Magnaporthe oryzae is the causal agent of rice blast. Glycosylation plays key roles in vegetative growth,development, and infection of M. oryzae. However, several glycosylation-related genes have not been characterize...Magnaporthe oryzae is the causal agent of rice blast. Glycosylation plays key roles in vegetative growth,development, and infection of M. oryzae. However, several glycosylation-related genes have not been characterized.In this study, we identified a Glyco_transf_22 domain-containing protein, MoAlg9, and found that MoAlg9 islocalized to the endoplasmic reticulum(ER). Deletion of MoALG9 significantly affected conidial production, normalappressorium formation, responses to stressors, and pathogenicity of M. oryzae. We also found that the ΔMoalg9mutant was defective in glycogen utilization, appressorial penetration, and invasive growth in host cells. Moreover,we further demonstrated that MoALG9 regulates the transcription of several target genes involved in conidiation,appressorium formation, and cell wall integrity. In addition, we found that the Glyco_transf_22 domain is essentialfor normal MoAlg9 function and localization. We also provide evidence that MoAlg9 is involved in N-glycosylationpathway in M. oryzae. Taken together, these results show that MoAlg9 is important for conidiation, appressoriumformation, maintenance of cell wall integrity, and the pathogenesis of M. oryzae.展开更多
Rice blast,caused by Magnaporthe oryzae,is a fungal disease that causes devastating damage to rice production worldwide.During infection,pathogens secrete effector proteins that modulate plant immunity.Disulfide bond ...Rice blast,caused by Magnaporthe oryzae,is a fungal disease that causes devastating damage to rice production worldwide.During infection,pathogens secrete effector proteins that modulate plant immunity.Disulfide bond formation catalyzed by protein disulfide isomerases(PDI)is essential for protein folding and maturation.However,the biological function of Pdi1 in M.oryzae has not yet been characterized.In this study,we identified the endoplasmic reticulum(ER)-located protein,MoPdi1,in M.oryzae.MoPdi1 regulates conidiation,cell wall stress,and pathogenicity of M.oryzae.Furthermore,the CGHC active sites in the a and a'redox domain of MoPdi1 were essential for the biological function of MoPDI1.Further tests demonstrated that MoPdi1 was involved in the regulation of ER stress and positively regulated ER phagy.We also found that MoPdi1 interacted with MoHut1.Deletion of MoPDI1 led to the bereft of MoHut1 dimerization,which depends on the formation of disulfide bonds.In addition,MoPdi1 affected the normal secretion of the cytoplasmic effector AVR-Pia.We provided evidence that MoHut1 is important for the vegetative growth,conidiation,and pathogenicity in M.oryzae.Therefore,our findings could provide a suitable target point for designing antifungal agrochemicals against rice blast fungus.展开更多
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
The fungal disease caused by Magnaporthe oryzae is one of the most devastating diseases that endanger many crops worldwide.Evidence shows that sexual reproduction can be advantageous for fungal diseases as hybridizati...The fungal disease caused by Magnaporthe oryzae is one of the most devastating diseases that endanger many crops worldwide.Evidence shows that sexual reproduction can be advantageous for fungal diseases as hybridization facilitates host-jumping.However,the pervasive clonal lineages of M.oryzae observed in natural fields contradict this expectation.A better understanding of the roles of recombination and the fungi-specific repeat-induced point mutation(RIP)in shaping its evolutionary trajectory is essential to bridge this knowledge gap.Here we systematically investigate the RIP and recombination landscapes in M.oryzae using a whole genome sequencing data from 252 population samples and 92 cross progenies.Our data reveal that the RIP can robustly capture the population history of M.oryzae,and we provide accurate estimations of the recombination and RIP rates across different M.oryzae clades.Significantly,our results highlight a parent-of-origin bias in both recombination and RIP rates,tightly associating with their sexual potential and variations of effector proteins.This bias suggests a critical trade-off between generating novel allelic combinations in the sexual cycle to facilitate host-jumping and stimulating transposon-associated diversification of effectors in the asexual cycle to facilitate host coevolution.These findings provide unique insights into understanding the evolution of blast fungus.展开更多
The conserved DNA damage repair complex,MMS21-SMC5/6(Methyl methane sulfonate 21-Structural maintenance of chromosomes 5/6),has been extensively studied in yeast,animals,and plants.However,its role in phytopathogenic ...The conserved DNA damage repair complex,MMS21-SMC5/6(Methyl methane sulfonate 21-Structural maintenance of chromosomes 5/6),has been extensively studied in yeast,animals,and plants.However,its role in phytopathogenic fungi,particularly in the highly destructive rice blast fungus Magnaporthe oryzae,remains unknown.In this study,we functionally characterized the homologues of this complex,MoMMS21 and MoSMC5,in M.oryzae.We first demonstrated the importance of DNA damage repair in M.oryzae by showing that the DNA damage inducer phleomycin inhibited vegetative growth,infection-related development and pathogenicity in this fungus.Additionally,we discovered that MoMMS21 and MoSMC5 interacted in the nuclei,suggesting that they also function as a complex in M.oryzae.Gene deletion experiments revealed that both MoMMS21 and MoSMC5 are required for infection-related development and pathogenicity in M.oryzae,while only MoMMS21 deletion affected growth and sensitivity to phleomycin,indicating its specific involvement in DNA damage repair.Overall,our results provide insights into the roles of MoMMS21 and MoSMC5 in M.oryzae,highlighting their functions beyond DNA damage repair.展开更多
