Bacterial blight of rice,caused by Xanthomonas oryzaepv.Oryzae(Xoo.),is one of the major rice diseases inChina.Making clear the shift of genetic diversity of the
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
由Xanthomonas oryzae pv.oryzae引起的水稻白叶枯病严重影响我国水稻安全生产,尤其在杂交稻上,损失高达40%~60%,在水稻种子市场监管过程中由于缺乏快速检测手段,造成带菌种子在市场中流通。利用SYBR Green I作为荧光指示剂,建立了水稻...由Xanthomonas oryzae pv.oryzae引起的水稻白叶枯病严重影响我国水稻安全生产,尤其在杂交稻上,损失高达40%~60%,在水稻种子市场监管过程中由于缺乏快速检测手段,造成带菌种子在市场中流通。利用SYBR Green I作为荧光指示剂,建立了水稻白叶枯病菌的环介导等温扩增(LAMP)可视化快速检测方法。以水稻白叶枯病菌为对象,通过Primer Explorer V4.0软件一共设计出四条特异性的LAMP引物以及两条环引物,优化并建立了水稻白叶枯病菌的可视化快速检测方法。结果表明:在65℃恒温条件下反应1 h,LAMP检测特异性和灵敏度,加入SYBR Green I荧光染料,水稻白叶枯病菌呈现荧光绿色,对照菌株则保持不变,依然呈橙色。LAMP反应检测灵敏度DNA的浓度大小为为100 fg/μL,菌悬液为3×103 cfu/mL。应用LAMP技术检测水稻白叶枯病菌Xanthomonas oryzae pv.oryzae具有特异性强、灵敏度高、操作简单快捷的特点,可用于市场监管过程中快速检测水稻白叶枯病菌。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
Xanthomonas oryzae pv.oryzae,the causal agent of bacterial blight in rice,interacts with rice plants in a gene-for-gene manner.The specificity of the interaction is dictated by avirulence(avr) genes in the pathogen an...Xanthomonas oryzae pv.oryzae,the causal agent of bacterial blight in rice,interacts with rice plants in a gene-for-gene manner.The specificity of the interaction is dictated by avirulence(avr) genes in the pathogen and resistance(R) genes in the host.To date,no avr genes that correspond to recessive R genes have been isolated.We isolated an avrBs3/pthA family gene,avrxa5,from our previously isolated clone p58,which was originally from strain JXOIII.The avrxa5 gene converted the PXO99A strain from compatible to incompatible in rice cultivars containing the recessive xa5 gene,but not in those containing the dominant Xa5 gene.Sequencing indicated that avrxa5,which is highly similar to members of the avrBs3/pthA family,encodes a protein of 1238 amino acid residues with a conserved carboxy-terminal region containing three nuclear localization signals and a transcription activation domain.It has 19.5 34-amino-acid direct repeats,but the 13th amino acid is missing in the fifth and ninth repetitive units.Domain swapping of the repetitive regions between avrxa5 and avrXa7 changed the avirulence specificity of the genes in xa5 and Xa7 rice lines,respectively.This indicates that avrxa5 is distinct from previously characterized avrBs3/pthA members.The specificity of avrxa5 toward recessive xa5 in rice could help us better understand the molecular mechanisms of plant-pathogen specific interactions.展开更多
The bacterial pathogen Xanthomonas oryzae pv.oryzae(Xoo),belonging to Xanthomonas sp.,causes one of the most destructive vascular diseases in rice worldwide,particularly in Asia and Africa.To better understand Xoo pat...The bacterial pathogen Xanthomonas oryzae pv.oryzae(Xoo),belonging to Xanthomonas sp.,causes one of the most destructive vascular diseases in rice worldwide,particularly in Asia and Africa.To better understand Xoo pathogenesis,we performed genome sequencing of the Korea race 1 strain DY89031(J18)and analyzed the phylogenetic tree of 63 Xoo strains.We found that the rich diversity of evolutionary features is likely associated with the rice cultivation regions.Further,virulence effector proteins secreted by the type III secretion system(T3SS)of Xoo showed pathogenesis divergence.The genome of DY89031 shows a remarkable difference from that of the widely prevailed Philippines race 6 strain PXO99A,which is avirulent to rice Xa21,a well-known disease resistance(R)gene that can be broken down by DY89031.Interestingly,plant inoculation experiments with the PXO99A transformants expressing the DY89031 genes enabled us to identify additional TAL(transcription activator-like)and non-TAL effectors that may support DY89031-specific virulence.Characterization of DY89031 genome and identification of new effectors will facilitate the investigation of the rice-Xoo interaction and new mechanisms involved.展开更多
