Avirulence effectors(Avrs),encoded by plant pathogens,can be recognized by plants harboring the corresponding resistance proteins,thereby initiating effector-triggered immunity(ETI).In susceptible plants,however,Avrs ...Avirulence effectors(Avrs),encoded by plant pathogens,can be recognized by plants harboring the corresponding resistance proteins,thereby initiating effector-triggered immunity(ETI).In susceptible plants,however,Avrs can function as effectors,facilitating infection via effector-triggered susceptibility(ETS).Mechanisms of Avr-mediated ETS remain largely unexplored.Here we report that the Magnaporthe oryzae effector Avr-PikD enters rice cells via the canonical cytoplasmic secretion pathway and suppresses rice basal defense.Avr-PikD interacts with an LSD1-like transcriptional activator AKIP30 of rice,and AKIP30 is also a positive regulator of rice immunity,whereas Avr-PikD impedes its nuclear localization and suppresses its transcriptional activity.In summary,M.oryzae delivers Avr-PikD into rice cells to facilitate ETS by inhibiting AKIP30-mediated transcriptional regulation of immune response against M.oryzae.展开更多
Background As demand for high quality animal feed continues to raise,it becomes increasingly important to mini-mize the environmental impact of feed production.An appealing sustainable approach to provide feed fractio...Background As demand for high quality animal feed continues to raise,it becomes increasingly important to mini-mize the environmental impact of feed production.An appealing sustainable approach to provide feed fractions is to use organic residues from agro-food industry.In this regard,volatile fatty acids(VFAs)such as acetic,propionic and butyric acids,derived from bioconversion of organic residues can be used as precursors for production of micro-bial protein with ruminant feed inclusion potential.This study aims to investigate the in vitro digestibility of the Asper-gillus oryzae edible fungal biomass cultivated on VFAs-derived from anaerobic digestion of residues.The produced fungal protein biomass,along with hay clover silage and rapeseed meal were subjected to various in vitro assays using two-stage Tilley and Terry(TT),gas,and bag methods to evaluate and compare its digestibility for application in ruminant feed.Results The produced fungal biomass contained a higher crude protein(CP)(41%–49%)and rather similar neutral detergent fiber(NDF)(41%–56%)compared to rapeseed meal.The rumen in vitro dry matter digestibility(IVDMD)of the fungal biomass in the TT method ranged from 82%to 88%(statistically similar to that of the gas method(72%to 85%)).The IVDMD of fungal biomass were up to 26%and 40%greater than that of hay clover silage and rapeseed meal,respectively.The type of substrate and bag method had pronounced effect on the fermentation products(ammonium-N(NH4+-N),total gas and VFAs).Fungal biomass digestion resulted in the highest release of NH4+-N(340–540 mg/L)and the ratio of acetate to propionate ratio(3.5)among subjected substrates.Conclusion The results indicate that gas method can be used as a reliable predictor for IVDMD as well as fermenta-tion products.Furthermore,the high IVDMD and fermentation product observed for Aspergillus oryzae fungal biomass digestion,suggest that the supplementation of fungal biomass will contribute to improving the rumen digestion by providing necessary nitrogen and energy to the ruminant and microbiota.展开更多
[Objectives]This study was conducted to screen out rice resources resistant to rice blast(Magnaporthe oryzae).[Methods]The qualitative and quantitative resistance of 1659 rice resources from 45 countries and regions t...[Objectives]This study was conducted to screen out rice resources resistant to rice blast(Magnaporthe oryzae).[Methods]The qualitative and quantitative resistance of 1659 rice resources from 45 countries and regions to rice blast were evaluated by disease nursery in upland condition and the test of the spectrum to rice blast isolates.