Maize as a New Host for Oat blue dwarf virus.

Oat blue dwarf virus (OBDV) is a marafivirus present throughout the North American Great Plains and is found at low incidence levels in barley, oats, and flax in the Upper Midwest United States. Cropping patterns in this region have changed considerably in recent years, with much greater acreages devoted to maize in areas historically planted to small grains. Considering that OBDV is endemic in small grains in this region and that its aster leafhopper vector (Macrosteles quadrilineatus) is commonly detected in maize (2), we questioned whether maize might be a host for OBDV. Although Westdal (4) reported that maize was not a host for OBDV, it is the primary host for the related Maize rayado fino virus (MRFV) transmitted by Dalbulus maidis. MRFV is the type member of the marafiviruses and ranges from South America to the southern United States. To reevaluate the ability of maize to serve as a host for OBDV, viruliferous aster leafhoppers were allowed to feed on young maize seedlings for a 7-day inoculation access period. Plants were assayed for the presence of OBDV by ELISA 21 days after leafhopper removal. Initially, open-pollinated Sunglow sweet corn was tested in three experiments, with infection rates of 17 to 100%. A test of hybrid Sunglow resulted in 63% infection. These are comparable to transmission levels obtained when oat and barley plants are fed upon by viruliferous leafhoppers. Subsequently, 10 additional varieties of sweet and field corn were tested to determine if they could serve as hosts for OBDV. Sweet corn included Silver Queen, Peaches and Cream, Morning Star, Sugar Dots, Kandy Korn, and Golden Cross Bantam. Field corn included four lines provided by the maize breeding program of M. Carena at North Dakota State University (056640, 056643, 056612, and 056652). At least one plant was infected in all sweet and field corn varieties except 056612. Infected plants were largely asymptomatic, although a very faint stipple-striping was observed on some plants. To determine whether leafhoppers could acquire OBDV from infected maize and transmit the virus to other hosts, healthy leafhoppers were fed upon detached leaves of infected maize for 4 days (approximately 30 days after initial infection) and subsequently transferred three times at weekly intervals to barley and oats. Infection rates of 42 to 55% were observed in target plants, indicating efficient transmission from maize to other cereal hosts. The extent of natural infection of maize by OBDV or economic loss, if any, remains to be determined. This discovery extends the host range of OBDV to include maize and suggests that maize has the potential to serve as a natural reservoir for OBDV. The identification of hosts for known and new marafiviruses, such as those recently detected in citrus (3) and grape (1), will expand our understanding of the ecology and epidemiology of viruses within this group. References: (1) M. Al Rwahnih. Virology 387:395, 2009. (2) J. L. Capinera. Page 320 in: Encyclopedia of Entomology. 2nd ed. Springer Publishing, New York, NY, 2008. (3) W. Maccheroni et al. J. Virol. 79:3028, 2005. (4) P. Westdal. Can. J. Bot. 46:1431, 1968.

First Report of Beet black scorch virus in the United States.

