小麦条锈病是由小麦条锈菌引起的重大病害,严重威胁我国粮食安全。夏孢子是条锈菌繁殖、传播和侵染的关键介质,但其产孢相关基因的调控机制尚不清楚。筛选并验证小麦条锈菌侵染前期高表达的候选基因PsCON6的功能,为解析其致病机制提供...小麦条锈病是由小麦条锈菌引起的重大病害,严重威胁我国粮食安全。夏孢子是条锈菌繁殖、传播和侵染的关键介质,但其产孢相关基因的调控机制尚不清楚。筛选并验证小麦条锈菌侵染前期高表达的候选基因PsCON6的功能,为解析其致病机制提供新线索。通过同源克隆从小麦条锈菌中获取PsCON6基因,利用qRT-PCR分析其在侵染前期的表达模式。通过生物信息学技术分析预测PsCON6蛋白的氨基酸序列、保守结构域及理化性质。通过寄主介导的基因沉默(BSMV-HIGS)技术瞬时沉默PsCON6,检测寄主活性氧面积、病原菌菌丝长度与面积及病原菌生物量。通过瞬时表达确定PsCON6蛋白的亚细胞定位。PsCON6在小麦条锈菌侵染前期显著高表达,其编码的蛋白含2个conidiation-specific protein 6保守结构域,由83个氨基酸组成。HIGS沉默PsCON6后,寄主活性氧水平显著升高,病原菌菌丝长度和面积显著减少,但孢子量未受显著影响。亚细胞定位表明,PsCON6定位于细胞膜。结果表明,PsCON6可能参与调控小麦条锈菌的菌丝生长过程,但对夏孢子形成无直接影响,其功能可能存在冗余,为揭示条锈菌致病机制提供了新靶点,并为后续深入解析其分子机制奠定了基础。展开更多
Sclerotinia sclerotiorum is generally considered one of the most economically damaging pathogens in oilseed rape(Brassica napus).Breeding for Sclerotinia resistance is challenging,as no immune germplasm available in B...Sclerotinia sclerotiorum is generally considered one of the most economically damaging pathogens in oilseed rape(Brassica napus).Breeding for Sclerotinia resistance is challenging,as no immune germplasm available in B.napus.It is desirable to develop new breeding strategies.In the present study,hostinduced gene silencing(HIGS),developed based on RNA interference(RNAi),was applied to protect B.napus from S.sclerotiorum infection.Three pathogenicity genes,the endo-polygalacturonase gene(Ss PG1),cellobiohydrolase gene(Ss CBH),and oxaloacetate acetylhydrolase gene(Ss OAH1),were chosen as HIGS targets.Co-incubation of synthesized double-stranded RNAs(ds RNAs)with S.sclerotiorum in liquid medium significantly reduced the transcript levels of the target genes.Application to plant surfaces of ds RNA targeting the three genes conferred effective protection against S.sclerotiorum.Stable transgenic B.napus plants expressing small interfering RNAs with sequence identity to Ss PG1,Ss CBH,and Ss OAH1 were generated.HIGS transgenic B.napus prevented the expression of S.sclerotiorum target genes,slowed pathogenicity-factor accumulation,impeded fungal growth,and suppressed appressorium formation,thereby conferring resistance to S.sclerotiorum.Simultaneous silencing of Ss PG1,Ss CBH,and Ss OAH1 by stable expression of a chimeric hairpin RNAi construct in B.napus led to enhanced protection phenotypes(with disease lesion size reduced by 36.8%–43.7%).We conclude that HIGS of pathogenic-factor genes of S.sclerotiorum is a promising strategy for controlling Sclerotinia rot in oilseed rape.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
对于难治性高血压的定义，2003年美国预防、检测、评估、与治疗高血压联合委员会第七次报告（the seventh report of the joint national committee on pre-vention, detection, evaluation and treatment of hig hblood pressure, JNC...对于难治性高血压的定义，2003年美国预防、检测、评估、与治疗高血压联合委员会第七次报告（the seventh report of the joint national committee on pre-vention, detection, evaluation and treatment of hig hblood pressure, JNC7）中强调了包括利尿剂的3种药物，但没有考虑改善生活方式。展开更多
Fungal pathogens represent a major group of plant invaders that are the causative agents of many notorious plant diseases. Large quantities of RNAs, especially small RNAs involved in gene silencing, have been found to...Fungal pathogens represent a major group of plant invaders that are the causative agents of many notorious plant diseases. Large quantities of RNAs, especially small RNAs involved in gene silencing, have been found to transmit bidirectionally between fungal pathogens and their hosts. Although host-induced gene silencing （HIGS） technology has been developed and applied to protect crops from fungal infections, the mechanisms of RNA transmission, especially small RNAs regulating trans- kingdom RNA silencing in plant immunity, are largely unknown. In this review, we summarize and discuss recent important findings regarding trans-kingdom sRNAs and RNA silencing in plant-fungal pathogen interactions compared with the well-known RNAi mechanisms in plants and fungi. We focus on the interactions between plant and fungal pathogens with broad hosts, represented by the vascular pathogen Verticillium dahliae and non-vascular pathogen Botrytis cinerea, and discuss the known instances of natural RNAi transmission between fungal pathogens and host plants. Given that HIGS has been developed and recently applied in controlling Verticillium wilt diseases, we propose an ideal research system exploiting plant vasculature-Verticillium interaction to further study trans-kingdom RNA silencing.展开更多
