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Aureobasidin A -For Yeast One/Two-Hybrid Library Screening System

Aureobasidin A (AbA), a cyclic depsipeptide isolated from the filamentous fungus Aureobasidium pullulans R106, is highly toxic to yeast, including Saccharomyces cerevisiaeSchizosaccharomyces pombeCandida glabrataAspergillus nidulans, and A. niger, even at low concentrations (0.1–0.5 µg/mL). AbA inhibits a yeast enzyme, inositol phosphorylceramide (IPC) synthase encoded by the AUR1 gene, hindering the synthesis of ceramide to inositol phosphorylamide, resulting in insufficient sphingolipids and rupture of cell membrane, and eventually killing the strain.

 

Fig.1 AbA Chemical Structure (CAS No. : 127785-64-2)

 

Mutations in the AUR1 gene of Saccharomyces cerevisiae confer resistance to AbA, so is its homolog in A. nidulans, the AURA gene. Both genes are necessary for inositol phosphorylceramide (IPC) synthase activity. AbA actually kills sensitive yeast cells, rather than merely retarding their growth, so AbA selection virtually eliminates the high numbers of background colonies. AbA resistance is also an ideal reporter in yeast one/two hybrid library screening system, which is compatible with yeast carrying corresponding resistance.

 

Yeast two Library Screening System

 

 

The yeast two-hybrid system (Y2H) was originally devised by Fields and Song as a protein interaction detection system in yeast and was widely used in studying the interaction between antigen and antibody, discovering new proteins and its function, screening drug targets, establishing genomic protein linkage map and so on. Eukaryotic transcription activators contain two different domains: DNA binding domain (DBD) and DNA transcription activation domain (AD). Provided that the two domains interact, transcription will be activated. And these two domains can be separated independently without affecting each other. In Y2H, both the DBD and the AD are fused with two other proteins (X and Y). These two hybrid proteins are referred to as the “bait” (DBD-X) and the “prey” (AD-Y). If the bait captures the prey—that is, if proteins X and Y interact—a complex will form and the gene transcription will be activated. A convenient reporter gene is used to monitor for a successful interaction (Fig. 2).

 


 

Fig.2 Principle of Y2H[1]

 

Yeast one Library Screening System

 

The yeast one-hybrid technique (Y1H), developed from the yeast two-hybrid technique, is based on the principle of binding of a DNA-binding protein (ie, a transcription factor) to DNA cis-acting elements to regulate the expression of a reporter gene. An efficient method for the specific binding of transcription factor genes. The system is applied to determine the interaction between known DNA and proteins, screen for novel genes binding to the cis regulatory element or other short DNA sequence of interest, identify the domain of the protein-bound DNA and accurately locate the DNA-binding protein sequence. Two components: a reporter construct with DNA of interest (the bait) cloned upstream of a gene encoding a reporter protein and an expression construct that generates a fusion between a TF of interest (the prey) and a yeast transcription activation domain (AD), are introduced into a suitable yeast strain, and if the TF binds the DNA in the yeast nucleus, the reporter protein expression will be introduced. Moreover, the yeast AD activates the reporter regardless of whether the TF is an activator or repressor, thus Y1H only reports on the physical interaction between protein and DNA (Fig. 3).

 

Fig.3 Principle of Y1H[2]

 

【New Arrival】60231ES Aureobasidin A (AbA) for yeast one/two-hybrid studies!

 

Yeasen recommends AbA working concentration in yeast one/two hybrid experiments: 100-1000 ng/mL, depending on the host strains (Table 1)

 

Table 1 Aureobasidin A Minimum inhibitory concentration (MIC) against various yeasts

Strains

MIC (ng/mL)

S.cerevisiae

ATCC9763 (diploid)

200-400

SH3328 (haploid)

100

Sake yeast (diploid)

100-200

Shochu yeast (diploid)

100

Beer yeast (triploid or tetraploid)

100

Baker’s yeast (diploid)

200-400

Schizo.pombe

JY-745 (monoploid)

100

C.albicans

TIMM-0136 (diploid)

40

C.tropicalis

TIMM-0324 (diploid)

80

 

Product Case

In Dong’s study, to elucidate whether the GmWRKYs identified in this study could interact with the cis-element of W-box in the promotor region of soybean immune responsive genes, the Y1H assay was conducted. The optimum concentration of AbA (Yeasen Co., China) was determined to be 500 ng/mL.[3]

 


 

Fig.4 The interaction of GmWRKY31 proteins with yeast bait strains harboring fragment of either F16, F20, F73, delF16, delF20 or delF73.[3]

 

References

 

[1] Paiano A, et al. Yeast Two-Hybrid Assay to Identify Interacting Proteins.Curr Protoc Protein Sci. 2019 Feb;95(1):e70.

[2] John S, et al. Yeast one-hybrid assays: a historical and technical perspective.Methods. 2012 August;57(4): 441-447.

[3] Dong H, et al. Transcriptome analysis of soybean WRKY TFs in response to Peronospora manshurica infection. Genomics. 2019 Dec;111(6):1412-1422.