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
Only few glufosinate-tolerant genes,such as phosphinothricin acetyltransferase(PAT)and bialaphos resistance(bar)identified from Streptomyces,are currently available for developing genetically modified rice in agricult...Only few glufosinate-tolerant genes,such as phosphinothricin acetyltransferase(PAT)and bialaphos resistance(bar)identified from Streptomyces,are currently available for developing genetically modified rice in agricultural application.Following the rapid development of genome editing technology,generation of novel glufosinate-tolerant gene resources through artificial evolution of endogenous genes is more promising and highly desirable in rice molecular breeding program.In this study,the endogenous Glutamine synthetase1(OsGS1)was artificially evolved by base-editing-mediated gene evolution(BEMGE)in rice cells to create novel alleles conferring glufosinate tolerance in rice germplasms.Two novel glufosinate-tolerant OsGS1 alleles(OsGS1-AVPS and OsGS1-+AF)and one reported tolerant allele(OsGS1-SGTA)were successfully identified from approximately 4200 independent hygromycin-tolerant calli.Germination assays and spray tests revealed that these three OsGS1 alleles confer glufosinate tolerance in rice.Furthermore,OsGS1-AVPS and OsGS1-SGTA were quickly deployed into the elite rice cultivar Nangeng 46 through precise base editing.Overall,our results demonstrate the feasibility of developing glufosinate-tolerant rice by editing an endogenous rice gene in molecular breeding programs.展开更多
Plant viruses are a group of intracellular pathogens that persistently threaten global food security.Significant advances in plant virology have been achieved by Chinese scientists over the last 20 years,including bas...Plant viruses are a group of intracellular pathogens that persistently threaten global food security.Significant advances in plant virology have been achieved by Chinese scientists over the last 20 years,including basic research and technologies for preventing and controlling plant viral diseases.Here,we review these milestones and advances,including the identification of new crop-infecting viruses,dissection of pathogenic mechanisms of multiple viruses,examination of multilayered interactions among viruses,their host plants,and virus-transmitting arthropod vectors,and in-depth interrogation of plantencoded resistance and susceptibility determinants.Notably,various plant virus-based vectors have also been successfully developed for gene function studies and target gene expression in plants.We also recommend future plant virology studies in China.展开更多
N^(6)-methyladenosine(m^(6)A)is the most abundant eukaryotic mRNA modification and is involved in various biological processes.Increasing evidence has implicated that m^(6)Amodification is an important anti-viral defe...N^(6)-methyladenosine(m^(6)A)is the most abundant eukaryotic mRNA modification and is involved in various biological processes.Increasing evidence has implicated that m^(6)Amodification is an important anti-viral defense mechanism in mammals and plants,but it is largely unknown how m^(6)Aregulates viral infection in plants.Here we report the dynamic changes and functional anatomy of m^(6)Ain Nicotiana benthamiana and Solanum lycopersicum during Pepino mosaic virus(PepMV)infection.m^(6)Amodification in the PepMV RNA genome is conserved in these two species.Overexpression of the m^(6)Awriters,mRNA adenosine methylase A(MTA),and HAKAI inhibit the PepMV RNA accumulation accompanied by increased viral m^(6)Amodifications,whereas deficiency of these writers decreases the viral RNA m^(6)Alevels but enhances virus infection.Further study reveals that the cytoplasmic YTH-domain family protein NbECT2A/2B/2C as m^(6)Areaders are involved in anti-viral immunity.Protein-protein interactions indicate that NbECT2A/2B/2C interact with nonsense-mediated mRNA decay(NMD)-related proteins,including NbUPF3 and NbSMG7,but not with NbUPF1.m^(6)Amodification-mediated restriction to PepMV infection is dependent on NMD-related factors.These findings provide new insights into the functionality of m^(6)Aanti-viral activity and reveal a distinct immune response that NMD factors recognize the m^(6)Areaders-viral m^(6)ARNA complex for viral RNA degradation to limit virus infection in plants.展开更多
Multilayered defense responses are activated upon pathogen attack.Viruses utilize a number of strategies to maximize the coding capacity of their small genomes and produce viral proteins for infection,including suppre...Multilayered defense responses are activated upon pathogen attack.Viruses utilize a number of strategies to maximize the coding capacity of their small genomes and produce viral proteins for infection,including suppression of host defense.Here,we reveal translation leakage as one of these strategies:two viral effec-tors encoded by tomato golden mosaic virus,chloroplast-localized C4(cC4)and membrane-associated C4(mC4),are translated from two in-frame start codons and function cooperatively to suppress defense.cC4 localizes in chloroplasts,to which it recruits NbPUB4 to induce ubiquitination of the outer membrane;as a result,this organelle is degraded,and chloroplast-mediated defenses are abrogated.However,chloroplast-localized cC4 induces the production of singlet oxygen(^(1)O_(2)),which in turn promotes translo-cation of the ^(1)O_(2) sensor NbMBS1 from the cytosol to the nucleus,where it activates expression of the CERK1 gene.Importantly,an antiviral effect exerted by CERK1 is countered by mC4,localized at the plasma membrane.mC4,like cC4,recruits NbPUB4 and promotes the ubiquitination and subsequent degradation of CERK1,suppressing membrane-based,receptor-like kinase-dependent defenses.Importantly,this translation leakage strategy seems to be conserved in multiple viral species and is related to host range.Thisfinding suggests that stacking of different cellular antiviral responses could be an effective way to abrogate viral infection and engineer sustainable resistance to major crop viral diseases in thefield.展开更多
