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R G Dietzgen

Publications and source records attributed to R G Dietzgen.

At least 19 recordsLinked to original sources
PubMed2005

Nucleocapsid gene variability reveals two subgroups of Lettuce necrotic yellows virus.

The complete nucleocapsid (N) genes of eight Australian isolates of Lettuce necrotic yellows virus (LNYV) were amplified by reverse transcription PCR, cloned and sequenced. Phylogenetic analyses of these sequences revealed two distinct subgroups of LNYV isolates. Nucleotide sequences within each subgroup were more than 96% identical but heterogeneity between groups was about 20% at the nucleotide sequence level. However, less than 4% heterogeneity was noted at the amino acid level, indicating mostly third nucleotide position changes and a strong conservation for N protein function. There was no obvious geographical or temporal separation of the subgroups in Australia.

Australia↗
PubMed2001

Promoters for pregenomic RNA of banana streak badnavirus are active for transgene expression in monocot and dicot plants.

Two putative promoters from Australian banana streak badnavirus (BSV) isolates were analysed for activity in different plant species. In transient expression systems the My (2105 bp) and Cv (1322 bp) fragments were both shown to have promoter activity in a wide range of plant species including monocots (maize, barley, banana, millet, wheat, sorghum), dicots (tobacco, canola, sunflower, Nicotiana benthamiana, tipu tree), gymnosperm (Pinus radiata) and fern (Nephrolepis cordifolia). Evaluation of the My and Cv promoters in transgenic sugarcane, banana and tobacco plants demonstrated that these promoters could drive high-level expression of either the green fluorescent protein (GFP) or the beta-glucuronidase (GUS) reporter gene (uidA) in vegetative plant cells. In transgenic sugarcane plants harbouring the Cv promoter, GFP expression levels were comparable or higher (up to 1.06% of total soluble leaf protein as GFP) than those of plants containing the maize ubiquitin promoter (up to 0.34% of total soluble leaf protein). GUS activities in transgenic in vitro-grown banana plants containing the My promoter were up to seven-fold stronger in leaf tissue and up to four-fold stronger in root and corm tissue than in plants harbouring the maize ubiquitin promoter. The Cv promoter showed activities that were similar to the maize ubiquitin promoter in in vitro-grown banana plants, but was significantly reduced in larger glasshouse-grown plants. In transgenic in vitro-grown tobacco plants, the My promoter reached activities close to those of the 35S promoter of cauliflower mosaic virus (CaMV), while the Cv promoter was about half as active as the CaMV 35S promoter. The BSV promoters for pregenomic RNA represent useful tools for the high-level expression of foreign genes in transgenic monocots.

Badnavirus↗
PubMed2000

Real-time RT-PCR fluorescent detection of tomato spotted wilt virus.

A real-time reverse transcription-polymerase chain reaction assay based on TaqMan chemistry was developed for the detection and quantification of tomato spotted wilt virus (TSWV). This method enabled sensitive, reproducible and specific detection of TSWV in 'leaf soak' and total RNA extracts from infected plants. TaqMan reliably detected TSWV in as little as 500 fg total RNA. The assay was 10-fold more sensitive than visualisation of ethidium bromide-stained bands following agarose gel electrophoresis. TSWV isolates from various crops and locations were detected with a cycle threshold of 20-26 in 1 ng total RNA extracted from fresh or freeze-dried leaves. A dilution series of in vitro transcripts from a cloned 628 base pair fragment of TSWV S RNA served as standard for quantification of viral template in infected leaf samples. The TaqMan assay detected reproducibly 1000 molecules of the target transcript.

Base Sequence↗
PubMed2000

Development of a multiplex immunocapture PCR with colourimetric detection for viruses of banana.

A multiplex, immunocapture PCR (M-IC-PCR) was developed for the simultaneous detection of three viruses from crude sap extracts of banana and plantain (Musa spp.). A reverse transcription step was required for Banana bract mosaic virus and Cucumber mosaic virus, which have ssRNA genomes. The detection of Banana bunchy top virus (ssDNA genome) was not adversely affected by inclusion in this step. All the three viruses could be detected simultaneously from a mixed infection. Identification and detection of individual viruses was achieved through the visualisation of discretely sized PCR amplicons by gel electrophoresis. Alternatively, a colourimetric microplate detection system utilising digoxigenin-labelled virus-specific probes was used. The latter assay was up to five times more sensitive than detection by gel electrophoresis and between 25 and 625 times more sensitive than ELISA for the various viruses. Careful selection of PCR primers was necessary to ensure the detection of a wide range of virus isolates and to avoid detrimental interactions between heterologous templates and primers.

