SUSPHIRE - Sustainable Bioproduction of Pheromones for Insect Pest Control in Agriculture

National Institute of Biology, Department of Biotechnology and Systems Biology projects

Short Name: T33_MonoterpenoidsFungi Title: Detection of Coccoidea pheromones or precursors in engineered fungi Description: Detection of lavandulol or lavandulyl acetate in different engineered fungi (SxF) lines by GC-MS-MS Phenodata: ./phenodata_20211223.txt pISA Investigation creation date: 2021-12-23 pISA Investigation creator: Sandra Vacas Principal investigator: Ismael Navarro License: CC BY 4.0 Sharing permission: Private Upload to FAIRDOMHub: Yes

Short Name: T32_CandidateGeneExpressionTesting Title: Candidate gene expression and testing Description: The purpose of this investigation is to express transiently candidate genes for Pcitri sex pheromone biosynthesis. This genes could act either as genes catalyzing steps related to the biosynthesis of precursors of the pheromone itself, either for the final steps of the biosynthesis. After expression, the monoterpenoid production function of those genes is checked by GCMS. Phenodata: ...

Short Name: T24_phero Title: Pheromone Content and EAG response Description: Investigate pheromone content and electrophysiological response of target insects to extracts of plants and fungi expressing Lepidoptera pheromones Phenodata: ./phenodata_21Sep21.txt pISA Investigation creation date: 21-Sep-21 pISA Investigation creator: Sandra Vacas Principal investigator: Ismael Navarro License: CC BY 4.0 Sharing permission: Private Upload to FAIRDOMHub: Yes

Short Name: T23_SxF Title: Moth pheromone production in filamentous fungi SxF Description: Demonstration of moth pheromone production in filamentous fungi by solvent extractions of liquid cultures and mycelial biomass and GC-MS/MS analysis. Constitutive and inducible expression of moth pheromones in filamentous fungi Penicillium species will be developed using a dCas9-based copper inducible system. An initial proof of concept will also be performed in P. digitatum using constitutive promoters. ...

Short Name: T22_SxPv2 Title: Sexy Plant version 2: inducible and regulable SxP versions Description: The purpose of this investigation is to optimize the SxP version 1, with a switchable expression and increased compound release. Phenodata: ./phenodata_20210920.txt Principal investigator: Diego Orzaez License: Creative Commons Attribution 4.0 Sharing permission: Private Upload to FAIRDOMHub: Yes Institutions involved: Instituto de Biología Molecular y Celular de Plantas (IBMCP), Spain; Institute ...

Short Name: T12_CuInducible Title: Production and testing of copper inducible dCas9EV system for moth pheromone production Description: dCas9EV system used to activate the synthetic promoters made in T11 was coupled to a copper-inducible promoter made of CBS repeats and a minimal DFR promoter. Such system will be induced via CUP2 protein, which binds to CBS motif in presence of copper. This cascade-like system was tested for production of the moth pheromone pathway. Phenodata: ./phenodata_20210107.txt ...

Short Name: T11_dCas9SynProm Title: Development of synthetic promoters inducible by dCasEV2.1 system Description: The aim of this work is to design a range of synthetic promoters with negligible basal expression that are activated by using the "dead" Cas9 activation system developed in our lab (dCasEV2.1, Selma et al., 2019). Such tool will then be used for creating synthetic regulatory cascades where the expression of the genes for the pheromone biosynthesis can be controlled by a single dCas9 ...

Short Name: T31_mealybug Title: Mealybug genome and transcriptome analyses Description: Searching for enzymes involved in the mealybug pheromone biosynthesis pathways. These pheromones are known to be released by virgin females. Phenodata: ./phenodata_20190523.txt Featuredata: Principal investigator: Heribert Warzecha, Špela Baebler License: Creative Commons Attribution 4.0 Sharing permission: Private Upload to FAIRDOMHub: Yes

Short Name: T21_SXPsysbio Title: Use a systems biology approach to identify regulatory bottlenecks in SxPv1 Description: Samples from SXPv1.0 plants as well as sister nulls (progeny from the original transgenic event in which the transgene has segregated) and wild type will be grown and leaf samples taken for RNA extraction and profiling of primary metabolites and volatiles (target pheromones as well as potential derivatives) (P1, P5). Phenotypic and GC-MS data will be obtained and analysed from ...