CRISPR-mediated base editors have been widely used to correct defective alleles and create novel alleles by artificial evolution for the rapid genetic improvement of crops.The editing capabilities of base editors stri...CRISPR-mediated base editors have been widely used to correct defective alleles and create novel alleles by artificial evolution for the rapid genetic improvement of crops.The editing capabilities of base editors strictly rely on the performance of various nucleotide modification enzymes.Compared with the welldeveloped adenine base editors(ABEs),cytosine base editors(CBEs)and dual base editors suffer from unstable editing efficiency and patterns at different genomic loci in rice,significantly limiting their application.Here,we comprehensively examined the base editing activities of multiple evolved TadA8e variants in rice.We found that both TadA-CDd and TadA-E27R/N46L achieved more robust C-to-T editing than previously reported hyperactive hAID*D,and TadA-CDd outperformed TadA-E27R/N46L.A C-to-G base editor(CGBE)engineered with TadA-CDd and OsUNG performed highly efficient C-to-G editing in rice compared with that of TadA-N46P.In addition,a dual base editor constructed with a single protein,TadDE,enabled simultaneous,highly efficient C-to-T and A-to-G editing in rice.Collectively,our results demonstrate that TadA8e derivatives improve both CBEs and dual base editors in rice,providing a powerful way to induce diverse nucleotide substitutions for plant genome editing.展开更多
Single nucleotide polymorphisms (SNPs) are widely present and related to desirable agronomic traits in crops.clustered regularly interspaced palindromic repeats(CRISPR)/CRISPR-associated nuclease 9 (Cas9)-mediated bas...Single nucleotide polymorphisms (SNPs) are widely present and related to desirable agronomic traits in crops.clustered regularly interspaced palindromic repeats(CRISPR)/CRISPR-associated nuclease 9 (Cas9)-mediated base editors have been frequently used to correct defective alleles and create novel alleles by artificial evolution for rapid crop genetic improvement (Ma et al.,2021).展开更多
Increasing evidence supports the notion that pattern-recognition-receptor-mediated immunity goes beyond plasma membrane-to-nucleus signaling.With the findings of the pathways linking plasma membrane and chloroplasts a...Increasing evidence supports the notion that pattern-recognition-receptor-mediated immunity goes beyond plasma membrane-to-nucleus signaling.With the findings of the pathways linking plasma membrane and chloroplasts and the functions of stromules and perinuclear chloroplast clustering(PCC)in plant defense,chloroplast immunity has emerged as a cornerstone of plant defense and a target of plant pathogens.展开更多
Dear Editor,This study reports a nuclease-mediated tiling deletion(NTD)method that uses LbCas12a nuclease with a tiling CRISPRderived RNA(crRNA)library to efficiently induce numerous nucleotide deletions in non-coding...Dear Editor,This study reports a nuclease-mediated tiling deletion(NTD)method that uses LbCas12a nuclease with a tiling CRISPRderived RNA(crRNA)library to efficiently induce numerous nucleotide deletions in non-coding regulatory regions of endogenous rice genes.This method was applied to non-coding regions of the Green Revolution gene SD1,generating 6 mutants with quantitative variations in plant height,which were then used to investigate associations between genotype and phenotype.NTD is thus a promising tool for molecular rice breeding.展开更多
Recently developed CRISPR-mediated base editors,which enable the generation of num erous nucleotide changes in target genomic regions,have been widely adopted for gene correction and generation of crop germ plasms con...Recently developed CRISPR-mediated base editors,which enable the generation of num erous nucleotide changes in target genomic regions,have been widely adopted for gene correction and generation of crop germ plasms containing im portant gain-of-function genetic variations.How ever,to engineer target genes with unknown functional SNPs remains challenging.To address this issue,we present here abase-e diting-mediated gene evolution(BEMGE)m ethod,employing both Cas9n-based cytosine and adenine base editors as well as a single-guide RNA(sgRNA)library tiling the full-length coding region,for developing novel rice germ plasm swith mutations in any endogenous gene.To this end,OsALS1 was artificially evolved in rice cells using BEMGE through both Agrobacterium-mediated and particle-bom bardment-mediated transform ation.Four different types of amino acid substitutions in the evolved OsALS1,derived from two sites that have never been targeted by natural or human selection during rice dom estication,were identified,conferring varying levels of tolerance to the herbicide bispyribac-sodium.Furtherm ore,the P171F substitution identified in a strong OsALS1 allele was quickly introduced into the commercial rice cultivar Nangeng 46 through precise base editing w ith the corresponding base editor and sgRNA.Collectively,these data indicate great potential of BEMGE in creating important genetic variants of target genes for crop improvement.展开更多
CRISPR technologies enabling precise genome manipulation are valuable for gene function studies and molecular crop breeding. However, the requirement of a protospacer adjacent motif (PAM)y such as NGG and TTN, for Cas...CRISPR technologies enabling precise genome manipulation are valuable for gene function studies and molecular crop breeding. However, the requirement of a protospacer adjacent motif (PAM)y such as NGG and TTN, for Cas protein recognition restricts the selection of targetable genomic loci in practical applications of CRISPR technologies. Recently Cas9-NG, which recognizes a minimal NG PAM, was reported to expand the targeting space of genome editing in human cells, but it remains unclear whether this Cas9 variant can be used in plants. In this study, we evaluated the nuclease activity of Cas9-NG toward various NGN PAMs by targeting endogenous genes in transgenic rice. We found that Cas9-NG edits all NGG, NGA, NGT, and NGC sites with impaired activity, while the gene-edited plants were dominated by monoallelic mutations. Cas9-NG-engineered base editors were then developed and used to generate O s B Z R I gainof- function plants that can not be created by other available Cas9-engineered base editors. Moreover, we showed that a Cas9-NG-based transcriptional activator efficiently upregulated the expression of endogenous target genes in rice. In addition, we discovered that Cas9-NG recognizes NAC, NTG, NTT, and NCG apart from NG PAM. Together, these findings demonstrate that Cas9-NG can greatly expand the targeting scope of genome-editing tools, showing great potential for targeted genome editing, base editing, and genome regulation in plants.展开更多
