plant growth and development PDF

ARTICLE ASSIGNMENT:
FU ET AL. 2020
PROTEIN CHEMISTRY
Prof. dr. Els Van Damme
Dr. Tibo De Coninck
Joppe Gebruers
Master cell and gene biotechnology
Year: 2021-2022

1. INTRODUCTION
This report reviews the article on the influence (plant growth and development) of exogenous Cry1Ab/c
protein on endogenous proteins from Fu, et al. (2020), is discussed.[1] Genes, which are not inherently
present in the genome (exogenous), are added to the genetic code, hence forming transgenic crops.
Making these transgenic organisms can have several goals e.g., higher yield to cost ratio, making it
economically beneficial to use these ‘enhanced’ crops. Therefore, the interest in these transgenic crops
is rising over the years. However, these exogenous proteins can interact with the already present
endogenous proteins, inducing unintended responses. A previous study, by the same group, showed that
the insect-resistant transgenic rice (Huahui-1) differs vastly (in plant height, biomass, etc.) from the
parental rice.[2] In the reviewed article, these protein-protein interactions are examined with the use of
Cry1Ab/c (protein from the insect-resistant rice Huahui-1) and the endogenous proteins of Minghui-63
(the parental rice). Fu, et al. (2020) suspect that the expressed CryAb/c in Huahui-1 interacts with
endogenous proteins, resulting in the previously observed phenotypical changes.
To analyze these interactions, multiple techniques are used. Firstly, a yeast two-hybrid assay is used to
get an idea of which endogenous proteins will interact with the Cry1Ab/c. 60 Proteins are identified as
Cry1Ab/c-interacting endogenous proteins. The researchers only focus on proteins involved in stress
resistance and photosynthesis assuming that these two factors lay on the basis of the phenotypical
difference. This premise provided a library reduction resulting in 14 interesting (and interacting)
endogenous proteins. For further identification and analysis of the interactions taking place, three
validation methods were used: subcellular co-localization, bimolecular fluorescence complementation
(BIFC) and co-immunoprecipitation (co-IP). The results of these three techniques were used to make
conclusions about the influence of the exogenous protein.
This report focuses on the protein chemistry side of the article, discussing the used techniques and
whether improvements are possible. The gene technological side of the research (e.g., the use of a
certain vector or a certain yeast species) is not discussed further.
2. USED TECHNIQUES
The first technique used in the article is the yeast two-hybrid assay (Y2H) to identify which endogenous
proteins will interact with the Cry1Ab/c protein. This system is based on a split transcription factor that is
reconstructed when two proteins interact with one another. The bait protein (in this case Cry1Ab/c) is
linked to a DNA binding domain, making it able to bind DNA specifically on a promotor sequence. The
prey on the other hand is linked with an activation domain, making it able to activate transcription of a
certain reporter gene. This can only occur if the activation domain is close enough to the gene.
Therefore, the full transcription factor (DNA binding domain and activation domain) needs to be present
to activate transcription. Only if the bait protein interacts with the prey protein, this will take place.
However, two potential pitfalls of this assay need to be verified. False positives can occur due to self-
activation of the bait protein, or no activation can occur due to cytotoxicity of the bait protein. These two
concerns were analyzed critically in the article. As a matter of fact, the former proves to be present. To
solve this issue, the researchers cleaved the Cry1Ab/c into three segments dependent on their separate
activity. These three domain-BD constructs were again tested on self-activation and cytotoxicity,
resulting in a doubly negative result. Thus, these constructs can be used to identify endogenous proteins,
adding them as bait for the Y2H screening. The assay resulted in 60 positive clones (meaning interacting
endogenous protein), but after selection for photosynthesis and stress resistance proteins, the library