Periodic Reporting for period 1 - CrysPINs (Crystal structures of PIN proteins - CrysPINs)
Reporting period: 2018-08-01 to 2020-07-31
The main goal of the “CrysPINS” project is to derive a model for the structure of PIN1 protein, and optionally other PIN proteins, using techniques of molecular biology, biochemistry and crystallography.
Members of the PIN family are plant-specific auxin transporters. Auxin is one of the key plant hormones and it is involved in the control of all growth and developmental responses, as well as how plants adapt to their changing environment. Therefore, it is vitally important that we understand how gradients of this hormone are generated and maintained. For these functions plant cells require auxin transporters, and amongst these the PIN proteins are principal players.
Although it is well known that PIN proteins drive polar auxin transport, the auxin research community is missing detailed functional and mechanistic models of these enigmatic transporters. The Fellow’s considerable experience from studying auxin metabolism and transport on the cellular and plant levels, combined with the expertise at the University of Warwick on auxin recognition and the structural biology of mammalian membrane transporter proteins, make it timely to move the science forward with a project focussed on the molecular structure of PINs. This goal will reveal how the PINs are energized, and create links with the auxin herbicide industries to explore opportunities for collaborations on agro-pharmaceutical compound design.
Societal benefit is very important. Synthetic auxins are used world-wide as herbicides in cereal and rice farms, as well as on amenity turf etc. Resistance to auxin herbicides is becoming increasingly frequent and we need to understand the mechanisms of these resistances so that we might develop strategies to maintain and grow food supply. Through our goal of determining the structure of the PIN auxin transporters we expect to be able to help design new auxins and advise on strategies to limit the damage of herbicide resistances in order to contribute to global food security.
Members of the PIN family are plant-specific auxin transporters. Auxin is one of the key plant hormones and it is involved in the control of all growth and developmental responses, as well as how plants adapt to their changing environment. Therefore, it is vitally important that we understand how gradients of this hormone are generated and maintained. For these functions plant cells require auxin transporters, and amongst these the PIN proteins are principal players.
Although it is well known that PIN proteins drive polar auxin transport, the auxin research community is missing detailed functional and mechanistic models of these enigmatic transporters. The Fellow’s considerable experience from studying auxin metabolism and transport on the cellular and plant levels, combined with the expertise at the University of Warwick on auxin recognition and the structural biology of mammalian membrane transporter proteins, make it timely to move the science forward with a project focussed on the molecular structure of PINs. This goal will reveal how the PINs are energized, and create links with the auxin herbicide industries to explore opportunities for collaborations on agro-pharmaceutical compound design.
Societal benefit is very important. Synthetic auxins are used world-wide as herbicides in cereal and rice farms, as well as on amenity turf etc. Resistance to auxin herbicides is becoming increasingly frequent and we need to understand the mechanisms of these resistances so that we might develop strategies to maintain and grow food supply. Through our goal of determining the structure of the PIN auxin transporters we expect to be able to help design new auxins and advise on strategies to limit the damage of herbicide resistances in order to contribute to global food security.
1. We have successfully expressed a set of Arabidopsis PINs in the baculovirus system. PIN1, PIN2, PIN5 and we are still working on PIN8. We have optimised expression, dropped PIN2, have a protocol for detergent extraction and purification using TALON. We have evaluated SMALPs as alternatives to detergents. Purification post-cleavage of the affinity tags remains problematic.
2. We have localised the membranes in which PINs accumulate in model insect cells. Most PIN5 accumulates in the ER and nuclear envelope, but in this overexpression system some also accumulates in the cell surface plasma membrane.
3. We have evaluated a series of activity assays for PINs to check that expressed proteins are active at auxin transport. Radiolabel assays on Sf9 cells indicate that PINs are active when expressed. We have developed assays for purified proteins using microscale thermophoresis (MST) and surface plasmon resonance (SPR), but as noted above purification is a problem.
4. We have evaluated methods for presentation of PINs for electron microscopy and made progress with negative staining protocols.
5. In an accessory project on the auxin receptors TIR1 and AFB5, significant progress has been made in the detailed molecular understanding of the earliest interactions between auxin and its binding pocket. This will lead to high profile publications on both the interaction of TIR1 with its co-receptor Aux/IAA degron and with the understanding of how mutations in and close to the degron can lead to natural resistances to auxins. This has serious implications on how this family of hormone analogues is used as commercail herbicides globally. Unfortunately, this work has not been concluded because of the laboratory closure.
6. To develop career skills as a supervisor the fellow has supervised an Erasmus research student in a project on how endogenous auxin metabolism is altered by application of auxin herbicides. This links directly the the fellow's research interests onauxin homeostasis by using methods of molecular biology and analytical chemistry.
7. The fellow has also continued to evaluate aspects of a set of novel synthetic auxin fluorescence molecules suitable for tracking tissue specific and subcellular distribution of auxin.
NOTE: the closure of the host's research laboratories has severely limited the delivery of this project and compromised its success as well as the career opportunities of the fellow
2. We have localised the membranes in which PINs accumulate in model insect cells. Most PIN5 accumulates in the ER and nuclear envelope, but in this overexpression system some also accumulates in the cell surface plasma membrane.