In October of 2005, sugar beet (Beta vulgaris L.) plants exhibiting symptoms of rhizomania caused by Beet necrotic yellow vein virus (BNYVV) (3) were observed in a production field near Greeley, CO. The roots of seven plants exhibiting moderate to severe symptoms characteristic of this disease were tested using double-antibody sandwich enzyme-linked immunosorbent assay with anti-BNYVV antiserum from rabbits. Of these, only two roots exhibiting the mildest symptoms tested positive for BNYVV (all roots tested negative for the presence of the related Beet soilborne mosaic virus (BSBMV). 'Hairy' lateral roots characteristic of the disease were combined from the remaining five roots, ground in phosphate buffer, and the supernatant from the suspension was mechanically applied to leaves of Chenopodium quinoa in an effort to isolate an infectious agent. Five days postinoculation (dpi), yellow lesions with necrotic centers were visible on inoculated leaves, well in advance of those typically observed for BNYVV or BSBMV. Lesions exhibiting a similar rate of development on C. quinoa subsequently were induced from extracts of root vascular tissue prepared from four of seven additional beet roots tested from this location. Transfer of the infection from the C. quinoa lesions to 32 healthy C. quinoa and 10 sugar beet plants (hybrid ACH9369; American Crystal Sugar Co., Moorhead MN) resulted in 100% infection. Inoculated leaves of C. quinoa exhibited a high density of necrotic local lesions within 3 dpi, whereas inoculated leaves of sugar beet exhibited pinpoint, necrotic to diffuse, chlorotic local lesions evident by 5 dpi. Electron microscopic examination of fixed, ultra-thin sections of symptomatic C. quinoa leaf tissue revealed aggregates of virus-like particles of icosahedral symmetry within the cell cytoplasm. Following a virus minipreparation procedure, nucleic acid extracted from the partially purified virus was found to be single-stranded RNA by ribonuclease digestion and alone was infectious when inoculated to C. quinoa leaves. The apparently monopartite RNA genome was 3.5 kb long and a candidate for the single coat protein (CP) had a mass of ˜25 kDa. The sole reference set found in the literature for a virus naturally occurring on sugar beet with similar characteristics was that for Beet black scorch virus (BBSV), a virus recently accepted by the ICTV into the genus Necrovirus within the family Tombusviridae (2). Prior to this communication, BBSV has only been reported in China where it was first documented affecting sugar beet in the late 1980s (1). Using the published sequence of BBSV (Genbank Accession No. AY626780), DNA primers directed to the 3' half of the BBSV genome were used in reverse transcription-polymerase chain reaction to produce an amplicon from the unknown virus. Sequencing the amplicon revealed 88.8% nucleotide sequence identity to the BBSV CP gene and 97% amino acid sequence identity to the predicted CP gene product. Combined, the nucleotide sequence and physical characteristics confirm the presence of BBSV in a U.S. sugarbeet field for the first time. To our knowledge, this is the first report of the occurrence of BBSV outside of China. References: (1) Y. Cao et al. Arch. Virol. 147:2431, 2002. (2) C. M. Fauquet et al. Eighth Report of the International Committee on the Taxonomy of Viruses. Academic Press, New York, 2005. (3) C. M. Rush. Ann. Rev. Phytopathol. 41:567, 2003.

Identification of Molecular Genetic Markers in Pyrenophora teres f. teres Associated with Low Virulence on 'Harbin' Barley.

ABSTRACT Two isolates of the barley net blotch pathogen (Pyrenophora teres f. teres), one possessing high virulence (0-1) and the other possessing low virulence (15A) on the barley cultivar Harbin, were crossed and the progeny of the mating were isolated. Conidia from cultures of the parent and progeny isolates were used as inoculum to determine the inheritance of virulence in the pathogen. Of the 82 progeny tested, 42 exhibited high virulence and 40 exhibited low virulence on 'Harbin' barley. The data support a model in which a single, major gene controls virulence in P. teres f. teres on this barley cultivar (1:1 ratio; chi(2) = 0.05, P = 0.83). Preparations of DNA were made from parental and progeny isolates, and the DNA was subjected to the random amplified polymorphic DNA (RAPD) technique in a search for molecular genetic markers associated with the virulence phenotype. Five RAPD markers were obtained that were associated in coupling with low virulence. The data indicate that the RAPD technique can be used to tag genetic determinants for virulence in P. teres f. teres.

Oat blue dwarf marafivirus resembles the tymoviruses in sequence, genome organization, and expression strategy.

The complete nucleotide sequence and genome organization of oat blue dwarf marafivirus (OBDV) were determined. The 6509 nucleotide RNA genome encodes a putative 227-kDa polyprotein (p227) with sequence motifs similar to the methyltransferase, papain-like protease, helicase, and polymerase motifs present in the nonstructural proteins of other positive strand RNA viruses. The 3' end of the open reading frame (ORF) that encodes p227 (ORF 227) also encodes the two capsid proteins: a 24-kDa capsid protein is presumably cleaved from the p227 polyprotein, whereas the 21-kDa capsid protein appears to be translated from a subgenomic RNA (sgRNA). Encoded amino acid and nucleotide sequence comparisons, as well as the OBDV genome expression strategy, show that OBDV closely resembles the tymoviruses. OBDV differs from the tymoviruses in its general biology, in its lack of a putative movement gene that overlaps the replication-associated genes, and in its fusion of the capsid gene sequences to the major ORF. OBDV also possesses a 3' poly(A) tail, as compared to the tRNA-like structures found in most tymoviral genomes. Due to the strong similarities in genome sequence and expression strategy, OBDV, and presumably the other marafiviruses, should be considered a member of the tymovirus lineage of the alpha-like plant viruses.

