Construction of a High-Quality Yeast Two-Hybrid Library and Its Application in Identification of Interacting Proteins with Brn1 in Curvularia lunata

Abstract

Curvularia lunata is an important maize foliar fungal pathogen that distributes widely in maize growing area in China, and several key pathogenic factors have been isolated. An yeast two-hybrid (Y2H) library is a very useful platform to further unravel novel pathogenic factors in C. lunata. To construct a high-quality full length-expression cDNA library from the C. lunata for application to pathogenesis-related protein-protein interaction screening, total RNA was extracted. The SMART (Switching Mechanism At 5′ end of the RNA Transcript) technique was used for cDNA synthesis. Double-stranded cDNA was ligated into the pGADT7-Rec vector with Herring Testes Carrier DNA using homologous recombination method. The ligation mixture was transformed into competent yeast AH109 cells to construct the primary cDNA library. Eventually, a high qualitative library was successfully established according to an evaluation on quality. The transformation efficiency was about 6.39 ×105 transformants/3 μg pGADT7-Rec. The titer of the primary cDNA library was 2.5×108 cfu/mL. The numbers for the cDNA library was 2.46×105. Randomly picked clones show that the recombination rate was 88.24%. Gel electrophoresis results indicated that the fragments ranged from 0.4 kb to 3.0 kb. Melanin synthesis protein Brn1 (1,3,8-hydroxynaphthalene reductase) was used as a “bait” to test the sufficiency of the Y2H library. As a result, a cDNA clone encoding VelB protein that was known to be involved in the regulation of diverse cellular processes, including control of secondary metabolism containing melanin and toxin production in many filamentous fungi was identified. Further study on the exact role of the VelB gene is underway.

Cytosolic TaGAPC2 Enhances Tolerance to Drought Stress in Transgenic Arabidopsis Plants

“…In order to explore the biology of a plant response mechanism—genes such as those responsible for survival, dehydration avoidance, and development—a full-length cDNA library was constructed using wheat leaves from a drought condition. The high-quality-expression cDNA library can offer molecular resources for the analysis of genes involved in biology and for studying its protein function [ 41 ]. Our library storage was 5.30 × 10 5 cfu/mL, and the titer was 3.62 × 10 7 cfu/mL.…”

Involvement of a Polyketide Synthetase ClPKS18 in the Regulation of Vegetative Growth, Melanin and Toxin Synthesis, and Virulence in Curvularia lunata

“…To uncover the pathogenicity mechanisms of CLS, most researches have focused on identifying virulence factors, such as melanin ( Gao et al, 2012 , 2015b ), and toxin ( Gao et al, 2014a ; Liu et al, 2009 ). Fungal melanins, formed by the oxidative polymerisation of phenolic compounds, are essential for enhance the mechanical strength of the infection into the host plant epidermis and contribute to virulence in numerous types of plant diseases ( Gao et al, 2015e ). Previous studies have also indicated that the albino C. lunata strains present in wild-type (WT) strains are weakly virulent to maize leaves, supporting the conclusion that melanin is a crucial virulence factor for C. lunata in susceptible maize leaves ( Yan et al, 2005 ).…”

Yeast two-hybrid screening for proteins that interact with PFT in wheat

“…A high-quality Y2H library can provide molecular resources to analyze protein functions and interactions based on a known protein to facilitate the construction of protein networks 19 . These proteins provide baseline information for further elucidating their regulatory network in the development process.…”