Bruno Gronenborn

Wheat dwarf virus, a geminivirus of graminaceous plants needs splicing for replication.

By analysing mRNAs with the polymerase chain reaction (PCR) and by studying in vitro generated mutants we have identified an intron in the genome of wheat dwarf virus (WDV), a geminivirus of cereals. Polypeptides whose expression is essential for the replication of the viral DNA have been defined. They are encoded by two distinct overlapping open reading frames (ORFs). The joining of these two ORFs by deletion of the intron as well as the introduction of a frameshift mutation within the intron do not prevent replication of the viral genome in suspension culture cells. In contrast to WDV, the geminiviruses of dicotyledonous plants possess a single continuous ORF, highly homologous to the two individual ones of WDV. We propose that mRNA splicing is a common feature of all geminiviruses of the Gramineae and might contribute to their host class specificity. The existence of a functional intron is a novel finding for the plant viruses.

Identification of the initiation sequence for viral-strand DNA synthesis of wheat dwarf virus.

The intergenic region of the circular single‐stranded DNA genome of geminiviruses contains a sequence potentially able to fold into a stem‐loop structure. This sequence has been reported to be involved in viral replication by serving as the origin for rolling‐circle replication. However, in wheat dwarf virus (WDV) a deletion of 128 bp, removing this sequence, surprisingly does not prevent de novo viral DNA synthesis, but instead abrogates the processing of replicative intermediates into monomeric genomes. This deletion mutant permitted us to study the initiation of viral‐strand DNA synthesis independently from its termination and also to identify the sequence within which rolling‐circle DNA replication of WDV begins. We have mapped the initiation site of replication to a pentanucleotide, TACCC, a sequence that occurs twice in the large intergenic region of WDV: it is found in the right half of the stem‐loop sequence and again 170 bases upstream where it is part of a 15 nucleotide sequence highly homologous to the right half of the stem‐loop sequence. Here we show that viral‐strand DNA synthesis efficiently initiates at both sequences.

Wheat dwarf virus vectors replicate and express foreign genes in cells of monocotyledonous plants.

Wheat dwarf virus (WDV) is a geminivirus that infects monocotyledonous plants. To exploit the potential of WDV as a replicative gene vector, we developed a transient replication and expression system based on the transfection of protoplasts derived from Triticum monococcum suspension culture cells. Cloned genomic copies of various WDV isolates as well as mutants constructed in vitro were introduced into the protoplasts and assayed for their ability to replicate. As a result, regions of the WDV genome necessary or dispensable for the viral DNA replication could be defined. In addition, the gene encoding the viral capsid protein was replaced by three different bacterial marker genes, neomycin phosphotransferase, chloramphenicol acetyltransferase, and beta-galactosidase. The beta-galactosidase gene doubled the size of the WDV genome. The replication of the recombinant WDV genomes and the expression of these genes were monitored in suspension culture cells of T. monococcum. The potential of replicative expression vectors based on the WDV genome is discussed.

DNA replication of wheat dwarf virus, a geminivirus, requires two cis-acting signals

Replication of the single-stranded DNA genome of wheat dwarf virus (WDV) leads to the accumulation of covalently closed double-stranded DNA of genome length in infected cells. By studying the replication properties of a naturally occurring deletion mutant of WDV isolated from infected plants and of deletion mutants constructed in vitro, we have defined cis-acting regions required for viral DNA replication. The results show that two distinct regions are required in cis to yield the normal replicative forms of WDV-DNA.

The Molecular Biology of Cauliflower Mosaic Virus and Its Application as Plant Gene Vector

The artificial introduction of new traits into living cells by means of molecular gene transfer techniques has been developed into a routine procedure, applicable to both prokaryotic and various eukaryotic organisms. For higher plants, nature itself has evolved an elegant and sophisticated gene transfer system in the Ti-plasmid of Agrobacterium tumefaciens. It ist now widely used to introduce and stably anchor new genes in the genome of susceptible host plants. (See Chapter 7 by S. Rogers, this volume.)