Multiplication Of Tobacco Mosaic Virus Research Articles

Genome-wide analysis of tobacco NtTOM1/TOM3 gene family and identification of NtTOM1a/ NtTOM3a response to tobacco mosaic virus

BackgroundTOBAMOVIRUS MULTIPLICATION 1 (TOM1) and its homolog TOBAMOVIRUS MULTIPLICATION 3 (TOM3) play a prominent role in the multiplication of tobacco mosaic virus (TMV) in higher plants. Although homologs of NtTOM1/TOM3 genes have been identified in several plant species, little is known about the characteristics and functions of NtTOM1/TOM3 at the genome-wide level in tobacco (Nicotiana tabacum L.).ResultsIn this study, we performed genome-wide identification and expression pattern analysis of the tobacco NtTOM1/TOM3 gene family. Twelve NtTOM1/TOM3 genes were identified and classified into four groups based on phylogenetic analysis. Sequence and conserved domain analyses showed that all these genes contained a specific DUF1084 domain. Expression pattern analysis showed that NtTOM1a, NtTOM1b, NtTOM1d, NtTOM3a, NtTOM3b, and NtTOM3d were induced by TMV at 1-, 3-, and 9 dpi, whereas the expression of other genes was not responsive to TMV at the early infection stage. TMV virion accumulation showed no obvious difference in either nttom1a or nttom3a mutants compared with the wild type. However, the virus propagation was significantly, but not completely, inhibited in the nttom1atom3a double mutant, indicating that other gene family members may function redundantly, such as NtTOM1b and NtTOM1d. In addition, overexpression of NtTOM1a or NtTOM3a also inhibited the TMV replication to some extent.ConclusionsThe present study performed genome-wide analysis of the NtTOM1/TOM3 gene family in tobacco, and identified NtTOM1a and NtTOM3a as important genes involved in TMV multiplication based on functional analysis. These results provide a theoretical basis for further improving TMV resistance in tobacco.

TOM1 family conservation within the plant kingdom for tobacco mosaic virus accumulation.

The susceptibility factor TOBAMOVIRUS MULTIPLICATION 1 (TOM1) is required for efficient multiplication of tobacco mosaic virus (TMV). Although some phylogenetic and functional analyses of the TOM1 family members have been conducted, a comprehensive analysis of the TOM1 homologues based on phylogeny from the most ancient to the youngest representatives within the plant kingdom, analysis of support for tobamovirus accumulation and interaction with other host and viral proteins has not been reported. In this study, using Nicotiana benthamiana and TMV as a model system, we functionally characterized the TOM1 homologues from N. benthamiana and other plant species from different plant lineages. We modified a multiplex genome editing tool and generated a sextuple mutant in which TMV multiplication was dramatically inhibited. We showed that TOM1 homologues from N. benthamiana exhibited variable capacities to support TMV multiplication. Evolutionary analysis revealed that the TOM1 family is restricted to the plant kingdom and probably originated in the Chlorophyta division, suggesting an ancient origin of the TOM1 family. We found that the TOM1 family acquired the ability to promote TMV multiplication after the divergence of moss and spikemoss. Moreover, the capacity of TOM1 orthologues from different plant species to promote TMV multiplication and the interactions between TOM1 and TOM2A and between TOM1 and TMV-encoded replication proteins are highly conserved, suggesting a conserved nature of the TOM2A-TOM1-TMV Hel module in promoting TMV multiplication. Our study not only revealed a conserved nature of a gene module to promote tobamovirus multiplication, but also provides a valuable strategy for TMV-resistant crop development.

Editing of TOM1 gene in tobacco using CRISPR/Cas9 confers resistance to Tobacco mosaic virus

Genome editing technology has become one of the excellent tools for precise plant breeding to develop novel plant germplasm. The Tobacco mosaic virus (TMV) is the most prominent pathogen that infects several Solanaceae plants, such as tobacco, tomato, and capsicum, which requires critical host factors for infection and replication of its genomic RNA in the host. The Tobamovirus multiplication (TOM) genes, such as TOM1, TOM2A, TOM2B, and TOM3, are involved in the multiplication of Tobamoviruses. TOM1 is a transmembrane protein necessary for efficient TMV multiplication in several plant species. The TOM genes are crucial recessive resistance genes that act against the tobamoviruses in various plant species. The single guided RNA (sgRNA) was designed to target the first exon of the NtTOM1 gene and cloned into the pHSE401 vector. The pHSE401-NtTOM1 vector was introduced into Agrobacterium tumefaciens strain LBA4404 and then transformed into tobacco plants. The analysis on T0 transgenic plants showed the presence of the hptII and Cas9 transgenes. The sequence analysis of the NtTOM1 from T0 plants showed the indels. Genotypic evaluation of the NtTOM1 mutant lines displayed the stable inheritance of the mutations in the subsequent generations of tobacco plants. The NtTOM1 mutant lines successfully conferred resistance to TMV. CRISPR/Cas genome editing is a reliable tool for investigating gene function and precision breeding across different plant species, especially the species in the Solanaceae family.

