid, glutathione and phenols) and enzymatic antioxidants (i.e., superoxide dismutase, peroxidase, polyphenol oxidase, catalase, lipoxygenase, ascorbate peroxidase, glutathione reductase, chitinase and ß-1,3 glucanase) of virus-infected plants were significantly increased. On the other hand, malondialdehyde and abscisic acid contents have been significantly reduced. Based on a gene expression study, all treated plants exhibited increased expression levels of some regulatory defense genes such as PR1 and PAL1. In conclusion, the combination of GB and CHT is the most effective treatment in alleviated virus infection. To our knowledge, this is the first report to demonstrate the induction of systemic resistance against CMV by using GB.

Application of Virus-Induced Gene Silencing to Arbuscular Mycorrhizal Fungi.

Downregulation of AM fungal genes using a plant viral vector is feasible. A partial sequence of a target fungal gene is cloned into the multicloning site of CMV2-A1 vector developed from RNA2 of Cucumber mosaic virus Y strain, and the RNA2, together with RNA1 and RNA3 of the virus, are in vitro-transcribed. Inoculation of Nicotiana benthamiana with these viral RNAs results in reconstitution of the virus in the plant, which triggers silencing of the fungal gene. Here, we describe not only the methods but also several tips for successful application of virus-induced gene silencing to AM fungi.

Ocurrence of pepper yellow mosaic virus and cucumber mosaic virus on Capsicum chinense in the state of Amazonas, Brazil / Ocorrência do pepper yellow mosaic virus e cucumber mosaic virus em Capsicum chinense no estado do Amazonas, Brasil

The habanero chilli pepper, Capsicum chinense is an important crop in the Amazon Basin, mainly grown by small-scale producers. Capsicum chinense plants in an experimental field in the northern Brazilian state of Amazonas were found exhibiting characteristic symptoms of viral infection. Leaf sap from symptomatic plants examined under a transmission electron microscope revealed the presence of elongated flexuous particles and isometric particles. Using molecular assays, the viruses were identified as pepper yellow mosaic virus (PepYMV) and cucumber mosaic virus (CMV). Aphids, identified as Aphis gossypii, were found colonizing the C. chinense plants in the field and may be the vector for both PepYMV and CMV. We report the first occurrence of these viruses infecting C. chinense in the state of Amazonas.

A pimenta-de-cheiro, Capsicum chinense é uma cultura importante na Bacia Amazônica, cultivada principalmente por pequenos produtores. Plantas de C. chinense em um campo experimental localizado no norte do estado brasileiro do Amazonas, foram encontradas apresentando sintomas característicos de infecção viral. Extratos de amostras de folhas sintomáticas examinados ao microscópio eletrônico de transmissão revelaram a presença de partículas alongadas e flexuosas e de partículas isométricas. Análises moleculares permitiram identificar a presença do pepper yellow mosaic virus (PepYMV) e do cucumber mosaic virus (CMV). Pulgões, identificados como Aphis gossypii foram encontrados colonizando pimenteiras-de-cheiro neste campo experimental e podem representar o provável vetor de PepYMV e CMV. Este trabalho relata a primeira ocorrência desses vírus infectando C. chinense no estado do Amazonas.

Cucumber mosaic virus 2b proteins inhibit virus-induced aphid resistance in tobacco.

Cucumber mosaic virus (CMV), which is vectored by aphids, has a tripartite RNA genome encoding five proteins. In tobacco (Nicotiana tabacum), a subgroup IA CMV strain, Fny-CMV, increases plant susceptibility to aphid infestation but a viral mutant unable to express the 2b protein (Fny-CMV∆2b) induces aphid resistance. We hypothesized that in tobacco, one or more of the four other Fny-CMV gene products (the 1a or 2a replication proteins, the movement protein, or the coat protein) are potential aphid resistance elicitors, whilst the 2b protein counteracts induction of aphid resistance. Mutation of the Fny-CMV 2b protein indicated that inhibition of virus-induced resistance to aphids (Myzus persicae) depends on amino acid sequences known to control nucleus-to-cytoplasm shuttling. LS-CMV (subgroup II) also increased susceptibility to aphid infestation but the LS-CMV∆2b mutant did not induce aphid resistance. Using reassortant viruses comprising different combinations of LS and Fny genomic RNAs, we showed that Fny-CMV RNA 1 but not LS-CMV RNA 1 conditions aphid resistance in tobacco, suggesting that the Fny-CMV 1a protein triggers resistance. However, the 2b proteins of both strains suppress aphid resistance, suggesting that the ability of 2b proteins to inhibit aphid resistance is conserved among divergent CMV strains.

Symptom recovery is affected by Cucumber mosaic virus coat protein phosphorylation.