文摘小麦条锈病是由小麦条锈菌引起的重大病害,严重威胁我国粮食安全。夏孢子是条锈菌繁殖、传播和侵染的关键介质,但其产孢相关基因的调控机制尚不清楚。筛选并验证小麦条锈菌侵染前期高表达的候选基因PsCON6的功能,为解析其致病机制提供新线索。通过同源克隆从小麦条锈菌中获取PsCON6基因,利用qRT-PCR分析其在侵染前期的表达模式。通过生物信息学技术分析预测PsCON6蛋白的氨基酸序列、保守结构域及理化性质。通过寄主介导的基因沉默(BSMV-HIGS)技术瞬时沉默PsCON6,检测寄主活性氧面积、病原菌菌丝长度与面积及病原菌生物量。通过瞬时表达确定PsCON6蛋白的亚细胞定位。PsCON6在小麦条锈菌侵染前期显著高表达,其编码的蛋白含2个conidiation-specific protein 6保守结构域,由83个氨基酸组成。HIGS沉默PsCON6后,寄主活性氧水平显著升高,病原菌菌丝长度和面积显著减少,但孢子量未受显著影响。亚细胞定位表明,PsCON6定位于细胞膜。结果表明,PsCON6可能参与调控小麦条锈菌的菌丝生长过程,但对夏孢子形成无直接影响,其功能可能存在冗余,为揭示条锈菌致病机制提供了新靶点,并为后续深入解析其分子机制奠定了基础。
基金the National Natural Science Foundation of China(32072020,U20A2028,and 31901504)the Jiangsu Agricultural Science and Technology Innovation Fund(CX(20)3120)+1 种基金the Project of Special Funding for Crop Science Discipline Development(yzuxk202006)the Priority Academic Program Development of Jiangsu Higher Education Institutions and the Qinglan Project of Yangzhou University。
文摘Sclerotinia sclerotiorum is generally considered one of the most economically damaging pathogens in oilseed rape(Brassica napus).Breeding for Sclerotinia resistance is challenging,as no immune germplasm available in B.napus.It is desirable to develop new breeding strategies.In the present study,hostinduced gene silencing(HIGS),developed based on RNA interference(RNAi),was applied to protect B.napus from S.sclerotiorum infection.Three pathogenicity genes,the endo-polygalacturonase gene(Ss PG1),cellobiohydrolase gene(Ss CBH),and oxaloacetate acetylhydrolase gene(Ss OAH1),were chosen as HIGS targets.Co-incubation of synthesized double-stranded RNAs(ds RNAs)with S.sclerotiorum in liquid medium significantly reduced the transcript levels of the target genes.Application to plant surfaces of ds RNA targeting the three genes conferred effective protection against S.sclerotiorum.Stable transgenic B.napus plants expressing small interfering RNAs with sequence identity to Ss PG1,Ss CBH,and Ss OAH1 were generated.HIGS transgenic B.napus prevented the expression of S.sclerotiorum target genes,slowed pathogenicity-factor accumulation,impeded fungal growth,and suppressed appressorium formation,thereby conferring resistance to S.sclerotiorum.Simultaneous silencing of Ss PG1,Ss CBH,and Ss OAH1 by stable expression of a chimeric hairpin RNAi construct in B.napus led to enhanced protection phenotypes(with disease lesion size reduced by 36.8%–43.7%).We conclude that HIGS of pathogenic-factor genes of S.sclerotiorum is a promising strategy for controlling Sclerotinia rot in oilseed rape.
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.
文摘对于难治性高血压的定义，2003年美国预防、检测、评估、与治疗高血压联合委员会第七次报告（the seventh report of the joint national committee on pre-vention, detection, evaluation and treatment of hig hblood pressure, JNC7）中强调了包括利尿剂的3种药物，但没有考虑改善生活方式。
基金This study was financially supported by the Strategic Priority Research Program of the Chinese Academy of Sciences （XDB11040500 to H.-S.G.）, National Natural Science Foundation （31730078 to H.-S.G., 31600124 to J.-H.Z., and 31500119 to C.H.）.
文摘Fungal pathogens represent a major group of plant invaders that are the causative agents of many notorious plant diseases. Large quantities of RNAs, especially small RNAs involved in gene silencing, have been found to transmit bidirectionally between fungal pathogens and their hosts. Although host-induced gene silencing （HIGS） technology has been developed and applied to protect crops from fungal infections, the mechanisms of RNA transmission, especially small RNAs regulating trans- kingdom RNA silencing in plant immunity, are largely unknown. In this review, we summarize and discuss recent important findings regarding trans-kingdom sRNAs and RNA silencing in plant-fungal pathogen interactions compared with the well-known RNAi mechanisms in plants and fungi. We focus on the interactions between plant and fungal pathogens with broad hosts, represented by the vascular pathogen Verticillium dahliae and non-vascular pathogen Botrytis cinerea, and discuss the known instances of natural RNAi transmission between fungal pathogens and host plants. Given that HIGS has been developed and recently applied in controlling Verticillium wilt diseases, we propose an ideal research system exploiting plant vasculature-Verticillium interaction to further study trans-kingdom RNA silencing.
