Ben Lockhart

Banana and Plantain

Banana and plantain (Musa spp.) are grown in more than 125 countries (Anon., 2001) and are a major source of carbohydrate for more than 400 million people (Swennen et al, 1995). The progenitors of almost all domesticated Musa are two wild species, Musa acuminata and M. balbisiana, whose genomes are represented using the letter codes A and B, respectively. The most commonly cultivated cultivars have the triploid genotypes AAA, AAB or ABB.

An analysis of the complete sequence of a sugarcane bacilliform virus genome infectious to banana and rice.

The genome of sugarcane bacilliform virus (ScBV), a badnavirus, consists of a circular dsDNA. The complete sequence of a cloned infective ScBV genome is reported here. The genome is 7568 bp in size and possesses a number of features suggesting that ScBV is a pararetrovirus. A tRNA(Met)-binding site that may serve as a primer for minus-strand synthesis is present. The plus-strand of the ScBV genome contains three open reading frames (ORFs) which are capable of encoding proteins with calculated M(r) values of 22K, 13K and 215K. The 215K protein has regions with similarity to the RNA-binding domains, aspartic proteases and replicases of retro-elements. In addition, the 215K protein also has a region with restricted similarity to the intercellular transport proteins of plant viruses. Comparisons with the other sequenced badnaviruses, Commelina yellow mottle (CoYMV) and rice tungro bacilliform (RTBV) viruses, indicate that the arrangement of the ORFs in these viruses is conserved. Located next to the putative RNA-binding domain is a cysteine-rich region that is unique to the badnaviruses. When the molecular relationships of a portion of the reverse transcriptases of plant pararetroviruses were determined, two badnaviruses, CoYMV and ScBV, form one distinct cluster, whereas three caulimoviruses, cauliflower mosaic virus, carnation etched ring virus and figwort mosaic virus, form a second cluster. The badnavirus RTBV and the caulimovirus soybean chlorotic mottle virus occupy intermediate positions between the clusters. When introduced by Agrobacterium-mediated inoculation, a construct containing 1.1 copies of the cloned ScBV genome is infectious to both rice and banana.

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.

Effect of Temperature on Symptom Expression and Reliability of Banana Streak Badnavirus Detection in Naturally Infected Plantain and Banana (Musa spp.)

The effect of temperature on symptom expression and detection of banana streak badnavirus (BSV) by immunosorbent electronmicroscopy (ISEM) and enzyme-linked immunosorbent assay of 12 in vitro-propagated plantain hybrids (genome AAB × AA), 3 ABB cooking banana, and 3 AAB plantain landraces was studied. Experiments were done for 2 years under two temperature regimes, 28 to 35°C in a screenhouse and 22°C in a temperature-controlled room. Most BSV-infected plants of plantain hybrids expressed symptoms under both conditions. Symptom expression was enhanced when plants were continuously grown at 22°C, but later became indiscernible when plants were continuously grown at 28 to 35°C. Plants grown at 22°C and showing severe symptoms contained significantly higher virus titer than plants grown at 28 to 35°C. When asymptomatic plants with very low virus titer at 28 to 35°C were transferred back to 22°C, there was a significant increase in both symptom severity and concentration of virus (greater than 3 to 5 times) in leaf tissues after 9 months. In contrast, the concentration of virus and symptom severity decreased in plants after transfer from 22°C to 28 to 35°C. Micropropagated plants of AAB plantain landrace cv. Mimi Abue and ABB cooking bananas (cvs. Bluggoe, Cardaba, and Pelipita) did not express visible symptoms under either temperature regime, but BSV was detected by ISEM in 23% of the plants. After 2 years at 22°C, virus was detected in 64% of the plants, but the concentration of virus remained low. Implications of these results on quarantine screening of in vitro plants and virus diagnosis are discussed.

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.

