The Salmonella pathogenicity islands(SPIs) play crucial roles in the progression of Salmonella infection. In this study, we constructed an improved λ Red homologous recombination system to prepare single and triple d...The Salmonella pathogenicity islands(SPIs) play crucial roles in the progression of Salmonella infection. In this study, we constructed an improved λ Red homologous recombination system to prepare single and triple deletion mutants of 3 prominent SPIs(SPI-1, 2, and 3), aiming at the impact of deletion on morphology, carbon source metabolism, adhesion and invasion capacity, in vivo colonization, and immune efficacy in chicks. Our examination revealed that the surface of the single deletion mutants(SM6ΔSPI1, ΔSPI2, and ΔSPI3) exhibited a more rugged texture and appeared to be enveloped in a layer of transparent colloid, whereas the morphology of the triple deletion mutant(SM6ΔSPI1&2&3) remained unaltered when compared to the parent strain. The carbon metabolic spectrum of the SPI mutants underwent profound alterations, with a notable and statistically significant modification observed in 30 out of 95 carbon sources, primarily carbohydrates(17 out of 30). Furthermore, the adhesion capacity of the 4 mutants to Caco-2 cells was significantly reduced when compared to that of the parent strain. Moreover,the invasion capacity of mutants SM6ΔSPI1 and SM6ΔSPI1&2&3 exhibited a substantial decrease, while it was enhanced to varying degrees for SM6ΔSPI3 and SM6ΔSPI2. Importantly, none of the 4 mutants induced any clinical symptoms in the chicks. However, they did transiently colonize the spleen and liver. Notably, the SM6ΔSPI1&2&3mutant was rapidly cleared from both the spleen and liver within 8 days post-infection and no notable pathological changes were observed in the organs. Additionally, when challenged, the mutants immunized groups displayed a significant increase in antibody levels and alterations in the CD3+CD4+ and CD3+CD8+ subpopulations, and the levels of IL-4 and IFN-γ cytokines in the SM6ΔSPI1&2&3 immunized chicken serum surpassed those of other groups.In summary, the successful construction of the 4 SPI mutants lays the groundwork for further exploration into the pathogenic(including metabolic) mechanisms of SPIs and the development of safe and effective live attenuated Salmonella vaccines or carriers.展开更多
Transcription factors play critical roles in the regulation of gene expression during maize kernel development.The maize endosperm,a large storage organ,accounting for nearly 90%of the dry weight of mature kernels,ser...Transcription factors play critical roles in the regulation of gene expression during maize kernel development.The maize endosperm,a large storage organ,accounting for nearly 90%of the dry weight of mature kernels,serves as the primary site for starch storage.In this study,we identify an endosperm-specific EREB gene,ZmEREB167,which encodes a nucleus-localized EREB protein.Knockout of ZmEREB167 significantly increases kernel size and weight,as well as starch and protein content,compared with the wild type.In situ hybridization experiments show that ZmEREB167 is highly expressed in the BETL as well as PED regions of maize kernels.Dual-luciferase assays show that ZmEREB167 exhibits transcriptionally repressor activity in maize protoplasts.Transcriptome analysis reveals that a large number of genes are up-regulated in the Zmereb167-C1 mutant compared with the wild type,including key genetic factors such as ZmMRP-1 and ZmMN1,as well as multiple transporters involved in maize endosperm development.Integration of RNA-seq and ChIP-seq results identify 68 target genes modulated by ZmEREB167.We find that ZmEREB167 directly targets OPAQUE2,ZmNRT1.1,ZmIAA12,ZmIAA19,and ZmbZIP20,repressing their expressions.Our study demonstrates that ZmEREB167 functions as a negative regulator in maize endosperm development and affects starch accumulation and kernel size.展开更多
基金supported by the National KeyR&DProgramof China(2022YFF0710500)the National Natural Science Foundation of China(32172853 and 32373013)the Central Public-interest Scientific Institution Basal Research Fund,China(1610302022001).
