Gibson assembly cloning

The Core35S-GFP construct was based on pAI101, a binary vector modified from pCambia1300 [28 (link),29 (link)]. An expression cassette consisting of the core 35S promoter (the last 99 nt of the cauliflower mosaic virus (CaMV) 35S promoter), the EGFP cDNA, and the poly-A signal of CaMV 35S RNA was inserted into pAI101 at the PstI site to create Core35S-GFP. The Core35S-GFP construct was then used as the backbone to create constructs in which the Core35S promoter was replaced by the sequences of the MYMV BV1 promoter, its counterpart in the soybean yellow mosaic virus (SYMV), and their mutated derivatives. The corresponding DNA fragments were custom-synthesized by Integrated DNA Technologies (IDT, Coralville, IA, USA) as gBlocks fragments and used to replace the Core35S sequence via Gibson Assembly cloning (New England Biolabs, Ipswich, MA, USA). The AC2-expressing construct was also made in pAI101, in which the AC2 cDNA was preceded by the CaMV 35S promoter with double enhancers (2X35S), plus a translational enhancer derived from the tobacco etch virus (TEV TE) [28 (link),29 (link),30 (link)]. The identity of all constructs was verified with Sanger sequencing.

Genes encoding RBD of SARS-CoV-2 Alpha, Beta, Gamma, Delta, and Omicron variants were cloned in-frame into the pcDNA3.4-SARS-CoV-2-spike RBD-his using Gibson Assembly cloning (NEB, Ipswich, MA, USA) [13 (link)]. SARS-CoV-2 antigens were produced in Expi293 cells (Thermo Fisher Scientific) and his–tagged SARS-CoV-2 antigens protein was purified using Ni-NTA agarose resin (Thermo Fisher Scientific) affinity chromatography, as described previously [13 (link)].
After 5 days of transfection, the supernatant was collected and passed over the Ni-NTA agarose resin column three times. First, the column was washed with 100 mL of 1× PBS to remove nonspecific bound proteins. Then, 3 mL of elution buffer (pH8.0, 50 mM sodium phosphate, 300 mM NaCl, and 250 mM imidazole) was added to elute the bound proteins. Finally, samples were buffer-exchanged into pH 7.4 PBS using Amicon Ultra-4 (Merck Millipore, Burlington, MA, USA) spin columns with a 10 kDa cutoff. The purity of purified samples was assessed by 14% SDS-PAGE gel (Additional file 1: Supplementary Fig. 1).

A vector (Figure 1) containing win3.12 promoter was constructed starting from the previously used CaMV35S‐OxO construct containing OxO driven by CaMV 35S promoter with ACT2 terminator (from A. thaliana), along with NPTII antibiotic selection gene (Zhang et al., 2013 (link)). The CaMV 35S promoter was removed via restriction digest and replaced with the win3.12 promoter via Gibson assembly cloning (New England Biolabs). The new vector was designated pwin3.12‐OxO. The pwin3.12‐OxO plasmid's promoter and coding region were sequenced via Sanger sequencing (SUNY Upstate Molecular Analysis Core Facility, Syracuse, NY, USA). The pwin3.12‐OxO plasmid was transformed into Agrobacterium tumefaciens EHA105 using electroporation for future C. dentata embryo transformations.