Pseudomonas syringae pv.actinidiae(Psa)causes destructive kiwifruit bacterial canker by invading vascular tissues across multiple plant organs.However,the cellular mechanism underlying its systemic transmission and ce...Pseudomonas syringae pv.actinidiae(Psa)causes destructive kiwifruit bacterial canker by invading vascular tissues across multiple plant organs.However,the cellular mechanism underlying its systemic transmission and cell-to-cell movement within these specialized vascular conduits remains unclear.In this study,a Psa-GFP strain and various microscopic techniques were used to investigate the interaction between kiwifruit and Psa.Our results reveal that Psa strategically exploits host vascular conduits for systemic movement,with the xylem vessel being the predominant avenue.In the phloem,Psa exhibits adaptive alteration in bacterial shape to traverse sieve pores,facilitating its systemic spread along sieve tubes and inducing phloem necrosis.Within the xylem,Psa breaches pit membranes to migrate between adjacent vessels.Furthermore,phloem fibers act as an initial barrier at the early stages of infection,delaying Psa's entry into vascular tissues during its journey to the xylem.Additionally,at the junctions of stemestem or stem-leaf,branch trace or leaf trace mediates the bacterial organ-to-organ translocation,thus facilitating the systemic progression of disease.In conclusion,our findings shed light on the cellular mechanism employed by Psa to exploit the woody plant's vascular network for infection,thereby enhancing a better understanding of the biology of this poorly defined bacterium.These insights carry implications for the pathogenesis of Psa and other vascular pathogens,offering theoretical guidance for effective control strategies.展开更多
A series of photoinduced palladium-catalyzed 1,3-diene-selective fluoroalkylamination derivatives was rationally synthesized based on diversity-oriented synthesis via cross coupling of 1,3-dienes,amines and fluoroalky...A series of photoinduced palladium-catalyzed 1,3-diene-selective fluoroalkylamination derivatives was rationally synthesized based on diversity-oriented synthesis via cross coupling of 1,3-dienes,amines and fluoroalkyl iodides.The reaction featured good function group tolerance and a broad substrate scope,which could be extended to the late-stage modification of bioactive molecules.Bactericidal activity of all the compounds against Xanthomonas oryzae pv.oryzae(Xoo)was evaluated.Among them,compound E14 showed significant activity against Xanthomonas oryzae pv.oryzae(Xoo)with half maximal effective concentration(EC50)value of 6.61μmol/mL.In pot experiments,the results showed that E14 could control rice bacterial blight with protective and curative efficiencies of 37.5%and 63.2%at 200μg/mL,respectively.Additionally,a plausible mechanism for antibacterial behavior of E14 was proposed by electron microscopy,flow cytometry,reactive oxygen species detection,and biofilm assay.In current work,it can promote the development of photoinduced palladium-catalyzed 1,3-diene-selective fluoroalkyl amination compounds as prospective antibacterial agent bearing an intriguing mode of action.展开更多
The polymerase chain reaction(PCR) is particularly useful for plant pathogen detection. In the present study, multiplex PCR and SYBR Green real-time PCR were developed to facilitate the simultaneous detection of three...The polymerase chain reaction(PCR) is particularly useful for plant pathogen detection. In the present study, multiplex PCR and SYBR Green real-time PCR were developed to facilitate the simultaneous detection of three important rice pathogens, Xanthomonas oryzae pv.oryzae, X. oryzae pv. oryzicola, and Burkholderia glumae. The unique PCR primer sets were designed from portions of a putative glycosyltransferase gene of X. oryzae pv. oryzae, an Avr Rxo gene of X. oryzae pv. oryzicola, and an internal transcribed spacer(ITS) sequence of B. glumae. Using a multiplex PCR assay, X. oryzae pv. oryzae, X. oryzae pv. oryzicola, and B. glumae were detected in one PCR reaction that contained the newly developed primer set mix. Using SYBR Green real-time PCR assays, X. oryzae pv. oryzae, X. oryzae pv. oryzicola, and B. glumae were detected at 1, 1, and 10 fg μL-1, respectively. These newly designed molecular assays are sensitive and could be reliable tools for pathogen detection and disease forecasting.展开更多
