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Biomedical subjects

Marilyn J Roossinck

Publications and source records attributed to Marilyn J Roossinck.

12 recordsLinked to original sources
PubMed2006

Rapid and efficient purification of Cowpea chlorotic mottle virus by sucrose cushion ultracentrifugation.

A simple, economical and efficient method for isolation of Cowpea chlorotic mottle virus (CCMV) particles was developed. Abundant viral particles could be obtained with three steps of 10 min duration each of conventional differential centrifugations and one sucrose cushion ultracentrifugation step of 2 h. Characterization of purified virus assessed by transmission electron microscopy, polyacrylamide gel electrophoresis (PAGE) and Western blot revealed the typical isometric particles of CCMV without any visible contamination or degradation. RNA extracted from purified CCMV particles showed all four RNA components by agarose gel electrophoresis. Both purified virus particles and RNA were biologically active and initiated successful infection upon inoculation to cowpea.

Blotting, Western↗
PubMed2006

Computational estimation and experimental verification of off-target silencing during posttranscriptional gene silencing in plants.

Successful application of posttranscriptional gene silencing (PTGS) for gene function study in both plants and animals depends on high target specificity and silencing efficiency. By computational analysis with genome and/or transcriptome sequences of 25 plant species, we predicted that about 50% to 70% of gene transcripts in plants have potential off-targets when used for PTGS that could obscure experimental results. We have developed a publicly available Web-based computational tool called siRNA Scan to identify potential off-targets during PTGS. Some of the potential off-targets obtained from this tool were tested by measuring the amount of off-target transcripts using quantitative reverse transcription-PCR. Up to 50% of the predicted off-target genes tested in plants were actually silenced when tested experimentally. Our results suggest that a high risk of off-target gene silencing exists during PTGS in plants. Our siRNA Scan tool is useful to design better constructs for PTGS by minimizing off-target gene silencing in both plants and animals.

Agrobacterium tumefaciens↗
PubMed2006

Comparative analysis of expressed sequences reveals a conserved pattern of optimal codon usage in plants.

Codon usage bias is a ubiquitous phenomenon, which may be caused by mutational bias, selection, or both. The patterns of codon usage in plants are not well understood. Datasets of expressed sequence tags (ESTs) available for many plant species provide the resources for large-scale comparative analysis of codon usage patterns. We developed a computational approach to translate EST or assembled contig sequences, and then used the coding information for comparative analysis of codon usage in 12 plant species, including 6 eudicots, 5 monocots and the green alga Chlamydomonas reinhardtii. While codon nucleotide composition is highly conserved within eudicots or monocots, there is a significant difference between these two major taxonomic groups of higher plants. The third nucleotide position of codons is AU-rich in the eudicot genomes (35-42% of G+C content), but GC-rich in the monocot genomes (59-61% of G+C content). To identify optimal codons in these species, we used EST counts to estimate gene transcript levels. It was demonstrated that codon usage bias is correlated positively with gene transcript levels. Interestingly, the use of optimal codons appears to be well conserved between eudicots and monocots, and to a lesser degree between the higher plants and C. reinhardtii. Most of the optimal codons end with a C or G base, regardless of the different nucleotide composition in these genomes. The results suggest that plant codon usage is affected by translational selection, and the selective pressure appears to be conserved in the plant kingdom.

Animals↗
PubMed2006

Analysis of genetic bottlenecks during horizontal transmission of Cucumber mosaic virus.

Genetic bottlenecks may occur in virus populations when only a few individuals are transferred horizontally from one host to another, or when a viral population moves systemically from the infection site. Genetic bottlenecks during the systemic movement of an RNA plant virus population were reported previously (H. Li and M. J. Roossinck, J. Virol. 78:10582-10587, 2004). In this study we mechanically inoculated an artificial population consisting of 12 restriction enzyme marker mutants of Cucumber mosaic virus (CMV) onto young leaves of squash plants and used two aphid species, Aphis gossypii and Myzus persicae, to transmit the virus populations from infected source plants to healthy squash plants. Horizontal transmission by aphids constituted a significant bottleneck, as the population in the aphid-inoculated plants contained far fewer mutants than the original inoculum source. Additional experiments demonstrated that genetic variation in the artificial population of CMV is not reduced during the acquisition of the virus but is significantly reduced during the inoculation period.

