Genomic characterization of cycad leaf necrosis virus, the first badnavirus identified in a gymnosperm.

A previously undescribed badnavirus was isolated from Zamia fischeri showing symptoms of chlorosis, necrosis, and ringspot. The virus has bacilliform virions 30 nm in diameter and averaging 120 nm in length. The viral genome is 9227 bp in length and contains three open reading frames characteristic of members of the genus Badnavirus. The largest open reading frame (ORF3) encodes a putative polyprotein, with predicted domains including zinc finger, aspartic protease, reverse transcriptase (RT) and RNase H. The virus is tentatively named "cycad leaf necrosis virus" (CLNV). Within the genus Badnavirus, CLNV was most closely related to sugarcane bacilliform Guadeloupe D virus (FJ439817), sharing 69% identity at the nucleotide level in the RT + RNase H region. This virus is the first badnavirus reported to infect cycads, and it has the largest genome among the currently characterized badnaviruses.

Complete genome sequence of aglaonema bacilliform virus (ABV).

Aglaonema bacilliform virus (ABV), a member of the genus Badnavirus in the family Caulimoviridae, is associated with leaf deformation and chlorosis in Aglaonema modestum. The complete genome sequence of a Minnesota isolate of ABV was determined. The ABV genome is 7,178 bp in length and similar in size and organization to those of the members of the genus Badnavirus, containing three open reading frames (ORFs) with the potential to encode three proteins of 14.92, 13.33 and 207.95 kDa, respectively. The last ORF (ORF3) encodes a putative polyprotein with conserved domains, including zinc finger, aspartic protease, reverse transcriptase (RT) and RNase H domains, in that order. Phylogenetic analysis using the amino acid sequence of the ORF3 polyprotein showed that ABV clusters with several isolates of taro bacilliform CH virus (TaBCHV). Pairwise alignment using the highly conserved RT/RNase H region reveals that ABV has the highest level of identity (71%) at the nucleotide level to a Hawaiian isolate of TaBCHV.

Complete genome sequence of a previously undescribed badnavirus occurring in Polyscias fruticosa L. (Ming aralia).

A previously undescribed badnavirus was identified in plants of Polyscias fruticosa (Ming aralia) showing symptoms of mild mosaic and leaf senescence. Characteristic bacilliform virions of the Polyscias badnavirus averaging 30 × 120 nm in size were observed by transmission electron microscopy in partially purified leaf tissue extracts from symptomatic but not asymptomatic plants collected in the USA and Nigeria. The isolate from the USA was complete sequenced. The genome is 7592 bp in length and contains three open reading frames with an arrangement similar to that of other members of the genus Badnavirus. The largest open reading frame (ORF3) encodes a putative polyprotein, with predicted domains including zinc finger, aspartic protease, reverse transcriptase (RT) and RNase H, in that order. The USA and Nigeria isolates of the virus had a high level (98%) of nucleotide sequence identity in the RT+RNase H region. Within the genus Badnavirus, these viruses were most closely related to schefflera ringspot virus (SRV), sharing 63% identity at the nucleotide level. Based on the ICTV species demarcation criteria for the genus Badnavirus (more than 20% nucleotide sequence divergence in the RT+RNase H region), the Polyscias virus is proposed to be a new member of the genus, and the name polyscias mosaic virus (PoMV) is proposed. The complete genome sequence was deposited in the NCBI GenBank database under accession no. MH475918.

Identification of secret agent as the O-GlcNAc transferase that participates in Plum pox virus infection.

Serine and threonine of many nuclear and cytoplasmic proteins are posttranslationally modified with O-linked N-acetylglucosamine (O-GlcNAc). This modification is made by O-linked N-acetylglucosamine transferases (OGTs). Genetic and biochemical data have demonstrated the existence of two OGTs of Arabidopsis thaliana, SECRET AGENT (SEC) and SPINDLY (SPY), with at least partly overlapping functions, but there is little information on their target proteins. The N terminus of the capsid protein (CP) of Plum pox virus (PPV) isolated from Nicotiana clevelandii is O-GlcNAc modified. We show here that O-GlcNAc modification of PPV CP also takes place in other plant hosts, N. benthamiana and Arabidopsis. PPV was able to infect the Arabidopsis OGT mutants sec-1, sec-2, and spy-3, but at early times of the infection, both rate of virus spread and accumulation were reduced in sec-1 and sec-2 relative to spy-3 and wild-type plants. By matrix-assisted laser desorption ionization-time of flight mass spectrometry, we determined that a 39-residue tryptic peptide from the N terminus of CP of PPV purified from the spy-3 mutant, but not sec-1 or sec-2, was O-GlcNAc modified, suggesting that SEC but not SPY modifies the capsid. While our results indicate that O-GlcNAc modification of PPV CP by SEC is not essential for infection, they show that the modification has a role(s) in the process.

