Generation of transgenic plants

The G. max XTH43 gene (XTH43) has been cloned [48]. The procedures used to generate XTH43-overexpressing (XTH43-OE) and XTH43-RNAi roots and used in the analyses here have been described, but are summarized here [48]. The pRAP15 plasmid has been used for XTH43 overexpression (XTH43-OE) [53]. The pRAP17 plasmid has been used for XTH43-RNAi [54]. XTH43 gene expression in both the pRAP15-OE and pRAP17-RNAi plasmids is driven by the figwort mosaic virus (FMV) sub-genomic transcript (Sgt) promoter [53–55]. The FMV-Sgt promoter consists of a 301 bp FMV Sgt promoter fragment (sequence -270 to +31 from the transcription start site (TSS) [55]. The RNAi cassette of pRAP17 is designed to produce a hairpin RNA having inverted repeats [54,56]. A solution of Agrobacterium rhizogenes strain K599 (K599) has been transformed with the appropriate XTH43-OE, pRAP15 (OE control), XTH43-RNAi or pRAP17 (RNAi control) vector. The genetically transformed K599 has been centrifuged to pellet the bacteria. The K599 has been re-suspended in Murashige and Skoog (MS) media, including vitamins (Duchefa Biochemie; The Netherlands) and 3.0% sucrose at a pH of 5.7 (MS media) [54,57]. The roots from 1 week old H. glycines_{[NL1-Rhg/HG-type 7/race 3]}-susceptible G. max_{[Williams 82/PI 518671]} (for XTH43-OE) or H. glycines_{[NL1-Rhg/HG-type 7/race 3]}-resistant G. max_{[Peking/PI 548402]} (for XTH43-RNAi) have been excised at the hypocotyl while in a Petri dish containing a slurry of K599 with a new, sterile razor blade that has been used for each individual tested genetic construct. The procedure allows the K599 access to the plant tissue through the wound site. Subsequently, 25 root-less plants have been placed in a 140 ml glass beaker containing 25 ml of transformed K599 in the MS media solution. The root-less plants, housed within the beaker containing the K599, have then been placed under a vacuum for 5 minutes. The plants have been left in the vacuum for 10 minutes, allowing the K599 ample time to gain entry to the plant tissue. The vacuum has then been slowly released, ensuring the transformed K599 remains in the plant tissue while also ensuring air bubbles do not enter the plant tissue. After this co-cultivation period, the cut ends of root-less G. max plants have been placed individually 3–4 cm deep into fresh coarse vermiculite (Palmetto Vermiculite) in 50-cell flats (T.O. Plastics). The 50-cell flats are placed in 61.9 (L) X 38.4 (W) X 15.6 (H) semi-clear plastic chambers (Sterlite^®). At this point, the plants have been covered to permit the build-up of humidity which prevents the root-less plants from drying out and time to regenerate roots. The humidity chambers have been placed at a distance of 20 cm from standard fluorescent cool white 4,100 K, 32 watt bulbs that emit 2,800 lumens (Sylvania) with a 16 h day/8 h night photoperiod ambient lab temperatures (22° C). The recovery period is 5 days at ambient lab temperatures. The plants have then been transferred to the greenhouse, then uncovered and cultured at its ambient temperatures (30° C day, 24° C night). The plants are allowed to grow for two weeks ambient temperatures. The visual selection of transgenic G. max roots has been performed with the enhanced green fluorescent protein (eGFP) reporter which is incorporated into the pRAP15 and pRAP17 vectors [50]. The eGFP reporting roots also possess the XTH43-OEor XTH43-RNAi cassette [48]. The eGFP reporter and expression cassette (OE or RNAi) each have their own promoter and terminator sequences. During this incubation period, K599 transfers the DNA cassettes that are located between the left and right borders of the pRAP15 and pRAP17 destination vectors into the root cell chromosomal DNA [53,54]. The result is a stable transformation event in the root somatic cell chromosome [58]. Roots subsequently develop from this transgenic cell located at the base of the shoot stock over a period of a few weeks, resulting in the generation of a genetically mosaic plant having a non-transgenic shoot with a transgenic root system. Consequently, in the experiments presented here, each individual transgenic root system is an independent transformant root. The transgenic plants have then been planted in a sandy (93.00% sand, 5.75% silt, and 1.25% clay) soil in SC10 Super cone-tainers (Stuewe and Sons^®, Inc.) secured in RL98 trays (Stuewe and Sons^® Inc.). The plants then recover for two weeks prior to the start of the experiment at ambient greenhouse temperatures and photoperiod [50]. The cDNA has been confirmed to not contain genomic DNA by using PCR of cupin (Glyma.20G148300) which amplifies across an intron (S1 Table). The eGFP has been confirmed by performing PCR on cDNA from transgenic and non-transgenic roots (S1 Table). The functionality of the genetic constructs in their ability to overexpress the gene of interest (increase the relative transcript abundance of XTH43) or undergo RNAi (decrease the relative transcript abundance of XTH43) in G. max has been confirmed by RT-qPCR (Please refer to RT-qPCR subheading of the Materials section). The controls for the transgenic experiments are stated here as follows. As the controls for the experiments, the un-engineered pRAP15 or pRAP17 vectors have the ccdB gene. The ccdB gene is located in the position where, otherwise, XTH43 has been incorporated during the original cloning procedure. This feature makes the un-engineered pRAP15-ccdB (OE control) and pRAP17-ccdB (RNAi control) vectors suitable controls for any non-specific effects caused by gene overexpression or RNAi [48,50,59].