AwSclB regulates a network for Aspergillus westerdijkiae asexual sporulation and secondary metabolism independent of the fungal light control.

As a prevalent pathogenic fungus, Aspergillus westerdijkiae poses a threat to both food safety and human health. The fungal growth, conidia production and ochratoxin A (OTA) in A. weterdijkiae are regulated by many factors especially transcription factors. In this study, a transcription factor AwSclB in A. westerdijkiae was identified and its function in asexual sporulation and OTA biosynthesis was investigated. In addition, the effect of light control on AwSclB regulation was also tested. The deletion of AwSclB gene could reduce conidia production by down-regulation of conidia genes and increase OTA biosynthesis by up-regulation of cluster genes, regardless under light or dark conditions. It is worth to note that the inhibitory effect of light on OTA biosynthesis was reversed by the knockout of AwSclB gene. The yeast one-hybrid assay indicated that AwSclB could interact with the promoters of BrlA, ConJ and OtaR1 genes. This result suggests that AwSclB in A. westerdijkiae can directly regulate asexual conidia formation by activating the central developmental pathway BrlA-AbaA-WetA through up-regulating the expression of AwBrlA, and promote the light response of the strain by activating ConJ. However, AwSclB itself is unable to respond to light regulation. This finding will deepen our understanding of the molecular regulation of A. westerdijkiae development and secondary metabolism, and provide potential targets for the development of new fungicides.

Bacillus amyloliquefaciens A-1 inhibiting fungal spoilage in agricultural products is improved by metabolic engineering of enhancing surfactin yield.

Fungi and subsequent mycotoxins contamination in agricultural products have caused enormous losses and great harm to human and animal health. Biological control has attracted the attention of researchers due to its advantages, including mild conditions, low cost, high efficiency and low nutrient loss. In this study, a newly isolated strain Bacillus amyloliquefaciens A-1 (A-1), was screened for its ability to inhibit the growth and Aflatoxin B1 (AFB1) production of Aspergillus flavus NRRL 3357. Electron microscopy results revealed that mycelium and conidia of A. flavus were destroyed by A-1, affecting hyphae, cell walls, cell membranes and organelles. RNA-seq analysis indicated disturbance in gene expression profiles of A. flavus, including amino acid degradation and starch and sucrose metabolism pathways. Importantly, the biosynthesis of AFB1 was significantly inhibited by the down-regulation of key regulatory genes, aflR and aflS, and the simultaneous down-regulation of most structural genes. Genome analysis predicted six secondary metabolites biosynthetic gene clusters. Then, four surfactin synthesized by cluster C were identified as the main active substance of A-1 using HPLC-Q-TOF-MS. The addition of alanine, threonine, Fe2+ increased surfactin production. Notably, the overexpression of comX also improved surfactin production. The vivo test results indicated that A-1 could significantly inhibit the decay of pear by Aspergillus westerdijkiae, and the mildew of maize and peanuts. Especially, the overexpression of comX in A-1 could enhance the inhibitory activity. In conclusion, the inhibition mechanism of A-1 was revealed, and comX was found can improve the production of surfactin and subsequent activities, which provides the scientific basis for the development of biocontrol agents to reduce spoilage in agricultural products.

The transcription factor VaMYC2 from Chinese wild Vitis amurensis enhances cold tolerance of grape (V. vinifera) by up-regulating VaCBF1 and VaP5CS.

Cultivated grapes, one of the most important fruit crops in the world, are sensitive to low temperature. Since Chinese wild grape Vitis amurensis is highly tolerant to cold, it is imperative to study and utilize its cold-tolerance genes for molecular breeding. Here, a VaMYC2 gene from V. amurensis was cloned, and its function was investigated by expressing VaMYC2 in the cold-sensitive V. vinifera cultivar 'Thompson Seedless'. The expression of VaMYC2 could be induced by cold stress, methyl jasmonate and ethylene treatment, but was inhibited by abscisic acid in leaves of V. amurensis. When transgenic grape lines expressing VaMYC2 were subjected to cold stress (-1 °C) for 41 h, the transgenic lines showed less freezing injury and lower electrolyte leakage and malondialdehyde content, but higher contents of soluble sugars, soluble proteins and proline, and antioxidant enzyme activities compared with wild-type. Moreover, the expression of some cold-tolerance related genes increased in transgenic lines. Besides, the interactions of VaMYC2 with VaJAZ1 and VaJAZ7B were confirmed by yeast two-hybrid and bimolecular fluorescence complementation assays. Yeast one-hybrid and dual luciferase assays showed that VaMYC2 can bind to the promoters of VaCBF1 and VaP5CS and activate their expressions. In conclusion, expression of VaMYC2 in V. vinifera enhances cold tolerance of transgenic grapes which is attributed to enhanced accumulation of osmotic regulatory substances, cell membrane stability, antioxidant enzyme activity, and expression of cold tolerance-related genes. Also, VaMYC2 interacts with VaJAZ1 and VaJAZ7, and activates the expression of VaCBF1 and VaP5CS to mediate cold tolerance in grapes.

VaMYB44 transcription factor from Chinese wild Vitis amurensis negatively regulates cold tolerance in transgenic Arabidopsis thaliana and V. vinifera.

