LAUSR

Most of the 41 approved COVID-19 vaccines and ~220 candidates currently undergoing clinical testing (Basta and Moodie, 2020) are designed around the SARS-CoV-2 spike (S) protein or the receptor-binding domain (RBD) within it, which facilitates virus uptake by binding to angiotensin-converting enzyme 2 (ACE2) on the cell surface. The S protein/RBD is also used as a component of diagnostic kits for the detection of antibodies and the quantification of antibody titers (Y€ uce et al., 2021). Plants have been evaluated as a large-scale production platform for COVID-19 vaccines and diagnostic reagents because they are more cost-effective than fermenter platforms (Capell et al., 2020), making them particularly suitable for lowand middle-income countries (LMICs). The transient expression has the advantage of speed and scalability and has been used to produce COVID-19 reagents (Makatsa et al., 2021) and one approved vaccine (Ward et al., 2020). However, transgenic rice plants provide a stable resource for larger-scale production over a longer term and have already been used to produce antiviral antibodies and lectins (Vamvaka et al., 2018). As proof of concept, we transformed mature-embryo-derived rice callus with constructs in which the RBD transgene was expressed under the control of either the constitutive maize ubiquitin promoter and first intron (Ubi-1) or the endospermspecific barley D-hordein promoter (Hord), each paired with the rice a-amylase signal peptide targeting the secretory pathway. We recovered 31 transgenic callus lines transformed with pUbi-RBD and 34 transformed with pHord-RBD. All 65 lines contained the corresponding gene of interest. The RBD content was evaluated by ELISA, using human ACE2 as the capture reagent to ensure that only the correctly folded and soluble formof the RBDwas detected. Functional RBD was present in 27/31 of the pUbi-RBD callus lines (~87%) and 22 of the positive lines (~82%) accumulated the correctly folded RBD at levels detectable by western blot. The highest yield of RBD (line 19) was 6.88 1.28 lg/g fresh callus weight (fw). However, only six lines (~15%) transformed with pHord-RBD produced a functional RBD protein and only five at levels detectable levels by western blot. The highest yield of RBD in the pHord-RBD callus was 2.20 0.9 lg/g fw (line 12), 3.12-fold lower than the best-performing pUbi-RBD line (Figure 1a). We regenerated 15 independent T0 transgenic rice plants containing pUbi-RBD and 14 containing pHord-RBD. Three independent T0 plants representing pUbi-RBD (lines 9, 19 and 26) and three representing pHord-RBD (lines 8, 12 and 14) were taken to maturity, and all six were shown to contain the gene of interest by PCR. Representative T1 seed extracts were tested by ELISA as described above. The maximum quantity of soluble and correctly folded RBD in the pUbi-RBD seeds was 5.31 0.50 lg/g dw in plant 19, whereas the maximum in the pHord-RBD seed extracts was 0.66 0.08 lg/g dw in plant 8 (Figure 1a). RDB was also detected in the leaves of nine pUbi-RBD plants (60%) and the maximum yield was 4.05 0.67 lg/g fw in line 9 (Figure 1a). We also analysed root extracts from plants 9, 19 and 26, and the highest yield was 5.25 0.90 lg/g fw in line 19. RBD also accumulated in the flag leaves of plants 9 (3.11 0.33 lg/g 1 fw) and 19 (4.44 1.01 lg/g 1 fw) but not plant 26, and accumulated in the seed coat of all three plants, with the highest level in plant 9 (3.54 0.44 lg/g fw). Western blot analysis revealed multiple bands in the flag leaf, leaf, seed