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RNA interference (RNAi) is a conserved mechanism against viruses in plants and animals. It is thought to inactivate the viral genome by producing virus-derived small interfering RNAs (vsiRNAs). Rice black-streaked dwarf virus (RBSDV) is transmitted to plants by the small brown planthopper (Laodelphax striatellus), and seriously threatens production of rice in East Asia, particularly Oryza sativa japonica subspecies. Through deep sequencing, genome-wide comparisons of RBSDV-derived vsiRNAs were made between the japonica variety Nipponbare, and the indica variety 9311. Four small RNA libraries were constructed from the leaves and shoots of each variety. We found 659,756 unique vsiRNAs in the four samples, and only 43,485 reads were commonly shared. The size distributions of vsiRNAs were mostly 21- and 22-nt long, and A/U bias (66-68%) existed at the first nucleotide of vsiRNAs. Additionally, vsiRNAs were continuously but heterogeneously distributed along S1-S10 segments of the RBSDV genome. Distribution profiles of vsiRNA hotspots were similar in different hosts and tissues, and the 5'- and 3'-terminal regions of S4, S5, and S8 had more hotspots. Distribution and abundance of RBSDV vsiRNAs could be useful in designing efficient targets for exploiting RNA interference for virus resistance. Degradome analysis found 25 and 11 host genes appeared to be targeted by vsiRNAs in 9311 and Nipponbare. We report for the first time vsiRNAs derived from RBSDV-infected rice., (© 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

In potato tubers showing pronounced corky ringspot symptoms, two related 'rule-breaking" tobacco rattle virus (TRV) RNA2s, named Da-2 and Db-2, were identified. Their coat protein (cp) genes are preceded on the 5' side by an additional gene for a 35 kDa protein for which no relationships with previously described TRV genes or their expression products were found. With estimated 4296 and 4247 nucleotides (nts) the two RNAs are the longest TRV RNA2s described so far. The difference in size between Da-2 and Db-2 is due to a duplication of a stretch of 49 nts in the 5' untranslated region of Da-2. An alignment of TRV coat proteins (cp) revealed that up to about amino acid (aa) 176 they form two rather uniform groups. The much shorter C-terminal parts of the cps, however, differ considerably in size and composition. With 56 aa this C-terminal part is much longer in the Da and Db sequences than in all other TRV cps. It differs in 18 positions in the two strains whereas their N-terminal 184 aa differ only in two positions. - In young potato plants developing from bud-cuttings of TRV Db-infected potato tubers which had been planted in soil free of virus and nematodes a gradual degradation of Db-2 was observed. In the newly formed rootlets already five days after planting a deletion of 80 nts was observed in the putative 2b gene which in other TRV strains encodes a protein necessary for nematode transmission. Thirty three days after planting the entire 2b gene, 119 nts at the 3'end of the cp gene and a portion of the original 3'untranslated region of Db-2 had been lost in the newly formed roots, leaves and stolons. The gene for the 35kDa protein was the only one which was not affected by deletions which seems to emphasize its importance for the virus. Fifty days after planting only TRV RNA1, but no TRV RNA2 were detectable., (Copyright © 2017 Elsevier B.V. All rights reserved.)

For a better understanding of the functionality and pathogenicity of beet soil-borne virus (BSBV), full-length cDNA clones have been constructed for the three genomic RNAs. With the aim of assessing their effectiveness and relative contribution to the virus housekeeping functions, transcripts were inoculated on Chenopodium quinoa and Beta macrocarpa leaves using five genome combinations. Both RNAs-1 (putative replicase) and -3 (putative movement proteins) proved to be essential for virus replication in planta and symptom production on C. quinoa, whereas RNA-2 (putative coat protein, CP, and a read-through domain, RT) was not. No symptoms were recorded on B. macrocarpa, but viral RNAs were detected. In both host plants, the 19 kDa CP was detected by Western blotting as well as a 115 kDa protein corresponding to the CP-RT.

The complete nucleotide sequence of Peanut stunt virus strain Mi (PSV-Mi) from China was determined and compared to other viruses of the genus Cucumovirus. The tripartite genome of PSV-Mi encoded five open reading frames (ORFs) typical of cucumoviruses. Distance analyses of four ORFs indicated that PSV-Mi differed sufficiently in nucleotide sequence from other PSV strains of subgroups I and II to warrant establishment of a third subgroup of PSV.

