1 Department of Biochemistry
2 Department of Plant Pathology, University of Kentucky, Lexington, Kentucky 40536
and 3 Department of Cell and Developmental Biology, Roche Institute of Molecular ...Biology, Nutley, New Jersey 07110, U.S.A.
Recombinant plasmids containing cDNA copies of the dsRNAs present in one hypovirulent strain of Endothia (Cryphonectria) parasitica , EP713, were constructed and analysed by restriction endonuclease mapping and Southern hybridization. Overlapping inserts of four plasmids were found to represent most of the large dsRNA (L-dsRNA) sequences. Inserts which represented the two termini of the L-dsRNAs, designated homopolymer and heteropolymer, were identified. These plasmids were used as probes in Northern hybridization experiments in an attempt to detect other RNAs having sequences related to those of the L-dsRNAs. No additional RNAs were detected that hybridized to plasmids representing the middle region of L-dsRNAs. However, plasmids representing the termini of L-dsRNAs hybridized to several RNAs in EP713 ranging in size from 300 to 1300 nucleotides. These RNAs were absent from EP155, the isogenic virulent strain of E. parasitica . RNAs related to the homopolymer terminus were more abundant than those related to the heteropolymer terminus. All were sensitive to digestion by S1 nuclease but resistant to RNase III, indicating that they were single-stranded. Only those ssRNAs related to the homopolymer terminus of L-dsRNAs were retained on oligo(dT)-cellulose. Single-stranded M13 phage DNAs containing the insert of one of the plasmids, in each orientation, were used as probes in Northern hybridization experiments. Hybridization to the ssRNAs was observed with only one of these probes, indicating that the transcripts are derived from only one strand of the L-dsRNAs. These results establish the existence of a set of poly(A)-containing ssRNAs that are 3'-coterminal with the homopolymer terminus of L-dsRNAs and have the same polarity as the poly(A)-containing strand of L-dsRNAs.
Keywords: chestnut blight, Endothia parasitica , cloning
Present address: Department of Entomology, University of California, Riverside, California 92521-1037, U.S.A.
Received 8 February 1988;
accepted 20 June 1988.
1 Department of Plant Pathology
and 2 Department of Biochemistry, University of Kentucky, Lexington, Kentucky 40546, U.S.A.
Dot blots of dsRNA from European and American hypovirulent (H) strains of ...the chestnut blight fungus, Endothia parasitica , were hybridized with 32 P-5'-end-labelled fragments of denatured dsRNA of French, Italian and American origins. Although dsRNA from European or American strains reacted well with labelled RNA probes from strains from the same continent there was little or no cross-hybridization between RNA from strains from different continents.
Keywords: dsRNA, hypovirulence, Endothia (Cryphonectria) parasitica , dot blot hybridization
Received 20 June 1984;
accepted 16 October 1984.
The female-sterile mutations, okra and spindle-B , cause variable axial patterning defects characteristic of defective signaling through the Gurken-Egfr pathway. A detailed characterization of the ...mutant phenotypes of these genes reveals that generation of the germline signal, Gurken, is affected at multiple levels. Localization of gurken mRNA is abnormal, and Gurken protein does not accumulate to wild type levels. Cloning of these two genes (in collaboration with R. Ray) has revealed that Okra and Spindle-B are homologous to yeast proteins known to function in the double strand break DNA repair pathway. To confirm the function of these genes in DNA repair, the frequency of recombination and non-disjunction was tested in the mutants. Consistent with a role for these genes in repair of the double strand breaks made during the initiation of meiotic recombination, the mutations cause a decrease in recombination frequencies and an increase in X-chromosome nondisjunction. I have determined that the production of patterning defects are a consequence of the failure to repair the double strand breaks created during the initiation of meiotic recombination—mutants which do not make these breaks suppress the okra and spindle-B mutant phenotypes. Furthermore, I found that double strand breaks do not directly impair Gurken-Egfr signaling, but rather that their presence activates a meiotic checkpoint pathway which seems to couple translation of oocyte specific proteins, such as Gurken, to progression through meiosis. Thus, mutations that bypass the checkpoint also suppress the okra and spindle-B patterning defects. Finally, I observed that the mobility of Vasa, an eIF4A-like translation factor, is altered in spindle-B mutants, suggesting that Vas is a downstream target of the meiotic checkpoint, and that modification of Vas by the checkpoint could mediate the checkpoint-dependent translational regulation of Gurken.
A change in permeability, indicated by a release of sodium and potassium ions, was detected in the roots of tobacco etch virus (TEV)-infected Tabasco pepper plants 24–48 hours before wilt symptoms ...occurred. The permeability change also preceded a decrease in the respiratory rate by 12–24 hours, and histological changes by 24–48 hours. None of these changes occurred in roots of noninoculated control plants, in roots of TEV-infectedpepper varieties which do not wilt, or in roots of Tabasco pepper infected with viruses which do not cause wilt. Since the change in permeability appeared to be specific for roots of TEV-infected Tabasco pepper, and since this preceded any other observable symptoms, a causal relationship between permeability change and wilting is suggested. No permeability changes were observed in systemically infected Tabasco pepper leaves.
In root extracts of TEV-infected Tabasco pepper plants at the first day of wilt, a decrease in ascorbic acid content, an elevated enzymatic oxidation of ascorbic acid, a pronounced accumulation of polyphenols, and an increase in peroxidase activity were detected, but polyphenol oxidase activity was unchanged.
Wilted plants recovered when their root tips were excised and the roots placed in water. This indicates that wilt results from the failure of water to reach the xylem rather than from blockage of water transport in the xylem itself.
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