Remorins are plant-specific membrane-associated proteins and were proposed to play crucial roles in plant-pathogen interactions. However, little is known about how pathogens counter remorin-mediated host responses. In...Remorins are plant-specific membrane-associated proteins and were proposed to play crucial roles in plant-pathogen interactions. However, little is known about how pathogens counter remorin-mediated host responses. In this study, by quantitative whole-proteome analysis we found that the remorin protein （NbREM1） is downregulated early in Rice stripe virus （RSV） infection. We further discovered that the turn- over of NbREM1 is regulated by S-acylation modification and its degradation is mediated mainly through the autophagy pathway. Interestingly, RSV can interfere with the S-acylation of NbREM1, which is required to negatively regulate RSV infection by restricting virus cell-to-cell trafficking. The disruption of NbREM1 S-acylation affects its targeting to the plasma membrane microdomain, and the resulting accumulation of non-targeted NbREM1 is subjected to autophagic degradation, causing downregulation of NbREMI. Moreover, we found that RSV-encoded movement protein, NSvc4, alone can interfere with NbREM1 S-acylation through binding with the C-terminal domain of NbREM1 the S-acylation of OsREM1.4, the homologous remorin of NbREM1, and thus remorin-mediated defense against RSV in rice, the original host of RSV, indicating that downregulation of the remorin protein level by interfering with its S-acylation is a common strategy adopted by RSV to overcome remorin-mediated inhibition of virus movement.展开更多
Dear Editor The newly developed CRISPR/Cas9-mediated base editing technology with cytosine deaminase is capable of precisely and efficiently introducing point mutations at the target genomic locus, which does not requ...Dear Editor The newly developed CRISPR/Cas9-mediated base editing technology with cytosine deaminase is capable of precisely and efficiently introducing point mutations at the target genomic locus, which does not require double-stranded DNA breaks or any donor templates and thus exhibit a great potential for gene correction and genetic diversification in yeasts, plants, and mammalian and human cells （Komor et al., 2016; Nishida et al., 2016; Lu and Zhu, 2017; Ren et al., 2017）.展开更多
Recently reported adenine base editors(ABEs)exhibit powerful potential for targeted gene correction as well as developing gain-of-function mutants and novel germplasms for both gene function studies and crop breeding....Recently reported adenine base editors(ABEs)exhibit powerful potential for targeted gene correction as well as developing gain-of-function mutants and novel germplasms for both gene function studies and crop breeding.However,editing efficiency varies significantly among different target sites.Here,we investigated the activities of three evolved E.coli adenosine deaminase TadA variants(TadA8e,TadA8.17,and TadA8.20)side-by-side in transgenic rice.We found that TadA8e outperforms TadA8.17 and TadA8.20,and induces efficient A-to-G conversion at all tested sites in the rice genome,including those that were un-editable by ABE7.10 in our previous experiments.Furthermore,V82S/Q154R mutations were incorporated into TadA8e,resulting in a new variant that we named TadA9.Our data show that TadA9 is broadly compatible with CRISPR/SpCas9,CRISPR/SpCas9-NG,and CRISPR/SpRY,as well as CRISPR/ScCas9 nickase systems,achieving comparable or enhanced editing in a larger editing window at diverse PAM sites as compared with TadA8e.Finally,TadA9 was used to simultaneously install novel SNPs in four endogenous herbicide target genes in the commercial rice cultivar Nangeng 46 for potential field application in.weed control.Collectively,we successfully generated a series of novel ABEs that can efficiently edit adenosines in the rice genome.Our findings suggest that TadA9 and TadA8e have great potentials in the development of plant base editors and crop molecular breeding.展开更多
The βC1 protein encoded by the Tomato yellow leaf curl China virus-associated betasatellite functions as a pathogenicity determinant. To better understand the molecular basis whereby βC1 functions in pathogenicity, ...The βC1 protein encoded by the Tomato yellow leaf curl China virus-associated betasatellite functions as a pathogenicity determinant. To better understand the molecular basis whereby βC1 functions in pathogenicity, a yeast two-hybrid screen of a tobacco cDNA library was carried out using βC1 as the bait. The screen revealed that βC1 interacts with a tobacco RING-finger protein designated NtRFP1, which was further confirmed by the bimolecular fluorescence complementation and co-immunoprecipitation assays in Nicotiana benthamiana cells. Expression of NtRFP1 was induced by βC1, and in vitro ubiquiti- nation assays showed that NtRFP1 is a functional E3 ubiquiUn ligase that mediates βC1 ubiquitination. In addition, βC1 was shown to be ubiquitinated in vivo and degraded by the plant 26S proteasome. After viral infection, plants overexpressing NtRFP1 developed attenuated symptoms, whereas plants with silenced expression of NtRFP1 showed severe symptoms. Other lines of evidence showed that NtRFP1 attenuates βC1-induced symptoms through promoting its degradation by the 26S proteasome. Taken together, our results suggest that tobacco RING E3 ligase NtRFP1 attenuates disease symptoms by interacting with βC1 to mediate its ubiquitination and degradation via the ubiquitin/26S proteasome system.展开更多
Geminiviruses are a group of plant viruses that cause severe diseases in many economically important crops worldwide, leading to devastating losses to agricultural production. Here we summarize the occurrence and dist...Geminiviruses are a group of plant viruses that cause severe diseases in many economically important crops worldwide, leading to devastating losses to agricultural production. Here we summarize the occurrence and distribution of geminiviruses in China,which provides valuable information for further epidemiological studies and supports the development of effective disease management strategies.展开更多