A single nucleotide substitution in the alpha a gene confers oat pathogenicity to barley stripe mosaic virus strain ND18.

A 236-nucleotide region from the alpha a gene of strain CV42 (pathogenic to oat), when substituted for the homologous region in strain ND18 (nonpathogenic to oat), was shown previously to confer a near wild-type oat pathogenicity to this strain (Weiland and Edwards, 1994, Virology 201: 116-126). The data suggested that six amino acid substitutions in the alpha a gene were responsible for the differences in oat pathogenicity, and that threonine-724, encoded by CV42, might be a critical amino acid in determining pathogenicity of barley stripe mosaic virus (BSMV) to oat. In the present work, codons specifying T-724, I-764, and N-785 (encoded by CV42 RNA alpha) were substituted individually and in combination for those coding for P-724, T-764, and K-785 (encoded by ND18 RNA alpha), respectively, by site-directed mutagenesis. The core K-733, T-734, and K-736 positions (CV42) were substituted for Q-733, S-734, and Q-736 (ND18) as a single block. The results of inoculations with these mutants indicate that the C2261-->A2261 nucleotide substitution (P-724-->T-724) by itself is sufficient to enable strain ND18 to infect oat plants, although poorly. Additional substitution of CV42 codons into ND18 RNA alpha at the remaining five positions altered symptom type, decreased the timing of the appearance of symptoms, and increased the percentage of plants infected per inoculation. Nonetheless, all mutants accumulated to similar levels in inoculated oat protoplasts after a 24-h period. Using a recombinant RNA beta from which beta-glucuronidase could be expressed, results were obtained suggesting that the multiplication of strain ND18 and the nonpathogenic variants generated in the study was restricted in the inoculated leaf. The data indicate a potential pathway by which pathogenicity to oat evolved in BSMV.

Coding density of the turnip yellow mosaic virus genome: roles of the overlapping coat protein and p206-readthrough coding regions.

More than one-third of the turnip yellow mosaic virus (TYMV) genome simultaneously encodes two ORFs. We have investigated the functions of the overlapping coat protein ORF and readthrough domain of ORF-206 in the 3' region of the genome. TYMC-206 RNA, in which a second stop codon has been positioned to prevent ORF-206 readthrough, induced infections in protoplasts and plants that were indistinguishable from wild type. ORF-206 readthrough is thus nonessential. Nevertheless, TYMV-221 RNA, in which the ORF-206 stop codon was replaced with a tyrosine codon to force readthrough, was infectious to protoplasts, suggesting that a role for ORF-206 readthrough under certain conditions is possible. TYMV RNA variants that produce truncated or no coat protein were used to show that the coat protein is dispensable for local movement but necessary for systemic spread of virus in plants. Studies in protoplasts showed that (-) RNA levels are normal in the absence of coat protein, but (+) strand levels are decreased about 10-fold relative to wild-type infections. A mutant with a short C-terminal coat protein extension that formed virions less stable than normal demonstrated the protective role of capsids toward genomic RNA. The evolutionary implications of the dense information content of the TYMV genome are discussed.

Evidence that the alpha a gene of barley stripe mosaic virus encodes determinants of pathogenicity to oat (Avena sativa).

