Periodic Reporting for period 1 - DAFNE (Delivery of Small RNAs by Functional Hydrogel Nanoparticles to Improve Osmotic strEss Tolerance in Plants)
Reporting period: 2023-10-01 to 2025-09-30
Climate change is having a huge impact on the worldwide crop yield with a considerable decreasing of crop production every year (between 20% to 40% of global crop production). Plant genetic improvement is continuously looking for novel approaches to obtain climate-ready crops, preserving the biodiversity and food safety. DAFNE aims to develop and validate a novel RNA interference (RNAi)-based technique for gene silencing, enabling the production of transgene-free plants while enhancing their tolerance to abiotic stress
This project positively contributed towards the creation of novel tools environmental-friendly to make a more sustainable crop, in line with EU policy objectives. RNAi has been already applied as a ‘green’ alternative for plant pest control and crop protection. In DAFNE a new approach is employed by using nanomaterials (NMs) functionalised with small RNA (siRNA) as vectors to trigger RNAi in plants and make them more tolerant to abiotic stress such as heat and drought. NMs represent a good platform to protect the siRNA from degradation and target the genome more specifically by playing with their surface charge. DAFNE is a multidisciplinary project that combines Nanotechnology and Plant Science with the following overall objectives:
1) Design, characterisation, and surface engineering of NMs used as nanocarriers for delivery of siRNA. The NMs considered for this objective are Au nanoparticles (AuNPs) and natural polymers such as alginate and chitosan in the form of emulsions or polyplexes.
2) RNA delivery and gene silencing in the model species Arabidopsis thaliana (At) and Nicotiana benthamiana (Nb) by using AuNPs as primary vectors along with responsive nanogels to control the release of siRNA.
3) Targeted downregulation of known genes to confer abiotic stress tolerance in plant model crop such as Potato.
This project positively contributed towards the creation of novel tools environmental-friendly to make a more sustainable crop, in line with EU policy objectives. RNAi has been already applied as a ‘green’ alternative for plant pest control and crop protection. In DAFNE a new approach is employed by using nanomaterials (NMs) functionalised with small RNA (siRNA) as vectors to trigger RNAi in plants and make them more tolerant to abiotic stress such as heat and drought. NMs represent a good platform to protect the siRNA from degradation and target the genome more specifically by playing with their surface charge. DAFNE is a multidisciplinary project that combines Nanotechnology and Plant Science with the following overall objectives:
1) Design, characterisation, and surface engineering of NMs used as nanocarriers for delivery of siRNA. The NMs considered for this objective are Au nanoparticles (AuNPs) and natural polymers such as alginate and chitosan in the form of emulsions or polyplexes.
2) RNA delivery and gene silencing in the model species Arabidopsis thaliana (At) and Nicotiana benthamiana (Nb) by using AuNPs as primary vectors along with responsive nanogels to control the release of siRNA.
3) Targeted downregulation of known genes to confer abiotic stress tolerance in plant model crop such as Potato.
The activities performed to achieve the main objectives of the first year of DAFNE are divided in two main work packages (WPs) with a secondment period of five months carried out at the Instituto de Nanociencia y Materiales de Aragón (INMA) hosted by Bionanosurf group for the synthesis of the NMs. All the activities foreseen are listed below:
1) WP1: a) Design of siRNA for the plant species Arabidopsis thaliana (At) and Nicotiana benthamiana (Nb) to target common reporter genes, including phytoene desaturase (PDS), whose silencing induces leaf bleaching, and green fluorescent protein (GFP)); b) synthesis and characterisation of alginate nanoemulsions in a core/shell structure water in oil in water (W/O/W) using homogenisation, prilling and vortexing, c) synthesis and characterisation of Au nanoparticles (AuNPs) of about 14 nm functionalised with 25% PEG coverage (50% of SH-EG(8)-(CH2)2-COOH and 50% of SH-(CH2)3-CONH-EG(6)-(CH2)2-N3); d) synthesis and characterisation of chitosan nanovesicles and chitosan polyplexes (CHPx) using vortexing methods; e) conjugation of siRNA on AuNPs and CHPx. The conjugation efficiency was evaluated using Ultraviolet-visible spectroscopy (UV-vis), dynamic light scattering (DLS) and gel electrophoresis.
