Agroinfection of transgenic plants leads to viable cauliflower mosaic virus by intermolecular recombination.

Intermolecular reconstitution of a plant virus has been detected in whole plants in a system using a defective cauliflower mosaic virus genome and transgenic host plants containing the missing viral gene. The information for the gene VI protein of the virus was integrated into the chromosome of host Brassica napus plants and leaves of these plants were inoculated with Agrobacterium tumefaciens containing the complementing viral sequences. In several cases, upper leaves contained replicating viral DNA which was able to incite CaMV symptoms on turnip plants. The sequence of the resultant recombinant viral molecules suggested that both DNA and RNA recombination events may have been involved in the production of functional virus, one event being gene targeting of the T-DNA.

Genomic homologous recombination in planta.

A system for monitoring intrachromosomal homologous recombination in whole plants is described. A multimer of cauliflower mosaic virus (CaMV) sequences, arranged such that CaMV could only be produced by recombination, was integrated into Brassica napus nuclear DNA. This set-up allowed scoring of recombination events by the appearance of viral symptoms. The repeated homologous regions were derived from two different strains of CaMV so that different recombinant viruses (i.e. different recombination events) could be distinguished. In most of the transgenic plants, a single major virus species was detected. About half of the transgenic plants contained viruses of the same type, suggesting a hotspot for recombination. The remainder of the plants contained viruses with cross-over sites distributed throughout the rest of the homologous sequence. Sequence analysis of two recombinant molecules suggest that mismatch repair is linked to the recombination process.

Differential inhibition of downstream gene expression by the cauliflower mosaic virus 35S RNA leader.

The effect of the 600 nucleotide-long CaMV 35S RNA 5' leader sequence on the expression of downstream genes was analyzed both in plant protoplasts and in vitro. For transient expression studies in protoplasts derived from host and nonhost plants, the bacterial chloramphenicol acetyl transferase (CAT) gene was fused to the initiation codon of ORF VII. The leader sequence reduced CAT expression two- to four-fold in protoplasts derived from three host species, but 10- to 50-fold in protoplasts derived from three different nonhost species. For in-vitro studies the 35S promoter was replaced by the SP6 promoter. The leader reduced in-vitro translation of SP6 transcripts approximately six-fold, indicating that at least part of the inhibition observed in protoplasts is directly due to the interference of the leader sequence with translation. Other steps in gene expression that may also be affected are discussed.

The leading sequence of caulimovirus large RNA can be folded into a large stem-loop structure.

The 600 nt long sequences preceeding the first large ORFs (ORF VII) of three caulimoviruses, although varying in primary sequence, can be folded into a large stem/loop structure centered around a conserved stretch of 36 nucleotides. Deletions of the conserved sequence delay symptom appearance considerably, but do not affect expression of a reporter gene in plant protoplasts. Another striking similarity between the leaders concerns the number and distribution of small open reading frames (sORF) they carry. Expression of two of these sORFs was tested by fusion of a reporter gene: both were expressed in plant protoplasts.

Genetic transformation of Brassica campestris var. rapa protoplasts with an engineered cauliflower mosaic virus genome.

A hybrid Cauliflower Mosaic Virus (CaMV) genome containing a selectable marker gene was constructed by replacing the gene VI coding region with the aminoglycoside (neomycin) phosphotransferase type II [APH(3')II] gene from Tn5. This modified viral genome was tested for its infectivity both in planta and in a protoplast transformation system of Brassica campestris var. rapa. Stable, genetically transformed cell lines of B. campestris var. rapa were obtained after transformation. DNA of the hybrid CaMV genome was found to be integrated into high molecular weight plant genomic DNA. Transformation was achieved only when the hybrid genome was supplied together with wild type viral DNA. A possible complementation of the modified CaMV genome with the wild type viral DNA as a helper molecule in planta and in the protoplast system is discussed.