Animals↗
PubMed2005

Symbiosis versus competition in plant virus evolution.

Darwin's theory of evolution by natural selection has been supported by molecular evidence and by experimental evolution of viruses. However, it might not account for the evolution of all life, and an alternative model of evolution through symbiotic relationships also has gained support. In this review, the evolution of plant viruses has been reinterpreted in light of these two seemingly opposing theories by using evidence from the earliest days of plant virology to the present. Both models of evolution probably apply in different circumstances, but evolution by symbiotic association (symbiogenesis) is the most likely model for many evolutionary events that have resulted in rapid changes or the formation of new species. In viruses, symbiogenesis results in genomic reassortment or recombination events among disparate species. These are most noticeable by phylogenetic comparisons of extant viruses from different taxonomic groups.

Evolution, Molecular↗
PubMed2004

Expression of antiapoptotic genes bcl-xL and ced-9 in tomato enhances tolerance to viral-induced necrosis and abiotic stress.

D satellite RNA (satRNA) is a strain of cucumber mosaic virus (CMV) satRNA that induces an epidemic lethal disease in tomato. No natural resistance or tolerance has ever been found. Previously, we demonstrated the involvement of programmed cell death in disease development. Here, transgenic tomato plants expressing animal antiapoptotic genes bcl-xL and ced-9 were generated through agrobacterium-mediated transformation. High expression of bcl-xL or ced-9 affected plant growth and seed development. Inoculation of seedlings with CMV/D satRNA at T(1) and T(2) generations resulted in delayed cell-death symptoms or absence of symptoms. The degree of symptom suppression was correlated with increasing expression levels of the transgenes. Survival rates were compared among inoculated transgenic lines expressing bcl-xL, ced-9, and bcl-xL (G138A), a loss-of-function mutant of bcl-xL. More than 80% of the bcl-xL and ced-9 T(1) transgenic lines showed higher survival rates than the average for bcl-xL (G138A) transgenic lines. Total RNA extracted from surviving plants contained D satRNA, indicating systemic accumulation of D satRNA. Thus, expression of bcl-xL and ced-9 improved tolerance to, rather than resistance to, CMV/D satRNA infection. In addition, expression of bcl-xL and ced-9 specifically abrogated the formation of necrotic lesions, but not other symptoms, in tomato leaves during chilling at 4 degrees C. At 7 degrees C, temperature-induced leaf senescence was dramatically delayed in bcl-xL and ced-9 transgenic plants, and high levels of anthocyanins accumulated, possibly limiting oxidative stress. Hence, expression of these animal antiapoptotic genes improved plant survival under abiotic or biotic stress.

Agrobacterium tumefaciens↗
PubMed2004

Genetic bottlenecks reduce population variation in an experimental RNA virus population.

Genetic bottlenecks are stochastic events that limit genetic variation in a population and result in founding populations that can lead to genetic drift. Evidence of past genetic bottlenecks in numerous biological systems, from mammals to viruses, has been described. In this study, we used an artificial population of Cucumber mosaic virus consisting of 12 restriction enzyme marker-bearing mutants. This population was inoculated onto young leaves of tobacco plants and monitored throughout the course of systemic infection. We show here that the genetic variation in a defined population of an RNA virus is significantly, stochastically, and reproducibly reduced during the systemic infection process, providing clear evidence of a genetic bottleneck.

Cucumovirus↗
PubMed2004

Plant virus satellite and defective interfering RNAs: new paradigms for a new century.