Discrete RNA secondary and higher-order structures, typically local in extent, play a fundamental role in RNA virus replication. Using new bioinformatics analysis methods, we have identified genome-scale ordered RNA structure (GORS) in many genera and families of positive-strand animal and plant RNA viruses. There was remarkably variability between genera that possess this characteristic; for example, hepaciviruses in the family Flaviviridae show evidence for extensive internal base-pairing throughout their coding sequences that was absent in both the related pestivirus and flavivirus genera. Similar genus-associated variability was observed in the Picornaviridae, the Caliciviridae, and many plant virus families. The similarity in replication strategies between genera in each of these families rules out a role for GORS in a fundamentally conserved aspect of this aspect of the virus life cycle. However, in the Picornaviridae, Flaviviridae, and Caliciviridae, the existence of GORS correlated strongly with the ability of each genus to persist in their natural hosts. This raises the intriguing possibility of a role for GORS in the modulation of innate intracellular defense mechanisms (and secondarily, the acquired immune system) triggered by double-stranded RNA, analogous in function to the expression of structured RNA transcripts by large DNA viruses. Irrespective of function, the observed evolutionary conservation of GORS in many viruses imposes a considerable constraint on genome plasticity and the consequent narrowing of sequence space in which neutral drift can occur. These findings potentially reconcile the rapid evolution of RNA viruses over short periods with the documented examples of extreme conservatism evident from their intimate coevolution with their hosts.

Analysis of virus spread using co-expressed reporter proteins has provided important details on cell-to-cell and long-distance movement of viruses in plants. However, most viruses cannot tolerate insertion of large non-viral segments or loss of any open-reading frames, procedures required to detect viruses non-evasively. A technique used to localize mRNAs intracellularly in yeast has been modified for detection of viral RNAs in whole plants. The technique makes use of the binding of the coat protein of MS2 bacteriophage (CPMS2) to a 19 base hairpin (hp). A fusion protein, consisting of the CPMS2, green fluorescent protein (GFP), and a nuclear localization signal (NLS), was nuclear-localized upon transient expression in protoplasts. However, addition of the hp to the 3' untranslated region of Turnip crinkle virus (TCV-hp) and co-transfection of the virus and fusion protein construct into protoplasts resulted in the re-location of GFP to the cytoplasm. Neither the insertion of the hp nor the interaction with the fusion protein impaired any viral functions. Transgenic plants expressing the GFP-NLS-CPMS2 fusion protein were generated, and GFP was detected in nuclei of young plant cells. Foci of GFP cytoplasmic fluorescence were detected in TCV-hp-inoculated leaves at 2 days post-inoculation. Later, GFP was detected in young leaves near the midvein and in the base (support) cells of trichomes in the vicinity of secondary and tertiary veins. In older leaves, cytoplasmic GFP could be visualized throughout many of the leaves. This technique should be amenable for detection of any virus with a transformable plant (or animal) host and may also prove useful for localizing properly engineered host RNAs.

Changes in glucose-6-phosphate dehydrogenase (G6P DH; EC 1.1.1.49) activity caused by infection of tobacco ( Nicotiana tabacum L.) leaves with potato virus Y (PVY), cucumber mosaic virus, potato virus X, tobacco rattle virus and turnip mosaic virus, the subcellular localisation of G6P DH isozymes in mesophyll protoplasts derived from healthy and PVY-infected tobacco leaves, as well as G6P DH control and the relationship of its isozymes with the degree of tobacco resistance to PVY multiplication, were studied. The activities of G6P DH were markedly increased in locally and systemically infected leaves and the time courses of the activity linearly correlated with those of virus multiplication. In leaves infected with PVY, the activity time courses of the crude and the partially purified G6P DH were coincident. This probably indicates the involvement of coarse regulation of the enzyme. PVY content linearly correlated with enhanced G6P DH activity in leaf discs derived from susceptible, tolerant and resistant cultivars of tobacco. The increased activity of the enzyme in infected protoplasts and plant tissues was predominantly caused by the increased activity of chloroplastic isozymes. This was confirmed by the specific staining of isozymes after electrophoretic separation of chloroplastic proteins of tobacco leaves. These findings enable the degree of resistance to virus multiplication to be quantified for the use of gene manipulation and breeding.

