PubMed Health⌕ Search

Biomedical subjects

R L Gilbertson

Publications and source records attributed to R L Gilbertson.

At least 19 recordsLinked to original sources
PubMed2003

Genetics of resistance to the geminivirus, Bean dwarf mosaic virus, and the role of the hypersensitive response in common bean.

Bean dwarf mosaic virus (BDMV) is a single-stranded DNA virus (genus: Begomovirus, family: Geminiviridae) that infects common bean ( Phaseolus vulgaris L.) and causes stunted plant growth, and mosaic and mottle symptoms in leaves. BDMV shows differential pathogenicity in common bean, infecting germplasm of the Andean gene pool (e.g., the snap bean cultivar Topcrop), but not that of the Middle American gene pool (e.g., the pinto bean cultivar Othello). Resistance to BDMV in Othello is associated with development of a hypersensitive response (HR) in vascular (phloem) tissues. In this study, Middle American germplasm representing the four recognized races (i.e., Durango, Guatemala, Jalisco, and Mesoamerica) and the parents of Othello were inoculated with BDMV and a BDMV-green fluorescent protein (GFP) reporter. All genotypes showed partial or complete resistance to BDMV and BDMV-GFP, indicating the widespread distribution of resistance in the Middle American gene pool. A number of BDMV-resistant germplasm did not show the HR, indicating it is not correlated with resistance. In the F(1), F(2), and F(3) of reciprocal crosses between Othello and Topcrop, a single dominant allele, Bdm, conferred BDMV resistance.

DNA, Viral↗
PubMed2001

Functional analysis of proteins involved in movement of the monopartite begomovirus, Tomato yellow leaf curl virus.

The functional properties of proteins [capsid protein (CP), V1, and C4] potentially involved with movement of the monopartite begomovirus, Tomato yellow leaf curl virus (TYLCV), were investigated using microinjection of Escherichia coli expressed proteins and transient expression of GFP fusion proteins. The TYLCV CP localized to the nucleus and nucleolus and acted as a nuclear shuttle, facilitating import and export of DNA. Thus, the CP serves as the functional homolog of the bipartite begomovirus BV1. The TYLCV V1 localized around the nucleus and at the cell periphery and colocalized with the endoplasmic reticulum, whereas C4 was localized to the cell periphery. Together, these patterns of localization were similar to that of the bipartite begomovirus BC1, known to mediate cell-to-cell movement. However, in contrast to BC1, V1 and C4, alone or in combination, had a limited capacity to move and mediate macromolecular trafficking through mesophyll or epidermal plasmodesmata. Immunolocalization and in situ PCR experiments, conducted with tomato plants at three stages of development, established that TYLCV infection was limited to phloem cells of shoot apical, leaf, stem, and floral tissues. Thus, the V1 and/or C4 may be analogs of the bipartite begomovirus BC1 that have evolved to mediate TYLCV movement within phloem tissue.

Active Transport, Cell Nucleus↗
PubMed2000

Bean dwarf mosaic virus BV1 protein is a determinant of the hypersensitive response and avirulence in Phaseolus vulgaris.

The capacities of the begomoviruses Bean dwarf mosaic virus (BDMV) and Bean golden yellow mosaic virus (BGYMV) to differeBean dwarf mosaic viru certain common bean (Phaseolus vulgaris) cultivars were used to identify viral determinants of the hypersensitive response (HR) and avirulence (avr) in BDMV. A series of hybrid DNA-B components, containing BDMV and BGYMV sequences, was constructed and coinoculated with BDMV DNA-A (BDMV-A) or BDMVA-green florescent protein into seedlings of cv. Topcrop (susceptible to BDMV and BGYMV) and the BDMV-resistant cvs. Othello and Black Turtle Soup T-39 (BTS). The BDMV avr determinant, in bean hypocotyl tissue, was mapped to the BDMV BV1 open reading frame and, most likely, to the BV1 protein. The BV1 also was identified as the determinant of the HR in Othello. However, the HR was not required for resistance in Othello nor was it associated with BDMV resistance in BTS. BDMV BV1, a nuclear shuttle protein that mediates viral DNA export from the nucleus, represents a new class of viral avr determinant. These results are discussed in terms of the relationship between the HR and resistance.

Carrier Proteins↗
PubMed2000

Transgenic plants expressing geminivirus movement proteins: abnormal phenotypes and delayed infection by Tomato mottle virus in transgenic tomatoes expressing the Bean dwarf mosaic virus BV1 or BC1 proteins.

