Summary: To explore the physiological significance of N‐glycan maturation in the plant Golgi apparatus, gnt1, a mutant with loss of N‐acetylglucosaminyltransferase I (GnTI) function, was isolated in Oryza sativa. gnt1 exhibited complete inhibition of N‐glycan maturation and accumulated high‐mannose N‐glycans. Phenotypic analyses revealed that gnt1 shows defective post‐seedling development and incomplete cell wall biosynthesis, leading to symptoms such as failure in tiller formation, brittle leaves, reduced cell wall thickness, and decreased cellulose content. The developmental defects of gnt1 ultimately resulted in early lethality without transition to the reproductive stage. However, callus induced from gnt1 seeds could be maintained for periods, although it exhibited a low proliferation rate, small size, and hypersensitivity to salt stress. Shoot regeneration and dark‐induced leaf senescence assays indicated that the loss of GnTI function results in reduced sensitivity to cytokinin in rice. Reduced expression of A‐type O. sativa response regulators that are rapidly induced by cytokinins in gnt1 confirmed that cytokinin signaling is impaired in the mutant. These results strongly support the proposed involvement of N‐glycan maturation in transport as well as in the function of membrane proteins that are synthesized via the endomembrane system.

Summary: In plants, α1,3‐fucosyltransferase (FucT) catalyzes the transfer of fucose from GDP‐fucose to asparagine‐linked GlcNAc of the N‐glycan core in the medial Golgi. To explore the physiological significance of this processing, we isolated two Oryza sativa (rice) mutants (fuct‐1 and fuct‐2) with loss of FucT function. Biochemical analyses of the N‐glycan structure confirmed that α1,3‐fucose is missing from the N‐glycans of allelic fuct‐1 and fuct‐2. Compared with the wild‐type cv Kitaake, fuct‐1 displayed a larger tiller angle, shorter internode and panicle lengths, and decreased grain filling as well as an increase in chalky grains with abnormal shape. The mutant allele fuct‐2 gave rise to similar developmental abnormalities, although they were milder than those of fuct‐1. Restoration of a normal tiller angle in fuct‐1 by complementation demonstrated that the phenotype is caused by the loss of FucT function. Both fuct‐1 and fuct‐2 plants exhibited reduced gravitropic responses. Expression of the genes involved in tiller and leaf angle control was also affected in the mutants. We demonstrate that reduced basipetal auxin transport and low auxin accumulation at the base of the shoot in fuct‐1 account for both the reduced gravitropic response and the increased tiller angle.

In plants, transgenes with inverted repeats are used to induce efficient RNA silencing, which is also frequently induced by highly transcribed sense transgenes. RNA silencing induced by sense transgenes is dependent on RNA-dependent RNA polymerase 6 (RDR6), which converts single-stranded (ss) RNA into double-stranded (ds) RNA. By contrast, it has been proposed that RNA silencing induced by self-complementary hairpin RNA (hpRNA) does not require RDR6, because the hpRNA can directly fold back on itself to form dsRNA. However, it is unclear whether RDR6 plays a role in hpRNA-induced RNA silencing by amplifying dsRNA to spread RNA silencing within the plant. To address the efficiency of hpRNA-induced RNA silencing in the presence or absence of RDR6, Wild type (WT, Col-0) and rdr6-11 Arabidopsis thaliana lines expressing green fluorescent protein (GFP) were generated and transformed with a GFP-RNA interference (RNAi) construct. Whereas most GFP-RNAi-transformed WT lines exhibited almost complete silencing of GFP expression in the T1 generation, various levels of GFP expression remained among the GFP-RNAi-transformed rdr6-11 lines. Homozygous expression of GFP-RNAi in the T3 generation was not sufficient to induce complete GFP silencing in several rdr6-11 lines. Our results indicate that RDR6 is required for efficient hpRNA-induced RNA silencing in plants.

The most abundant N-glycan in plants is the paucimannosidic N-glycan with core β1,2-xylose and α1,3-fucose residues (Man₃XylFuc(GlcNAc)₂). Here, we report a mechanism in Arabidopsis thaliana that efficiently produces the largest N-glycan in plants. Genetic and biochemical evidence indicates that the addition of the 6-arm β1,2-GlcNAc residue by N-acetylglucosaminyltransferase II (GnTII) is less effective than additions of the core β1,2-xylose and α1,3-fucose residues by XylT, FucTA, and FucTB in Arabidopsis. Furthermore, analysis of gnt2 mutant and 35S:GnTII transgenic plants shows that the addition of the 6-arm non-reducing GlcNAc residue to the common N-glycan acceptor GlcNAcMan₃(GlcNAc)₂ inhibits additions of the core β1,2-xylose and α1,3-fucose residues. Our findings indicate that plants limit the rate of the addition of the 6-arm GlcNAc residue to the common N-glycan acceptor as a mechanism to facilitate formation of the prevalent N-glycans with Man₃XylFuc(GlcNAc)₂ and (GlcNAc)₂Man₃XylFuc(GlcNAc)₂ structures.

A major discovery in human etiology recognized that cervical cancer is a consequence of an infection caused by some mucosatropic types of human papillomavirus (HPV). Since L1 protein of HPV is able to induce the formation of neutralizing antibodies, it becomes a protein target to develop HPV vaccines. Therefore, this study aims to obtain and analyze the expression of HPV subunit recombinant protein, namely L1 HPV 52 in E. coli BL21 DE3. The raw material used was L1 HPV 52 protein, while the synthetic gene, which is measured at 1473 bp in pD451-MR plasmid, was codon-optimized (ATUM) and successfully integrated into 5643 base pairs (bps) of pETSUMO. Bioinformatic studies were also conducted to analyze B cell epitope, T cell epitope, and immunogenicity prediction for L1HPV52 protein. The pETSUMO-L1HPV52 construct was successfully obtained in a correct ligation size when it was cut with EcoRI. Digestion by EcoRI revealed a size of 5953 and 1160 bps for both TA cloning petSUMO vector and gene of interest, respectively. Furthermore, the right direction of construct pETSUMO-L1HPV52 was proven by PCR techniques using specific primer pairs then followed by sequencing, which shows 147 base pairs. Characterization of L1 HPV 52 by SDS-PAGE analysis confirms the presence of a protein band at a size of ~55 kDa with 6.12 mg/L of total protein concentration. Observation under by transmission electron microscope demonstrates the formation of VLP-L1 at a size between 30 and 40 nm in assembly buffer under the condition of pH 5.4. Based on bioinformatics studies, we found that there are three B cell epitopes (GFPDTSFYNPET, DYLQMASEPY, KEKFSADLDQFP) and four T cell epitopes (YLQMASEPY, PYGDSLFFF, DSLFFFLRR, MFVRHFFNR). Moreover, an immunogenicity study shows that among all the T cell epitopes, the one that has the highest affinity value is DSLFFFLRR for Indonesian HLAs. Regarding the achievement on successful formation of L1 HPV52-VLPs, followed by some possibilities found from bioinformatics studies, this study suggests promising results for future development of L1 HPV type 52 vaccine in Indonesia.