Eighteen blast isolates were obtained from hybrid combination Wuyou308 using the Magnaporthe oryzae pathogen isolation method.Race identification of these isolates was conducted based on seven Chinese blast differenti...Eighteen blast isolates were obtained from hybrid combination Wuyou308 using the Magnaporthe oryzae pathogen isolation method.Race identification of these isolates was conducted based on seven Chinese blast differentials and 11 blast monogenic lines.The results indicated that the isolates were identified as the races of ZB13,ZB15 and ZC13,accounting for 66.67%,27.78%,5.56%,respectively,and the resistance genes including Pi-ta2 and Pi-sh,Pi-i were highly susceptible to these isolates,while the resistance genes like Pi-kh,Pi-1,Pi2,Pi-9 and Pi-50 showed good resistance to tested pathogens.All isolates were compatible to the original rice hybrid Wuyou308.Three isolates including GDHY-308-1401 were used for testing their pathogenicity to 45 local varieties.The results demonstrated that 13 varieties appeared highly susceptible to the tested isolates,accounting for 28.89%;two varieties appeared moderately susceptible to the tested isolates,accounting for 4.44%;30 varieties showed moderately/highly resistance,accounting for 66.67%.Among them,some of new hybrid combinations such as Wufengyou 9802,Wuyou 613,Wuyou 1179 showed good resistance to the inoculated strains,and they were recommended to be candidates in the rice region where Wuyou308 showed susceptibility.展开更多
By mRNA differential display, eight induced cDNAs were obtained from rice leaves infected with an incompatible race 131 of Magnaporthe grisea, and one of these cDNAs was highly similar to salt-induced mannose-binding ...By mRNA differential display, eight induced cDNAs were obtained from rice leaves infected with an incompatible race 131 of Magnaporthe grisea, and one of these cDNAs was highly similar to salt-induced mannose-binding lectin gene. Using this fragment as a probe, a full length cDNA was isolated from a nice cDNA library, which was constructed using mRNA from the incompatible race-infected leaves. Sequence analysis indicates that the cDNA encodes a protein of 15 kD with 145 amino, acids and shares 96% identity at nucleotide level with MRL and salT, but is identical to MRL at amino acid level. Genomic Southern blotting shows that there are two mannose-binding lectin genes in rice genome. Northern blotting analysis indicates that the gene was strongly and specifically induced in rice leaves infected with the incompatible race, suggesting that the lectin induction be involved in the defense of rice to M. grisea.展开更多
In this study the MTP1 gene, encoding a type III integral transmembrane protein, was isolated from the rice blast fungus Magnaporthe oryzae. The Mtp 1 protein is 520 amino acids long and is comparable to the Ytp 1 pro...In this study the MTP1 gene, encoding a type III integral transmembrane protein, was isolated from the rice blast fungus Magnaporthe oryzae. The Mtp 1 protein is 520 amino acids long and is comparable to the Ytp 1 protein of Saccharomyces cerevisiae with 46% sequence similarity. Prediction programs and MTP1-GFP （green fluorescent protein） fusion expression results indicate that Mtp 1 is a protein located at several membranes in the cytoplasm. The functions of the MTP1 gene in the growth and development of the fungus were studied using an MTP1 gene knockout mutant. The MTP1 gene was primarily expressed at the hyphal and conidial stages and is necessary for conidiation and conidial germination, but is not required for pathogenicity. The Amtpl mutant grew more efficiently than the wild type strain on non-fermentable carbon sources, implying that the MTP1 gene has a unique role in respiratory growth and carbon source use.展开更多
Single-spore isolates were obtained from rice-growing fields of Yuan＇an in Hubei Province where rice blast seriously occurs in some years. DNA fingerprints were divided into 112 haplotypes and 14 lineages at 73% gene...Single-spore isolates were obtained from rice-growing fields of Yuan＇an in Hubei Province where rice blast seriously occurs in some years. DNA fingerprints were divided into 112 haplotypes and 14 lineages at 73% genetic similarity level. Among the lineages, no dominant lineages were found. The population genetic structures of Magnaporthe oryzae were not distinctly different in different years. The analysis also showed that there wasn＇t obvious simple relationship between patho- types and fingerprint groups.展开更多
Rice blast, caused by Magnaporthe oryzae, is one of the most devastating diseases. Using map-based strategy and in silico approach we isolated a new rice （Oryza sativa L.） blast resistance allele of Pid3, designated...Rice blast, caused by Magnaporthe oryzae, is one of the most devastating diseases. Using map-based strategy and in silico approach we isolated a new rice （Oryza sativa L.） blast resistance allele of Pid3, designated Pi25, from a stable blast resistance cultivar Gumei2. Overexpression analysis and complementation test showed that Pi25 conferred blast resistance to M. oryzae isolate js001-20. Sequence analysis showed that Pi25 was an intronless gene of 2772 nucleotides with single nucleotide substitution in comparison to Pid3 at the nucleotide position 459 and predicatively encoded a typical coiled coil-nucleotide binding site-leucine rich repeat （CC-NBS-LRR） protein of 924 amino acid residuals with 100% identity to Pid3 putative protein. The susceptible allele pi25 in Nipponbare contained a nonsense mutation at the nucleotide position 2209 resulting in a truncated protein with 736 amino acid residuals. In addition, 14 nucleotide substitutions resulting in 10 amino acid substitutions were identified between Pi25 and pi25 upstream the premature stop codon in the susceptible allele. Although the mechanism of Pi25/Pid3-mediated resistance needs to be further investigated, the isolation of the allele would facilitate the utilization of Pi25/Pid3 in rice blast resistance breeding program via transgenic approach and marker assisted selection.展开更多