Transgenic tomato plants expressing wild-type or mutated BV1 or BC1 movement proteins from Bean dwarf mosaic virus (BDMV) were generated and examined for phenotypic effects and resistance to Tomato mottle virus (ToMoV). Fewer transgenic plants were recovered with the wild-type or mutated BC1 genes, compared with the wild-type or mutated BV1 genes. Transgenic tomato plants expressing the wild-type or mutated BV1 proteins appeared normal. Interestingly, although BDMV induces only a symptomless infection in tomato (i.e., BDMV is not well adapted to tomato), transgenic tomato plants expressing the BDMV BC1 protein showed a viral disease-like phenotype (i.e., stunted growth, and leaf mottling, curling, and distortion). This suggests that the symptomless phenotype of BDMV in tomato is not due to a host-specific defect in the BC1 protein. One transgenic line expressing the BC1 gene did not show the viral disease-like phenotype. This was associated with a deletion in the 3' region of the gene, which resulted in expression of a truncated BC1 protein. Several R0 plants, expressing either wild-type or mutated BV1 or BC1 proteins, showed a significant delay in ToMoV infection, compared with non-transformed plants. R1 progeny plants also showed a significant delay in ToMoV infection, but this delay was less than that in the R0 parents. These results also demonstrate that expression of viral movement proteins, in transgenic plants, can have deleterious effects on various aspects of plant development.

Blotting, Northern↗
PubMed1998

Bean Dwarf mosaic geminivirus movement proteins recognize DNA in a form- and size-specific manner.

Plant viral movement proteins mediate the cell-to-cell movement of nucleic acids. This involves either a direct interaction between the viral movement protein and the nucleic acid or an indirect interaction involving host factors. The bipartite geminiviruses possess two movement proteins, BV1 and BC1, that coordinate movement of viral DNA across nuclear and plasmodesmal boundaries, respectively. Here, we demonstrate that both BV1 and BC1 interact directly with DNA and, in addition, that they have the unique property to recognize DNA on the basis of form and size rather than sequence. This is a novel feature for plant virus movement proteins and raises the possibility that BV1 and BC1 may be determinants of genome size in the bipartite geminiviruses.

DNA, Circular↗
PubMed1997

Capsid protein and helper component-proteinase function as potyvirus cell-to-cell movement proteins.

The role of bean common mosaic necrosis potyvirus (BCMNV) and lettuce mosaic potyvirus (LMV) proteins was investigated in terms of their capacity to function as viral movement proteins (MPs). Using Escherichia coli-expressed proteins and microinjection techniques, direct evidence was obtained that both the potyviral capsid protein (CP) and helper component- proteinase (HC-Pro) function in this capacity, in that both proteins (a) trafficked from cell to cell, (b) induced an increase in plasmodesmal size exclusion limit, and (c) facilitated cell-to-cell movement of viral RNA. CP and HC-Pro mutants were also produced and used in microinjection experiments. Mutations in the core region of the CP either impaired (single and double amino acid substitution mutants) or abolished (triple amino acid substitution mutant) cell-to-cell movement, as did C-terminal deletion mutants in HC-Pro. The BCMNV P1, CI, NIa, and NIb proteins did not exhibit viral MP properties, but NIa and NIb proteins were found to accumulate within the nuclei of injected cells. These results further establish the multifunctional nature of the potyvirus CP and HC-Pro.

Capsid↗
PubMed1996

Increased pathogenicity in a pseudorecombinant bipartite geminivirus correlates with intermolecular recombination.

Most whitefly-transmitted geminiviruses possess bipartite DNA genomes, and this feature may facilitate viral evolution through pseudorecombination and/or recombination. To test this hypothesis, the DNA-A and DNA-B components of the geminiviruses bean dwarf mosaic virus (BDMV) and tomato mottle virus (ToMoV) were exchanged, and the resultant pseudorecombinants were serially passaged through plants. Both pseudorecombinants were infectious in Nicotiana benthamiana but induced attenuated symptoms and had reduced DNA-B levels. Serial passage experiments revealed that the BDMV DNA-A plus ToMoV DNA-B pseudorecombinant could not be maintained beyond three passages. In contrast, the ToMoV DNA-A plus BDMV DNA-B pseudorecombinant was maintained during serial passage through N. benthamiana and Phaseolus vulgaris and, after three to five passages, became highly pathogenic. Furthermore, the increased pathogenicity of this pseudorecombinant was consistently associated with an increased level of DNA-B, which eventuated in equivalent levels of both components. Sequence analysis of the DNA-B component of the more pathogenic pseudorecombinant revealed that intermolecular recombination had taken place in which most of the BDMV DNA-B common region was replaced with the ToMoV DNA-A common region. This recombinant DNA-B component, which contained the ToMoV origin of replication, was the predominant DNA-B component associated with the more pathogenic pseudorecombinant. These results provide the first demonstration of recombination between distinct bipartite geminiviruses and establish that the bipartite genome can facilitate viral evolution through pseudorecombination and intermolecular recombination.

