Xanthomonas oryzae pv.oryzae(Xoo)causes bacterial blight in rice,which reduces crop yield and leads to significant economic losses.Bacterial sigma(σ)factors are highly specialized proteins that allow RNA polymerase t...Xanthomonas oryzae pv.oryzae(Xoo)causes bacterial blight in rice,which reduces crop yield and leads to significant economic losses.Bacterial sigma(σ)factors are highly specialized proteins that allow RNA polymerase to recognize and bind to specific promoters.σ^(70) factors also regulate the expression of genes involved in stress response and virulence.However,the role of RpoD in Xoo is still unclear.In this study,we found thatσ^(70) factor RpoD is quite conservative among phytopathogenic bacteria,especially in Xanthomonas sp.In Xoo,PXO_RpoD plays an important role in oxidative stress tolerance and cell motility,as well as being essential for full virulence.Cleavage under targets and tagmentation(CUT&Tag)analyses indicated that RpoD mediates the type three secretion system(T3SS)by regulating the regulation of hrpG and hrpX.By performing bacterial one-hybrid and electrophoretic mobility assay(EMSA),we observed that RpoD directly bound to the promoters of hrpG and hrpX.Collectively,these results demonstrate the transcriptional mechanism and pathogenic functions of RpoD in regulating cell motility and oxidative stress response,providing novel insights into potential targets for disease control.展开更多
A nuclear-encoded sigma(σ) factor is essential for the transcriptional regulation of plant chloroplastencoded genes. Five putative maize r factors have been identified by database searches, but their functions are un...A nuclear-encoded sigma(σ) factor is essential for the transcriptional regulation of plant chloroplastencoded genes. Five putative maize r factors have been identified by database searches, but their functions are unknown. We report a maize leaf color mutant etiolated/albino leaf 1(eal1) that was derived from space mutation breeding. The eal1 mutant displays etiolated or albino leaves that then gradually turn to normal green at the seedling stage. The changes in eal1 leaf color are associated with changes in photosynthetic pigment content and chloroplast development. Map-based cloning revealed that a single amino-acid deletion changing Val_(480)-Val_(481)-Val_(482) to Val_(480)-Val_(481), in the C-terminal domain σ_(4) of the putative σ factor ZmSig2A, is responsible for the eal1 mutation. In comparison with the expression level of the wild-type(WT) allele ZmSig2A^(+) in WT plants, much higher expression of the mutant allele ZmSig2A^(⊿V) in eal1 plants was detected before the eal1 plants turned to normal green. ZmSig2A shows the highest similarity to rice OsSig2A and Arabidopsis SIG2. Ectopic expression of ZmSig2A^(+) or ZmSig2A^(⊿V) driven by the cauliflower mosaic virus 35 S promoter rescued the pale green leaf of the sig2 mutant, but ectopic expression of ZmSig2A^(⊿V) driven by the SIG2 promoter did not. We propose that the Val deletion generated a new weak allele of ZmSig2A that cannot completely abolish the ZmSig2A function. Some genes involved in chloroplast development and photosynthesis-associated nuclear genes showed significant expression differences between eal1 and WT plants. We conclude that ZmSig2A encoding a r factor is essential for maize chloroplast development. The eal1 mutant with a weak allele of ZmSig2A represents a valuable genetic resource for investigating the regulation of ZmSig2A-mediated chloroplast development in maize.The eal1 mutation may be useful as a marker for early identification and elimination of false hybrids or transgene transmission in the application of genetic male sterility to commercial hybrid seed production.展开更多