2) WP2: a) phytotoxicity tests performed using the materials synthesized in WP1 upon different administration routes (e.g. root uptake, seed priming and leaf spray/infiltration); b) RNA extraction, RT-PCR and qRT-PCR to evaluate at the molecular level the effect of the silencing; c) optical microscopy to measure the stomata density, foliar and root uptake of the NMs; d) fluorescent microscopy and confocal microscopy to study the root uptake of chitosan nanovesicles; e) computed tomography (CT) used to study the interaction of NMs with the plant cell tissue in vivo using Zeiss Xradia 510 system on At leaves. This work was supported by the National Research Facility for Lab X-ray CT (NXCT) at the µ-VIS X-ray Imaging Centre, University of Southampton, through EPSRC grant EP/T02593X.
1) WP1: a) Design of siRNA for the plant species Arabidopsis thaliana (At) and Nicotiana benthamiana (Nb) to target common reporter genes, including phytoene desaturase (PDS), whose silencing induces leaf bleaching, and green fluorescent protein (GFP)); b) synthesis and characterisation of alginate nanoemulsions in a core/shell structure water in oil in water (W/O/W) using homogenisation, prilling and vortexing, c) synthesis and characterisation of Au nanoparticles (AuNPs) of about 14 nm functionalised with 25% PEG coverage (50% of SH-EG(8)-(CH2)2-COOH and 50% of SH-(CH2)3-CONH-EG(6)-(CH2)2-N3); d) synthesis and characterisation of chitosan nanovesicles and chitosan polyplexes (CHPx) using vortexing methods; e) conjugation of siRNA on AuNPs and CHPx. The conjugation efficiency was evaluated using Ultraviolet-visible spectroscopy (UV-vis), dynamic light scattering (DLS) and gel electrophoresis.
2) WP2: a) phytotoxicity tests performed using the materials synthesized in WP1 upon different administration routes (e.g. root uptake, seed priming and leaf spray/infiltration); b) RNA extraction, RT-PCR and qRT-PCR to evaluate at the molecular level the effect of the silencing; c) optical microscopy to measure the stomata density, foliar and root uptake of the NMs; d) fluorescent microscopy and confocal microscopy to study the root uptake of chitosan nanovesicles; e) computed tomography (CT) used to study the interaction of NMs with the plant cell tissue in vivo using Zeiss Xradia 510 system on At leaves. This work was supported by the National Research Facility for Lab X-ray CT (NXCT) at the µ-VIS X-ray Imaging Centre, University of Southampton, through EPSRC grant EP/T02593X.
In this project we firstly synthesised and characterised alginate emulsions, CHPx and AuNPs and then validated an effective siRNA delivery and gene silencing in mature plants. The alginate nanoemulsions, however, exhibited significant instability and were deemed unsuitable for plant uptake due to the presence of oil, which demonstrated phytotoxic effects on plant tissues. On the other hand, AuNPs and CHPx were considered ideal for plant uptake and silencing because not phytotoxic. Therefore, these two types of NMs were used for the tests with plants.
Regarding the plant species, At was employed for toxicity and uptake tests while Nb was considered for the infiltration studies.
The main challenge of the project is the demonstration that NMs tagged with siRNA are protected from RNase degradation while the complex is small enough to overcome the plant leaf cell wall. The interaction of NMs with plant cell tissue was evaluated as first step using confocal microscopy for root uptake and X-ray CT scan with optical microscopy for leaf uptake. These techniques revealed that only a small amount of nanomaterials (NMs) is absorbed by root cells, while foliar uptake is likely restricted by the cuticle. Preliminary RNAi experiments demonstrated that PDS siRNA-tagged NMs infiltrated into Nicotiana benthamiana leaves neither caused toxic effects nor induced the anticipated phenotypic changes, possibly due to the inefficient delivery of siRNAs into plant cells.
Regarding the plant species, At was employed for toxicity and uptake tests while Nb was considered for the infiltration studies.
The main challenge of the project is the demonstration that NMs tagged with siRNA are protected from RNase degradation while the complex is small enough to overcome the plant leaf cell wall. The interaction of NMs with plant cell tissue was evaluated as first step using confocal microscopy for root uptake and X-ray CT scan with optical microscopy for leaf uptake. These techniques revealed that only a small amount of nanomaterials (NMs) is absorbed by root cells, while foliar uptake is likely restricted by the cuticle. Preliminary RNAi experiments demonstrated that PDS siRNA-tagged NMs infiltrated into Nicotiana benthamiana leaves neither caused toxic effects nor induced the anticipated phenotypic changes, possibly due to the inefficient delivery of siRNAs into plant cells.