KEY MESSAGE: Heterologous expression of VaMYB44 gene in Arabidopsis and V. vinifera cv. 'Thompson Seedless' increases cold sensitivity, which is mediated by the interaction of VaMYC2 and VaTIFY5A with VaMYB44 MYB transcription factors play critical roles in plant stress response. However, the function of MYB44 under low temperature stress is largely unknown in grapes. Here, we isolated a VaMYB44 gene from Chinese wild Vitis amurensis acc. 'Shuangyou' (cold-resistant). The VaMYB44 is expressed in various organs and has lower expression levels in stems and young leaves. Exposure of the cold-sensitive V. vinifera cv. 'Thompson Seedless' and cold-resistant 'Shuangyou' grapevines to cold stress (-1 °C) resulted in differential expression of MYB44 in leaves with the former reaching 14 folds of the latter after 3 h of cold stress. Moreover, the expression of VaMYB44 was induced by exogenous ethylene, abscisic acid, and methyl jasmonate in the leaves of 'Shuangyou'. Notably, the subcellular localization assay identified VaMYB44 in the nucleus. Interestingly, heterologous expression of VaMYB44 in Arabidopsis and 'Thompson Seedless' grape increased freezing-induced damage compared to their wild-type counterparts. Accordingly, the transgenic lines had higher malondialdehyde content and electrolyte permeability, and lower activities of superoxide dismutase, peroxidase, and catalase. Moreover, the expression levels of some cold resistance-related genes decreased in transgenic lines. Protein interaction assays identified VaMYC2 and VaTIFY5A as VaMYB44 interacting proteins, and VaMYC2 could bind to the VaMYB44 promoter and promote its transcription. In conclusion, the study reveals VaMYB44 as the negative regulator of cold tolerance in transgenic Arabidopsis and transgenic grapes, and VaMYC2 and VaTIFY5A are involved in the cold sensitivity of plants by interacting with VaMYB44.

Genome-wide analysis of the Universal stress protein A gene family in Vitis and expression in response to abiotic stress.

Universal Stress Protein A (USPA) plays critical roles in the regulation of growth, development and response to abiotic stress in plants. To date, most research related to the role of USPA in plants has been carried out in herbaceous models such as Arabidopsis, rice and soybean. Here, we used bioinformatics approaches to identify 21 USPA genes in the genome of Vitis vinifera L. Phylogenetic analysis revealed that VvUSPAs could be divided into eight clades. Based on predicted chromosomal locations, we identified 16 pairs of syntenic, orthologous genes between A. thaliana and V. vinifera. Further promoter cis-elements analysis, together with identification of potential microRNA (miRNA) binding sites, suggested that at least some of the VvUSPAs participate in response to phytohormones and abiotic stress. To add support for this, we analyzed the developmental and stress-responsive expression patterns of the homologous USPA genes in the drought-resistant wild Vitis yeshanensis accession 'Yanshan-1' and the drought-sensitive Vitis riparia accession 'He'an'. Most of the USPA genes were upregulated in different degrees in the two genotypes after drought stress and exposure to ethephon (ETH), abscisic acid (ABA) and methyl jasmonate (MeJA). Individual USPA genes showed various tissue-specific expression patterns. Heterologous expression of five selected genes (VvUSPA2, VvUSPA3, VvUSPA11, VvUSPA13 and VvUSPA16) in Escherichia coli (E. coli) enhanced resistance to drought stress. Our study provides a model for mapping gene function in response to abiotic stress and identified three candidate genes, VvUSPA3, VvUSPA11 and VvUSPA16, as regulators of drought response in V. vinifera.

L. johnsonii, L. plantarum, and L. rhamnosus alleviated Enterohaemorrhagic Escherichia coli-induced diarrhoea in mice by regulating gut microbiota.

Enterohaemorrhagic Escherichia coli (EHEC) is a prominent foodborne pathogen that causes infectious intestinal diarrhoea. Lactobacillus is a recognized probiotic that inhibits intestinal pathogens and maintains the balance of the intestinal flora. The purpose of this study was to investigate the regulatory effects of three Lactobacillus strains, L. johnsonii, L. plantarum, and L. rhamnosus, on the intestinal flora of EHEC-infected mice. The initial weight and diarrhoea index of the mice were recorded. After 21 days, the faeces of the mice were subjected to 16S rDNA high-throughput sequencing. The diarrhoea index of mice treated with Lactobacillus improved, their body weight continued to rise, and their liver index gradually decreased. The α diversity analysis showed that the intestinal flora diversity and abundance were lower in mice infected with EHEC than in healthy mice. L. plantarum, L. johnsonii, and L. rhamnosus significantly improved the diversity of the flora species. In terms of flora composition, the three main phyla present were Bacteroidetes, Firmicutes, and Proteobacteria. The abundance of these three phyla was reduced to 93.81% after infection and restored to over 96.30% after treatment. At the genus level, Lactobacillus reduced the abundance of Bacteroides, Helicobacter pylori, and Shigella, while increasing the abundance of butyric acid-producing bacteria and Lactobacillus. Finally, a heat map and non-metric multidimensional scaling analysis showed that the intestinal flora structures in the L. johnsonii, L. plantarum, and L. rhamnosus treatment groups were closest to those of healthy mice. In conclusion, L. johnsonii, L. plantarum, and L. rhamnosus regulated and improved the structure of intestinal flora and relieved diarrhoea caused by EHEC infection.