The application of the valuable natural product thaxtomin A,a potent bioherbicide from the potato scab pathogenic Streptomyces strains,has been greatly hindered by the low yields from its native producers.Here,we deve...The application of the valuable natural product thaxtomin A,a potent bioherbicide from the potato scab pathogenic Streptomyces strains,has been greatly hindered by the low yields from its native producers.Here,we developed an orthogonal transcription system,leveraging extra-cytoplasmic function(ECF)sigma(σ)factor 17(ECF17)and its cognate promoter Pecf17,to express the thaxtomin gene cluster and improve the production of thaxtomin A.The minimal Pecf17 promoter was determined,and a Pecf17 promoter library with a wide range of strengths was constructed.Furthermore,a cumate inducible system was developed for precise temporal control of the ECF17 transcription system in S.venezuelae ISP5230.Theoretically,the switchable ECF17 transcription system could reduce the unwanted influences from host and alleviate the burdens introduced by overexpression of heterologous genes.The yield of thaxtomin A was significantly improved to 202.1±15.3μg/mL using the switchable ECF17 transcription system for heterologous expression of the thaxtomin gene cluster in S.venezuelae ISP5230.Besides,the applicability of this transcription system was also tested in Streptomyces albus J1074,and the titer of thaxtomin A was raised to as high as 239.3±30.6μg/mL.Therefore,the inducible ECF17 transcription system could serve as a complement of the generally used transcription systems based on strong native constitutive promoters and housekeepingσfactors for the heterologous expression of valuable products in diverse Streptomyces hosts.展开更多
Listeria monocytogenes is an important zoonotic foodborne pathogen that can tolerate a number of environmental stresses. Rsb R, an upstream regulator of the sigma B(Sig B) factor, is thought to sense environmental cha...Listeria monocytogenes is an important zoonotic foodborne pathogen that can tolerate a number of environmental stresses. Rsb R, an upstream regulator of the sigma B(Sig B) factor, is thought to sense environmental challenges and trigger the SigB pathway. In Bacillus subtilis, two phosphorylation sites in RsbR are involved in activating the SigB pathway and a feedback mechanism, respectively. In this study, the role of RsbR in L. monocytogenes under mild and severe stresses was investigated. Strains with genetic deletion(Δrsb R), complementation(C-Δrsb R), and phosphorylation site mutations in the rsbR(RsbR-T175 A, RsbR-T209 A, and RsbR-T175 A-T209 A) were constructed to evaluate the roles of these RsbR sequences in listerial growth and survival. SigB was examined at the transcriptional and translational levels. Deletion of rsb R reduced listerial growxth and survival in response to acidic stress. Substitution of the phosphorylation residue RsbR-T175 A disabled RsbR complementation, while RsbR-T209 A significantly upregulated SigB expression and listerial survival. Our results provide clear evidence that two phosphorylation sites of Rsb R are functional in L. monocytogenes under acidic conditions, similar to the situation in B. subtilis.展开更多
基金supported by the National Natural Science Foundation of China(32072379,32001865 and 32202259)。
文摘Xanthomonas oryzae pv.oryzae(Xoo)causes bacterial blight in rice,which reduces crop yield and leads to significant economic losses.Bacterial sigma(σ)factors are highly specialized proteins that allow RNA polymerase to recognize and bind to specific promoters.σ^(70) factors also regulate the expression of genes involved in stress response and virulence.However,the role of RpoD in Xoo is still unclear.In this study,we found thatσ^(70) factor RpoD is quite conservative among phytopathogenic bacteria,especially in Xanthomonas sp.In Xoo,PXO_RpoD plays an important role in oxidative stress tolerance and cell motility,as well as being essential for full virulence.Cleavage under targets and tagmentation(CUT&Tag)analyses indicated that RpoD mediates the type three secretion system(T3SS)by regulating the regulation of hrpG and hrpX.By performing bacterial one-hybrid and electrophoretic mobility assay(EMSA),we observed that RpoD directly bound to the promoters of hrpG and hrpX.Collectively,these results demonstrate the transcriptional mechanism and pathogenic functions of RpoD in regulating cell motility and oxidative stress response,providing novel insights into potential targets for disease control.
