Fig. 2: Interactions between CHIL and flavonoid enzymes in snapdragon and soybean as determined in yeast assay systems. | Nature Communications

Fig. 2: Interactions between CHIL and flavonoid enzymes in snapdragon and soybean as determined in yeast assay systems.

From: A conserved strategy of chalcone isomerase-like protein to rectify promiscuous chalcone synthase specificity

Fig. 2

a Interactions between CHIL and cytochromes P450 in snapdragon (i–iii) and soybean (iv–vi) were analyzed with the split-ubiquitin yeast two-hybrid systems. Regarding the protein names, the initials of the scientific name of plant species are followed by the abbreviated enzyme/protein names, with homologs indicated with a hyphen. For example, GmCHS-1 refers to isozyme 1 of a soybean (Glycine max) CHS. (i–iii) Growth of yeast cells co-expressing X-Cub-LexA-VP16 with NubG-AmCHIL, where X refers to (i) nothing, (ii) flavone synthase II (AmFNSII), and (iii) flavonoid 3′-hydroxylase (AmF3′H). (iv–vi) Growth of yeast cells co-expressing X-Cub-LexA-VP16 with NubG-GmCHIL, where X refers to (iv) nothing, (v) isozyme 1 of 2-hydroxyisoflavanone synthase (GmIFS-1), and (vi) isozyme 2 of GmIFS (GmIFS-2). In the NubG-Y column of each panel, P.C. refers to the positive control [transformed cells harboring pOst1-NubI (a plasmid expressing a fusion protein comprising the yeast resident ER protein Ost1 and the wild-type Nub portion of yeast ubiquitin) and derivatives of pBT3-SUC]. “None” refers to the negative control (transformed cells harboring the empty pPR3-N vector and derivatives of pBT3-SUC). b Interactions between CHIL and soluble flavonoid enzymes in (i, ii) snapdragon and (iii, iv) soybean were analyzed with GAL4-based yeast two-hybrid systems. The growth of yeast cells co-expressing CHIL and flavonoid enzymes as fusions with the activation domain (AD, AD-Y column) and the DNA-binding domain (BD, BD-X column) of the yeast GAL4 transcription factor is presented. (i) BD-AmCHIL was co-expressed with AD-Y, where Y refers to CHS (AmCHS), CHI (AmCHI), AmCHIL, dihydroflavonol 4-reductase (AmDFR), or flavanone 3-hydroxylase (AmF3H). “None” refers to yeast cells co-expressing AD (without a fused protein) and BD-AmCHIL. (ii) AD-AmCHIL was co-expressed with BD-X, where X refers to AmCHS, AmCHI, AmDFR, or AmF3H. “None” refers to yeast cells co-expressing BD (without a fused protein) and AD-AmCHIL. (iii) BD-GmCHIL was co-expressed with AD-Y, where Y refers to CHS isozymes (GmCHS-1 and GmCHS-7), chalcone reductase (CHR) isozymes (GmCHR-1 and GmCHR-5), CHI isozymes (GmCHI-1A, GmCHI-1B2, and GmCHI-2), GmCHIL, or a 2-hydroxyisoflavanone dehydratase isozyme (GmHID-1). “None” refers to yeast cells co-expressing AD (without a fused protein) and BD-GmCHIL. (iv) AD-GmCHIL was co-expressed with BD-X, where X refers to GmCHS-1, GmCHS-7, GmCHR-1, GmCHR-5, GmCHI-1A, or GmCHI-2. “None” refers to yeast cells co-expressing BD (without a fused protein) and AD-GmCHIL. Abbreviated growth media names are as follows: −WL, SD agar medium lacking tryptophan and leucine; −WLH, −WL medium lacking histidine; −WLHAde, −WLH medium lacking adenine.

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