Cucurbit aphid-borne yellows virus(CABYV),melon aphid-borne yellows virus(MABYV)and suakwa aphid-borne yellows virus(SABYV)are three poleroviruses that infect cucurbit crops.Developing specific antisera against such v...Cucurbit aphid-borne yellows virus(CABYV),melon aphid-borne yellows virus(MABYV)and suakwa aphid-borne yellows virus(SABYV)are three poleroviruses that infect cucurbit crops.Developing specific antisera against such viruses is crucial for their detection and functional understanding of related genes.However,no studies have yet reported viral detection using antisera against movement proteins(MP)in these three viruses.In this study,we generated plasmids expressing three viral MP genes,and transformed them into the Escherichia coli strain,Rosetta,to recombinantly express and purify fusion proteins.Then,polyclonal antisera were derived by immunizing New Zealand white rabbits,after which western blotting was used to determine the titer,sensitivity and specificity of the antisera.The antisera titers against MP^(CABYV),MP^(MABYV) and MP^(SABYV) were 1:512000,1:256000 and 1:256000,respectively.The optimized working concentrations for the three antisera ranged between 1:10000 and 1:64000.Additionally,antisera against MP^(CABYV) and MP^(MABYV) only reacted with their corresponding MP proteins.Antiserum against MP^(SABYV) not only had the strongest reaction with its MP,but also reacted weakly with MP^(CABYV) and MP^(MABYV).All three antisera exerted no serological reactions with other poleroviruses.Furthermore,our data showed that all antisera specifically detected MPs in both Nicotiana benthamiana and cucumber leaves.Thus,we have established a system that sensitively detects three poleroviruses infecting cucurbits,using antisera against MPs.We provide a foundation for future research on the serological detection of these viruses,and interaction mechanisms between viruses and host plants.展开更多
Beclin 1/ATG6 plays a critical role in the biogenesis of autophagosomes and various cellular processes,but how Beclin 1-dependent autophagy is activated in plants remains elusive.In this study,we found that the cystei...Beclin 1/ATG6 plays a critical role in the biogenesis of autophagosomes and various cellular processes,but how Beclin 1-dependent autophagy is activated in plants remains elusive.In this study,we found that the cysteine protease metacaspase 1(MC1)functions as a new positive regulator of autophagy and cleaves Beclin 1 behind residues R97 and R99 in tobacco(Nicotiana benthamiana).Genetic analysis further demonstrated that the MC1-Beclin 1 cleavage module is both necessary and sufficient for the activation of plant autophagy.Mechanistically,this cleavage releases the N-terminal fragment of Beclin 1(aa 1-97),which exhibits intrinsic autophagy-inducing activity and is dependent on the phosphatidylinositol 3-kinase complex.Moreover,the activated autophagy boosts broad-spectrum antiviral responses,while the barley stripe mosaic virus-encodedγb protein targets MC1 and suppresses MC1-mediated Beclin 1 cleavage,thereby optimizing viral infection.The cleavage of Beclin 1 is thought to abolish its autophagic function in mammals;however,our findings unveil a distinctive plant autophagy mechanism whereby Beclin 1 activation necessitates MC1-mediated cleavage to drive antiviral autophagy.展开更多
The serological method is one of the most important techniques extensively used in crop production to detect different pathogens,especially plant viruses.An antiserum is essential for serological tests.The 17 kDa move...The serological method is one of the most important techniques extensively used in crop production to detect different pathogens,especially plant viruses.An antiserum is essential for serological tests.The 17 kDa movement protein(MP)of Potato leafroll virus(PLRV)is related to the membranous structures and localized to the plasmodesmata,but there is no report on preparation of PLRV-MP antiserum for detection of PLRV.To prepare PLRV-MP antiserum,reverse transcription polymerase chain reaction(RT-PCR)was carried out to amplify the PLRV-MP gene,which was constructed into a prokaryotic vector to express the protein in Escherichia coli for immunization of rabbits,after purification.Western blotting revealed that this developed antiserum could effectively detect PLRV,but with better results from the perspective of color development and economics by using antiserum at the ratio range of 1:10000 to 1:40000,presenting high sensitivity and specificity to PLRV.The serological detection results for PLRV of the field samples were identical to the RT-PCR detection.This is the first report on the development of PLRV-MP antiserum that has been successfully used for both laboratory and field detection of PLRV.The results provide a fundamental tool for further research on the function of PLRV-MP.展开更多
Wheat （Triticum aestivum L.） yellow mosaic virus （WYMV） is transmitted by a fungal vector through soil and causes serious wheat yield losses due to yellow mosaic disease, with yellow-streaked leaves and stunted pl...Wheat （Triticum aestivum L.） yellow mosaic virus （WYMV） is transmitted by a fungal vector through soil and causes serious wheat yield losses due to yellow mosaic disease, with yellow-streaked leaves and stunted plants. In the present study, the amplified fragment length polymorphisms （AFLP） and simple sequence repeat （SSR） were used to identify the molecular linkages with the resistance gene against WYMV. Bulked segregant analysis was performed with an F2 population derived from the cross of cultivar Ningmai 9 （resistant） × cultivar Yangmai 10 （susceptible）. By screening among the resistant or susceptible parents, the F2 pools and the individuals in the F2 population with 64 combined selective AFLP primers （EcoRI/MseI） or 290 reported SSR primers, a polymorphic DNA segment （approximately 120 bp） was amplified using the primer pair E2/M5, and an SSR marker （approximately 180 bp） was located on wheat chromosome 2A using the primer Xgwm328. Analysis with MAPMAKER/Exp Version 3.0b （Whitehead institute for Biomedical Research, Cambridge, MA, USA） indicated that these two markers were dominantly associated with the resistance gene at distances of 5.4 cM or 17.6 cM, respectively. The resistance gene to WYMV derived from Ningmai 9, is temporarily named YmNM, and was mapped to wheat chromosome 2A.展开更多
基金supported by the National Natural Science Foundation of China(31671995 and 31000840).
