Construction of a yeast two-hybrid library derived from Curvularia lunata grown in fries 3 medium and its application in the identification of Clt-1 interacting proteins

发布日期：2021-01-31

Author：Gao Jin-Xin; Chen Jie*

Abstract：

The Clt-1 protein may interact with other functional proteins via a BTB (Drosophila transcription factors Bric-abrac, Tramtrack and Broad Complex) domain to regulate methyl 5-(hydroxymethyl) furan-2-carboxylate toxin production in the fungal pathogen Curvularia lunata. To date, however, the proteins that interact with Clt-1 are unknown. In this study, a high-quality yeast two-hybrid (Y2H) library was successfully constructed from Curvularia. lunata grown in toxin-inducing Fries 3 medium. The transformation efficiency was 4.0 × 1010 cfu/μg DNA. The titer of the Y2H library was 1.0 × 107 cfu/ml, and the numbers for the Y2H library were 1.2 × 107 . Electrophoresis results showed that the inserted fragments ranged from 0.5 kb to 3.0 kb, and sequence analysis of random clones revealed a recombination rate of 100%. Clt-1 was used as a bait to screen for interacting proteins in the Y2H library. A cDNA clone encoding acetylxylan esterase (CIAxe56), which is known to be involved in the regulation of xylan catabolism, was identified. Bimolecular fluorescence complementation (BiFC) confirmed that CIAxe56 can interact with Clt-1, suggesting that CIAxe56 may potentially play roles in toxin and melanin synthesis. Further work aimed at elucidating the exact role of CIAxe56 in pathogenesis is ongoing.

Key Words：Acetylxylan esterase . Clt-1 . Curvularia leaf spot . M5HF2C

Source：Tropical plant pathology (2018) 43:85–89