Short Name: P5_SxF_lav_ac Title: Detection of lavandulyl acetate in extracts of engineered fungi Description: Detection of lavandulyl acetate in extracts of engineered fungi (SxF) by GC-MS-MS Raw Data: pISA Study creation date: 2021-12-23 pISA Study creator: Sandra Vacas Principal investigator: Ismael Navarro License: CC BY 4.0 Sharing permission: Private Upload to FAIRDOMHub: Yes

Short Name: P5_SxF_lavandulol Title: Detection of lavandulol in SxF extracts Description: Detection of lavandulol in extracts of engineered fungi by GC-MS-MS Raw Data: pISA Study creation date: 2021-12-23 pISA Study creator: Sandra Vacas Principal investigator: Ismael Navarro License: CC BY 4.0 Sharing permission: Private Upload to FAIRDOMHub: Yes

Investigation files _I_T33_MonoterpenoidsFungi

Investigation files _I_T32_CandidateGeneExpressionTesting

Short Name: TransientLPPSCPPS Title: Transient expression of lavandulyl pirophosphate synthase (LPPS) and chrysanthemyl pirophhosphate synthase (CPPS) Description: The purpose of this study is to perform the transient expression of LPPS and CPPS gene for the production of lavandulol and chrysanthemol, respectively. Production is measured by GCMS of samples. Raw Data: pISA Study creation date: 2021-12-03 pISA Study creator: RMF Principal investigator: Diego Orzaez License: Creative Commons Attribution ...

Short Name: P5_PhContentPlants Title: Quantification of total moth pheromone contents in SxP Description: Quantification of the total pheromone content in SxPv1.2 plants Raw Data: pISA Study creation date: 21-Sep-21 pISA Study creator: Sandra Vacas Principal investigator: Ismael Navarro License: CC BY 4.0 Sharing permission: Private Upload to FAIRDOMHub: Yes

Short Name: P5_PhEmissionPlants Title: Quantification of the pheromone emitted by SxP plants Description: Quantification of the quantity of pheromone emitted by SxPv1.2 plants by volatile collection and GC-MS/MS analysis Raw Data: pISA Study creation date: 21-Sep-21 pISA Study creator: Sandra Vacas Principal investigator: Ismael Navarro License: CC BY 4.0 Sharing permission: Private Upload to FAIRDOMHub: Yes

Investigation files _I_T24_phero

Short Name: P5_EAGresponse Title: Electrophysiological response of moths to SxP extracts Description: Electrophysiological response of Sesamia nonagrioides males to selected fractions of the SxPv1.2 extracts Raw Data: pISA Study creation date: 21-Sep-21 pISA Study creator: Sandra Vacas Principal investigator: Ismael Navarro License: CC BY 4.0 Sharing permission: Private Upload to FAIRDOMHub: Yes

Short Name: P1_ConstExp_Pdigitatum Title: Constitutive expression of moth pheromone pathway in Penicillium digitatum. Description: For this Proof of Concept, the three enzymes of the moth pheromones route were assembled in TUs using fungal promoters and terminators, and transformed into Penicillium digitatum. Pheromone production of the selected transformants will be assessed via GC-MS. Raw Data: pISA Study creation date: 2020-18-04 pISA Study creator: ElenaMG Principal investigator: Diego Orzaez ...

