Part:BBa_K2575000

BS3 Resistance Gene, plant reporter under control of Gal1P

The BS3 resistance gene is an avirulence initiator protein from Capsicum annuum that promotes cell death in response to its activation. This part is attached to a Gal1P to enable activation in response to Gal4, thus enabling easy activation in plant lines through co-transformation with Gal4. The BS3 resistance gene encodes a flavin monooxygenase that is activated by AvrBS3, and results in plant hypertrophy to stop the invading pathogen. This hypertrophy can be used as a reporter of gene expression.

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  COMPATIBLE WITH RFC[1000]

Characterization: UGA 2019

The 2019 UGA iGEM team cotransformed this part with a GAL4BD-VP16 transcription factor into agrobacterium. Upon a successful transformation, the team transfected Nicotiana benthamiana and observed the expression of the BS3 resistance gene.

In the figures that follow, the results of the transfection of GAL4BD-VP16 and 1XUAS-BS3 into Nicotiana benthamiana can be seen. When doing our infiltrations, we had 5 different spots on the leaf. Here is the Key:

Infiltration Spots - Key

1. BS3 in pGWB2 (contains a constitutive P35s Promoter)
2. Yellow Fluorescent Protein (YFP)
3. Gal4BD-VP16
4. BS3 in pGWB1 (does not contain a constitutive P35s Promoter)
5. BS3 in pGWB1 + Gal4BD-VP16

Based on these results, it is clear that the control part works as expected. Ideally, the only spot where any sort of cell death should be present is spot 5. However, some cell death was observed in the experimental spots that contain only BS3, either in pGWB1 (spot 4) or pGWB2 (spot 1). This was likely due to “leaky” expression occurring in the plant. In all three trials, the greatest amount of cell death was observed in spot 5, followed by spot 4, and then lastly spot 1. This was consistent with our expectations. We expected the greatest amount of cell death in spot 5 because the Gal4BD-VP16 transcriptional factor should be driving a strong downstream expression of BS3. Background BS3 expression was also expected to be higher in pGWB2 than in pGWB1 due to the presence of a constitutive p35S promoter. This is evident as spot 4 displayed higher levels of cell death than spot 1. Finally, we confirmed that the control part functions properly because there was no observed cell death in spot 3 (where Gal4BD-VP16 is infiltrated on its own), but there was observed cell death in spot 5 (where Gal4BD-VP16 is infiltrated in conjunction with 1xUAS-BS3).

As discussed above, the 2019 UGA iGEM team was able to validate the function of the GAL4BD-VP16 part. Additionally, our sequencing was confirmed through Europhins when the part was inserted into a gateway binary vector (pGWB2). Due to the availability of our resources, the UGA iGEM team was only able to validate this part through its ability to drive expression of a reporter gene (BS3). In the future, we intend to test our GAL4BD-VP16 transcription factor on other UAS-reporter genes, in an effort to further validate its function as a versatile and strong transcription factor. The UGA iGEM team is also developing a 6xUAS-BS3 gene to generate a stronger expression of cell death with the GAL4BD-VP16 part.