Cucumber mosaic virus induces specific recovery phenotype, namely cyclic mosaic symptoms on tobacco plants. We provide further evidence that besides the 2b suppressor protein, the coat protein (CP) also has a role in symptom recovery and it is connected to its phosphorylation. We analyzed the impact of the phosphorylated (S148D) and the non-phosphorylated (S148A) state of CP148 Ser on symptom formation, virion stability and the effect of CP and its mutants on 2b-mediated local GFP-silencing. We demonstrated that a single aa change could be responsible for preventing the recovery phenomenon as replacing the phosphorylatable Ser with Ala in the 148aa position abolishing the cyclic phenomenon. CP/S148A mutation equilibrates the accumulation of the virus during the infection both at RNA and protein level in N. tabacum L. cv Xanthi plants. In summary, we determined a regulatory effect of the CMV CP on the self-attenuation mechanism and downregulation of the suppressor effect of the 2b protein.

Activation of biochemical factors in CMV-infected tobacco by ningnanmycin.

Cucumber mosaic virus (CMV) is a plant virus with one of the largest host ranges, the widest distribution, and economic importance, and ningnanmycin (NNM) is a commercial antiviral agent. Studies have shown that NNM induces and promotes pathogenesis-related proteins in tobacco mosaic virus-inoculated tobacco. In the present study, the defense enzymes and the biochemical factors of CMV-inoculated tobacco treated with NNM were measured. The biochemical factors of CMV-inoculated tobacco leaves treated with NNM were analyzed. Results showed that the phenylalanine ammonia-lyase, peroxidase, polypheuoloxidase, and superoxide in the CMV-inoculated tobacco leaves treated with NNM were higher than those in non-treated tobacco leaves. Furthermore, NNM activated the oxidation-reduction process, metabolic process, and oxidoreductase activity in the CMV-infected tobacco.

Inhibition of Satellite RNA Associated Cucumber Mosaic Virus Infection by Essential Oil of Micromeria croatica (Pers.) Schott.

The present results dealing with the antiphytoviral activity of essential oil indicate that these plant metabolites can trigger a response to viral infection. The essential oil from Micromeria croatica and the main oil components ß-caryophyllene and caryophyllene oxide were tested for antiphytoviral activity on plants infected with satellite RNA associated cucumber mosaic virus. Simultaneous inoculation of virus with essential oil or with the dominant components of oil, and the treatment of plants prior to virus inoculation, resulted in a reduction of virus infection in the local and systemic host plants. Treatment with essential oil changed the level of alternative oxidase gene expression in infected Arabidopsis plants indicating a connection between the essential oil treatment, aox gene expression and the development of viral infection.

Large-Scale Synonymous Substitutions in Cucumber Mosaic Virus RNA 3 Facilitate Amino Acid Mutations in the Coat Protein.

The effect of large-scale synonymous substitutions in a small icosahedral, single-stranded RNA viral genome on virulence, viral titer, and protein evolution were analyzed. The coat protein (CP) gene of the Fny stain of cucumber mosaic virus (CMV) was modified. We created four CP mutants in which all the codons of nine amino acids in the 5' or 3' half of the CP gene were replaced by either the most frequently or the least frequently used synonymous codons in monocot plants. When the dicot host (Nicotiana benthamiana) was inoculated with these four CP mutants, viral RNA titers in uninoculated symptomatic leaves decreased, while all mutants eventually showed mosaic symptoms similar to those for the wild type. The codon adaptation index of these four CP mutants against dicot genes was similar to those of the wild-type CP gene, indicating that the reduction of viral RNA titer was due to deleterious changes of the secondary structure of RNAs 3 and 4. When two 5' mutants were serially passaged in N. benthamiana, viral RNA titers were rapidly restored but competitive fitness remained decreased. Although no nucleic acid changes were observed in the passaged wild-type CMV, one to three amino acid changes were observed in the synonymously mutated CP of each passaged virus, which were involved in recovery of viral RNA titer of 5' mutants. Thus, we demonstrated that deleterious effects of the large-scale synonymous substitutions in the RNA viral genome facilitated the rapid amino acid mutation(s) in the CP to restore the viral RNA titer.IMPORTANCE Recently, it has been known that synonymous substitutions in RNA virus genes affect viral pathogenicity and competitive fitness by alteration of global or local RNA secondary structure of the viral genome. We confirmed that large-scale synonymous substitutions in the CP gene of CMV resulted in decreased viral RNA titer. Importantly, when viral evolution was stimulated by serial-passage inoculation, viral RNA titer was rapidly restored, concurrent with a few amino acid changes in the CP. This novel finding indicates that the deleterious effects of large-scale nucleic acid mutations on viral RNA secondary structure are readily tolerated by structural changes in the CP, demonstrating a novel part of the adaptive evolution of an RNA viral genome. In addition, our experimental system for serial inoculation of large-scale synonymous mutants could uncover a role for new amino acid residues in the viral protein that have not been observed in the wild-type virus strains.