The peroxisomal matrix proteins involved in many important biological metabolism pathways in eukaryotic cells are encoded by nucleal genes, synthesized in the cytoplasm and then transported into the organelles. Target...The peroxisomal matrix proteins involved in many important biological metabolism pathways in eukaryotic cells are encoded by nucleal genes, synthesized in the cytoplasm and then transported into the organelles. Targeting and import of these proteins depend on their two peroxisomal targeting signals （PTS 1 and PTS2） in sequence as we have known so far. The vectors of the fluorescent fusions with PTS, i.e., green fluorescence protein （GFP）-PTS1, GFP-PTS2 and red fluorescence protein （RFP）-PTS1, were constructed and introduced into Magnaporthe oryzae Guy ll cells. Transformants containing these fusions emitted fluorescence in a punctate pattern, and the locations of the red and green fluorescence overlapped exactly in RFP-PTS 1 and GFP-PTS2 co-transformed strains. These data indicated that both PTS1 and PTS2 fusions were imported into peroxisomes. A probable higher efficiency of PTS1 machinery was revealed by comparing the fluorescence backgrotmds in GFP-PTS1 and GFP-PTS2 transformants. By introducing both RFP-PTS1 and GFP-PTS2 into Amgpex6 mutants, the involvement of MGPEX6 gene in both PTS1 and PTS2 pathways was proved. In addition, using these transformants, the inducement ofperoxisomes and the dynamic of peroxisomal number during the pre-penetration processes were investigated as well. In summary, by the localization and co-localization of PTS1 and PTS2, we provided a useful tool to evaluate the biological roles of the peroxisomes and the related genes.展开更多
A mature appressorium cDNA library of rice blast fungus, Magnaporthe grisea, was constructed in a λTriplEx2 vector by SMART?cDNA library containing 2.37×106 independent clones about 100% of which harbor foreign ...A mature appressorium cDNA library of rice blast fungus, Magnaporthe grisea, was constructed in a λTriplEx2 vector by SMART?cDNA library containing 2.37×106 independent clones about 100% of which harbor foreign cDNA inserts with average size of 660 bp. Of 9 randomly selected clones, 2 expressed sequence tags (ESTs) sequences did not have homologous EST sequences of M grisea in GenBank. The appressorium cDNA library is suitable for gene expression analysis and function analysis of the late stages of appressorium formation and the early stages of penetration of M grisea.展开更多
Wheat blast,caused by the fungus Magnaporthe oryzae Triticum(MoT)pathotype,is a devastating disease persistent in South America and Bangladesh.Since MoT generally fails to cause visual symptoms in wheat until the head...Wheat blast,caused by the fungus Magnaporthe oryzae Triticum(MoT)pathotype,is a devastating disease persistent in South America and Bangladesh.Since MoT generally fails to cause visual symptoms in wheat until the heading stage when the infection would have advanced,disease control by fungicide application solely based on the detection of visual symptoms is ineffective.To develop an accurate and sensitive method to detect MoT at the seedling and vegetative stages for disease control,we sequenced the genomes of two MoT isolates from Brazil and identified two DNA fragments,MoT-6098 and MoT-6099,that are present in the MoT genome but not in the genome of the rice-infecting Magnaporthe oryzae Oryzae(MoO)pathotype.Using polymerase chain reaction(PCR),we confirmed the specificity of the two markers in 53 MoT and MoO isolates from South America and Bangladesh.To test the efficiency of the two markers,we first established a loop-mediated isothermal amplification(LAMP)method to detect MoT at isothermal conditions,without the use of a PCR machine.Following this,we used the Cas12a protein and guide RNAs(gRNAs)to target the MoT-6098 and MoT-6099 sequences.The activated Cas12a showed indiscriminate single-stranded deoxyribonuclease(ssDNase)activity.We then combined targetdependent Cas12a ssDNase activation with recombinase polymerase amplification(RPA)and nucleic acid lateral flow immunoassay(NALFIA)to develop a method that accurately,sensitively,and cost-effectively detects MoT-specific DNA sequences in infected wheat plants.This novel technique can be easily adapted for the rapid detection of wheat blast and other important plant diseases in the field.展开更多
377 isolates of Magnaporthe grisea were collected from 17 provinces in China and their geographic distribution of mating types and their fertility was tested with four standard isolates, KA3 and TH12 (Mat1.1) and Guy1...377 isolates of Magnaporthe grisea were collected from 17 provinces in China and their geographic distribution of mating types and their fertility was tested with four standard isolates, KA3 and TH12 (Mat1.1) and Guy11 and TH16 (Mat1.2) provided by CIRAD. 73 fertile isolates were tested with SCAR markers of 13 pairs of primers. Preliminary results showed that the geographic distribution of M.grisea existed among isolates collected from the same location as well as different locations and the genetic relationship between fertile isolates of the fungus in China. The existence of sexual reproduction of M .grisea was explored in the field as well.展开更多
Phenolamide(PA)metabolites play important roles in the interaction between plants and pathogens.The putrescine hydroxycinnamoyl transferase genes Os PHT3 and Os PHT4 positively regulate rice cell death and resistance ...Phenolamide(PA)metabolites play important roles in the interaction between plants and pathogens.The putrescine hydroxycinnamoyl transferase genes Os PHT3 and Os PHT4 positively regulate rice cell death and resistance to Magnaporthe oryzae.The b ZIP transcription factor APIP5,a negative regulator of cell death and rice immunity,directly binds to the Os PHT4 promoter to regulate putrescine-derived PAs.Whether other hydroxycinnamoyl transferase(HT)genes also participate in APIP5-mediated immunity remains unclear.Surprisingly,we find that genes encoding agmatine hydroxycinnamoyl transferases Os AHT1 and Os AHT2,tryptamine hydroxycinnamoyl transferases Os TBT1 and Os TBT2,and tyramine hydroxycinnamoyl transferases Os THT1 and Os THT2,responsible for the biosynthesis of polyamine-derived PAs are all up-regulated in APIP5-RNAi transgenic plants compared with segregated wild-type rice.Furthermore,both Os AHT1/2 and Os TBT1/2 are induced during M.oryzae infection,showing expression patterns similar to those previously reported for Os THT1/2 and Os PHT3/4.Transgenic plants overexpressing either Os AHT2-GFP or Os TBT1-GFP show enhanced resistance against M.oryzae and accumulated more PA metabolites and lignin compared with wild-type plants.Interestingly,as demonstrated for Os PHT4,APIP5 directly binds to the promoters of Os AHT1/2,Os TBT1/2,and Os THT1/2,repressing their transcription.Together,these results indicate that the HT genes are common targets of APIP5 and that PAs play critical roles in rice immunity.展开更多
