Biological activity of SeMNPV, AcMNPV, and three AcMNPV deletion mutants against Spodoptera exigua larvae (Lepidoptera: noctuidae).

Virulence and speed of action, as related to dose, are important effectiveness-determining properties of insect-pathogenic biocontrol agents. We used the droplet-feeding bioassay to compare dose responses between two wild-type baculoviruses, Autographa californica multicapsid nucleopolyhedrovirus (AcMNPV) and Spodoptera exigua MNPV (SeMNPV), and three deletion mutants of AcMNPV in S. exigua larvae. In each mutant one gene was deleted by genetic engineering: pp34, coding for the polyhedral membrane; egt, coding for ecdysteroid UDP-glucosyltransferase; or p10, coding for fibrillar structures in infected insect cells. SeMNPV had the lowest median lethal dose (LD(50)) as well as the highest speed of action (LT(50)) of all viruses investigated. In our comparative bioassays the only significant effect of gene deletions in AcMNPV was a slightly lower speed of action for the p10 deletion mutant. Otherwise, wild-type and recombinant AcMNPVs had similar biological activities. Our results suggest, in contrast to what is generally assumed, that gene deletions in AcMNPV for improved insecticidal activity should be critically assessed in each host system prior to further implementation as a control agent. Insertion of foreign genes coding for entomotoxins is less questionable and more promising in this respect.

Mitochondrial DNA acts as potential promoter of the baculovirus RNA polymerase.

We have examined whether mitochondrial DNA could act as target of the RNA polymerase encoded by the baculovirus Autographa californica multicapsid nuclear polyhedrosis virus, because the baculovirus late promoters and the control region of host mitochondrial DNA show a high degree of sequence similarity. In vitro transcription using mitochondrial DNA from Spodoptera frugiperda cells and nuclear extracts prepared from baculovirus infected cells demonstrates that mitochondrial DNA is recognized by the viral RNA polymerase. Transcriptional initiation occurs at TAAG sequences, although not all of the six TAAG motifs present in the mitochondrial DNA fragment are recognized. The TAAG motif in the control region served as weak transcriptional start site, but some of the TAAG motifs in the coding sequences of the adjacent tRNA and rRNA genes are recognized efficiently. The sequences flanking the TAAG motifs used as transcriptional start sites have a lower helix stability than the flanking sequences of the nonfunctional TAAG motifs. These results support the view that helix stability rather than sequence specificity is an important factor for recognition of TAAG motifs by the viral RNA polymerase.

In vitro transcription of pe38/polyhedrin hybrid promoters reveals sequences essential for recognition by the baculovirus-induced RNA polymerase and for the strength of very late viral promoters.

In vitro transcription was used to analyze the promoter specificity of the alpha-amanitin-resistant RNA polymerase that is induced late during infection of Autographa californica multicapsid nuclear polyhedrosis virus. By modifying the preparation of crude nuclear extracts, we have established an assay that permits differentiation between weak late and strong very late viral promoters. The virus-induced RNA polymerase initiates at a TAAG sequence motif in both late and very late promoters. Based on the sensitivity of our in vitro transcription system, we have investigated the sequences responsible for a functional TAAG motif and their putative role with respect to the strength of very late promoters. By constructing hybrid promoters between the early pe38 and the very late polyhedrin promoters, we demonstrated that the replacement of 7 nucleotides upstream of the nonfunctional TAAG sequences in the pe38 promoter with the corresponding sequences of the polyhedrin promoter was sufficient for recognition by the virus-induced RNA polymerase. The strength of the very late polyhedrin promoter was established after replacing the 5' untranslated sequences of the pe38 promoter by those of the polyhedrin promoter in addition to the 7 nucleotides upstream of the TAAG motif.

Sequence and transcriptional analysis of the ubiquitin gene cluster in the genome of Spodoptera exigua nucleopolyhedrovirus.

The nucleotide sequence of a 1200 bp DNA fragment of Spodoptera exigua nucleopolyhedrovirus (SeMNPV) was determined. This sequence contained a cluster of two open reding frames (ORFs), one coding for a viral ubiquitin (v-ubi) and another with homology to orf2 of Autographa californica (Ac) MNPV and Orgyia pseudotsugata (Op) MNPV. The vubi ORF is 240 nucleotides (nt) long, potentially encoding a protein of 80 amino acids with a predicted molecular mass of 9.4 kDa. The amino acid sequence of the v-ubi gene in SeMNPV has 75% and 81.6% identity with the v-ubi gene of AcMNPV and OpMNPV and approximately 84% with cellular ubiquitins. Northern blot analysis revealed three major small transcripts late in infection, of about 690, 550 and 400 nt long. Primer extension analysis showed that transcription started from within two consensus late promoter elements (TAAG), located at positions -6 and -30. The start site at position -4/-5 precedes the shortest leader reported to date for a baculovirus gene. The other ORF, xb187, was identified in the opposite orientation immediately upstream of the v-ubi gene. This ORF potentially encodes a 22 kDa protein with unknown function and about 60% amino acid similarity to the products of the orf2 genes of AcMNPV and OpMNPV. The SeMNPV xb187 ORF is transcribed late in infection via two transcripts, 1.2 kb and 770 nt long. The v-ubi-xb187 gene cluster is located at map unit (m.u.) 89 on the genome of SeMNPV. This is different from the position of an identical cluster in the AcMNPV and OpMNPV genomes, located at relative m.u. 20.