Post-transcriptional gene silencing (PTGS) is a sequence-specific degradation mechanism that operates in almost all eukaryotic cells. In plants, double-stranded RNA triggers PTGS, generating 21- to 25-nucleotide guide RNAs responsible for specific degradation of cognate mRNA. The double stranded RNA intermediates of replicating plant viruses often induce PTGS, leading to symptom attenuation. Here we demonstrate the role of PTGS in defective interfering (DI) RNA-mediated symptom attenuation in plants infected with Cymbidium ringspot tombusvirus (CymRSV). Analysis of 21- to 25-nucleotide RNAs in Nicotiana benthamiana infected with CymRSV indicated that PTGS was not spread homogeneously along the viral genome. The 21- to 25-nucleotide RNAs derived mainly from plus-stranded RNA and likely arose from local basepaired structures. In contrast to helper viral RNA, short DI RNAs were not accessible to helper virus-induced RNA degradation guided by the 21- to 25-nucleotide RNAs. Our results suggest a model in which PTGS plays an important role in the selective accumulation and symptom attenuation mediated by DI RNAs. Because PTGS operates in a wide variety of different organisms, this model is applicable to DI RNA generation and accumulation in both plant and animal cells.

The RNA genome of tobacco rattle virus (TRV) is bipartite. RNA 2 of the nematode-transmissible TRV isolate PPK20 encodes the viral coat protein (cp) and proteins with molecular weights of 29,400 and 32,800 (29.4K and 32.8K proteins). When this isolate was serially passaged in tobacco by using phenol-extracted RNA as the inoculum in each transfer, defective interfering (DI) RNAs rapidly accumulated. A number of these DI RNAs were cloned. Six DI RNAs had single internal deletions in RNA 2 that removed most of the cp gene, the 29.4K gene, and the 5' half of the 32.8K gene. The borders of the deletions in these DI RNAs were found to be flanked in the genomic RNA 2 by short nucleotide repeats or sequences resembling the 5' end of TRV genomic and subgenomic RNAs. Two DI RNAs were found to be recombinants containing a 5' sequence derived from RNA 2 and a 3' sequence derived from RNA 1. When serial passage of TRV isolate PPK20 was carried out by using leaf homogenates as inocula in each transfer, accumulation of a DI RNA (designated D7) with a functional cp gene was observed. The deletion in D7 covered the 3' end of the cp gene, the 29.4K gene, and the 5' half of the 32.8K gene. An infectious cDNA clone of D7 RNA was made. In mixed infections, D7 RNA rapidly outcompeted RNA 2 but did not compete with RNA 1. The deletion in D7 RNA abolished the nematode transmissibility of the PPK20 isolate. These results may explain the observation that many laboratory isolates of tobraviruses have lost their nematode transmissibility and contain RNA 2 molecules of widely different lengths.

The coat protein gene of pea early browning virus (PEBV) was replaced with that of another tobravirus, tobacco rattle virus (TRV strain PPK20). The recombinant virus multiplied efficiently in the systemic host Nicotiana benthamiana and, on the local lesion host Phaseolus vulgaris, produced symptoms typical of PEBV rather than TRV showing that viral coat protein is not a determinant for lesion morphology. Both viral RNAs were encapsidated by TRV coat protein although the shorter particles (encapsidated RNA-2) did not form a discrete population. Evidence is presented to suggest involvement of nucleotide sequences upstream of the coat protein gene in virus particle assembly.

Several strains of the broad bean mottle virus (BBMV), an icosahedral tripartite plant RNA virus, which show distinct reactions on certain plant hosts have been described (K. M. Makkouk et al., Neth. J. Plant Pathol. 94, 195-212, 1988). Here we report defective interfering (DI) RNAs encapsidated in two BBMV strains from Morocco and Tunisia. While not effective in some plants, these DI RNAs exacerbated the severity of symptoms in others. The most dramatic, lethal effect of DI RNAs has been found on pea (Pisum sativum, cv. Rondo) seedlings. Sequence analysis has revealed that the DI RNAs were derived by single in-frame central deletions of 448 to 537 nt in the corresponding genomic RNA2 components. A comparison of the intensities of full-length RNA2 bands from DI molecule-containing and DI molecule-deficient virion RNA preparations revealed that the DI RNAs decreased the level of RNA2 components in total RNA preparations. The differences between corresponding virion RNAs were much smaller. This suggests an interference with RNA replication. In vitro assays and an analysis of the polyribosomal RNA fractions confirmed the translational activity of DI RNAs. This paper reports the first description of natural DI RNAs in tripartite isometric plant RNA viruses.

