Cloning and expression analysis of Phytoplasma protein translocation genes.

Genes encoding SecA and SecY proteins, essential components of the Sec protein translocation system, were cloned from onion yellows phytoplasma, an unculturable plant pathogenic bacterium. The secA gene consists of 2,505 nucleotides encoding an 835 amino acid protein (95.7 kDa) and shows the highest similarity with SecA of Bacillus subtilis. Anti-SecA rabbit antibody was prepared from a purified partial SecA protein, with a histidine tag expressed in Escherichia coli. Western blot analysis confirmed that SecA protein (approximately 96 kDa) is produced in phytoplasma-infected plants. Immunohistochemical thin sections observed by optical microscopy showed that SecA is characteristically present in plant phloem tissues infected with phytoplasma. The secY gene consists of 1,239 nucleotides encoding a 413 amino acid protein (45.9 kDa) and shows the highest similarity with SecY of B. subtilis. These results suggest the presence of a functional Sec system in phytoplasmas. Because phytoplasmas are endocellular bacteria lacking cell walls, this system might secrete bacterial proteins directly into the host cytoplasm. This study is what we believe to be the first report of the sequence and expression analysis of phytoplasma genes encoding membrane proteins with a predicted function.

A plasmid isolated from phytopathogenic onion yellows phytoplasma and its heterogeneity in the pathogenic phytoplasma mutant.

A 3.6-kbp DNA fragment was cloned from the extrachromosomal DNA of a pathogenic plant mollicute, onion yellows phytoplasma (OY-W). Sequence analysis of the fragment revealed an open reading frame (ORF) encoding the replication (Rep) protein of rolling-circle replication (RCR)-type plasmids. This result suggests the existence of a plasmid (pOYW1) in OY-W that uses the RCR mechanism. This assumption was confirmed by detecting the single-stranded DNA (ssDNA) of a replication intermediate that is specifically produced by the RCR mechanism. This is the first report on the identification of the replication system of this plasmid and the genes encoded in it. With a DNA fragment including the Rep gene region of pOYW1 used as a probe, Southern and Northern (RNA) blot hybridizations were employed to examine the heterogeneity between the plasmids found in OY-W and a pathogenic mutant (OY-M) isolated from OY-W. Multiple bands were detected in the DNA and RNA extracted from both OY-W and OY-M infected plants, although the banding patterns were different. Moreover, the copy number of plasmids from OY-W was about 4.2 times greater than that from OY-M. These results indicate constructive heterogeneity between OY-W and OY-M plasmids, and the possibility of a relationship between the plasmid-encoded genes and the pathogenicity of the phytoplasma was suggested.

Complete nucleotide sequence of wheat yellow mosaic bymovirus genomic RNAs.

The complete sequences of wheat yellow mosaic bymovirus (WYMV) RNA1 and RNA2 were determined. RNA1 is 7636 nucleotides long [excluding the 3'-poly(A)], and codes for a 269 kDa polyprotein of 2,404 amino acids which contains the capsid protein (CP) at the C terminus and seven putative nonstructural proteins. RNA2 is 3,659 nucleotides long and codes for a polyprotein of 904 amino acids which contains a 28 kDa putative proteinase and a 73 kDa polypeptide. These functional proteins are arranged as in RNA1 and RNA2 of barley yellow mosaic bymovirus (BaYMV). Comparisons with the sequence reported for the 3' half of RNA1 of wheat spindle streak mosaic bymovirus (WSSMV) from Southern France show that WYMV and WSSMV have a similar genetic organization. However, WYMV and WSSMV share only 77% amino acid sequence identity in their deduced CPs in spite of their close serological relationship, and 74% nucleotide sequence identity in their 3' non-coding regions. Thus, the sequence data indicate that WYMV and WSSMV are not strains of the same virus, which has long been suggested, but are distinct virus species within the genus Bymovirus of the family Potyviridae.

Complete sequence of an infectious full-length cDNA clone of citrus tatter leaf capillovirus: comparative sequence analysis of capillovirus genomes.