基金supported by the National Key Research and Development Program of China (2016YFD0102104)Platform for Mutation Breeding by Radiation of Sichuan (2016NZ0106)Applied Basic Research Program of Sichuan Provincial Science and Technology Department (2020YJ0249)。
文摘A nuclear-encoded sigma(σ) factor is essential for the transcriptional regulation of plant chloroplastencoded genes. Five putative maize r factors have been identified by database searches, but their functions are unknown. We report a maize leaf color mutant etiolated/albino leaf 1(eal1) that was derived from space mutation breeding. The eal1 mutant displays etiolated or albino leaves that then gradually turn to normal green at the seedling stage. The changes in eal1 leaf color are associated with changes in photosynthetic pigment content and chloroplast development. Map-based cloning revealed that a single amino-acid deletion changing Val_(480)-Val_(481)-Val_(482) to Val_(480)-Val_(481), in the C-terminal domain σ_(4) of the putative σ factor ZmSig2A, is responsible for the eal1 mutation. In comparison with the expression level of the wild-type(WT) allele ZmSig2A^(+) in WT plants, much higher expression of the mutant allele ZmSig2A^(⊿V) in eal1 plants was detected before the eal1 plants turned to normal green. ZmSig2A shows the highest similarity to rice OsSig2A and Arabidopsis SIG2. Ectopic expression of ZmSig2A^(+) or ZmSig2A^(⊿V) driven by the cauliflower mosaic virus 35 S promoter rescued the pale green leaf of the sig2 mutant, but ectopic expression of ZmSig2A^(⊿V) driven by the SIG2 promoter did not. We propose that the Val deletion generated a new weak allele of ZmSig2A that cannot completely abolish the ZmSig2A function. Some genes involved in chloroplast development and photosynthesis-associated nuclear genes showed significant expression differences between eal1 and WT plants. We conclude that ZmSig2A encoding a r factor is essential for maize chloroplast development. The eal1 mutant with a weak allele of ZmSig2A represents a valuable genetic resource for investigating the regulation of ZmSig2A-mediated chloroplast development in maize.The eal1 mutation may be useful as a marker for early identification and elimination of false hybrids or transgene transmission in the application of genetic male sterility to commercial hybrid seed production.
基金supported by the National Key Research and Development Program of China[2018YFA0900700]Natural Science Foundation of China[31900901 and 31500069]+1 种基金the Chinese Academy of Sciences[No.QYZDB-SSW-SMC050,No.XDPB1801 of the Strategic Priority Research Program]the Shenzhen Science and Technology Innovation Committee[No.JCYJ20180507182241844,JCHZ20200005,DWKF20190009].
文摘The application of the valuable natural product thaxtomin A,a potent bioherbicide from the potato scab pathogenic Streptomyces strains,has been greatly hindered by the low yields from its native producers.Here,we developed an orthogonal transcription system,leveraging extra-cytoplasmic function(ECF)sigma(σ)factor 17(ECF17)and its cognate promoter Pecf17,to express the thaxtomin gene cluster and improve the production of thaxtomin A.The minimal Pecf17 promoter was determined,and a Pecf17 promoter library with a wide range of strengths was constructed.Furthermore,a cumate inducible system was developed for precise temporal control of the ECF17 transcription system in S.venezuelae ISP5230.Theoretically,the switchable ECF17 transcription system could reduce the unwanted influences from host and alleviate the burdens introduced by overexpression of heterologous genes.The yield of thaxtomin A was significantly improved to 202.1±15.3μg/mL using the switchable ECF17 transcription system for heterologous expression of the thaxtomin gene cluster in S.venezuelae ISP5230.Besides,the applicability of this transcription system was also tested in Streptomyces albus J1074,and the titer of thaxtomin A was raised to as high as 239.3±30.6μg/mL.Therefore,the inducible ECF17 transcription system could serve as a complement of the generally used transcription systems based on strong native constitutive promoters and housekeepingσfactors for the heterologous expression of valuable products in diverse Streptomyces hosts.
基金Project supported by the National Natural Science Foundation of China(Nos.31702250 and 31600292)the Open Fund Project by Key Laboratory of Animal Preventive Medicine of Zhejiang Province(No.ZPKLPVM2017KFKT001),China
文摘Listeria monocytogenes is an important zoonotic foodborne pathogen that can tolerate a number of environmental stresses. Rsb R, an upstream regulator of the sigma B(Sig B) factor, is thought to sense environmental challenges and trigger the SigB pathway. In Bacillus subtilis, two phosphorylation sites in RsbR are involved in activating the SigB pathway and a feedback mechanism, respectively. In this study, the role of RsbR in L. monocytogenes under mild and severe stresses was investigated. Strains with genetic deletion(Δrsb R), complementation(C-Δrsb R), and phosphorylation site mutations in the rsbR(RsbR-T175 A, RsbR-T209 A, and RsbR-T175 A-T209 A) were constructed to evaluate the roles of these RsbR sequences in listerial growth and survival. SigB was examined at the transcriptional and translational levels. Deletion of rsb R reduced listerial growxth and survival in response to acidic stress. Substitution of the phosphorylation residue RsbR-T175 A disabled RsbR complementation, while RsbR-T209 A significantly upregulated SigB expression and listerial survival. Our results provide clear evidence that two phosphorylation sites of Rsb R are functional in L. monocytogenes under acidic conditions, similar to the situation in B. subtilis.
