Periodic Reporting for period 1 - MossTOR (Investigating the TORC1 signaling pathway in the moss Physcomitrella and its application for enhanced production of valuable pharmaceutical compounds)
Reporting period: 2022-08-01 to 2025-08-31
Summary of the context and overall objectives of the project
TORC1 (Target of Rapamycin Complex 1) is a highly conserved kinase complex that plays a central and pivotal role in a signalling pathway that coordinates eukaryotic cell growth according to diverse extrinsic and intrinsic cues. It was identified and characterized first in yeast, then in animals, humans, and finally in seed plants and algae, and it is assumed to be present in all eukaryotes1–3. Its mode of action has been largely dissected. TORC1 operates by integrating diverse upstream signals such as nutrients, growth factors, hormones and energy, which act positively on its activity, to modulate by phosphorylation a wide variety of effector proteins that promote anabolic functions (e.g. protein and RNA synthesis, lipid and nucleotide synthesis, ribosome biogenesis, and in plants chloroplast biogenesis), thus promoting cell growth. Other proteins phosphorylated by TORC1 inhibit autophagy and mRNA degradation, and such processes are thus triggered when TORC1 is inhibited (e.g. under nutrient deprivation conditions or environmental stresses such as osmotic stress or some pathogens)3–6. These coordinated actions are crucial for the control of cell growth and survival. Consistently, deregulation of this pathway in humans is associated with a number of pathological conditions including cancer, and understanding how TORC1 works might help to develop novel therapies7. This is why studies on TORC1 and its regulation have been growing exponentially during the last years. Added to this are the findings that its activity could be tuned to improve diverse plant biotechnological applications such as enhancing crop biomass, yield and resistance to stress, as reported in several recent studies8–10.
The TOR protein that is part of TORC1 is an atypical serine/threonine kinase and a member of the phosphatidylinositol (PI) 3-kinase-related protein kinases (PIKKs) family. It was originally identified in a genetic selection for Saccharomyces cerevisiae mutants that could grow in the presence of rapamycin, a macrolide from Streptomyces hygroscopicus11. Rapamycin inhibits TOR by forming a ternary complex with the highly conserved 12-kDa FK506-binding protein (FKBP12) and the FKBP12-rapamycin binding (FRB) domain in TOR, thus restricting its kinase domain12,13. Other conserved regions in TOR are the HEAT repeats and FAT located amino-terminal to the FRB and kinase domains, and the FATC located carboxy-terminal14. TOR uses its HEAT repeats and the kinase domain to interact with the two proteins Raptor and Lst8, found in all eukaryotes. Apart from bringing TORC1 to its final functional conformation, these proteins play an important role in regulating TOR’s activity either by integrating direct information or by bridging TOR to its substrates4,5. In yeast and animals, TOR is also part of another complex with a distinct function known as TORC2. Except for TOR and Lst8, there are no homologs of TORC2 subunits in plants, and therefore it is assumed to be absent15.
Besides elucidating the central role that TORC1 plays in plant development, as it appeared to be from the work on the seed plant (angiosperm) model Arabidopsis thaliana5,16,17, a growing interest of plant physiologists is to study TORC1-function in plant growth, yield and resistance to stress. These are strikingly often correlated with an increase in TORC1 activity, thus opening the door for innovative plant biotechnology applications. Studies on Arabidopsis and on rice (Oryza sativa ssp. indica) interestingly revealed that transgenic plants with hyperactive TORC1 signalling, triggered by over-expressing the TOR kinase or the Tap46 inhibitory subunit of the TORC1 downstream negative effector protein phosphatase 2A, exhibit enhanced shoot growth, leaf and cell size, inflorescence size, seed size and weight, chlorophyll content, and tolerance to abiotic stresses such as to ABA and drought8–10. Comparative transcriptome analysis showed a high correlation between these phenotypes and the expressed genes in these transgenic plants, where genes implicated in processes such as transcription activation, ribosome biogenesis, protein translation, cell wall and lignin biosynthesis, chloroplast biogenesis, nitrogen assimilation and stress resistance are upregulated, whereas genes triggering degradational processes such as autophagy and lipid degradation are downregulated16,18,9,19,20,10. Inhibiting TORC1 results in an opposite phenotype and in algae this lead to triacylglycerol accumulation, a strategy that could be used to improve biofuel production from these organisms6,21.