hrp mutants were produced from strain JXOIII of Xanthomonas oryzae pv. oryzae (Xoo) and strain RS105 of X.o. pv. oryzicola (Xooc), respectively, by using diethyl sulfate (DES) as a mutagenic che ...hrp mutants were produced from strain JXOIII of Xanthomonas oryzae pv. oryzae (Xoo) and strain RS105 of X.o. pv. oryzicola (Xooc), respectively, by using diethyl sulfate (DES) as a mutagenic che mical. All the hrp mutants lost their pathogenicity on a susceptible host plant, rice (Shanyou63), and elicitation of the hypersensitive response (HR) on a nonhost plant, tobacco (NC89). Extracellular enzyme (amy lase, pectate lyase, proteinase, cellulase and lipase) activities of all the hrp mutants were similar to those of the corresponding wild type strains. The response of tobacco to cell sonicated integrations of the wild type strains and the hrp mutants demonstrated that there existed an HR eliciting substance which was heat stable and sensitive to protease. No HR appeared on tobacco after infiltration of the lipopolysaccharide (LPS) of both the wild strains and hrp mutants into tobacco leaves. The ability of the Xooc hrp mutants to induce HR on tobacco and cause streak disease on rice was restored by complementation with pUHRX245 from JXOIII genomic DNA library and by pUHRS138 from RS105 genomic DNA library, respectively. Subcloning of a 38.6 kb hrp fragment insert in pUHRX245 and a 39.3 kb insert in pUHRS138 revealed that a 3.3 kb Sac Ⅰ fragment from pUHRX245 and a 4.5 kb Bam HⅠ Kpn Ⅰ fragment from pUHRS138 were the minimal functional portions required for restoration of the ability of Xooc hrp mutants to induce HR on tobacco and cause disease on rice. The disease symptom caused by the conjugant (M1005 plus 3.3 kb) on rice was similar to that caused by the wild type of Xooc. It suggests that the two fragments contain the same hrp gene(s) and are responsible reciprocally for HR induction on tobacco and pathogenicity on rice.展开更多
Phenazines are secondary metabolites with broad spectrum antibiotic activity and thus show high potential in biological control of pathogens. In this study, we identified phenazine biosynthesis （phz） genes in two ge...Phenazines are secondary metabolites with broad spectrum antibiotic activity and thus show high potential in biological control of pathogens. In this study, we identified phenazine biosynthesis （phz） genes in two genome-completed plant pathogenic bacteria Pseudomonas syringae pv. tomato （Pst） DC3000 and Xanthomonas oryzae pv. oryzae （Xoo） PXO99A. Unlike the phz genes in typical phenazine-producing pseudomonads, phz homologs in Pst DC3000 and Xoo PXO99A consisted of phzC/D/E/F/G and phzC/E1/E2/F/G, respectively, and the both were not organized into an operon. Detection experiments demonstrated that phenazine-l-carboxylic acid （PCA） of Pst DC3000 accumulated to 13.4 IJg L-1, while that of Xoo PXO99A was almost undetectable. Moreover, Pst DC3000 was resistant to 1 mg mL-1 PCA, while Xoo PXO99A was sensitive to 50 IJg mL ~ PCA. Furthermore, mutation of phzF blocked the PCA production and significantly reduced the pathogenicity of Pst DC3000 in tomato, while the complementary strains restored these phenotypes. These results revealed that Pst DC3000 produces low level of and is resistant to phenazines and thus is unable to be biologically controlled by phenazines. Additionally, phz-mediated PCA production is required for full pathogenicity of Pst DC3000. To our knowledge, this is the first report of PCA production and its function in pathogenicity of a plant pathogenic P. syringae strain.展开更多
[ Objective ] The paper was to confirrm the effect of hrpZpsg12 gene on the pathogenicity of Pseudomonas syringae pv. glycinea. [ Method ] hrpZpsg12 gene was cloned from P. syringae using PCR method. The knockout plas...[ Objective ] The paper was to confirrm the effect of hrpZpsg12 gene on the pathogenicity of Pseudomonas syringae pv. glycinea. [ Method ] hrpZpsg12 gene was cloned from P. syringae using PCR method. The knockout plasmid pKNOCK-Cm with suicide characteristics and cosmid pUFR034 with complementation func- tion were used to construct the mutation vector pKNOCK477-7 and complementary vector pUFR1026-68 of hrpZpsg12 gene, the mutant 477-1 and the functional com- plementation unit 1026-5 of the gene was also screened out. Three strains including wild-type Psg12, mutant 477-1 and complementary unit 1026-5 were simultane- ously inoculated into soybean leaves and tobacco leaves, then pathogenicity determination and hypersensitive reaction analysis were carried out. [ Result] All the inoculated leaves of soybean and tobacco produced reaction lesion. However, the sizes of reaction lesion were different. The lesion in the leaves inoculated with Psgl2 was relatively large, while the lesion in the leaves inoculated with 477-1 was relatively small; the lesion of complementary unit 1026-5 was similar to wild- type Psgl2. Analysis of reproduction quantity of bacteria in lesions showed that the reproduction quantity of wild-type Psg12 was the highest, while that of mutant 477-1 was the lowest. The reproduction quantity of complementary unit 1026-5 was similar to that of wild-type Psg12. [ Conclusion] hrpZpsg12 gene could enhance the pathogenicity of P. syrimgae on Soybean and produce hypersensitive response in tobacco.展开更多
