Silencing of a Nicotiana benthamiana ascorbate oxidase gene reveals its involvement in resistance against cucumber mosaic virus

BackgroundAphids, 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.ResultsAnalysis 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.ConclusionsCMV 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.

Phlox paniculata L., a perennial plant from the family Polemoniaceae, is cultivated as an ornamental in gardens and for cut-flower production. In spring 2003, two types of symptoms were observed in P. paniculata plants grown for cut flowers on a farm in the Var department, France. Some plants showed a mild leaf mosaic while others showed leaf browning and delayed growth. In plants showing mild mosaic, Cucumber mosaic virus (CMV) was detected on the basis of the symptoms exhibited by a range of inoculated plants, the observation of isometric particles (approximately 30 nm) with the electron microscope in crude sap preparations from the infected plants, and the positive reaction in double-antibody sandwich (DAS)-ELISA to polyclonal antibodies raised against CMV (1). In double-immunodiffusion analysis, the five tested isolates were shown to belong to group II of CMV strains. To determine if CMV was responsible for the symptoms observed, one isolate was multiplied in Nicotiana tabacum cv. Xanthi-nc plants after isolation from local lesions on Vigna unguiculata and mechanically inoculated to 12 1-year-old P. paniculata plants. At 3 months post inoculation (mpi), all plants showed mild mosaic and CMV was detected by DAS-ELISA. In sap preparations from P. paniculata plants showing leaf browning symptoms, rod-shaped particles with two distinct sizes of 190 to 210 and 70 to 90 nm long, typical of those associated with tobraviruses, were revealed using electron microscopy. Local lesions typical of Tobacco rattle virus (TRV) were observed after inoculation of N. tabacum cv. Xanthi-nc, Chenopodium amaranticolor, and C. quinoa. Total nucleic acid preparations were prepared from symptomatic plants, and amplicons of the expected size (463 bp) were generated by reverse-transcription (RT)-PCR using primers specific to TRV RNA 1 (4). The nucleotide sequence of one amplicon was 93.6% identical to the sequence of a reference TRV isolate (GenBank Accession No. AJ586803). Twelve 1-year-old P. paniculata plants were mechanically inoculated with an extract of infected tissues from one symptomatic P. paniculata plant. TRV was detected 2 to 6 mpi in apical leaves of all inoculated plants by RT-PCR, although the plants did not express symptoms. Since no other pathogens were detected in the source plants, it is plausible that the lack of symptoms in back-inoculated plants is either due to a long incubation period or an interaction with particular environmental factors such as cold conditions. The survey of approximately 200 plants revealed that approximately 7, 10, and 1% were infected by TRV, CMV, or by both viruses, respectively. CMV and TRV were previously detected in P. paniculata in Latvian SSR and in Lithuania (2,3). These results show that sanitary selection of P. paniculata prior to vegetative propagation should include a screening for TRV and CMV infections.

The current study focuses on the effects of Cucumber mosaic virus (CMV) infection on phytochemical changes and pathogenesis- and phenylpropanoid pathway-associated gene activities in squash (Cucurbita pepo L.) plants during a time course of 2 to 12 days post inoculation (dpi). The identity of the CMV isolate was confirmed by DAS-ELISA, TEM, and coat protein gene sequence. The CMV infection initially boosts and then suppresses transcript levels of the defense-related genes PR-1, PR-2, PAL, HQT, and CHS during the investigated time course compared to controls. The expression profile during the time-course study indicated that early, transient induction of PR-1 occurs during CMV infection, while CMV induced the expression of PR-2 in systemically infected squash tissues at all time points and suppressed the expression of PAL and HQT at 8-12 dpi. CHS transcript levels fluctuated between up- and down-regulation, but by 12 dpi, CHS expression reached its peak. The HPLC and GC–MS analyses of CMV-infected squash extracts revealed that different phenolic, flavonoid, and fatty acid compounds could be induced or suppressed upon CMV infection. In particular, CMV could suppress the synthesis of most phenolic compounds, specifically chlorogenic acid, possibly leading to the virus’s rapid spread.

