Page 291 - PC2019 Program & Proceedings

PLANT CANADA 2019

TOPIC 19: Molecular Plant Improvement and Genome Editing
(Posters P155-P158)

P155. Characterization of Arabidopsis thaliana MYB transcription factor complexes and their roles
in the regulation of suberin biosynthetic genes
*
Tapp, K. ; S. Khalil; O. Rowland
Carleton University

Terrestrial plants deposit cell wall-associated, lipid-based polymeric barriers in specific tissue layers to
help protect themselves from various environmental stresses (e.g. drought, salinity, insects, and
pathogens). One of these barriers, suberin, is found in various underground and aerial tissues, including
root endodermis, tuber periderms, and bark (cork). Suberin mediates water uptake, ion transport, and gas
exchange within roots. The degree of root suberization influences water relations and stress tolerance.
Some of the Myeloblastosis (MYB)-type transcription factors (TFs) have been identified in regulating
suberin biosynthetic genes. The transcriptional activities of MYBs are known to be influenced by specific
protein-protein interactions forming larger regulatory complexes. Therefore, the focus of this study is to
characterize the protein-protein interaction network of MYBs that up-regulate suberin biosynthesis. Here,
we used yeast two-hybrid screening of bioinformatics-guided gene candidates and an Arabidopsis
thaliana cDNA library to identify the potential protein interactors of suberin-associated MYB TFs. These
binding partners are being further investigated by a combination of reverse genetic, biochemical, and
bioinformatics approaches. This study will be important for the future generation of sustainable
metabolically engineered crops that are more stress resistant, via enhancement of suberin deposition along
their cell walls. Environmental stress can cause major yield loss of harvested crops, and improvement of
stress tolerance can mitigate, and potentially eliminate these problems.

Kyle Tapp (kyle.tapp@carleton.ca)

P156. Targeted mutagenesis in soybean using CRISPR-Cas9 system
Lu, M.; L. Tian
Agriculture and Agri-Food Canada

Genome editing using CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats/CRISPR-
associated 9) has become a popular approach to induce targeted mutations for crop trait
improvement. Soybean (Glycine max) is an economically important crop worldwide. As the first step to
develop CRISPR technology to modify soybean genes for soybean cyst nematode resistance, phytoene
desaturase (PDS) genes were selected as targets. Two PDS genes are present in soybean:
Glyma.18G003900 (GmPDS18g) and Glyma.11G253000 (GmPDS11g). Constructs specifically targeting
each gene, as well as simultaneously targeting both in the conserved regions were created. Each construct
contains 35S promoter driving the expression of Cas9 translationally fused to green fluorescent protein
(eGFP), and Arabidopsis AtU6 promoter driving the expression of single-guide RNA (sgRNA) containing
a 20-nucleotide guide sequence. Agrobacterium-mediated genetic transformation of soybean cultivar
Williams 82 were carried out using 2 constructs targeting different sites in GmPDS18g. Plantlets were
recovered from tissue culture. Polymerase chain reaction (PCR) using vector-specific primers verified
transformation in 33% and 100% of regenerated plantlets introduced with each construct. Among the
positive transformants, insertion and deletion mutations at desired sites were detected from leaf tissues,
validating genome editing in soybean using CRISPR/Cas9 system. Genetic transformation with
additional constructs targeting PDS genes is underway. This technology will be subsequently employed
to modify genes involved in soybean cyst nematode infection, towards potential disease resistance.

Mimmie Lu (mimmie.lu@canada.ca)

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