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The inositol requiring enzyme (IRE1) is an endoplasmic reticulum (ER) stress sensor. When activated, it splices the bZIP60 mRNA, producing a truncated transcription factor that upregulates genes involved in the unfolded protein response. Bax inhibitor 1 (BI-1) is another ER stress sensor that regulates cell death in response to environmental assaults. The potyvirus 6K2 and potexvirus TGB3 proteins are known to reside in the ER, serving, respectively, as anchors for the viral replicase and movement protein complex. This study used green fluorescent protein (GFP)-tagged Turnip mosaic virus (TuMV), Plantago asiatica mosaic virus (PlAMV), Potato virus Y (PVY), and Potato virus X (PVX) to determine that the IRE1/bZIP60 pathway and BI-1 machinery are induced early in virus infection in Arabidopsis thaliana, Nicotiana benthamiana, and Solanum tuberosum. Agrodelivery of only the potyvirus 6K2 or TGB3 genes into plant cells activated bZIP60 and BI-1 expression in Arabidopsis thaliana, N. benthamiana, and S. tuberosum. Homozygous ire1a-2, ire1b-4, and ire1a-2/ire1b-4 mutant Arabidopsis plants were inoculated with TuMV-GFP or PlAMV-GFP. PlAMV accumulates to a higher level in ire1a-2 or ire1a-2/ire1b-4 mutant plants than in ire1b-4 or wild-type plants. TuMV-GFP accumulates to a higher level in ire1a-2, ire1b-4, or ire1a-2/ire1b-4 compared with wild-type plants, suggesting that both isoforms contribute to TuMV-GFP infection. Gene silencing was used to knock down bZIP60 and BI-1 expression in N. benthamiana. PVX-GFP and PVY-GFP accumulation was significantly elevated in these silenced plants compared with control plants. This study demonstrates that two ER stress pathways, namely IRE1/bZIP60 and the BI-1 pathway, limit systemic accumulation of potyvirus and potexvirus infection. Silencing BI-1 expression also resulted in systemic necrosis. These data suggest that ER stress-activated pathways, led by IRE1 and BI-1, respond to invading potyvirus and potexviruses to restrict virus infection and enable physiological changes enabling plants to tolerate virus assault.

Phosphomannomutase (PMM) catalyzes the interconversion of mannose-6-phosphate and mannose-1-phosphate. However, systematic molecular and functional investigations on PMM from higher plants have hitherto not been reported. In this work, PMM cDNAs were isolated from Arabidopsis, Nicotiana benthamiana, soybean, tomato, rice and wheat. Amino acid sequence comparisons indicated that plant PMM proteins exhibited significant identity to their fungal and mammalian orthologs. In line with the similarity in primary structure, plant PMM complemented the sec53-6 temperature sensitive mutant of Saccharomyces cerevisiae. Histidine-tagged Arabidopsis PMM (AtPMM) purified from Escherichia coli converted mannose-1-phosphate into mannose-6-phosphate and glucose-1-phosphate into glucose-6-phosphate, with the former reaction being more efficient than the latter one. In Arabidopsis and N. benthamiana, PMM was constitutively expressed in both vegetative and reproductive organs. Reducing the PMM expression level through virus-induced gene silencing caused a substantial decrease in ascorbic acid (AsA) content in N. benthamiana leaves. Conversely, raising the PMM expression level in N. benthamiana using viral-vector-mediated ectopic expression led to a 20-50% increase in AsA content. Consistent with this finding, transgenic expression of an AtPMM-GFP fusion protein in Arabidopsis also increased AsA content by 25-33%. Collectively, this study improves our understanding on the molecular and functional properties of plant PMM and provides genetic evidence on the involvement of PMM in the biosynthesis of AsA in Arabidopsis and N. benthamiana plants.

Phosphomannomutase (PMM) catalyzes the interconversion of mannose-6-phosphate and mannose-1-phosphate. However, systematic molecular and functional investigations on PMM from higher plants have hitherto not been reported. In this work, PMM cDNAs were isolated from Arabidopsis, Nicotiana benthamiana, soybean, tomato, rice and wheat. Amino acid sequence comparisons indicated that plant PMM proteins exhibited significant identity to their fungal and mammalian orthologs. In line with the similarity in primary structure, plant PMM complemented the sec53-6 temperature sensitive mutant of Saccharomyces cerevisiae. Histidine-tagged Arabidopsis PMM (AtPMM) purified from Escherichia coli converted mannose-1-phosphate into mannose-6-phosphate and glucose-1-phosphate into glucose-6-phosphate, with the former reaction being more efficient than the latter one. In Arabidopsis and N. benthamiana, PMM was constitutively expressed in both vegetative and reproductive organs. Reducing the PMM expression level through virus-induced gene silencing caused a substantial decrease in ascorbic acid (AsA) content in N. benthamiana leaves. Conversely, raising the PMM expression level in N. benthamiana using viral-vector-mediated ectopic expression led to a 20–50% increase in AsA content. Consistent with this finding, transgenic expression of an AtPMM–GFP fusion protein in Arabidopsis also increased AsA content by 25–33%. Collectively, this study improves our understanding on the molecular and functional properties of plant PMM and provides genetic evidence on the involvement of PMM in the biosynthesis of AsA in Arabidopsis and N. benthamiana plants. [ABSTRACT FROM AUTHOR]