Badnavirus↗
PubMed1999

A promoter from sugarcane bacilliform badnavirus drives transgene expression in banana and other monocot and dicot plants.

A 1369 bp DNA fragment (Sc) was isolated from a full-length clone of sugarcane bacilliform badnavirus (ScBV) and was shown to have promoter activity in transient expression assays using monocot (banana, maize, millet and sorghum) and dicot plant species (tobacco, sunflower, canola and Nicotiana benthamiana). This promoter was also tested for stable expression in transgenic banana and tobacco plants. These experiments showed that this promoter could drive high-level expression of the beta-glucuronidase (GUS) reporter gene in most plant cells. The expression level was comparable to the maize ubiquitin promoter in standardised transient assays in maize. In transgenic banana plants the expression levels were variable for different transgenic lines but was generally comparable with the activities of both the maize ubiquitin promoter and the enhanced cauliflower mosaic virus (CaMV) 35S promoter. The Sc promoter appears to express in a near-constitutive manner in transgenic banana and tobacco plants. The promoter from sugarcane bacilliform virus represents a useful tool for the high-level expression of foreign genes in both monocot and dicot transgenic plants that could be used similarly to the CaMV 35S or maize polyubiquitin promoter.

Badnavirus↗
PubMed1998

Sequences of the coat protein gene of five peanut stripe virus (PStV) strains from Thailand and their evolutionary relationship with other bean common mosaic virus sequences.

The coat protein gene and part of the 3' non-coding region of five strains of peanut stripe virus (PStV) from Thailand have been cloned and sequenced. Phylogenetic comparisons of these strains, known as T1, T3, T5, T6 and T7, and related sequences showed that these strains are indeed strains of PStV. Further, PStV strains appear to be related to each other according to their geographic origin. That is, the Thai strains are more closely related to each other than they are to strains from the USA or Indonesia, despite the variety of symptoms caused by these strains and the overlap of symptom types between the strains from different locations. Like other PStV strains, PStV-Thai can be considered strains of bean common mosaic virus (BCMV) but can be distinguished from bean-infecting strains of BCMV and blackeye cowpea mosaic virus (B1CMV) through sequence and host range. No evidence was found that PStV-Thai strains, unlike PStV-Ib, are recombinants of PStV and B1CMV, although the T3 strain may be a recombinant of different PStV sequences. Phylogenetic analyses of viruses of the BCMV group suggest that acquisition of the ability to infect peanut may have occurred only once.

Amino Acid Sequence↗
PubMed1998

Retrotransposon-like sequences integrated into the genome of pineapple, Ananas comosus.

Retrotransposon-like sequences have been serendipitously detected in the genome of commercial pineapple, Ananas comosus. The sequence from a 2.6 kb cloned fragment of this element had greatest similarity to the del1 Lilium henryi retrotransposon and the gypsy/Ty3 group of retroelements. The order of the genes from 5' to 3' was reverse transcriptase, ribonuclease H and integrase. The integrase domain contained the amino acid sequence motifs which have been associated with recognition of the long terminal repeats and with the cutting/joining reactions required for integration of similar retroelements into the host genome. The retrotransposon existed as a population of variable sequences which were dispersed throughout the genome of pineapple. Southern hybridisation showed that the retrotransposon had integrated repeatedly into the pineapple genome. The reading frame of the element was not interrupted by stop codons, suggesting that it is still potentially capable of transposing. This is the first report of a retrotransposon in pineapple, which we have called deal (for dispersed element of Ananas).

Amino Acid Sequence↗
PubMed1995

Detection of DNA and RNA plant viruses by PCR and RT-PCR using a rapid virus release protocol without tissue homogenization.

A simple, single-step plant tissue preparation protocol suitable for the detection of viruses by the polymerase chain reaction and reverse transcription-polymerase chain reaction is described. The effect of buffer components and pH, and the incubation temperature for the release of virus from plant material was evaluated. A small amount of plant tissue was heated in a solution containing 100 mM Tris-HCl, pH 7.4 or 8.4, 1 M KCl and 10 mM EDTA for 10 min at 95 degrees C and the supernatant used for enzymatic amplification. This protocol was suitable for the detection of both DNA and RNA viruses in a variety of plant species and tissues and reduced plant inhibitory factors which may interfere with PCR. The application of this method was demonstrated for the detection of banana bunchy top virus in banana leaves, root and corn, zucchini yellow mosaic potyvirus in squash leaves and lettuce necrotic yellows rhabdovirus in lettuce and Nicotiana glutinosa leaves.