文摘The Salmonella pathogenicity islands(SPIs) play crucial roles in the progression of Salmonella infection. In this study, we constructed an improved λ Red homologous recombination system to prepare single and triple deletion mutants of 3 prominent SPIs(SPI-1, 2, and 3), aiming at the impact of deletion on morphology, carbon source metabolism, adhesion and invasion capacity, in vivo colonization, and immune efficacy in chicks. Our examination revealed that the surface of the single deletion mutants(SM6ΔSPI1, ΔSPI2, and ΔSPI3) exhibited a more rugged texture and appeared to be enveloped in a layer of transparent colloid, whereas the morphology of the triple deletion mutant(SM6ΔSPI1&2&3) remained unaltered when compared to the parent strain. The carbon metabolic spectrum of the SPI mutants underwent profound alterations, with a notable and statistically significant modification observed in 30 out of 95 carbon sources, primarily carbohydrates(17 out of 30). Furthermore, the adhesion capacity of the 4 mutants to Caco-2 cells was significantly reduced when compared to that of the parent strain. Moreover,the invasion capacity of mutants SM6ΔSPI1 and SM6ΔSPI1&2&3 exhibited a substantial decrease, while it was enhanced to varying degrees for SM6ΔSPI3 and SM6ΔSPI2. Importantly, none of the 4 mutants induced any clinical symptoms in the chicks. However, they did transiently colonize the spleen and liver. Notably, the SM6ΔSPI1&2&3mutant was rapidly cleared from both the spleen and liver within 8 days post-infection and no notable pathological changes were observed in the organs. Additionally, when challenged, the mutants immunized groups displayed a significant increase in antibody levels and alterations in the CD3+CD4+ and CD3+CD8+ subpopulations, and the levels of IL-4 and IFN-γ cytokines in the SM6ΔSPI1&2&3 immunized chicken serum surpassed those of other groups.In summary, the successful construction of the 4 SPI mutants lays the groundwork for further exploration into the pathogenic(including metabolic) mechanisms of SPIs and the development of safe and effective live attenuated Salmonella vaccines or carriers.
基金supported by STI 2030-Major Project(2023ZD04069)National Key Research and Development Program of China(2023YFD1202900)+3 种基金The National Science Fund for Distinguished Young Scholars(32425041)The“Breakthrough”Science and Technology Project of Tongliao(TL2024TW001)Science and Technology Demonstration Project of Shandong Province(2024SFGC0402)Pinduoduo-China Agricultural University Research Fund(PC2023A01004).
文摘Transcription factors play critical roles in the regulation of gene expression during maize kernel development.The maize endosperm,a large storage organ,accounting for nearly 90%of the dry weight of mature kernels,serves as the primary site for starch storage.In this study,we identify an endosperm-specific EREB gene,ZmEREB167,which encodes a nucleus-localized EREB protein.Knockout of ZmEREB167 significantly increases kernel size and weight,as well as starch and protein content,compared with the wild type.In situ hybridization experiments show that ZmEREB167 is highly expressed in the BETL as well as PED regions of maize kernels.Dual-luciferase assays show that ZmEREB167 exhibits transcriptionally repressor activity in maize protoplasts.Transcriptome analysis reveals that a large number of genes are up-regulated in the Zmereb167-C1 mutant compared with the wild type,including key genetic factors such as ZmMRP-1 and ZmMN1,as well as multiple transporters involved in maize endosperm development.Integration of RNA-seq and ChIP-seq results identify 68 target genes modulated by ZmEREB167.We find that ZmEREB167 directly targets OPAQUE2,ZmNRT1.1,ZmIAA12,ZmIAA19,and ZmbZIP20,repressing their expressions.Our study demonstrates that ZmEREB167 functions as a negative regulator in maize endosperm development and affects starch accumulation and kernel size.