Complementary DNA clones from which infectious RNA can be transcribed were used to map the genetic determinants for oat (Avena sativa L.) pathogenicity of barley stripe mosaic virus (BSMV). Pseudorecombinant analysis of BSMV strains ND18 (nonpathogenic to oat) and CV42 (pathogenic to oat) indicated that the ability to systemically infect oat mapped to RNA alpha. A homologous recombinant of ND18 (18 alpha TKTKIN), possessing nucleotides 2218 to 2454 from CV42 RNA alpha, induced symptoms on oat similar to those generated by wild-type CV42. Six amino acids encoded by the alpha a gene differ between ND18 (PQSQTK) and CV42 (TKTKIN) in this region. Fine structure recombinants that encoded subsets of the six amino acid changes either were slow in their infection phenotype (18 alpha TKTK and 18 alpha TQSQIN) relative to recombinant 18 alpha TKTKIN or were not infectious (18 alpha KTKIN and 18 alpha KTK) to inoculated oat plants. Neither coat protein antigen nor viral RNA was detected in inoculated plants that did not display symptoms. All recombinants infected isolated oat protoplasts, however, as determined by Northern and Western blots of extracts from inoculated oat protoplasts. The accumulation of viral genomic RNAs in protoplasts among the recombinants tested was similar, except for that of recombinant 18 alpha TKTK which was reduced by 30%. The data support an earlier suggestion (Y. Zheng, and M. C. Edwards, 1990, J. Gen. Virol. 71, 1865-1868) that resistance of oat to BSMV strain ND18 is due to restricted virus movement in planta and provide evidence that the alpha a protein itself may be involved in the movement of BSMV in the infected plant.

Preferential replication of defective turnip yellow mosaic virus RNAs that express the 150-kDa protein in cis.

The turnip yellow mosaic virus genome encodes two proteins (the 150-kDa and 70-kDa proteins) that are proteolytically released from a single precursor and which are essential for RNA replication. Genomes with mutations in either of these coding regions were defective for independent replication in turnip protoplasts. The replication in trans of genomes with mutations in each region was studied by coinoculation with either a helper genome that carries a deletion in the coat protein gene, or with a second defective RNA that carries a mutation in the region encoding the other essential protein. Inefficient trans-replication of the defective RNAs was observed in most cases. In contrast, a defective RNA with a large deletion in the 70-kDa protein coding region could be replicated efficiently in trans, demonstrating that the cis-preference of replication can be overcome in some cases. Defective RNAs encoding wild type 150-kDa protein and defective 70-kDa protein were more efficiently replicated in trans than those encoding defective 150-kDa protein and wild type 70-kDa protein. The results suggest a model in which the 150-kDa and 70-kDa proteins form a relatively stable complex in cis on the viral RNA template.

Cis-preferential replication of the turnip yellow mosaic virus RNA genome.

The largest open reading frame of the turnip yellow mosaic virus RNA genome encodes a 206-kDa protein that is cleaved to yield N-terminal 150-kDa (p150) and C-terminal 70-kDa (p70) proteins. Using a genomic cDNA clone capable of generating infectious transcripts in vitro, we have introduced substitution, frameshift, and in-frame deletion mutations into the regions encoding both proteins. None of the mutant RNAs was able to replicate independently in turnip protoplasts, indicating that p150 and p70 are both essential. The replication in protoplasts of most of these defective RNAs was poorly supported in trans by a coinoculated helper genome with a deletion in the coat protein gene; replication could also be supported in trans by certain defective RNAs, but this complementation was likewise inefficient in most cases. The replication in trans was more efficient for defective RNAs encoding wild-type p150 and defective p70 than for those encoding defective p150 and wild-type p70. One defective RNA with a large deletion in the p70 coding region was able to replicate efficiently, both when inoculated with the helper genome and when inoculated with a second complementing defective RNA that supplied a wild-type p70. Thus, the cis preference of replication can be overcome in some cases. A model in which p150 and p70 form a complex with the 3' end of the RNA is proposed to explain the cis-preferential replication of turnip yellow mosaic virus RNA.

Expression of ORF-69 of turnip yellow mosaic virus is necessary for viral spread in plants.

