Identification and Host Relations of Turnip ringspot virus, A Novel Comovirus from Ohio.

Viruslike chlorotic ring spot symptoms and line patterns of unknown origin were observed on a greenhouse-grown turnip plant. The suspected virus was mechanically transmissible to plants in the Brassicaceae. Electron microscopic analysis revealed icosahedral particles approximately 28 nm in diameter. Reverse transcriptase-polymerase chain reaction (RT-PCR) analyses suggested that the pathogen is a comovirus, an observation that was confirmed by analysis of portions of the genomic sequence. This virus was provisionally named Turnip ringspot virus (TuRSV). Based on the RNA 1 sequence, TuRSV is most similar to Radish mosaic virus, another pathogen that infects members of the Brassicaceae. Arabidopsis thaliana is susceptible to TuRSV, and 12 out of the 23 ecotypes studied showed symptoms when inoculated with the virus. TuRSV induced a variety of responses on ecotypes from death to no infection. Some ecotypes showed one or two rounds of symptom display followed by recovery when inoculated with TuRSV. About half of the ecotypes (11/23) analyzed showed no symptoms when inoculated with TuRSV. Col-0 plants showed no symptoms, and infectious virus was not recovered from systemic leaves, although it could be detected by RT-PCR. Col-0 plants harboring mutations impairing the ethylene, jasmonic acid, or salicylic acid signaling pathways did not show symptoms when inoculated with TuRSV.

Third position codon composition suggests two classes of genes within the Cauliflower mosaic virus genome.

The translation of viral mRNAs by host ribosomes is essential for infection. Hence, codon usage of virus genes may influence efficiency of infection. In addition, composition of nucleotides in the third position within codons of genes can reflect evolutionary relationships. In this study, third position codon composition was examined for the seven genes of eight Cauliflower mosaic virus isolates. Genes IV-VII had similar codon composition values and were termed Class 1 genes. Genes I-III possessed corresponding codon composition values and were termed Class 2 genes. The codon composition values of Class 1 and genes differed significantly. Neither Class 1 nor Class 2 genes had codon composition values identical to that of the host plant, Arabidopsis thaliana. However, Class 1 genes possessed codon composition values closer to those of the host than Class 2 genes. Examination of the genomes of three Rous sarcoma virus isolates indicated that codon composition values were similar for the gag, pol, and env genes but these genes differed significantly from the src genes. Since codon composition values for Rous sarcoma virus distinguished a "foreign" gene from the rest of the viral genome, it is possible that the Cauliflower mosaic virus genome is composed of genes from two different sources. Others have suggested that Cauliflower mosaic virus evolved in this manner and our data provide support for this hypothesis.

The 5' Third of Cauliflower mosaic virus Gene VI Conditions Resistance Breakage in Arabidopsis Ecotype Tsu-0.

ABSTRACT Arabidopsis thaliana ecotypes vary in their responses to viruses. In this study, we analyzed the variation in response of A. thaliana ecotype Tsu-0 to Cauliflower mosaic virus (CaMV). This ecotype was previously reported to be resistant to two CaMV isolates (CM1841 and CM4-184), but susceptible to W260. In this study, we show that Tsu-0 is resistant to four additional CaMV isolates. CaMV propagated within the rosette leaves of Tsu-0 plants, but did not appear to spread systemically into the inflorescence. However, virus viability in rosette leaves of Tsu-0 plants apparently was not compromised because infectious CaMV could be recovered from these organs. W260 overcomes Tsu-0 resistance by a passive mechanism (i.e., this virus avoids activating plant defenses). The portion of the viral genome responsible for W260 resistance breakage was mapped to the 5' third of gene VI, which we have termed RBR-1. This region is also responsible for controlling the ability of CaMV to infect different types of solanaceous plants. Hence, the pathways by which plants of different families interact with CaMV may be conserved through evolution.

Cauliflower Mosaic Virus Isolate NY8153 Breaks Resistance in Arabidopsis Ecotype En-2.