Many geminivirus C4 proteins induce severe developmental abnormalities in plants.We previously demon- strated that Tomato leaf curl Yunnan virus (TLCYnV)C4 induces plant developmental abnormalities at least partically...Many geminivirus C4 proteins induce severe developmental abnormalities in plants.We previously demon- strated that Tomato leaf curl Yunnan virus (TLCYnV)C4 induces plant developmental abnormalities at least partically by decreasing the accumulation of NbSKη,an ortholog of Arabidopsis BIN2 kinase involved in the brassinosteroid signaling pathway,in the nucleus through directing it to the plasma membrane.However, the molecular mechanism by which the membrane-associated C4 modifies the localization of NbSKη in the host cell remains unclear.Here,we show that TLCYnV C4 is a nucleocytoplasmic shuttle protein,and that C4 shuttling is accompanied by nuclear export of NbSKTI.TLCYnV C4 is phosphorylated by NbSKη in the nucleus,which promotes myristoylation of the viral protein.Myristoylation of phosphorylated C4 favors its interaction with exportin-α(XPO I);which in turn facilitates nuclear export of the C4/NbSKTI complex. Supporting this model,chemical inhibition of N-myristoyltransferases or exportin-α enhanced nuclear retention of C4,and mutations of the putative phosphorylation or myristoylation sites in C4 resulted in increased nuclear retention ofrC4 and thus decreased severity of C4-induced developmental abnormalities. The impact of C4 on development is also lessened when a nuclear localization signal or a nuclear export signal is added to its C-terminus,restricting it to a specific cellular niche and therefore impairing nucleocytoplasmic shuttling.Taken together,our results suggest that nucleocytoplasmic shuttling of TLCYnV C4,enabled by phosphorylation by NbSKη,myristoylation,and interaction with exportin-α is critical for its function as a pathogenicity factor.展开更多
Citrus tristeza virus （CTV） is one of the most economically important citrus viruses and harms the citrus industry worldwide. To develop reliable and effective serological detection assays of CTV, the major capsid p...Citrus tristeza virus （CTV） is one of the most economically important citrus viruses and harms the citrus industry worldwide. To develop reliable and effective serological detection assays of CTV, the major capsid protein （CP） gene of CTV was expressed in Escherichia coli BL21 （DE3） using the expression vector pET-28a and purified through Ni＊-NTA affinity chromatography. The recombinant protein was used to immunize BALB/c mice. Four hybridoma cell lines （14B10, 14Hll, 20D5, and 20G12） secreting monoclonal antibodies （MAbs） against CTV were obtained through conventional hybridoma technology. The titers of MAb-containing ascitic fluids secreted by the four hybridoma lines ranged from 10-6 to 10.7 in indirect enzyme-linked immunosorbent assay （ELISA）. Western blots showed that all four MAbs could specifically react with CTV CP. Using the prepared MAbs, dot-ELISA, Tissue print-ELISA, and triple antibody sandwich （TAS）-ELISA were developed to detect CTV in tree nurseries and epidemiological studies. The developed dot-ELISA and TAS-ELISA methods could detect CTV in crude extracts of infected citrus leaves with dilutions of 1：2560 and 1：10, 240 （w/v, g/mL）, respectively. Tissue print-ELISA was particularly useful for large-scale field sample detection, mainly owing to its simplicity and lack of sample preparation requirements. The field survey revealed that CTV is prevalent on citrus trees in the Chongqing Municipality, Jiangxi Province, and Zhejiang Province of China. The coincidence rate of serological and RT-PCR test results reached more than 99.5%. The prepared MAbs against CTV and established sensitive and specific serological assays have a significant role in the detection and prevention and control of CTV in our country.展开更多
Rice stripe mosaic virus(RSMV) is a rhabdovirus recently found in southern part of China and can cause severe reduction in rice production. To establish serological methods for RSMV epidemiological studies and to esta...Rice stripe mosaic virus(RSMV) is a rhabdovirus recently found in southern part of China and can cause severe reduction in rice production. To establish serological methods for RSMV epidemiological studies and to establish a control strategy for this virus, we first purified RSMV virions from infected rice plants and then used them as an immunogen to produce four RSMV-specific monoclonal antibodies(MAbs)(i.e.,1D4, 4A8, 8E4 and 11F11). With these MAbs, we have developed a highly specific and sensitive antigen-coated plate enzyme-linked immunosorbent assay(ACP-ELISA), a Dot-ELISA and a Tissue print-ELISA for rapid detections of RSMV infection in rice plants or in leafhoppers. Our results showed that RSMV can be readily detected in RSMV-infected rice plant tissue crude extracts diluted at 1:20,971,520(w/v, g/m L)through ACP-ELISA or diluted at 1:327,680(w/v, g/m L) through Dot-ELISA. Both ACP-ELISA and Dot-ELISA can also be used to detect RSMV infection in individual RSMV viruliferous leafhopper(Recilia dorsalis) homogenate diluted at 1:307,200 and 1:163,840(individual leafhopper/l L), respectively. Detection of RSMV infection in field-collected rice samples or in RSMV viruliferous leafhoppers indicated that the three serological methods can produce same results with that produced by RT-PCR(19 of the 33 rice samples and 5 of the 16 leafhoppers were RSMV-positive). We consider that the four MAbs produced in this study are very specific and sensitive, and the three new serological methods are very useful for detections of RSMV infection in rice plants or in leafhoppers and the establishment of the disease control strategies.展开更多
Geminiviruses are a family of plant viruses that cause devastating diseases in many economically important crops worldwide. These pathogens encapsidate circular,single-stranded DNAs (ssDNAs)of 2.5-3.0 kb that replicat...Geminiviruses are a family of plant viruses that cause devastating diseases in many economically important crops worldwide. These pathogens encapsidate circular,single-stranded DNAs (ssDNAs)of 2.5-3.0 kb that replicate through double-stranded DNA (dsDNA)intermediates.Members of the genus Begomovirus,which comprises by far the largest number of species in the family,are transmitted by whiteflies and have genomes consisting of one or two DNAs.展开更多
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.
基金supported by grants from the Shenzhen Science and Technology Program(KQTD20180411143628272)Special Funds for Science Technology Innovation and Industrial Development of Shenzhen Dapeng New District(PT202101-02)+3 种基金the Hainan Yazhou Bay Seed Lab(B21HJ0215),the National Natural Science Foundation of China(32102294)the China National Postdoctoral Program for Innovative Talents(BX2020378)the China Postdoctoral Science Foundation(2020M672902)the Central Publicinterest Scientific Institution Basal Research Fund(Y2022PT24).