Short Name: P5_SxFliquids Title: Pheromone detection in liquid extracts of SxF Description: Pheromone detection in liquid extracts of modified filamentous fungi (SxF) by liquid-liquid solvent extraction and GC-MS/MS analysis Raw Data: pISA Study creation date: 21-Sep-22 pISA Study creator: Sandra Vacas Principal investigator: * License: CC BY 4.0 Sharing permission: Private Upload to FAIRDOMHub: Yes

Short Name: P5_SxFmycelia Title: Pheromone detection in SxF mycelial biomass Description: Pheromone detection in the mycelial biomass of modified filamentous fungi by solid-liquid solvent extraction and GC-MS/MS analysis Raw Data: pISA Study creation date: 21-Sep-22 pISA Study creator: Sandra Vacas Principal investigator: * License: CC BY 4.0 Sharing permission: Private Upload to FAIRDOMHub: Yes

Investigation files _I_T23_SxF

Investigation files _I_T22_SxPv2

Short Name: P1_TransgenicStableSxPv2 Title: Generation of Sexy Plant version 2: transgenic plants with the moth sex pheromones biosynthetic pathway genes regulated by synthetic promotors. Description: The purpose of this study is to generate transgenic stable lines of Nicotiana benthamiana plants ready to express the moth sex pheromone biosynthetic pathway in a regulable way. The moth sex pheromones biosynthetic pathway genes are regulated by synthetic promotors, so in a basal state, the plants ...

Investigation files _I_T12_CuInducible

Short Name: P1_TransientExp_Nb Title: Agroinfiltration of copper inducible system in Nicotiana benthamiana Description: The copper inducible dCasEV was tested transiently in Nicotiana benthamiana leaves together with the moth pheromone pathway under the regulation of the synthetic promoters created in T11 Raw Data: https://zenodo.org/communities/susphire/search?page=1&size=20 pISA Study creation date: 2020-10-28 pISA Study creator: ElenaMG Principal investigator: Diego Orzaez License: Creative ...

Investigation files _I_T11_dCas9SynProm

Short Name: P1_SynProm_Nbenthamiana Title: Transient expression in Nicotiana benthamiana of the synthetic promoters developed Description: Different A1 and A2 Different A1 and A2 promoter pieces were designed containing as much random sequence as possible, and the target sequence for the guideRNA 1 of SlDFR promoter. In the same way, some minimal promoters containing only the TATA-Box signal and the 5'UTR region were designed as "core promoter" pieces. Different synthetic promoters were then ...

Short Name: P4_Pcitri_tr1tr2combo Title: Combining tr1 and tr2 P. citri de novo transcriptomes into one transcriptome set Description: Production of one set of transcripts that will be easier for the readers of the planned publication Raw Data: Principal investigator: Špela Baebler License: Creative Commons Attribution 4.0 Sharing permission: Private Upload to FAIRDOMHub: Yes

Files connected to investigation: _I_T11_dCas9SynProm

Files connected to study: _S_P1_SynProm_Nbenthamiana

Short Name: 20190208-LUM Assay Class: WET Assay Type: LUM Title: Luciferase assay first trial Description: Promoter series containing random sequences and the recognition sequence for guideRNA1 DFR (P1.X series) fused to the developed core promoter minDFR and to luciferase for measuring the expression in the presence or absence of such guideRNA1 DFR. A promoter containing the target sequence in a different position (P1.7.1) and a promoter with the target sequence repeated twice (P2.1) were also ...

Short Name: 20190717-LUM Assay Class: WET Assay Type: LUM Title: Luciferase assay second trial Description: New 1.X and P2.X promoter sequences, and also a P3.1 promoter containing three times the target sequence for guideRNA1 DFR pISA Assay creation date: 2019-07-17 pISA Assay creator: ElenaMG Lab manager: DO Operator: ElenaMG Link to SynBio DM: https://docs.google.com/spreadsheets/d/1KqJ2s7WZOro2pTCjGuUDpnSRfum26omN1TRFDl6-gAI/edit#gid=1915967703 Link to protocols: ../protocols/LUCIFERASE ...

Short Name: 20191209-LUM Assay Class: WET Assay Type: LUM Title: Luciferase assay third trial Description: In order to imitate better the native DFR promoter, all upstream promoter sequences were re-designed as A2 pieces and A1 pieces were ordered which consisted on just random sequences witout any promoter signals. Assembling these pieces created synthetic promoters of the same length than the original SlDFR. Due to some assembly issues, all new constructs were made using 35s terminator. pISA ...