Deletion of the baculovirus ecdysteroid UDP-glucosyltransferase gene induces early degeneration of Malpighian tubules in infected insects.

Deletion of the ecdysteroid UDP-glucosyltransferase gene (egt) from the Autographa californica nuclear polyhedrosis virus (AcNPV) genome increases the speed of killing of this virus (D. R. O'Reilly and L. K. Miller, Bio/Technology 9:1086-1089, 1991). Second-instar Spodoptera exigua larvae are killed more rapidly by the egt deletion mutant of AcNPV than by wild-type AcNPV. Unlike wild-type AcNPV-infected larvae, larvae infected with an egt deletion mutant molt and resume feeding as mock-infected larvae do. Wild-type AcNPV and egt deletion mutant recombinants marked with a lacZ gene were used to study their pathogenesis in insects. Histopathological investigation revealed that early degeneration of the Malpighian tubules, not the molting per se, may be the cause of this increased speed of killing by AcNPV.

Mutational analysis of the pseudoknot in the tRNA-like structure of turnip yellow mosaic virus RNA. Aminoacylation efficiency and RNA pseudoknot stability.

Site-directed mutations were introduced in the connecting loops and one of the two stem regions of the RNA pseudoknot in the tRNA-like structure of turnip yellow mosaic virus RNA. The kinetic parameters of valylation for each mutated RNA were determined in a cell-free extract from wheat germ. Structure mapping was performed on most mutants with enzymic probes, like RNase T1, nuclease S1 and cobra venom ribonuclease. An insertion of four A residues in the four-membered connecting loop L1 that crosses the deep groove of the pseudoknot reduces aminoacylation efficiency. Deletions up to three nucleotides do not affect aminoacylation or RNA pseudoknot formation. Deletion of the entire loop abolishes aminoacylation. Although elimination of the pseudoknot is presumed, this could not be demonstrated. Unlike the mutations in loop L1, all mutations in the three-membered connecting loop L2 that crosses the shallow groove of the RNA pseudoknot decrease the aminoacylation efficiency considerably. Nonetheless, the RNA pseudoknot is still present in most mutated RNAs. These results indicate that a number of mutations can be introduced in both loops without abolishing aminoacylation. Results obtained with the introduction of mismatches and A.U base-pairs in stem S1 of the pseudoknot, containing three G.C base-pairs in wild-type RNA, indicate that the pseudoknot is only marginally stable. Our estimation of the gain of free energy due to the pseudoknot formation is at most 2.0 kcal/mol. The pseudoknot structure can, however, be stabilized upon binding the valyl-tRNA synthetase.

Aminoacylation of 3' terminal tRNA-like fragments of turnip yellow mosaic virus RNA: the influence of 5' nonviral sequences.

The present model of the L-shaped tRNA-like structure of turnip yellow mosaic virus (TYMV) RNA encompasses 82 nucleotides. A previous kinetic study on 3' terminal TYMV RNA fragments that contain the tRNA-like structure and a 5' nonviral GGGAGA sequence, suggested that viral sequences upstream of the tRNA-like domain, i.e., upstream of nucleotide 82, increase the rate of aminoacylation (Dreher et al. (1988) Biochimie 70, 1719-1727). Here we report an increase in the aminoacylation rate when the number of nonviral nucleotides at the 5' end of TYMV RNA transcripts was reduced. The influence of these 5' proximal nonviral sequences on the conformation of the RNA molecule was investigated by structure mapping experiments. A structure that deviates from the tRNA-like structure was found in some of the transcripts. The formation of this alternative structure is dependent upon: (1) the nature and number of the nonviral nucleotides; (2) the number and secondary structure of viral nucleotides between the nonviral nucleotides and the tRNA-like domain. Footprinting experiments with valyl-tRNA synthetase from yeast suggest that the enzyme does not recognize the alternative structure.

Interaction of RNase P from Escherichia coli with pseudoknotted structures in viral RNAs.