[Results]There were 292 entries which accounted for 17.6%showed high blast resistance(0 and 1 disease scale),68 entries(counted for 4.1%)showed resistance to blast(3 disease scale);and the number of the entries showed intermediate resistance,intermediate susceptible and susceptible were 208(with the corresponding percentage of 12.5%),471(28.4%),620(37.4%)respectively.Among the tested entries,27 entries including BG1222,BL122,BTX,IR37704-131-2-3-2,and LEBONNET had showed broad-spectrum blast resistance with the resistance frequency of higher than 90%,Quantitative resistance evaluation was conducted on some key resources,and 14 entries,of which are BR27,DRAGO,IR100,QINLIUAI、SERIBU GANTANG,YUEXIANGZHAN and so on,showed good quantitative resistances,and 8 entries had higher quantitative resistances than IR36.[Conclusions]This study provides important blast resistance resources for the local rice breeding program and has a significant value for the discovery of new blast resistance genes and its application in the blast resistance breeding.展开更多
One of the most devastating diseases of rice worldwide is bacterial blight (BLB) caused by Xanthomonas oryzae pv. Oryzae (Xoo). In Benin, Xoo was first described in 2013 on wild rice Oryzae longistaminata. So far, no ...One of the most devastating diseases of rice worldwide is bacterial blight (BLB) caused by Xanthomonas oryzae pv. Oryzae (Xoo). In Benin, Xoo was first described in 2013 on wild rice Oryzae longistaminata. So far, no study has been done on Beninese Xoo strains. We do not know whether the pathogen has already passed into the rice varieties grown, or if they are exposed to other bacteria. Whereas the use of resistant varieties, carrying resistance genes, is the only highly effective and environmentally friendly way to control this disease, no information is available on these Xoo resistance genes in rice varieties grown in Benin apart from the one we recently. This study aims to identify Beninese Xoo strains, causing BLB and screen rice varieties grown in Benin for the main resistance genes. Diseased rice leaves showing typical symptoms of fire blight collected from different rice fields in the three phytogeographic areas of Benin were analyzed by PCR for Xoo-specific sequence identification. Furthermore, seventy-five collected rice accessions were screened to identify xa5, Xa7, xa13, and Xa21 resistance genes to Xoo. The results reveal that Xanthomonas oryzae was identified in two fields in Banikouara and one in Malanville. On the other hand, Sphingomonas sp. has been identified in several other rice fields in Benin. Forty-seven of seventy-five rice accessions examined (62.66%) carried Xoo resistance genes with 3 (4%) and 40 (53.33%) of xa5 and Xa21 respectively. None of the accessions had either Xa7 or xa13 resistance genes. Three accessions possess both xa5 and Xa21 genes. Isogenic lines IRBB60 and IRBB21, supposed to be a positive control, presented a Xoo sensitivity allele. These results indicate that Xoo has moved from the wild rice variety to the cultivated variety in northern Benin and varietal improvement programs must be implemented with varieties having several resistance genes for the efficient response against a possible BLB pandemic in Benin.展开更多
In this study, three wheat varieties were tested to determine seed germination and the incidence of Magnaporthe oryzae triticum (MoT). Among these varieties, BARI Gom 24 (Prodip) wheat seed exhibited the highest seed ...In this study, three wheat varieties were tested to determine seed germination and the incidence of Magnaporthe oryzae triticum (MoT). Among these varieties, BARI Gom 24 (Prodip) wheat seed exhibited the highest seed germination rate (93%) but also had the highest incidence (30%) of MoT. To manage blast disease in an ecofriendly manner, seven treatments were employed: T<sub>1</sub> = Control, T<sub>2</sub> = Garlic clove extracts, T<sub>3</sub> = Aloe vera leaf extracts, T<sub>4</sub> = Black cumin seed extracts, T<sub>5</sub> = Neem leaf extracts, T<sub>6</sub> = Nativo 75 WG, and T<sub>7</sub> = Provax 200 WP. The experiment was conducted using a Randomized Complete Block Design (RCBD) layout with three replications using Prodip wheat variety that exhibited highest MoT infection severity based on laboratory analysis among collected varieties. Data were collected on blast disease incidence (%), disease severity, and various growth and yield parameters of wheat. The experiment’s