Base Sequence↗
PubMed1995

Mutational analysis of a putative NTP-binding domain in the replication-associated protein (AC1) of bean golden mosaic geminivirus.

Bean golden mosaic virus (BGMV) is a whitefly-transmitted, ssDNA geminivirus with a bipartite genome. AC1 is the only ORF required for geminiviral replication. A putative NTP-binding motif, EGX4GKTX32DD, was present in the derived amino acid sequence of the replication-associated protein from the AC1 ORF for 13 geminiviruses including BGMV-GA (Guatemalan isolate, amino acids 221-263). We analyzed the phenotypes of mutations within this domain using a rapid and sensitive PCR-based assay for geminiviral replication developed for these studies. Replication in tobacco cells (NT-1 suspension cells) and infection of beans were abolished when codons were changed from K228 to H or D262 to R within the putative NTP-binding site. A temperature-sensitive replication phenotype was conferred by changing E221 to R within the putative NTP-binding domain. Replication was unaffected by changing a nonconserved codon near the putative NTP-binding domain from 1190 to R. Our results demonstrate that the putative NTP-binding domain is required for geminiviral replication. The role of NTP hydrolysis and the possible value of these mutants in a trans-dominant interference scheme for virus-derived resistance are discussed.

Amino Acid Sequence↗
PubMed1995

The complete nucleotide sequence and genome organization of bean common mosaic virus (NL3 strain).

The nucleotide sequences of 3 cDNA clones corresponding to entire RNA genome of bean common mosaic virus NL3 strain have been determined. The RNA is 9612 nucleotides long, excluding a 3'-terminal poly(A) tail. A putative start codon located at nucleotide positions 170-172 initiates one large open reading frame that is terminated with a UAA codon at position 9368-9370. The predicted polyprotein has 3066 amino acids and an M(r) of 340.3 kDa. The positions of putative protein cleavage sites have been determined by analogy to consensus sequences in other potyviruses. The nucleotide sequences of the non-translated regions and the predicted amino acid sequences of BCMV NL3, were compared with those of other potyviruses. Comparison of the BCMV NL3 proteins with those of other potyviruses indicated a similar genomic organization, and high percentage of amino acid sequence identity in the cylindrical inclusion protein, nuclear inclusion 'b' protein and coat protein. BCMV NL3 displays the highest amino acid sequence identity with soybean mosaic virus.

Amino Acid Sequence↗
PubMed1995

Effects of Incubation Time and Temperature on In Vitro Selective Delignification of Silver Leaf Oak by Ganoderma colossum.

The effects of incubation time and temperature on the ability of isolates of the chlamydosporic and thermophilic fungus Ganoderma colossum (Fr.) C. F. Baker to cause selective delignification of Quercus hypoleucoides A. Camus were evaluated by standard in vitro agar block tests. Chemical and scanning electron microscopy studies of decayed wood were used to determine the extent of selective delignification or simultaneous decay caused by each fungal isolate. At 35 deg C, the percent weight loss increased from 6.1% after 4 weeks to a maximum of 32.5 to 33.0% after 16 and 20 weeks of incubation. The average percent Klason lignin-chlorite holocellulose ratios (PKL/CHC) decreased from 0.35 in the control wood block to 0.22 in wood blocks incubated for 12 weeks; this indicated selective delignification. The average PKL/CHC increased for the 16- and 20-week incubation periods, indicating greater removal of polysaccharides during longer incubation periods. In temperature studies, the percent weight loss after 12 weeks was 26 to 27% between 30 and 40 deg C and less than 16% for the 25 and 45 deg C treatments. The average PKL/CHC ranged from 0.18 to 0.16 between 35 and 40 deg C, whereas they were 0.23 and 0.31 for the 25 and 45 deg C treatments, respectively. Scanning electron microscopy confirmed an optimum temperature range near 35 to 40 deg C and incubation times of 8 to 12 weeks for selective delignification. Under these conditions, ray parenchyma, fiber tracheids, and vessels were devoid of middle lamella; pit regions of cells were visible with significantly enlarged apertures; and individual cells were separated and clearly delimited. Extensive delignification of wood occurred throughout the wood blocks evaluated. Incubation times longer than 12 weeks resulted in greater degradation of wood cell walls and thus in greater removal of the polysaccharide component of the wood. For incubation times of 4 weeks or a temperature of 25 deg C, limited to no degradation of cells was observed. At 45 deg C, walls of fiber tracheids were eroded and ray parenchymal cells were extensively degraded, indicating that simultaneous degradation of cell walls occurred. Thus, the incubation temperature influenced the type of decay by G. colossum observed on oak wood blocks: extensive selective delignification at 35 to 40 deg C after more than 8 weeks of incubation or simultaneous decay at 45 deg C with 14% weight loss after 12 weeks of incubation. Isolates of G. colossum may prove useful in studies on mechanisms of delignification and biotechnological applications (e.g., biopulping) of lignin-degrading fungi.