Epigenetic Changes in the Regulation of Nicotiana tabacum Response to Cucumber Mosaic Virus Infection and Symptom Recovery through Single-Base Resolution Methylomes.

Plants have evolved multiple mechanisms to respond to viral infection. These responses have been studied in detail at the level of host immune response and antiviral RNA silencing (RNAi). However, the possibility of epigenetic reprogramming has not been thoroughly investigated. Here, we identified the role of DNA methylation during viral infection and performed reduced representation bisulfite sequencing (RRBS) on tissues of Cucumber mosaic virus (CMV)-infected Nicotiana tabacum at various developmental stages. Differential methylated regions are enriched with CHH sequence contexts, 80% of which are located on the gene body to regulate gene expression in a temporal style. The methylated genes depressed by methyltransferase inhibition largely overlapped with methylated genes in response to viral invasion. Activation in the argonaute protein and depression in methyl donor synthase revealed the important role of dynamic methylation changes in modulating viral clearance and resistance signaling. Methylation-expression relationships were found to be required for the immune response and cellular components are necessary for the proper defense response to infection and symptom recovery.

Genome-Wide Analysis of DCL, AGO, and RDR Gene Families in Pepper (Capsicum Annuum L.).

RNA silencing is an evolutionarily conserved mechanism that regulates variety of cellular processes in plants. Argonaute protein (AGO), Dicer-like protein (DCL) and RNA-dependent RNA polymerase (RDR) are critical components of RNA silencing. These efficient and indispensable components of the RNAi pathway have not been identified and characterized in pepper. In this study, we identified 12 CaAGO, 4 CaDCL and 6 CaRDR genes in pepper and compared them with those of Arabidopsis, tobacco, potato and tomato. Detailed phylogenetic analyses revealed that each CaAGO, CaDCL and CaRDR protein family were classified into four clades. The tissue specific expression and respond to abiotic or biotic stress were studied. The real-time quantitative polymerase chain reaction (PCR) results demonstrated that CaAGO2, CaAGO10b, CaDCL2 and CaDCL4 were upregulated with cucumber mosaic virus (CMV), potato virus Y (PVY) and tobacco mosaic virus (TMV) infections, whereas they showed difference expression patterns in response to abiotic stress. In addition, we found that many of the candidate genes were induced by phytohormones and H2O2 treatment. Our results provide useful information for further elucidation of gene silencing pathways and RNAi-mediated host immunity in pepper.

Cucumber mosaic virus and its 2b protein alter emission of host volatile organic compounds but not aphid vector settling in tobacco.

BACKGROUND: Aphids, including the generalist herbivore Myzus persicae, transmit cucumber mosaic virus (CMV). CMV (strain Fny) infection affects M. persicae feeding behavior and performance on tobacco (Nicotiana tabacum), Arabidopsis thaliana and cucurbits in varying ways. In Arabidopsis and cucurbits, CMV decreases host quality and inhibits prolonged feeding by aphids, which may enhance virus transmission rates. CMV-infected cucurbits also emit deceptive, aphid-attracting volatiles, which may favor virus acquisition. In contrast, aphids on CMV-infected tobacco (cv. Xanthi) exhibit increased survival and reproduction. This may not increase transmission but might increase virus and vector persistence within plant communities. The CMV 2b counter-defense protein diminishes resistance to aphid infestation in CMV-infected tobacco plants. We hypothesised that in tobacco CMV and its 2b protein might also alter the emission of volatile organic compounds that would influence aphid behavior. RESULTS: Analysis of headspace volatiles emitted from tobacco plants showed that CMV infection both increased the total quantity and altered the blend produced. Furthermore, experiments with a CMV 2b gene deletion mutant (CMV∆2b) showed that the 2b counter-defense protein influences volatile emission. Free choice bioassays were conducted where wingless M. persicae could choose to settle on infected or mock-inoculated plants under a normal day/night regime or in continual darkness. Settling was recorded at 15 min, 1 h and 24 h post-release. Statistical analysis indicated that aphids showed no marked preference to settle on mock-inoculated versus infected plants, except for a marginally greater settlement of aphids on mock-inoculated over CMV-infected plants under normal illumination. CONCLUSIONS: CMV infection of tobacco plants induced quantitative and qualitative changes in host volatile emission and these changes depended in part on the activity of the 2b counter-defense protein. However, CMV-induced alterations in tobacco plant volatile emission did not have marked effects on the settling of aphids on infected versus mock-inoculated plants even though CMV-infected plants are higher quality hosts for M. persicae.

RNA-seq approach to analysis of gene expression profiles in dark green islands and light green tissues of Cucumber mosaic virus-infected Nicotiana tabacum.