文摘Only few glufosinate-tolerant genes,such as phosphinothricin acetyltransferase(PAT)and bialaphos resistance(bar)identified from Streptomyces,are currently available for developing genetically modified rice in agricultural application.Following the rapid development of genome editing technology,generation of novel glufosinate-tolerant gene resources through artificial evolution of endogenous genes is more promising and highly desirable in rice molecular breeding program.In this study,the endogenous Glutamine synthetase1(OsGS1)was artificially evolved by base-editing-mediated gene evolution(BEMGE)in rice cells to create novel alleles conferring glufosinate tolerance in rice germplasms.Two novel glufosinate-tolerant OsGS1 alleles(OsGS1-AVPS and OsGS1-+AF)and one reported tolerant allele(OsGS1-SGTA)were successfully identified from approximately 4200 independent hygromycin-tolerant calli.Germination assays and spray tests revealed that these three OsGS1 alleles confer glufosinate tolerance in rice.Furthermore,OsGS1-AVPS and OsGS1-SGTA were quickly deployed into the elite rice cultivar Nangeng 46 through precise base editing.Overall,our results demonstrate the feasibility of developing glufosinate-tolerant rice by editing an endogenous rice gene in molecular breeding programs.
基金the National Natural Science Foundation of China for financial support(31530062 and 32025031)。
文摘Plant viruses are a group of intracellular pathogens that persistently threaten global food security.Significant advances in plant virology have been achieved by Chinese scientists over the last 20 years,including basic research and technologies for preventing and controlling plant viral diseases.Here,we review these milestones and advances,including the identification of new crop-infecting viruses,dissection of pathogenic mechanisms of multiple viruses,examination of multilayered interactions among viruses,their host plants,and virus-transmitting arthropod vectors,and in-depth interrogation of plantencoded resistance and susceptibility determinants.Notably,various plant virus-based vectors have also been successfully developed for gene function studies and target gene expression in plants.We also recommend future plant virology studies in China.
基金supported by the National Key Research and Development Program of China (2021YFD1400400) to Fangfang Lithe National Natural Science Foundation of China (32172385 and 31930089) to Fangfang Li and Xueping Zhou, respectively
文摘N^(6)-methyladenosine(m^(6)A)is the most abundant eukaryotic mRNA modification and is involved in various biological processes.Increasing evidence has implicated that m^(6)Amodification is an important anti-viral defense mechanism in mammals and plants,but it is largely unknown how m^(6)Aregulates viral infection in plants.Here we report the dynamic changes and functional anatomy of m^(6)Ain Nicotiana benthamiana and Solanum lycopersicum during Pepino mosaic virus(PepMV)infection.m^(6)Amodification in the PepMV RNA genome is conserved in these two species.Overexpression of the m^(6)Awriters,mRNA adenosine methylase A(MTA),and HAKAI inhibit the PepMV RNA accumulation accompanied by increased viral m^(6)Amodifications,whereas deficiency of these writers decreases the viral RNA m^(6)Alevels but enhances virus infection.Further study reveals that the cytoplasmic YTH-domain family protein NbECT2A/2B/2C as m^(6)Areaders are involved in anti-viral immunity.Protein-protein interactions indicate that NbECT2A/2B/2C interact with nonsense-mediated mRNA decay(NMD)-related proteins,including NbUPF3 and NbSMG7,but not with NbUPF1.m^(6)Amodification-mediated restriction to PepMV infection is dependent on NMD-related factors.These findings provide new insights into the functionality of m^(6)Aanti-viral activity and reveal a distinct immune response that NMD factors recognize the m^(6)Areaders-viral m^(6)ARNA complex for viral RNA degradation to limit virus infection in plants.
基金funded by grants from the National Natural Science Foundation of China (31930089)the National Key Research and Development Program of China (2021YFD1400400)the Yunnan Zhouxueping Expert Workstation (No.202205AF150047).
文摘Multilayered defense responses are activated upon pathogen attack.Viruses utilize a number of strategies to maximize the coding capacity of their small genomes and produce viral proteins for infection,including suppression of host defense.Here,we reveal translation leakage as one of these strategies:two viral effec-tors encoded by tomato golden mosaic virus,chloroplast-localized C4(cC4)and membrane-associated C4(mC4),are translated from two in-frame start codons and function cooperatively to suppress defense.cC4 localizes in chloroplasts,to which it recruits NbPUB4 to induce ubiquitination of the outer membrane;as a result,this organelle is degraded,and chloroplast-mediated defenses are abrogated.However,chloroplast-localized cC4 induces the production of singlet oxygen(^(1)O_(2)),which in turn promotes translo-cation of the ^(1)O_(2) sensor NbMBS1 from the cytosol to the nucleus,where it activates expression of the CERK1 gene.Importantly,an antiviral effect exerted by CERK1 is countered by mC4,localized at the plasma membrane.mC4,like cC4,recruits NbPUB4 and promotes the ubiquitination and subsequent degradation of CERK1,suppressing membrane-based,receptor-like kinase-dependent defenses.Importantly,this translation leakage strategy seems to be conserved in multiple viral species and is related to host range.Thisfinding suggests that stacking of different cellular antiviral responses could be an effective way to abrogate viral infection and engineer sustainable resistance to major crop viral diseases in thefield.
基金supported by the STI 2030-Major Projects(2023ZD04074)the National Key Research and Development Program of China(2023YFD1202900)+2 种基金the Nanfan special project of the Chinese Academy of Agricultural Sciences(YBXM2313)the Hainan Seed Industry Laboratory(project of B23CJ0208)the Agricultural Science and Technology Innovation Program of the Chinese Academy of Agricultural Sciences.