The paper aimed to establish a duplex PCR method for simultaneous detection of Pseudomonas savastanoi pv. Phaseolicola （Psp） and Curtobaeterium /accumfadens pv. Flaccumfaciens （Cff）. Based on the argK gene of Psp ...The paper aimed to establish a duplex PCR method for simultaneous detection of Pseudomonas savastanoi pv. Phaseolicola （Psp） and Curtobaeterium /accumfadens pv. Flaccumfaciens （Cff）. Based on the argK gene of Psp in GenBank, the primers PSPF1/PSPR2 were designed. The duplex PCR assay was dereloped using the combined primers PSPF1/PSPR2 and CflF1/CffR2, which were specific primers for Cff. The reaction conditions were optimized and specificity md sensitivity of the duplex PCR were tested. The expected DNA fragment was specifically amplified from the genomic DNA of Psp and Cff. Specificity was conirmed in the artificially inoculated soybean samples imparted. Thus, the duplex PCR developed in this study could be used for the simultaneous detection of Psp md Cff from imported soybean.展开更多
Xanthomonas oryzae pv.oryzicola （Xoc）,the critical pathogen causing bacterial leaf streak in rice,possesses a hrp cluster that is responsible for triggering hypersensitive response （HR） in non-host tobacco and pat...Xanthomonas oryzae pv.oryzicola （Xoc）,the critical pathogen causing bacterial leaf streak in rice,possesses a hrp cluster that is responsible for triggering hypersensitive response （HR） in non-host tobacco and pathogenicity in host rice,and is considered to be one of the model pathogens in the rice model plant.Here,we developed a high-throughput mutagenesis system using a two-step integration mediated by a novel suicide vector pKMS1.It was used to generate single or poly-gene mutants of hpa1,hpa2,hrcV,hrpE,hpaB,and hrpF gene for functional analysis.In total,five single,four double,and two triple hrp gene mutants were constructed.The double and triple hrp gene deletion mutants triggered novel phenotypes in planta.Our data suggest that pKMS1 is a useful tool for non-marker mutagenesis of multiple genes in Xoc.展开更多
基金supported by grants from the National Key Research and Development Program of China(Grant No.2022YFD1400200)the Special Support Plan for High-Level Talent of Shaanxi Provincethe First-Class Universities and Academic Programs of Northwest A&F University.
文摘Pseudomonas syringae pv.actinidiae(Psa)causes destructive kiwifruit bacterial canker by invading vascular tissues across multiple plant organs.However,the cellular mechanism underlying its systemic transmission and cell-to-cell movement within these specialized vascular conduits remains unclear.In this study,a Psa-GFP strain and various microscopic techniques were used to investigate the interaction between kiwifruit and Psa.Our results reveal that Psa strategically exploits host vascular conduits for systemic movement,with the xylem vessel being the predominant avenue.In the phloem,Psa exhibits adaptive alteration in bacterial shape to traverse sieve pores,facilitating its systemic spread along sieve tubes and inducing phloem necrosis.Within the xylem,Psa breaches pit membranes to migrate between adjacent vessels.Furthermore,phloem fibers act as an initial barrier at the early stages of infection,delaying Psa's entry into vascular tissues during its journey to the xylem.Additionally,at the junctions of stemestem or stem-leaf,branch trace or leaf trace mediates the bacterial organ-to-organ translocation,thus facilitating the systemic progression of disease.In conclusion,our findings shed light on the cellular mechanism employed by Psa to exploit the woody plant's vascular network for infection,thereby enhancing a better understanding of the biology of this poorly defined bacterium.These insights carry implications for the pathogenesis of Psa and other vascular pathogens,offering theoretical guidance for effective control strategies.
基金the National Natural Science Foundation of China(No.32072450)the National Science Fund for Distinguished Young Scholars of Guangdong Province(No.2021B1515020107)the International Science and Technology Cooperation Program in Guangdong(Nos.2020A0505100048 and 2022A0505050060).