Inhibitory effect of protein Y3 from Coprinus comatus on tobacco mosaic virus

The antiviral protein Y3 produced by the edible mushroom Coprinus comatus disrupts the tobacco mosaic virus (TMV) and inhibits the multiplication of TMV in Nicotiana tabacum; however, the detailed mechanism of its activity remains unclear. In this study, Y3 was demonstrated to interact with TMV coat protein (TMV-CP) in vitro as well as in tobacco plants by using a yeast two-hybrid system and bimolecular fluorescence complementation (BiFC). To explore the interaction site between Y3 and TMV-CP, the phenylalanine (Phe) at the 43rd and arginine (Arg) at the 55th amino acid of Y3 were individually converted to cysteine (Cys) and serine (Ser), named Y3T1 and Y3T2, respectively, and were then used in BiFC assays. Based on the information obtained about disulfide bonds in the protein structure, the two mutations were predicted to change the protein's disulfide bonds. The results showed Y3T1 lost the ability to interact with TMV-CP, suggesting that a specific Phe of Y3 is critical for the interaction between Y3 and CP in plants. Furthermore, a prokaryotic expression system was used to test the antiviral activities of protein Y3 (PY3) and two other mutated proteins (P-Y3T1, P-Y3T2). The results showed that recombinant protein P-Y3 had a slightly lower inhibitory effect against TMV than Y3 extracted directly from mushrooms; further, P-Y3T1 decreased antiviral activity in the tobacco plant significantly compared with P-Y3, suggesting that the anti-TMV effect of Y3 was directly related to the Y3-CP interaction. In contrast, P-Y3T2 was able to still interact with TMV-CP in the tobacco plant, and it increased the ability of the plant to resist TMV compared with PY3, indicating that PY3-T2 could be a candidate peptide for plant protection against TMV and that Y3 may have other inhibitory mechanisms against TMV in addition to its interaction with TMV-CP.

Azelaic acid accumulates in phloem exudates of TMV-infected tobacco leaves, but its application does not induce local or systemic resistance against selected viral and bacterial pathogens

Local and systemic effects of azelaic acid (AzA) pretreatments on local necrotic lesion formation induced by Tobacco mosaic virus (TMV) were studied in Nicotiana tabacum cv. Xanthi nc plants as measured by a semi-automated, computer-assisted method. Local application of AzA (0.2–1.0 mM) showed no or limited influence (either increase or decrease) on lesion size of TMV-inoculated leaves. In addition, AzA pretreatment did not modify the multiplication of TMV detected by semiquantitative RT-PCR. No significant systemic effect of AzA on lesion size of TMV was detectable in distant leaves. Moreover, AzA treatment had no considerable local and systemic effect on symptom expression and multiplication of incompatible (P. syringae pv. tomato DC3000) and compatible (P. syringae pv. tabaci) bacteria. The response of the viral and bacterial pathogens to AzA was not different if tobacco plants were exposed to light or darkness after AzA treatment. However, the amount of AzA, measured by HPLC–MS, was doubled in phloem exudates of TMV infected leaves as compared to the control leaves, but concentrated exudates containing AzA did not induce local resistance response. In addition, the amounts of two other short-chain dicarboxylic acids (suberic acid and sebacic acid) increased in phloem exudates of virus-infected leaves. All these results suggest that, at least in tobacco, AzA does not induce considerable local or systemic effects on viral and bacterial infections. Therefore, its formerly reported role in signal transduction and/or induction of systemic acquired resistance (SAR) in Arabidopsis cannot be confirmed in tobacco.