文摘CRISPR-mediated base editors have been widely used to correct defective alleles and create novel alleles by artificial evolution for the rapid genetic improvement of crops.The editing capabilities of base editors strictly rely on the performance of various nucleotide modification enzymes.Compared with the welldeveloped adenine base editors(ABEs),cytosine base editors(CBEs)and dual base editors suffer from unstable editing efficiency and patterns at different genomic loci in rice,significantly limiting their application.Here,we comprehensively examined the base editing activities of multiple evolved TadA8e variants in rice.We found that both TadA-CDd and TadA-E27R/N46L achieved more robust C-to-T editing than previously reported hyperactive hAID*D,and TadA-CDd outperformed TadA-E27R/N46L.A C-to-G base editor(CGBE)engineered with TadA-CDd and OsUNG performed highly efficient C-to-G editing in rice compared with that of TadA-N46P.In addition,a dual base editor constructed with a single protein,TadDE,enabled simultaneous,highly efficient C-to-T and A-to-G editing in rice.Collectively,our results demonstrate that TadA8e derivatives improve both CBEs and dual base editors in rice,providing a powerful way to induce diverse nucleotide substitutions for plant genome editing.
基金supported by the Biological Breeding-Major Projects (2023ZD04074)the Nanfan special project of the Chinese Academy of Agricultural Sciences (YBXM2313)+1 种基金the Hainan Seed Industry Laboratory (B23CJ0208)the Agricultural Science and Technology Innovation Program of the Chinese Academy of Agricultural Sciences。
文摘Single nucleotide polymorphisms (SNPs) are widely present and related to desirable agronomic traits in crops.clustered regularly interspaced palindromic repeats(CRISPR)/CRISPR-associated nuclease 9 (Cas9)-mediated base editors have been frequently used to correct defective alleles and create novel alleles by artificial evolution for rapid crop genetic improvement (Ma et al.,2021).
基金supported by grants from the National Key Research and Development Program of China(2021YFD1400400)the Natural Science Foundation of China(32320103010,32172385,and 31930089).
文摘Increasing evidence supports the notion that pattern-recognition-receptor-mediated immunity goes beyond plasma membrane-to-nucleus signaling.With the findings of the pathways linking plasma membrane and chloroplasts and the functions of stromules and perinuclear chloroplast clustering(PCC)in plant defense,chloroplast immunity has emerged as a cornerstone of plant defense and a target of plant pathogens.
基金supported by grants from the National Natural Science Foundation of China,China (31871948)the STI 2030–Major Projects (2023ZD04074)+3 种基金the Hainan Yazhou Bay Seed Lab (B21HJ0215)the Central Public-interest Scientific Institution Basal Research Fund (Y2022QC03)the Key Research and Development Program of Shandong Province,China (Agricultural Seed Improvement Project,2022LZGC012)the Agricultural Science and Technology Innovation Program of the Chinese Academy of Agricultural Sciences,China。
文摘Dear Editor,This study reports a nuclease-mediated tiling deletion(NTD)method that uses LbCas12a nuclease with a tiling CRISPRderived RNA(crRNA)library to efficiently induce numerous nucleotide deletions in non-coding regulatory regions of endogenous rice genes.This method was applied to non-coding regions of the Green Revolution gene SD1,generating 6 mutants with quantitative variations in plant height,which were then used to investigate associations between genotype and phenotype.NTD is thus a promising tool for molecular rice breeding.
基金This work was supported by grants from the National Natural Science Foundation of China(31871948)the Fundamental Research Funds,and the Agricultural Science and Technology Innovation Program of the Chinese Academy of Agricultural Sciences to H.Z.a grant from the Fundamental Research Funds for the Central Universities to S.L.
文摘Recently developed CRISPR-mediated base editors,which enable the generation of num erous nucleotide changes in target genomic regions,have been widely adopted for gene correction and generation of crop germ plasms containing im portant gain-of-function genetic variations.How ever,to engineer target genes with unknown functional SNPs remains challenging.To address this issue,we present here abase-e diting-mediated gene evolution(BEMGE)m ethod,employing both Cas9n-based cytosine and adenine base editors as well as a single-guide RNA(sgRNA)library tiling the full-length coding region,for developing novel rice germ plasm swith mutations in any endogenous gene.To this end,OsALS1 was artificially evolved in rice cells using BEMGE through both Agrobacterium-mediated and particle-bom bardment-mediated transform ation.Four different types of amino acid substitutions in the evolved OsALS1,derived from two sites that have never been targeted by natural or human selection during rice dom estication,were identified,conferring varying levels of tolerance to the herbicide bispyribac-sodium.Furtherm ore,the P171F substitution identified in a strong OsALS1 allele was quickly introduced into the commercial rice cultivar Nangeng 46 through precise base editing w ith the corresponding base editor and sgRNA.Collectively,these data indicate great potential of BEMGE in creating important genetic variants of target genes for crop improvement.
基金National Natural Science Foundation of China (31871948)the National Key Research and Development Program of China (2017YFD0200900)the Agricultural Science and Technology Innovation Program of the Chinese Academy of Agricultural Sciences to H.Z.
文摘CRISPR technologies enabling precise genome manipulation are valuable for gene function studies and molecular crop breeding. However, the requirement of a protospacer adjacent motif (PAM)y such as NGG and TTN, for Cas protein recognition restricts the selection of targetable genomic loci in practical applications of CRISPR technologies. Recently Cas9-NG, which recognizes a minimal NG PAM, was reported to expand the targeting space of genome editing in human cells, but it remains unclear whether this Cas9 variant can be used in plants. In this study, we evaluated the nuclease activity of Cas9-NG toward various NGN PAMs by targeting endogenous genes in transgenic rice. We found that Cas9-NG edits all NGG, NGA, NGT, and NGC sites with impaired activity, while the gene-edited plants were dominated by monoallelic mutations. Cas9-NG-engineered base editors were then developed and used to generate O s B Z R I gainof- function plants that can not be created by other available Cas9-engineered base editors. Moreover, we showed that a Cas9-NG-based transcriptional activator efficiently upregulated the expression of endogenous target genes in rice. In addition, we discovered that Cas9-NG recognizes NAC, NTG, NTT, and NCG apart from NG PAM. Together, these findings demonstrate that Cas9-NG can greatly expand the targeting scope of genome-editing tools, showing great potential for targeted genome editing, base editing, and genome regulation in plants.
