Spectinomycin

All plasmids listed in Table S1 were created using circular polymerase extension cloning (CPEC)24 (link). The primers listed in Table S2 were used to create linear dsDNA products that were subsequently DpnI digested for at least 15 minutes and then gel-purified. The DNA was eluted in 6 ul of dH₂O and mixed with corresponding linear product. These products were then used as template for a reaction with Q5 polymerase for 15 cycles. The product was then used to directly transform chemically competent E. coli DH5α or NEB Turbo cells. The following plasmids and maps have been deposited to Addgene (Cambridge, MA); pCas9cr4 (Plasmid #62655), pKDsg-ack (Plasmid #62654), and pKDsg-p15 (Plasmid #62656).
The 20 bp targeting sequences of the sgRNA were re-targeted by CPEC cloning of two linear PCR fragments. The re-targeting primers listed in Table S2 were approximately 40-mers that had overlapping protospacer sequences. The primer pair protospacerF and gamR were used to yield a 3 kb product. The protospacer R primer was paired with pKDseq1F to yield a product of about 4 kb. This design yielded PCR product with about 280 bp of overlapping homology between the gam and araC (Fig. 2), as well as 20 bp of overlap in the protospacer. PCR products were gel purified, mixed together in equal volumes and CPEC cloned with Q5 polymerase. The mixture was used to transform chemically competent DH5α or NEB Turbo cells (New England Biolabs), recovered for 1 hour in super optimal broth with catabolite repression (SOC), and then plated on LB with 50 mg L⁻¹ spectinomycin (spec) and incubated at 30 °C. A step-by-step protocol for primer design and retargeting is available as part of the no-SCAR protocol at Addgene.

For combined cloning of plasmids and subsequent expression of proteins thereof, AQUA Cloning was performed using 25 μL of BL21 (DE3), or TOP10 E. coli cells as a control, as described above. After transformation, cells were grown overnight in 10 mL LB-medium containing 50 μg/μL spectinomycin at 37°C, 150 r.p.m. The next morning, the overnight culture was used to inoculate 200 mL fresh, antibiotic-containing LB-medium which was induced with 1 mM IPTG when OD₆₀₀ = 0.9 was reached at 37°C, 150 r.p.m. Cells were harvested 6 h post induction by centrifugation at 5,000 g for 5 min, washed with PBS and resuspended in 2 mL PBS before an image was acquired.

The bacterial transformation of the plasmids for exogenous expression of FLAG-KRAS (pMDS-TetOn3G-kozak-FLAG-KRAS WT/mutants) was performed using the One-Shot Stbl3 (C737303; Invitrogen) chemically competent bacterial cells to replicate each plasmid following the manufacturer’s instructions. Subsequently, 100 μl of the bacteria–plasmid solutions was plated into LB selective agar plates containing the antibiotic spectinomycin (50 μg/ml). Plates were incubated at 37°C, overnight. The next day, individual bacterial colonies were selected from a LB agar plate and grown in 4 ml LB broth with the corresponding antibiotics for 6–12 h at 37°C in a shaker-incubator at 250 rpm. After incubation, several aliquots of this original starter culture were used to generate a bacterial glycerol stock for long-term storage at −80°C (1 ml transformed bacteria in 1 ml 50% glycerol). The remainder of the original starter culture was then used to grow at a large scale the transformed bacteria under selective antibiotics overnight in 500 ml of LB medium at 37°C in a shaker-incubator at 250 rpm. The HiSpeed Plasmid Maxi Kit (QIAGEN) was used to generate a larger amount of the FLAG-KRAS plasmids. The kit was used following the manufacturer’s instructions. The final DNA was eluted in 400 μl of TE buffer and allowed to resuspend overnight to ensure homogeneity. The next day, concentrations and purities were measured on the Implen NanoPhotometer NP80, and plasmid DNA was stored at −20°C.

The mutagenesis of effectors in HA4-1 strain was performed as previously described (Cheng et al., 2021 (link)). To generate a mutant of the R. solanacearum target effector gene, the effector gene was replaced with a cassette harboring spectinomycin resistance. The genome fragment containing the target gene and its two flanking regions was amplified from the strain HA4-1 genome and then was cloned into pCE2-TA-Blunt-Zero vector (Vazyme 5min™ TA/Blunt-Zero Cloning Kit C601-01/02). A reverse amplification of the above recombinant vector was performed to delete the target gene and generate a linear vector only with the flanking region of the effector. A spectinomycin resistance gene (Spe) was connected with the linear to replace the target gene. The mutant plasmid was successfully constructed. Then the spectinomycin cassette with the effector flanking regions was generated, which was used to be transformed into R. solanacearum competent cells to replace the target effector gene.
To generate the effector complementation strain, the coding sequences with their native promoters and a Km^r cassette were cloned into the plasmid pH7C and then the fragments containing the coding sequences with the effector promoters and the antibiotic gene were amplified and inserted into a permissive chromosome site by transferred into the mutant competent cell, as described previously (Monteiro et al., 2012 (link)).