So far, TORC1 has not been studied in bryophytes and the Chair Plant Biotechnology is particularly interested in researching if TORC1 can be tuned to improve bryophyte-based biotechnological applications (Bryotechnology). Possible applications are the production of valuable compounds as active pharmaceutical ingredients (e.g. paclitaxel, the precursor for the anticancer diterpene, and the anti-malarial drug artemisinin), or recombinant pharmaceutical proteins (e.g. Factor H for patients with complement disorders)22–24. Bryophytes (comprising liverworts, hornworts and mosses) are small non-vascular, non-seed plants that resemble in phenotype the first plants that conquered land about 600 million years ago and are separated from seed plants by about 450 million years of evolution. The the Chair Plant Biotechnology developed the moss Physcomitrella (new botanical name: Physcomitrium patens) to a model species for basic biology and biotechnology22–25. Research on Physcomitrella provided ground-breaking new insights into the evolution of developmental processes26–28. To better understand the functional evolution of TORC1 and its role in plant development, Physcomitrella needs to be added to the list of model organisms.
The goal of this project is first to characterize the TORC1 signalling pathway in Physcomitrella in order to address another important goal, which is to test whether TORC1 could be tuned to improve moss-based biotechnological applications as indicated above. To reach these goals, the project is organized in four scientific work packages (WP). The sensitivity of Physcomitrella to rapamycin will be determined, and the PpTOR, PpRaptor, and PpLst8 proteins will be identified and characterized both functionally and structurally. A TORC1 activity assay will be established to evaluate whether Physcomitrella TORC1 responds to conserved signals. Furthermore, Physcomitrella lines with hyperactive TORC1 signalling will be developed and characterized, and their potential use for the production of valuable compounds will be assessed in collaboration with the industry.
Bibliography
1. Loewith, R. et al. Two TOR complexes, only one of which is rapamycin sensitive, have distinct roles in cell growth control. Mol. Cell 10, 457 (2002).
2. Crespo, J. L., Díaz-Troya, S. & Florencio, F. J. Inhibition of target of rapamycin signaling by rapamycin in the unicellular green alga Chlamydomonas reinhardtii. Plant Physiol 139, 1736 (2005).
3. Wullschleger, S., Loewith, R. & Hall, M. N. TOR signaling in growth and metabolism. Cell 124, 471 (2006).
4. González, A. & Hall, M. N. Nutrient sensing and TOR signaling in yeast and mammals. EMBO J. 36, 397 (2017).
5. Shi, L., Wu, Y. & Sheen, J. TOR signaling in plants: conservation and innovation. Development 145, dev160887 (2018).
6. Pancha, I. et al. Microalgal Target of Rapamycin (TOR): a central regulatory hub for growth, stress response and biomass production. Plant Cell Physiol 61, 675 (2020).
7. Saxton, R. A. & Sabatini, D. M. mTOR signaling in growth, metabolism, and disease. Cell 168, 960 (2017).
8. Deprost, D. et al. The Arabidopsis TOR kinase links plant growth, yield, stress resistance and mRNA translation. EMBO Rep 8, 864 (2007).
9. Ahn, C. S., Ahn, H.-K. & Pai, H.-S. Overexpression of the PP2A regulatory subunit Tap46 leads to enhanced plant growth through stimulation of the TOR signalling pathway. J Exp Bot 66, 827 (2015).
10. Bakshi, A. et al. Ectopic expression of Arabidopsis Target of Rapamycin (AtTOR) improves water-use efficiency and yield potential in rice. Sci Rep 7, 42835 (2017).
11. Heitman, J., Movva, N. R. & Hall, M. N. Targets for cell cycle arrest by the immunosuppressant rapamycin in yeast. Science 253, 905 (1991).
12. Heitman, J. et al. FK 506-binding protein proline rotamase is a target for the immunosuppressive agent FK 506 in Saccharomyces cerevisiae. Proc. Natl. Acad. Sci. U.S.A. 88, 1948 (1991).
13. Choi, J. et al. Structure of the FKBP12-rapamycin complex interacting with the binding domain of human FRAP. Science 273, 239 (1996).