Inhibitory effect of sulfated lentinan and lentinan against tobacco mosaic virus (TMV) in tobacco seedlings

The antiviral activities of sulfated lentinan (sLNT) and lentinan (LNT) against tobacco mosaic virus (TMV) in tobacco seedlings and the underlying mechanism were investigated. Compared with LNT, sLNT showed significantly higher inhibitory effects on viral infection and TMV multiplication in a dose-dependent way, which might be due to its binding with TMV coat protein. In addition, both sLNT and LNT induced the transient production of H2O2 and expression of some defense-related genes (stilbene synthase, glucanase, acidic chitinase class IV, phenylalanine ammonia-lyase and 5-epi-aristolochene synthase) both locally and systemically. These results suggested that sLNT and LNT could control TMV incidence and the action mechanism might be associated with the affinity towards TMV coat protein and activation of some defense genes.

Inhibitory effect of esterified lactoferin and lactoferin against tobacco mosaic virus (TMV) in tobacco seedlings

The inhibitory effects of esterified lactoferrin (ELF) and lactoferrin (LF) against tobacco mosaic virus (TMV) in tobacco seedlings and the underlying mechanism were investigated. ELF and LF significantly inhibited viral infection and TMV multiplication in tobacco plants. ELF showed a higher inhibition effect against TMV than LF treatment in a dose and time-dependent way. Moreover, ELF induced a higher increase in the levels of transcription of pathogenesis-related (PR) protein genes [acidic PRs (PR-1a, PR-2, PR-3, PR-5) and basic PR-1] and defense-related enzymes [phenylalanine ammonia lyase (PAL, EC 4.3.1.5), and 5-epi-aristolochene synthase (EAS, EC 2.5.1.35)] both locally and systemically, in correlation with the induction of resistance against tobacco mosaic virus. Furthermore, ELF also induced accumulation of salicylic acid, SA 2-O-β-d-glucoside and H2O2. These results suggested that ELF and LF could control TMV incidence and the mechanism might attribute to activate the expression of a number of defense genes.

New asperxanthone and asperbiphenyl from the marine fungus Aspergillus sp.

Marine fungi are of great importance as potential sources of agricultural pesticide leads. To continue work on new and biologically active metabolites from marine fungi, culture extracts of selected isolates were screened for inhibitory activity against tobacco mosaic virus (TMV), a typical member of the tobamovirus group of plant viruses. A new difuranxanthone, asperxanthone (1), and a new biphenyl, asperbiphenyl (2), as well as seven known unsaturated fatty acid glycerol esters, were isolated from a selected marine fungus, identified as Aspergillus sp. (MF-93), collected in the Quan-Zhou Gulf. Two-dimensional NMR methods and FAB-MS established the structures of the new compounds. Among the compounds, 1 and 2 showed moderate activity (inhibitory rates 62.9 and 35.5% respectively) in inhibiting multiplication of TMV, higher than that of the other seven known lipids but much lower than that of the extract at the identical concentration of 0.2 mg mL(-1). Two new compounds, asperxanthone and asperbiphenyl, were identified as active components from Aspergillus sp. (MF-93).

Decreased level of ferredoxin I in Tobacco mosaic virus-infected tobacco is associated with development of the mosaic symptom

A decrease of ferredoxin I (Fd I) level was detected in the Tobacco mosaic virus (TMV)-infected tobacco ( Nicotiana tabacum) leaves and the extent of decrease was uneven in the leaves showing mosaic symptoms. The Fd I level in the dark-green regions which contain less TMV was higher than that in the chlorotic regions of the same leaf, but was lower than that in the healthy control plants. The N. benthamiana plants with the Fed-1 gene being silenced by a chimeric viral vector showed chlorotic and yellowish phenotype in which H 2O 2 accumulated to a high level. In antisense Fed-1 transgenic N. benthamiana plants, the multiplication of TMV in infected plants and the accumulation of the viral coat protein in the chloroplasts were enhanced by the decrease of Fd I. Taken together, these results suggest that the unevenly decreased level of Fd I in TMV-infected tobacco leaves may contribute to the expression of chlorosis and mosaic symptoms.

Purification and Characterization of a New Ribosome Inactivating Protein from Cinchonaglycoside C‐treated Tobacco Leaves

Abstract A new ribosome‐inactivating protein (RIP) with a molecular weight of 31 kDa induced by Cinchonaglycoside C (1) designated CIP31, was isolated from tobacco leaves. Analysis of this protein sequence indicated that it belongs to the RIP family and it was distinct from the other plant RIPs reported previously at its N‐terminal amino acid sequence. CIP31 can directly impair synthesis of coat protein (CP) of tobacco mosaic virus (TMV), which resulted in inhibition of TMV long distance movement and multiplication in tobacco plants at concentrations of ng/mL. Furthermore, no toxicity was shown to the growth and fertility of the plants. CIP31 was synthesized only in the presence of Cinchonaglycoside C (1) and was independent of the salicylic acid (SA) signal pathway. We provided evidence for the SA‐independent biological induction of resistance.