Genomic RNA of cymbidium ringspot tombusvirus (CyRSV) contains five large open reading frames (ORFs). The two 5' proximal ORFs encode proteins of 33 and 92 kDa and the three 3' proximal ORFs encode proteins of 41, 22, and 19 kDa. In addition, a small reading frame encoding a protein of 4 kDa was recently observed by computer analysis of the 3' nontranslated region of CyRSV and other tombusvirus RNAs (ORF 6). The function of putative gene products was investigated with the use of several mutants. Mutants in ORFs 1 and 2 were not viable indicating that both 33- and 92-kDa proteins are required for replication. Deletion of a large portion of the coat protein gene, encoding a 41-kDa protein, did not prevent replication of viral RNA and cell-to-cell movement, but interfered severely with long-distance translocation. No virus particles or viral RNA could be detected in inoculated or upper leaves of plants inoculated with transcripts obtained from mutants not expressing the 22-kDa protein. However, replication and encapsidation occurred normally in inoculated protoplasts indicating that this gene product is a transport protein. Conversely, no role could be assigned to putative gene products of ORF5 (19-kDa protein) and ORF6. It was also shown that factors other than gene expression can influence the replication of RNA mutants, probably due to instability of RNA molecules.

The hammerhead self-cleavage reaction is generally considered to be involved in the in vivo production of (+) and (-) monomeric RNAs of the viroid-like, satellite RNA or virusoid of lucerne transient streak virus (vLTSV) from multimeric replicative intermediates, as part of the symmetrical rolling circle mechanism. To test this, three cDNA clones of vLTSV RNA, mutated at sites that inactivate in vitro self-cleavage of (-) RNA, were inoculated as excised plasmid cDNA inserts, together with helper virus LTSV, on Nicotiana clevelandii plants. As was predicted if hammerhead self-cleavage is involved in in vivo cleavage of (-) RNAs, high molecular weight (-) vLTSV RNAs were present in total RNA extracts from these mutant inoculated plants, but not in wild-type inoculated plants. Surprisingly however, the mutated virusoids also produced monomeric (-) RNAs in vivo. Sequence analysis of cDNA clones prepared from progeny virusoid RNA revealed 8-20% of progeny contained reversions and pseudo-reversions of the introduced mutations. Hence, monomeric (-) RNAs were most likely produced by restoration of in vivo self-cleavage activity in the (-) RNA. Overall, the results indicate that mutations which disrupted self-cleavage in vitro also abolished the production of monomeric (-) RNAs in vivo and that hammerhead self-cleavage is involved in the cleavage of multimeric (-) RNAs to monomers. The observation that greater than 50% of the progeny cDNA clones generated from plants inoculated with mutant vLTSV contained base changes, compared to 20% from wild-type inoculated plants, may reflect an interesting adaptive response on the part of the mutated virusoids.

Cocksfoot mottle sobemovirus supports replication and encapsidation of the viroid-like satellite RNA (sat-RNA) of lucerne transient streak virus (LTSV) in two monocotyledonous species, Triticum aestivum and Dactylis glomerata. Additionally, LTSV sat-RNA replicates effectively in the presence of turnip rosette sobemovirus in Brassica rapa, Raphanus raphanistrum and Sinapsis arvensis, but not in Thlaspi arvense or Nicotiana bigelovii, indicating that host species markedly influence this interaction. Previous reports of the association between LTSV sat-RNA and helper sobemoviruses were limited to dicotyledonous hosts. Our results demonstrate that the biological interaction between these two entities spans divergent dicotyledonous and monocotyledonous species.

