VIGS(Virus-induced gene silencing),a method for posttranscriptional gene silencing,is an effective technique for investigating the activities of genes in plants.Since there is no report for available VIGS system in St...VIGS(Virus-induced gene silencing),a method for posttranscriptional gene silencing,is an effective technique for investigating the activities of genes in plants.Since there is no report for available VIGS system in Styrax japonicus,the application of a VIGS approach that results in a gene knockdown to study gene function is limited.In this study,we compared the characteristics that could affect the viability of VIGS in S.japonicus,including the acetosyringone(AS)concentration,the Agrobacterium’s optical density and the inoculation method.The stable reference genes of S.japonicus were selected to validate the gene’s knockdown by quantitative PCR.As a result,we successfully constructed 2 VIGS systems based on TRV virus:vacuum with AS concentration of 200μmol·L^(-1)and OD600of 0.5,and friction-osmosis with AS concentration of 200μmol·L^(-1)and OD600of 1.0,which silencing efficiency was 83.33%and 74.19%,respectively.The successfully applied VIGS method provides a rapid and effective reverse gene functional analysis approach in S.japonicus to identify unknown gene functions.展开更多
Verticillium wilt(VW),induced by the soil-borne fungus Verticillium dahliae(Vd),poses a substantial threat to a diverse array of plant species.Employing molecular breeding technology for the development of cotton vari...Verticillium wilt(VW),induced by the soil-borne fungus Verticillium dahliae(Vd),poses a substantial threat to a diverse array of plant species.Employing molecular breeding technology for the development of cotton varieties with heightened resistance to VW stands out as one of the most efficacious protective measures.In this study,we successfully generated two stable transgenic lines of cotton(Gossypium hirsutum L.),VdThitRNAi-1 and VdThit-RNAi-2,using host-induced gene silencing(HIGS)technology to introduce double-stranded RNA(dsRNA)targeting the thiamine transporter protein gene(VdThit).Southern blot analysis confirmed the presence of a single-copy insertion in each line.Microscopic examination showed marked reductions in the colonization and spread of Vd-mCherry in the roots of VdThit-RNAi cotton compared to wild type(WT).The corresponding disease index and fungal biomass of VdThit-RNAi-1/2 also exhibited significant reductions.Real-time quantitative PCR(qRT-PCR)analysis demonstrated a substantial inhibition of VdThit expression following prolonged inoculation of VdThit-RNAi cotton.Small RNA sequencing(sRNA-Seq)analysis revealed the generation of a substantial number of VdThit-specific siRNAs in the VdThit-RNAi transgenic lines.Additionally,the silencing of VdThit by the siVdThit produced by VdThit-RNAi-1/2 resulted in the elevated expression of multiple genes involved in the thiamine biosynthesis pathway in Vd.Under field conditions,VdThit-RNAi transgenic cotton exhibited significantly enhanced disease resistance and yield compared with WT.In summary,our findings underscore the efficacy of HIGS targeting VdThit in restraining the infection and spread of Vd in cotton,thereby potentially enabling the development of cotton breeding as a promising strategy for managing VW.展开更多
本研究旨在提高黄瓜绿斑驳花叶病毒(cucumber green mottle mosaic virus,CGMMV)介导的基因沉默(VIGS)在瓜类作物中应用效率。以黄瓜、甜瓜和西瓜优良种质为实验材料,采用不同的CGMMV病毒载体接种方式、并设置不同的培养温度和相对湿度...本研究旨在提高黄瓜绿斑驳花叶病毒(cucumber green mottle mosaic virus,CGMMV)介导的基因沉默(VIGS)在瓜类作物中应用效率。以黄瓜、甜瓜和西瓜优良种质为实验材料,采用不同的CGMMV病毒载体接种方式、并设置不同的培养温度和相对湿度,以测试基因沉默的有效性。结果表明,真空渗透和种子吸胀的接种方式能够在瓜类作物中产生最高的基因沉默率(FGS)。在22℃和25℃的培养环境下,黄瓜、甜瓜和西瓜PDS沉默有效性(EGSL)较高。黄瓜、甜瓜和西瓜生长1个月后,EGSL达到100%,显著高于30℃的培养环境下EGSL。生长3个月后,甜瓜和西瓜在22℃培养环境下EGSL分别为89.7%和95%;在25℃培养环境下EGSL分别为85.6%和86.1%,均显著性高于30℃下EGSL。在相对湿度为30%和50%环境下,黄瓜的EGSL分别为70%和72%,甜瓜的EGSL分别为76%和73%,显著高于相对湿度为80%的培养环境下EGSL。西瓜在相对湿度为30%的培养环境下的EGSL为69%,显著高于相对湿度为50%时的EGSL(38%)和相对湿度在80%时的EGSL(33%)。综上所述,通过优化了瓜类作物的CGMMV-VIGS的技术体系中接种方式和培养环境参数,提高了基因沉默率和有效性,并且能够快速获得整个植株基因沉默的种质资源,为作物优质和抗逆基因功能的研究提供有效途径。展开更多