Magnaporthe oryzae has been used as a primary model organism for investigating fungus-plant interaction. Many researches focused on molecular mechanisms of appressorium formation to restrain this fungal pathogen. Auto...Magnaporthe oryzae has been used as a primary model organism for investigating fungus-plant interaction. Many researches focused on molecular mechanisms of appressorium formation to restrain this fungal pathogen. Autophagy is a very high conserved process in eukaryotic cells. Recently, autophagy has been considered as a key process in development and differentia-tion in M. oryzae. In this report, we present and discuss the current state of our knowledge on gene expression in appressorium formation and the progress in autophagy of rice blast fungi.展开更多
Appressorium formation is an important event in establishing a successful interaction between the rice blast fungus, Magnaporthe oryzae, and its host plant, rice. An understanding of molecular events occurring in appr...Appressorium formation is an important event in establishing a successful interaction between the rice blast fungus, Magnaporthe oryzae, and its host plant, rice. An understanding of molecular events occurring in appressorium differentiation will give new strategies to control rice blast. A quick and reliable method to extract total RNA from appressorium is essential for studying gene expression during appressorium formation and its mechanism. We found that duplicate film is an efficient substratum for appressorium formation, even when inoculated with high density conidia. When inoculated with conidia at 1 × 106 ml^-1, the percentages of conidium germination and appressorium formation were （97.98±0.67）% and （97.88±0.45）%, respectively. We applied Trizol before appressorium collection for total RNA isolation, and as much as 113.6 lag total RNA was isolated from the mature appressoria at 24 h after inoculation. Functional analysis of two genes, MNH6 and MgATG1, isolated from the cDNA subtractive library, revealed that the quantity of RNA was good enough to construct a cDNA （complementary DNA） library or a cDNA subtractive library. This method may be also applicable for the appressorium RNA isolation of other pathogenic fungi in which conidia differentiate into appressoria in the early stages of host infection.展开更多
Plants utilize multiple layers of defense mechanisms to fight against the invasion of diverse pathogens.The R gene mediates resistance,in most cases,dependent on the co-existence of its cognate pathogen-derived avirul...Plants utilize multiple layers of defense mechanisms to fight against the invasion of diverse pathogens.The R gene mediates resistance,in most cases,dependent on the co-existence of its cognate pathogen-derived avirulence (Avr) gene.The rice blast R gene Piz-t corresponds in gene-for-gene fashion to the Magnaporthe oryzae Avr gene AvrPiz-t.In this study,we determined and compared the genomic sequences surrounding the AvrPiz-t gene in both avirulent and virulent isolates,designating as AvrPiz-t-ZB15 and avrPiz-t-70-15 regions,respectively.The sequence of the AvrPiz-t-ZB15 region is 120966 bp whereas avrPiz-t-70-15 is 146292 bp in length.The extreme sequence similarity and good synteny in gene order and content along with the absence of two predicted genes in the avrPiz-t-70-15 region were observed in the predicted protein-coding regions in the AvrPiz-t locus.Nevertheless,frequent presence/absence and highly dynamic organization of transposable elements (TEs) were identified,representing the major variation of the AvrPiz-t locus between different isolates.Moreover,TEs constitute 27.3% and 43.2% of the genomic contents of the AvrPiz-t-ZB15 and avrPiz-t-70-15 regions,respectively,indicating that TEs contribute largely to the organization and evolution of AvrPiz-t locus.The findings of this study suggest that M.oryzae could benefit in an evolutionary sense from the presence of active TEs in genes conferring avirulence and provide an ability to rapidly change and thus to overcome host R genes.展开更多
Appressorium is an infection structure of the phytopathogenic fungus Magnaporthe grisea. Analysis of gene expression profiles ofappressorium development provides insight into the molecular basis of pathogenicity and c...Appressorium is an infection structure of the phytopathogenic fungus Magnaporthe grisea. Analysis of gene expression profiles ofappressorium development provides insight into the molecular basis of pathogenicity and control of this fungal plant disease. A cDNA array representing 2927 unique genes based on a large EST （expressed sequence tag） database ofM. grisea strain Y34 was constructed and used to profile the gene expression patterns at mycelium and appressorium maturation stages. Compared with mycelia, 55 up-regulated and 22 down-regulated genes were identified in mature appressoria. Among 77 genes, 16 genes showed no similarity to the genome sequences of M. grisea. A novel homologue of peptidyl-prolyl cis-trans isomerase was found to be expressed at low-level in mature appressoria of M. grisea. The results indicated that the genes such as pyruvate carboxylase, phospholipid metabolism-related protein and glyceraldehyde 3-phosphate dehydrogenase involved in gluconeogenesis, lipid metabolism and glycolysis, showed differential expression in mature appressoria. Furthermore, genes such as PTHll, beta subunit of G protein and SGTI involved in cell signalling, were expressed differentially in mature appressoria. Northern blot analysis was used to confirm the cDNA array results.展开更多
Jacalin-related lectins （JRLs） are widely distributed carbohydrate-binding proteins in the plant kingdom, which play key roles in development and pathogen defense. In this study, we profiled evolutionary trajectory ...Jacalin-related lectins （JRLs） are widely distributed carbohydrate-binding proteins in the plant kingdom, which play key roles in development and pathogen defense. In this study, we profiled evolutionary trajectory of JRLs family in 30 plant species and identified domain diversification and recombination leading to different responsive patterns of JRLs in rice during defense against rice blast. All of 30 plant species analyzed in our study have two types of JRLs by containing either a single jacalin or repeated jacalin domains, while chimeric jacalins exist in more than half of the species, especially in the Poaceae family. Moreover, Poaceae species have evolved two types of unique chimeric JRLs by fusing the jacalin domain（s） with dirigent or NB_ARC domain, some of which positively regulate plant immunity. Seven Poaceae-specific JRLs are found in the rice genome. We further found expression of rice JRLs, including four Poaceae-specific JRLs, are induced by Magnaporthe oryzae infections at either early or late infection stages. Overall, the results present the evolutionary trajectory of JRLs in plant and highlight essential roles of Poaceae specific JRLs against pathogen attacks in rice.展开更多