The complete nucleotide sequence of citrus tatter leaf capillovirus (CTLV lily strain) was determined. It is 6496 nucleotides long, excluding the 3'-terminal poly(A) tract, and contains two putative overlapping open reading frames (ORFs). ORF1 (positions 37-6354) encodes a potential polyprotein of molecular mass 242 kDa. ORF2 (positions 4788-5750) codes for a 36 kDa protein. The 242 kDa polypeptide contains several non-structural protein domains (i.e. methyltransferase, NTP-binding helicase, papain-like proteinase and polymerase) and, at its C terminus, the putative coat protein. The N-terminal region of the 36 kDa protein displays sequence similarity to the cell-to-cell movement proteins of the '30 K superfamily'. Such a genome structure is conserved between CTLV and apple stem grooving capillovirus. Capped transcripts from a plasmid containing the complete sequence of CTLV, with a T7 RNA promoter, successfully infected Chenopodium quinoa plants and caused symptoms characteristic of CTLV. Uncapped transcripts were noninfectious.

Nucleotide sequence of the coat protein coding region of tulip breaking virus.

cDNA of tulip breaking virus-tulip (TBV-tulip) RNA was synthesized and cloned in E. coli. One clone that contains a 4.5 kb insert was identified by restriction enzyme analysis, dot immunobinding assay (DIBA), and partial sequencing. Then 1479 nucleotides of the 3'-terminus of the clone were sequenced and revealed that the sequence contains one open reading frame (ORF), followed by an untranslated region of 255 nucleotides and a poly(A) tract. The deduced amino acid sequence was found to include the C terminus of the predicted RNA-dependent RNA polymerase and the coat protein. A glutamine-alanine dipeptide was identified as a putative NIa protease cleavage site at the N terminus of the coat protein.

Nucleotide sequence of the 3'-terminal region of citrus tatter leaf virus RNA.

The 3'-terminal sequence of citrus tatter leaf virus lily isolate (CTLV-L) was determined from cloned cDNA. The sequence contains two open reading frames (ORFs). ORF1 encodes a protein that contains consensus sequences associated with the RNA-dependent RNA polymerase. ORF2, which is in a different reading frame within ORF1, can encode a 36 kD protein, putatively identified as a movement protein. CTLV-L coat protein (CP) was found to be located in the C-terminal region of the polyprotein encoded by ORF1. Evolutionary relationships and classification of capilloviruses is discussed.

Phylogenetic diversity of phytopathogenic mycoplasmalike organisms.

By using specific primers, the 16S rRNA genes of Japanese mycoplasmalike organisms (MLOs) were amplified by polymerase chain reactions from MLO-enriched fractions of plants infected with each of six different MLOs. Each of the polymerase chain reaction fragments (length, 1,370 nucleotides) was directly sequenced in both strands by using 17 oligonucleotide primers. A phylogenetic tree constructed by using the sequence data showed that these Japanese MLOs are phylogenetically diverse microorganisms that fall into three groups, group I (onion yellows, tomato yellows, mulberry dwarf, and paulownia witches' broom MLOs), group II (tsuwabuki witches' broom MLO), and group III (rice yellow dwarf MLO). A high level of sequence homology (99%) between the Oenothera hookeri MLO and the severe strain of the western aster yellows MLO on the one hand and group I MLOs on the other indicates that the O. hookeri MLO and the severe strain of the western aster yellows MLO belong to group I and suggests that these MLOs, isolated from two geographically separated locations, descended from a very similar ancestor. Although group I contains phylogenetically identical MLOs, the organisms are transmitted by diverse insect vectors. The three MLO groups are more closely related to Acholeplasma laidlawii than to Mycoplasma gallisepticum. Thus, although MLOs are phylogenetically diverse, they are evolutionarily distant from other mollicutes. These data, together with other information (including phylogenetic relationships, vector specificity, plant-pathogenic properties, and habitat in plant phloem sieve tubes), suggest that MLOs could be classified into at least three phylogenetic groups (groups I through III).

Electrotransfection of protoplasts from tomato, wild tomato, barley and chrysanthemum with tobacco mosaic virus RNA.