Rice sheath blight, caused by Rhizoctonia solani AG1-IA, is a major disease in rice-growing areas worldwide. Effectors of phytopathogenic fungi play important roles during the infection process of fungal pathogens ont...Rice sheath blight, caused by Rhizoctonia solani AG1-IA, is a major disease in rice-growing areas worldwide. Effectors of phytopathogenic fungi play important roles during the infection process of fungal pathogens onto their host plants. However, the molecular mechanisms by which R. solani effectors regulate rice immunity are not well understood. Through prediction, 78 candidate effector molecules were identified. Using the tobacco rattle virus-host induced gene silencing(TRV-HIGS) system, 45 RNAi constructs of effector genes were infiltrated into Nicotiana benthamiana leaves. The results revealed that eight of these constructs resulted in a significant reduction in necrosis caused by infection with the AG1-IA strain GD-118. Additionally, stable rice transformants carrying the double-stranded RNA construct for one of the effector genes, AGLIP1, were generated to further verify the function of this gene. The suppression of the AGLIP1 gene increased the resistance of both N. benthamiana and rice against GD-118, and also affected the growth rate of GD-118, indicating that AGLIP1 is a key pathogenic factor. Small RNA sequencing showed that the HIGS vectors were processed into si RNAs within the plants and then translocated to the fungi, leading to the silencing of the target genes. As a result, AGLIP1 might be an excellent candidate for HIGS, thereby enhancing crop resistance against the pathogen and contributing to the control of R. solani infection.展开更多
Virus-induced gene silencing(VIGS)and clustered regularly interspaced short palindromic repeats/CRISPR-associated protein(CRISPR/Cas)systems are effective technologies for rapid and accurate gene function verification...Virus-induced gene silencing(VIGS)and clustered regularly interspaced short palindromic repeats/CRISPR-associated protein(CRISPR/Cas)systems are effective technologies for rapid and accurate gene function verification in modern plant biotechnology.However,the investigation of gene silencing and editing in radish remains limited.In this study,a bleaching phenotype was generated through the knockdown of RsPDS using tobacco rattle virus(TRV)-and turnip yellow mosaic virus(TYMV)-mediated gene silencing vectors.The TYMV-mediated gene silencing efficiency was higher than the TRV-based VIGS system in radish.The expression level of RsPDS was significantly inhibited using VIGS in'NAU-067'radish leaves.The rootless seedlings of‘NAU-067'were infected with Agrobacterium rhizogenes using the 2300GN-Ubi-RsPDS-Cas9 vector with two target sequences.Nine adventitious roots were blue with GUs staining,and four of these adventitious roots were edited at target sequence 1 of the RsPDS gene as indicated by Sanger sequencing.Furthermore,albino lines were generated with A.tumefaciens-mediated transformation of radish cotyledons.Five base substitutions and three base deletions occurred at target sequence 2 in Line 1,and three base insertions and three base substitutions occurred at target sequence 1 in Line 2.This study shows that VIGS and CRISPR/Cas9 techniques can be employed to precisely verify the biological functions of genes in radish,which will facilitate the genetic improvement of vital horticultural traits in radish breeding programs.展开更多
Stress response pathways detect and alleviate adverse conditions to safeguard cell and tissue homeostasis,yet their prolonged activation induces apoptosis and disrupts organismal health1-3.How stress responses are tur...Stress response pathways detect and alleviate adverse conditions to safeguard cell and tissue homeostasis,yet their prolonged activation induces apoptosis and disrupts organismal health1-3.How stress responses are turned off at the right time and place remains poorly understood.Here we report a ubiquitin-dependent mechanism that silences the cellular response to mitochondrial protein import stress.展开更多