Complete nucleotide sequence of Rosa rugosa leaf distortion virus, a new member of the family Tombusviridae

This report describes the complete nucleotide sequence and genome organization of Rosa rugosa leaf distortion virus (RrLDV), the causal agent of a previously undescribed virus disease of Rosa rugosa. The RrLDV genome is a positive-sense ssRNA, 3971 nucleotides in length, containing five open reading frames (ORFs). ORF1 encodes a 27-kDa peptide (p27). ORF2 shares a common start codon with ORF1 and continues through the amber stop codon of p27 to produce an 87-kDa protein (p87) with amino acid sequence similarity to the RNA-dependent RNA polymerases (RdRp) of members of the family Tombusviridae. ORF3 encodes a protein of 8 kDa with no significant similarity to known viral sequences. ORF4 encodes a 6-kDa protein (p6) with similarity to the p13 movement proteins of members of the family Tombusviridae. ORF5 has no conventional start codon and overlaps with p6. A putative +1 frame shift mechanism allows p6 translation to continue through the stop codon and results in a 12-kDa protein with high homology to the carmovirus p13 movement protein. The 37-kDa protein encoded by ORF6 has amino acid sequence similarity to coat proteins (CPs) of members of the family Tombusviridae. Phylogenetic analyses of the RdRp and CP amino acid sequences placed RrLDV in a subgroup close to members of the genus Carmovirus of the family Tombusviridae.

Sequencing, Improved Detection, and a Novel Form of Kalanchoë top-spotting virus

Virions of Kalanchoë top-spotting virus (KTSV) were purified from infected leaf tissue of Kalanchoë blossfeldiana using a procedure that prevented loss of virus in the initial extraction step. The double-stranded DNA viral genome was cloned and sequenced. The KTSV genome was 7,591 bp in size and contained three open reading frames capable of encoding proteins of 21, 14, and 223 kDa, respectively. The size and organization of the KTSV genome were similar to those of other mealybug-transmitted badnaviruses. Several oligonucleotide primer pairs, based on the KTSV genomic sequence, were used to efficiently detect the virus in plants, thereby removing a major constraint to reliable screening of kalanchoë propagating stock and breeding lines for KTSV infection. Two KTSV sequences, one symptom-inducing and the other not, were identified and differentiated by polymerase chain reaction (PCR) amplification and digestion of the resulting amplicon with restriction endonucleases. Preliminary results from graft-transmission tests and PCR indexing suggest that the nonsymptomatic form of KTSV may represent an integrated viral element. The occurrence of such integrated pararetroviral elements poses practical problems for routine PCR indexing of breeding and propagating stock, and also raises the possibility of symptomatic episomal infections arising from these viral integrants.

Ortervirales: a new viral order unifying five families of reverse-transcribing viruses

Reverse-transcribing viruses, which synthesize a copy of genomic DNA from an RNA template, are widespread in animals, plants, algae and fungi (1, 2).….

Complete nucleotide sequence of clematis chlorotic mottle virus, a new member of the family Tombusviridae

Clematis chlorotic mottle virus (ClCMV) is a previously undescribed virus associated with symptoms of yellow mottling and veining, chlorotic ring spots, line pattern mosaics, and flower distortion and discoloration on ornamental Clematis. The ClCMV genome is 3,880 nt in length with five open reading frames (ORFs) encoding a 27-kDa protein (ORF 1), an 87-kDa replicase protein (ORF 2), two centrally located movement proteins (ORF 3 and 4), and a 37-kDa capsid protein (ORF 5). Based on morphological, genomic, and phylogenetic analysis, ClCMV is predicted to be a member of the genus Pelarspovirus in the family Tombusviridae.

Complete nucleotide sequence of rose yellow leaf virus, a new member of the family Tombusviridae

The genome of the rose yellow leaf virus (RYLV) has been determined to be 3918 nucleotides long and to contain seven open reading frames (ORFs). ORF1 encodes a 27-kDa peptide (p27). ORF2 shares a common start codon with ORF1 and continues through the amber stop codon of p27 to encode an 87-kDa (p87) protein that has amino acid similarity to the RNA-dependent RNA polymerase (RdRp) of members of the family Tombusviridae. ORFs 3 and 4 have no significant amino acid similarity to known functional viral ORFs. ORF5 encodes a 6-kDa (p6) protein that has similarity to movement proteins of members of the Tombusviridae. ORF5A has no conventional start codon and overlaps with p6. A putative +1 frameshift mechanism allows p6 translation to continue through the stop codon and results in a 12-kDa protein that has high homology to the carmovirus p13 movement protein. The 37-kDa protein encoded by ORF6 has amino acid sequence similarity to coat proteins (CP) of members of the Tombusviridae. ORF7 has no significant amino acid similarity to known viral ORFs. Phylogenetic analysis of the RdRp amino acid sequences grouped RYLV together with the unclassified Rosa rugosa leaf distortion virus (RrLDV), pelargonium line pattern virus (PLPV), and pelargonium chlorotic ring pattern virus (PCRPV) in a distinct subgroup of the family Tombusviridae.