results indicated that among all the treatments, T<sub>7</sub> (Seed treatment with Provax 200 WP) and T<sub>5</sub> (Foliar spraying with Neem leaf extract) performed better in controlling blast disease in wheat. The lowest blast disease incidence (%) was observed with T<sub>7</sub> (Provax 200 WP), with values of 7.86, 9.86, and 10.19 recorded during the milking stage, soft dough stage, and hard dough stage of wheat, respectively. T<sub>5</sub> (Neem leaf extract) also demonstrated a statistically equivalent reduction in blast disease incidence (%). In terms of disease severity, T<sub>7</sub> (Seed treatment with Provax 200 WP) showed the lowest values of 1.03, 1.23, and 1.63 during the milking stage, soft dough stage, and hard dough stage of wheat, respectively. Foliar spraying with neem leaf extract also exhibited similar result as of Provax 200 WP regarding panicle blast severity. As a result of these findings, it can be concluded that T<sub>5</sub> (Neem leaf extract) is recommended as an ecofriendly management approach for blast disease in wheat.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
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
Saikuzuo (N, N’-methylene-di (2-amino-5-sulfurhydrogen-1, 3, 4-thiodiazole)) a good bactericideagainst rice bacterial blight disease, has been usedin China for more than 20 years. In this study,the sensitivity of X. ...Saikuzuo (N, N’-methylene-di (2-amino-5-sulfurhydrogen-1, 3, 4-thiodiazole)) a good bactericideagainst rice bacterial blight disease, has been usedin China for more than 20 years. In this study,the sensitivity of X. oryzae pv. oryzae (Xoo) tosaikuzuo was tested in vitro and in vivo. Seventy-seven and 11 isolates were collected from Hexian,Anhui Province, and Liuhe, Jiangsu Province, re-展开更多
Rice blast disease, caused by Magnaporthe oryzae, threatens global food security. The rice blast pathosystem is a longstanding model system for understanding plant-microbe interactions. In order to elucidate the coevo...Rice blast disease, caused by Magnaporthe oryzae, threatens global food security. The rice blast pathosystem is a longstanding model system for understanding plant-microbe interactions. In order to elucidate the coevolution of the host and pathogen, and provide the appropriate methods for preventing or controlling rice blast disease, researchers have focused on the evolution of virulence factors and resistance genes. Thus far, more than 30 rice blast resistance(R) genes and 12 avirulence(Avr) genes have been cloned. This review summarizes the cloned rice blast R genes, cloned Avr genes of M. oryzae and the interaction between them. This discussion also considers some of the major unanswered questions concerning this pathosystem and the opportunities for future investigations.展开更多
基金supported by grants from the National Natural Science Foundation of China(31401692,31901960,32272513,32001976)the Natural Science Foundation of Fujian Province(2019J01766,2023J011418,2020J05177)+3 种基金Fujian Provincial Science and Technology Key Project(2022NZ030014)External Cooperation Program of Fujian Academy of Agricultural Sciences(DWHZ-2024-23)State Key Laboratory of Ecological Pest Control for Fujian and Taiwan Crop Opening Project(SKL2019005)Project of Fujian Provincial Department of Education(JAT190627)。
文摘Avirulence effectors(Avrs),encoded by plant pathogens,can be recognized by plants harboring the corresponding resistance proteins,thereby initiating effector-triggered immunity(ETI).In susceptible plants,however,Avrs can function as effectors,facilitating infection via effector-triggered susceptibility(ETS).Mechanisms of Avr-mediated ETS remain largely unexplored.Here we report that the Magnaporthe oryzae effector Avr-PikD enters rice cells via the canonical cytoplasmic secretion pathway and suppresses rice basal defense.Avr-PikD interacts with an LSD1-like transcriptional activator AKIP30 of rice,and AKIP30 is also a positive regulator of rice immunity,whereas Avr-PikD impedes its nuclear localization and suppresses its transcriptional activity.In summary,M.oryzae delivers Avr-PikD into rice cells to facilitate ETS by inhibiting AKIP30-mediated transcriptional regulation of immune response against M.oryzae.
