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
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
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
The phenomenon and potential mechanisms of trans-kingdom RNA silencing(or RNA interference,RNAi)are among the most exciting topics in science today.Based on trans-kingdom RNAi,host-induced gene silencing(HIGS)has been...The phenomenon and potential mechanisms of trans-kingdom RNA silencing(or RNA interference,RNAi)are among the most exciting topics in science today.Based on trans-kingdom RNAi,host-induced gene silencing(HIGS)has been widely applied to create crops with resistance to various pests and pathogens,overcoming the limitations of resistant cultivars.However,a lack of transformation technology in many crops limits the application of HIGS.Here,we describe the various fates of trans-kingdom RNAs in recipient organisms.Based on the assumption that small RNAs can be transferred between the host and its microbiome or among microbiome members,we propose a possible alternative strategy for plant protection against pathogens without the need for crop genetic modification.展开更多
Small RNAs(sRNAs)are a class of short,non-coding regulatory RNAs that have emerged as critical components of defense regulatory networks across plant kingdoms.Many sRNA-based technologies,such as host-induced gene sil...Small RNAs(sRNAs)are a class of short,non-coding regulatory RNAs that have emerged as critical components of defense regulatory networks across plant kingdoms.Many sRNA-based technologies,such as host-induced gene silencing(HIGS),spray-induced gene silencing(SIGS),virus-induced gene silencing(VIGS),artificial microRNA(amiRNA)and synthetic trans-acting siRNA(syn-tasiRNA)-mediated RNA interference(RNAi),have been developed as disease control strategies in both monocot and dicot plants,particularly in crops.This review aims to highlight our current understanding of the roles of sRNAs including miRNAs,heterochromatic siRNAs(hc-siRNAs),phased,secondary siRNAs(phasiRNAs)and natural antisense siRNAs(nat-siRNAs)in disease resistance,and sRNAs-mediated trade-offs between defense and growth in crops.In particular,we focus on the diverse functions of sRNAs in defense responses to bacterial and fungal pathogens,oomycete and virus in crops.Further,we highlight the application of sRNA-based technologies in protecting crops from pathogens.Further research perspectives are proposed to develop new sRNAs-based efficient strategies to breed non-genetically modified(GMO),diseasetolerant crops for sustainable agriculture.展开更多
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.
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.
基金supported by the National Natural Science Foundation of China(no.31730078).
文摘The phenomenon and potential mechanisms of trans-kingdom RNA silencing(or RNA interference,RNAi)are among the most exciting topics in science today.Based on trans-kingdom RNAi,host-induced gene silencing(HIGS)has been widely applied to create crops with resistance to various pests and pathogens,overcoming the limitations of resistant cultivars.However,a lack of transformation technology in many crops limits the application of HIGS.Here,we describe the various fates of trans-kingdom RNAs in recipient organisms.Based on the assumption that small RNAs can be transferred between the host and its microbiome or among microbiome members,we propose a possible alternative strategy for plant protection against pathogens without the need for crop genetic modification.
基金supported by funding from the National Natural Science Foundation of China(91940301 to Z.H.,32070564 and 31600207 to J.L.)and Yunnan Fundamental Research Projects(202101AW070002 to J.L.).
文摘Small RNAs(sRNAs)are a class of short,non-coding regulatory RNAs that have emerged as critical components of defense regulatory networks across plant kingdoms.Many sRNA-based technologies,such as host-induced gene silencing(HIGS),spray-induced gene silencing(SIGS),virus-induced gene silencing(VIGS),artificial microRNA(amiRNA)and synthetic trans-acting siRNA(syn-tasiRNA)-mediated RNA interference(RNAi),have been developed as disease control strategies in both monocot and dicot plants,particularly in crops.This review aims to highlight our current understanding of the roles of sRNAs including miRNAs,heterochromatic siRNAs(hc-siRNAs),phased,secondary siRNAs(phasiRNAs)and natural antisense siRNAs(nat-siRNAs)in disease resistance,and sRNAs-mediated trade-offs between defense and growth in crops.In particular,we focus on the diverse functions of sRNAs in defense responses to bacterial and fungal pathogens,oomycete and virus in crops.Further,we highlight the application of sRNA-based technologies in protecting crops from pathogens.Further research perspectives are proposed to develop new sRNAs-based efficient strategies to breed non-genetically modified(GMO),diseasetolerant crops for sustainable agriculture.
