Figures & data
Figure 1 The pHg/pSILBAγ vector system. The ihpRNA expression cassette is constructed in pSILBAγ under A. bisporus gpdII promoter by two directed cloning steps. Laccaria Lbnr intron forms the spacer for the inverted repeats. The hygromycin B resistance cassette is located in the T-DNA of the binary vector pHg. Joining of pSILBAγ and pHg creates the AMT/silencing/rescue-vector with a predicted T-DNA structure.
Figure 2 Plasmid rescue of genomic T-DNA integration sites from pHg/pSILBAγ-transformed Laccaria strains by RB rescue with BamHI l and ampicilline selection in E. coli. Untruncated integrations generate rescue plasmids of a minimal size of ∼3.1 kb and linearizable with BamHI. Fungal genomic sequences can be resolved by sequencing with the universal primers T3 promoter/pUC or M13/pUC-reverse (−26) 17 mer. HRC, hygromycin B resistance cassette; SC, silencing cassette.
Figure 3 Different uses of the pHg/pSILBAγ vector system. The figure represents the T-DNAs of binary vectors for (A) ihpRNA triggering without the plasmid rescue motif; (B) simultaneous silencing of two different genes or boosting ihpRNA triggering of a single target; (C) the use in transgene expression studies with an intronic sequence included into the expression cassette. HRC, hygromycin B resistance cassette; SC, silencing cassette; EC, expression cassette.