Journal Article↗
PubMed1994

Two proteins of a plant DNA virus coordinate nuclear and plasmodesmal transport.

Plant viruses establish a systemic infection by moving through plasmodesmata, but little is known of the mechanism(s) involved. The roles of two movement-associated proteins of a single-stranded DNA virus were investigated in vivo, using functional proteins expressed in E. coli and microinjection into plant cells. We report here that the BL1 protein of bean dwarf mosaic geminivirus moves extensively from cell to cell, increases mesophyll plasmodesmal size exclusion limit, and potentiates the movement of double-stranded DNA from cell to cell. Movement of single- and double-stranded DNA out of the nucleus is mediated by the BR1 protein. These results provide direct experimental evidence for intercellular macro-molecular transport in plants, and suggest that the BR1 and BL1 proteins coordinate the movement of viral DNA across both nuclear and plasmodesmal boundaries.

Base Sequence↗
PubMed1993

Toward an integrated linkage map of common bean. III. Mapping genetic factors controlling host-bacteria interactions.

Restriction fragment length polymorphism (RFLP)-based genetic linkage maps allow us to dissect the genetic control of quantitative traits (QT) by locating individual quantitative trait loci (QTLs) on the linkage map and determining their type of gene action and the magnitude of their contribution to the phenotype of the QT. We have performed such an analysis for two traits in common bean, involving interactions between the plant host and bacteria, namely Rhizobium nodule number (NN) and resistance to common bacterial blight (CBB) caused by Xanthomonas campestris pv. phaseoli. Analyses were conducted in the progeny of a cross between BAT93 (fewer nodules; moderately resistant to CBB) and Jalo EEP558 (more nodules; susceptible to CBB). An RFLP-based linkage map for common bean based on 152 markers had previously been derived in the F2 of this cross. Seventy F2-derived F3 families were inoculated in separate greenhouse experiments with Rhizobium tropici strain UMR1899 or X. c. pv. phaseoli isolate isolate W18. Regression and interval mapping analyses were used to identify genomic regions involved in the genetic control of these traits. These two methods identified the same genomic regions for each trait, with a few exceptions. For each trait, at least four putative QTLs were identified, which accounted for approximately 50% and 75% of the phenotypic variation in NN and CBB resistance, respectively. A chromosome region on linkage group D7 carried factor(s) influencing both traits. In all other cases, the putative QTLs affecting NN and CBB were located in different linkage groups or in the same linkage group, but far apart (more than 50 cM). Both BAT93 and Jalo EEP558 contributed alleles associated with higher NN, whereas CBB resistance was always associated with BAT93 alleles. Further investigations are needed to determine whether the QTLs for NN and CBB on linkage group D7 represent linked genes or the same gene with pleiotropic effects. Identification of the QTLs raises the possibility of initiating map-based cloning and marker-assisted selection for these traits.

Alleles↗
PubMed1993

Pseudorecombination between infectious cloned DNA components of tomato mottle and bean dwarf mosaic geminiviruses.

A newly described whitefly-transmitted geminivirus infecting tomato plants in Florida induces yellow mottling symptoms on leaves, and stunted and distorted growth. The DNA-A and DNA-B components were cloned from extracts of field-infected tomato tissue; excised monomers or uncut tandem dimers of these clones were infectious when co-inoculated on to Nicotiana benthamiana by rub-inoculation. Tomato plants inoculated directly with the DNA-A and DNA-B dimers, or indirectly by sap or graft transmission from N. benthamiana plants previously infected with the dimers, developed symptoms similar to those observed in field-infected plants. This tomato geminivirus is different from previously characterized geminiviruses, and has been named tomato mottle geminivirus (ToMoV). DNA sequence comparisons revealed that ToMoV is closely related to bean dwarf mosaic geminivirus (BDMV) and abutilon mosaic geminivirus. Infectious pseudorecombinants were made by exchanging the cloned infectious DNA components of ToMoV and BDMV and inoculating N. benthamiana plants. The presence of the inoculated DNA components in systemically infected plants was confirmed by characterization of DNA-A and DNA-B fragments amplified by the polymerase chain reaction. This is the first report of pseudorecombination between two distinct geminiviruses. The implications of this finding in geminivirus evolution are discussed.