A set of full-length cDNA clones of the satellite RNA of grapevine fanleaf nepovirus isolate F13 (GFLV-F13) was constructed with a variable number of additional, non-viral nucleotides at the 5' and 3' ends. The biological activity of the RNAs transcribed from these constructs was tested in Chenopodium quinoa protoplasts using a helper virus. When inoculated with arabis mosaic virus S (ArMV-S) RNA as helper, transcripts with 33 non-viral nucleotides at the 5' end (tr45p4) did not replicate, whereas transcripts with only one non-viral nucleotide at the 5' end (tr3S and tr3M) did replicate. Capping of the transcripts enhanced their replication. On the other hand, the presence of extra nucleotides at the 3' end had little influence on the biological activity of the in vitro transcripts. In contrast with ArMV-S, GFLV isolate 24 was not a helper for tr3M transcripts, indicating a specific interaction between the helper strain and the satellite RNA.

Transgenic tobacco plants which express untranslatable sense or antisense forms of the tobacco etch virus potyvirus (TEV) coat protein (CP) gene sequence have been generated. One of seven transgenic plant lines expressing a CP gene antisense transcript showed an attenuation of symptoms when inoculated with TEV. Three of ten transgenic plant lines expressing untranslatable sense transcripts did not develop symptoms when inoculated with TEV. These lines were resistant to either aphid or mechanically transmitted TEV. In contrast to CP-mediated resistance reported for other viruses, resistance was (1) mediated by an RNA molecule; (2) TEV-specific (i.e., "broad-spectrum resistance" was not observed); (3) independent of inoculum levels; (4) not dependent on plant size and; (5) due to decreased levels of virus replication. Protoplast experiments were used to demonstrate that resistant plant lines did not support the production of virus protein and progeny virus at wild-type levels.

Full-length cDNA of beet western yellows virus genomic RNA has been cloned behind the bacteriophage T7 RNA polymerase promoter of the transcription vector BS(-). The in vitro run-off transcription product obtained in the presence of T7 RNA polymerase and m7GpppG cap has the same messenger properties as natural viral RNA in in vitro translation systems. The full-length transcript was also able to infect Chenopodium quinoa protoplasts inoculated by electroporation. Infection could be followed by the appearance of viral coat protein in the inoculated protoplasts and the de novo synthesis of viral RNA. Site-directed mutagenesis experiments revealed that expression of beet western yellows virus open reading frame 1 and the C-terminal portion of open reading frame 6 were not required for infection of protoplasts. Additional experiments with these mutants and mutants in the other viral open reading frames should provide information concerning the requirements for beet western yellows virus replication and, ultimately, the role of virus genes in other important steps in the virus infection cycle, such as aphid transmission.

Turnip crinkle virus is one of several single-stranded RNA plant viruses associated with defective interfering (DI) RNAs. A complete cDNA copy of a 344-base DI RNA (DI RNA G) was cloned downstream from a T7 RNA polymerase promoter. Transcripts synthesized in vitro were infectious when inoculated with helper virus on turnip plants. Studies of the infectivity of DI transcripts containing deletions, insertions, and single-base changes suggest that (i) in general, only the 5' two-thirds of the molecule can tolerate mutations; (ii) between 52 and 67 bases of terminal 5' sequence are required for infectivity; (iii) nucleotides in positions 68 to 138 are not specifically involved in RNA infectivity; (iv) DI RNA G molecules smaller than 327 bases are not amplified efficiently in plants.

Full-length cDNA clones of both RNAs of pea early browning virus have been constructed. Synthetic transcripts derived in vitro from these clones are infectious when inoculated onto plants. Electron microscopy revealed the presence of virions in transcript-inoculated plants, and both purified RNA and virions isolated from such plants could be used to infect other plants. Transcripts of RNA1 alone were able to replicate and spread systemically which is a characteristic of members of the tobravirus group of plant viruses.

Carnation latent virus was shown to direct the synthesis of virus-specific polypeptides in both reticulocyte lysate and wheat germ in vitro translation systems. The L-(4,5-3H)-leucine-labeled products ranged in molecular mass from Mr 190 to 33 kD. The 33 kD product, synthesized after only 15 min incubation, was the only major polypeptide that immunoprecipitated with antiserum to CarLV. Coat-protein synthesis does not occur as a result of proteolytic processing, but may arise as a result of translation of a subgenomic RNA species. Subgenomic RNA species were not detected by Northern hybridization of CarLV cDNA to either viral RNA or total nucleic acid from systemically infected plants, although CarLV-specific dsRNA species equivalent to 1.6 and 2.1 kb were detected.