Characterisation of Banana streak Mysore virus and evidence that its DNA is integrated in the B genome of cultivated Musa.

We have sequenced the complete genome of an isolate of Banana streak virus from banana cv. 'Mysore' and show that it is sufficiently different from a previously characterised isolate from cv. 'Obino l'Ewai' to warrant recognition as a distinct species, for which the name Banana streak Mysore virus (BSMysV) is proposed. The structure of the BSMysV genome was typical of badnaviruses in general, although ORF I had a non-conventional start codon. Evidence that at least part of the BSMysV genome is integrated in the B genome of cultivated Musa is presented and transmissibility by the mealybug Planococcus citri also demonstrated.

The role of SPY and its TPR domain in the regulation of gibberellin action throughout the life cycle of Petunia hybrida plants.

SPY acts as a negative regulator of gibberellin (GA) action in Arabidopsis, but its mode of action and regulation are still unknown. SPY over-expression in transgenic petunia plants affected various GA-regulated processes, including seed germination, shoot elongation, flower initiation, flower development and the expression of a GA-induced gene, GIP. A similar phenotype was obtained when wild-type petunia plants were treated with the GA-biosynthesis inhibitor, paclobutrazol. The N-terminus of SPY contains tetratricopeptide repeats (TPR). TPR motifs participate in protein-protein interactions, suggesting that SPY is part of a multiprotein complex. To test this hypothesis, we over-expressed the SPY's TPR region without the catalytic domain in transgenic petunia and generated a dominant-negative SPY mutant. The transgenic seeds were able to germinate on paclobutrazol, suggesting an enhanced GA signal. We cloned the petunia SPY homologue, PhSPY, and showed that its mRNA level is not affected by GA or ABA. The results of this study support the role of SPY as a negative regulator of GA action, suggest that the TPR domain is required for the interaction with other proteins to form an active complex and indicate that different plants use similar mechanisms to transduce the GA signal.

Altered expression of SPINDLY affects gibberellin response and plant development.

Gibberellins (GAs) are plant hormones with diverse roles in plant growth and development. SPINDLY (SPY) is one of several genes identified in Arabidopsis that are involved in GA response and it is thought to encode an O-GlcNAc transferase. Genetic analysis suggests that SPY negatively regulates GA response. To test the hypothesis that SPY acts specifically as a negatively acting component of GA signal transduction, spy mutants and plants containing a 35S:SPY construct have been examined. A detailed investigation of the spy mutant phenotype suggests that SPY may play a role in plant development beyond its role in GA signaling. Consistent with this suggestion, the analysis of spy er plants suggests that the ERECTA (ER) gene, which has not been implicated as having a role in GA signaling, appears to enhance the non-GA spy mutant phenotypes. Arabidopsis plants containing a 35S:SPY construct possess reduced GA response at seed germination, but also possess phenotypes consistent with increased GA response, although not identical to spy mutants, during later vegetative and reproductive development. Based on these results, the hypothesis that SPY is specific for GA signaling is rejected. Instead, it is proposed that SPY is a negative regulator of GA response that has additional roles in plant development.

Ectopic expression of the tetratricopeptide repeat domain of SPINDLY causes defects in gibberellin response.

The SPINDLY (SPY) protein of Arabidopsis is a negative regulator of gibberellin (GA) response. The SPY protein has 10 copies of the tetratricopeptide repeat (TPR) at the N terminus. TPR motifs function as protein-protein interaction domains. Several spy alleles are affected only in the TPR region suggesting that protein-protein interactions mediated by this domain are important for proper GA signaling. We have used a reverse genetics approach to further investigate the role of the TPR domain. The TPR domain of SPY was overexpressed in wild-type, gai, and spy plants. Expression of the TPR domain alone is not sufficient to rescue spy mutants. Expression of the TPR domain in a wild-type background produces phenotypes similar to those caused by loss-of-function spy mutants including resistance to GA biosynthesis inhibitors, short hypocotyl length, and early flowering. The dwarfing of the floral shoot internodes caused by the gai mutation was suppressed by expression of the TRP domain. Expression of the TPR domain had no effect on the abundance of endogenous SPY mRNA. The TPR domain was found to interact with SPY both in vitro and in yeast two-hybrid assays. These data indicate that the TPR domain of SPY can participate in protein-protein interactions and that these interactions are important for the proper functioning of SPY.