14. Maegawa, K. et al. Evolutionary conservation of TORC1 components, TOR, Raptor, and LST8, between rice and yeast. Mol Genet Genomics 290, 2019 (2015).
15. Dobrenel, T. et al. TOR signaling and nutrient sensing. Annu Rev Plant Biol 67, 261 (2016).
16. Moreau, M. et al. Mutations in the Arabidopsis homolog of LST8/GβL, a partner of the target of Rapamycin kinase, impair plant growth, flowering, and metabolic adaptation to long days. Plant Cell 24, 463 (2012).
17. Ingargiola, C. et al. The plant Target of Rapamycin: a Conduc TOR of nutrition and metabolism in photosynthetic organisms. Genes 11, E1285 (2020).
18. Xiong, Y. et al. Glucose-TOR signalling reprograms the transcriptome and activates meristems. Nature 496, 181 (2013).
19. Xiong, F. et al. Tomato FK506 Binding Protein 12KD (FKBP12) mediates the interaction between rapamycin and Target of Rapamycin (TOR). Front Plant Sci 7, 1746 (2016).
20. Dobrenel, T. et al. The Arabidopsis TOR kinase specifically regulates the expression of nuclear genes coding for plastidic ribosomal proteins and the phosphorylation of the cytosolic ribosomal protein S6. Front Plant Sci 7, 1611 (2016).
21. Pérez-Pérez, M. E., Couso, I. & Crespo, J. L. The TOR signaling network in the model unicellular green alga Chlamydomonas reinhardtii. Biomolecules 7, E54 (2017).
22. Reski, R., Bae, H. & Simonsen, H. T. Physcomitrella patens, a versatile synthetic biology chassis. Plant Cell Rep 37, 1409 (2018).
23. Decker, E. L. & Reski, R. Mosses in biotechnology. Curr Opin Biotechnol 61, 21 (2020).
24. Horn, A. et al. Natural products from bryophytes: from basic biology to biotechnological applications. Crit. Rev. Plant Sci. 40, 191 (2021).
25. Reski, R. Development, genetics and molecular biology of mosses. Botanica Acta 111, 1 (1998).
26. Khraiwesh, B. et al. Transcriptional control of gene expression by microRNAs. Cell 140, 111 (2010).
27. Horst, N. A. et al. A single homeobox gene triggers phase transition, embryogenesis and asexual reproduction. Nat. Plants 2, 1 (2016).
28. Resemann, H. C. et al. Convergence of sphingolipid desaturation across over 500 million years of plant evolution. Nat. Plants 7, 219 (2021).
The TOR protein that is part of TORC1 is an atypical serine/threonine kinase and a member of the phosphatidylinositol (PI) 3-kinase-related protein kinases (PIKKs) family. It was originally identified in a genetic selection for Saccharomyces cerevisiae mutants that could grow in the presence of rapamycin, a macrolide from Streptomyces hygroscopicus11. Rapamycin inhibits TOR by forming a ternary complex with the highly conserved 12-kDa FK506-binding protein (FKBP12) and the FKBP12-rapamycin binding (FRB) domain in TOR, thus restricting its kinase domain12,13. Other conserved regions in TOR are the HEAT repeats and FAT located amino-terminal to the FRB and kinase domains, and the FATC located carboxy-terminal14. TOR uses its HEAT repeats and the kinase domain to interact with the two proteins Raptor and Lst8, found in all eukaryotes. Apart from bringing TORC1 to its final functional conformation, these proteins play an important role in regulating TOR’s activity either by integrating direct information or by bridging TOR to its substrates4,5. In yeast and animals, TOR is also part of another complex with a distinct function known as TORC2. Except for TOR and Lst8, there are no homologs of TORC2 subunits in plants, and therefore it is assumed to be absent15.