Exogenous melatonin(MT)was found to be an interesting tool for enhancing the resistance of rice to Xanthomonasoryzaepv.oryzae(Xoo)-caused bacterial blight(BB).However,the accurate comparison of the expression levels a...Exogenous melatonin(MT)was found to be an interesting tool for enhancing the resistance of rice to Xanthomonasoryzaepv.oryzae(Xoo)-caused bacterial blight(BB).However,the accurate comparison of the expression levels across samples was a challenging task.In this work,the stability of 10 common used housekeeping genes under Xoo-infection and MT supplementation in rice was analyzed using quantitative real-time PCR(qRT-PCR),and algorithms geNorm,NormFinder and BestKeeper.Our results indicated that most reference genes remained stable in Xoo-infected rice plants,while a number of reference genes were affected by MT supplementation.Among all studied genes,the transcript levels of 18S(18S ribosomal RNA)and UBC(Ubiquitin-conjugating enzyme E2)remained unaltered by Xoo infection,while UBC and UBQ5(Ubiquitin 5)were the most stable genes when examining simultaneous Xoo-infection and MT supplementation,demonstrating that UBC is a suitable reference gene for qRT-PCR data normalization in rice under Xoo-infection and MT supplementation.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
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
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 oryzae pv. oryzicola (Xoc) causes bacterial leaf streak, a devastating disease in rice-growing regions worldwide. A Tn5-insertion mutant in Xoc_3248, encoding an inner membrane protein (Imp), showed re...Xanthomonas oryzae pv. oryzicola (Xoc) causes bacterial leaf streak, a devastating disease in rice-growing regions worldwide. A Tn5-insertion mutant in Xoc_3248, encoding an inner membrane protein (Imp), showed reduced virulence in rice. To explore the potential function of this gene in virulence, a deletion mutant R?imp was constructed in the wild-type RS105. The R?imp mutant was signiifcantly impaired for bacterial virulence and growth in planta. The mutation in imp made the pathogen insufifciently utilize glucose, fructose, mannose or pyruvate as a sole carbon source, leading to less extracellular polysaccharide (EPS) production and reduced motility. The deifciencies noted for the mutant were restored to wild-type levels when imp was introduced in trans. Transcription of imp was signiifcantly declined when hrpG and hrpX was mutated and the expression of hrpG and hrpX was also signiifcantly declined when imp was deleted. Cell sublocalization in planta showed Imp membrane-binding feature. These results suggest that Imp is a virulence factor with roles in the catabolism of sugars, EPS production, and bacterial motility.展开更多
Ninety one isolates of Xanthomonas oryzae pv. oryzae were collected from different rice- growing regions in China and determined for their virulence on 24 rice near-isogenic lines containing single resistance gene a...Ninety one isolates of Xanthomonas oryzae pv. oryzae were collected from different rice- growing regions in China and determined for their virulence on 24 rice near-isogenic lines containing single resistance gene and 2-4 genes: IRBB1 (Xa1), IRBB2 (Xa2), IRBB3 (Xa3), IRBB4 (Xa4), IRBB5 (xa5), IRBB7 (Xa7), IRBB8 (xa8), IRBB10 (Xa10), IRBB11 (Xa11), IRBB13 (xa13), IRBB14 (Xa14), IRBB21 (Xa21), IR24 (Xa18), IRBB50 (Xa4 + xa5), IRBB51 (Xa4 + xa13), IRBB52 (Xa4 + Xa21), IRBB53 (xa5 + xa13), IRBB54 (xa5 + Xa21), IRBB55 (xa13 + Xa21), IRBB56 (Xa4 + xa5 + xa13), IRBB57 (Xa4 + xa5 + Xa21), IRBB58 (Xa4 + xa13 + Xa21), IRBB59 (xa5 + xa13 + Xa21) and IRBB60 (Xa4 + xa5 + xa13 + Xa21). The results showed that most isolates were less virulent on lines with more than one genes pyramided than those with single resistance gene. The isolates tested were more virulent on IR24 and IRBB10, less virulent on IRBB5, IRBB7 and IRBB21. Based on interactions between isolates and rice near-isogenic lines, 7 cultivars with single gene (IRBB5, IRBB4, IRBB3, IRBB14, IRBB2, IRBB1 and IR24) were chosen as the differentials, and the tested isolates were classified into 7 virulence groups. The reaction patterns of the 7 groups in order were: RRRRRRR, RRRRRRS, RRRRRSS, RR/SRRSSS, RRRSSSS, RRSSSSS, RSSSSSS. The virulence frequencies were 7.69, 6.59, 14.29, 12.09, 14.29, 28.57 and 16.48% respectively. The elementary system for races identification has been established in China based on the results. It will be possible to compare with races in other countries, and the results will facilitate the development of rice resistance breeding to bacterial blight in China.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
