TITLE: Simple Seed-vacuum Protocol for Agrobacterium-mediated Virus Induced Gene Silencing (VIGS) in Sunflower Helianthus annuus L.
AUTHORS: Majd Mardini, Mikhail Kazantsev, Elina Ivoilova, Victoria Utkina, Anastasia Vlasova, Ilya Kirov

[DESCRIPTION]
Virus-Induced Gene Silencing (VIGS) stands out as one of the most powerful tools for functional genomic research. The underlying principle of the VIGS system is based on harnessing the innate defense mechanism of the studied species to induce post-transcriptional gene silencing. In brief, the process involves introducing a modified viral constructs carrying a fragment of the host targeted gene (300-500 bp). The introduced virus will prompt a group of enzymes within the plant's defense system, setting off the degradation of viral RNA products together with any similar sequences in the cell, including the host’s own mRNA. As a result, the expression level of the targeted gene is altered or, in optimal scenarios, completely silenced. 
The introduction of the viral constructs is typically achieved through agrobacterium-mediated transformation. Numerous agrobacterium-VIGS systems have been firmly established for a number of crops and model plant species (Zhang Y. et al., 2019; Ramegowda V. et al., 2014). However, it is important to note that the infiltration procedure and viral construct often exhibit strong species-specificity. And as research advances, customizing new protocols becomes imperative for successful implementation across diverse plant species. 
Here, we represent a simple and reproducible agrobacterium-mediated VIGS protocol for sunflower (Helianthus annuus L.). Our methodology is seed vacuum-based without the necessity for pre-germination, MS medium recovery, or even seeds sterilization. Within this protocol, we provide comprehensive instructions that ensure the consistent replication of results. For demonstration we used PHYTOENE DESATURASE gene (PDS) of Helianthus annuus as a target for VIGS. The viral constructs are derived from Tobacco Rattle Virus (TRV) (Liu Y. et al., 2002). Information on vectors assembly and post-experimental validation are also provided. The protocol's reproducibility has been rigorously tested on a breeding line and various commercial sunflower cultivars.

[BEFORE_START]
The VIGS constructs should be prepared beforehand and introduced into agrobacterium cells using the standard transformation method employed in your laboratory.
The constructs utilized for demonstration in this protocol have been developed based on TRV vectors. The primary target gene for VIGS is the PDS of sunflower (Helianthus annuus L.). This gene serves as a common positive control in VIGS experiments due to its easily detectable phenotypic characteristics. Silencing effects manifest as color-bleaching of the leaves.
The PDS fragment for VIGS (siRNA) was designed utilizing the open-access web server https://www.zhaolab.org/pssRNAit/. The PDS fragment used within these VIGS constructs was amplified from sunflower genomic DNA using these primers: 5'-taattctagaATGGCATTTTTAGATGGCAGCCC-3' and 5'-taatggatccTGGAGTAGCAAATACATAAGCATCCCC-3'. Subsequently, the resulting amplicon was ligated into the pYL156 TRV RNA2 vector following standard procedure. The resulting construct was introduced into Agrobacterium tumefaciens strain GV3101 using a standard electroporation procedure.

TRV2 constructs used in this protocol: 
  
  

PDS gene in sunflower and the location of primers:
  

link for pYL192 (TRV1): https://www.addgene.org/148968/
link for pYL156 (TRV RNA2): https://www.addgene.org/148969/

Primers information for cultures validation and results analysis:
 



[STEPS]
SECTION: Infiltration suspension preparation
1. Streak previously prepared glycerol stocks of agrobacterium carrying the required VIGS constructs (TRV1 and TRV2) on agar-LB plates. For each plate: 20 mL LB medium + 1% agar + 50 μg/mL kanamycin + 10 μg/mL gentamycin + 100 μg/mL rifampicin.
 

SECTION: Infiltration suspension preparation
2. Incubate at 28°C. Individual, well-separated colonies will start to emerge within 24-38 hours. 

 

 

SECTION: Infiltration suspension preparation
3. To prepare the starting culture, inoculate 5 mL of liquid LB containing antibiotics with single colony and incubated at 28°C/180rpm for another 24-38 hours. 
 

SECTION: Performing seed-vacuum agrobacterium infiltration
9. After removing the outer coat of sunflower seeds immerse them in tap water for approximately 2 hours. This will facilitate the removal of the inner membrane.
SECTION: Infiltration suspension preparation
4. To prepare the infiltration culture, transfer the starting culture to a sterile flask containing 50mL of LB (10 times the volume of the starting culture) suplemented with antibiotics, MES (10 mM), and acetosyringone (200 µM) . 
SECTION: Infiltration suspension preparation
5. Incubate at 28°C/180rpm and monitor OD600 every 1-1.5 hour. Remove from the incubator when OD reaches 1.5.
 

SECTION: Infiltration suspension preparation
6. Transfer the infiltration culture to a 50-mL tube and collect the agrobacterium cells by centrifuging at 3000 rcf for 10 minute. 
SECTION: Infiltration suspension preparation
7. Remove the supernatant and wash agrobacterium cells in distilled water for a couple of times. 
SECTION: Infiltration suspension preparation
8. Re-suspend the cells in the infiltration buffer to achieve a volume that sustains an OD of 1.5. The infiltration buffer is a water-based solution comprising MES (10 mM), acetosyringone (200 µM), and MgCl2 (10mM). After resuspending, allow the infiltration suspension to sit at room temperature for 2-3 hours.
SECTION: Performing seed-vacuum agrobacterium infiltration
10. Transfer the seeds to a sterile Petri dish. Add a 1:1 ratio of TRV1:TRV2 infiltration suspension. Each Petri dish should contain a total volume of 20-25 mL of the infiltration suspension (TRV1 + TRV2).

In our example, three different Petri dishes for different VIGS variants: 
TRV1 + TRV2-empty 
TRV1 + TRV2-PDS
Infiltration buffer (negative control) 
SECTION: Performing seed-vacuum agrobacterium infiltration
11. In each Petri dish, introduce a 2-3 g of autoclave-sterile silica sand. In order to produce wounds in the surface of the seeds, gently rub using index and middle fingers (wearing gloves). The rubbing should be executed carefully in a circular motion, taking extra care not to fracture the seeds.
 

SECTION: Performing seed-vacuum agrobacterium infiltration
12. Initiate the vacuum process as follows: Apply vacuum for 3 minutes, followed by a 3-minute pause, and then another 3 minutes of vacuum.
SECTION: Performing seed-vacuum agrobacterium infiltration
13. Seal the Petri dishes and incubate at 28°C/50rpm for 6 hours.
 

SECTION: Performing seed-vacuum agrobacterium infiltration
14. Plant the treated seeds in clean, humid soil. Subsequently, cover the pots with plastic wrap and transfer them to a dark location for a period of 2 days.
SECTION: Performing seed-vacuum agrobacterium infiltration
15. After 2 days, remove the plastic wrap and transfer the pots to a greenhouse environment. Maintain a light/dark cycle of 16 hours of light and 8 hours of darkness for optimal growth conditions. 
The PDS silencing symptoms (color-bleaching of leaves) will start to be visible within 11-13 days.