Besides elucidating the central role that TORC1 plays in plant development, as it appeared to be from the work on the seed plant (angiosperm) model Arabidopsis thaliana5,16,17, a growing interest of plant physiologists is to study TORC1-function in plant growth, yield and resistance to stress. These are strikingly often correlated with an increase in TORC1 activity, thus opening the door for innovative plant biotechnology applications. Studies on Arabidopsis and on rice (Oryza sativa ssp. indica) interestingly revealed that transgenic plants with hyperactive TORC1 signalling, triggered by over-expressing the TOR kinase or the Tap46 inhibitory subunit of the TORC1 downstream negative effector protein phosphatase 2A, exhibit enhanced shoot growth, leaf and cell size, inflorescence size, seed size and weight, chlorophyll content, and tolerance to abiotic stresses such as to ABA and drought8–10. Comparative transcriptome analysis showed a high correlation between these phenotypes and the expressed genes in these transgenic plants, where genes implicated in processes such as transcription activation, ribosome biogenesis, protein translation, cell wall and lignin biosynthesis, chloroplast biogenesis, nitrogen assimilation and stress resistance are upregulated, whereas genes triggering degradational processes such as autophagy and lipid degradation are downregulated16,18,9,19,20,10. Inhibiting TORC1 results in an opposite phenotype and in algae this lead to triacylglycerol accumulation, a strategy that could be used to improve biofuel production from these organisms6,21.
So far, TORC1 has not been studied in bryophytes and the Chair Plant Biotechnology is particularly interested in researching if TORC1 can be tuned to improve bryophyte-based biotechnological applications (Bryotechnology). Possible applications are the production of valuable compounds as active pharmaceutical ingredients (e.g. paclitaxel, the precursor for the anticancer diterpene, and the anti-malarial drug artemisinin), or recombinant pharmaceutical proteins (e.g. Factor H for patients with complement disorders)22–24. Bryophytes (comprising liverworts, hornworts and mosses) are small non-vascular, non-seed plants that resemble in phenotype the first plants that conquered land about 600 million years ago and are separated from seed plants by about 450 million years of evolution. The the Chair Plant Biotechnology developed the moss Physcomitrella (new botanical name: Physcomitrium patens) to a model species for basic biology and biotechnology22–25. Research on Physcomitrella provided ground-breaking new insights into the evolution of developmental processes26–28. To better understand the functional evolution of TORC1 and its role in plant development, Physcomitrella needs to be added to the list of model organisms.
The goal of this project is first to characterize the TORC1 signalling pathway in Physcomitrella in order to address another important goal, which is to test whether TORC1 could be tuned to improve moss-based biotechnological applications as indicated above. To reach these goals, the project is organized in four scientific work packages (WP). The sensitivity of Physcomitrella to rapamycin will be determined, and the PpTOR, PpRaptor, and PpLst8 proteins will be identified and characterized both functionally and structurally. A TORC1 activity assay will be established to evaluate whether Physcomitrella TORC1 responds to conserved signals. Furthermore, Physcomitrella lines with hyperactive TORC1 signalling will be developed and characterized, and their potential use for the production of valuable compounds will be assessed in collaboration with the industry.
Bibliography
1. Loewith, R. et al. Two TOR complexes, only one of which is rapamycin sensitive, have distinct roles in cell growth control. Mol. Cell 10, 457 (2002).
2. Crespo, J. L., Díaz-Troya, S. & Florencio, F. J. Inhibition of target of rapamycin signaling by rapamycin in the unicellular green alga Chlamydomonas reinhardtii. Plant Physiol 139, 1736 (2005).
3. Wullschleger, S., Loewith, R. & Hall, M. N. TOR signaling in growth and metabolism. Cell 124, 471 (2006).
4. González, A. & Hall, M. N. Nutrient sensing and TOR signaling in yeast and mammals. EMBO J. 36, 397 (2017).
5. Shi, L., Wu, Y. & Sheen, J. TOR signaling in plants: conservation and innovation. Development 145, dev160887 (2018).
6. Pancha, I. et al. Microalgal Target of Rapamycin (TOR): a central regulatory hub for growth, stress response and biomass production. Plant Cell Physiol 61, 675 (2020).
7. Saxton, R. A. & Sabatini, D. M. mTOR signaling in growth, metabolism, and disease. Cell 168, 960 (2017).
8. Deprost, D. et al. The Arabidopsis TOR kinase links plant growth, yield, stress resistance and mRNA translation. EMBO Rep 8, 864 (2007).
9. Ahn, C. S., Ahn, H.-K. & Pai, H.-S. Overexpression of the PP2A regulatory subunit Tap46 leads to enhanced plant growth through stimulation of the TOR signalling pathway. J Exp Bot 66, 827 (2015).