Newly synthesized virions of flock house virus (FHV), an insect nodavirus, were detected in plant cells inoculated with FHV RNA. FHV was found in whole plants of barley (Hordeum vulgare), cowpea (Vigna sinensis), chenopodium (Chenopodium hybridum), tobacco (Nicotiana tabacum), and Nicotiana benthamiana and in protoplasts derived from barley leaves. Virions produced in plants contained newly synthesized RNA as well as newly synthesized capsid protein. These results show that the intracellular environment in these plants is suitable for synthesis of a virus normally indigenous only to insects. Such synthesis involves, minimally, translation of viral RNA, RNA replication, and virion assembly. Inoculation of barley protoplasts with FHV virions resulted in synthesis of small amounts of progeny virions, suggesting that FHV virions are capable of releasing their RNA in plant cells. In N. benthamiana, virions resulting from inoculation with RNA were detected not only in inoculated leaves but also in other leaves of inoculated plants, suggesting that virions could move in this plant species. Such movement probably occurs by a passive transport through the vascular system rather than by an active transport involving mechanisms that have evolved for plant viruses.

Synthesis of circular (RNA 2) and linear (RNA 3) molecules of velvet tobacco mottle virus (VTMoV) satellite RNA (sat RNA) has been studied by incubating strips of tissues excised from systemically infected Nicotiana clevelandii in solutions of [14C]uridine. After a short lag, RNA and virus synthesis proceeded at a constant rate for at least 24 hr, during which time most of the synthesis was directed to the production of RNAs 2 and 3. The kinetics of [14C]uridine incorporation into the sat RNA molecules after increasing times of incubation and during pulses of [14C]uridine followed by chase incubation with excess [12C]uridine suggest that RNA 3 is a percursor of RNA 2. However, not all the RNA 3 synthesized was shown to end up as RNA 2, even after 72 hr of incubation. Several lines of evidence are presented supporting the conclusion that VTMoV-infected cells contain large pools of unencapsidated sat RNA. It is suggested that the sat RNA may have a greater affinity for the VTMoV replicase than the helper viral RNA which results in copious production of the sat RNA.

Plants mixedly infected with the U2 strain of tobacco mosaic virus (T2MV) and sunnhemp mosaic virus (SHMV) and grown at 35 degrees, yield particles of the same modal lengths (300 and 40 nm) as those found in plants singly infected with SHMV, but not in plants infected with T2MV, which yield only the long particles. At least some of the particles produced in mixedly infected plants contain coat proteins of both viruses. When RNAs from these particles are translated in vitro the coat proteins of both viruses are produced, although when a mixture of RNAs from particles of SHMV and T2MV, grown separately, are translated in vitro only SHMV protein is produced. These and other results suggest that the short particles produced in mixedly infected plants contain both coat protein messengers.

Genome-length cDNA clones of turnip crinkle virus (TCV) were constructed with SmaI and XbaI restriction sites engineered at the 5' and 3' termini, respectively. The genome-length cDNAs were positioned downstream of modified lambda and T7 phage promoters such that in vitro transcription resulted in RNAs with 5 extra nucleotides at the 3' end, and 1, 2, or 14 extra nucleotides at the 5' end depending on the construction. Transcripts with 14 extraviral 5' nucleotides were not infectious, while transcripts with 1 or 2 additional 5' nucleotides, with or without 5'-cap analog included in transcription reactions, were biologically active. These were approximately an order of magnitude less infectious than RNA extracted from TCV virions. The additional 5' nucleotides were not maintained in progeny viral RNAs isolated from plants.

In a synchronous infection of cowpea chlorotic mottle virus in cowpea leaves, the three largest viral RNA components were synthesized throughout the infection at relatively constant ratios of two molecules of component 3 to one each of components 1 and 2. Component 4 (the coat protein message) was produced minimally during the initial stages of the infection, and as the infection progressed the proportion of component 4 continuously increased. The replicative forms of components 1, 2, and 3 were produced with kinetics similar to those of the corresponding single-stranded molecules.