文摘Remorins are plant-specific membrane-associated proteins and were proposed to play crucial roles in plant-pathogen interactions. However, little is known about how pathogens counter remorin-mediated host responses. In this study, by quantitative whole-proteome analysis we found that the remorin protein （NbREM1） is downregulated early in Rice stripe virus （RSV） infection. We further discovered that the turn- over of NbREM1 is regulated by S-acylation modification and its degradation is mediated mainly through the autophagy pathway. Interestingly, RSV can interfere with the S-acylation of NbREM1, which is required to negatively regulate RSV infection by restricting virus cell-to-cell trafficking. The disruption of NbREM1 S-acylation affects its targeting to the plasma membrane microdomain, and the resulting accumulation of non-targeted NbREM1 is subjected to autophagic degradation, causing downregulation of NbREMI. Moreover, we found that RSV-encoded movement protein, NSvc4, alone can interfere with NbREM1 S-acylation through binding with the C-terminal domain of NbREM1 the S-acylation of OsREM1.4, the homologous remorin of NbREM1, and thus remorin-mediated defense against RSV in rice, the original host of RSV, indicating that downregulation of the remorin protein level by interfering with its S-acylation is a common strategy adopted by RSV to overcome remorin-mediated inhibition of virus movement.
基金This study was supported by grants from the National Key Research and Development Program of China （2017YFD0200900） and the Agricultural Science and Technology Innovation Program of The Chinese Academy of Agricultural Sciences to H.Z., and a grant from the National Natural Science Foundation of China （31701780） to F.Y.
文摘Dear Editor The newly developed CRISPR/Cas9-mediated base editing technology with cytosine deaminase is capable of precisely and efficiently introducing point mutations at the target genomic locus, which does not require double-stranded DNA breaks or any donor templates and thus exhibit a great potential for gene correction and genetic diversification in yeasts, plants, and mammalian and human cells （Komor et al., 2016; Nishida et al., 2016; Lu and Zhu, 2017; Ren et al., 2017）.
基金supported by grants from the National Transgenic Science and Technology Program of China(2019ZX08010-003)to F.Y.the National Natural Science Foundation of China(31871948)the Fundamental Research Funds,and the Agricultural Science and Technology Innovation Program of the Chinese Academy of Agricultural Sciences(Y2020PT26)to H.Z.
文摘Recently reported adenine base editors(ABEs)exhibit powerful potential for targeted gene correction as well as developing gain-of-function mutants and novel germplasms for both gene function studies and crop breeding.However,editing efficiency varies significantly among different target sites.Here,we investigated the activities of three evolved E.coli adenosine deaminase TadA variants(TadA8e,TadA8.17,and TadA8.20)side-by-side in transgenic rice.We found that TadA8e outperforms TadA8.17 and TadA8.20,and induces efficient A-to-G conversion at all tested sites in the rice genome,including those that were un-editable by ABE7.10 in our previous experiments.Furthermore,V82S/Q154R mutations were incorporated into TadA8e,resulting in a new variant that we named TadA9.Our data show that TadA9 is broadly compatible with CRISPR/SpCas9,CRISPR/SpCas9-NG,and CRISPR/SpRY,as well as CRISPR/ScCas9 nickase systems,achieving comparable or enhanced editing in a larger editing window at diverse PAM sites as compared with TadA8e.Finally,TadA9 was used to simultaneously install novel SNPs in four endogenous herbicide target genes in the commercial rice cultivar Nangeng 46 for potential field application in.weed control.Collectively,we successfully generated a series of novel ABEs that can efficiently edit adenosines in the rice genome.Our findings suggest that TadA9 and TadA8e have great potentials in the development of plant base editors and crop molecular breeding.
文摘The βC1 protein encoded by the Tomato yellow leaf curl China virus-associated betasatellite functions as a pathogenicity determinant. To better understand the molecular basis whereby βC1 functions in pathogenicity, a yeast two-hybrid screen of a tobacco cDNA library was carried out using βC1 as the bait. The screen revealed that βC1 interacts with a tobacco RING-finger protein designated NtRFP1, which was further confirmed by the bimolecular fluorescence complementation and co-immunoprecipitation assays in Nicotiana benthamiana cells. Expression of NtRFP1 was induced by βC1, and in vitro ubiquiti- nation assays showed that NtRFP1 is a functional E3 ubiquiUn ligase that mediates βC1 ubiquitination. In addition, βC1 was shown to be ubiquitinated in vivo and degraded by the plant 26S proteasome. After viral infection, plants overexpressing NtRFP1 developed attenuated symptoms, whereas plants with silenced expression of NtRFP1 showed severe symptoms. Other lines of evidence showed that NtRFP1 attenuates βC1-induced symptoms through promoting its degradation by the 26S proteasome. Taken together, our results suggest that tobacco RING E3 ligase NtRFP1 attenuates disease symptoms by interacting with βC1 to mediate its ubiquitination and degradation via the ubiquitin/26S proteasome system.
基金supported by the National Natural Science Foundation of China (31720103914, 32172385)the National Key Research and Development Program of China (2021YFD1400400)the China National Tobacco Corporation (110202001030(JY-13))。
文摘Geminiviruses are a group of plant viruses that cause severe diseases in many economically important crops worldwide, leading to devastating losses to agricultural production. Here we summarize the occurrence and distribution of geminiviruses in China,which provides valuable information for further epidemiological studies and supports the development of effective disease management strategies.
基金grants from the National Natural Science Foundation of China (31720103914 and 31390422).