10. Bakshi, A. et al. Ectopic expression of Arabidopsis Target of Rapamycin (AtTOR) improves water-use efficiency and yield potential in rice. Sci Rep 7, 42835 (2017).
11. Heitman, J., Movva, N. R. & Hall, M. N. Targets for cell cycle arrest by the immunosuppressant rapamycin in yeast. Science 253, 905 (1991).
12. Heitman, J. et al. FK 506-binding protein proline rotamase is a target for the immunosuppressive agent FK 506 in Saccharomyces cerevisiae. Proc. Natl. Acad. Sci. U.S.A. 88, 1948 (1991).
13. Choi, J. et al. Structure of the FKBP12-rapamycin complex interacting with the binding domain of human FRAP. Science 273, 239 (1996).
14. Maegawa, K. et al. Evolutionary conservation of TORC1 components, TOR, Raptor, and LST8, between rice and yeast. Mol Genet Genomics 290, 2019 (2015).
15. Dobrenel, T. et al. TOR signaling and nutrient sensing. Annu Rev Plant Biol 67, 261 (2016).
16. Moreau, M. et al. Mutations in the Arabidopsis homolog of LST8/GβL, a partner of the target of Rapamycin kinase, impair plant growth, flowering, and metabolic adaptation to long days. Plant Cell 24, 463 (2012).
17. Ingargiola, C. et al. The plant Target of Rapamycin: a Conduc TOR of nutrition and metabolism in photosynthetic organisms. Genes 11, E1285 (2020).
18. Xiong, Y. et al. Glucose-TOR signalling reprograms the transcriptome and activates meristems. Nature 496, 181 (2013).
19. Xiong, F. et al. Tomato FK506 Binding Protein 12KD (FKBP12) mediates the interaction between rapamycin and Target of Rapamycin (TOR). Front Plant Sci 7, 1746 (2016).
20. Dobrenel, T. et al. The Arabidopsis TOR kinase specifically regulates the expression of nuclear genes coding for plastidic ribosomal proteins and the phosphorylation of the cytosolic ribosomal protein S6. Front Plant Sci 7, 1611 (2016).
21. Pérez-Pérez, M. E., Couso, I. & Crespo, J. L. The TOR signaling network in the model unicellular green alga Chlamydomonas reinhardtii. Biomolecules 7, E54 (2017).
22. Reski, R., Bae, H. & Simonsen, H. T. Physcomitrella patens, a versatile synthetic biology chassis. Plant Cell Rep 37, 1409 (2018).
23. Decker, E. L. & Reski, R. Mosses in biotechnology. Curr Opin Biotechnol 61, 21 (2020).
24. Horn, A. et al. Natural products from bryophytes: from basic biology to biotechnological applications. Crit. Rev. Plant Sci. 40, 191 (2021).
25. Reski, R. Development, genetics and molecular biology of mosses. Botanica Acta 111, 1 (1998).
26. Khraiwesh, B. et al. Transcriptional control of gene expression by microRNAs. Cell 140, 111 (2010).
27. Horst, N. A. et al. A single homeobox gene triggers phase transition, embryogenesis and asexual reproduction. Nat. Plants 2, 1 (2016).
28. Resemann, H. C. et al. Convergence of sphingolipid desaturation across over 500 million years of plant evolution. Nat. Plants 7, 219 (2021).
Work performed from the beginning of the project to the end of the period covered by the report and main results achieved so far
We have found that Physcomitrella is sensitive to AZD8055 in a dose-dependent manner. A dose of 10 µM completely inhibited Physcomitrella growth.
We have found that Physcomitrella is resistant to rapamycin. On the other hand, the FKBP12 protein of Physcomitrella completely conferred sensitivity to rapamycin of yeast cells lacking endogenous FKBP12. This result has indicated that Physcomitrella sensitivity to the drug is due to low levels of the endogenous FKBP12 protein.
We isolated a Physcomitrella line with multiple integration of a DNA cassette expressing PpFKBP12 under the 35S promoter. This line showed 75 % sensitivity to 10 µM rapamycin (milestone M1).
We have also found that both rapamycin and AZD8055 trigger chlorosis, inhibit photosynthesis, delay the cell cycle, and affect total protein and chlorophyll levels. These data are first in describing TOR signalling in the moss Physcomitrella.