ABSTRACT Arabidopsis thaliana ecotype En-2 was previously shown to be resistant to cauliflower mosaic caulimovirus (CaMV) isolate CM4-184. In this study, En-2 plants were screened with eight other isolates of CaMV to identify viruses capable of overcoming resistance and to determine if the mechanism of resistance was the same for each virus. En-2 resistance to most CaMV isolates was mediated by the same mechanism, i.e., preventing virus long-distance movement. One CaMV isolate, NY8153, was found that produced a severe systemic infection on En-2 plants. In addition, the CM1841 isolate was able to spread systemically through En-2 plants, to a limited extent, without producing visible symptoms. These data indicate that the resistance shown by En-2 plants is not an all-or-none phenomenon. En-2 plants were susceptible to turnip mosaic potyvirus, suggesting that resistance is specific to CaMV.

Movement of virus and photoassimilate in the phloem: a comparative analysis.

Recent progress in the study of short-distance (cell-to-cell) movement of plant virus, facilitated by 'movement proteins', has led to a resurgence of interest in long-distance virus transport in the phloem. Relatively little is known about phloem-specific barriers to virus movement or about the form in which virus enters, travels within and exists this tissue. Progress in understanding virus and photoassimilate transport is limited by a paucity of information on the substructure and properties of plasmodesmata at specific interfaces. The direction of virus movement, once it has entered the phloem, can be understood by following photoassimilate translocation, a complex and dynamic process influenced by plant growth, development and vascular topology.

Long-distance movement of viruses in plants.

During systemic infections, plant viruses move long distances through the plant vasculature. Leaf age, the rate of plant development, plant anatomy and the direction of nutrient flow in the vasculature influence the pattern and extent of systemic spread of the virus, and, in turn, these factors are major determinants of virus resistance.

Effects of host plant development and genetic determinants on the long-distance movement of cauliflower mosaic virus in Arabidopsis.

During systemic infections, viruses move long distances through the plant vascular system. The long-distance movement of cauliflower mosaic virus (CaMV) in Arabidopsis has been examined using a whole plant in situ hybridization technique called plant skeleton hybridization. CaMV moves long distance through the phloem largely following the flow of photoassimilates from source to sink leaves. During the course of plant development, sink-source relationships change and the region of the plant that CaMV can invade is progressively reduced. In Arabidopsis, we have found that conditions that influence the rate of plant development dramatically impact the long-distance movement of CaMV, because under normal conditions the rate of plant development is closely matched to the kinetics of virus movement. Ecotypes and mutants of Arabidopsis that flower early show a form of resistance to systemic CaMV infection, which we call "developmental resistance." Developmental resistance results from the fact that the rosette leaves mature early in the life of an early flowering plant and become inaccessible to virus. On the other hand, if the development of early flowering plants is retarded by suboptimal growth conditions, inoculated plants appear more susceptible to the virus and systemic infections become more widespread. We have found that other Arabidopsis ecotypes, such as Enkheim-2 (En-2), show another form of resistance to virus movement that is not based on developmental or growth conditions. The virus resistance in ecotype En-2 is largely conditioned by a dominant trait at a single locus.

Multiple domains exist within the upstream activator sequence of the octopine synthase gene.

It is known that a 16-base pair palindrome (ACGTAAGCGCTTACGT) located upstream of the ocs gene can activate a maize adh1 promoter in a transient expression system [Ellis et al. (1987). EMBO J. 6, 11-16; Ellis et al. (1987). EMBO J. 6, 3203-3208]. We have determined that this palindrome is also essential for ocs promoter activity in tobacco calli. In addition, sequences immediately adjacent to this palindrome, both 5' and 3', modulate its activity. The palindrome is sensitive to the differentiated state of the plant cells in which it resides; it is active in calli and the leaves of small shoots but is inactive in the leaves of rooted plants. We have tested upstream sequences from two other T-DNA genes that have homology to this palindrome for their ability to activate the octopine synthase promoter in tobacco calli. The upstream region from the mannopine synthase gene can activate the octopine synthase promoter, but an upstream region from the gene implicated in octopine and nopaline secretion cannot activate the promoter.

Structure of the octopine synthase upstream activator sequence.

