Transcriptome profiling reveals the underlying mechanism of grape post-harvest pathogen Penicillium olsonii against the metabolites of Bacillus velezensis

IntroductionPathogen infection influences the post-harvest shelf life of grape berries. In a preliminary study, metabolites produced by Bacillus velezensis significantly inhibited the growth of the grape postharvest pathogen Penicillium olsonii.MethodsTo investigate the mechanism of interaction between B. velezensis and P. olsonii, a draft genome was generated for P. olsonii WHG5 using the Illumina NovaSeq platform, and the transcriptomic changes in WHG5 were analyzed in response to the exposure to B. velezensis metabolites (10% v/v).ResultsThe expression levels of genes associated with sporulation, including GCY1, brlA, and abaA, were down-regulated compared with those of the control. In addition, spore deformation and abnormal swelling of the conidiophore were observed. The expression of crucial enzymes, including fructose 2,6-bisphosphate and mannitol-2-dehydrogenase, was down-regulated, indicating that the glycolytic pathway of WHG5 was adversely affected by B. velezensis metabolites. The KEGG pathway enrichment analysis revealed that glutathione metabolism and the antioxidant enzyme system were involved in the response to B. velezensis metabolites. The down-regulation of the pathogenesis-related genes, PG1 and POT1, suggested that B. velezensis metabolites decreased the pathogenicity of P. olsonii. B. velezensis metabolites disrupted the homeostasis of reactive oxygen species in P. olsonii by affecting glucose metabolism, resulting in spore deformation and disruption of growth. In addition, the expression of key pathogenesis-related genes was down-regulated, thereby reducing the pathogenicity of P. olsonii.DisscusionThis study provides insights into the responses of P. olsonii to B. velezensis metabolites and identifies potential target genes that may be useful in biocontrol strategies for the suppression of post-harvest spoilage in grapes

An ENU-Induced Mutation of Nrg1 Causes Dilated Pupils and a Reduction in Muscarinic Receptors in the Sphincter Pupillae

BACKGROUND: N-ethyl-N-nitrosourea (ENU)-induced mutagenesis is a powerful tool for the study of gene function and the generation of human disease models. A large number of mouse mutants obtained by ENU-induced mutagenesis with a variety of phenotypes have been recovered. However, after genetic confirmation testing, only approximately 50% of the abnormal phenotypes were found to be heritable. METHODOLOGY/PRINCIPAL FINDINGS: A mouse mutant, Dp1, with a dilated pupil phenotype was induced with an N-ethyl-N-nitrosourea (ENU) mutagenesis strategy. Sequence analysis for Nrg1 reveals a G>A base substitution that flanks exon E59, encoding for an EGFβ domain, in the 5' splice donor site. The mutation affects but does not abolish the splicing of EGFβ-type Nrg1 mRNA in Dp1 mice and produces several different transcripts by activating other, cryptic splice sites. These types of protein isoforms are expected, and the result shows that, in the mutant, the effect is a decrease in but not an elimination of the high affinity EGFβ-type Nrg1 isoforms. This is partially compensated for by an increase in expression of the low affinity alpha forms or inactive proteins, suggesting that the mutation results in a hypomorphic allele. Interestingly, genetic model testing shows that Dp1 is a mutation that results in a dilated pupil phenotype that is inherited with very low penetrance when heterozygous and with complete penetrance when homozygous. Pharmacological and immunohistochemical tests show a reduction of muscarinic (M) receptors in the sphincter pupillae of Dp1 mice, which is a major cause of dilated pupils. CONCLUSIONS/SIGNIFICANCE: This study is the first report of an Nrg1 mutation being associated with a dilated pupil phenotype and the reduction of M receptors. This report may help in establishing more mutant mouse lines and models of human genetic disease and can be applied to other organisms. Dp1 mice are a valuable resource for the further clarification of Nrg1 biological function

Advantages of Nanchong City in Developing Late-maturing Citrus and Countermeasures

Nanchong City has the advantages of developing late-maturing citrus in ecology, no-quarantine diseases and insect pests, base construction starting taking shape, production and management technology becoming increasingly mature, leaders paying close attention and market recognition, and significant income increase of fruit growers. However, there are also problems such as weak infrastructure and weak anti-disaster ability, inadequate technology extension system, unmatched cold chain warehouses, and uneven quality of seedlings. Therefore, it is recommended to make scientific plan in accordance with actual local situations, regulate fine variety breeding and market supervision, build orchards with high standard and strengthen management, do a good job of propaganda and branding, improve post-harvest processing, extend the industrial chain, and issue preferential policies to promote the development of citrus industry in Nanchong City

Transcripts of <i>CRD-Nrg1</i> and the expected protein isoforms in wild-type and <i>Dp1/Dp1</i> homozygous mice.