Analysis of the distribution and structure of integrated Banana streak virus DNA in a range of Musa cultivars.

Summary Banana streak virus strain OL (BSV-OL) commonly infects new Musa hybrids, and this infection is thought to arise de novo from integrated virus sequences present in the nuclear genome of the plant. Integrated DNA (Musa6+8 sequence) containing the whole genome of the virus has previously been cloned from cv. Obino l'Ewai (Musa AAB group), a parent of many of the hybrids. Using a Southern blot hybridization assay, we have examined the distribution and structure of integrated BSV-OL sequences in a range of Musa cultivars. For cv. Obino l'Ewai, almost every restriction fragment hybridizing to BSV-OL was predicted from the Musa6+8 sequence, suggesting that this is the predominant type of BSV-OL integrant in the genome. Furthermore, since only two junction fragments of Musa/BSV sequence were detected, and the Musa6+8 sequence is believed to be integrated as multiple copies in a tandem array, then the internal Musa spacer sequences must be highly conserved. Similarly sized restriction fragments were detected in four BB group cultivars, but not in six AA or AAA group cultivars, suggesting that the BSV-OL sequences are linked to the B-genome of Musa. We also provide evidence that cv. Williams (Musa AAA group) contains a distinct badnavirus integrant that is closely related to the 'dead' virus integrant previously characterized from Calcutta 4 (Musa acuminata ssp. burmannicoides). Our results suggest that the virus integrant from cv. Williams is linked to the A-genome, and the complexity of the hybridization patterns suggest multiple sites of integration and/or variation in sequence and structure of the integrants.

Characterization and genomic analysis of tobacco vein clearing virus, a plant pararetrovirus that is transmitted vertically and related to sequences integrated in the host genome.

A previously undescribed caulimo-like virus was identified in the hybrid tobacco species Nicotiana edwardsonii, and was named tobacco vein clearing virus (TVCV) after the symptoms associated with its occurrence in this plant. The virions of TVCV are 50 nm in diameter and are composed of a 45 kDa capsid protein and a 7767 bp dsDNA genome. Each strand of the genome is interrupted by a site-specific discontinuity. In genome sequence and arrangement of ORFs TVCV was most similar to cassava vein mosaic virus, indicating that TVCV is a pararetrovirus. No serological relationship was detected between TVCV and any other caulimoviruses, including petunia vein clearing virus, which has similar biological properties. In N. edwardsonii TVCV was seed-transmitted to 100% of progeny plants, but was not transmitted by mechanical inoculation, grafting or Myzus persicae to any of seven other Nicotiana spp. Genomic DNA of TVCV hybridized to genomic DNA of N. edwardsonii and of N. glutinosa, its male parent, but not to genomic DNA of N. clevelandii, the female parent. TVCV has 78% sequence identity with pararetrovirus-like sequences that are present in high copy number in the N. tabacum genome, and TVCV genomic DNA hybridized to genomic DNA of N. tabacum and N. rustica. These observations suggest that the episomal form of TVCV may arise from integrated pararetroviral elements present in N. edwardsonii, that these integrants were inherited from the male parent N. glutinosa, and that these elements are related but not identical to pararetroviral elements occurring in other Nicotiana spp.

Evidence that badnavirus infection in Musa can originate from integrated pararetroviral sequences.

When some virus- and disease-free Musa spp. (banana and plantain) are propagated by tissue culture, the resulting plants develop infections with banana streak badnavirus (BSV), a pararetrovirus. In sharp contrast to the virion DNA recovered from natural infections, the virion DNA from tissue culture-associated infections of different Musa spp. was highly similar if not identical. Although BSV does not employ integration during the infection cycle, BSV DNA was found to be integrated into the Musa genome. While one integration consisted of a partial BSV genome, a second contained more than one complete genome that was almost identical to BSV recovered from tissue culture-derived plants. The arrangement of this integrated BSV DNA suggests that it can yield an infectious episomal genome via homologous recombination. This report documents the first instance of integrated DNA of a nonintegrating virus giving rise to an episomal viral infection and identifies tissue culture as a possible trigger for the infection, raising the question of whether similar activatable viral sequences exist in the genomes of other plants and animals.

Gibberellin and abscisic acid regulate GAST1 expression at the level of transcription.