We have found that Physcomitrella LST8 can functionally substitute its yeast homolog in a complementation assay.
Physcomitrella TOR and its four RAPTOR paralogs couldn’t substitute their yeast homologs in complementation assays.
We isolated Physcomitrella lines in which LST8, TOR, or the four RAPTORs can be downregulated via estradiol-inducible RNA interference systems (milestone M2).
We have found that the downregulation of the above-mentioned genes impacts Physcomitrella growth and development.
Because the Physcomitrella line overexpressing FKBP12 carried a C-terminal Myc-tag, we immunoprecipitated FKBP12-Myc using anti-Myc beads from rapamycin-treated cells and analysed the eluates by LC-MS/MS to test whether TOR, LST8, or any of the four RAPTOR isoforms co-purified. This experiment aimed to provide evidence that Physcomitrella TOR, LST8, and RAPTOR form a complex in vivo, but no such interactions were detected under the tested conditions.
We used an antibody against phosphorylated Thr389 in human S6K1 to detect phosphorylated ribosomal S6 kinases in Physcomitrella, which also recognizes phosphorylated Thr449 and Thr455 in Arabidopsis S6K1 and S6K2, respectively. It did not work. This is highly likely to be due to low expression levels of Physcomitrella S6Ks.
As an alternative, we used the antibody that recognizes phosphorylated Ser240 in Arabidopsis RPS6 to detect phosphorylated PpRPS6 proteins, but it also did not work. This was not due to low expression level of the PpRPS6 proteins, since the total form could be detected, but to a lack of conservation of the sequence window around the known phosphorylation site of AtRPS6A.
To determine which serine in PpRPS6 is phosphorylated in a TOR-dependent manner, we phospho-enriched peptides from Physcomitrella samples treated with AZD8055 and subjected them to LC-MS/MS analyses. We have found that Ser241 in PpRPS6 is phosphorylated in a TOR-dependent manner. This work was carried out in collaboration with the Biochemistry and Functional Proteomics Laboratory of the University of Freiburg.
As the milestone (M3) to develop a Physcomitrella PpTORC1 activity assay via Western blot could not be achieved, we could not test TORC1 responses to different environmental cues.
It was previously found in the host laboratory that synthetic auxin enhances the yield in Physcomitrella of a recombinant pharmaceutical protein called MFHR1. Since auxin is one of the major activators of TORC1 in plants, we repeated the latter experiment, but this time in the presence of AZD8055 (which inhibits TORC1). We found that auxin did not have an effect on MFHR1 yield. This result indicated that by modulating TORC1 activity we can affect the yield of recombinant pharmaceutical proteins.
We cloned PpTOR on plasmid vectors under the expression of the Arabidopsis Ubiqtuin10 or the 35S promoter. The linearized plasmids were used to isolate Physcomitrella lines, already producing MFHR1 or MFHR13 (another recombinant pharmaceutical protein), with single or multiple integrations (stable transformants) of the PpTOR expression constructs. Our methodology was correct but the first trial to isolate these lines has failed. Due to limited time, we could not repeat this experiment. Therefore, milestone 4 could not be achieved, neither objectives 1 and 2.
As an alternative to the above strategy, we checked if the yield of MFHR13 could be enhanced in transformed protoplasts with circular plasmids of the PpTOR expression constructs. Using ELISA as a protocol, we found that MFHR13’s yield increased by 44% and 50.54% when PpTOR was transiently expressed from the Ubiquitin10 or the 35S promoter, respectively. These results indicate that by tuning TORC1, we can enhance the yield of recombinant pharmaceutical protein in transiently transfected Physcomitrella, which supports our hypothesis.
We have found that Physcomitrella is resistant to rapamycin. On the other hand, the FKBP12 protein of Physcomitrella completely conferred sensitivity to rapamycin of yeast cells lacking endogenous FKBP12. This result has indicated that Physcomitrella sensitivity to the drug is due to low levels of the endogenous FKBP12 protein.
We isolated a Physcomitrella line with multiple integration of a DNA cassette expressing PpFKBP12 under the 35S promoter. This line showed 75 % sensitivity to 10 µM rapamycin (milestone M1).