We have identified a transcriptional activating element within the 5' flanking sequence of the Agrobacterium tumefaciens octopine synthase (ocs) gene that is necessary for ocs expression in transformed tobacco calli. This element is located between 333 and 116 base pairs upstream from the transcription initiation site and functions independent of orientation when placed upstream of the ocs gene. It does not function in either orientation when placed downstream of the gene, nor can it activate its promoter when separated by a distance of 608 base pairs. Deletion analysis indicates that sequences essential for activator function are localized between 222 and 177 base pairs upstream of the transcription initiation site. Another region, located between 333 and 249 base pairs upstream of the transcription initiation site, does not as a monomer activate the ocs promoter, but it can as a dimer.

Localization of cytokinin biosynthetic sites in pea plants and carrot roots.

The biosynthesis of cytokinins was examined in pea (Pisum sativum L.) plant organs and carrot (Daucus carota L.) root tissues. When pea roots, stems, and leaves were grown separately for three weeks on a culture medium containing [8-(14)C]adenine without an exogenous supply of cytokinin and auxin, radioactive cytokinins were synthesized by each of these organs. Incubation of carrot root cambium and noncambium tissues for three days in a liquid culture medium containing [8-(14)C]adenine without cytokinin demonstrates that radioactive cytokinins were synthesized in the cambium but not in the noncambium tissue preparation. The radioactive cytokinins extracted from each of these tissues were analyzed by Sephadex LH-20 columns, reverse phase high pressure liquid chromatography, paper chromatography in various solvent systems, and paper electrophoresis. The main species of cytokinins detectable by these methods are N(6)-(Delta(2)-isopentyl_adenine-5'-monophosphate, 6-(4-hydroxy-3-methyl-2-butenyl-amino)-9-beta-ribofuranosylpurine-5'- monophosphate, N(6)-(Delta(2)-isopentenyl)adenosine, 6-(4-hydroxy-3-methyl-2-butenylamino)-9-beta-ribofuranosylpurine, N(6)-(Delta(2)-isopentenyl)adenine, and 6-(4-hydroxy-3-methyl-2-butenylamino)purine. On the basis of the amounts of cytokinin synthesized per gram fresh tissues, these results indicate that the root is the major site, but not the only site, of cytokinin biosynthesis. Furthermore, cambium and possibly all actively dividing tissues are responsible for the synthesis of this group of plant hormones.

Cytokinin-modulated gene expression in excised pumpkin cotyledons.

Comparison of two-dimensional polyacrylamide gel electrophoretic maps of proteins isolated from benzyladenine-treated and untreated pumpkin (Cucurbita pepo L. cv Halloween) cotyledons showed that the expression of certain proteins is enhanced, induced, or suppressed by the cytokinin treatment. The amount of poly(A)(+) mRNA isolated from cotyledons incubated with 10(-4) molar benzyladenine for five days was about four-fold over the water-incubated control. The activity of hydroxypyruvate reductase prepared from purified cotyledonous microbodies and analyzed by native gel electrophoresis is proportionally enhanced by sequentially higher concentrations (10(-9) to 10(-4) molar) of benzyladenine. Ethidium bromide (1 microgram per milliliter) did not inhibit hydroxypyruvate reductase activity; thus, the enzyme synthesis does not appear to be controlled by organelle genes. Hydroxypyruvate reductase synthesis is inhibited by cycloheximide, cordycepin, and to a certain degree by actinomycin D. These data support the view of a close association between cytokinin action and gene expression.

Modification of cytokinins by cauliflower microsomal enzymes.

N(6)-(Delta(2)-isopentenyl)Adenine and N(6)-(Delta(2)-isopentenyl)adenosine were hydroxylated, respectively, to 6-(4-hydroxy-3-methyl-trans-2-butenylamino)purine and 6-(4-hydroxy-3-methyl-trans-2-butenylamino) -9-beta-ribofuranosylpurine in the presence of NADPH and the microsomal fraction from cauliflowers (Brassica oleracea L.). The hydroxylating reaction was completely inhibited by 10 millimolars metyrapone and partially inactivated by 10-minute treatment of the microsomal preparation with ethylene. The cytokinins were also dealkylated by the microsomal enzymes and formed adenine from cytokinin base and adenosine from cytokinin nucleoside. These results suggest that plant cytochrome P-450 is involved in the conversion of one type of cytokinin to another, and in the modification of cytokinin molecules.