文摘Cucurbit aphid-borne yellows virus(CABYV),melon aphid-borne yellows virus(MABYV)and suakwa aphid-borne yellows virus(SABYV)are three poleroviruses that infect cucurbit crops.Developing specific antisera against such viruses is crucial for their detection and functional understanding of related genes.However,no studies have yet reported viral detection using antisera against movement proteins(MP)in these three viruses.In this study,we generated plasmids expressing three viral MP genes,and transformed them into the Escherichia coli strain,Rosetta,to recombinantly express and purify fusion proteins.Then,polyclonal antisera were derived by immunizing New Zealand white rabbits,after which western blotting was used to determine the titer,sensitivity and specificity of the antisera.The antisera titers against MP^(CABYV),MP^(MABYV) and MP^(SABYV) were 1:512000,1:256000 and 1:256000,respectively.The optimized working concentrations for the three antisera ranged between 1:10000 and 1:64000.Additionally,antisera against MP^(CABYV) and MP^(MABYV) only reacted with their corresponding MP proteins.Antiserum against MP^(SABYV) not only had the strongest reaction with its MP,but also reacted weakly with MP^(CABYV) and MP^(MABYV).All three antisera exerted no serological reactions with other poleroviruses.Furthermore,our data showed that all antisera specifically detected MPs in both Nicotiana benthamiana and cucumber leaves.Thus,we have established a system that sensitively detects three poleroviruses infecting cucurbits,using antisera against MPs.We provide a foundation for future research on the serological detection of these viruses,and interaction mechanisms between viruses and host plants.
基金supported by the National Natural Science Foundation of China(32330086 and 32270168)to D.L.the Young Elite Scientists Sponsorship Program by CAST(2022QNRC001)to M.Y.the 2115 Talent Development Program of China Agricultural University.
文摘Beclin 1/ATG6 plays a critical role in the biogenesis of autophagosomes and various cellular processes,but how Beclin 1-dependent autophagy is activated in plants remains elusive.In this study,we found that the cysteine protease metacaspase 1(MC1)functions as a new positive regulator of autophagy and cleaves Beclin 1 behind residues R97 and R99 in tobacco(Nicotiana benthamiana).Genetic analysis further demonstrated that the MC1-Beclin 1 cleavage module is both necessary and sufficient for the activation of plant autophagy.Mechanistically,this cleavage releases the N-terminal fragment of Beclin 1(aa 1-97),which exhibits intrinsic autophagy-inducing activity and is dependent on the phosphatidylinositol 3-kinase complex.Moreover,the activated autophagy boosts broad-spectrum antiviral responses,while the barley stripe mosaic virus-encodedγb protein targets MC1 and suppresses MC1-mediated Beclin 1 cleavage,thereby optimizing viral infection.The cleavage of Beclin 1 is thought to abolish its autophagic function in mammals;however,our findings unveil a distinctive plant autophagy mechanism whereby Beclin 1 activation necessitates MC1-mediated cleavage to drive antiviral autophagy.
基金supported by the National Natural Science Foundation of China(31671995 and 31371909)the 111 project(B13006).
文摘The serological method is one of the most important techniques extensively used in crop production to detect different pathogens,especially plant viruses.An antiserum is essential for serological tests.The 17 kDa movement protein(MP)of Potato leafroll virus(PLRV)is related to the membranous structures and localized to the plasmodesmata,but there is no report on preparation of PLRV-MP antiserum for detection of PLRV.To prepare PLRV-MP antiserum,reverse transcription polymerase chain reaction(RT-PCR)was carried out to amplify the PLRV-MP gene,which was constructed into a prokaryotic vector to express the protein in Escherichia coli for immunization of rabbits,after purification.Western blotting revealed that this developed antiserum could effectively detect PLRV,but with better results from the perspective of color development and economics by using antiserum at the ratio range of 1:10000 to 1:40000,presenting high sensitivity and specificity to PLRV.The serological detection results for PLRV of the field samples were identical to the RT-PCR detection.This is the first report on the development of PLRV-MP antiserum that has been successfully used for both laboratory and field detection of PLRV.The results provide a fundamental tool for further research on the function of PLRV-MP.
文摘Wheat （Triticum aestivum L.） yellow mosaic virus （WYMV） is transmitted by a fungal vector through soil and causes serious wheat yield losses due to yellow mosaic disease, with yellow-streaked leaves and stunted plants. In the present study, the amplified fragment length polymorphisms （AFLP） and simple sequence repeat （SSR） were used to identify the molecular linkages with the resistance gene against WYMV. Bulked segregant analysis was performed with an F2 population derived from the cross of cultivar Ningmai 9 （resistant） × cultivar Yangmai 10 （susceptible）. By screening among the resistant or susceptible parents, the F2 pools and the individuals in the F2 population with 64 combined selective AFLP primers （EcoRI/MseI） or 290 reported SSR primers, a polymorphic DNA segment （approximately 120 bp） was amplified using the primer pair E2/M5, and an SSR marker （approximately 180 bp） was located on wheat chromosome 2A using the primer Xgwm328. Analysis with MAPMAKER/Exp Version 3.0b （Whitehead institute for Biomedical Research, Cambridge, MA, USA） indicated that these two markers were dominantly associated with the resistance gene at distances of 5.4 cM or 17.6 cM, respectively. The resistance gene to WYMV derived from Ningmai 9, is temporarily named YmNM, and was mapped to wheat chromosome 2A.
