Fig 1.
Agrobacterium-mediated transformation of C. elegans.
(a) T-binary system. DNA to be integrated into the target genome is cloned between T-DNA borders (LB, and RB) in a T-binary vector, which is introduced to Agrobacterium by electroporation. Induction of virulence genes on a Vir helper plasmid is triggered with acetosyringone, resulting in cleavage of T-DNA and its delivery to the nucleus of the target species for insertion in the genome. (b) Anatomy of a C. elegans hermaphrodite, indicating reproductive components (labelled in blue), and suggested sites for Agrobacterium access (labelled in black).
Fig 2.
Appearance of C. elegans fed on Agrobacterium.
Adult C. elegans fed on Agrobacterium or E. coli were monitored for (a) length and (b) speed (Mann–Whitney test, n > 72. Error bars represent mean + /- SD). Widefield microscopy of C. elegans fed (c) GFP expressing Agrobacterium or (d) non fluorescent Agrobacterium.
Fig 3.
Autofluorescence of Agrobacterium infected C. elegans.
C. elegans fed on Agrobacterium control or Agrobacterium containing T-binary vectors (Prpl-28::wrmScarlet::let-858 or Prpl-28:BFP::let-858) induced to transfer T-DNA to host, were monitored for fluorescence using widefield microscopy in the (a) DsRed channel or (b) blue channel. (c) Under certain conditions C. elegans fed Agrobacterium with the T-binary vector Prpl-28::wrmScarlet::let-858 or Pmyo3::mcherry::unc-54 showed strong fluorescence in specific regions.
Fig 4.
Locomotion of C. elegans fed on Agrobacterium control strain, or strains containing T-binary vectors engineered to paralyse C. elegans.
(a) Frequency of forward motion, (b) frequency of paused motion (Kruskal–Wallis test with Dunn’s post hoc test, n ≥ 6, boxes represent IQR, line shows median value). Each independent replicate represents an average of 3 worms per well.
Fig 5.
Schematic of the transformation protocol parameters varied during testing for C. elegans transformation using Agrobacterium.
Expanded details of experimental conditions are provided in S1 Table.
Table 1.
Constructs for testing Agrobacterium-mediated transformation.