基金supported by the National Natural Science Foundation of China (32202253)the Natural Science Foundation of Anhui Higher Education Institutions, China (KJ2020A0102)the Talent Research Project of Anhui Agricultural University, China (rc342001)。
文摘Magnaporthe oryzae is the causal agent of rice blast. Glycosylation plays key roles in vegetative growth,development, and infection of M. oryzae. However, several glycosylation-related genes have not been characterized.In this study, we identified a Glyco_transf_22 domain-containing protein, MoAlg9, and found that MoAlg9 islocalized to the endoplasmic reticulum(ER). Deletion of MoALG9 significantly affected conidial production, normalappressorium formation, responses to stressors, and pathogenicity of M. oryzae. We also found that the ΔMoalg9mutant was defective in glycogen utilization, appressorial penetration, and invasive growth in host cells. Moreover,we further demonstrated that MoALG9 regulates the transcription of several target genes involved in conidiation,appressorium formation, and cell wall integrity. In addition, we found that the Glyco_transf_22 domain is essentialfor normal MoAlg9 function and localization. We also provide evidence that MoAlg9 is involved in N-glycosylationpathway in M. oryzae. Taken together, these results show that MoAlg9 is important for conidiation, appressoriumformation, maintenance of cell wall integrity, and the pathogenesis of M. oryzae.
基金supported by the National Natural Science Foundation of China(32202253)the Natural Science Foundation of Anhui Higher Education Institutions,China(KJ2020A0102)the Talent Research Project of Anhui Agricultural University,China(rc342001)。
文摘Rice blast,caused by Magnaporthe oryzae,is a fungal disease that causes devastating damage to rice production worldwide.During infection,pathogens secrete effector proteins that modulate plant immunity.Disulfide bond formation catalyzed by protein disulfide isomerases(PDI)is essential for protein folding and maturation.However,the biological function of Pdi1 in M.oryzae has not yet been characterized.In this study,we identified the endoplasmic reticulum(ER)-located protein,MoPdi1,in M.oryzae.MoPdi1 regulates conidiation,cell wall stress,and pathogenicity of M.oryzae.Furthermore,the CGHC active sites in the a and a'redox domain of MoPdi1 were essential for the biological function of MoPDI1.Further tests demonstrated that MoPdi1 was involved in the regulation of ER stress and positively regulated ER phagy.We also found that MoPdi1 interacted with MoHut1.Deletion of MoPDI1 led to the bereft of MoHut1 dimerization,which depends on the formation of disulfide bonds.In addition,MoPdi1 affected the normal secretion of the cytoplasmic effector AVR-Pia.We provided evidence that MoHut1 is important for the vegetative growth,conidiation,and pathogenicity in M.oryzae.Therefore,our findings could provide a suitable target point for designing antifungal agrochemicals against rice blast fungus.
基金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funded by the National Natural Science Foundation of China(32270664 and 32170327)the National Key Research and Development Program of China(2023YFD2200102 and 2023YFD2200104)Jiangsu Collaborative Innovation Center for Modern Crop Production。
文摘The fungal disease caused by Magnaporthe oryzae is one of the most devastating diseases that endanger many crops worldwide.Evidence shows that sexual reproduction can be advantageous for fungal diseases as hybridization facilitates host-jumping.However,the pervasive clonal lineages of M.oryzae observed in natural fields contradict this expectation.A better understanding of the roles of recombination and the fungi-specific repeat-induced point mutation(RIP)in shaping its evolutionary trajectory is essential to bridge this knowledge gap.Here we systematically investigate the RIP and recombination landscapes in M.oryzae using a whole genome sequencing data from 252 population samples and 92 cross progenies.Our data reveal that the RIP can robustly capture the population history of M.oryzae,and we provide accurate estimations of the recombination and RIP rates across different M.oryzae clades.Significantly,our results highlight a parent-of-origin bias in both recombination and RIP rates,tightly associating with their sexual potential and variations of effector proteins.This bias suggests a critical trade-off between generating novel allelic combinations in the sexual cycle to facilitate host-jumping and stimulating transposon-associated diversification of effectors in the asexual cycle to facilitate host coevolution.These findings provide unique insights into understanding the evolution of blast fungus.
基金Research and Development Program of China(2023YFD1400200)the Natural Science Foundation of Fujian Province,China(2022J01125)+2 种基金the Fujian Key Laboratory for Monitoring and Integrated Management of Crop Pests,China(MIMCP-202301)the Fujian Provincial Science and Technology Key Project,China(2022NZ030014)the National Natural Science Foundation of China(NSFC31871914).
文摘The conserved DNA damage repair complex,MMS21-SMC5/6(Methyl methane sulfonate 21-Structural maintenance of chromosomes 5/6),has been extensively studied in yeast,animals,and plants.However,its role in phytopathogenic fungi,particularly in the highly destructive rice blast fungus Magnaporthe oryzae,remains unknown.In this study,we functionally characterized the homologues of this complex,MoMMS21 and MoSMC5,in M.oryzae.We first demonstrated the importance of DNA damage repair in M.oryzae by showing that the DNA damage inducer phleomycin inhibited vegetative growth,infection-related development and pathogenicity in this fungus.Additionally,we discovered that MoMMS21 and MoSMC5 interacted in the nuclei,suggesting that they also function as a complex in M.oryzae.Gene deletion experiments revealed that both MoMMS21 and MoSMC5 are required for infection-related development and pathogenicity in M.oryzae,while only MoMMS21 deletion affected growth and sensitivity to phleomycin,indicating its specific involvement in DNA damage repair.Overall,our results provide insights into the roles of MoMMS21 and MoSMC5 in M.oryzae,highlighting their functions beyond DNA damage repair.