基金Open access funding provided by University of Boras
文摘Background As demand for high quality animal feed continues to raise,it becomes increasingly important to mini-mize the environmental impact of feed production.An appealing sustainable approach to provide feed fractions is to use organic residues from agro-food industry.In this regard,volatile fatty acids(VFAs)such as acetic,propionic and butyric acids,derived from bioconversion of organic residues can be used as precursors for production of micro-bial protein with ruminant feed inclusion potential.This study aims to investigate the in vitro digestibility of the Asper-gillus oryzae edible fungal biomass cultivated on VFAs-derived from anaerobic digestion of residues.The produced fungal protein biomass,along with hay clover silage and rapeseed meal were subjected to various in vitro assays using two-stage Tilley and Terry(TT),gas,and bag methods to evaluate and compare its digestibility for application in ruminant feed.Results The produced fungal biomass contained a higher crude protein(CP)(41%–49%)and rather similar neutral detergent fiber(NDF)(41%–56%)compared to rapeseed meal.The rumen in vitro dry matter digestibility(IVDMD)of the fungal biomass in the TT method ranged from 82%to 88%(statistically similar to that of the gas method(72%to 85%)).The IVDMD of fungal biomass were up to 26%and 40%greater than that of hay clover silage and rapeseed meal,respectively.The type of substrate and bag method had pronounced effect on the fermentation products(ammonium-N(NH4+-N),total gas and VFAs).Fungal biomass digestion resulted in the highest release of NH4+-N(340–540 mg/L)and the ratio of acetate to propionate ratio(3.5)among subjected substrates.Conclusion The results indicate that gas method can be used as a reliable predictor for IVDMD as well as fermenta-tion products.Furthermore,the high IVDMD and fermentation product observed for Aspergillus oryzae fungal biomass digestion,suggest that the supplementation of fungal biomass will contribute to improving the rumen digestion by providing necessary nitrogen and energy to the ruminant and microbiota.
基金Supported by Science and Technology Plan Project of Shaoguan(210804164531395)Special Fund for Modern Agricultural Industry Technology System(CARS-01-32,2022KJ105)"14thFive-Year Plan"New Discipline Team Building Project of Guangdong Academy of Agricultural Sciences(202116TD)。
文摘[Objectives]This study was conducted to screen out rice resources resistant to rice blast(Magnaporthe oryzae).[Methods]The qualitative and quantitative resistance of 1659 rice resources from 45 countries and regions to rice blast were evaluated by disease nursery in upland condition and the test of the spectrum to rice blast isolates.[Results]There were 292 entries which accounted for 17.6%showed high blast resistance(0 and 1 disease scale),68 entries(counted for 4.1%)showed resistance to blast(3 disease scale);and the number of the entries showed intermediate resistance,intermediate susceptible and susceptible were 208(with the corresponding percentage of 12.5%),471(28.4%),620(37.4%)respectively.Among the tested entries,27 entries including BG1222,BL122,BTX,IR37704-131-2-3-2,and LEBONNET had showed broad-spectrum blast resistance with the resistance frequency of higher than 90%,Quantitative resistance evaluation was conducted on some key resources,and 14 entries,of which are BR27,DRAGO,IR100,QINLIUAI、SERIBU GANTANG,YUEXIANGZHAN and so on,showed good quantitative resistances,and 8 entries had higher quantitative resistances than IR36.[Conclusions]This study provides important blast resistance resources for the local rice breeding program and has a significant value for the discovery of new blast resistance genes and its application in the blast resistance breeding.
文摘One of the most devastating diseases of rice worldwide is bacterial blight (BLB) caused by Xanthomonas oryzae pv. Oryzae (Xoo). In Benin, Xoo was first described in 2013 on wild rice Oryzae longistaminata. So far, no study has been done on Beninese Xoo strains. We do not know whether the pathogen has already passed into the rice varieties grown, or if they are exposed to other bacteria. Whereas the use of resistant varieties, carrying resistance genes, is the only highly effective and environmentally friendly way to control this disease, no information is available on these Xoo resistance genes in rice varieties grown in Benin apart from the one we recently. This study aims to identify Beninese Xoo strains, causing BLB and screen rice varieties grown in Benin for the main resistance genes. Diseased rice leaves showing typical symptoms of fire blight collected from different rice fields in the three phytogeographic areas of Benin were analyzed by PCR for Xoo-specific sequence identification. Furthermore, seventy-five collected rice accessions were screened to identify xa5, Xa7, xa13, and Xa21 resistance genes to Xoo. The results reveal that Xanthomonas oryzae was identified in two fields in Banikouara and one in Malanville. On the other hand, Sphingomonas sp. has been identified in several other rice fields in Benin. Forty-seven of seventy-five rice accessions examined (62.66%) carried Xoo resistance genes with 3 (4%) and 40 (53.33%) of xa5 and Xa21 respectively. None of the accessions had either Xa7 or xa13 resistance genes. Three accessions possess both xa5 and Xa21 genes. Isogenic lines IRBB60 and IRBB21, supposed to be a positive control, presented a Xoo sensitivity allele. These results indicate that Xoo has moved from the wild rice variety to the cultivated variety in northern Benin and varietal improvement programs must be implemented with varieties having several resistance genes for the efficient response against a possible BLB pandemic in Benin.