Both gibberellic acid (GA3) and abscisic acid (ABA) regulate the expression of the GAST1 gene of tomato. Treatment with GA3 increases the abundance of GAST1 RNA while treatment with ABA blocks this effect. In this study, the effects of GA3 and ABA on the rate of transcription of the GAST1 gene and the stability of GAST1 RNA were examined. Nuclear run-on analyses detected an increase in transcription of the GAST1 gene 1 h after GA3 treatment with transcription increasing to a maximum at 9 h after treatment. The half-life of GAST1 RNA in GA3-treated leaves was similar to that in control leaves. In addition, the extent of overexpression of GAST1 RNA in transgenic tomato plants containing the CaMV 35S promoter driving the expression of the GAST1 transcribed region was largely unaffected by GA3. These results suggest that GA3 stimulates the expression of the GAST1 gene by acting only at the level of transcription. ABA treatment dramatically reduced the abundance of GAST1 RNA in gib1 shoots through an effect at the level of transcription and did not appear to affect the stability of this RNA. Midcourse ABA addition to the GA3-incubated shoots reversed the GA3-mediated increase in the transcription of GAST1 gene within 15 min. Transgenic plants that either overexpressed or underexpressed GAST1 RNA exhibited no phenotypic differences from wild type.

The sugarcane bacilliform badnavirus promoter is active in both monocots and dicots.

Regions of the sugarcane bacilliform badnavirus genome were tested for promoter activity. The genomic region spanning nucleotides 5999-7420 was shown to possess promoter activity as exemplified by its ability to drive the expression of the coding region of the uidA gene of Escherichia coli, in both Avena sativa and Arabidopsis thaliana. In A. sativa, the promoter was active in all organs examined and, with the exception of the anthers where the expression was localized, this activity was constitutive. In A. thaliana, the promoter activity was constitutive in the rosette leaf, stem, stamen, and root and limited primarily to vascular tissue in the sepal and the silique. The transgene was inherited and active in progeny plants of both A. sativa and A. thaliana.

Identification of a negative regulator of gibberellin action, HvSPY, in barley.

To broaden our understanding of the molecular mechanisms of gibberellin (GA) action, we isolated a spindly clone (HvSPY) from barley cultivar Himalaya and tested whether the HvSPY protein would modulate GA action in barley aleurone. The HvSPY cDNA showed high sequence identity to Arabidopsis SPY along its entire length, and the barley protein functionally complemented the spy-3 mutation. HvSPY and SPY proteins showed sequence relatedness with animal O-linked N-acetylglucosamine transferases (OGTs), suggesting that they may also have OGT activity. HvSPY has a locus distinct from that of Sln, a mutation that causes the constitutive GA responses of slender barley, which phenotypically resembles Arabidopsis spy mutants. The possibility that the HvSPY gene encodes a negative regulator of GA action was tested by expressing HvSPY in a barley aleurone transient assay system. HvSPY coexpression largely abolished GA3-induced activity of an alpha-amylase promoter. Surprisingly, HvSPY coexpression increased reporter gene activity from an abscisic acid (ABA)-inducible gene promoter (dehydrin), even in the absence of exogenous ABA. These results show that HvSPY modulates the transcriptional activities of two hormonally regulated promoters: negatively for a GA-induced promoter and positively for an ABA-induced promoter.

Tubules containing virions are present in plant tissues infected with Commelina yellow mottle badnavirus.

Tubular structures containing bacilliform virions were observed in cell-free extracts of Commelina diffusa infected with Commelina yellow mottle badnavirus (CoYMV). The exterior of the tubule reacted with antibodies to CoYMV movement protein, but not with antibodies to virus coat protein. Similar tubular structures containing bacilliform particles were also observed in ultrathin sections of CoYMV-infected C. diffusa. These tubular structures traversed the cell wall at points where this was thickened or protruded. No similar structures were observed in healthy C. diffusa. These observations support the hypothesis that the virion-containing tubular structures observed in cell-free extracts are the same as those observed in situ, that these structures are composed, at least in part, of virus movement protein, and that they play a role in the cell-to-cell trafficking of virions of CoYMV.

SPINDLY's role in the gibberellin response pathway.