<p>(A) RT-PCR amplified region of <i>CRD-Nrg1</i> transcripts. The primers used for RT-PCR are indicated; the arrow indicates the approximate primer position within the exon. (B) Transcripts of <i>CRD-Nrg1</i> produced by wild-type mice. (C) Transcripts of <i>CRD-Nrg1</i> produced by <i>Dp1/Dp1</i> homozygous mice. In band a, one transcript contains a new exon, E104 (which is expected to produce a truncated protein without the EGFc, EGFβ, and EGFα domains), and another transcript (accounting for 6/7 of the clones) transcribes through exon E59 into the adjacent 112 bp sequence (which is expected to produce CRD-β3 type isoforms). In band b, one transcript (accounting for 4/7 of the clones) bypasses the mutated splice donor site by splicing over exon E59 (which is expected to produce CRD-α2 type isoforms).</p

Genetic mapping places the mutant in the region between markers rs32829041 and D8Mit4.

<p>Primary markers used to refine the map position are listed on the left from the centromere (top), and the number of animals in each genotypic class is shown at the bottom. 1^a and 1^b: mice with unilateral and partially dilated pupil phenotypes. The others are mice with a severe bilaterally dilated pupil phenotype.</p

Genetic model testing.

<p>In the test, all affected heterozygous offspring have a unilaterally and partially dilated pupil phenotype, but the size of the pupil varies with each individual.</p><p>Of the 152 homozygous offspring, 148 have a severe bilaterally dilated pupil phenotype, 3 show a bilateral dilated pupil phenotype with one completely and one partially dilated pupil, and 1 has a severe unilaterally dilated pupil phenotype.</p

<i>Nrg1</i> gene structure and mutation identification.

<p>(A) Mouse <i>Nrg1</i> gene structure. Exons are represented as vertical bars and are numbered as per Steinthorsdottir et al. [2004], Paul et al. [2006] and Chen et al. [2008]. In the absence of an agreed or definitive exon numbering system for NRG1, exons are labeled as described by Steinthorsdottir et al. [2004] with the number denoting their length in nucleotides. The 5′ exons, which define the “types” of NRG1, are in black with the corresponding Roman numeral above (including three additional 5′ exons found in humans that encode type IV, V, and VI). In mice, exon E171 may encode isoforms with a “3” stalk (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0025176#pone-0025176-g004" target="_blank">Figure 4</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0025176#pone-0025176-g005" target="_blank">5</a>). (B) Sequence analysis of the <i>Nrg1</i> gene product obtained using PCR of genomic DNA discovered a G to A transition mutation in <i>Dp1/Dp1</i> homozygous mice compared to wild-type B6 mice. (C) The mutation located in the 5′ splice donor site that flanks exon E59 encoding the EGFβ domain. (D) Electrophoresis results using primers specific for CRD-Nrg1 and Ig-Nrg1 from homozygous <i>Dp1</i> and wild-type B6 mice, respectively. M, molecular weight markers. GAPDH, glyceraldehyde-3-phosphate dehydrogenase. (E) Quantitative analysis of <i>Nrg1</i> mRNA levels in band b by densitometry. Ratio: ratio of volume (intensity) of respective Nrg1 and GAPDH mRNA RT-PCR product. **p<0.01 vs. respective <i>Dp1/Dp1</i> group (n = 4).</p

Immunohistochemistry results reveal the severe reduction of the ChRM3 receptor in the iris constrictor muscles of <i>Dp1</i> homozygotes.

<p>Representative iris sections from wild-type and homozygous mice. Staining of an anti-ChRM3 receptor antibody is shown in red and is indicated by an arrow. Scale bars: 50 µm.</p