文摘In this study, three wheat varieties were tested to determine seed germination and the incidence of Magnaporthe oryzae triticum (MoT). Among these varieties, BARI Gom 24 (Prodip) wheat seed exhibited the highest seed germination rate (93%) but also had the highest incidence (30%) of MoT. To manage blast disease in an ecofriendly manner, seven treatments were employed: T<sub>1</sub> = Control, T<sub>2</sub> = Garlic clove extracts, T<sub>3</sub> = Aloe vera leaf extracts, T<sub>4</sub> = Black cumin seed extracts, T<sub>5</sub> = Neem leaf extracts, T<sub>6</sub> = Nativo 75 WG, and T<sub>7</sub> = Provax 200 WP. The experiment was conducted using a Randomized Complete Block Design (RCBD) layout with three replications using Prodip wheat variety that exhibited highest MoT infection severity based on laboratory analysis among collected varieties. Data were collected on blast disease incidence (%), disease severity, and various growth and yield parameters of wheat. The experiment’s results indicated that among all the treatments, T<sub>7</sub> (Seed treatment with Provax 200 WP) and T<sub>5</sub> (Foliar spraying with Neem leaf extract) performed better in controlling blast disease in wheat. The lowest blast disease incidence (%) was observed with T<sub>7</sub> (Provax 200 WP), with values of 7.86, 9.86, and 10.19 recorded during the milking stage, soft dough stage, and hard dough stage of wheat, respectively. T<sub>5</sub> (Neem leaf extract) also demonstrated a statistically equivalent reduction in blast disease incidence (%). In terms of disease severity, T<sub>7</sub> (Seed treatment with Provax 200 WP) showed the lowest values of 1.03, 1.23, and 1.63 during the milking stage, soft dough stage, and hard dough stage of wheat, respectively. Foliar spraying with neem leaf extract also exhibited similar result as of Provax 200 WP regarding panicle blast severity. As a result of these findings, it can be concluded that T<sub>5</sub> (Neem leaf extract) is recommended as an ecofriendly management approach for blast disease in wheat.
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.
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Saikuzuo (N, N’-methylene-di (2-amino-5-sulfurhydrogen-1, 3, 4-thiodiazole)) a good bactericideagainst rice bacterial blight disease, has been usedin China for more than 20 years. In this study,the sensitivity of X. oryzae pv. oryzae (Xoo) tosaikuzuo was tested in vitro and in vivo. Seventy-seven and 11 isolates were collected from Hexian,Anhui Province, and Liuhe, Jiangsu Province, re-
基金support from the National Natural Science Foundation of China (U1405212)the National Key Research and Development Program of China (2016YFD0300707)+1 种基金the Natural Science Foundation of Fujian Province, China (2017J01618)the 100 Talent Project from Fujian Province to Dr.Daniel J.Ebbole (Texas A&M University, USA)
文摘Rice blast disease, caused by Magnaporthe oryzae, threatens global food security. The rice blast pathosystem is a longstanding model system for understanding plant-microbe interactions. In order to elucidate the coevolution of the host and pathogen, and provide the appropriate methods for preventing or controlling rice blast disease, researchers have focused on the evolution of virulence factors and resistance genes. Thus far, more than 30 rice blast resistance(R) genes and 12 avirulence(Avr) genes have been cloned. This review summarizes the cloned rice blast R genes, cloned Avr genes of M. oryzae and the interaction between them. This discussion also considers some of the major unanswered questions concerning this pathosystem and the opportunities for future investigations.
