Plant-based transient overexpression systems enable rapid and scalable production of subunit vaccines. Previously, we have shown that cholera toxin B subunit (CTB), an oral cholera vaccine antigen, is N-glycosylated upon expression in transgenic Nicotiana benthamiana. Here, we found that overexpression of aglycosylated CTB by agroinfiltration of a tobamoviral vector causes massive tissue necrosis and poor accumulation unless retained in the endoplasmic reticulum (ER). However, the re-introduction of N-glycosylation to its original or an alternative site significantly relieved the necrosis and provided a high CTB yield without ER retention. Quantitative gene expression analysis of PDI, BiP, bZIP60, SKP1, 26Sα proteasome and PR1a, and the detection of ubiquitinated CTB polypeptides revealed that N-glycosylation significantly relieved ER stress and hypersensitive response, and facilitated the folding/assembly of CTB. The glycosylated CTB (gCTB) was characterized for potential vaccine use. Glycan profiling revealed that gCTB contained approximately 38% plant-specific glycans. gCTB retained nanomolar affinity to GM1-ganglioside with only marginal reduction of physicochemical stability and induced an anti-cholera holotoxin antibody response comparable to native CTB in a mouse oral immunization study. These findings demonstrated gCTB's potential as an oral immunogen and point to a potential role of N-glycosylation in increasing recombinant protein yields in plants.

• Premise of the study: Pterospora andromedea (Ericaceae) is a mycoheterotrophic plant endemic to North America with a disjunct distribution. Eastern populations are in decline compared to western populations. Microsatellite loci will allow comparison of genetic diversity in endangered to nonthreatened populations.• Methods and Results: Illumina MiSeq sequencing resulted in development of 12 polymorphic microsatellite loci from 63 perfect microsatellite loci tested. One polymorphic locus was obtained from a traditional enrichment method. These 13 loci were screened across two western and two eastern populations. For western and eastern populations, respectively, number of alleles ranged from one to 10 and one to four, and observed heterozygosity ranged from 0.000 to 0.389 and 0.000 to 0.143.• Conclusions: These are the first microsatellite loci developed for Pterospora. They will be useful in conservation efforts of the eastern populations and for examination of population genetic parameters at different geographic scales and comparison with mycorrhizal fungal hosts.

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