Potato virus X-induced gene silencing in leaves and tubers of potato.

Virus induced gene silencing (VIGS) is increasingly used to generate transient loss-of-function assays and has potential as a powerful reverse-genetics tool in functional genomic programs as a more rapid alternative to stable transformation. A previously described potato virus X (PVX) VIGS vector has been shown to trigger silencing in the permissive host Nicotiana benthamiana. This paper demonstrates that a PVX-based VIGS vector is also effective in triggering a VIGS response in both diploid and cultivated tetraploid Solanum species. We show that systemic silencing of a phytoene desaturase gene is observed and maintained throughout the foliar tissues of potato plants and was also observed in tubers. Here we report that VIGS can be triggered and sustained on in vitro micropropagated tetraploid potato for several cycles and on in vitro generated microtubers. This approach will facilitate large-scale functional analysis of potato expressed sequence tags and provide a noninvasive reverse-genetic approach to study mechanisms involved in tuber and microtuber development.

Regulated expression of a novel TCP domain transcription factor indicates an involvement in the control of meristem activation processes in Solanum tuberosum.

In this study, the aim was to determine whether TCP transcription factors are implicated in meristem activation in potato (Solanum tuberosum). By searching a database of potato EST sequences, with a sequence characteristically conserved in TCP domains, a potato tcp gene was identified. A BAC clone containing the tcp sequence was isolated and the genomic sequence was determined. Using a CAPS marker assay, the potato tcp gene (sttcp1) was mapped to chromosome 8. In dormant buds, relatively high levels of sttcp1-specific transcript were detected by in situ hybridization. By contrast, in sprouting buds, no expression of the sttcp1 could be detected. Furthermore, an inverse relationship between axillary bud size and the steady-state level of the sstcp1 transcript was demonstrated. In non-growing buds exhibiting correlative inhibition, sttcpI-specific transcript levels were also relatively high, but rapidly decreased when apical dominance was removed by excision of the apical bud.

Changes in gene expression during meristem activation processes in Solanum tuberosum with a focus on the regulation of an auxin response factor gene.

A suppression subtractive hybridization approach (SSH) was used to generate a cDNA library enriched in clones representing genes that are up-regulated in the potato tuber apical bud on dormancy release. The sequences of cDNAs representing 385 different genes were determined. This study focuses on the characterization of one of these cDNAs. On the basis of sequence similarity, the cDNA was identified as encoding a member of the auxin response factor family (ARF6). The expression pattern of potato ARF6 was determined by in situ hybridization. In apical tuber buds in the early stages of sprouting, relatively high levels of ARF6-specific transcripts were detected, especially in the peripheral zones of the tunica and corpus of the apical meristems. Expression was also detected in procambial and early vascular tissues, both subtending the meristem and in adjacent leaf primordia. By contrast, in dormant buds no expression of ARF6 could be detected. The expression pattern was also determined during the tuberization process; steady-state expression levels decreased c. 10-fold in the apical region as tuberization proceeded. In non-growing buds, exhibiting correlative inhibition, ARF6-specific transcript levels were relatively low, but rapidly increased when apical dominance was removed by excision of the apical bud. The effects of gibberellin and auxin on axillary bud growth and ARF6 expression are described.