The SPINDLY (SPY) locus of Arabidopsis thaliana is believed to be involved in gibberellin (GA) signal transduction. The six known mutations at this locus cause a phenotype that is consistent with constitutive activation of the GA signal transduction pathway. spy alleles are epistatic to gai, a mutation conferring gibberellin-insensitivity, indicating that SPY acts as a negative regulator of GA signal transduction, downstream of GAI. SPY was cloned using a T-DNA insertion in the spy-4 allele. SPY encodes a 914 amino acid protein with an N-terminal TPR region (a likely protein-protein interaction domain) and a novel C-terminal domain. The spy mutants show that both the N- and C-terminal domains of SPY are functionally important, spy-4 is likely to be a null allele and displays some morphological defects not seen in the other alleles. A 35S:SPY construct rescues the spy mutant phenotype, but does not show any gain-of-function SPY phenotypes. Smaller constructs overexpressing different domains of the SPY protein have no effect on plant development.

Expression of the Commelina yellow mottle virus promoter in transgenic oat.

The Commelina yellow mottle virus (CoYMV) infects the monocot weed Commelina diffusa. The objective of this study was to investigate the transgene expression conferred by the CoYMV promoter in a monocot species. Friable, embryogenic oat (Avena sativa L.) tissue cultures were stably transformed with the CoYMV promoter fused to the coding region of E. coli ß-glucuronidase (uidA, GUS). Developmental and tissue-specific expression of the CoYMV-GUS construct was investigated in regenerated plants and their progeny. Histochemical GUS staining was primarily localized in the vascular tissues of shoots, leaves, floral bracts and in roots. While ovaries stained intensely, no staining was detected in anthers or the endosperm in mature seed. The scutellum of mature and germinating seed exhibited GUS activity.

The N-terminal portion of the 216-kDa polyprotein of Commelina yellow mottle badnavirus is required for virus movement but not for replication.

Commelina yellow mottle virus (CoYMV) is the type member of the badnaviruses, a genus of plant pararetroviruses. The N-terminus of the polyprotein encoded by ORF III has limited similarity to known cell-to-cell movement proteins. To test the hypothesis that the N-terminus is required for viral movement, the phenotypes caused by mutations constructed in this region were determined. Similar to mutants affected in the reverse transcriptase, mutants affected in the putative movement protein were unable to cause a systemic infection. However, when the abilities of the mutated viral genomes to direct virion assembly and replication were tested using an in vitro stem-culture system, the mutants affected in the putative movement protein were found to assemble virions, whereas the reverse transcriptase mutants were unable to do so. Moreover, the putative movement protein mutants were shown to be replication competent by detection and mapping of one of the genomic discontinuities that are the hallmark of replication by reverse transcription. Thus the N-terminal region of ORF III is required for the systemic movement but not for the replication of CoYMV.

Isolation and characterization of abscisic acid-deficient Arabidopsis mutants at two new loci.

Novel Arabidopsis mutants with lowered levels of endogenous abscisic acid (ABA) were isolated. These were selected in a screen for germination in the presence of the gibberellin biosynthesis inhibitor paclobutrazol. Another mutant was isolated in a screen for NaCl tolerance. The ABA-deficiency was caused by two monogenic, recessive mutations, aba2 and aba3, that were both located on chromosome 1. The mutants showed a phenotype that is known to be characteristic for ABA-deficiency: a reduced seed dormancy and excessive water loss, leading to a wilty phenotype. Double mutant analysis, combining different aba mutations, indicated the leaky nature of the mutations.

The ORF I and II proteins of Commelina yellow mottle virus are virion-associated.

Antibodies were prepared against bacterially expressed Commelina yellow mottle badnavirus (CoYMV) proteins. Antiserum against purified virions and antiserum against the C-terminus of the putative coat protein-encoding region of ORF III detected the same virus-specific proteins, indicating that the CoYMV coat protein is encoded in ORF III. In addition to the two major forms of the coat protein (37 and 39 kDa), several high molecular weight virus-specific proteins were detected when virions were isolated without chloroform treatment. These proteins are possible ORF III polyprotein processing intermediates and might be associated with "immature" virions which are eliminated by chloroform treatment. As predicted by the genomic sequence, a 20-kDa virus-specific protein was detected by an antiserum raised against the C-terminus of the putative ORF I protein. Results of filtration experiments suggest that the ORF I protein is equally associated with virions and with plant component(s). The association between the ORF I protein and the virions was further confirmed using immunosorbent electron microscopy and immunogold labeling. The ORF I protein was not detected in virus preparations treated with chloroform, and colocalized with virions containing immature coat protein on sucrose-cesium sulfate density gradients, suggesting that it is associated with immature virions. An antiserum raised against the putative ORF II gene product detected a 15-kDa virus-specific protein whose association with the virions was unaffected by chloroform treatment. The ORF II protein was found to be sensitive to some protease(s) that copurified with the virions, and protease inhibitors preventing this degradation have been identified.