We have also found that both rapamycin and AZD8055 trigger chlorosis, inhibit photosynthesis, delay the cell cycle, and affect total protein and chlorophyll levels. These data are first in describing TOR signalling in the moss Physcomitrella.
We have found that Physcomitrella LST8 can functionally substitute its yeast homolog in a complementation assay.
Physcomitrella TOR and its four RAPTOR paralogs couldn’t substitute their yeast homologs in complementation assays.
We isolated Physcomitrella lines in which LST8, TOR, or the four RAPTORs can be downregulated via estradiol-inducible RNA interference systems (milestone M2).
We have found that the downregulation of the above-mentioned genes impacts Physcomitrella growth and development.
Because the Physcomitrella line overexpressing FKBP12 carried a C-terminal Myc-tag, we immunoprecipitated FKBP12-Myc using anti-Myc beads from rapamycin-treated cells and analysed the eluates by LC-MS/MS to test whether TOR, LST8, or any of the four RAPTOR isoforms co-purified. This experiment aimed to provide evidence that Physcomitrella TOR, LST8, and RAPTOR form a complex in vivo, but no such interactions were detected under the tested conditions.
We used an antibody against phosphorylated Thr389 in human S6K1 to detect phosphorylated ribosomal S6 kinases in Physcomitrella, which also recognizes phosphorylated Thr449 and Thr455 in Arabidopsis S6K1 and S6K2, respectively. It did not work. This is highly likely to be due to low expression levels of Physcomitrella S6Ks.
As an alternative, we used the antibody that recognizes phosphorylated Ser240 in Arabidopsis RPS6 to detect phosphorylated PpRPS6 proteins, but it also did not work. This was not due to low expression level of the PpRPS6 proteins, since the total form could be detected, but to a lack of conservation of the sequence window around the known phosphorylation site of AtRPS6A.
To determine which serine in PpRPS6 is phosphorylated in a TOR-dependent manner, we phospho-enriched peptides from Physcomitrella samples treated with AZD8055 and subjected them to LC-MS/MS analyses. We have found that Ser241 in PpRPS6 is phosphorylated in a TOR-dependent manner. This work was carried out in collaboration with the Biochemistry and Functional Proteomics Laboratory of the University of Freiburg.
As the milestone (M3) to develop a Physcomitrella PpTORC1 activity assay via Western blot could not be achieved, we could not test TORC1 responses to different environmental cues.
It was previously found in the host laboratory that synthetic auxin enhances the yield in Physcomitrella of a recombinant pharmaceutical protein called MFHR1. Since auxin is one of the major activators of TORC1 in plants, we repeated the latter experiment, but this time in the presence of AZD8055 (which inhibits TORC1). We found that auxin did not have an effect on MFHR1 yield. This result indicated that by modulating TORC1 activity we can affect the yield of recombinant pharmaceutical proteins.
We cloned PpTOR on plasmid vectors under the expression of the Arabidopsis Ubiqtuin10 or the 35S promoter. The linearized plasmids were used to isolate Physcomitrella lines, already producing MFHR1 or MFHR13 (another recombinant pharmaceutical protein), with single or multiple integrations (stable transformants) of the PpTOR expression constructs. Our methodology was correct but the first trial to isolate these lines has failed. Due to limited time, we could not repeat this experiment. Therefore, milestone 4 could not be achieved, neither objectives 1 and 2.
As an alternative to the above strategy, we checked if the yield of MFHR13 could be enhanced in transformed protoplasts with circular plasmids of the PpTOR expression constructs. Using ELISA as a protocol, we found that MFHR13’s yield increased by 44% and 50.54% when PpTOR was transiently expressed from the Ubiquitin10 or the 35S promoter, respectively. These results indicate that by tuning TORC1, we can enhance the yield of recombinant pharmaceutical protein in transiently transfected Physcomitrella, which supports our hypothesis.
Progress beyond the state of the art and expected potential impact (including the socio-economic impact and the wider societal implications of the project so far)
The work conducted during the action describes for the first time the dissection of TOR signalling in the moss Physcomitrella. The developed methodology constitutes a basis to further characterise this pathway in Physcomitrella, which will improve understanding of TOR in general and especially its evolution. The findings that by overexpressing TOR we can enhance the yield of recombinant pharmaceutical proteins is of high interest for the biopharmaceutical-producing industry.