基金Supported by Project of Collaborative Innovation Center of GDAAS-XTXM202202(XT202211)The Introduction of Icientific and Iechnological Ialents of Guangdong Academy of Agricultural Sciences(R2021YJ-YB3020).
文摘Eighteen blast isolates were obtained from hybrid combination Wuyou308 using the Magnaporthe oryzae pathogen isolation method.Race identification of these isolates was conducted based on seven Chinese blast differentials and 11 blast monogenic lines.The results indicated that the isolates were identified as the races of ZB13,ZB15 and ZC13,accounting for 66.67%,27.78%,5.56%,respectively,and the resistance genes including Pi-ta2 and Pi-sh,Pi-i were highly susceptible to these isolates,while the resistance genes like Pi-kh,Pi-1,Pi2,Pi-9 and Pi-50 showed good resistance to tested pathogens.All isolates were compatible to the original rice hybrid Wuyou308.Three isolates including GDHY-308-1401 were used for testing their pathogenicity to 45 local varieties.The results demonstrated that 13 varieties appeared highly susceptible to the tested isolates,accounting for 28.89%;two varieties appeared moderately susceptible to the tested isolates,accounting for 4.44%;30 varieties showed moderately/highly resistance,accounting for 66.67%.Among them,some of new hybrid combinations such as Wufengyou 9802,Wuyou 613,Wuyou 1179 showed good resistance to the inoculated strains,and they were recommended to be candidates in the rice region where Wuyou308 showed susceptibility.
文摘By mRNA differential display, eight induced cDNAs were obtained from rice leaves infected with an incompatible race 131 of Magnaporthe grisea, and one of these cDNAs was highly similar to salt-induced mannose-binding lectin gene. Using this fragment as a probe, a full length cDNA was isolated from a nice cDNA library, which was constructed using mRNA from the incompatible race-infected leaves. Sequence analysis indicates that the cDNA encodes a protein of 15 kD with 145 amino, acids and shares 96% identity at nucleotide level with MRL and salT, but is identical to MRL at amino acid level. Genomic Southern blotting shows that there are two mannose-binding lectin genes in rice genome. Northern blotting analysis indicates that the gene was strongly and specifically induced in rice leaves infected with the incompatible race, suggesting that the lectin induction be involved in the defense of rice to M. grisea.
基金the National Natural Science Foundation of China (Nos. 30671351 and 30470064)the Natural Science Foun-dation of Zhejiang Province, China (No. Y304211)
文摘In this study the MTP1 gene, encoding a type III integral transmembrane protein, was isolated from the rice blast fungus Magnaporthe oryzae. The Mtp 1 protein is 520 amino acids long and is comparable to the Ytp 1 protein of Saccharomyces cerevisiae with 46% sequence similarity. Prediction programs and MTP1-GFP （green fluorescent protein） fusion expression results indicate that Mtp 1 is a protein located at several membranes in the cytoplasm. The functions of the MTP1 gene in the growth and development of the fungus were studied using an MTP1 gene knockout mutant. The MTP1 gene was primarily expressed at the hyphal and conidial stages and is necessary for conidiation and conidial germination, but is not required for pathogenicity. The Amtpl mutant grew more efficiently than the wild type strain on non-fermentable carbon sources, implying that the MTP1 gene has a unique role in respiratory growth and carbon source use.
基金Supported by Competitive Project of Hubei Academy of Agricultural Sciences(2016jzxjh010)Major Research and Development Program of China(2016YFD0200807-1)~~
文摘Single-spore isolates were obtained from rice-growing fields of Yuan＇an in Hubei Province where rice blast seriously occurs in some years. DNA fingerprints were divided into 112 haplotypes and 14 lineages at 73% genetic similarity level. Among the lineages, no dominant lineages were found. The population genetic structures of Magnaporthe oryzae were not distinctly different in different years. The analysis also showed that there wasn＇t obvious simple relationship between patho- types and fingerprint groups.
基金supported by the Zhejiang Natural Science Foundation(No.R307131 and No.Y3080528)the National High Technology Research and Development Program of China(No.2009AA101101)
文摘Rice blast, caused by Magnaporthe oryzae, is one of the most devastating diseases. Using map-based strategy and in silico approach we isolated a new rice （Oryza sativa L.） blast resistance allele of Pid3, designated Pi25, from a stable blast resistance cultivar Gumei2. Overexpression analysis and complementation test showed that Pi25 conferred blast resistance to M. oryzae isolate js001-20. Sequence analysis showed that Pi25 was an intronless gene of 2772 nucleotides with single nucleotide substitution in comparison to Pid3 at the nucleotide position 459 and predicatively encoded a typical coiled coil-nucleotide binding site-leucine rich repeat （CC-NBS-LRR） protein of 924 amino acid residuals with 100% identity to Pid3 putative protein. The susceptible allele pi25 in Nipponbare contained a nonsense mutation at the nucleotide position 2209 resulting in a truncated protein with 736 amino acid residuals. In addition, 14 nucleotide substitutions resulting in 10 amino acid substitutions were identified between Pi25 and pi25 upstream the premature stop codon in the susceptible allele. Although the mechanism of Pi25/Pid3-mediated resistance needs to be further investigated, the isolation of the allele would facilitate the utilization of Pi25/Pid3 in rice blast resistance breeding program via transgenic approach and marker assisted selection.