Xanthomonas oryzae pv.oryzicola(Xoc) causes a destructive bacterial leaf streak disease in rice.Some of the gene products annotated as hypothetical proteins in the genome of Xoc may contribute to its virulence in ri...Xanthomonas oryzae pv.oryzicola(Xoc) causes a destructive bacterial leaf streak disease in rice.Some of the gene products annotated as hypothetical proteins in the genome of Xoc may contribute to its virulence in rice.A mutant,Mxoc1679,screened from our previous Tn5-tagged mutant library for Xoc strain RS105,showed reduced virulence in rice.In this mutant,a gene named as Xoryp_08180 was disrupted by Tn5 insertion.Xoryp_08180 encodes a 1 306-aa hypothetical protein which is highly conserved in Xanthomonas spp.Non-polar mutation of Xoryp_08180 in RS105 strain led to a significant reduction in bacterial virulence and growth in rice,a delayed hypersensitive response(HR) in non-host tobacco,and a decrease in extracellular protease activity.The deficiencies above were restored to wild-type level in the complementary strain by expressing Xoryp_08180 in trans.In addition,the expression of Xoryp_08180 was repressed in hrpG and hrpX mutants in planta but not in a nutrient-rich condition.These results suggested that Xoryp_08180 is a virulence factor required for extracellular protease production,HR induction and full virulence of Xoc.展开更多
文摘Bacterial blight of rice,caused by Xanthomonas oryzaepv.Oryzae(Xoo.),is one of the major rice diseases inChina.Making clear the shift of genetic diversity of the
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.
文摘由Xanthomonas oryzae pv.oryzae引起的水稻白叶枯病严重影响我国水稻安全生产,尤其在杂交稻上,损失高达40%~60%,在水稻种子市场监管过程中由于缺乏快速检测手段,造成带菌种子在市场中流通。利用SYBR Green I作为荧光指示剂,建立了水稻白叶枯病菌的环介导等温扩增(LAMP)可视化快速检测方法。以水稻白叶枯病菌为对象,通过Primer Explorer V4.0软件一共设计出四条特异性的LAMP引物以及两条环引物,优化并建立了水稻白叶枯病菌的可视化快速检测方法。结果表明:在65℃恒温条件下反应1 h,LAMP检测特异性和灵敏度,加入SYBR Green I荧光染料,水稻白叶枯病菌呈现荧光绿色,对照菌株则保持不变,依然呈橙色。LAMP反应检测灵敏度DNA的浓度大小为为100 fg/μL,菌悬液为3×103 cfu/mL。应用LAMP技术检测水稻白叶枯病菌Xanthomonas oryzae pv.oryzae具有特异性强、灵敏度高、操作简单快捷的特点,可用于市场监管过程中快速检测水稻白叶枯病菌。
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supported by the State Key Basic Research and Development Project of China (Grant No. 2006CB101902)the National Natural Science Foundation of China (Grant Nos. 30710103902 and 30671354)the Ministry of Agriculture of China (Grant No. NYHYZX07-056)
文摘Xanthomonas oryzae pv.oryzae,the causal agent of bacterial blight in rice,interacts with rice plants in a gene-for-gene manner.The specificity of the interaction is dictated by avirulence(avr) genes in the pathogen and resistance(R) genes in the host.To date,no avr genes that correspond to recessive R genes have been isolated.We isolated an avrBs3/pthA family gene,avrxa5,from our previously isolated clone p58,which was originally from strain JXOIII.The avrxa5 gene converted the PXO99A strain from compatible to incompatible in rice cultivars containing the recessive xa5 gene,but not in those containing the dominant Xa5 gene.Sequencing indicated that avrxa5,which is highly similar to members of the avrBs3/pthA family,encodes a protein of 1238 amino acid residues with a conserved carboxy-terminal region containing three nuclear localization signals and a transcription activation domain.It has 19.5 34-amino-acid direct repeats,but the 13th amino acid is missing in the fifth and ninth repetitive units.Domain swapping of the repetitive regions between avrxa5 and avrXa7 changed the avirulence specificity of the genes in xa5 and Xa7 rice lines,respectively.This indicates that avrxa5 is distinct from previously characterized avrBs3/pthA members.The specificity of avrxa5 toward recessive xa5 in rice could help us better understand the molecular mechanisms of plant-pathogen specific interactions.
