Magnaporthe oryzae is the causal agent of rice blast. Glycosylation plays key roles in vegetative growth,development, and infection of M. oryzae. However, several glycosylation-related genes have not been characterize...Magnaporthe oryzae is the causal agent of rice blast. Glycosylation plays key roles in vegetative growth,development, and infection of M. oryzae. However, several glycosylation-related genes have not been characterized.In this study, we identified a Glyco_transf_22 domain-containing protein, MoAlg9, and found that MoAlg9 islocalized to the endoplasmic reticulum(ER). Deletion of MoALG9 significantly affected conidial production, normalappressorium formation, responses to stressors, and pathogenicity of M. oryzae. We also found that the ΔMoalg9mutant was defective in glycogen utilization, appressorial penetration, and invasive growth in host cells. Moreover,we further demonstrated that MoALG9 regulates the transcription of several target genes involved in conidiation,appressorium formation, and cell wall integrity. In addition, we found that the Glyco_transf_22 domain is essentialfor normal MoAlg9 function and localization. We also provide evidence that MoAlg9 is involved in N-glycosylationpathway in M. oryzae. Taken together, these results show that MoAlg9 is important for conidiation, appressoriumformation, maintenance of cell wall integrity, and the pathogenesis of M. oryzae.展开更多
Rice blast,caused by Magnaporthe oryzae,is a fungal disease that causes devastating damage to rice production worldwide.During infection,pathogens secrete effector proteins that modulate plant immunity.Disulfide bond ...Rice blast,caused by Magnaporthe oryzae,is a fungal disease that causes devastating damage to rice production worldwide.During infection,pathogens secrete effector proteins that modulate plant immunity.Disulfide bond formation catalyzed by protein disulfide isomerases(PDI)is essential for protein folding and maturation.However,the biological function of Pdi1 in M.oryzae has not yet been characterized.In this study,we identified the endoplasmic reticulum(ER)-located protein,MoPdi1,in M.oryzae.MoPdi1 regulates conidiation,cell wall stress,and pathogenicity of M.oryzae.Furthermore,the CGHC active sites in the a and a'redox domain of MoPdi1 were essential for the biological function of MoPDI1.Further tests demonstrated that MoPdi1 was involved in the regulation of ER stress and positively regulated ER phagy.We also found that MoPdi1 interacted with MoHut1.Deletion of MoPDI1 led to the bereft of MoHut1 dimerization,which depends on the formation of disulfide bonds.In addition,MoPdi1 affected the normal secretion of the cytoplasmic effector AVR-Pia.We provided evidence that MoHut1 is important for the vegetative growth,conidiation,and pathogenicity in M.oryzae.Therefore,our findings could provide a suitable target point for designing antifungal agrochemicals against rice blast fungus.展开更多
Oxidation of self-stored carbohydrates and lipids provides the energy for the rapid morphogenetic transformation during asexual and infection-related development in Pyricularia oryzae,which results in intracellular ac...Oxidation of self-stored carbohydrates and lipids provides the energy for the rapid morphogenetic transformation during asexual and infection-related development in Pyricularia oryzae,which results in intracellular accumulation of reducing equivalents NADH and FADH_(2),requiring a cytosolic shuttling machinery towards mitochondria.Our previous studies identified the mitochondrial D-lactate dehydrogenase MoDld1 as a regulator to channel the metabolite flow in conjunction with redox homeostasis.However,the regulator(s)facilitating the cytosolic redox balance and the importance in propelling nutrient metabolite flow remain unknown.The G-3-P shuttle is a conserved machinery transporting the cytosolic reducing power to mitochondria.In P.oryzae,the mitochondrial G-3-P dehydrogenase Gpd2 was required for cellular NAD^(+)/NADH balance and fungal virulence.In this study,we relocate the mitochondrial G-3-P dehydrogenase Gpd2 to the cytosol for disturbing cytosolic redox status.Our results showed overexpression of cytosolic gpd2^(Δmts)without the mitochondrial targeted signal(MTS)driven by Ribosomal protein 27 promoter(PR27)exerted conflicting regulation of cellular oxidoreductase activities compared to theΔModld1 deletion mutant by RNA-seq and prevented the conidiation and pathogenicity of P.oryzae.Moreover,overexpression of gpd2^(Δmts)caused defects in glycogen and lipid mobilization underlying asexual and infectious structural development associated with decreased cellular NADH production and weakened anti-oxidation activities.RNA-seq and non-targeted metabolic profiling revealed down-regulated transcriptional activities of carbohydrate metabolism and lower abundance of fatty acids and secondary metabolites in RP27:gpd2^(Δmts).Thus,our studies indicate the essential role of cytosolic redox control in nutrient metabolism fueling the asexual and infection-related development in P.oryzae.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
