The mycoviral double-stranded-RNAs of Periconia circinata

Yutaka Matsumoto, Purdue University

Abstract

Three types of mycoviruses, unrelated to each other in the nucleotide sequence of their dsRNAs, were identified in natural isolates of the fungal plant pathogen, Periconia circinata. By Northern hybridization and electrophoretic analysis of dsRNA composition, it was established that, in a given isolate of the fungus, these viral types could exist alone or in combination with another type. Some of the relationships among the dsRNAs comprising the viral types were studied. For example, in some fungal isolates, short dsRNAs homologous to the terminal sequences of longer dsRNAs were detected. Thus, these truncated dsRNAs resemble the genomic remnant RNAs described in other viruses. Major effort was directed toward determining the sequence and structure of a 2.0 kb dsRNA (M2) and its 1.8 kb dsRNA satellite (M3). Both dsRNAs were cloned and partially sequenced. A 1.5 kb overlap was established among the cDNA inserts representing the M2 dsRNA, and the sequence of 766 bp was determined. This region contained a contiguous open reading frame, but potential coding regions were not found in the other frames or in the complementary strand. The sequences of both termini of the M2 dsRNA were determined by direct RNA sequencing. In the fast-strand, a translational frame begins and extends internally without disruption. Upstream from the translational initiation site, a putative 5.8 S/18 S interaction site and a 14-bp A/U tract were observed. The structure of the M2 dsRNA was analyzed by its electrophoretic migration as influenced by a variety of conditions, including the matrix, temperature, and buffer (solute) concentration. These analyses established that the M2 dsRNA is more complex than a pliable linear duplex. At elevated temperature (55 C) a fraction of the 2.0 kb dsRNA molecules assumed an altered structure which co-migrated with at two-unit length (4 kb) or three-unit-length (6 kb) DNA marker, depending upon the buffer solute concentration. The thermally induced form was interconvertible and reverted to the original structure when the temperature was lowered. These results suggested that the structure of the M2 dsRNA in solution is not a uniform A$\sp\prime$-Form helix and that the molecule contains regions which undergo structural transitions under conditions that are known to induce A$\sp\prime$-Form to Z-Form transitions of poly(r(G:C)) /poly(r(G:C)) duplexes.

Degree

Ph.D.

Advisors

Dunkle, Purdue University.

Subject Area

Botany|Microbiology|Genetics

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