Base Sequence↗
PubMed1992

Toxicity of pentachlorophenol to six species of white rot fungi as a function of chemical dose.

The growth of six species of white rot fungi was a function of pentachlorophenol (PCP) dose, expressed as mass of PCP per mass of mycelia, at PCP doses </=35 mug mg of mycelium, and not concentration. At higher doses, Inonotus dryophilus, Perenniporia medulla-panis, and Ganoderma oregonense removed less PCP than three other species of white rot fungi. Phanerochaete chrysosporium grown under nitrogen-deficient conditions was inactivated at PCP doses that under nitrogen-sufficient conditions resulted in only 2-day lag periods in growth. Trametes versicolor was the fastest-growing species that remained viable at higher PCP doses. Both Trametes versicolor and Phellinus badius were able to degrade PCP at higher PCP doses.

Journal Article↗
PubMed1991

Use of the asymmetric polymerase chain reaction and DNA sequencing to determine genetic variability of bean golden mosaic geminivirus in the Dominican Republic.

A combination of the polymerase chain reaction (PCR), asymmetric PCR (A-PCR) and DNA sequencing was used to determine the nucleotide sequence of a hypervariable region of the bipartite genome of bean golden mosaic geminivirus (BGMV). This region, which was part of the intergenic region of the DNA-B component, was amplified using primers designed from the nucleotide sequence of a DNA-B component clone (pDRB1) of an isolate of BGMV from the Dominican Republic (BGMV-DR). pDRB1 is infectious on beans when coinoculated with the DNA-A component of BGMV-DR (pDRA1), and typical bean golden mosaic symptoms are observed on infected plants. Bean leaf tissue infected with BGMV was collected at five separate field locations in the Dominican Republic and the hypervariable region was amplified by PCR, ssDNA was produced using A-PCR, and partial nucleotide sequences were determined. The sequences of the hypervariable region from the field-collected samples ranged from 95% (one sample) to 98% (four samples) identical to the sequence of pDRB1. This contrasts with sequence identities of 86, 75 and 46% between the pDRB1 hypervariable region and the hypervariable regions of BGMV isolates from Guatemala, Puerto Rico and Brazil respectively, and 42% with bean dwarf mosaic geminivirus. These results indicate that Dominican Republic isolates of BGMV are very similar and should be considered isolates of the same virus (BGMV-DR), and that the infectious clones of BGMV-DR are representative of BGMV isolates in the Dominican Republic. The procedures described for DNA extraction from leaf tissue and for production of high quality ssDNA using PCR and A-PCR are rapid and efficient and could be applied to studies of variability and epidemiology of other viruses.

Base Sequence↗
PubMed1990

Comparative studies of delignification caused by ganoderma species.

Isolates of six species of Ganoderma in the G. lucidum complex were evaluated for their ability to decay wood of Quercus hypoleucoides A. Camus and Abies concolor (Gord. and Glend.) Lindl. ex. Hildebr. by using in vitro agar block decay tests. Morphological, ultrastructural, and chemical studies of decayed wood were used to determine the extent of delignification or simultaneous decay caused by each species of Ganoderma. All species decayed both white fir and oak wood; however, less percent weight loss (%WL) occurred in white fir than oak. In white fir, isolates of two undescribed Ganoderma species (RLG16161, RLG16162, JEA615, and JEA625) caused significantly higher%WL (21 to 26%) than that in G. colossum, G. oregonense, G. meredithiae, and G. zonatum (10 to 16%). Only Ganoderma sp. isolates JEA615 and JEA625 caused delignification, with JEA615 causing a lignin-to-glucose gram loss ratio of 1.6:1. Morphological and ultrastructural studies confirmed delignification by this fungus and showed that some delignification had occurred by all of the species, although areas of delignification were limited to small regions adjacent to simultaneously decayed cells. In oak, G. colossum caused significantly less%WL (22 to 35%) than the other species (38 to 52%). All of the species, except G. meredithiae, caused delignification with lignin-to-glucose gram loss ratios ranging from 1.4 to 4.9:1. Extensive delignification by isolates of G. colossum and G. oregonense was observed; moderate delignification was caused by the other species. Ganoderma meredithiae caused a simultaneous decay, with only small localized regions of cells delignified, while delignification by G. zonatum was irregular, with specific zones within the cell wall delignified. The thermophilic and chlamydosporic G. colossum has the capacity to cause extensive delignification and appears ideally suited for use in lignin degradation studies and biotechnological applications of lignin-degrading fungi.

Journal Article↗