[Objective] The research aimed to isolate the glyphosate-degraded strain and study its degradation characteristics.[Method] A glyphosate-degraded fungal strain A-F02 was isolated from sludge in an aeration tank of a g...[Objective] The research aimed to isolate the glyphosate-degraded strain and study its degradation characteristics.[Method] A glyphosate-degraded fungal strain A-F02 was isolated from sludge in an aeration tank of a glyphosate manufacture.The fungal strain A-F02 was identified according to morphological characteristics and internal transcribed spacer（ITS）region of nuclear ribosomal DNA sequence analysis.The glyphosate-biodegraded characteristics of strain A-F02 and the influencing factors were studied.[Result] The fungal strain A-F02 was identified as Aspergillus oryzae sp..The glyphosate-biodegraded rate was 86.82% in the mineral salt medium with 1 000 mg/L of glyphosate as the sole source of carbon,after being incubated at 30 ℃ and 150 rpm for 7 d.The biodegradation rates and biomass of the A-F02 were the highest under the culture conditions with glucose（0.5%,w/v）,pH 7.5,30 ℃ and glyphosate（1 500 mg/L）.[Conclusion] The research provided the experimental basis for glyphosate-biodegraded enzyme purification.展开更多
[ Objective] This study was to breed rice cultivars with multi-resistance to Orseolia oryzae （Wood-Mason）. [ Method] The Guangxi local cultivar GX-M001 （Jiangchao） with high resistance to Orseolia oryzae （Wood-Ma...[ Objective] This study was to breed rice cultivars with multi-resistance to Orseolia oryzae （Wood-Mason）. [ Method] The Guangxi local cultivar GX-M001 （Jiangchao） with high resistance to Orseolia oryzae （Wood-Mason） was used to hybrid with the known resistance cultivars ＂Kangwenqingzhan＂ （harboring GM5 gene）, OB677（ harboring GM3 gene） from Sri Lanka, HT1350 and high yield end quality cultivar ＂ Guiruanzhan＂. [ Result] Through pyramiding the multi-resistant genes via routine hybridization, the general resistances of the hybrids were remarkably enhanced. The grades of resistance were also improved, many of the combinations were endowed with a resistance at immune level （grade 0） ; and interestingly, the respective hybridization of GX-M001 （high resistance） with OB677（ medium resistance） and HT1350（suscepti- ble） also generate two lines at immune level, which is probably the effects of additive effects of genes.[ Conclusion] By routine hybridization, multiple genes were successfully pyramided, thus generating novel rice lines with multiple resistances. For the rice breeding scientists at the grass-roots level, the resistance-resistance pyramiding is an effective approach to breed high resistance cultivars.展开更多
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
Bacterial blight, caused by Xanthomonas oryzae pv. oryzae(Xoo), is one of the most destructive diseases of rice(Oryza sativa L.) worldwide. The type III secretion system(T3SS) of Xoo, encoded by the hrp(hypersensitive...Bacterial blight, caused by Xanthomonas oryzae pv. oryzae(Xoo), is one of the most destructive diseases of rice(Oryza sativa L.) worldwide. The type III secretion system(T3SS) of Xoo, encoded by the hrp(hypersensitive response and pathogenicity) genes, plays critical roles in conferring pathogenicity in host rice and triggering a hypersensitive response(HR) in non-host plants. To investigate the major genes conferring the pathogenicity and avirulence of Xoo, we previously constructed a random Tn5-insertion mutant library of Xoo strain PXO99A. We report here the isolation and characterization of a Tn5-insertion mutant PXM69. Tn5-insertion mutants were screened on indica rice JG30, which is highly susceptible to PXO99A, by leaf-cutting inoculation.Four mutants with reduced virulence were obtained after two rounds of screening. Among them, the mutant PXM69 had completely lost virulence to the rice host and ability to elicit HR in non-host tobacco. Southern blotting analysis showed a single copy of a Tn5-insertion in the genome of PXM69. PCR walking and sequencing analysis revealed that the Tn5 transposon was inserted at nucleotide position 70,192–70,201 in the genome of PXO99A, disrupting the type III hrc(hrp-conserved) gene hrcQ, the first gene in the D operon of the hrp