No abstract

Bacteriophages represent one prospect for preventing and treating multi-drug-resistant Escherichia coli. In this study, we have isolated a novel E. coli-specific bacteriophage and characterised its biological properties. vB_EcoM-ep3 has a broad host range and was able to lyse 9 out of 15 clinical isolates of multi-drug-resistant pathogenic E. coli from chickens. The optimal multiplicity of infection for vB_EcoM-ep3 in host bacteria was 0.01. vB_EcoM-ep3 was thermostable at temperatures below 50 °C for up to 60 min. Electron microscopy demonstrated that vB_EcoM-ep3 belongs to Myoviridae. The vB_EcoM-ep3 genome contained 42,351 pairs of nucleotides with a GC content of 53.35 %. There were 52 predicted open reading frames that appeared to overlap and have a modular structure. Phylogenetic analysis indicates that the closest evolutionary relative to vB_EcoM-ep3 is the previously reported E. coli phage vB_EcoM_ECO1230-10. However, there was no homology between reported E. coli phage lysins and the vB_EcoM-ep3 lysin gene. Lysep3 was 58 % similar to the Pseudomonas phage PPpW-3 lysin despite showing no similarities at the gene sequence level. And Lysep3 has good lysis activity.

Bacteriophage endolysin is one of the most promising antibiotic substitutes, but in Gram-negative bacteria, the outer membrane prevents the lysin from hydrolyzing peptidoglycans and blocks the development of lysin applications. The prime strategy for new antibiotic substitutes is allowing lysin to access the peptidoglycan from outside of the bacteria by reformation of the lysin. In this study, the novel Escherichia coli (E. coli) phage lyase lysep3, which lacks outside-in catalytic ability, was fused with the N-terminal region of the Bacillus amyloliquefaciens lysin including its cell wall binding domain D8 through the best manner of protein fusion based on the predicted tertiary structure of lysep3-D8 to obtain an engineered lysin that can lyse bacteria from the outside. Our results showed that lysep3-D8 could lyse both Gramnegative and Gram-positive bacteria, whereas lysep3 and D8 have no impact on bacterial growth. The MIC of lysep3-D8 on E. coli CVCC1418 is 60 μg/ml; lysep3-D8 can inhibit the growth of bacteria up to 12 h at this concentration. The bactericidal spectrum of lysep3-D8 is broad, as it can lyse of all of 14 E. coli strains, 3 P. aeruginosa strains, 1 Acinetobacter baumannii strain, and 1 Streptococcus strain. Lysep3-D8 has sufficient bactericidal effects on the 14 E. coli strains tested at the concentration of 100 μg/ml. The cell wall binding domain of the engineered lysin can destroy the integrity of the outer membrane of bacteria, thus allowing the catalytic domain to reach its target, peptidoglycan, to lyse the bacteria. Lysep3-D8 can be used as a preservative in fodder to benefit the health of animals. The method we used here proved to be a successful exploration of the reformation of phage lysin.

Southern corn rust (SCR), which is caused by the fungal pathogen Puccinia polysora Underw, is a prevalent foliar disease in maize. Breeding for resistant cultivars is a desirable way for the efficient control of this disease. To identify quantitative trait loci (QTL) for conferring resistance to SCR, a recombinant inbred population including 138 lines (RILs) derived from the SCR‐resistant line CML496 and susceptible line Lx9801 was evaluated for phenotypic reaction to SCR in three trials in two locations over 2 years. The population was genotyped with the maize 9.4K SNP Genotyping Array marker platform. A total of 3 QTL were mapped on chromosomes 6, 9 and 10, respectively. One major QTL on chromosome 10 (bin 10.00/10.01), RppCML496, was consistently detected across environments, which explained 43–78% of the total phenotypic variation. Using a fine mapping strategy, we delimited RppCML496 to an interval of 128 Kb. Genome mining of this region suggests two candidate genes, and a NBS‐LRR gene is the promising one for RppCML496 against SCR. The tightly linked molecular markers developed in this study can be used for molecular breeding of resistance to SCR in maize.

Chrysanthemum morifolium cv. ‘Huaihuang’ has ornamental, edible, medicinal, and tea product uses. However, its field growth, yield, and quality are negatively affected by black spot disease caused by Alternaria sp. (Strain: HQJH10092301; GenBank accession number: KF688111). In this study, we transcriptionally and transgenically characterized a new cultivar, ‘Huaiju 2#’ (Henan Traditional Chinese Medicine Plant Cultivar identification number: 2016002), which was bred from ‘Huaihuang’ and shows resistance to Alternaria sp. Numerous ‘Huaiju 2#’ plants were inoculated with Alternaria sp. for three or five days. Metabolic analysis showed increases in both salicylic acid (SA) and jasmonic acid (JA) in infected plants compared to the control. Protein activity analysis also revealed a significant increase in defense enzyme activities in infected plants. RNA-Seq of plants infected for 3 or 5 days produced a total of 58.6 GB of clean reads. Among these reads, 16,550 and 13,559 differentially expressed genes (DEGs) were identified in Cm_3 dpi (sample from 3 days post-inoculation labeled as Cm_3 dpi) and Cm_5 dpi (sample from 5 days post-inoculation labeled as Cm_5 dpi), respectively, compared with their controls (Cm_0 d: a mixture samples from 0 d (before inoculation) and those treated with sterile distilled water at 3 dpi and 5 dpi). Gene annotation and cluster analysis of the DEGs revealed a variety of defense responses to Alternaria sp. infection, which were characterized by increases in resistance (R) proteins and the reactive oxygen species (ROS), Ca2+, mitogen-activated protein kinase (MAPK), and JA signaling pathways. In particular, SA signaling was highly responsive to Alternaria sp. infection. The qPCR analysis of 12 DEG candidates supported their differential expression characterized by using the RNA-Seq data. One candidate was CmNPR1 (nonexpressor of pathogenesis-related gene 1), an important positive regulator of SA in systemic acquired resistance (SAR). Overexpression of CmNPR1 in ‘Huaiju 2#’ increased the resistance of transgenic plants to black spot. These findings indicate that the SA response pathway is likely involved in the defense of ‘Huaiju 2#’ against Alternaria sp. pathogens.