BackgroundGrey mould is an important plant disease worldwide, caused by Botrytis cinerea, resulting in serious economic loss. Wuyiencin, a low toxicity, high efficiency, and broad-spectrum agricultural antibiotic, has been demonstrated effectiveness against B. cinerea.ResultsWuyiencin treatment inhibited growth and sporulation of B. cinerea, specifically altering hypha morphology and intracellular structures. These changes were accompanied by differential expression (fold change > 2.0) of 316 proteins identified by iTRAQ-labelling LC-MS/MS analysis (P < 0.05). Up-regulation of 14 proteins, including carbohydrate metabolism proteins and cell wall stabilization proteins, was validated by parallel reaction monitoring (PRM). Down-regulation of 13 proteins was validated by PRM, including regulators of energy metabolism, nucleotide/protein synthesis, and the biosynthesis of mediators of plant stress and decay.ConclusionOur results confirm the inhibitory biological effects of wuyiencin on B. cinereal and elaborate on the differentially expressed proteins and associated pathways implicated in the capacity of wuyiencin to debilitate the growth and pathogenicity of grey mould. This study provides validated candidates for further targeted exploration with the goal of optimizing wuyiencin as a safe, low-toxicity agent for biological control.

Aim: The aim of the present work was to investigate the overexpression of the wysR gene in Streptomyces albulus var. wuyiensis strain CK-15 based on the DwysR3 mutant strain including the effect on morphological development, wuyiencin production and antibacterial activity. At the same time, we report a new rapid method for producing genetically engineered strains for industrial production of wuyiencin. Methods and Results: We developed a method to create a wysR overexpression strain based on the DwysR3 mutant strain by direct transformation. In this method, the desired gene fragment to be overexpressed was amplified by polymerase chain reaction (PCR) using Phusion High Fidelity DNA polymerase and fused with the linearized pSETC integrative plasmid by Gibson assembly. The resulting recombinant plasmid was transformed into DwysR3 mutant strain by the intergeneric conjugation method. The plasmid was then integrated into the chromosome and the resulting apramycinresistant overexpression strain was confirmed by PCR using the Apra-F and Apra-R primers. Finally, we successfully screened the genetically engineered strain with overexpression of wysR gene in DwysR3 mutant. Conclusion: We can conclude that overexpression of wysR gene in DwysR3 mutant strain proved to be an effective strategy for significantly increasing wuyiencin production together with faster morphological development. Quantitative real-time RT-PCR analysis showed that wysR regulated wuyiencin biosynthesis by modulating other putative regulatory genes and bld, whi, chp, rdl and ram family genes are crucial for the morphological development. Significance and Impact of the Study: Overexpression of wysR gene in the DwysR3 mutant strain named OoWysR strain may increase the efficiency in the industrial fermentation processes for wuyiencin production. The mechanism by which wysR overexpression promotes rapid sporulation and a high yield of wuyiencin production is likely related to modulation of other putative regulatory genes.

Grey mould is caused by the ascomycetes Botrytis cinerea in a range of crop hosts. As a biological control agent, the nucleoside antibiotic wuyiencin has been industrially produced and widely used as an effective fungicide. To elucidate the effects of wuyiencin on the transcriptional regulation in B. cinerea, we, for the first time, report a genome-wide transcriptomic analysis of B. cinerea treated with wuyiencin. 2067 genes were differentially expressed, of them, 886 and 1181 genes were significantly upregulated and downregulated, respectively. Functional categorization indicated that transcript levels of genes involved in amino acid metabolism and those encoding putative secreted proteins were altered in response to wuyiencin treatment. Moreover, the expression of genes involved in protein synthesis and energy metabolism (oxidative phosphorylation) and of those encoding ATP-binding cassette transporters was markedly upregulated, whereas that of genes participating in DNA replication, cell cycle, and stress response was downregulated. Furthermore, wuyiencin resulted in mycelial malformation and negatively influenced cell growth rate and conidial yield in B. cinerea. Our results suggest that this nucleoside antibiotic regulates all aspects of cell growth and differentiation in B. cinerea. To summarize, some new candidate pathways and target genes that may related to the protective and antagonistic mechanisms in B. cinerea were identified underlying the action of biological control agents.