A simple procedure for the isolation of double-stranded (ds) RNA from virus-infected plants is described. The method is based on grinding plant tissue in 4% p-aminosalicylic acid and recovery of ds RNA by phenol extraction and precipitation with 30% ethanol. The presence of both negative and positive virus RNA strands in RNA fractionated in agarose gels was verified by Northern blot hybridization with polynucleotide kinase labelled genomic RNA or complementary DNA (cDNA) probes. The procedure enabled detection of three major ds RNA species (MWs 4.2, 1.05 and 0.48 X 10(6)) and at least 4 minor bands with estimated MWs of 3.5, 2.5, 2.2 and 2.0 X 10(6) in Nicotiana tabacum plants systemically infected with tobacco mosaic virus (TMV). Cucumber mosaic virus (CMV)-infected Pachystachys coccinea plants contained 2 minor bands of MWs 0.49 and 0.35 X 10(6) in addition to the previously described 4 major ds RNAs and ds CARNA 5 (MW 0.22 X 10(6)). The patterns of ds RNA are useful for diagnosing natural infections of CMV and TMV in N. glauca plants and of citrus tristeza virus in Citrus spp.

Brome mosaic virus (BMV) RNA that had both termini chemically modified by periodate oxidation and aniline-catalyzed cleavage of the terminal nucleotide had drastically reduced infectivity. BMV RNA that was first enzymatically tyrosylated to protect the 3' terminus from modification, and then modified at the 5' terminus by periodate oxidation and aniline cleavage, had a similar reduction in infectivity. Tyrosylation followed by acetylation modifies only the 3' terminus. Nevertheless, acetylated tyrosyl-BMV RNA was less than one-fourth as infectious as a control sample subjected to procedures that differed only by the presence of tyrosinol (which prevents aminoacylation and subsequent acetylation). For each modified form of viral RNA, care was taken to test the infectivity of appropriate control samples. The integrity of the modified RNAs was examined by gel electrophoresis and by biological translation and aminoacylation assays. We conclude that, in different ways, both the 5'- and 3'-terminal structures of BMV RNA play important roles during infection of the host.

Beet necrotic yellow vein virus (BNYVV) has a quadripartite plus-strand RNA genome in which the two smallest genome components, RNA 3 and 4, are not necessary for virus multiplication in leaves. Infectious transcripts of BNYVV RNA 3 and 4 have already been described (V. Ziegler-Graff, S. Bouzoubaa, I. Jupin, H. Guilley, G. Jonard, and K. Richards (1988) J. Gen. Virol. 69, 2347-2357). In this paper we describe synthesis of a full-length RNA-1 transcript by bacteriophage T7 RNA polymerase-directed run-off transcription of cloned viral cDNA. A recombinant plasmid containing a full-length cDNA insert of RNA 2 could not be maintained in Escherichia coli. Therefore full-length transcript of RNA 2 was produced by transcription of cDNA ligation products without amplification in bacteria. When inoculated together to leaves of Chenopodium quinoa or Tetragonia expansa the RNA 1 and 2 transcripts were infectious; they also supported multiplication of the BNYVV RNA 3 and 4 transcripts, providing a totally synthetic inoculum of the virus. In one recombinant clone of RNA 2 a point mutation causing an arginine to serine substitution at position 119 of the viral coat protein was discovered. The mutation was detected because the resulting coat protein had altered electrophoretic mobility. RNA 2 transcripts containing this mutation were infectious but viral RNA was not encapsidated. The mutation also interfered with long distance movement of the virus in spinach, presumably as a consequence of the packaging deficiency.

Molecular hybridization by the Northern blot technique in combination with 125I-labeled PSTV (+) RNA and 32P-labeled PSTV cDNA as probes has been applied to detect viroid-specific sequences in healthy and viroid(PSTV)-infected tomato plants. Conditions are described which allow differentiation of (+) and (-) viroid sequences on the basis of the different thermostabilities of the corresponding hybrid molecules. By this experimental approach, it is documented that no viroid-specific DNA sequences can be detected and that viroid replication proceeds via complementary RNA intermediates. Out of the seven (-) RNA species found, six are apparently larger than the circular viroid (+) RNA and one is about the same size as the linear (+) RNA molecule.