Figure 1.
Stable and transient transformation of P. japonicum mediated by A. rhizogenes.
Transformation induced by A. rhizogenes ATCC15834. A. Five-week-old plants showing accumulation of black substance(s) at the wound site after hypocotyl-cutting infection method. B. Transformed roots emerged from cotyledons 5 weeks after A. rhizogenes inoculation by the SAAT method. The black arrow indicates transformed roots. C and D GFP-fluorescing transformed roots observed under bright field (C) or fluorescent (D) microscopy. The black arrows point to fluorescent roots and the white arrow to non-fluorescent root. E and F Transiently-transformed cotyledons observed under bright field (E) or fluorescent (F) microscopy. G and H Confocal micrograph of cotyledon leaves. Non-transformed (G) and transiently-transformed (H). Red color corresponds to autofluorescence from chlorophyll. White bars correspond to 2 mm and yellow bars to 20 µm.
Figure 2.
Detection of transgenes by PCR and RT-PCR.
A-B PCR analysis of genomic DNA isolated from P. japonicum fluorescent hairy roots (F) and non-transformed tissues (NT). A. Amplification of GFP and rolB fragments with expected sizes (380 bp and 780 bp, respectively). B. No amplification of virD1 fragment (450 bp) in fluorescent tissues (F), as positive control a diluted ATCC15834 bacterial suspension (Ar) was used. C. Southern blot of genomic DNA extracted from fluorescent (F) and non-transformed (NT) P. japonicum roots. DNA was digested with EcoRI. The positive control (Pl) corresponds to linearised pBCR101 plasmid (30 ng). D. RT-PCR analysis of the rolB gene using total RNAs extracted from P. japonicum fluorescent roots (F) and non-transformed tissues (NT).
Figure 3.
Effect of sonication treatment on P. japonicum plants.
A Transformation efficiency with A. rhizogenes strain ATCC15834 and sonication (0, 10, 50 and 100 s) followed by application of vacuum for 5 min. Bars represent the means and standard error of at least 3 independent experiments with 20–40 plants each. Statistical significance is marked by * (P < 0.1). B and C Photographs of 5-week-old plants without sonication treatment (B) and submitted to 100 s sonication at the age of 3 days old(C). D to G Scanning electron micrographs of a 3-day-old cotyledons without sonication (D) and cotyledons after 10 s (E), 50 s (F) and 100 s (G) sonication. Arrows point to damage on cotyledon surfaces caused by sonication. Bars correspond to 125 µm
Figure 4.
Optimization of factors influencing stable transformation of P. japonicum.
Frequency of stable transformation in P. japonicum seedlings submitted to different treatments. A Transformation efficiency in 3-day-old seedlings infected with strains ATCC15834, LBA1334 and AR1193 and co-cultivated for 2 or 7 days. The data show representative results from one of two independent experiments using 20 to 60 plants each. B Effect of acetosyringone (AS) on transformation efficiency. Seedlings were co-cultivated with A. rhizogenes strains ATCC15834 and LBA1334 in media with or without 100 µM AS for 2 days for LBA1334, and 7 days for ATCC15834. 4–5 weeks after the inoculation the transformation frequency was scored. The data show representative results from one of at least two independent experiments using 20 to 60 plants each.
Figure 5.
A flowchart for hairy root transformation in P. japonicum.
Table 1.
Agrobacterium rhizogenes strains.
Table 2.
Efficiency of stable transformation after the addition of Silwet L-77 and/or NAA into bacterial suspension.
Figure 6.
Transgenic root retains parasitic competence.
A and B. Haustorium development in transgenic hairy roots following 2-day exposure to 10 µM DMBQ observed under bright field (A) and fluorescent (B) microscopy. The white arrows point to haustoria developing on a transformed root and the black arrow points to a haustorium in a non-transformed root. C and D. Haustorial connection with host rice observed under bright field (C) and fluorescence (D) microscopy. E and F. Haustorial connection with host maize observed under bright field (E) and fluorescence (F) microscopy. White arrows indicate haustorial connection of transgenic roots to hosts. H: host, P: parasite. Bars correspond to 0.5 mm.
Figure 7.
Cell division is induced during early haustorium development.
Hairy root transformed with CYCB1;2 pro::YFP treated or not treated with 10 µM DMBQ. A to C. Transformed root without DMBQ treatment. YFP-fluorescing transgenic roots observed under bright field (A) or fluorescence (B) stereoscopy. In magnified view of root tip under fluorescence (C), roots were stained with 400 µg/ml propidium iodide (PI) to highlight root cell morphology. D to F. Transformed root after 24 h on DMBQ-containing agar (w/v 0.7%). Haustorium region in bright field (D) and under fluorescence (E) stereoscopy. PI-stained haustorium observed under fluorescence (F). The long arrows point to haustoria and the arrowheads to root tips. Red color indicates PI staining and green indicates GFP fluorescence. Yellow bar corresponds to 0.25 mm and white bar to 50 µm.