Molecular cloning and characterisation of banana fruit polyphenol oxidase.

Polyphenol oxidase (PPO; EC 1.10.3.2) is the enzyme thought to be responsible for browning in banana [Musa cavendishii (AAA group, Cavendish subgroup) cv. Williams] fruit. Banana flesh was high in PPO activity throughout growth and ripening. Peel showed high levels of activity early in development but activity declined until ripening started and then remained constant. PPO activity in fruit was not substantially induced after wounding or treatment with 5-methyl jasmonate. Banana flowers and unexpanded leaf roll had high PPO activities with lower activities observed in mature leaves, roots and stem. Four different PPO cDNA clones were amplified from banana fruit (BPO1, BPO11, BPO34 and BPO35). Full-length cDNA and genomic clones were isolated for the most abundant sequence (BPO1) and the genomic clone was found to contain an 85-bp intron. Introns have not been previously found in PPO genes. Northern analysis revealed the presence of BPO1 mRNA in banana flesh early in development but little BPO1 mRNA was detected at the same stage in banana peel. BPO11 transcript was only detected in very young flesh and there was no detectable expression of BPO34 or BPO35 in developing fruit samples. PPO transcripts were also low throughout ripening in both flesh and peel. BPO1 transcripts were readily detected in flowers, stem, roots and leaf roll samples but were not detected in mature leaves. BPO11 showed a similar pattern of expression to BPO1 in these tissues but transcript levels were much lower. BPO34 and BPO35 mRNAs were only detected at a low level in flowers and roots and BPO34 transcript was detected in mature leaves, the only clone to do so. The results suggest that browning of banana fruit during ripening results from release of pre-existing PPO enzyme, which is synthesised very early in fruit development.

Construct design for efficient, effective and high-throughput gene silencing in plants.

Post-transcriptional silencing of plant genes using anti-sense or co-suppression constructs usually results in only a modest proportion of silenced individuals. Recent work has demonstrated the potential for constructs encoding self-complementary 'hairpin' RNA (hpRNA) to efficiently silence genes. In this study we examine design rules for efficient gene silencing, in terms of both the proportion of independent transgenic plants showing silencing, and the degree of silencing. Using hpRNA constructs containing sense/anti-sense arms ranging from 98 to 853 nt gave efficient silencing in a wide range of plant species, and inclusion of an intron in these constructs had a consistently enhancing effect. Intron-containing constructs (ihpRNA) generally gave 90-100% of independent transgenic plants showing silencing. The degree of silencing with these constructs was much greater than that obtained using either co-suppression or anti-sense constructs. We have made a generic vector, pHANNIBAL, that allows a simple, single PCR product from a gene of interest to be easily converted into a highly effective ihpRNA silencing construct. We have also created a high-throughput vector, pHELLSGATE, that should facilitate the cloning of gene libraries or large numbers of defined genes, such as those in EST collections, using an in vitro recombinase system. This system may facilitate the large-scale determination and discovery of plant gene functions in the same way as RNAi is being used to examine gene function in Caenorhabditis elegans.

Plant cell-directed control of virion sense gene expression in wheat dwarf virus.

We have used particle bombardment (biolistics) to deliver replication-competent wheat dwarf virus (WDV)-based constructs, carrying reporter gene sequences fused to the virion sense promoter (Pv) or the CaMV 35S promoter, to suspension culture cells and immature zygotic embryos of wheat. While the replication of WDV double-stranded DNA forms (replicons) was equivalent between wheat suspension culture cells and embryos, GUS reporter gene activity was 20-40 times higher in the embryo cultures. Maximum expression of WDV replicons occurred in the embryonic axis tissue of wheat embryos but their expression in suspension cells was compromised, compared with transiently maintained input plasmid DNA containing the same sequences. From these studies, we propose that WDV replicons are subject to a host cell-controlled competency for virion sense transcription. The term competency is used to distinguish between the phenomenon described here and control of gene expression by specific transcription factors. Control of competency is independent of Pv, the replacement 35S promoter and of the complementary sense control of virion sense expression involving specific sequences in Pv. We propose that factors controlling the competency for replicon expression may be present in cells which, as well as maintaining high rates of DNA synthesis, are totipotent. Cell type control of active chromatin, methylation of specific sequences in WDV minichromosomes and/or interaction of virus-encoded proteins with specific host factors are considered as possible mechanisms.

A simple system for pea transformation.

The lateral cotyledonary meristems of germinatingPisum sativum cv. Puget seeds were used to develop a reproducibleAgrobacterium tumefaciens-mediated transformation system. This procedure exhibits distinct advantages over those previously reported, in that it uses dry seed as starting material, and the highly regenerable cotyledonary meristems rapidly produce transgenic shoots without an intermediate callus phase. This transformation regime facilitates the rapid generation of phenotypically normal, self-fertile plants containing functional transgenes inherited in a Mendelian fashion.

The two nonstructural proteins from wheat dwarf virus involved in viral gene expression and replication are retinoblastoma-binding proteins.

Tumor-inducing viruses like simian virus 40 or the human adenovirus produce oncoproteins which interfere with the cellular retinoblastoma (Rb) tumor-suppressor protein to create an appropriate molecular environment in the nucleus for viral transcription and replication. Such a strategy has been considered to be restricted to animal viruses. Here we demonstrate that plant viruses may use similar mechanisms for recruiting host factors. Wheat dwarf virus (WDV) encodes two potential nonstructural proteins, C1 and C1:C2, both containing the consensus Rb-binding motif LeuXCysXGlu that allows the oncoproteins from animal viruses to inactivate Rb. C1:C2 is a key determinant of viral replication and V(virion)-sense expression. Using a yeast two-hybrid protein assay, we demonstrate for the first time that the C1:C2 protein from WDV interacts with a retinoblastoma protein, providing an explanation for the previously observed dependence of viral replication on an intact Rb-binding motif. We also show that C1, for which no function had been demonstrated, is required for V-sense gene expression. This suggests that V-sense expression might be dependent on the interaction of C1 with Rb. Our findings provide further evidence for the presence of transforming-like proteins in a plant virus and will help to explain the production of symptoms in a plant viral infection through a mechanism mediated by a key regulator of cell cycle and differentiation.