A TOM1 homologue is required for multiplication of Tobacco mosaic virus in Nicotiana benthamiana

The AtTOM1 gene of Arabidopsis thaliana had been shown to be essential for the efficient multiplication of Tobacco mosaic virus (TMV) in A. thaliana. In this study, we cloned an AtTOM1-like gene from Nicotiana benthamiana named as NbTOM1. Sequence alignment showed that NbTOM1 is closely related to AtTOM1 homologues of N. tabacum and Lycopersicon esculentum with 97.2% and 92.6% nucleotide sequence identities, respectively. Silencing of NbTOM1 by a modified viral satellite DNA-based vector resulted in complete inhibition of the multiplication of TMV in N. benthamiana. The result suggests that inhibition of NbTOM1 via RNA silencing is a potentially useful method for generating TMV-resistant plants.

RDR6 Has a Broad-Spectrum but Temperature-Dependent Antiviral Defense Role in Nicotiana benthamiana

SDE1/SGS2/RDR6, a putative RNA-dependent RNA polymerase (RdRP) from Arabidopsis thaliana, has previously been found to be indispensable for maintaining the posttranscriptional silencing of transgenes, but it is seemingly redundant for antiviral defense. To elucidate the antiviral role of this RdRP in a different host plant and to evaluate whether plant growth conditions affect its role, we down-regulated expression of the Nicotiana benthamiana homolog, NbRDR6, and examined the plants for altered susceptibility to various viruses at different growth temperatures. The results we describe here clearly show that plants with reduced expression of NbRDR6 were more susceptible to all viruses tested and that this effect was more pronounced at higher growth temperatures. Diminished expression of NbRDR6 also permitted efficient multiplication of tobacco mosaic virus in the shoot apices, leading to serious disruption with microRNA-mediated developmental regulation. Based on these results, we propose that NbRDR6 participates in the antiviral RNA silencing pathway that is stimulated by rising temperatures but suppressed by virus-encoded silencing suppressors. The relative strengths of these two factors, along with other plant defense components, critically influence the outcome of virus infections.

Isolation of pathogenesis-related proteins from TMV-infected tobacco and their influence on infectivity of TMV

The composition of pathogenesis-related proteins (PR-proteins) in the intercellular fluid (ICF) and leaf tissue of the hypersensitive tobacco cultivar Xanthi-nc inoculated with Tobacco mosaic virus (TMV), and their inhibitory influence on TMV multiplication were studied. The ICF PR-proteins of infected plants were separated after solubilisation by decreasing gradient of ammonium sulphate, the cell PR-proteins were separated after acidic homogenisation of leaf tissues. The ICF and cell PR-proteins were further purified by ion exchange chromatography on DEAE cellulose. Using discontinuous non-denaturating polyacrylamide gel electrophoresis of DEAE cellulose fractions the PR-proteins were detected. Their molecular weights were estimated by SDS-PAGE. The ICF and cell proteins of infected leaves included PR-proteins of the molecular weights 15–16 kDa (Group 1), 27–28 kDa (Group 3: chitinases) and 36–40 kDa (Group 2a: &#1704-1,3-glucanases). Fractions with different PR-proteins were tested for their effect on infectivity of TMV. Particularly the PR3 and PR2a proteins seem to decrease the infectivity of TMV.

Pokeweed Antiviral Protein Inhibits Brome Mosaic Virus Replication in Plant Cells

Pokeweed antiviral protein (PAP) is a ribosome-inactivating protein isolated from the pokeweed plant (Phytolacca americana) that inhibits the proliferation of several plant and animal viruses. We have shown previously that PAP and nontoxic mutants of PAP can directly depurinate brome mosaic virus (BMV) RNA in vitro, resulting in reduced viral protein translation. Here we expand on these initial studies and, using a barley protoplast system, demonstrate that recombinant PAP and nontoxic mutants isolated from E. coli are able to reduce the accumulation of BMV RNAs in vivo. Pretreatment of only BMV RNA3 with PAP prior to transfection of barley protoplasts reduced the accumulation of all BMV RNAs, with a more severe effect on subgenomic RNA4 levels. Using in vitro RNA synthesis assays, we show that a depurinated template causes the BMV replicase to stall at the template nucleotide adjacent to the missing base. These results provide new insight into the antiviral mechanism of PAP, namely that PAP depurination of BMV RNA impedes both RNA replication and subgenomic RNA transcription. These novel activities are distinct from the PAP-induced reduction of viral RNA translation and represent new targets for the inhibition of viral infection.

