Deciphering the role of ACTIN-RELATED PROTEIN8 in Arabidopsis thaliana using CRISPR-Cas9 mutagenesis and coimmunoprecipitation (Co-IP) Open Access

Kim, Kyeong Sik "Jacob" (Spring 2021)

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The actin protein family consists of cytoskeletal actin as well as actin related proteins (ARPs). The role of cytoskeletal ARP is well known, but the role of nuclear ARPs is not as clear. Nuclear ARPs are known to be related to nuclear actin in that they play a role in transcriptional regulation and are often found as components of chromatin remodeling complexes. Arabidopsis thaliana encodes six nuclear ARP proteins, one of which is ARP8. ARP8 is a plant-specific nuclear ARP, and it is unique in being localized exclusively in the nucleolus. It also contain an F-box domain, which is involved in protein degradation via the recruitment of ubiquitin ligase, and is not found in any other ARPs. Despite its unique characteristics, the role of Arabidopsis ARP8 is unclear. Here, we generated Arabidopsis ARP8 homozygous mutants using CRISPR/Cas9 to introduce a premature stop codon and examine whether the loss of ARP8 protein affects plant development. Yeast-two hybrid screening had previously identified ASK1 and RPL14p proteins as potentially interacting with ARP8. ASK1 protein is a component of ubiquitin ligase and RPL14p protein is a ribosomal protein, so determining ARP8’s interaction with these proteins would help us better understand its potential function. We therefore tested for direct protein-protein interaction between ARP8 and the proteins ASK1 and RPL14p by performing in vitro coimmunoprecipitation (Co-IP) to get a better understanding of the molecular role of ARP8. We found that phenotypes of ARP8 homozygous mutant plants are variable, with some appearing highly similar to wild-type and others showing reduced size and potentially late flowering. The ARP8 transcript level has not been affected in the mutant plants, but the plants likely lack the ARP8 protein due to the premature stop codon. Moreover, the co-IP suggests that ARP8’s interaction with ASK1 is weak, but the results are inconclusive. Future studies including determination of ARP8 protein levels in the mutant plants and in vivo protein interaction of ARP8 with ASK1 and RPL14p would further assist in characterizing ARP8’s potential role in Arabidopsis thaliana.

Table of Contents

Introduction 1

Result 4

Generating homozygous ARP8 mutant Arabidopsis thaliana plant using CRISPR/Cas9 mutagenesis 4

Phenotypes of homozygous ARP8 mutant plants vary compared to wild-type plants 7

mRNA levels in homozygous ARP8 mutant plants do not differ from wild-type plants 9

Results of in vitro protein-protein interaction testing between ARP8 and ASK1 or RPL14p are inconclusive 10

Discussion 14

Methods 18

References 22

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