Elimination of mixed infection of Cucumber mosaic and Tomato aspermy virus from Chrysanthemum morifolium Ramat. cv. Pooja by shoot meristem culture

ABSTRACTMore than 100 alkaloids have been found in periwinkle, of which vincristine and vinblastine are the most notable for the treatment of diseases such as leukaemia. In this study, characterization of naturally occurring Cucumber mosaic virus (CMV) infection showed mosaic, leaf deformation and stunting of plants. Viral identification was confirmed by double-antibody sandwich enzyme-linked immunosorbent assay (DAS-ELISA) with a specific CMV polyclonal antibody and reverse transcription PCR with a specific primer for the CMV-RdRp-gene, which yielded a 513 base pair DNA fragment. The effect of CMV infection on the antineoplastic alkaloids in periwinkle leaves was determined. We also studied the effect of using plant-growth-promoting rhizobacteria (PGPR) isolates against virus infection to stimulate resistance induction in host plants. Bacillus subtilis 281 and B. pumilus 293 were examined individually (B1 and B2) and in a mixture (B1&B2) for their effectiveness against infection with CMV. The results of greenhouse experiments were confirmed by artificial mechanical inoculation. The PGPR strain treatments and results were reinforced by analysing the protein patterns as well as determining the total phenol, total flavonoid and total alkaloid contents. PGPR treatment evidently lowered the virus concentrations, the percentage of infected plants and the disease severity compared with healthy and infected controls. Seedlings treated with the B1&B2 strain mixture yielded significantly lower levels of virus infection than B1 or B2 individually in all experiments compared to controls. Our findings demonstrate the possibility of using selected Bacillus spp. strains to induce systemic resistance for CMV infection control.

Transcription factors (TFs) play an important role in plant development; however, their role during viral infection largely remains unknown. The present study was designed to uncover the role transcription factors play in Cucumber mosaic virus (CMV) infection. During the screening of an Arabidopsis thaliana (Col-0) transcription factor library, using the CMV 2b protein as bait in the yeast two-hybrid system, the 2b protein interacted with Homeobox protein 27 (HB27). HB27 belongs to the zinc finger homeodomain family and is known to have a regulatory role in flower development, and responses to biotic and abiotic stress. The interaction between CMV 2b and HB27 proteins was further validated using in planta (bimolecular fluorescence complementation assay) and in vitro far-Western blotting (FWB) methods. In the bimolecular fluorescence complementation assay, these proteins reconstituted YFP fluorescence in the nucleus and the cytoplasmic region as small fluorescent dots. In FWB, positive interaction was detected using bait anti-MYC antibody on the target HB27-HA protein. During CMV infection, upregulation (~3-fold) of the HB27 transcript was observed at 14 days post-infection (dpi) in A. thaliana plants, and expression declined to the same as healthy plants at 21 dpi. To understand the role of the HB27 protein during CMV infection, virus accumulation was determined in HB27-overexpressing (HB27 OE) and knockout mutants. In HB27-overexpressing lines, infected plants developed mild symptoms, accumulating a lower virus titer at 21 dpi compared to wild-type plants. Additionally, knockout HB27 mutants had more severe symptoms and a higher viral accumulation than wild-type plants. These results indicate that HB27 plays an important role in the regulation of plant defense against plant virus infection.

Plant virus-based gene-silencing vectors have been extensively and successfully used to elucidate functional genomics in plants. However, only limited virus-induced gene-silencing (VIGS) vectors can be used in both monocot and dicot plants. Here, we established a dual gene-silencing vector system based on Bamboo mosaic virus (BaMV) and its satellite RNA (satBaMV). Both BaMV and satBaMV vectors could effectively silence endogenous genes in Nicotiana benthamiana and Brachypodium distachyon. The satBaMV vector could also silence the green fluorescent protein (GFP) transgene in GFP transgenic N.benthamiana. GFP transgenic plants co-agro-inoculated with BaMV and satBaMV vectors carrying sulphur and GFP genes, respectively, could simultaneously silence both genes. Moreover, the silenced plants could still survive with the silencing of genes essential for plant development such as heat-shock protein 90 (Hsp90) and Hsp70. In addition, the satBaMV- but not BaMV-based vector could enhance gene-silencing efficiency in newly emerging leaves of N.benthamiana deficient in RNA-dependant RNA polymerase 6. The dual gene-silencing vector system of BaMV and satBaMV provides a novel tool for comparative functional studies in monocot and dicot plants.