3. We have evaluated a series of activity assays for PINs to check that expressed proteins are active at auxin transport. Radiolabel assays on Sf9 cells indicate that PINs are active when expressed. We have developed assays for purified proteins using microscale thermophoresis (MST) and surface plasmon resonance (SPR), but as noted above purification is a problem.
4. We have evaluated methods for presentation of PINs for electron microscopy and made progress with negative staining protocols.
5. In an accessory project on the auxin receptors TIR1 and AFB5, significant progress has been made in the detailed molecular understanding of the earliest interactions between auxin and its binding pocket. This will lead to high profile publications on both the interaction of TIR1 with its co-receptor Aux/IAA degron and with the understanding of how mutations in and close to the degron can lead to natural resistances to auxins. This has serious implications on how this family of hormone analogues is used as commercail herbicides globally. Unfortunately, this work has not been concluded because of the laboratory closure.
6. To develop career skills as a supervisor the fellow has supervised an Erasmus research student in a project on how endogenous auxin metabolism is altered by application of auxin herbicides. This links directly the the fellow's research interests onauxin homeostasis by using methods of molecular biology and analytical chemistry.
7. The fellow has also continued to evaluate aspects of a set of novel synthetic auxin fluorescence molecules suitable for tracking tissue specific and subcellular distribution of auxin.
NOTE: the closure of the host's research laboratories has severely limited the delivery of this project and compromised its success as well as the career opportunities of the fellow
The final 6 months of this fellowship suffered becuase the laboratories were closed by Covid-19. This has severely compromised the success of the project and the career opportunities of the fellow.
The most outstanding achievements have been:
1. the development of a patch-clamp-based assay for phytopharmacology of PIN proteins in collaboration with the Warwick neuropharmacology group. This is incomplete because of Covid.
2. The advanced molecular understanding of the primary molecular events in auxin binding to its receptor TIR1. This may lead to 2 high-impact publications, but is severely compromised and delayed because of Covid closures.
Amongst the stories to come out will be a detailed molecular understanding of how a naturally-occurring resistance to auxin herbicides is manifested at the receptor. This will help change how herbicides and resistance to auxins are managed globally.
List of manuscripts currently under revision or in preparation supported by the CrysPINs project
o Harborough S.R. Ramans S., Kalverda A.P. Thompson G.S. Kieffer M., Kubes M., Quareshy M., Uzunova V., Prusinkska J.M. Hayashi K., Napier R., Manfield I.W. Kepinski S.: A fuzzy encounter complex precedes formation of the fully-engaged TIR1-Aux/IAA auxin co-receptor system. bioRxiv preprint doi: https://doi.org/10.1101/781922.
o Todd O.E. Figueiredo M.R.A. Morran S., Soni N., Preston Ch., Kubes M.F. Napier R., Gaines T.A.: Synthetic auxin herbicides: finding the lock and key to weed resistance. Plant Science, under revision.
o Parizkova B., Zukauskaitė A., Vain T., Grones P., Raggi S., Kubes M., Kieffer M., Strnad M., Kepinski S., Napier R., Dolezal K., Robert S., Novak O.: New fluorescent auxin probes visualize tissue-specific and subcellular distributions of auxin in Arabidopsis. New Phytologist, under revision.
o Draft: Figueiredo M.R.A. Morran S., Soni N., Preston Ch., Todd O.E. Kubes M.F. Napier R., Gaines T.A.: Field-based resistance to dicamba is conferred by deletion of the degron tail of AUX/IAA27 in Indian mustard.
The most outstanding achievements have been:
1. the development of a patch-clamp-based assay for phytopharmacology of PIN proteins in collaboration with the Warwick neuropharmacology group. This is incomplete because of Covid.
2. The advanced molecular understanding of the primary molecular events in auxin binding to its receptor TIR1. This may lead to 2 high-impact publications, but is severely compromised and delayed because of Covid closures.
Amongst the stories to come out will be a detailed molecular understanding of how a naturally-occurring resistance to auxin herbicides is manifested at the receptor. This will help change how herbicides and resistance to auxins are managed globally.
List of manuscripts currently under revision or in preparation supported by the CrysPINs project
o Harborough S.R. Ramans S., Kalverda A.P. Thompson G.S. Kieffer M., Kubes M., Quareshy M., Uzunova V., Prusinkska J.M. Hayashi K., Napier R., Manfield I.W. Kepinski S.: A fuzzy encounter complex precedes formation of the fully-engaged TIR1-Aux/IAA auxin co-receptor system. bioRxiv preprint doi: https://doi.org/10.1101/781922.
o Todd O.E. Figueiredo M.R.A. Morran S., Soni N., Preston Ch., Kubes M.F. Napier R., Gaines T.A.: Synthetic auxin herbicides: finding the lock and key to weed resistance. Plant Science, under revision.
o Parizkova B., Zukauskaitė A., Vain T., Grones P., Raggi S., Kubes M., Kieffer M., Strnad M., Kepinski S., Napier R., Dolezal K., Robert S., Novak O.: New fluorescent auxin probes visualize tissue-specific and subcellular distributions of auxin in Arabidopsis. New Phytologist, under revision.
o Draft: Figueiredo M.R.A. Morran S., Soni N., Preston Ch., Todd O.E. Kubes M.F. Napier R., Gaines T.A.: Field-based resistance to dicamba is conferred by deletion of the degron tail of AUX/IAA27 in Indian mustard.