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
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
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
To demonstrate the expression profiling of Xanthomonas oryzae pv.oryzae(Xoo) in vitro and in planta,DNA microarrays of 371 genes potentially associated with pathogenicity and virulence were used to compare the transcr...To demonstrate the expression profiling of Xanthomonas oryzae pv.oryzae(Xoo) in vitro and in planta,DNA microarrays of 371 genes potentially associated with pathogenicity and virulence were used to compare the transcriptional level alteration of the wild-type strain PXO99A and gene deletion mutants ΔgacAxoo and ΔfleQxoo of Xoo grown in the rich medium NBY vs.hrp-inducing minimal medium XOM2 or leaf tissues of rice.Results indicated that 17 and 38 genes of PXO99A were differentially expressed in XOM2 and the leaf tissues of rice relative to NBY,respectively.Twenty-eight genes of ΔgacAxoo grown in XOM2 and 12 genes of ΔfleQxoo in NBY were differentially expressed relative to PXO99A.The identification of differentially-expressed genes,GacAxoo-and FleQxoo-regulons and novel candidate genes of Xoo strains would provide us the target genes for further functional analysis in pathogenesis of Xoo.展开更多
Xanthomonas oryzae pv.oryzae(Xoo),the causal agent of bacterial blight of rice,employs the transcription activator-like effectors(TALEs)to induce the expression of the OsSWEET family of putative sugar transporter gene...Xanthomonas oryzae pv.oryzae(Xoo),the causal agent of bacterial blight of rice,employs the transcription activator-like effectors(TALEs)to induce the expression of the OsSWEET family of putative sugar transporter genes,which function in conferring disease susceptibility(S)in rice plants.To engineer broadspectrum bacterial blight resistance,we used CRISPR/Cas9-mediated gene editing to disrupt the TALEbinding elements(EBEs)of two S genes,OsSWEETH and OsSWEET14,in rice cv.Kitaake,which harbors the recessive resistance allele of Xa25/OsSWEET13.The engineered rice line MS14K exhibited broadspectrum resistance to most Xoo strains with a few exceptions,suggesting that the compatible strains may contain new TALEs.We identified two PthXo2-like TALEs,Tal5LN18 and Tal7PX061,as major virulence factors in the compatible Xoo strains LN18 and PX061,respectively,and found that Xoo encodes at least five types of PthXo2-like effectors.Given that PthXo2/PthXo2.1 target OsSlVEETf3 for transcriptional activation,the genomes of 3000 rice varieties were analyzed for EBE variationsin the OsSWEET13 promoter,and 10Xa25-like haplotypes were identified.We found that Tal5LN18 and Tal7PX〇6i bind slightly different EBE sequences in the OsSWEET13 promoter to activate its expression.CRISPR/Cas9 technology was then used to generate InDels in the EBE of the OsSWEET13 promoter in MS14K to creat a new germplasm with three edited OsSWEET EBEs and broad-spectrum resistance against all Xoo strains tested.Collectively,our findings illustrate how to disarm TALE-S co-evolved loci to generate broad-spectrum resistance through the loss of effector-triggered susceptibility in plants.展开更多
Rice blast and bacterial blight are important diseases of rice(Oryza sativa)caused by the fungus Magnaporthe oryzae and the bacterium Xanthomonas oryzae pv.oryzae(Xoo),respectively.Breeding rice varieties for broadspe...Rice blast and bacterial blight are important diseases of rice(Oryza sativa)caused by the fungus Magnaporthe oryzae and the bacterium Xanthomonas oryzae pv.oryzae(Xoo),respectively.Breeding rice varieties for broadspectrum resistance is considered the most effective and sustainable approach to controlling both diseases.Although dominant resistance genes have been extensively used in rice breeding and production,generating diseaseresistant varieties by altering susceptibility(S)genes that facilitate pathogen compatibility remains unexplored.Here,using CRISPR/Cas9 technology,we generated loss-of-function mutants of the S genes Pi21 and Bsr-d1 and showed that they had increased resistance to M.oryzae.We also generated a knockout mutant of the S gene Xa5 that showed increased resistance to Xoo.Remarkably,a triple mutant of all three S genes had significantly enhanced resistance to both M.oryzae and Xoo.Moreover,the triple mutant was comparable to the wild type in regard to key agronomic traits,including plant height,effective panicle number per plant,grain number per panicle,seed setting rate,and thousand-grain weight.These results demonstrate that the simultaneous editing of multiple S genes is a powerful strategy for generating new rice varieties with broadspectrum resistance.展开更多
