Expression of recombinant EARLI1, a hybrid proline-rich protein of Arabidopsis, in Escherichia coli and its inhibition effect to the growth of fungal pathogens and Saccharomyces cerevisiae

Abstract

EARLI1 is an Arabidopsis gene with pleiotropic effects previously shown to have auxiliary functions in protecting plants against freezing-induced cellular damage and promoting germinability under low-temperature and salinity stresses. Here we determined whether recombinant EARLI1 protein has anti-fungal activity. Recombinant EARLI1 protein lacking its signal peptide was produced in Escherichia coli BL21(DE3) using isopropyl β-d-1-thiogalactopyranoside (IPTG) induction and the prokaryotic expression vector pET28a. Expression of EARLI1 was analyzed by Western blotting and the protein was purified using affinity chromatography. Recombinant EARLI1 protein was applied to fungal cultures of Saccharomyces cerevisiae, Botrytis cinerea and Fusarium oxysporum, and membrane permeability was determined using SYTOX green. Full-length EARLI1 was expressed in S. cerevisiae from the GAL1 promoter using 2% galactose and yeast cell viability was compared to control cells. Our results indicated that application of recombinant EARLI1 protein to B. cinerea and F. oxysporum could inhibit the growth of the necrotrophic fungi. Besides, addition of the recombinant protein to liquid cultures of S. cerevisiae significantly suppressed yeast growth and cell viability by increasing membrane permeability, and in vivo expression of the secreted form of EARLI1 in S. cerevisiae also had a remarkable inhibition effect on the growth of yeast cells.

Salt Stress in Arabidopsis: Lipid Transfer Protein AZI1 and Its Control by Mitogen-Activated Protein Kinase MPK3

“…Future studies shall help to clarify whether Arabidopsis EARLI proteins are recognized and modified by stress-induced yeast MAPKs to reach their full protecting activity. In engineered plants, the targeted exchange of critical residues (phosphorylation sites; MAPK binding sites) may be a means to manipulate reported HyPRP-controlled processes such as fruit ripening (Blanco-Portales et al, 2004), fungal growth inhibition (Li et al, 2012), or freezing tolerance (Zhang and Schlappi, 2007; Xu et al, 2011b). Based on its gene expression profile (induced by numerous stressors), functions of AZI1 as a protectant towards other types of stress seem likely.…”

Overexpression of Camellia sinensis H1 histone gene confers abiotic stress tolerance in transgenic tobacco

“…empty vector were transfected into competent BL21(DE3) E. coli cells following the method of Sambrook and Russell (2001). The fusion protein was expressed according to the methods described by Li et al (2012). Furthermore, the fusion protein was collected and analyzed by 12 % sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE).…”

Streamlined assessment of membrane permeability and its application to membrane engineering of Escherichia coli for octanoic acid tolerance

“…Theoretically, this approach is expected to work with other cell types. SYTOX Green has previously been used to characterize not just membrane permeability of E. coli, but also Bacillus species [32,33], Saccharomyces cerevisiae [39,40], Candida albicans [38], Fusarium graminearum [38], Drosophila neuronal cells [36], and human breast cancer cells [37]. This approach can possibly also be extended to other DNA-binding dyes used to assess the status of the microbial cell membrane, such as propidium iodide [58].…”

“…Most mechanistic studies of cell permeability use microscopic-based imaging of SYTOX permeability at the single-cell level [32,33]. At larger scales, quantification of SYTOX Green permeability is typically performed by flow cytometry, with the sorting of cells as either SYTOX positive or SYTOX negative or a population distribution [16,20,25,[34][35][36][37][38][39][40]. A high-throughput assay using SYTOX Green to measure cell viability has been reported [41], though other reports have recommended that SYTOX Green should be used cautiously in the distinguishing of live and dead cells [42].…”