文摘A series of photoinduced palladium-catalyzed 1,3-diene-selective fluoroalkylamination derivatives was rationally synthesized based on diversity-oriented synthesis via cross coupling of 1,3-dienes,amines and fluoroalkyl iodides.The reaction featured good function group tolerance and a broad substrate scope,which could be extended to the late-stage modification of bioactive molecules.Bactericidal activity of all the compounds against Xanthomonas oryzae pv.oryzae(Xoo)was evaluated.Among them,compound E14 showed significant activity against Xanthomonas oryzae pv.oryzae(Xoo)with half maximal effective concentration(EC50)value of 6.61μmol/mL.In pot experiments,the results showed that E14 could control rice bacterial blight with protective and curative efficiencies of 37.5%and 63.2%at 200μg/mL,respectively.Additionally,a plausible mechanism for antibacterial behavior of E14 was proposed by electron microscopy,flow cytometry,reactive oxygen species detection,and biofilm assay.In current work,it can promote the development of photoinduced palladium-catalyzed 1,3-diene-selective fluoroalkyl amination compounds as prospective antibacterial agent bearing an intriguing mode of action.
基金support of the National 863 Project (2012AA021601)the New Seedling program for graduate students of Zhejiang Province (2012R409012)
文摘The polymerase chain reaction(PCR) is particularly useful for plant pathogen detection. In the present study, multiplex PCR and SYBR Green real-time PCR were developed to facilitate the simultaneous detection of three important rice pathogens, Xanthomonas oryzae pv.oryzae, X. oryzae pv. oryzicola, and Burkholderia glumae. The unique PCR primer sets were designed from portions of a putative glycosyltransferase gene of X. oryzae pv. oryzae, an Avr Rxo gene of X. oryzae pv. oryzicola, and an internal transcribed spacer(ITS) sequence of B. glumae. Using a multiplex PCR assay, X. oryzae pv. oryzae, X. oryzae pv. oryzicola, and B. glumae were detected in one PCR reaction that contained the newly developed primer set mix. Using SYBR Green real-time PCR assays, X. oryzae pv. oryzae, X. oryzae pv. oryzicola, and B. glumae were detected at 1, 1, and 10 fg μL-1, respectively. These newly designed molecular assays are sensitive and could be reliable tools for pathogen detection and disease forecasting.
文摘hrp mutants were produced from strain JXOIII of Xanthomonas oryzae pv. oryzae (Xoo) and strain RS105 of X.o. pv. oryzicola (Xooc), respectively, by using diethyl sulfate (DES) as a mutagenic che mical. All the hrp mutants lost their pathogenicity on a susceptible host plant, rice (Shanyou63), and elicitation of the hypersensitive response (HR) on a nonhost plant, tobacco (NC89). Extracellular enzyme (amy lase, pectate lyase, proteinase, cellulase and lipase) activities of all the hrp mutants were similar to those of the corresponding wild type strains. The response of tobacco to cell sonicated integrations of the wild type strains and the hrp mutants demonstrated that there existed an HR eliciting substance which was heat stable and sensitive to protease. No HR appeared on tobacco after infiltration of the lipopolysaccharide (LPS) of both the wild strains and hrp mutants into tobacco leaves. The ability of the Xooc hrp mutants to induce HR on tobacco and cause streak disease on rice was restored by complementation with pUHRX245 from JXOIII genomic DNA library and by pUHRS138 from RS105 genomic DNA library, respectively. Subcloning of a 38.6 kb hrp fragment insert in pUHRX245 and a 39.3 kb insert in pUHRS138 revealed that a 3.3 kb Sac Ⅰ fragment from pUHRX245 and a 4.5 kb Bam HⅠ Kpn Ⅰ fragment from pUHRS138 were the minimal functional portions required for restoration of the ability of Xooc hrp mutants to induce HR on tobacco and cause disease on rice. The disease symptom caused by the conjugant (M1005 plus 3.3 kb) on rice was similar to that caused by the wild type of Xooc. It suggests that the two fragments contain the same hrp gene(s) and are responsible reciprocally for HR induction on tobacco and pathogenicity on rice.
基金supported by the grants from the Genetically Modified Organisms Breeding Major Projects, China (2014ZX0800905B)the Fundamental Research Funds for the Central Universities, Chinathe Program for New Century 151 Talents of Zhejiang Province, China
文摘Phenazines are secondary metabolites with broad spectrum antibiotic activity and thus show high potential in biological control of pathogens. In this study, we identified phenazine biosynthesis （phz） genes in two genome-completed plant pathogenic bacteria Pseudomonas syringae pv. tomato （Pst） DC3000 and Xanthomonas oryzae pv. oryzae （Xoo） PXO99A. Unlike the phz genes in typical phenazine-producing pseudomonads, phz homologs in Pst DC3000 and Xoo PXO99A consisted of phzC/D/E/F/G and phzC/E1/E2/F/G, respectively, and the both were not organized into an operon. Detection experiments demonstrated that phenazine-l-carboxylic acid （PCA） of Pst DC3000 accumulated to 13.4 IJg L-1, while that of Xoo PXO99A was almost undetectable. Moreover, Pst DC3000 was resistant to 1 mg mL-1 PCA, while Xoo PXO99A was sensitive to 50 IJg mL ~ PCA. Furthermore, mutation of phzF blocked the PCA production and significantly reduced the pathogenicity of Pst DC3000 in tomato, while the complementary strains restored these phenotypes. These results revealed that Pst DC3000 produces low level of and is resistant to phenazines and thus is unable to be biologically controlled by phenazines. Additionally, phz-mediated PCA production is required for full pathogenicity of Pst DC3000. To our knowledge, this is the first report of PCA production and its function in pathogenicity of a plant pathogenic P. syringae strain.