文摘Many geminivirus C4 proteins induce severe developmental abnormalities in plants.We previously demon- strated that Tomato leaf curl Yunnan virus (TLCYnV)C4 induces plant developmental abnormalities at least partically by decreasing the accumulation of NbSKη,an ortholog of Arabidopsis BIN2 kinase involved in the brassinosteroid signaling pathway,in the nucleus through directing it to the plasma membrane.However, the molecular mechanism by which the membrane-associated C4 modifies the localization of NbSKη in the host cell remains unclear.Here,we show that TLCYnV C4 is a nucleocytoplasmic shuttle protein,and that C4 shuttling is accompanied by nuclear export of NbSKTI.TLCYnV C4 is phosphorylated by NbSKη in the nucleus,which promotes myristoylation of the viral protein.Myristoylation of phosphorylated C4 favors its interaction with exportin-α(XPO I);which in turn facilitates nuclear export of the C4/NbSKTI complex. Supporting this model,chemical inhibition of N-myristoyltransferases or exportin-α enhanced nuclear retention of C4,and mutations of the putative phosphorylation or myristoylation sites in C4 resulted in increased nuclear retention ofrC4 and thus decreased severity of C4-induced developmental abnormalities. The impact of C4 on development is also lessened when a nuclear localization signal or a nuclear export signal is added to its C-terminus,restricting it to a specific cellular niche and therefore impairing nucleocytoplasmic shuttling.Taken together,our results suggest that nucleocytoplasmic shuttling of TLCYnV C4,enabled by phosphorylation by NbSKη,myristoylation,and interaction with exportin-α is critical for its function as a pathogenicity factor.
基金supported by Public Science and Technology Research Funds Projects of Agriculture (20120307605)
文摘Citrus tristeza virus （CTV） is one of the most economically important citrus viruses and harms the citrus industry worldwide. To develop reliable and effective serological detection assays of CTV, the major capsid protein （CP） gene of CTV was expressed in Escherichia coli BL21 （DE3） using the expression vector pET-28a and purified through Ni＊-NTA affinity chromatography. The recombinant protein was used to immunize BALB/c mice. Four hybridoma cell lines （14B10, 14Hll, 20D5, and 20G12） secreting monoclonal antibodies （MAbs） against CTV were obtained through conventional hybridoma technology. The titers of MAb-containing ascitic fluids secreted by the four hybridoma lines ranged from 10-6 to 10.7 in indirect enzyme-linked immunosorbent assay （ELISA）. Western blots showed that all four MAbs could specifically react with CTV CP. Using the prepared MAbs, dot-ELISA, Tissue print-ELISA, and triple antibody sandwich （TAS）-ELISA were developed to detect CTV in tree nurseries and epidemiological studies. The developed dot-ELISA and TAS-ELISA methods could detect CTV in crude extracts of infected citrus leaves with dilutions of 1：2560 and 1：10, 240 （w/v, g/mL）, respectively. Tissue print-ELISA was particularly useful for large-scale field sample detection, mainly owing to its simplicity and lack of sample preparation requirements. The field survey revealed that CTV is prevalent on citrus trees in the Chongqing Municipality, Jiangxi Province, and Zhejiang Province of China. The coincidence rate of serological and RT-PCR test results reached more than 99.5%. The prepared MAbs against CTV and established sensitive and specific serological assays have a significant role in the detection and prevention and control of CTV in our country.
基金Project was supported by the Ministry of Agriculture of China(No.2016ZX08009003-001)the National Key Research and Development Program of China(No.2016YFD0300706)+1 种基金the National Natural Science Foundation of China(No.31571976)the Earmarked Fund for China Agriculture Research System(No.nycytx-001).
文摘Rice stripe mosaic virus(RSMV) is a rhabdovirus recently found in southern part of China and can cause severe reduction in rice production. To establish serological methods for RSMV epidemiological studies and to establish a control strategy for this virus, we first purified RSMV virions from infected rice plants and then used them as an immunogen to produce four RSMV-specific monoclonal antibodies(MAbs)(i.e.,1D4, 4A8, 8E4 and 11F11). With these MAbs, we have developed a highly specific and sensitive antigen-coated plate enzyme-linked immunosorbent assay(ACP-ELISA), a Dot-ELISA and a Tissue print-ELISA for rapid detections of RSMV infection in rice plants or in leafhoppers. Our results showed that RSMV can be readily detected in RSMV-infected rice plant tissue crude extracts diluted at 1:20,971,520(w/v, g/m L)through ACP-ELISA or diluted at 1:327,680(w/v, g/m L) through Dot-ELISA. Both ACP-ELISA and Dot-ELISA can also be used to detect RSMV infection in individual RSMV viruliferous leafhopper(Recilia dorsalis) homogenate diluted at 1:307,200 and 1:163,840(individual leafhopper/l L), respectively. Detection of RSMV infection in field-collected rice samples or in RSMV viruliferous leafhoppers indicated that the three serological methods can produce same results with that produced by RT-PCR(19 of the 33 rice samples and 5 of the 16 leafhoppers were RSMV-positive). We consider that the four MAbs produced in this study are very specific and sensitive, and the three new serological methods are very useful for detections of RSMV infection in rice plants or in leafhoppers and the establishment of the disease control strategies.
基金The National Natural Science Foundation of China (31390422 and 31720103914)The Bisaro laboratory is supported by grants from the US National Science Foundation (NSF IOS-1354636)the US Department of Agriculture and National Institute of Food and Agriculture (USDA/NIFA 2015-6703-22999).
文摘Geminiviruses are a family of plant viruses that cause devastating diseases in many economically important crops worldwide. These pathogens encapsidate circular,single-stranded DNAs (ssDNAs)of 2.5-3.0 kb that replicate through double-stranded DNA (dsDNA)intermediates.Members of the genus Begomovirus,which comprises by far the largest number of species in the family,are transmitted by whiteflies and have genomes consisting of one or two DNAs.