Dark green islands (DGIs) surrounded by light green tissues (LGTs) are common leaf symptoms of plants that are systemically infected by various viruses that induce leaf mosaic in infected plants. The inoculation of Cucumber mosaic virus (CMV) in Nicotiana tabacum produced a commonly occurring sequence of classic patterns of DGIs and LGTs. Previous studies confirmed that there are significant differences between DGIs and LGTs in terms of physiology, biochemistry and molecular biology, but the mechanisms by which DGIs form remain unclear. To investigate the global gene expression changes that occur in these special tissues, individual differential gene expression tag libraries were constructed from three total RNA samples isolated from DGIs, LGTs and control plants (CK) and were sequenced using an Illumina HiSeqTM 2000. An analysis of differentially expressed genes (DEGs) and Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis were performed. These analyses revealed the differences between DGIs, LGTs and CK. GO enrichment and KEGG pathway analyses suggested that several pathways related to photosynthesis and chlorophyll metabolism were enriched in DGIs compared to LGTs and CK. Several pathways related to apoptosis were significantly up-regulated in LGTs compared to DGIs. Additionally, we identified sets of DEGs that may be related to the formation or development of DGIs and LGTs. Our systematic analyses provide comprehensive transcriptomic information regarding DGIs and LGTs in CMV-infected N. tabacum. These data will help characterize the detailed mechanisms of DGI and LGT formation.

Prosystemin overexpression induces transcriptional modifications of defense-related and receptor-like kinase genes and reduces the susceptibility to Cucumber mosaic virus and its satellite RNAs in transgenic tomato plants.

Systemin is a plant signal peptide hormone involved in the responses to wounding and insect damage in the Solanaceae family. It works in the same signaling pathway of jasmonic acid (JA) and enhances the expression of proteinase inhibitors. With the aim of studying a role for systemin in plant antiviral responses, a tomato (Solanum lycopersicum) transgenic line overexpressing the prosystemin cDNA, i.e. the systemin precursor, was inoculated with Cucumber mosaic virus (CMV) strain Fny supporting either a necrogenic or a non-necrogenic satellite RNA (satRNA) variant. Transgenic plants showed reduced susceptibility to both CMV/satRNA combinations. While symptoms of the non-necrogenic inoculum were completely suppressed, a delayed onset of lethal disease occurred in about half of plants challenged with the necrogenic inoculum. RT-qPCR analysis showed a correlation between the systemin-mediated reduced susceptibility and the JA biosynthetic and signaling pathways (e.g. transcriptional alteration of lipoxygenase D and proteinase inhibitor II). Moreover, transgenically overexpressed systemin modulated the expression of a selected set of receptor-like protein kinase (RLK) genes, including some playing a known role in plant innate immunity. A significant correlation was found between the expression profiles of some RLKs and the systemin-mediated reduced susceptibility to CMV/satRNA. These results show that systemin can increase plant defenses against CMV/satRNA through transcriptional reprogramming of diverse signaling pathways.

Complete nucleotide sequences and construction of full-length infectious cDNA clones of cucumber green mottle mosaic virus (CGMMV) in a versatile newly developed binary vector including both 35S and T7 promoters.

Seed-transmitted viruses have caused significant damage to watermelon crops in Korea in recent years, with cucumber green mottle mosaic virus (CGMMV) infection widespread as a result of infected seed lots. To determine the likely origin of CGMMV infection, we collected CGMMV isolates from watermelon and melon fields and generated full-length infectious cDNA clones. The full-length cDNAs were cloned into newly constructed binary vector pJY, which includes both the 35S and T7 promoters for versatile usage (agroinfiltration and in vitro RNA transcription) and a modified hepatitis delta virus ribozyme sequence to precisely cleave RNA transcripts at the 3' end of the tobamovirus genome. Three CGMMV isolates (OMpj, Wpj, and Mpj) were separately evaluated for infectivity in Nicotiana benthamiana, demonstrated by either Agroinfiltration or inoculation with in vitro RNA transcripts. CGMMV nucleotide identities to other tobamoviruses were calculated from pairwise alignments using DNAMAN. CGMMV identities were 49.89% to tobacco mosaic virus; 49.85% to pepper mild mottle virus; 50.47% to tomato mosaic virus; 60.9% to zucchini green mottle mosaic virus; and 60.96% to kyuri green mottle mosaic virus, confirming that CGMMV is a distinct species most similar to other cucurbit-infecting tobamoviruses. We further performed phylogenetic analysis to determine relationships of our new Korean CGMMV isolates to previously characterized isolates from Canada, China, India, Israel, Japan, Korea, Russia, Spain, and Taiwan available from NCBI. Analysis of CGMMV amino acid sequences showed three major clades, broadly typified as 'Russian,' 'Israeli,' and 'Asian' groups. All of our new Korean isolates fell within the 'Asian' clade. Neither the 128 nor 186 kDa RdRps of the three new isolates showed any detectable gene silencing suppressor function.