Effect of κ/ß-Carrageenan from red alga Tichocarpus crinitus (Tichocarpaceae) on infection of detached tobacco leaves with tobacco mosaic virus

It is shown that κ/s-carrageenan obtained from red marine alga Tichocarpus crinitus possesses antiviral activity against Tobacco mosaic virus (TMV). Two days after inoculation of Nicotiana tabacum L. cv. ‘Samsun’ detached leaves with a mixture of TMV (1 μ/ml) and carrageenan (1 mg/ml), the infectivity and virus content in the leaves as well as the intracellular accumulation of virus particles were significantly inhibited in comparison with leaves inoculated with TMV only. The carrageenan applied 24 h prior to inoculation with TMV also exhibited the antiviral effects but to a lower degree than when the virus and polysaccharide were inoculated together. As the infection developed, the inhibitory effects of carrageenan decreased. Treatment of the leaves with carrageenan affected the formation of TMV-specific intracellular inclusions (granular and tubule) known to consist of the virus-encoded protein components of the viral replicase. A characteristic of the infection development in the presence of carrageenan, 4 days after infection of the leaves with TMV, was the predominant formation in cells of granular inclusions famous to appear at early stages of TMV multiplication. At the same time, cells of the leaves inoculated with TMV alone contained for the most part tubule inclusions considered to arise from granular ones on the advanced infection stages. Taken together, our observations indicate that the κ/s-carrageenan inhibits TMV infection in detached tobacco leaves at early stages.

Changes in Glucose-6-Phosphate Dehydrogenase, Ribonucleases, Esterases and Contents of Viruses in Potato Virus Y Infected Tobacco Superinfected with Tobacco Mosaic Virus

Effects of the superinfection with tobacco mosaic virus (TMV) on susceptible tobacco plants infected with potato virus Y (PVY) were determined. Dynamic changes in the TMV and/or PVY contents, the ribonucleases (RNases), the phosphomonoesterase (PME), the phosphodiesterase (PDE) and the glucose-6-phosphate dehydrogenase (G6P DH) activities were studied. The PVY infection caused a substantial reduction in the multiplication of TMV. The content of TMV in the PVY inoculated leaves amounts to 6 and 9 % in the PVY systemically infected leaves when compared with single TMV. Surprisingly, the challenging virus (TMV) enhanced the content of inducing virus (PVY) in the locally inoculated leaves up to 130 – 141 %. In contrast, the reduction of PVY content down to 35 – 40 % by TMV was seen in the PVY systemically infected leaves. The activities of the RNase, the PME, the PDE and the G6P DH were increased (when compared with the healthy plants) during the acute phase of single virus multiplication (PVY or TMV). The increase in the activities of the enzymes in the leaves with mixed infection was at least as high as the sum of the increases of single infections. Moreover, a higher increase than the sum was seen for G6P DH and PDE (by about 20 – 35 %).

Glucose-6-Phosphate Dehydrogenase, Ribonucleases and Esterases upon Tobacco Mosaic Virus Infection and Benzothiodiazole Treatment in Tobacco

Effect of the benzothiodiazole (BTH) pre-treatment was monitored during the acute infection stage in the susceptible and the hypersensitive tobacco plants infected with the tobacco mosaic virus (TMV). Dynamic changes in the contents of chlorophyll, the total proteins, and the pathogenesis related proteins (PR-proteins), and activities of ribonucleases (RNase), phosphomonoesterase (PME), phosphodiesterase (PDE), and glucose-6-phosphate dehydrogenase (G6P DH) were studied. Neither the protein nor the chlorophyll contents were significantly changed by the TMV infection and/or the BTH treatment. The BTH pre-treatment caused a substantial reduction in the multiplication of TMV in the locally-infected leaves of the hypersensitive cultivar Xanthi-nc (to 15.1%). A lesser decrease (to 50.3%) was observed in the locally-infected leaves of susceptible cultivar Samsun. But in the systemically-infected leaves of this cultivar, only a 4-d delay in the multiplication of TMV was found. In the locally-infected leaves of both cultivars, the activities of the RNase, PME, PDE and G6P DH were sharply increased during the acute phase of TMV multiplication (when compared with the healthy plants) and the curves of these activities correlated with the multiplication curves of TMV. The BTH alone also strongly enhanced the activities of these enzymes early after application. Only low additional increases in some enzymes and even slight declines in the others were observed when the inoculation of leaves of cultivar Xanthi-nc followed the pre-treatment with the BTH. No inhibition of the enzymes was observed when the direct effect of different concentration of the BTH (1 – 1000 μM) was examined in vitro during a measurement of the activity. The analysis of intercellular proteins by PAGE under native conditions shows the similar spectrum of the proteins extracted from either the BTH-treated or the TMV-infected tobacco cv. Xanthi-nc.