Plants have developed various mechanisms for avoiding pathogen invasion,including resistance(R)genes.Most R genes encode nucleotide-binding domain and leucine-rich repeat containing proteins(NLRs).Here,we report the i...Plants have developed various mechanisms for avoiding pathogen invasion,including resistance(R)genes.Most R genes encode nucleotide-binding domain and leucine-rich repeat containing proteins(NLRs).Here,we report the isolation of three new bacterial blight R genes in rice,Xa1-2,Xa14,and Xa31(t),which were allelic to Xa1 and encoded atypical NLRs with unique central tandem repeats(CTRs).We also found that Xa31(t)was the same gene as Xa1-2.Although Xa1-2 and Xa14 conferred different resistance spectra,their performance could be attenuated by iTALEs,as has previously been reported for Xa1.XA1,XA1-2,XA14,and non-resistant RGAF differed mainly in the substructure of the leucine-rich repeat domain.They all contained unique CTRs and belonged to the CTR-NLRs,which existed only in Gramineae.We also found that interactions among these genes led to differing resistance performance.In conclusion,our results uncover a unique locus in rice consisting of at least three multiple alleles(Xa1,Xa1-2,and Xa14)that encode CTRNLRs and confer resistance to Xanthomonas oryzae pv.oryzae(Xoo).展开更多
Xanthomonas oryzae pv.oryzae(Xoo),the causal agent of bacterial leaf blight in rice,delivers transcription activator-like effector(TALE)proteins into host cells to activate susceptibility or resistance(R)genes that pr...Xanthomonas oryzae pv.oryzae(Xoo),the causal agent of bacterial leaf blight in rice,delivers transcription activator-like effector(TALE)proteins into host cells to activate susceptibility or resistance(R)genes that promote disease or immunity,respectively.Nonhost plants serve as potential reservoirs of R genes;consequently,nonhost R genes may trap TALEs to trigger an immune response.In this study,we screened 17 Xoo TALEs for their ability to induce a hypersensitive response(HR)in the nonhost plant Nicotiana benthamiana(Nb);only AvrXa10 elicited an HR when transiently expressed in Nb.The HR generated by AvrXa10 required both the central repeat region and the activation domain,suggesting a specific interaction between AvrXa10 and a potential R-like gene in nonhost plants.Evans blue staining and ion leakage measurements confirmed that the AvrXa10-triggered HR was a form of cell death,and the transient expression of AvrXa10 in Nb induced immune responses.Genes targeted by AvrXa10 in the Nb genome were identified by transcriptome profiling and prediction of effector binding sites.Using several approaches(in vivo reporter assays,electrophoretic mobility-shift assays,targeted designer TALEs,and on-spot gene silencing),we confirmed that AvrXa10 targets NbZnFP1,a C2H2-type zinc finger protein that resides in the nucleus.Functional analysis indicated that overexpression of NbZnFP1 and its rice orthologs triggered cell death in rice protoplasts.An NbZnFP1 ortholog was also identified in tomato and was specifically activated by AvrXa10.These results demonstrate that NbZnFP1 is a nonhost R gene that traps AvrXa10 to promote plant immunity in Nb.展开更多
XA21 encodes a rice immune receptor that confers robust resistance to most strains of the Gram-negative bacterium Xanthomonas oryzae pv.oryzae(Xoo).XA21-mediated immunity is triggered by recognition of a small protein...XA21 encodes a rice immune receptor that confers robust resistance to most strains of the Gram-negative bacterium Xanthomonas oryzae pv.oryzae(Xoo).XA21-mediated immunity is triggered by recognition of a small protein called RaxX-sY(required for activation of XA21-mediated immunity X,tyrosine-sulfated)secreted by Xoo.To identify components regulating XA21-mediated immunity,we generated and screened a mutant population of fast-neutron-mutagenized rice expressing Ubi:Myc-XA21 for those susceptible to Xoo.Here,we report the characterization of one of these rice mutants,named sxi2(suppressor of XA21-mediated immunity-2).Whole-genome sequencing revealed that sxi2 carries a deletion of the PALADIN(PALD)gene encoding a protein with three putative protein tyrosine phosphatase-like domains(PTP-A,-B,and-C).Expression of PALD in the sxi2 genetic background was sufficient to complement the susceptible phenotype,which requires the catalytic cysteine of the PTP-A active site to restore resistance.PALD coimmunoprecipitated with the full-length XA21 protein,whose levels are positively regulated by the presence of the PALD transgene.Furthermore,we foundd that sxi2 retains many hallmarks of XA21-mediated immunity,similar to the wild type.These results reveal that PALD,a previously uncharacterized class of phosphatase,functions in rice innate immunity,and suggest that the conserved cysteine in the PTP-A domain of PALD is required for its immune function.展开更多
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 (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.