The contribution aims to examine the co-implications between Serres’work and posthumanist ideas of body and subjectivity.This scope is pursued primarily through the methodological choice of a gerund,such as silencing...The contribution aims to examine the co-implications between Serres’work and posthumanist ideas of body and subjectivity.This scope is pursued primarily through the methodological choice of a gerund,such as silencing,and the harnessing of its performative,processual,and relational value.Building on Serres’conception of silence as a dilation of the me the paper will follow Serresian anti-Cartesian reflections on the interchangeability of subject and object,his conception of the pre-positional body,and his thematization of the soul-body relationship.In close inter-implication with the employment of silencing is then the choice,again as a methodological device,of the preposition trans,made to act in order to explore the affinities/overlaps/assonances between Serres’theorization of the body,dimension of the human,and posthumanist conceptions of body/subjectivity.展开更多
Virus-induced gene silencing(VIGS)is a natural defense mechanism of plants,which can cause sequence-specific degradation of viral RNA and is often used for studying the functional genome.At present,the VIGS system med...Virus-induced gene silencing(VIGS)is a natural defense mechanism of plants,which can cause sequence-specific degradation of viral RNA and is often used for studying the functional genome.At present,the VIGS system mediated by Tobacco rattle virus(TRV)has been successfully established in many plant species.However,the VIGS system in Chinese narcissus had not been reported yet.In this paper,the first use of the TRV-VIGS system in Chinese narcissus is described in detail.By injecting pTRV2-GFP into narcissus leaves,we confirmed that TRV could infect narcissus and move in narcissus plants.After the inoculation of pTRV2-Nt PDS by two methods,phenomena such as photo-bleaching appeared in narcissus leaves and qRT-PCR results proved that PDS gene of narcissus was successfully silenced.Besides,pTRV2-NtMYB3 was inoculated into basal plates of narcissus to study the function of NtMYB3;the results showed that NtMYB3 is an inhibitor of flavonoids biosynthesis,which can increase the content of proanthocyanidins in basal plates of narcissus by repressing the expression level of NtFLS.These results indicate that the TRV-VIGS system has been successfully established in Chinese narcissus and successfully applied to the functional study of NtMYB3.展开更多
Virus-induced gene silencing (VIGS) technique, which is developed in recent years, is a rapid identification of plant gene function from reverse genetics. It is a manifestation of post-transcriptional gene silencing m...Virus-induced gene silencing (VIGS) technique, which is developed in recent years, is a rapid identification of plant gene function from reverse genetics. It is a manifestation of post-transcriptional gene silencing mechanism. Compared with the traditional transgenic technology, VIGS is a transient expression system, which can achieve good results in a short time. At present, it is widely used to study the function of plant genes, but most of them are model plants, and the experiments are carried out always in the indoor environment with controlled light and temperature conditions. In this study, we creatively provided a method to establish VIGS system using perennial Rosa plants as experimental materials under field conditions. The recombinant virus vector was constructed with RrGT1 gene as reporter gene and modified TRV-GFP virus as vector, and the perennial R. rugosa “Zizhi” and R. davurica were used as experimental verification materials. According to the growth conditions of Rosa plants, the natural environment in the field and the optimal conditions for the occurrence of VIGS, the technical problems such as the confirmation of the inoculation period, the preparation of the infective fluid, the inoculation technology of the virus vector and the light and temperature conditions of plant materials cultured after inoculation were solved one by one. When the RrGT1 gene was silenced, the Rosa plants showed a pale petal color phenotype. By detection, it was found that the expression of endogenous RrGT1 gene was significantly down-regulated, and the content of all anthocyanins also decreased significantly. Therefore, we believed that the attempt to establish VIGS system in perennial Rosa plants under field conditions was very successful.展开更多