基金the National Natural Science Foundation of China (Nos. 30671351 and 30810033the Natural Science Foundation of Zhejiang Province of China (No. Y306638)
文摘The peroxisomal matrix proteins involved in many important biological metabolism pathways in eukaryotic cells are encoded by nucleal genes, synthesized in the cytoplasm and then transported into the organelles. Targeting and import of these proteins depend on their two peroxisomal targeting signals （PTS 1 and PTS2） in sequence as we have known so far. The vectors of the fluorescent fusions with PTS, i.e., green fluorescence protein （GFP）-PTS1, GFP-PTS2 and red fluorescence protein （RFP）-PTS1, were constructed and introduced into Magnaporthe oryzae Guy ll cells. Transformants containing these fusions emitted fluorescence in a punctate pattern, and the locations of the red and green fluorescence overlapped exactly in RFP-PTS 1 and GFP-PTS2 co-transformed strains. These data indicated that both PTS1 and PTS2 fusions were imported into peroxisomes. A probable higher efficiency of PTS1 machinery was revealed by comparing the fluorescence backgrotmds in GFP-PTS1 and GFP-PTS2 transformants. By introducing both RFP-PTS1 and GFP-PTS2 into Amgpex6 mutants, the involvement of MGPEX6 gene in both PTS1 and PTS2 pathways was proved. In addition, using these transformants, the inducement ofperoxisomes and the dynamic of peroxisomal number during the pre-penetration processes were investigated as well. In summary, by the localization and co-localization of PTS1 and PTS2, we provided a useful tool to evaluate the biological roles of the peroxisomes and the related genes.
文摘A mature appressorium cDNA library of rice blast fungus, Magnaporthe grisea, was constructed in a λTriplEx2 vector by SMART?cDNA library containing 2.37×106 independent clones about 100% of which harbor foreign cDNA inserts with average size of 660 bp. Of 9 randomly selected clones, 2 expressed sequence tags (ESTs) sequences did not have homologous EST sequences of M grisea in GenBank. The appressorium cDNA library is suitable for gene expression analysis and function analysis of the late stages of appressorium formation and the early stages of penetration of M grisea.
基金The data that support the findings of this study have being submitted to GenBank and the accession numbers are JAAXMV000000000 and JAAXMU000000000.
文摘Wheat blast,caused by the fungus Magnaporthe oryzae Triticum(MoT)pathotype,is a devastating disease persistent in South America and Bangladesh.Since MoT generally fails to cause visual symptoms in wheat until the heading stage when the infection would have advanced,disease control by fungicide application solely based on the detection of visual symptoms is ineffective.To develop an accurate and sensitive method to detect MoT at the seedling and vegetative stages for disease control,we sequenced the genomes of two MoT isolates from Brazil and identified two DNA fragments,MoT-6098 and MoT-6099,that are present in the MoT genome but not in the genome of the rice-infecting Magnaporthe oryzae Oryzae(MoO)pathotype.Using polymerase chain reaction(PCR),we confirmed the specificity of the two markers in 53 MoT and MoO isolates from South America and Bangladesh.To test the efficiency of the two markers,we first established a loop-mediated isothermal amplification(LAMP)method to detect MoT at isothermal conditions,without the use of a PCR machine.Following this,we used the Cas12a protein and guide RNAs(gRNAs)to target the MoT-6098 and MoT-6099 sequences.The activated Cas12a showed indiscriminate single-stranded deoxyribonuclease(ssDNase)activity.We then combined targetdependent Cas12a ssDNase activation with recombinase polymerase amplification(RPA)and nucleic acid lateral flow immunoassay(NALFIA)to develop a method that accurately,sensitively,and cost-effectively detects MoT-specific DNA sequences in infected wheat plants.This novel technique can be easily adapted for the rapid detection of wheat blast and other important plant diseases in the field.
文摘377 isolates of Magnaporthe grisea were collected from 17 provinces in China and their geographic distribution of mating types and their fertility was tested with four standard isolates, KA3 and TH12 (Mat1.1) and Guy11 and TH16 (Mat1.2) provided by CIRAD. 73 fertile isolates were tested with SCAR markers of 13 pairs of primers. Preliminary results showed that the geographic distribution of M.grisea existed among isolates collected from the same location as well as different locations and the genetic relationship between fertile isolates of the fungus in China. The existence of sexual reproduction of M .grisea was explored in the field as well.
基金supported by grants from the National Natural Science Foundation of China (32161143009, 31822041 and 31972225) to Y.N.the National Natural Science Foundation of China (U20A2021) to R.W.the National Natural Science Foundation of China (31801692) to F.Z
文摘Phenolamide(PA)metabolites play important roles in the interaction between plants and pathogens.The putrescine hydroxycinnamoyl transferase genes Os PHT3 and Os PHT4 positively regulate rice cell death and resistance to Magnaporthe oryzae.The b ZIP transcription factor APIP5,a negative regulator of cell death and rice immunity,directly binds to the Os PHT4 promoter to regulate putrescine-derived PAs.Whether other hydroxycinnamoyl transferase(HT)genes also participate in APIP5-mediated immunity remains unclear.Surprisingly,we find that genes encoding agmatine hydroxycinnamoyl transferases Os AHT1 and Os AHT2,tryptamine hydroxycinnamoyl transferases Os TBT1 and Os TBT2,and tyramine hydroxycinnamoyl transferases Os THT1 and Os THT2,responsible for the biosynthesis of polyamine-derived PAs are all up-regulated in APIP5-RNAi transgenic plants compared with segregated wild-type rice.Furthermore,both Os AHT1/2 and Os TBT1/2 are induced during M.oryzae infection,showing expression patterns similar to those previously reported for Os THT1/2 and Os PHT3/4.Transgenic plants overexpressing either Os AHT2-GFP or Os TBT1-GFP show enhanced resistance against M.oryzae and accumulated more PA metabolites and lignin compared with wild-type plants.Interestingly,as demonstrated for Os PHT4,APIP5 directly binds to the promoters of Os AHT1/2,Os TBT1/2,and Os THT1/2,repressing their transcription.Together,these results indicate that the HT genes are common targets of APIP5 and that PAs play critical roles in rice immunity.