基金supported by Chinese Academy of Sciences(XDB27040201)the National Natural Science Foundation of China(3181101746)。
文摘The bacterial pathogen Xanthomonas oryzae pv.oryzae(Xoo),belonging to Xanthomonas sp.,causes one of the most destructive vascular diseases in rice worldwide,particularly in Asia and Africa.To better understand Xoo pathogenesis,we performed genome sequencing of the Korea race 1 strain DY89031(J18)and analyzed the phylogenetic tree of 63 Xoo strains.We found that the rich diversity of evolutionary features is likely associated with the rice cultivation regions.Further,virulence effector proteins secreted by the type III secretion system(T3SS)of Xoo showed pathogenesis divergence.The genome of DY89031 shows a remarkable difference from that of the widely prevailed Philippines race 6 strain PXO99A,which is avirulent to rice Xa21,a well-known disease resistance(R)gene that can be broken down by DY89031.Interestingly,plant inoculation experiments with the PXO99A transformants expressing the DY89031 genes enabled us to identify additional TAL(transcription activator-like)and non-TAL effectors that may support DY89031-specific virulence.Characterization of DY89031 genome and identification of new effectors will facilitate the investigation of the rice-Xoo interaction and new mechanisms involved.
基金supported by grants from the State Key Laboratory for Managing Biotic and Chemical Threats to the Quality and Safety of Agroproducts(2010DS700124-KF2007)the National Natural Science Foundation of China(31571974)+2 种基金the Special Fund for Agroscientific Research in the Public Interest(201303015)the National Key R&D Program of China(2017YFD0200900)the Natural Science Foundation of Jiangsu Province of China(BK20170606).
文摘Exogenous melatonin(MT)was found to be an interesting tool for enhancing the resistance of rice to Xanthomonasoryzaepv.oryzae(Xoo)-caused bacterial blight(BB).However,the accurate comparison of the expression levels across samples was a challenging task.In this work,the stability of 10 common used housekeeping genes under Xoo-infection and MT supplementation in rice was analyzed using quantitative real-time PCR(qRT-PCR),and algorithms geNorm,NormFinder and BestKeeper.Our results indicated that most reference genes remained stable in Xoo-infected rice plants,while a number of reference genes were affected by MT supplementation.Among all studied genes,the transcript levels of 18S(18S ribosomal RNA)and UBC(Ubiquitin-conjugating enzyme E2)remained unaltered by Xoo infection,while UBC and UBQ5(Ubiquitin 5)were the most stable genes when examining simultaneous Xoo-infection and MT supplementation,demonstrating that UBC is a suitable reference gene for qRT-PCR data normalization in rice under Xoo-infection and MT supplementation.
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.
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.