cluster in Xoo. To confirm the relationship between the Tn5-insertion and the avirulence phenotype of PXM69, we used the marker exchange mutagenesis to create a PXO99Amutant, ΔhrcQ::KAN, in which the hrcQ was disrupted by a kanamycin-encoding gene cassette at the same site as that of the Tn5-insertion. ΔhrcQ::KAN showed the same phenotype as mutant PXM69. Reintroduction of the wild-type hrcQ gene partially complemented the pathogenic function of PXM69. RT-PCR and cellulase secretion assays showed that the Tn5-disruption of hrcQ did not affect transcription of downstream genes in the D operon and function of the type II secretion system. Our results provide new insights into the pathogenic functions of clustered hrp genes in Xoo.展开更多
Xanthomonas oryzea pv.oryzae(Xoo)is the causal agent of bacterial blight of rice,which is a significant threat to many of rice-growing regions.The type Ⅲ secretion system(T3SS)is an essential virulence factor in Xoo....Xanthomonas oryzea pv.oryzae(Xoo)is the causal agent of bacterial blight of rice,which is a significant threat to many of rice-growing regions.The type Ⅲ secretion system(T3SS)is an essential virulence factor in Xoo.Expression of the T3SS is often induced in the host environment or in hrp-inducing medium but is repressed in nutrient-rich medium.The elucidation of molecular mechanism underlying induction of T3SS genes expression is a very important step to lift the veil on global virulence regulation network in Xoo.Thus,an efficient and reliable genetic tool system is required for detection of the T3SS proteins.In this study,we constructed a protein expression vector pH3-flag based on the backbone of pHM1,a most widely used vector in Xoo strains,especially a model strain PXO99A.This vector contains a synthesized MCS-FLAG cassette that consists of a multiple cloning site(MCS),containing a modified pUC18 polylinker,and Flag as a C-terminal tag.The cassette is flanked by transcriptional terminators to eliminate interference of external transcription enabling detection of accurate protein expression.We evaluated the potential of this expression vector as T3SS proteins detection system and demonstrated it is applicable in the study of T3SS genes expression regulation in Xoo.This improved expression system could be very effectively used as a molecular tool in understanding some virulence genes expression and regulation in Xoo and other Xanthomonas spp.展开更多
[Objective] To explore the efficient way for the preparation of Aspergillus oryzae protoplast.[Method] The statistics of protoplast yield by the enzymolysis of mycelia with different culture time,different osmotic sta...[Objective] To explore the efficient way for the preparation of Aspergillus oryzae protoplast.[Method] The statistics of protoplast yield by the enzymolysis of mycelia with different culture time,different osmotic stabilizers and different concentrations of enzyme system was carried out in this study.[Result] The protoplast yield was higher when the mycelium was cultivated for 108 h,and the osmotic stabilizer was 0.8 mol/L of NaCl.With the enzymes system of 2 mg/ml of lysozyme,6 mg/ml of cellulose and 6 mg/ml of glusulase,the protoplast yield achieved its highest,which was up to 9.0×105 per ml.[Conclusion] The yield of protoplast was affected by the situation of mycelia themselves and external conditions.This study had provided a basis for the preparation of a large number of active protoplasts and the further researches on cell fusion.展开更多
Single-spore isolates were obtained from rice-growing fields of Yuan＇an in Hubei Province where rice blast seriously occurs in some years. DNA fingerprints were divided into 112 haplotypes and 14 lineages at 73% gene...Single-spore isolates were obtained from rice-growing fields of Yuan＇an in Hubei Province where rice blast seriously occurs in some years. DNA fingerprints were divided into 112 haplotypes and 14 lineages at 73% genetic similarity level. Among the lineages, no dominant lineages were found. The population genetic structures of Magnaporthe oryzae were not distinctly different in different years. The analysis also showed that there wasn＇t obvious simple relationship between patho- types and fingerprint groups.展开更多
基金supported by the National Natural Science Foundation of China (32202253)the Natural Science Foundation of Anhui Higher Education Institutions, China (KJ2020A0102)the Talent Research Project of Anhui Agricultural University, China (rc342001)。