Protoplasts isolated from tomato, wild tomato, barley and chrysanthemum were electrotransfected with tobacco mosaic virus (TMV) RNA under almost the same optimum electric conditions: five square DC pulses of 50 microseconds duration at 500 to 800 V/cm, with the protoplasts suspended at 2 x 10(5)/ml in 0.5 M-mannitol containing 100 microM-MgCl2 and 10 to 20 micrograms/ml TMV RNA. ELISAs of these transfected protoplasts showed that the yields and the growth curves of the virus were quite similar, indicating a lack of host specificity in the initially infected cells of these plants.

Comparison of nonradioactive cDNA probes for detection of potato spindle tuber viroid by dot-blot hybridization assay.

Six nonradioactive cDNA probes were compared for their sensitivities for detecting potato spindle tuber viroid (PSTVd) by dot-blot hybridization assay. Three biotinylated PSTVd cDNA probes, labeled by photoactivation with photobiotin, by nick translation or by random priming with biotinylated deoxyribonucleotides, were all capable of detecting 20 pg of purified PSTVd by a colorimetric assay and 2-20 pg by a chemiluminescent assay. Digoxigenin-labeled probe was able to detect 200 pg of purified PSTVd. Two biotinylated probes prepared with polymerase chain reaction (PCR) incorporating biotinylated dUTP or dATP were the most sensitive: 0.2-2 pg of PSTVd was detectable by both assays. All six probes could detect PSTVd also in extracts of infected tomato leaves at a dilution of up to 1/250-1/1250. These nonradioactive probes are equal to radioactive probes in their sensitivity, and the biotinylated probes produced with PCR amplification are particularly suitable for practical diagnosis, as they are sensitive and rapidly prepared in large quantities.

Nucleotide sequence of the capsid protein gene of barley yellow mosaic virus.

The sequence of the 3'-terminal 1370 nucleotides of barley yellow mosaic virus (BaYMV) RNA 1 was determined. The sequence contains a long open reading frame (ORF) of 1137 nucleotides and a non-coding region of 231 nucleotides upstream of the poly(A) tail. Mapping of the partial amino acid sequences of the capsid protein onto the putative translational product of the ORF indicates that the 3'-proximal region of RNA 1 encodes the capsid protein which consists of 297 amino acids with an Mr of 32334; the capsid protein is produced by proteolytic processing from a precursor polypeptide at a glutamine-alanine dipeptide. The removal of the N- and C-terminal regions of the capsid protein by mild proteolysis of intact virus particles indicates that both terminal regions are exposed on the external surfaces of virus particles. Alignment of the BaYMV capsid protein sequence with those of some potyviruses showed only small blocks of homology which contrast with the extensive matches among potyviruses. This fact together with the genome organization and the vector specificity clearly distinguishes BaYMV from potyviruses.

The outer capsid protein of rice dwarf virus is encoded by genome segment S8.

The nucleotide sequence of DNA complementary to the eighth largest (S8) of the 12 genome segments of rice dwarf virus was determined. This genome segment is 1424 nucleotides in length and has a single long open reading frame extending 1260 nucleotides from the first AUG triplet (residues 24 to 26). The predicted translational product comprises 420 amino acids and has an Mr of 46,422. The amino acid sequences of several peptide fragments of the major outer capsid protein were found to be contained in the predicted translational product of the above nucleotide sequence. This protein, previously reported to be 43K, is encoded by genome segment S8 and therefore renamed the 46K protein.

Nucleotide sequence of segment S10 of the rice dwarf virus genome.

DNA complementary to the tenth largest (S10) of the 12 genome segments of rice dwarf virus (RDV) was cloned and its sequence was determined. This genome segment is 1319 nucleotides in length and has a single long open reading frame extending for 1059 nucleotides from the first AUG triplet (residues 27 to 29). The predicted translation product comprises 352 amino acids and has a mol. wt. of 39094. RDV transcripts synthesized in vitro have the same polarity as the plus strand of the genome. Terminal sequences were (+) 5' GGUA---UGAU 3' and (-) 3' CCAU---ACUA 5' which are similar to those of wound tumour virus RNA.