基金Supported by Scientific Research Foundation Project of Jilin Agricultural University" hrpZ Psg12 Protein Function of Pseudomonas syringae pv.glycinea" (384)Major Project of Cultivation of Genetically Modified Biological New Varieties of "Eleventh Five-Year Plan" of Ministry of Agriculture"Cultivation of New Transgenic Varieties of Soybean with Diseases and Pests Resistance"(2008ZX08004-004)~~
文摘[ Objective ] The paper was to confirrm the effect of hrpZpsg12 gene on the pathogenicity of Pseudomonas syringae pv. glycinea. [ Method ] hrpZpsg12 gene was cloned from P. syringae using PCR method. The knockout plasmid pKNOCK-Cm with suicide characteristics and cosmid pUFR034 with complementation func- tion were used to construct the mutation vector pKNOCK477-7 and complementary vector pUFR1026-68 of hrpZpsg12 gene, the mutant 477-1 and the functional com- plementation unit 1026-5 of the gene was also screened out. Three strains including wild-type Psg12, mutant 477-1 and complementary unit 1026-5 were simultane- ously inoculated into soybean leaves and tobacco leaves, then pathogenicity determination and hypersensitive reaction analysis were carried out. [ Result] All the inoculated leaves of soybean and tobacco produced reaction lesion. However, the sizes of reaction lesion were different. The lesion in the leaves inoculated with Psgl2 was relatively large, while the lesion in the leaves inoculated with 477-1 was relatively small; the lesion of complementary unit 1026-5 was similar to wild- type Psgl2. Analysis of reproduction quantity of bacteria in lesions showed that the reproduction quantity of wild-type Psg12 was the highest, while that of mutant 477-1 was the lowest. The reproduction quantity of complementary unit 1026-5 was similar to that of wild-type Psg12. [ Conclusion] hrpZpsg12 gene could enhance the pathogenicity of P. syrimgae on Soybean and produce hypersensitive response in tobacco.
基金Supported by Science and Technology Project of AQSIQ(2013IK277)National Rice Industry System Development Program
文摘The paper aimed to establish a duplex PCR method for simultaneous detection of Pseudomonas savastanoi pv. Phaseolicola （Psp） and Curtobaeterium /accumfadens pv. Flaccumfaciens （Cff）. Based on the argK gene of Psp in GenBank, the primers PSPF1/PSPR2 were designed. The duplex PCR assay was dereloped using the combined primers PSPF1/PSPR2 and CflF1/CffR2, which were specific primers for Cff. The reaction conditions were optimized and specificity md sensitivity of the duplex PCR were tested. The expected DNA fragment was specifically amplified from the genomic DNA of Psp and Cff. Specificity was conirmed in the artificially inoculated soybean samples imparted. Thus, the duplex PCR developed in this study could be used for the simultaneous detection of Psp md Cff from imported soybean.
基金supported by the National Natural Science Foundation of China (30710103902,31071656)the Ph D Programs Foundation of Ministry of Education of China (20100073110045)
文摘Xanthomonas oryzae pv.oryzicola （Xoc）,the critical pathogen causing bacterial leaf streak in rice,possesses a hrp cluster that is responsible for triggering hypersensitive response （HR） in non-host tobacco and pathogenicity in host rice,and is considered to be one of the model pathogens in the rice model plant.Here,we developed a high-throughput mutagenesis system using a two-step integration mediated by a novel suicide vector pKMS1.It was used to generate single or poly-gene mutants of hpa1,hpa2,hrcV,hrpE,hpaB,and hrpF gene for functional analysis.In total,five single,four double,and two triple hrp gene mutants were constructed.The double and triple hrp gene deletion mutants triggered novel phenotypes in planta.Our data suggest that pKMS1 is a useful tool for non-marker mutagenesis of multiple genes in Xoc.