Constitutive expression of interferon-induced human MxA protein in transgenic tobacco plants does not confer resistance to a variety of RNA viruses.

MxA is a key component in the interferon-induced antiviral defense in humans. After viral infections, MxA is rapidly induced and accumulates in the cytoplasm. The multiplication of many RNA viruses, including all bunyaviruses tested so far, is inhibited by MxA. These findings prompted us to express MxA in plants in an attempt to create resistance to tospoviruses. Here, we report the generation of transgenic tobacco plants that constitutively express MxA under the control of the 35 S cauliflower mosaic virus promotor. Northern and western blot analysis confirmed the expression of MxA in several transgenic plant lines. MxA expression had no obvious detrimental effects on plant growth and fertility. However, challenge experiments with tomato spotted wilt virus, tomato chlorotic spot virus, and groundnut ringspot virus revealed no increased resistance of MxA-transgenic tobacco plants to tospovirus infections. Neither was the multiplication of tobacco mosaic virus, cucumber mosaic virus and potato virus Y inhibited in MxA-transgenic plants. The results indicate that the expression of human MxA alone does not enhance virus resistance in planta.

Isolation of a Mutant ofArabidopsis thalianaCarrying Two Simultaneous Mutations Affecting Tobacco Mosaic Virus Multiplication within a Single Cell

Tobacco mosaic virus strain Cg (TMV-Cg) infectsA. thalianasystemically. In order to identify host factors involved in the multiplication of TMV-Cg, we isolated a mutant ofA. thalianafrom an M2 population mutagenized by fast neutron irradiation, in which the accumulation of the coat protein in upper systemic leaves was reduced to low levels. The phenotype of the mutant, YS241, was controlled primarily by a single nuclear recessive mutation namedtom2-1,which was distinct fromtom1, a separate mutation which also affects TMV-Cg multiplication. Thetom2-1mutation affected the accumulation of TMV-related RNAs in protoplasts in a tobamovirus-specific manner, suggesting that the wild-typeTOM2gene product is necessary for efficient amplification of TMV-related RNAs within a single cell, through specific interaction with virus-coded factors. Furthermore, we found that YS241 contained a single dominant modifier namedttm1,which increased the efficiency of multiplication of TMV-Cg and a tomato strain of TMV in atom2-1genetic background, both in plants and in protoplasts. We propose that thettm1element might be a translocated form of theTOM2gene.

Isolation of an Arabidopsis thaliana mutant in which accumulation of cucumber mosaic virus coat protein is delayed

Cucumber mosaic virus (CMV) is known to systemically infect Arabidopsis thaliana ecotype Columbia plants. In order to identify the host factors involved in the multiplication of CMV, we isolated an A. thaliana mutant in which the accumulation of the coat protein (CP) of CMV in upper uninoculated leaves was delayed. Genetic analyses suggested that the phenotype of delayed accumulation of CMV CP in the mutant plants was caused by a single, nuclear and recessive mutation designated cum1-1, which was located on chromosome IV. The cum1-1 mutation did not affect the multiplication of tobacco mosaic virus, turnip crinkle virus or turnip yellow mosaic virus, which belong to different taxonomic groups from CMV. Accumulation of CMV CP in the inoculated leaves of cum1-1 plants was also delayed either when CMV virion or CMV virion RNA was inoculated. On the other hand, when cum1-1 and the wild-type Col-0 protoplasts were inoculated with CMV virion RNA by electroporation, the accumulations of CMV-related RNAs and the coat protein were similar. These results suggest that the cum1-1 mutation did not affect the uncoating of CMV virion and subsequent replication in an initially infected cell but affected the spreading of CMV within an infected leaf, possibly the cell-to-cell movement of CMV in a virus-specific manner.