文摘Magnaporthe oryzae is the causal agent of rice blast. Glycosylation plays key roles in vegetative growth,development, and infection of M. oryzae. However, several glycosylation-related genes have not been characterized.In this study, we identified a Glyco_transf_22 domain-containing protein, MoAlg9, and found that MoAlg9 islocalized to the endoplasmic reticulum(ER). Deletion of MoALG9 significantly affected conidial production, normalappressorium formation, responses to stressors, and pathogenicity of M. oryzae. We also found that the ΔMoalg9mutant was defective in glycogen utilization, appressorial penetration, and invasive growth in host cells. Moreover,we further demonstrated that MoALG9 regulates the transcription of several target genes involved in conidiation,appressorium formation, and cell wall integrity. In addition, we found that the Glyco_transf_22 domain is essentialfor normal MoAlg9 function and localization. We also provide evidence that MoAlg9 is involved in N-glycosylationpathway in M. oryzae. Taken together, these results show that MoAlg9 is important for conidiation, appressoriumformation, maintenance of cell wall integrity, and the pathogenesis of M. oryzae.
基金supported by the National Natural Science Foundation of China(32202253)the Natural Science Foundation of Anhui Higher Education Institutions,China(KJ2020A0102)the Talent Research Project of Anhui Agricultural University,China(rc342001)。
文摘Rice blast,caused by Magnaporthe oryzae,is a fungal disease that causes devastating damage to rice production worldwide.During infection,pathogens secrete effector proteins that modulate plant immunity.Disulfide bond formation catalyzed by protein disulfide isomerases(PDI)is essential for protein folding and maturation.However,the biological function of Pdi1 in M.oryzae has not yet been characterized.In this study,we identified the endoplasmic reticulum(ER)-located protein,MoPdi1,in M.oryzae.MoPdi1 regulates conidiation,cell wall stress,and pathogenicity of M.oryzae.Furthermore,the CGHC active sites in the a and a'redox domain of MoPdi1 were essential for the biological function of MoPDI1.Further tests demonstrated that MoPdi1 was involved in the regulation of ER stress and positively regulated ER phagy.We also found that MoPdi1 interacted with MoHut1.Deletion of MoPDI1 led to the bereft of MoHut1 dimerization,which depends on the formation of disulfide bonds.In addition,MoPdi1 affected the normal secretion of the cytoplasmic effector AVR-Pia.We provided evidence that MoHut1 is important for the vegetative growth,conidiation,and pathogenicity in M.oryzae.Therefore,our findings could provide a suitable target point for designing antifungal agrochemicals against rice blast fungus.
基金funded by the National Natural Science Foundation of China(32272513 and 31770156)the Natural Sciences and Engineering Research Council of Canada(Discovery Grant,RGPIN-2016-05356)the Canadian Foundation for Innovation(Discovery Grant,227398-2011)。
文摘Oxidation of self-stored carbohydrates and lipids provides the energy for the rapid morphogenetic transformation during asexual and infection-related development in Pyricularia oryzae,which results in intracellular accumulation of reducing equivalents NADH and FADH_(2),requiring a cytosolic shuttling machinery towards mitochondria.Our previous studies identified the mitochondrial D-lactate dehydrogenase MoDld1 as a regulator to channel the metabolite flow in conjunction with redox homeostasis.However,the regulator(s)facilitating the cytosolic redox balance and the importance in propelling nutrient metabolite flow remain unknown.The G-3-P shuttle is a conserved machinery transporting the cytosolic reducing power to mitochondria.In P.oryzae,the mitochondrial G-3-P dehydrogenase Gpd2 was required for cellular NAD^(+)/NADH balance and fungal virulence.In this study,we relocate the mitochondrial G-3-P dehydrogenase Gpd2 to the cytosol for disturbing cytosolic redox status.Our results showed overexpression of cytosolic gpd2^(Δmts)without the mitochondrial targeted signal(MTS)driven by Ribosomal protein 27 promoter(PR27)exerted conflicting regulation of cellular oxidoreductase activities compared to theΔModld1 deletion mutant by RNA-seq and prevented the conidiation and pathogenicity of P.oryzae.Moreover,overexpression of gpd2^(Δmts)caused defects in glycogen and lipid mobilization underlying asexual and infectious structural development associated with decreased cellular NADH production and weakened anti-oxidation activities.RNA-seq and non-targeted metabolic profiling revealed down-regulated transcriptional activities of carbohydrate metabolism and lower abundance of fatty acids and secondary metabolites in RP27:gpd2^(Δmts).Thus,our studies indicate the essential role of cytosolic redox control in nutrient metabolism fueling the asexual and infection-related development in P.oryzae.