Turnip yellow mosaic virus (TYMV) RNA has two extensively overlapping open reading frames (ORFs) encoding noncapsid proteins. The longest of these, ORF-206, is essential for RNA replication. We have investigated the expression and role of ORF-69 (encoding a protein with a MW of 69K) using specific antiserum and mutant genomic RNAs designed to interrupt ORF-69 expression. TYC69 antiserum immunoprecipitated a protein with an apparent MW of 75K (p69) from in vitro translations of TYMV RNA. Mutant RNAs with stop codons inserted at nucleotides 139, 178, and 178/224 in ORF-69 expressed ORF-69 at very low levels in vitro. These mutants replicated to wild-type levels in turnip or Chinese cabbage protoplasts, but were not recovered from any leaves of inoculated plants. These results suggest that ORF-69 products are dispensible for replication, but are required for viral spread. Revertant and pseudorevertant viruses with uninterrupted ORF-69 were recovered from plants showing delayed onset of symptoms after inoculation with two of the above mutants, indicating the importance of ORF-69 expression in establishing a systemic infection in plants. ORF-69 expression could be detected using Western blots in extracts from young, symptomatic leaves of infected plants. A single band with an apparent MW of 75-80K was detected in leaves infected with cDNA-derived viral RNAs, while a doublet was detected after infection with type strain viral RNA, suggesting the presence of two ORF-69 alleles or differential post-translational modification.

Proteolytic maturation of the 206-kDa nonstructural protein encoded by turnip yellow mosaic virus RNA.

The longest open reading frame of turnip yellow mosaic virus genomic RNA (ORF-206) encodes a 206-kDa nonstructural protein. The most prominent in vitro translation products of ORF-206 are the full-length p206 and a shorter N-coterminal 150-kDa protein. We have confirmed these assignments by immunoprecipitation of in vitro translation products with antisera raised to N-terminal and C-terminal regions encoded by ORF-206. The mechanism by which the 150-kDa protein arises from ORF-206 was investigated by in vitro translation of deletion and substitution derivatives transcribed from pTYMC, a cDNA clone of TYMV RNA. The following observations demonstrate that the 150-kDa protein and a C-terminal 70-kDa protein arise from ORF-206 by autoproteolysis: (1) Two regions encoded by ORF-206 were necessary for the formation of the 150-kDa protein: a domain between amino acids 555 and 1051, postulated to encode a protease, and the region between amino acids 1253 and 1261, thought to constitute the protease recognition and/or cleavage site. (2) Mutants with substitutions between amino acids 1253 and 1261 that produce low levels of the 150-kDa protein in in vitro translations also have high levels of p206 and low levels of the 70-kDa protein. (3) The rate of formation of the 150-kDa protein is dilution insensitive, suggesting that proteolysis occurs mainly in cis.

Infectious TYMV RNA from cloned cDNA: effects in vitro and in vivo of point substitutions in the initiation codons of two extensively overlapping ORFs.

Full-length cDNA of the 6.3 kb turnip yellow mosaic virus (TYMV) genome was placed between a T7 promoter and a unique Hind III site. In vitro transcription of Hind III-linearized DNA of clone pTYMC yielded full-length RNA transcripts. In inoculations of Chinese cabbage protoplasts and plants, capped transcripts and virion RNA had similar specific infectivities and produced similar systemic symptoms. We have used the pTYMC clone in studies of the expression of two overlapping open reading frames (1.9 kb and 5.5 kb ORFs) by making mutants with alterations in the initiation codons. Evidence is presented from in vitro translations of mutant and wild type RNAs that both ORFs are expressed from TYMV RNA. A mutant in the initiation codon of the 5.5 kb ORF did not replicate in protoplasts, while mutants in the initiation codon of the 1.9 kb ORF replicated at low levels. The two groups of mutants were not able to complement each other.

Isolation and characterization of Caenorhabditis elegans DNA sequences homologous to the v-abl oncogene.

DNA sequences homologous to the v-abl oncogene were isolated from a Caenorhabditis elegans genomic library by their ability to hybridize with a v-src probe. The DNA sequence of 2465 nucleotides of one clone was determined. This region corresponds to the 5' protein kinase domain of v-abl plus approximately equal to 375 base pairs toward the 3' end. Four potential introns were identified. The homology between the deduced amino acid sequence of the C. elegans clone and that of the 1.2-kilobase-pair protein kinase region of v-abl is 62%. The tyrosine residue corresponding to the tyrosine that is phosphorylated in the v-src protein is conserved in the C. elegans sequence. When 95 amino acids around this tyrosine were compared with the corresponding sequences of Drosophila c-abl, v-abl, and v-src, the identities were 83%, 79%, and 56%, respectively. Hybridization of the cloned DNA with C. elegans poly(A)+ RNA revealed a major transcript of 4.4 kilobases.