基金supported by the Subject of Key R&D Plan of Shandong Province(Major Scientific and Technological Innovation Project)“Mining and Accurate Identification of Forest Tree Germplasm Resources”(Grant Nos.2021LZGC02303)Science&Technology Specific Projects in Agricultural High-tech Industrial Demonstration Area of the Yellow River Delta(Grant No.022SZX16)。
文摘VIGS(Virus-induced gene silencing),a method for posttranscriptional gene silencing,is an effective technique for investigating the activities of genes in plants.Since there is no report for available VIGS system in Styrax japonicus,the application of a VIGS approach that results in a gene knockdown to study gene function is limited.In this study,we compared the characteristics that could affect the viability of VIGS in S.japonicus,including the acetosyringone(AS)concentration,the Agrobacterium’s optical density and the inoculation method.The stable reference genes of S.japonicus were selected to validate the gene’s knockdown by quantitative PCR.As a result,we successfully constructed 2 VIGS systems based on TRV virus:vacuum with AS concentration of 200μmol·L^(-1)and OD600of 0.5,and friction-osmosis with AS concentration of 200μmol·L^(-1)and OD600of 1.0,which silencing efficiency was 83.33%and 74.19%,respectively.The successfully applied VIGS method provides a rapid and effective reverse gene functional analysis approach in S.japonicus to identify unknown gene functions.
基金supported by the National Key Research and Development Program of China(2022YFD1200300)the National Natural Science Foundation of China(32072376 and 32372515)+3 种基金Winall Hi-tech Seed Co.,Ltd.,China(GMLM2023)the Nanfan Special Project of Chinese Academy of Agricultural Sciences(CAAS)(ZDXM2303 and YBXM2415)the Natural Science Foundation of Hebei Province,China(C2022204205)the Agricultural Science and Technology Innovation Program of CAAS。
文摘Verticillium wilt(VW),induced by the soil-borne fungus Verticillium dahliae(Vd),poses a substantial threat to a diverse array of plant species.Employing molecular breeding technology for the development of cotton varieties with heightened resistance to VW stands out as one of the most efficacious protective measures.In this study,we successfully generated two stable transgenic lines of cotton(Gossypium hirsutum L.),VdThitRNAi-1 and VdThit-RNAi-2,using host-induced gene silencing(HIGS)technology to introduce double-stranded RNA(dsRNA)targeting the thiamine transporter protein gene(VdThit).Southern blot analysis confirmed the presence of a single-copy insertion in each line.Microscopic examination showed marked reductions in the colonization and spread of Vd-mCherry in the roots of VdThit-RNAi cotton compared to wild type(WT).The corresponding disease index and fungal biomass of VdThit-RNAi-1/2 also exhibited significant reductions.Real-time quantitative PCR(qRT-PCR)analysis demonstrated a substantial inhibition of VdThit expression following prolonged inoculation of VdThit-RNAi cotton.Small RNA sequencing(sRNA-Seq)analysis revealed the generation of a substantial number of VdThit-specific siRNAs in the VdThit-RNAi transgenic lines.Additionally,the silencing of VdThit by the siVdThit produced by VdThit-RNAi-1/2 resulted in the elevated expression of multiple genes involved in the thiamine biosynthesis pathway in Vd.Under field conditions,VdThit-RNAi transgenic cotton exhibited significantly enhanced disease resistance and yield compared with WT.In summary,our findings underscore the efficacy of HIGS targeting VdThit in restraining the infection and spread of Vd in cotton,thereby potentially enabling the development of cotton breeding as a promising strategy for managing VW.