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[Objective] The research aimed to isolate the glyphosate-degraded strain and study its degradation characteristics.[Method] A glyphosate-degraded fungal strain A-F02 was isolated from sludge in an aeration tank of a glyphosate manufacture.The fungal strain A-F02 was identified according to morphological characteristics and internal transcribed spacer（ITS）region of nuclear ribosomal DNA sequence analysis.The glyphosate-biodegraded characteristics of strain A-F02 and the influencing factors were studied.[Result] The fungal strain A-F02 was identified as Aspergillus oryzae sp..The glyphosate-biodegraded rate was 86.82% in the mineral salt medium with 1 000 mg/L of glyphosate as the sole source of carbon,after being incubated at 30 ℃ and 150 rpm for 7 d.The biodegradation rates and biomass of the A-F02 were the highest under the culture conditions with glucose（0.5%,w/v）,pH 7.5,30 ℃ and glyphosate（1 500 mg/L）.[Conclusion] The research provided the experimental basis for glyphosate-biodegraded enzyme purification.
基金Supported by National Natural Science Foundation of China(30760117)National Key Technology R &D Program (2007BAD68B01)~~
文摘[ Objective] This study was to breed rice cultivars with multi-resistance to Orseolia oryzae （Wood-Mason）. [ Method] The Guangxi local cultivar GX-M001 （Jiangchao） with high resistance to Orseolia oryzae （Wood-Mason） was used to hybrid with the known resistance cultivars ＂Kangwenqingzhan＂ （harboring GM5 gene）, OB677（ harboring GM3 gene） from Sri Lanka, HT1350 and high yield end quality cultivar ＂ Guiruanzhan＂. [ Result] Through pyramiding the multi-resistant genes via routine hybridization, the general resistances of the hybrids were remarkably enhanced. The grades of resistance were also improved, many of the combinations were endowed with a resistance at immune level （grade 0） ; and interestingly, the respective hybridization of GX-M001 （high resistance） with OB677（ medium resistance） and HT1350（suscepti- ble） also generate two lines at immune level, which is probably the effects of additive effects of genes.[ Conclusion] By routine hybridization, multiple genes were successfully pyramided, thus generating novel rice lines with multiple resistances. For the rice breeding scientists at the grass-roots level, the resistance-resistance pyramiding is an effective approach to breed high resistance cultivars.