To protect against infection, plants have evolved multi-layered defense systems such as RNA silencing, salicylic acid (SA)-mediated resistance, resistance (R) protein-conferred resistance and defense priming. Antiviral defense systems seem to act in a coordinated manner in response to viral infection, but the underlying mechanisms are unknown except in a limited number of specific virus–plant combinations. Cucumber mosaic virus (CMV) infection of Arabidopsis thaliana is a well-known sophisticated pathosystem often used to investigate antiviral defense reactions. On the basis of extensive studies on the interaction between CMV and A. thaliana, RNA silencing plays a key role in suppressing CMV infection in the host plant. However, several strains of CMV are able to overcome the RNA silencing system by producing an RNA silencing suppressor protein. If the virulent CMV strain is indeed able to overcome basal antiviral RNA silencing, host plants carrying the R gene exhibit a resistance response, generally accompanied by necrotic local lesions at the primary infection sites. RCY1, which encodes an NB-LRR class R protein, was isolated from A. thaliana and identified as the first R gene conferring resistance to CMV. SA-dependent and -independent signaling pathways also seem to be associated with CMV resistance, while in A. thaliana, resistance induced by a plant activator treatment is thought to activate defense priming against a broad range of pathogens. In this review, we summarize current knowledge on the multiple defense systems of A. thaliana against CMV infection.

Eukaryotic translation elongation factors are implicated in protein synthesis across different living organisms, but their biological functions in the pathogenesis of cucumber mosaic virus (CMV) and tobacco rattle virus (TRV) infections are poorly understood. Here, we isolated and characterized a cDNA clone, LreEF1A4, encoding the alpha subunit of elongation factor 1, from a CMV-elicited suppression subtractive hybridization library of Lilium regale. The infection tests using CMV remarkably increased transcript abundance of LreEF1A4; however, it also led to inconsistent expression profiles of three other LreEF1A homologs (LreEF1A1–3). Protein modelling analysis revealed that the amino acid substitutions among four LreEF1As may not affect their enzymatic functions. LreEF1A4 was ectopically overexpressed in petunia (Petunia hybrida), and transgenic plants exhibited delayed leaf and flower senescence, concomitant with increased transcription of photosynthesis-related genes and reduced expression of senescence-associated genes, respectively. A compromised resistance to CMV and TRV infections was found in transgenic petunia plants overexpressing LreEF1A4, whereas its overexpression resulted in an enhanced tolerance to salt and drought stresses. Taken together, our data demonstrate that LreEF1A4 functions as a positive regulator in viral multiplication and plant adaption to high salinity and dehydration.

