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Green mold, caused by Penicillium digitatum, is the major cause of citrus postharvest decay. Currently, the application of sterol demethylation inhibitor (DMI) fungicide is one of the main control measures to prevent green mold. However, the fungicide-resistance problem in the pathogen P. digitatum is growing. The regulatory mechanism of DMI fungicide resistance in P. digitatum is poorly understood. Here, we first performed transcriptomic analysis of the P. digitatum strain Pdw03 treated with imazalil (IMZ) for 2 and 12 h. A total of 1338 genes were up-regulated and 1635 were down-regulated under IMZ treatment for 2 h compared to control while 1700 were up-regulated and 1661 down-regulated under IMZ treatment for 12 h. The expression of about half of the genes in the ergosterol biosynthesis pathway was affected during IMZ stress. Further analysis identified that 84 of 320 transcription factors (TFs) were differentially expressed at both conditions, making them potential regulators in DMI resistance. To confirm their roles, three differentially expressed TFs were selected to generate disruption mutants using the CRISPR/Cas9 technology. The results showed that two of them had no response to IMZ stress while ∆PdflbC was more sensitive compared with the wild type. However, disruption of PdflbC did not affect the ergosterol content. The defect in IMZ sensitivity of ∆PdflbC was restored by genetic complementation of the mutant with a functional copy of PdflbC. Taken together, our results offer a rich source of information to identify novel regulators in DMI resistance. [ABSTRACT FROM AUTHOR]

Physalis macrophysa (PMA) and Physalis ixocarpa (PIX) have significant economic, medicinal, and ornamental value and are often used in the fields of edible fruits, medicinal herbs, and ornamental plants. In the present study, we obtained the complete chloroplast (cp) genome sequences of PMA and PIX, compared to the cp genome sequences of 10 Physalis species, and constructed the phylogenetic tree among the tribe Physaleae. The results showed that the cp genomes of PMA and PIX consisted of a large single copy (LSC) region (87,115 bp and 87,103 bp, respectively), a small single copy (SSC) region (18,412 bp and 18,420 bp, respectively), and a pair of same-length inverted-repeat (IRa and IRb) regions (25,604 bp and 25,674 bp, respectively). The two species contained 132 genes, including 87 encoding proteins, eight encoding ribosomal RNAs (rRNAs), and 37 encoding transfer RNAs (tRNAs), which indicated that the two species have strong similarities with respect to genome structure and gene content. PMA and PIX contained repeat sequences (35 and 40, respectively) and simple-sequence repeats (SSRs) (61 and 60, respectively). Nine regions with considerable nucleotide divergence were found, most of which were located in the LSC and SSC regions. The gene selective pressure analysis indicated that eight genes were affected by positive selection, the Ka/Ks values of which were greater than one. Our phylogenetic results indicated that PMA and PIX had the closest genetic relationship and are closely adjacent to Physalis philadelphica (PPH) in the subtribe Physalinae. Our analysis of the cp genomes in both Physalis species will be beneficial for further research into species identification, phylogeny, evolution, and the potential for germplasm resource exploitation in Physalis. [ABSTRACT FROM AUTHOR]

Dechlorination is the key step in biological degradation of dichloromethane (DCM), which is catalyzed by the enzyme DCM dehalogenase. In this study, the detailed structure and function of DCM dehalogenase in Methylobacterium rhodesianum H13 were investigated. Our results indicate that DCM dehalogenase (C1505H2255N403O430S7) is a stable hydrophilic protein localized in the cytoplasm, with an isoelectric point of 6.35. They also indicate that DCM dehalogenase is a frizzled protein with 52.43% of amino acid residues exposed on its surface, and that alpha-helices are a main structural component (53.47%). As one of the theta class glutathione S-transferases (GSTs), DCM dehalogenase presents as a dimeric structure and shows strong substrate selectivity. The catalytic tunnel radius and length of DCM dehalogenase are both 1.4 Å. Glu42 and Val43 are two of the essential amino acids in both the catalytic tunnel and catalytic pockets. The amino acid sequence of DCM dehalogenase was predicted to be homologous among various microorganisms, and its active site was conserved with eight potential sites. The recombinant DCM dehalogenase was expressed in E. coli BL21 strain. The optimum conditions for DCM dehalogenase are pH 8, temperature of 30 °C and presence of Mn2+. All the obtained information of DCM dehalogenase will lay the foundation for further study of its structural transformation and functional improvement. [ABSTRACT FROM AUTHOR]