文摘本研究旨在提高黄瓜绿斑驳花叶病毒(cucumber green mottle mosaic virus,CGMMV)介导的基因沉默(VIGS)在瓜类作物中应用效率。以黄瓜、甜瓜和西瓜优良种质为实验材料,采用不同的CGMMV病毒载体接种方式、并设置不同的培养温度和相对湿度,以测试基因沉默的有效性。结果表明,真空渗透和种子吸胀的接种方式能够在瓜类作物中产生最高的基因沉默率(FGS)。在22℃和25℃的培养环境下,黄瓜、甜瓜和西瓜PDS沉默有效性(EGSL)较高。黄瓜、甜瓜和西瓜生长1个月后,EGSL达到100%,显著高于30℃的培养环境下EGSL。生长3个月后,甜瓜和西瓜在22℃培养环境下EGSL分别为89.7%和95%;在25℃培养环境下EGSL分别为85.6%和86.1%,均显著性高于30℃下EGSL。在相对湿度为30%和50%环境下,黄瓜的EGSL分别为70%和72%,甜瓜的EGSL分别为76%和73%,显著高于相对湿度为80%的培养环境下EGSL。西瓜在相对湿度为30%的培养环境下的EGSL为69%,显著高于相对湿度为50%时的EGSL(38%)和相对湿度在80%时的EGSL(33%)。综上所述,通过优化了瓜类作物的CGMMV-VIGS的技术体系中接种方式和培养环境参数,提高了基因沉默率和有效性,并且能够快速获得整个植株基因沉默的种质资源,为作物优质和抗逆基因功能的研究提供有效途径。
基金supported by the Henan Province Science and Technology Research Project, China (Grant No. 242102110232)the National Natural Science Foundation of China (Grant No. 31801677)the Major Program of Guangdong Basic and Applied Basic Research, China (Grant No. 2019B030302006)。
文摘Rice sheath blight, caused by Rhizoctonia solani AG1-IA, is a major disease in rice-growing areas worldwide. Effectors of phytopathogenic fungi play important roles during the infection process of fungal pathogens onto their host plants. However, the molecular mechanisms by which R. solani effectors regulate rice immunity are not well understood. Through prediction, 78 candidate effector molecules were identified. Using the tobacco rattle virus-host induced gene silencing(TRV-HIGS) system, 45 RNAi constructs of effector genes were infiltrated into Nicotiana benthamiana leaves. The results revealed that eight of these constructs resulted in a significant reduction in necrosis caused by infection with the AG1-IA strain GD-118. Additionally, stable rice transformants carrying the double-stranded RNA construct for one of the effector genes, AGLIP1, were generated to further verify the function of this gene. The suppression of the AGLIP1 gene increased the resistance of both N. benthamiana and rice against GD-118, and also affected the growth rate of GD-118, indicating that AGLIP1 is a key pathogenic factor. Small RNA sequencing showed that the HIGS vectors were processed into si RNAs within the plants and then translocated to the fungi, leading to the silencing of the target genes. As a result, AGLIP1 might be an excellent candidate for HIGS, thereby enhancing crop resistance against the pathogen and contributing to the control of R. solani infection.
基金This work was supported by Jiangsu Seed Industry Revitalization Project,China[JBGS(2021)071]Fundamental Research Funds for the Central Universities,China(YDZX2023019)+3 种基金the National Natural Science Foundation of China(32172579)the earmarked fund for Jiangsu Agricultural Industry Technology System,China[JATS(2023)421]the Jiangsu Postgraduate Scientific Research Innovation Plan,China(KYCX21_0610-2021)the Project Founded by the Priority Academic Program Development of Jiangsu Higher Education Institutions,China(PAPD).
文摘Virus-induced gene silencing(VIGS)and clustered regularly interspaced short palindromic repeats/CRISPR-associated protein(CRISPR/Cas)systems are effective technologies for rapid and accurate gene function verification in modern plant biotechnology.However,the investigation of gene silencing and editing in radish remains limited.In this study,a bleaching phenotype was generated through the knockdown of RsPDS using tobacco rattle virus(TRV)-and turnip yellow mosaic virus(TYMV)-mediated gene silencing vectors.The TYMV-mediated gene silencing efficiency was higher than the TRV-based VIGS system in radish.The expression level of RsPDS was significantly inhibited using VIGS in'NAU-067'radish leaves.The rootless seedlings of‘NAU-067'were infected with Agrobacterium rhizogenes using the 2300GN-Ubi-RsPDS-Cas9 vector with two target sequences.Nine adventitious roots were blue with GUs staining,and four of these adventitious roots were edited at target sequence 1 of the RsPDS gene as indicated by Sanger sequencing.Furthermore,albino lines were generated with A.tumefaciens-mediated transformation of radish cotyledons.Five base substitutions and three base deletions occurred at target sequence 2 in Line 1,and three base insertions and three base substitutions occurred at target sequence 1 in Line 2.This study shows that VIGS and CRISPR/Cas9 techniques can be employed to precisely verify the biological functions of genes in radish,which will facilitate the genetic improvement of vital horticultural traits in radish breeding programs.