Base Sequence↗
PubMed1995

Identification of Zucchini yellow mosaic potyvirus by RT-PCR and analysis of sequence variability.

A reverse transcription-polymerase chain reaction (RT-PCR) method was used to identify Zucchini yellow mosaic virus (ZYMV) in leaves of infected cucurbits. Oligonucleotide primers which annealed to regions in the nuclear inclusion body (NIb) and the coat protein (CP) genes, generated a 300-bp product from ZYMV and also from the closely related watermelon mosaic virus type 2 (WMV-2). However, no product was obtained from papaya ringspot potyvirus which also infects cucurbits. ZYMV and WMV-2 were differentiated using a third primer which was complementary to a sequence in the 3'-untranslated region; a 1186-bp amplified product was obtained for ZYMV only. Nucleotide sequence analysis of the 300-bp fragments of Australian ZYMV and WMV-2 strains revealed 93.7-100% sequence identity between ZYMV strains. Multiple sequence alignments indicated that the nucleotide sequence which codes for the N-terminus of the CP was 74-100% identical for different isolates of ZYMV. The Australian isolate of WMV-2 was 43-46% identical to all isolates of ZYMV and was 84.6% identical to a Florida isolate of WMV-2.

Amino Acid Sequence↗
PubMed1994

Analysis of the nucleocapsid gene of lettuce necrotic yellows rhabdovirus.

The complete nucleotide sequence of the nucleocapsid (N) gene of lettuce necrotic yellows virus (LNYV), the type member of the genus Cytorhabdovirus of plant rhabdoviruses, has been determined from cDNA clones of both the viral genomic and messenger RNAs. The previously identified N gene was adjacent to the 3' leader RNA and began at position 91 from the 3' end of the LNYV genome. Analysis of the sequence showed that the N mRNA contained a 78-nt 5' untranslated region, followed by a 1377-nt open reading frame (ORF) encoding a 459-amino-acid protein. This ORF was similar in size to the N protein ORFs of other rhabdoviruses. However, little if any direct sequence homology was found with N protein sequences of other rhabdoviruses. Short amino acid sequences were found to be conserved between the N proteins of LNYV and sonchus yellow net virus (SYNV), a member of the genus Nucleorhabdovirus of plant rhabdoviruses. Among them, a sequence of 24 amino acids which was similar between LNYV and SYNV N proteins corresponded to a region with high homology between isolates of vesicular stomatitis virus and rabies virus N protein sequences, the type members of the genera Vesiculovirus and Lyssavirus of animal rhabdoviruses, respectively.

Amino Acid Sequence↗
PubMed1994

Genomic organization of lettuce necrotic yellows rhabdovirus.

We have mapped the genome of lettuce necrotic yellows virus (LNYV), the type member of the genus cytorhabdovirus of the family Rhabdoviridae. We have cloned and sequenced all intergenic regions and the 3' leader and 5' trailer of the negative-sense, single-stranded RNA genome of LNYV. The LNYV genome appears to contain six genes, the five expected genes coding for the virion proteins, and a sixth gene of unknown function, as for sonchus yellow net virus (SYNV), a member of the genus nucleorhabdovirus. The proposed LNYV genomic map is 3'-N-4a-4b-M-G-L-5', where N is the nucleocapsid protein gene; 4a and 4b are two genes, one of which codes for the proposed phosphoprotein P and the other for a putative protein of unknown function; M is the proposed matrix protein gene; G is the proposed glycoprotein gene; and L is the proposed transcriptase gene. The different LNYV intergenic regions have highly conserved consensus sequences, which could be divided into three components: the sequences corresponding to the 3' end of the mRNAs, intergenic sequences of variable length, and the sequences corresponding to the 5' end of the mRNAs. A leader sequence of 84 nucleotides (nt) at the 3' end of the LNYV genomic RNA preceeded the N gene. A trailer sequence of 187 nt at the 5' end of the genomic RNA followed the L gene. A comparison between LNYV leader and trailer sequences revealed complementary 3' and 5' ends, which could give rise to a putative "panhandle" structure with a two bases overhang in the leader sequence. We have compared these sequences to the corresponding sequences of SYNV as well as to vesicular stomatitis virus (VSV) and rabies virus (RV), the type members of the vesiculovirus and lyssavirus genera, respectively, of animal rhabdoviruses. Homologies were found in the intergenic regions between LNYV, SYNV, VSV, and RV, at the 3' ends of the mRNAs. LNYV intergenic sequences were of variable lengths, as were those found in RV. The consensus sequences found at the 5' ends of LNYV mRNAs differed from the highly conserved consensus transcription start sequence UUGU/A found in SYNV, VSV, and RV. Conserved sequences were also found in the first 30 nt of the leader and the last 30 nt of the trailer, between LNYV, SYNV, VSV, and RV.