SUMMARYFour field trials were done with narrow‐leafed lupins (Lupinus angustifolius) in 1988 ‐ 1989, to examine the effect of sowing seed with 5% and 0.5% cucumber mosaic virus (CMV) infection on subsequent virus spread, grain yield and percentage of infection in harvested seed. A proportion of the CM V‐infected seed failed to produce established plants and thus, plots sown with 5% and 0.5% infected seed contained 1.5‐2.9% and 0.2‐0.3% of seed‐infected plants respectively. The rate of virus spread by aphids was faster and resulted in more extensive infection at maturity in plots sown with 5% infected seed than with 0.5% infected seed. In three trials, sowing 5% infected seed resulted in yield losses of 34 ‐ 53% and CMV infection in the seed harvested of 6 ‐ 13%. The spread of CMV infection resulting from sowing 0.5% infected seed did not significantly decrease yield. However, late CMV spread in these plots caused > 1% seed infection. In the fourth trial, which was badly affected by drought, CMV spread only slowly, there was no significant effect of CMV on grain yield and the percentage of infected seed harvested was 3–5 times less than that in the seed sown.When CMV‐infected seed was sown at different depths, target depths of 8 and 11 cm decreased the incidence of seed‐infected plants by c. 15% and c. 50% respectively compared with sowing at 5 cm. However, in glasshouse tests, treatment with the pre‐emergence herbicide simazine failed to selectively cull out seed‐infected plants.The field trials were colonised by green peach (Myzus persicae), blue‐green (Acyrthosiphon kondoi) and cowpea (Aphis craccivora) aphids. When the abilities of these aphid species and of the turnip aphid (Lipaphis erysimi) in transmitting CMV from lupins to lupins were examined in glasshouse tests, short acquisition access times favoured transmission. With 5–10 min acquisition access times, overall transmission efficiencies were 10.8%, 9.4%, 6.1% and 3.9% for the green peach, cowpea, blue‐green and turnip aphids respectively.

This study was conducted to determine the effect of cucumber mosaic virus (CMV) infection on the levels of some mineral elements and plant hormones in some melon genotypes. All experiments mentioned in this research were conducted in the Laboratory of plant virology and Greenhouses belonging to the Plant Protection Department/ College of the Agriculture/ University of Kerbala. PCR amplification using the complementary DNA (cDNA), synthesized from RNA isolated from an infected plant, produced a 650bp PCR product. A BLAST search using the nucleotide sequence of the PCR product demonstrated that this CMV isolate was previously registered at the National Centre for Biotechnology Information (NCBI) with a similarity of 100% with some CMV isolates identified in Hungary (AJ517802), Australia (U22821), and Slovenia (OL142046). It was also found that all melon genotypes evaluated in this study were susceptible to CMV, and the genotypes HA-144 and HA-609 were the most susceptible to CMV among the other tested genotypes. Results also showed that the CMV infection significantly reduced the levels of some minerals (calcium, magnesium, and manganese), and the genotypes HA-144, GM-3034, and GA-1534 were the most affected by the virus.; whereas the mineral elements sodium and potassium were significantly increased in their levels in infected plants, especially in the genotype HA-609. The CMV infection also clearly reduced the level of the hormone gibberellin in genotypes and the GA-1534 genotype was the most impacted and significantly different from their levels in the uninfected plants. However, the CMVinfected genotypes showed a significant increase in the level of the hormones cytokinin.

Cucumber mosaic virus (CMV) is vectored by aphids. Infection of Arabidopsis thaliana plants with CMV affects their attractiveness to aphids (Myzus persicae) and the performance of aphids confined on these plants. CMV-induced changes in plant-aphid interactions ('viral manipulation') may promote transmission. M. persicae, an efficient CMV vector is a 'generalist', i.e., it has many plant hosts. A. thaliana is also exploited by crucifer-specialist aphids including Lipaphis erysimi (an efficient CMV vector) and Brevicoryne brassicae (a poor CMV vector). We explored the hypothesis that CMV-induced viral manipulation of aphid behaviour would exert stronger effects on M. persicae than on crucifer-specialists. M. persicae, B. brassicae and L. erysimi were released in microcosms and allowed to choose to settle on either CMV-infected or mock-inoculated plants. Initial experiments showed that as systemic CMV infection developed in A. thaliana, aphids of M. persicae were decreasingly likely to settle on infected plants. In subsequent experiments, using plants at 14 days post-infection, it was found that aphids of M. persicae were faster to choose between infected and uninfected plants than specialist aphids, but that both the generalist and specialists were less likely to settle on CMV-infected plants. Olfactometry showed that volatiles emitted by CMV-infected plants attracted M. persicae, and although the specialists showed no significant preferences, greater numbers of aphids of all three species responded when CMV-infected plant volatiles were presented to them. As CMV infection develops, A. thaliana becomes less susceptible to aphid colonisation, however, plants continue to emit attractive olfactory cues. This is consistent with a model in which aphids are attracted to infected plants but discouraged from settling (e.g., by gustatory cues), which encourages aphids to carry CMV to non-infected plants. CMV appears to be more successful in manipulating the interactions of A. thaliana with the generalist aphid M. persicae, than with the crucifer specialists B. brassicae or L. erysimi.