文摘To demonstrate the expression profiling of Xanthomonas oryzae pv.oryzae(Xoo) in vitro and in planta,DNA microarrays of 371 genes potentially associated with pathogenicity and virulence were used to compare the transcriptional level alteration of the wild-type strain PXO99A and gene deletion mutants ΔgacAxoo and ΔfleQxoo of Xoo grown in the rich medium NBY vs.hrp-inducing minimal medium XOM2 or leaf tissues of rice.Results indicated that 17 and 38 genes of PXO99A were differentially expressed in XOM2 and the leaf tissues of rice relative to NBY,respectively.Twenty-eight genes of ΔgacAxoo grown in XOM2 and 12 genes of ΔfleQxoo in NBY were differentially expressed relative to PXO99A.The identification of differentially-expressed genes,GacAxoo-and FleQxoo-regulons and novel candidate genes of Xoo strains would provide us the target genes for further functional analysis in pathogenesis of Xoo.
基金This research was supported by the National Key Research and Development Program of China(2016YFD0100601)the National Natural Science Foundation of China(31830072)the National Transgenic Major Program(2016ZX08001-002).
文摘Xanthomonas oryzae pv.oryzae(Xoo),the causal agent of bacterial blight of rice,employs the transcription activator-like effectors(TALEs)to induce the expression of the OsSWEET family of putative sugar transporter genes,which function in conferring disease susceptibility(S)in rice plants.To engineer broadspectrum bacterial blight resistance,we used CRISPR/Cas9-mediated gene editing to disrupt the TALEbinding elements(EBEs)of two S genes,OsSWEETH and OsSWEET14,in rice cv.Kitaake,which harbors the recessive resistance allele of Xa25/OsSWEET13.The engineered rice line MS14K exhibited broadspectrum resistance to most Xoo strains with a few exceptions,suggesting that the compatible strains may contain new TALEs.We identified two PthXo2-like TALEs,Tal5LN18 and Tal7PX061,as major virulence factors in the compatible Xoo strains LN18 and PX061,respectively,and found that Xoo encodes at least five types of PthXo2-like effectors.Given that PthXo2/PthXo2.1 target OsSlVEETf3 for transcriptional activation,the genomes of 3000 rice varieties were analyzed for EBE variationsin the OsSWEET13 promoter,and 10Xa25-like haplotypes were identified.We found that Tal5LN18 and Tal7PX〇6i bind slightly different EBE sequences in the OsSWEET13 promoter to activate its expression.CRISPR/Cas9 technology was then used to generate InDels in the EBE of the OsSWEET13 promoter in MS14K to creat a new germplasm with three edited OsSWEET EBEs and broad-spectrum resistance against all Xoo strains tested.Collectively,our findings illustrate how to disarm TALE-S co-evolved loci to generate broad-spectrum resistance through the loss of effector-triggered susceptibility in plants.
基金supported by grants from the National Natural Science Foundation of China(31822041,31972225)the National Natural Science Foundation of China(U20A2021)to R.W+1 种基金the National Natural Science Foundation of China(31801692)to F.Zthe National Key Research and Development Program of China(2016YFD0100600)to Y.N.
文摘Rice blast and bacterial blight are important diseases of rice(Oryza sativa)caused by the fungus Magnaporthe oryzae and the bacterium Xanthomonas oryzae pv.oryzae(Xoo),respectively.Breeding rice varieties for broadspectrum resistance is considered the most effective and sustainable approach to controlling both diseases.Although dominant resistance genes have been extensively used in rice breeding and production,generating diseaseresistant varieties by altering susceptibility(S)genes that facilitate pathogen compatibility remains unexplored.Here,using CRISPR/Cas9 technology,we generated loss-of-function mutants of the S genes Pi21 and Bsr-d1 and showed that they had increased resistance to M.oryzae.We also generated a knockout mutant of the S gene Xa5 that showed increased resistance to Xoo.Remarkably,a triple mutant of all three S genes had significantly enhanced resistance to both M.oryzae and Xoo.Moreover,the triple mutant was comparable to the wild type in regard to key agronomic traits,including plant height,effective panicle number per plant,grain number per panicle,seed setting rate,and thousand-grain weight.These results demonstrate that the simultaneous editing of multiple S genes is a powerful strategy for generating new rice varieties with broadspectrum resistance.
基金supported by grants from the National Natural Science Foundation of China(grant nos.31821005,31772145,and 31200912)the China Scholarship Council(file no.201908420054).
