- Calendar
- Lab
- Protocol

Protection

Design

After P. infestans penetrates the cell wall of potato, it will exploit the potato and in turn infect potatoes nearby within 3 days. Since it will infect other potatoes in such a short time that there is no effective biological method to react and inhibit the development of the disease, we decide to prevent the disease at the very beginning by rendering the potatoes the ability to prevent the invasion of P. infestans. Under certain conditions, the zoospores of P. infestans will attach to the surface of potato leaves, penetrate the cell wall by high turgor pressure and some enzymes, and secrete some effector protein, such as Avr3, into potato cells. The effector protein needs to bind to a transmembrane receptor called PI3P, which can mediate its entry into potato cell to translocate into host cell. It will suppress plant resistance gene-based immunity so that P. infestans can enter potato cells without any resistance. To stop the effector protein from entering potato cell, we found through literature research that reduction in translocated effector is a promising way to decrease the virulence of pathogens and improve disease resistance in potatoes. In research done by other scientists, FYVE protein domain from Hrs or EEA1 can also bind to PI3P receptor strongly in animal cells. We then decided to construct a FYVE protein domain with high affinity that can compete with the effector protein to inhibit the entry of P. infestans.

FYVE protein domain

FYVE protein domain is well conserved PI3P binding domain in various organisms with only 141 amino acids. FYVE protein domain originally existed in EEA1 and Hrs proteins in human and mouse, respectively. However, EEA1 and Hrs protein are too large and may take long time for the plant to degrade, we then decided to extract the protein domain from Hrs protein. On the other hand, monomeric FYVE has far lower affinity to PI3P than Hrs and it is not so stable. Therefore, we decided to construct a dimeric FYVE which has a higher affinity and is much more stable than monomeric FYVE.

Promoter choice

Though the depletion of PI3P may be an effective way to prevent p/infestans from invading the potato, the constitutive expression of FYVE protein domain may cause some physiological effect to potatoes since PI3P is an important receptor to plants. Thus the timing of the expression of FYVE is the key to successful implementation of this technology.

We choose a promoter, Gst1, in potato that will be activated within 24 hours when p.infestans infect the potato and the production of the promoter will down-regulate after the infection. Also, this promotor will be activated mildly when the plant is wounded, in which the plant is vulnerable to late blight.

Circuit design

Our goal in this part is to inhibit the entry of P. infestans effector protein, so the dimeric FYVE will be constitutively expressed in plant cell. The circuit will have a viral constitutive promoter CAMV35S following the coding sequence of dimeric FYVE.
The circuit is shown on the right.

Experiment