基金the National Natural Science Foundation of China (Nos. 30671351 and 30870101)the Hi-Tech Research and Development Program (863) of China (No. 2002AA245041)
文摘Magnaporthe oryzae has been used as a primary model organism for investigating fungus-plant interaction. Many researches focused on molecular mechanisms of appressorium formation to restrain this fungal pathogen. Autophagy is a very high conserved process in eukaryotic cells. Recently, autophagy has been considered as a key process in development and differentia-tion in M. oryzae. In this report, we present and discuss the current state of our knowledge on gene expression in appressorium formation and the progress in autophagy of rice blast fungi.
基金Project (Nos. 30671351 and 30870101) supported by the National Natural Science Foundation of China
文摘Appressorium formation is an important event in establishing a successful interaction between the rice blast fungus, Magnaporthe oryzae, and its host plant, rice. An understanding of molecular events occurring in appressorium differentiation will give new strategies to control rice blast. A quick and reliable method to extract total RNA from appressorium is essential for studying gene expression during appressorium formation and its mechanism. We found that duplicate film is an efficient substratum for appressorium formation, even when inoculated with high density conidia. When inoculated with conidia at 1 × 106 ml^-1, the percentages of conidium germination and appressorium formation were （97.98±0.67）% and （97.88±0.45）%, respectively. We applied Trizol before appressorium collection for total RNA isolation, and as much as 113.6 lag total RNA was isolated from the mature appressoria at 24 h after inoculation. Functional analysis of two genes, MNH6 and MgATG1, isolated from the cDNA subtractive library, revealed that the quantity of RNA was good enough to construct a cDNA （complementary DNA） library or a cDNA subtractive library. This method may be also applicable for the appressorium RNA isolation of other pathogenic fungi in which conidia differentiate into appressoria in the early stages of host infection.
基金Project supported by the National Natural Science Foundation of China (Nos.30700450 and 30971878)the Fundamental Research Funds for the Central Universities (No.2009QNA6024),China
文摘Plants utilize multiple layers of defense mechanisms to fight against the invasion of diverse pathogens.The R gene mediates resistance,in most cases,dependent on the co-existence of its cognate pathogen-derived avirulence (Avr) gene.The rice blast R gene Piz-t corresponds in gene-for-gene fashion to the Magnaporthe oryzae Avr gene AvrPiz-t.In this study,we determined and compared the genomic sequences surrounding the AvrPiz-t gene in both avirulent and virulent isolates,designating as AvrPiz-t-ZB15 and avrPiz-t-70-15 regions,respectively.The sequence of the AvrPiz-t-ZB15 region is 120966 bp whereas avrPiz-t-70-15 is 146292 bp in length.The extreme sequence similarity and good synteny in gene order and content along with the absence of two predicted genes in the avrPiz-t-70-15 region were observed in the predicted protein-coding regions in the AvrPiz-t locus.Nevertheless,frequent presence/absence and highly dynamic organization of transposable elements (TEs) were identified,representing the major variation of the AvrPiz-t locus between different isolates.Moreover,TEs constitute 27.3% and 43.2% of the genomic contents of the AvrPiz-t-ZB15 and avrPiz-t-70-15 regions,respectively,indicating that TEs contribute largely to the organization and evolution of AvrPiz-t locus.The findings of this study suggest that M.oryzae could benefit in an evolutionary sense from the presence of active TEs in genes conferring avirulence and provide an ability to rapidly change and thus to overcome host R genes.
基金Project (No. 2002BA711A15) supported by the National Hi-Tech Research and Development Program (863) of China
文摘Appressorium is an infection structure of the phytopathogenic fungus Magnaporthe grisea. Analysis of gene expression profiles ofappressorium development provides insight into the molecular basis of pathogenicity and control of this fungal plant disease. A cDNA array representing 2927 unique genes based on a large EST （expressed sequence tag） database ofM. grisea strain Y34 was constructed and used to profile the gene expression patterns at mycelium and appressorium maturation stages. Compared with mycelia, 55 up-regulated and 22 down-regulated genes were identified in mature appressoria. Among 77 genes, 16 genes showed no similarity to the genome sequences of M. grisea. A novel homologue of peptidyl-prolyl cis-trans isomerase was found to be expressed at low-level in mature appressoria of M. grisea. The results indicated that the genes such as pyruvate carboxylase, phospholipid metabolism-related protein and glyceraldehyde 3-phosphate dehydrogenase involved in gluconeogenesis, lipid metabolism and glycolysis, showed differential expression in mature appressoria. Furthermore, genes such as PTHll, beta subunit of G protein and SGTI involved in cell signalling, were expressed differentially in mature appressoria. Northern blot analysis was used to confirm the cDNA array results.
基金funded by the National Key Research and Development Program of China(2016YFD0100600)the National Natural Science Foundation of China(U1405212)
文摘Jacalin-related lectins （JRLs） are widely distributed carbohydrate-binding proteins in the plant kingdom, which play key roles in development and pathogen defense. In this study, we profiled evolutionary trajectory of JRLs family in 30 plant species and identified domain diversification and recombination leading to different responsive patterns of JRLs in rice during defense against rice blast. All of 30 plant species analyzed in our study have two types of JRLs by containing either a single jacalin or repeated jacalin domains, while chimeric jacalins exist in more than half of the species, especially in the Poaceae family. Moreover, Poaceae species have evolved two types of unique chimeric JRLs by fusing the jacalin domain（s） with dirigent or NB_ARC domain, some of which positively regulate plant immunity. Seven Poaceae-specific JRLs are found in the rice genome. We further found expression of rice JRLs, including four Poaceae-specific JRLs, are induced by Magnaporthe oryzae infections at either early or late infection stages. Overall, the results present the evolutionary trajectory of JRLs in plant and highlight essential roles of Poaceae specific JRLs against pathogen attacks in rice.