Gerbera spp., which is in the Asteraceae family, is the most widely cultivated cut flower ornamental plant in Türkiye after carnation. Approximately 74% of Türkiye's production is met from Antalya province. Virus diseases are a problem in the cultivation of Gerbera as they significantly reduce the yield and quality. In this study, which was carried out to detect some virus diseases seen in Gerbera production areas in Antalya province, surveys were carried out in commercial production areas in the 2021-2022 production season. For this purpose, a total of 112 leaf samples, which are thought to be infected with viruses, were collected from 100 Gerbera plants showing symptoms similar to virus disease symptoms and 12 selected weeds regardless of the presence of symptoms. These leaf samples, which were later brought to the laboratory, then were tested by the Double Antibody Sandwich Enzyme-linked Immunosorbent Assay (DAS-ELISA) method to detect infections of Tomato spotted wilt virus (TSWV), Cucumber mosaic virus (CMV), Tobacco streak virus (TSV) and Impatiens necrotic spot virus (INSV). As a result of the tests, TSWV (41.9%), CMV (1.7%) and TSV (37.5%) infections were detected in Gerbera plant samples. In addition, TSWV (25%) infections were most common in weed samples, followed by TSV (16%) and CMV (8.3%) infections. The presence of INSV virus was not detected in any of the leaf samples tested by DAS-ELISA. With this study, TSWV, TSV and CMV infections in Gerbera production areas in Türkiye were revealed for the first time.

Viral symptoms are frequently observed in production fields of ranunculus (Ranunculus asiaticus L.) in Japan. Based on incidence of diseases caused by a large number of ranunculus-infective viruses, ranunculus mild mosaic virus (RanMMV), tomato spotted wilt virus (TSWV) and cucumber mosaic virus (CMV) infections were the focus of an epidemiological field survey in Japan. To efficiently investigate the incidence and distribution of the three viruses, we first developed a new multiplex reverse transcription-polymerase chain reaction method that enables simultaneous detection of RanMMV, TSWV, and CMV in ranunculus. A field survey of virus infections in ranunculus production fields in Tohoku and Kyushu regions revealed that the infection rate of RanMMV was much higher than that of the other viruses in all fields sampled. Interestingly, the infection rate of RanMMV showed an increasing trend in proportion to the number of vegetative propagation cycles of ranunculus, implying virus transmission by aphids. Taken together, this method was proven to be effective for simultaneous detection of RanMMV, TSWV and CMV in ranunculus plants, and RanMMV was recognized as one of the most prevalent plant viruses in ranunculus fields in Japan.

Paeonia officinalis (family Paeoniaceae), known as the garden peony, is a very popular flowering plant with large, showy flowers grown in many gardens in Serbia. In May 2021, peony plants showing chlorotic ringspot and severe mosaic of leaves were observed in a private garden in Zemun (District of Belgrade, Serbia). Symptomatic leaves from two plants were collected and analysed for the presence of cucumber mosaic virus (CMV), tobacco rattle virus (TRV), alfalfa mosaic virus (AMV) and tomato spotted wilt virus (TSWV) using commercial ELISA kits. CMV was detected serologically in both peony samples and no other plant virus was identified. The causal agents from both ELISA-positive samples were successfully mechanically transmissible to Chenopodium quinoa and Nicotiana glutinosa plants. CMV infection in symptomatic garden peony plants was also confirmed by RT-PCR with CMV-specific primers amplifying the complete coat protein (CP) gene and parts of the 3′- and 5′-UTRs. Selected ELISA-positive sample (318-21) was Sanger sequenced using the same primer as in RT-PCR and phylogenetic tree based on complete CP sequences showed that Serbian CMV isolate from garden peony belongs to the CMV subgroup IA.