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 Natural Science Foundation of China (No. 31171812)
文摘Bacterial blight, caused by Xanthomonas oryzae pv. oryzae(Xoo), is one of the most destructive diseases of rice(Oryza sativa L.) worldwide. The type III secretion system(T3SS) of Xoo, encoded by the hrp(hypersensitive response and pathogenicity) genes, plays critical roles in conferring pathogenicity in host rice and triggering a hypersensitive response(HR) in non-host plants. To investigate the major genes conferring the pathogenicity and avirulence of Xoo, we previously constructed a random Tn5-insertion mutant library of Xoo strain PXO99A. We report here the isolation and characterization of a Tn5-insertion mutant PXM69. Tn5-insertion mutants were screened on indica rice JG30, which is highly susceptible to PXO99A, by leaf-cutting inoculation.Four mutants with reduced virulence were obtained after two rounds of screening. Among them, the mutant PXM69 had completely lost virulence to the rice host and ability to elicit HR in non-host tobacco. Southern blotting analysis showed a single copy of a Tn5-insertion in the genome of PXM69. PCR walking and sequencing analysis revealed that the Tn5 transposon was inserted at nucleotide position 70,192–70,201 in the genome of PXO99A, disrupting the type III hrc(hrp-conserved) gene hrcQ, the first gene in the D operon of the hrp cluster in Xoo. To confirm the relationship between the Tn5-insertion and the avirulence phenotype of PXM69, we used the marker exchange mutagenesis to create a PXO99Amutant, ΔhrcQ::KAN, in which the hrcQ was disrupted by a kanamycin-encoding gene cassette at the same site as that of the Tn5-insertion. ΔhrcQ::KAN showed the same phenotype as mutant PXM69. Reintroduction of the wild-type hrcQ gene partially complemented the pathogenic function of PXM69. RT-PCR and cellulase secretion assays showed that the Tn5-disruption of hrcQ did not affect transcription of downstream genes in the D operon and function of the type II secretion system. Our results provide new insights into the pathogenic functions of clustered hrp genes in Xoo.
基金supported by the National Key R&D Program of China (2017YFD0200400)the National Natural Science Foundation of China (31772122 and 31470235)
文摘Xanthomonas oryzea pv.oryzae(Xoo)is the causal agent of bacterial blight of rice,which is a significant threat to many of rice-growing regions.The type Ⅲ secretion system(T3SS)is an essential virulence factor in Xoo.Expression of the T3SS is often induced in the host environment or in hrp-inducing medium but is repressed in nutrient-rich medium.The elucidation of molecular mechanism underlying induction of T3SS genes expression is a very important step to lift the veil on global virulence regulation network in Xoo.Thus,an efficient and reliable genetic tool system is required for detection of the T3SS proteins.In this study,we constructed a protein expression vector pH3-flag based on the backbone of pHM1,a most widely used vector in Xoo strains,especially a model strain PXO99A.This vector contains a synthesized MCS-FLAG cassette that consists of a multiple cloning site(MCS),containing a modified pUC18 polylinker,and Flag as a C-terminal tag.The cassette is flanked by transcriptional terminators to eliminate interference of external transcription enabling detection of accurate protein expression.We evaluated the potential of this expression vector as T3SS proteins detection system and demonstrated it is applicable in the study of T3SS genes expression regulation in Xoo.This improved expression system could be very effectively used as a molecular tool in understanding some virulence genes expression and regulation in Xoo and other Xanthomonas spp.
基金the National Science-Technology Supporting Project for the 11th Five-Year Plan (Nos. 2006BAD27B09-6 and 2007BAK36B03)the Key Technology R&D Program of Guangdong Province (Nos. 2007A010900001 and 2008A010900001)
文摘[Objective] To explore the efficient way for the preparation of Aspergillus oryzae protoplast.[Method] The statistics of protoplast yield by the enzymolysis of mycelia with different culture time,different osmotic stabilizers and different concentrations of enzyme system was carried out in this study.[Result] The protoplast yield was higher when the mycelium was cultivated for 108 h,and the osmotic stabilizer was 0.8 mol/L of NaCl.With the enzymes system of 2 mg/ml of lysozyme,6 mg/ml of cellulose and 6 mg/ml of glusulase,the protoplast yield achieved its highest,which was up to 9.0×105 per ml.[Conclusion] The yield of protoplast was affected by the situation of mycelia themselves and external conditions.This study had provided a basis for the preparation of a large number of active protoplasts and the further researches on cell fusion.
基金Supported by Competitive Project of Hubei Academy of Agricultural Sciences(2016jzxjh010)Major Research and Development Program of China(2016YFD0200807-1)~~
文摘Single-spore isolates were obtained from rice-growing fields of Yuan＇an in Hubei Province where rice blast seriously occurs in some years. DNA fingerprints were divided into 112 haplotypes and 14 lineages at 73% genetic similarity level. Among the lineages, no dominant lineages were found. The population genetic structures of Magnaporthe oryzae were not distinctly different in different years. The analysis also showed that there wasn＇t obvious simple relationship between patho- types and fingerprint groups.