文摘Stress response pathways detect and alleviate adverse conditions to safeguard cell and tissue homeostasis,yet their prolonged activation induces apoptosis and disrupts organismal health1-3.How stress responses are turned off at the right time and place remains poorly understood.Here we report a ubiquitin-dependent mechanism that silences the cellular response to mitochondrial protein import stress.
文摘The contribution aims to examine the co-implications between Serres’work and posthumanist ideas of body and subjectivity.This scope is pursued primarily through the methodological choice of a gerund,such as silencing,and the harnessing of its performative,processual,and relational value.Building on Serres’conception of silence as a dilation of the me the paper will follow Serresian anti-Cartesian reflections on the interchangeability of subject and object,his conception of the pre-positional body,and his thematization of the soul-body relationship.In close inter-implication with the employment of silencing is then the choice,again as a methodological device,of the preposition trans,made to act in order to explore the affinities/overlaps/assonances between Serres’theorization of the body,dimension of the human,and posthumanist conceptions of body/subjectivity.
基金the Science and technology innovation fund of Fujian Agriculture and Forestry University(Grant Nos.KFA17352A,KFA17602A)。
文摘Virus-induced gene silencing(VIGS)is a natural defense mechanism of plants,which can cause sequence-specific degradation of viral RNA and is often used for studying the functional genome.At present,the VIGS system mediated by Tobacco rattle virus(TRV)has been successfully established in many plant species.However,the VIGS system in Chinese narcissus had not been reported yet.In this paper,the first use of the TRV-VIGS system in Chinese narcissus is described in detail.By injecting pTRV2-GFP into narcissus leaves,we confirmed that TRV could infect narcissus and move in narcissus plants.After the inoculation of pTRV2-Nt PDS by two methods,phenomena such as photo-bleaching appeared in narcissus leaves and qRT-PCR results proved that PDS gene of narcissus was successfully silenced.Besides,pTRV2-NtMYB3 was inoculated into basal plates of narcissus to study the function of NtMYB3;the results showed that NtMYB3 is an inhibitor of flavonoids biosynthesis,which can increase the content of proanthocyanidins in basal plates of narcissus by repressing the expression level of NtFLS.These results indicate that the TRV-VIGS system has been successfully established in Chinese narcissus and successfully applied to the functional study of NtMYB3.
文摘Virus-induced gene silencing (VIGS) technique, which is developed in recent years, is a rapid identification of plant gene function from reverse genetics. It is a manifestation of post-transcriptional gene silencing mechanism. Compared with the traditional transgenic technology, VIGS is a transient expression system, which can achieve good results in a short time. At present, it is widely used to study the function of plant genes, but most of them are model plants, and the experiments are carried out always in the indoor environment with controlled light and temperature conditions. In this study, we creatively provided a method to establish VIGS system using perennial Rosa plants as experimental materials under field conditions. The recombinant virus vector was constructed with RrGT1 gene as reporter gene and modified TRV-GFP virus as vector, and the perennial R. rugosa “Zizhi” and R. davurica were used as experimental verification materials. According to the growth conditions of Rosa plants, the natural environment in the field and the optimal conditions for the occurrence of VIGS, the technical problems such as the confirmation of the inoculation period, the preparation of the infective fluid, the inoculation technology of the virus vector and the light and temperature conditions of plant materials cultured after inoculation were solved one by one. When the RrGT1 gene was silenced, the Rosa plants showed a pale petal color phenotype. By detection, it was found that the expression of endogenous RrGT1 gene was significantly down-regulated, and the content of all anthocyanins also decreased significantly. Therefore, we believed that the attempt to establish VIGS system in perennial Rosa plants under field conditions was very successful.