In a previous study it was shown that RNase P from E. coli cleaves the tRNA-like structure of turnip yellow mosaic virus (TYMV) RNA in vitro (Guerrier-Takada et al. (1988) Cell, 53, 267-272). Cleavage takes place at the 3' side of the loop that crosses the deep groove of the pseudoknot structure present in the aminoacyl acceptor domain. In the present study fragments of TYMV RNA with mutations in the pseudoknot, generated by transcription in vitro, were tested for susceptibility to cleavage by RNase P. Changes in the specificity with respect to the site of cleavage and decreases in the rate of cleavage were observed with most of these substrates. The behaviour of various mutants in the reaction catalyzed by RNase P is in agreement with the present model of the TYMV RNA pseudoknot (Dumas et al. (1987), J. Biomol. Struct. Dyn. 263, 652-657). Base substitutions in the loop that crosses the shallow groove of the pseudoknot structure resulted, however, in an unexpected decrease in the rate of cleavage, probably due to conformational changes in the substrates. Studies on other tRNA-like structures revealed an important role in the reaction with RNase P for both the nucleotide at the 3' side of the loop that spans the deep groove and the nucleotide at position 4, which correspond to positions--1 and 73, respectively, in tRNA precursors.

Conformational and thermodynamic consequences of the introduction of a nick in duplexed DNA fragments: an NMR study augmented by biochemical experiments.

NMR studies were carried out on various equimolar mixtures consisting of a combination of oligomers: d(ACGGCT) (I). d(pACGGCT) (Ia), d(TGCAGT) (II), d(AGCCGTACTGCA) (III), d(TGCAGTACGGCT) (IV). It is shown that I + II + III (MI) and Ia + II + III (M2) form stable duplexes with nicks in the centre of the respective double helices. A close analysis of the NOESY experiments of M1 and M2 revealed that these fragments form B-DNA type duplex structures. A comparison of the chemical-shift data of the nicked duplexes with those of the intact duplex of III + IV (M3) demonstrated that only small local distortions occur when a nick is introduced. The chemical-shift profiles of M1 and M3 were used to obtain the thermodynamic data for the duplex/coil transitions. The profiles of M1 were analysed by means of a new thermodynamic model (TRIDUP). From the calculated thermodynamic data of M1 and M3 it is concluded that the melting behaviour of M1 occurs cooperatively. A ligation experiment demonstrated that the relatively small substrate (M2) was almost completely joined after an overnight incubation at 14 degrees C.

Structure and expression of DNA transferred to tobacco via transformation of protoplasts with Ti-plasmid DNA: co-transfer of T-DNA and non T-DNA sequences.

The T-DNA structure and organization in tissues obtained via transformation of tobacco protoplasts with Ti-plasmid DNA was found to be completely different from the T-DNA introduced via Agrobacterium tumefaciens. It is often fragmented. Overlapping copies of T-DNA, having various sizes, as well as separated fragments of T-DNA were detected. The border sequences of 23 basepairs (bp), flanking the T-region in the Ti-plasmid as direct repeats are not used as preferred sequences for integration. Similar results were obtained with a T-region clone lacking one of the TL-borders. This clone, which carried the cytokinin locus and only the right border sequence of TL and the left border sequence of TR, still had the capacity to transform protoplasts. Also the Vir-region of the Ti-plasmid is not required for integration of foreign DNA via DNA transformation. This is demonstrated by the results with the T-region clone mentioned and by the transforming capacity of a Ti-plasmid carrying a mutated Vir-region. Nevertheless, in a number of Ti-plasmid DNA transformants Vir-region fragments were found to be stably integrated. Furthermore, it has been established that co-transformation can occur with plant cells. Besides the detection of Ti-plasmid fragments from outside the T-region also DNA sequences originating from two DNA sources, which were both independently present in transformation experiments, have been found in some DNA transformants, e.g. calf thymus DNA, which was used as carrier DNA. No expression of the co-transferred DNA was observed. In total three phenotypical classes of DNA transformants were isolated. Although the T-DNA was often scrambled, polyA(+) mRNA studies indicated that the different phenotypes studied can be explained by the presence of active T-DNA genes with known functions.

Transformation of plant protoplasts with DNA: cotransformation of non-selected calf thymus carrier DNA and meiotic segregation of transforming DNA sequences.

With the DNA transformation procedure developed in our laboratory (13) several transformed tobacco SR1 tissues were obtained which, apart from selected and non-selected pTi sequences (T(+)), also had acquired non-selected calf thymus carrier DNA sequences (C(+)), being integrated in their nuclear genomes. From one such tissue (cNT4), with a shooty crown gall phenotype and expressing mannopine synthesis activity (Mas(+)), shoots were grafted and mature, flowering plants (gNT4) were obtained. After cross pollination with wild type SR1 tobacco pollen 49% of the seedlings obtained, had the maternal NT4-like crown gall phenotype and 51% showed wild type (SR1) features. The mannopine locus segregated independently from the locus determining the crown gall phenotype. When screened for integrated ('transforming') foreign DNA sequences 97% of the NT4-like seedlings turned out to be C(+)T(+). Most of the SR1-like seedlings, having a wild type tobacco morphology, proved to be transformed as well: roughly a 1:1:1:1 ratio as found for C(+)T(+):C(-)T(+): C(+)T:C T SR1-like seedlings. Based on the segregation of transforming sequences during meiosis a model is presented showing the integration of these sequences in three different host chromosomes.