Although many subviral RNAs reduce or intensify disease symptoms caused by the helper virus, only recently have clues concerning the mechanism of disease modulation been revealed. New models for DI RNA-mediated reduction in helper virus levels and symptom attenuation include DI RNA enhancement of posttranscriptional gene silencing (PTGS), which is an antiviral defense mechanism in plants. Symptom enhancement by the satRNA of Cucumber mosaic virus is caused by minus-strand induction of the programmed cell death pathway. In contrast, symptom enhancement by satC of Turnip crinkle virus is due to satC interference with virion formation, leading to increased levels of free coat protein, which is the viral suppressor of PTGS. Mutualism between satRNA and helper virus can be seen for the satRNA of Groundnut rosette virus, which contributes to the virus by allowing virion assembly. These novel findings are leading to re-evaluation of the relationships between subviral RNAs, helper viruses, and hosts.

Animals↗
PubMed2003

Plant RNA virus evolution.

RNA viruses are the most common viruses of plants, and the evolution of these viruses has been studied both experimentally and phylogenetically. The basic molecular mechanisms for plant virus evolution are similar to those of other viruses, with some notable exceptions. Recent advances include new insights into the origins of plant viruses, analyses of quasispecies and mutation frequencies, population studies on field isolates and practical studies on the importance of virus evolution to agriculture.

Biological Evolution↗
PubMed2003

In spite of induced multiple defense responses, tomato plants infected with Cucumber mosaic virus and D satellite RNA succumb to systemic necrosis.

Cucumber mosaic virus (CMV) D satellite RNA (satRNA) attenuates the symptoms induced by CMV in most plants, but causes leaf epinasty and systemic necrosis in tomato plants, where programmed cell death (PCD) is involved. However, our understanding of the cellular and molecular responses to the infection of CMV D satRNA that result in this lethal disease remains limited. In this article, we show for the first time, by histochemical and molecular analysis, that multiple defense responses are specifically induced in CMV and D satRNA (CMV/D satRNA)-infected tomato plants but not in mock-inoculated or CMV-infected plants. These responses include callose deposition and hydrogen peroxide accumulation in infected plants. Furthermore, the transcription of several tomato defense-related genes (e.g., PR-1a1, PR-1b1, PR-2, and PR-10) were activated, and the expression of tomato PR-5 and some abiotic and biotic stress-responsive genes (e.g., catalase II and tomato analogs of Arabidopsis AtBI-1 and tobacco hsr203j) are enhanced. The activation and increase in expression of these genes is correlated with the appearance of leaf epinasty and the development of systemic necrosis in infected tomato plants, while increased expression of the hsr203j analog precedes the development of any disease symptoms. The spatial and temporal expression patterns of these genes as detected by RNA in situ hybridization point to the involvement of a complex developmental program that accompanies disease development resulting from CMV/D satRNA infection.

Arabidopsis Proteins↗
PubMed2002

Evolutionary history of Cucumber mosaic virus deduced by phylogenetic analyses.

Cucumber mosaic virus (CMV) is an RNA plant virus with a tripartite genome and an extremely broad host range. Previous evolutionary analyses with the coat protein (CP) and 5' nontranslated region (NTR) of RNA 3 suggested subdivision of the virus into three groups, subgroups IA, IB, and II. In this study 15 strains of CMV whose nucleotide sequences have been determined were used for a complete phylogenetic analysis of the virus. The trees estimated for open reading frames (ORFs) located on the different RNAs were not congruent and did not completely support the subgrouping indicated by the CP ORF, indicating that different RNAs had independent evolutionary histories. This is consistent with a reassortment mechanism playing an important role in the evolution of the virus. The evolutionary trees of the 1a and 3a ORFs were more compact and displayed more branching than did those of the 2a and CP ORFs. This may reflect more rigid host-interactive constraints exerted on the 1a and 3a ORFs. In addition, analysis of the 3' NTR that is conserved among all RNAs indicated that evolutionary constraints on this region are specific to the RNA component rather than the virus isolate. This indicates that functions other than replication are encoded in the 3' NTR. Reassortment may have led to the genetic diversity found among CMV strains and contributed to its enormous evolutionary success.

3' Untranslated Regions↗