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 Ministry of Agriculture of China (201303015)the Key Basic Research Project of Shanghai Committee of Science and Technology, China (11JC1406300)the Ph D Programs Foundation of Ministry of Education of China (20100073110045)
文摘Xanthomonas oryzae pv. oryzicola (Xoc) causes bacterial leaf streak, a devastating disease in rice-growing regions worldwide. A Tn5-insertion mutant in Xoc_3248, encoding an inner membrane protein (Imp), showed reduced virulence in rice. To explore the potential function of this gene in virulence, a deletion mutant R?imp was constructed in the wild-type RS105. The R?imp mutant was signiifcantly impaired for bacterial virulence and growth in planta. The mutation in imp made the pathogen insufifciently utilize glucose, fructose, mannose or pyruvate as a sole carbon source, leading to less extracellular polysaccharide (EPS) production and reduced motility. The deifciencies noted for the mutant were restored to wild-type levels when imp was introduced in trans. Transcription of imp was signiifcantly declined when hrpG and hrpX was mutated and the expression of hrpG and hrpX was also signiifcantly declined when imp was deleted. Cell sublocalization in planta showed Imp membrane-binding feature. These results suggest that Imp is a virulence factor with roles in the catabolism of sugars, EPS production, and bacterial motility.
基金This study was supported by the National Natural Science Foundation of China(30070497)National 863 Program of China(2002AA245041).
文摘Ninety one isolates of Xanthomonas oryzae pv. oryzae were collected from different rice- growing regions in China and determined for their virulence on 24 rice near-isogenic lines containing single resistance gene and 2-4 genes: IRBB1 (Xa1), IRBB2 (Xa2), IRBB3 (Xa3), IRBB4 (Xa4), IRBB5 (xa5), IRBB7 (Xa7), IRBB8 (xa8), IRBB10 (Xa10), IRBB11 (Xa11), IRBB13 (xa13), IRBB14 (Xa14), IRBB21 (Xa21), IR24 (Xa18), IRBB50 (Xa4 + xa5), IRBB51 (Xa4 + xa13), IRBB52 (Xa4 + Xa21), IRBB53 (xa5 + xa13), IRBB54 (xa5 + Xa21), IRBB55 (xa13 + Xa21), IRBB56 (Xa4 + xa5 + xa13), IRBB57 (Xa4 + xa5 + Xa21), IRBB58 (Xa4 + xa13 + Xa21), IRBB59 (xa5 + xa13 + Xa21) and IRBB60 (Xa4 + xa5 + xa13 + Xa21). The results showed that most isolates were less virulent on lines with more than one genes pyramided than those with single resistance gene. The isolates tested were more virulent on IR24 and IRBB10, less virulent on IRBB5, IRBB7 and IRBB21. Based on interactions between isolates and rice near-isogenic lines, 7 cultivars with single gene (IRBB5, IRBB4, IRBB3, IRBB14, IRBB2, IRBB1 and IR24) were chosen as the differentials, and the tested isolates were classified into 7 virulence groups. The reaction patterns of the 7 groups in order were: RRRRRRR, RRRRRRS, RRRRRSS, RR/SRRSSS, RRRSSSS, RRSSSSS, RSSSSSS. The virulence frequencies were 7.69, 6.59, 14.29, 12.09, 14.29, 28.57 and 16.48% respectively. The elementary system for races identification has been established in China based on the results. It will be possible to compare with races in other countries, and the results will facilitate the development of rice resistance breeding to bacterial blight in China.
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supported by the National Natural Science Foundation of China(31071656,31000071)the National Transgenic Major Program,China(2008ZX08001-002)the Special Fund for Agro-scientific Research in the Public Interest,China(NYHYZX07-056)
文摘Xanthomonas oryzae pv.oryzicola(Xoc) causes a destructive bacterial leaf streak disease in rice.Some of the gene products annotated as hypothetical proteins in the genome of Xoc may contribute to its virulence in rice.A mutant,Mxoc1679,screened from our previous Tn5-tagged mutant library for Xoc strain RS105,showed reduced virulence in rice.In this mutant,a gene named as Xoryp_08180 was disrupted by Tn5 insertion.Xoryp_08180 encodes a 1 306-aa hypothetical protein which is highly conserved in Xanthomonas spp.Non-polar mutation of Xoryp_08180 in RS105 strain led to a significant reduction in bacterial virulence and growth in rice,a delayed hypersensitive response(HR) in non-host tobacco,and a decrease in extracellular protease activity.The deficiencies above were restored to wild-type level in the complementary strain by expressing Xoryp_08180 in trans.In addition,the expression of Xoryp_08180 was repressed in hrpG and hrpX mutants in planta but not in a nutrient-rich condition.These results suggested that Xoryp_08180 is a virulence factor required for extracellular protease production,HR induction and full virulence of Xoc.