cDNA-derived hypovirus RNA in transformed chestnut blight fungus is spliced and trimmed of vector nucleotides.

Unencapsidated double-stranded viral RNAs belonging to the genus Hypovirus attenuate virulence of the chestnut blight fungus, Cryphonectria parasitica. A full-length cDNA clone of hypovirus CHV1-713 double-stranded RNA was recently shown to be infectious when introduced into the C. parasitica genome by DNA-mediated transformation. In this study, we show that the viral RNA derived from the chromosomally integrated cDNA copy is trimmed of extraneous vector nucleotide sequences. The cDNA-derived viral RNA was also found to contain a 73-bp deletion located within the 5'-noncoding leader sequence as a result of a pre-mRNA splicing event. Implications of these results are discussed in terms of hypovirus RNA replication and anticipated field studies involving engineered hypovirulent C. parasitica strains.

Escherichia coli-Salmonella typhimurium hybrid nusA genes: identification of a short motif required for action of the lambda N transcription antitermination protein.

The Escherichia coli nusA gene, nusAEc, encodes an essential protein that influences transcription elongation. Derivatives of E. coli in which the Salmonella typhimurium nusA gene, nusASt, has replaced nusAEc are viable. Thus, NusASt can substitute for NusAEc in supporting essential bacterial activities. However, hybrid E. coli strains with the nusASt substitution do not effectively support transcription antitermination mediated by the N gene product of phage lambda. We report the DNA sequence of nusASt, showing that the derived amino acid sequence is 95% identical to the derived amino acid sequence of nusAEc. The alignment of the amino acid sequences reveals scattered single amino acid differences and one region of significant heterogeneity. In this region, called 449, NusAEc has four amino acids and NusASt has nine amino acids. Functional studies of hybrid nusA genes, constructed from nusAEc and nusASt, show that the 449 region of the NusAEc protein is important for lambda N-mediated transcription antitermination. A hybrid that has a substitution of the four E. coli codons for the nine S. typhimurium codons, but is otherwise nusASt, supports the action of the N antitermination protein. The 449 region and, presumably, adjacent sequences appear to compose a functional domain of NusAEc important for the action of the N transcription antitermination protein of phage lambda.

Papain-like protease p29 as a symptom determinant encoded by a hypovirulence-associated virus of the chestnut blight fungus.

Viral double-stranded RNAs (dsRNAs) responsible for virulence attenuation (hypovirulence) of the chestnut blight fungus, Cryphonectria parasitica, profoundly influence a range of host functions in addition to virulence. The 5'-proximal open reading frame, A, of the prototypical hypovirulence-associated viral dsRNA, L-dsRNA, present in hypovirulent strain EP713, was recently shown by DNA-mediated transformation analysis to suppress fungal sporulation, pigmentation, and accumulation of the enzyme laccase (G. H. Choi and D. L. Nuss, EMBO J. 11:473-477, 1992). We mapped this suppressive activity to the autocatalytic papain-like protease, p29, present within the amino-terminal portion of open reading frame A-encoded polyprotein p69. Mutational analysis revealed that the ability of p29 to alter fungal phenotype is dependent upon release from the polyprotein precursor but is independent of intrinsic proteolytic activity. Deletion of the p29-coding domain within the context of an infectious L-dsRNA cDNA clone resulted in a replication-competent viral dsRNA that exhibited intermediate suppressive activity while retaining the ability to confer hypovirulence. Thus, p29 is necessary but not sufficient for the level of virus-mediated suppression of fungal pigmentation, sporulation, and laccase accumulation observed for wild-type hypovirulent strain EP713 and is nonessential for viral RNA replication and virulence attenuation. These results also illustrate the feasibility of engineering infectious viral cDNA for construction of hypovirulent fungal strains with specific phenotypic traits.

Identification of the rph (RNase PH) gene of Bacillus subtilis: evidence for suppression of cold-sensitive mutations in Escherichia coli.

A shotgun cloning of Bacillus subtilis DNA into pBR322 yielded a 2-kb fragment that suppresses the cold-sensitive defect of the nusA10(Cs) Escherichia coli mutant. The responsible gene encodes an open reading frame that is greater than 50% identical at the amino acid level to the E. coli rph gene, which was formerly called orfE. This B. subtilis gene is located at 251 degrees adjacent to the gerM gene on the B. subtilis genetic map. It has been named rph because, like its E. coli analog, it encodes a phosphate-dependent exoribonuclease activity, RNase PH, that removes the 3' nucleotides from precursor tRNAs. The cloned B. subtilis rph gene also suppresses the cold-sensitive phenotype of other unrelated cold-sensitive mutants of E. coli, but not the temperature-sensitive phenotype of three temperature-sensitive mutants, including the nusA11(Ts) mutant, that were tested.

Analysis of the Escherichia coli nusA10(Cs) allele: relating nucleotide changes to phenotypes.

The Escherichia coli nusA gene product, known to influence transcription elongation, is essential for both bacterial viability and growth of lambdoid phages. We report the cloning and sequencing of the conditionally lethal nusA10(Cs) allele. Changes from nusA+ were observed at nucleotides 311 and 634. Functional studies showed that both nucleotide changes are necessary for the cold-sensitive phenotype, although bacteria with the change at 634 grew more slowly at 30 degrees C than the nusA+ controls. The mutant nusA10(Cs) gene product is not as active as nusA+ in supporting transcription antitermination mediated by the N regulatory protein of bacteriophage lambda. The change at nucleotide 634 is shown to be solely responsible for this phenotype. Four differences were found between the nusA+ gene that we sequenced and the published nusA sequence. These changes alter the reading frame of nusA in a functionally important domain [as identified by the nusA1 and nusA11(Ts) mutations], resulting in an arginine-rich region that may be involved with RNA binding.

Transcription-dependent competition for a host factor: the function and optimal sequence of the phage lambda boxA transcription antitermination signal.

Ordered development of lambdoid phages relies on systems of transcription termination and antitermination. The phage-encoded N early regulatory proteins, acting with the Nus proteins of Escherichia coli, modify RNA polymerase to a form that overrides many transcription termination signals. These modifications require cis-acting sites, nut, located downstream of the early phage promoters. The nut sites in phages lambda, 21, and P22, which share similarities but are not identical, contain two signals, boxA and boxB. We demonstrate that although a consensus sequence for the boxA signal (boxAcon), 5'CGCTCTTTA, is found only in P22, changes to consensus in the nutR sites of lambda and 21 create more effective antitermination signals than the wild-type signals. An in vivo competition assay demonstrates that a lambda nut region with boxAcon outcompetes nut regions with wild-type, as well as other variations of the boxA sequence, for the host NusB protein. This suggests that boxA influences NusB activity in N-mediated antitermination. Successful competition by boxAcon requires transcription of the nut site as well as N activation. Nucleotide replacement further demonstrates that bases at both ends of boxA are important for antitermination.