Short Name: 20200219-LUM Assay Class: WET Assay Type: LUM Title: Luciferase assay of different A1 random sequences Description: With the aim of making three promoters as different as possible for the moth pheromone enzymes, three A1 pieces containing random sequences withous any promoter signal were assembled and tested using luciferase assay. pISA Assay creation date: 2020-02-19 pISA Assay creator: ElenaMG Lab manager: DO Operator: ElenaMG Link to SynBio DM: ...

Short Name: 20200302-LUM Assay Class: WET Assay Type: LUM Title: Luciferase Assay US1.7.X and US3.X pieces Description: Analysis of new US pieces with three recognition sites for gRNA1 DFR (US3.X) and US pieces with the recognition site in different positions (US1.7.1-US1.7.4), as well as another US part with the recognition site of another gRNA (US1.7.5, for gRNA4 from NOS promoter). pISA Assay creation date: 2020-02-03 pISA Assay creator: ElenaMG Lab manager: DO Operator: ElenaMG Link to SynBio ...

Short Name: 20200818-LUM Assay Class: WET Assay Type: LUM Title: Luciferase assay of new minimal promoters Description: New minimal promoters were designed and tested to increase the battery of pieces available for creating synthetic promoters pISA Assay creation date: 2020-08-18 pISA Assay creator: ElenaMG Lab manager: DO Operator: ElenaMG Link to SynBio DM: https://docs.google.com/spreadsheets/d/1KqJ2s7WZOro2pTCjGuUDpnSRfum26omN1TRFDl6-gAI/edit#gid=1915967703 Link to protocols: ../protocols/LUCIFERASE ...

Short Name: 20201112-LUM Assay Class: WET Assay Type: LUM Title: Luciferase assay re-testing US6.X Description: The US6.X series was tested again using separated plasmids for the gRNAs and the dCas9EV system (so far it was used a plasmid containig the system and the guide together) pISA Assay creation date: 2020-11-12 pISA Assay creator: ElenaMG Lab manager: DO Operator: ElenaMG Link to SynBio DM: https://docs.google.com/spreadsheets/d/1KqJ2s7WZOro2pTCjGuUDpnSRfum26omN1TRFDl6-gAI/edit#gid=1915967703 ...

Short Name: 20211221-LUM Assay Class: WET Assay Type: LUM Title: dCasEV synthetic promoters with NanoLuc reporter system Description: Test for dCasEV synthetic promoters using EI reporter system based on Nanoluciferase as reporter and luciferase as reference signal. pISA Assay creation date: 2021-12-22 pISA Assay creator: ElenaMG Lab manager: DO Operator: ElenaMG Link to SynBio DM: https://docs.google.com/spreadsheets/d/1KqJ2s7WZOro2pTCjGuUDpnSRfum26omN1TRFDl6-gAI/edit#gid=1915967703 Link to ...

Files connected to investigation: _I_T12_CuInducible

Files connected to study: _S_P1_TransientExp_Nb

Short Name: AgroinfFinalConstructsdCas9EV-GCMS Assay Class: WET Assay Type: GCMS Title: Agroinfiltration of final constructs for copper-inducible pathway in Nicotiana benthamiana leaves Description: Two constructs were made containing the three genes of the moth pheromone pathway under the regulation of synthetic promoters made in T11, nptII selection marker and with/without the gRNA1 for activation. In parallel, another construct containing the copper-inducible dCas9EV system with/without the ...

Short Name: AgroinfGuidedPathwaywithConstitutivedCas9EV-GCMS Assay Class: WET Assay Type: GCMS Title: Agroinfiltration of guided pathway for moth pheromones production with constitutive dCas9EV system in Nicotiana benthamiana leaves Description: Two constructs were made that comprised the three genes required for moth pheromone production (AtrD11, HarFar and EaDAct/ATF1) under the regulation of the synthetic promoters created in T11. In the constructs it was also included the guideRNA1 and nptII ...