Base Sequence↗
PubMed1994

Cloning and sequence analysis of the coat protein genes of an Australian strain of peanut mottle and an Indonesian 'blotch' strain of peanut stripe potyviruses.

We have analysed the coat protein gene sequences of two potyviruses infecting peanut. The 3' terminal 1247 nucleotides (nt) of an Australian strain of peanut mottle virus (PeMoV-AU) and the 3' terminal 1388 nt of an Indonesian 'blotch' strain of peanut stripe virus (PStV-Ib) were cloned and sequenced. Those regions included the 861 and 864 nt encoding the respective putative coat proteins as well as the 285 and 253 nt, respectively of 3' non-coding sequences. Comparison of the nucleotide sequences of PeMoV-AU and PStV-Ib revealed a sequence similarity of 64.4% for the coat protein gene and 34.6% for the 3' non-coding region. The deduced amino acid sequences of PeMoV-AU and PStV-Ib coat proteins are 66.7% identical. These results provide further evidence that PeMoV and PStV are distinct viruses. Comparisons of the 3' terminal sequences of PeMoV-AU and PStV-Ib with those of the genomic RNA of other strains of PeMoV and PStV and with other potyviruses are discussed.

Amino Acid Sequence↗
PubMed1994

Alleged reaction in gel-immunodiffusion of an IgM monoclonal antibody with alfalfa mosaic virus and cucumber mosaic virus is an artefact.

A previously reported spurious serological cross-reaction between alfalfa mosaic virus (AMV) and cucumber mosaic virus (CMV), which had been defined by the reaction in gel-immunodiffusion tests of a single IgM monoclonal antibody (MAb), MAb 8, was no longer detected in the presence of 0.1 M-NaCl. The non-specific reactivity of this IgM was also confirmed in Western blotting assays. When skimmed milk was used as a blocking agent and as a diluent of antibodies, MAb 8 failed to recognize AMV and CMV coat proteins. Hence, it is concluded that the alleged cross-reaction between AMV and CMV is due to non-specific binding of MAb 8 and that there is no evidence for a serological relationship between these two viruses.

Alfalfa mosaic virus↗
PubMed1993

Nucleotide sequence of one component of the banana bunchy top virus genome contains a putative replicase gene.

One DNA component of the banana bunchy top virus (BBTV) genome was cloned and sequenced. This component is present as a circular, ssDNA in the virions and consists of 1111 nucleotides. It contains one large open reading frame (ORF) of 858 nucleotides in the virion sense; this ORF encodes a putative replicase based on the presence of a dNTP-binding motif (GGEGKT). Two smaller ORFs (249 and 366 nucleotides), in the complementary orientation, could not be assigned any obvious function. Neither of these ORFs had significant sequence homology with any known DNA plant virus gene or gene product. Computer analysis of this component-predicted a strong stem-loop structure in the virion sense putative untranslated region; a nonanucleotide sequence in the loop was nearly identical to the nonanucleotide invariant loop sequence of geminiviruses and coconut foliar decay virus. There is strong evidence that the genome of BBTV consists of more than one component because no ORF was found that would encode a protein the size of the BBTV coat protein. BBTV has some characteristics in common with geminiviruses but cannot be classified as one. Rather, BBTV probably belongs to an undescribed plant virus group which could also include subterranean clover stunt virus and coconut foliar decay virus.

Amino Acid Sequence↗
PubMed1991

Alleged common antigenic determinant of tobacco mosaic virus coat protein and the host protein ribulose-1,5-bisphosphate carboxylase is an artifact of indirect ELISA and western blotting.

We have reported the detection of an antigenic determinant shared by the tobacco mosaic virus coat protein and the large subunit of ribulose-1,5-bisphosphate carboxylase, a host protein (R.G. Dietzgen and M. Zaitlin Virology 155, 262-266, 1986). This conclusion was questioned by D. Zimmermann and M.H.V. Van Regenmortel (Arch. Virol. 106, 15-22, 1989). Thus we have reinvestigated this unexpected serological cross-reaction in Western immunoblotting and indirect ELISA. We found that when skimmed milk instead of bovine serum albumin was used as a blocking agent and as a diluent for antibodies and alkaline phosphatase conjugates, the alleged cross-reaction was abolished. In light of these findings, we retract our previous conclusions.

Animals↗