文摘Plants have developed various mechanisms for avoiding pathogen invasion,including resistance(R)genes.Most R genes encode nucleotide-binding domain and leucine-rich repeat containing proteins(NLRs).Here,we report the isolation of three new bacterial blight R genes in rice,Xa1-2,Xa14,and Xa31(t),which were allelic to Xa1 and encoded atypical NLRs with unique central tandem repeats(CTRs).We also found that Xa31(t)was the same gene as Xa1-2.Although Xa1-2 and Xa14 conferred different resistance spectra,their performance could be attenuated by iTALEs,as has previously been reported for Xa1.XA1,XA1-2,XA14,and non-resistant RGAF differed mainly in the substructure of the leucine-rich repeat domain.They all contained unique CTRs and belonged to the CTR-NLRs,which existed only in Gramineae.We also found that interactions among these genes led to differing resistance performance.In conclusion,our results uncover a unique locus in rice consisting of at least three multiple alleles(Xa1,Xa1-2,and Xa14)that encode CTRNLRs and confer resistance to Xanthomonas oryzae pv.oryzae(Xoo).
基金This work was supported by the National Natural Science Foundation of China(31830072)the National Key Research and Development Program of China(2016YFD0100601)the National Transgenic Major Program(2016ZX08001-002).
文摘Xanthomonas oryzae pv.oryzae(Xoo),the causal agent of bacterial leaf blight in rice,delivers transcription activator-like effector(TALE)proteins into host cells to activate susceptibility or resistance(R)genes that promote disease or immunity,respectively.Nonhost plants serve as potential reservoirs of R genes;consequently,nonhost R genes may trap TALEs to trigger an immune response.In this study,we screened 17 Xoo TALEs for their ability to induce a hypersensitive response(HR)in the nonhost plant Nicotiana benthamiana(Nb);only AvrXa10 elicited an HR when transiently expressed in Nb.The HR generated by AvrXa10 required both the central repeat region and the activation domain,suggesting a specific interaction between AvrXa10 and a potential R-like gene in nonhost plants.Evans blue staining and ion leakage measurements confirmed that the AvrXa10-triggered HR was a form of cell death,and the transient expression of AvrXa10 in Nb induced immune responses.Genes targeted by AvrXa10 in the Nb genome were identified by transcriptome profiling and prediction of effector binding sites.Using several approaches(in vivo reporter assays,electrophoretic mobility-shift assays,targeted designer TALEs,and on-spot gene silencing),we confirmed that AvrXa10 targets NbZnFP1,a C2H2-type zinc finger protein that resides in the nucleus.Functional analysis indicated that overexpression of NbZnFP1 and its rice orthologs triggered cell death in rice protoplasts.An NbZnFP1 ortholog was also identified in tomato and was specifically activated by AvrXa10.These results demonstrate that NbZnFP1 is a nonhost R gene that traps AvrXa10 to promote plant immunity in Nb.
基金supported by the following grants to P.R.:NIH no.GM59962,NIH no.GM122968,NSF no.1237975,NSF IOS-1656501,and NSF-NIFA no.2017-03128supported by the following grant to T.-C.C.:a Taiwan Government Scholarship.Support for M.S.+1 种基金provided by the Corteva Open Innovations Programsupported by the Office of Science,Office of Biological and Environmental Research of the U.S.Department of Energy under contract no.DE-AC02-05CH11231.
文摘XA21 encodes a rice immune receptor that confers robust resistance to most strains of the Gram-negative bacterium Xanthomonas oryzae pv.oryzae(Xoo).XA21-mediated immunity is triggered by recognition of a small protein called RaxX-sY(required for activation of XA21-mediated immunity X,tyrosine-sulfated)secreted by Xoo.To identify components regulating XA21-mediated immunity,we generated and screened a mutant population of fast-neutron-mutagenized rice expressing Ubi:Myc-XA21 for those susceptible to Xoo.Here,we report the characterization of one of these rice mutants,named sxi2(suppressor of XA21-mediated immunity-2).Whole-genome sequencing revealed that sxi2 carries a deletion of the PALADIN(PALD)gene encoding a protein with three putative protein tyrosine phosphatase-like domains(PTP-A,-B,and-C).Expression of PALD in the sxi2 genetic background was sufficient to complement the susceptible phenotype,which requires the catalytic cysteine of the PTP-A active site to restore resistance.PALD coimmunoprecipitated with the full-length XA21 protein,whose levels are positively regulated by the presence of the PALD transgene.Furthermore,we foundd that sxi2 retains many hallmarks of XA21-mediated immunity,similar to the wild type.These results reveal that PALD,a previously uncharacterized class of phosphatase,functions in rice innate immunity,and suggest that the conserved cysteine in the PTP-A domain of PALD is required for its immune function.