Short Name: AgroinfNonGuidedPathwaywithCopperInducibledCas9EV-GCMS Assay Class: WET Assay Type: GCMS Title: Agroinfiltration of non-guided pathway for moth pheromones production with copper-inducible dCas9EV system in Nicotiana benthamiana leaves Description: Two constructs were made that comprised the three genes required for moth pheromone production (AtrD11, HarFar and EaDAct/ATF1) under the regulation of the synthetic promoters created in T11. The constructs were first tested transiently in ...

Short Name: AgroinfNonGuidedPathwayywithConstitutivedCas9EV-GCMS Assay Class: WET Assay Type: GCMS Title: Agroinfiltration of nonguided pathway for moth pheromones production with constitutive dCAs9EV system in Nicotiana benthamiana leaves Description: Two constructs were made that comprised the three genes required for moth pheromone production (AtrD11, HarFar and EaDAct/ATF1) under the regulation of the synthetic promoters created in T11. The constructs were first tested transiently in Nicotiana ...

Files connected to investigation: _I_T21_SXPsysbio

Files connected to study: _S_P1_SPv10T0andT1

Files connected to study: _S_P1_SPv10T2andT3

Files connected to study: _S_P1_SPv1TransientExp

_p_SUSPHIRE/_I_T33_MonoterpenoidsFungi/_S_P5_SxF_lavandulol/_A_L_myc-GC-MS/

_p_SUSPHIRE/_I_T33_MonoterpenoidsFungi/_S_P5_SxF_lavandulol/_A_L_myc-GC-MS/

_p_SUSPHIRE/_I_T33_MonoterpenoidsFungi/_S_P5_SxF_lavandulol/_A_L_liq-GC-MS/

_p_SUSPHIRE/_I_T33_MonoterpenoidsFungi/_S_P5_SxF_lavandulol/_A_L_myc-GC-MS/

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_p_SUSPHIRE/_I_T33_MonoterpenoidsFungi/_S_P5_SxF_lavandulol/_A_L_liq-GC-MS/

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_p_SUSPHIRE/_I_T33_MonoterpenoidsFungi/_S_P5_SxF_lavandulol/

_p_SUSPHIRE/_I_T33_MonoterpenoidsFungi/_S_P5_SxF_lavandulol/

_p_SUSPHIRE/_I_T33_MonoterpenoidsFungi/_S_P5_SxF_lavandulol/_A_L_liq-GC-MS/

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_p_SUSPHIRE/_I_T33_MonoterpenoidsFungi/_S_P5_SxF_lav_ac/_A_LA_liq-GC-MS/

_p_SUSPHIRE/_I_T33_MonoterpenoidsFungi/

_p_SUSPHIRE/_I_T33_MonoterpenoidsFungi/

_p_SUSPHIRE/_I_T33_MonoterpenoidsFungi/_S_P5_SxF_lav_ac/_A_LA_liq-GC-MS/

_p_SUSPHIRE/_I_T33_MonoterpenoidsFungi/_S_P5_SxF_lav_ac/_A_LA_myc-GC-MS/

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_p_SUSPHIRE/_I_T33_MonoterpenoidsFungi/_S_P5_SxF_lavandulol/_A_L_myc-GC-MS/

_p_SUSPHIRE/_I_T32_CandidateGeneExpressionTesting/_S_TransientLPPSCPPS/

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_p_SUSPHIRE/_I_T24_phero/_S_P5_EAGresponse/_A_GC-MS-EAD-EAG/

_p_SUSPHIRE/_I_T24_phero/_S_P5_EAGresponse/_A_GC-MS-EAD-EAG/

_p_SUSPHIRE/_I_T24_phero/_S_P5_PhContentPlants/_A_PhCont_fr20-GC-MS/

_p_SUSPHIRE/_I_T24_phero/_S_P5_PhContentPlants/_A_PhCont_fr80-GC-MS/

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_p_SUSPHIRE/_I_T24_phero/

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_p_SUSPHIRE/_I_T24_phero/_S_P5_EAGresponse/_A_EAG_manual-EAG/