CRISPR/Cas9 mutation of MYB134 and MYB115 to study regulation and functions of proanthocyanidins in poplar roots




Liu, Yalin

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Secondary metabolites play important roles in tree defense. Proanthocyanidins (PAs), one of the most common secondary metabolites, are widely distributed in trees and woody plants, and are abundant in poplar. In my research, molecular biology and biochemistry techniques were used to investigate the function of two important transcription factors, MYB115 and MYB134, in regulating the PA pathway in hybrid poplars. The importance of these transcription factors in regulating PA synthesis in leaves has recently emerged, but their roles in roots are not known. MYB134- and MYB115-overexpressing transgenic poplars showed a strong high-PA phenotype in leaves, but how these two regulators interact in vivo is still a mystery. This research aims to test the function of both MYBs in the regulation of PAs in poplar roots, and to explore the antimicrobial functions of root PAs. Both alleles of the MYB genes were sequenced in wild type poplars to design gRNAs for creating transgenic poplars with knocked-out (KO) MYB115 and MYB134 using the CRISPR Cas9 system. Both hairy root and whole plant transgenics with respective single- and double knock-outs were generated. Chemical and genetic characterization of both mutant types showed reduced PA content and down-regulated flavonoid genes in leaves. In poplar roots, only double-KOs showed a significant change in PA and salicinoid metabolism. These results indicated that the regulatory pathways for PA biosynthesis may differ in poplar leaves and roots. Significant PA concentrations remained in double-KO plants, suggesting other transcription factors for PA regulation are active. Because poplars accumulate large amounts of PAs in roots, potential functions of root tannins were also investigated. Antimicrobial activity of PAs was tested by disc inhibition assay in vitro and mycorrhizal co-culture sandwich assay in vivo. Pure PAs showed no inhibition towards the pathogenic fungi Armillaria ostoyae and A. sinapina but displayed slight inhibition to the mycorrhiza fungus Laccaria bicolor. These results provide preliminary insight into the functions of PAs in roots.



CRISPR, MYB, PAs, poplar