Wheat dwarf disease caused by wheat dwarf virus(WDV) is currently present in wheat growing regions in China and causes serious losses in wheat yield. To develop reliable and effective serological detection methods for...Wheat dwarf disease caused by wheat dwarf virus(WDV) is currently present in wheat growing regions in China and causes serious losses in wheat yield. To develop reliable and effective serological detection methods for WDV, the coat protein(CP) gene of WDV was cloned and expressed in Escherichia coli. The purified recombinant CP protein was immunized to BALB/c mice, and four hybridoma cell lines(i.e. 18G10, 9G4, 23F4 and 22A10) secreting anti-WDV monoclonal antibodies(MAbs) were obtained through the hybridoma technique. Using the prepared MAbs, an antigencoated-plate enzyme-linked immunosorbent assay(ACP-ELISA) and a dot-ELISA were established for detecting WDV in wheat samples. The most sensitive ACP-ELISA based on MAb 23F4 or 22A10 was able to detect WDV in 1:163,840(w/v,g/mL) diluted WDV-infected wheat plant crude extracts. The dot-ELISA based on MAb 23F4 was the most sensitive and able to detect the virus in 1:5,120(w/v, g/mL) diluted wheat plant crude extracts. A total of 128 wheat samples were collected from wheat growing regions in the Shaanxi and Qinghai provinces, China, and were screened for the presence of WDV using two developed serological assays. Results from the survey showed that approximately 62% of the samples were infected with WDV. PCR followed by DNA sequencing and sequence alignment validated the results from the two serological assays. Therefore, we consider that these two serological detection methods can be significantly useful for the control of WDV in China.展开更多
High-throughput deep-sequencing technology and bioinformatics analysis of the small RNA(sRNA)population isolated from plants allows universal virus detection and complete virome reconstruction for a given sample.In th...High-throughput deep-sequencing technology and bioinformatics analysis of the small RNA(sRNA)population isolated from plants allows universal virus detection and complete virome reconstruction for a given sample.In the present sRNA deep-sequencing analysis of virus-infected wheat samples in the Czech Republic,samples were firstly tested for barley yellow dwarf viruses(BYDVs),wheat streak mosaic virus(WSMV)and wheat dwarf virus(WDV)using ELISA,RT-PCR and PCR.Subsequent sRNA sequencing of these samples yielded more than^60 million single-end 50-bp reads with high confidence for nine field samples of wheat.Overall,16.5%of reads were virus-specific and 83.5%were mapped to the host.More 21-nt reads(-7.7E+06 reads)were found than 24-nt(~6.20E+06 reads)or 22-nt(-4.30E+06 reads)reads.De novo assembly of the high-quality contigs revealed the presence of three earlier repoted viruses in the Czech Republic:BYDVs(31.48%),WSMV(24.23%)and WDV(2666%).We also showed the presence of cereal yellow dwarf virus(14.33%;two species CYDV-RPS and CYDV-RPV family Luteoviridae/Polerovirus)and wheat yellow dwarf virus(WYDV,3.30%;Luteoviridae).Phylogenetic analysis showed CYDV and WYDV grouped separately from BYDVs.Furthermore,several recombination breakpoints were found among the groups of yellow dwarf viruses(BYDVs,CYDV,and WYDV).Using RNA deep sequencing,we confirmed the presence of the three known viruses(BYDVs,WSMV,and WDV)and the first record of two species of CYDV and WYDV in wheat in the Czech Republic.展开更多
Wheat dwarf virus （WDV）, an important cereal pathogen, is closely related to Maize streak virus （MSV）, a model virus of the Mastrevirus genus. Based on its similarity to known MSV resistance strategies, a truncate...Wheat dwarf virus （WDV）, an important cereal pathogen, is closely related to Maize streak virus （MSV）, a model virus of the Mastrevirus genus. Based on its similarity to known MSV resistance strategies, a truncated part of the WDV replication- associated （RepA） gene （WDVRepA215） and the WDV RepA gene with a mutated retinoblastoma-related protein （RBR） interaction domain （WDVRepA215RBRre^t） were cloned into the plPKb002 expression vector and transformed into immature embryos of spring barley cv. Golden Promise plants through Agrobacterium-mediated transformation. A detailed study of T1-generation plants infected by leafhoppers （Psammotettix alienus） fed on infection sources of variable strength was performed over a 5-week period encompassing the initial stages of virus infection. A DNA WDV TaqMan qPCR assay normalized using the DNA puroindoline-b SYBR Green qPCR assay for samples on a per week basis revealed an approximately 2-week delay in WDVRepA215RBR^mut plants to WDVRepA215 plants before significant increases in the WDV viral levels occurred. Both WDVRepA215 and WDVRepA215RBR^mut plants showed similar levels of transgenic transcripts over the screened period; however, the transgenic plants also showed increased numbers of infected plants compared to the control plants.展开更多
基金supported by Public Science and Technology Research Funds Projects of Agriculture (201303021)the National Basic Research Program (973) of China (No.2014CB138400)
文摘Wheat dwarf disease caused by wheat dwarf virus(WDV) is currently present in wheat growing regions in China and causes serious losses in wheat yield. To develop reliable and effective serological detection methods for WDV, the coat protein(CP) gene of WDV was cloned and expressed in Escherichia coli. The purified recombinant CP protein was immunized to BALB/c mice, and four hybridoma cell lines(i.e. 18G10, 9G4, 23F4 and 22A10) secreting anti-WDV monoclonal antibodies(MAbs) were obtained through the hybridoma technique. Using the prepared MAbs, an antigencoated-plate enzyme-linked immunosorbent assay(ACP-ELISA) and a dot-ELISA were established for detecting WDV in wheat samples. The most sensitive ACP-ELISA based on MAb 23F4 or 22A10 was able to detect WDV in 1:163,840(w/v,g/mL) diluted WDV-infected wheat plant crude extracts. The dot-ELISA based on MAb 23F4 was the most sensitive and able to detect the virus in 1:5,120(w/v, g/mL) diluted wheat plant crude extracts. A total of 128 wheat samples were collected from wheat growing regions in the Shaanxi and Qinghai provinces, China, and were screened for the presence of WDV using two developed serological assays. Results from the survey showed that approximately 62% of the samples were infected with WDV. PCR followed by DNA sequencing and sequence alignment validated the results from the two serological assays. Therefore, we consider that these two serological detection methods can be significantly useful for the control of WDV in China.
基金We thank Ms.Michaela Brozenska(Plant Virus and Vector Interactions Group,Division of Crop Protection and Plant Health,Crop Research Institute,Czech Republic)for her excellent technical assistance.We also thank Dr.Beth E.Hazen(Willows End Scientific Editing and Writing Cortland,NY,USA)for editing the English of the manuscript.This work was supported by a grant from the Technology Agency of the Czech Republic(TF02000056).
文摘High-throughput deep-sequencing technology and bioinformatics analysis of the small RNA(sRNA)population isolated from plants allows universal virus detection and complete virome reconstruction for a given sample.In the present sRNA deep-sequencing analysis of virus-infected wheat samples in the Czech Republic,samples were firstly tested for barley yellow dwarf viruses(BYDVs),wheat streak mosaic virus(WSMV)and wheat dwarf virus(WDV)using ELISA,RT-PCR and PCR.Subsequent sRNA sequencing of these samples yielded more than^60 million single-end 50-bp reads with high confidence for nine field samples of wheat.Overall,16.5%of reads were virus-specific and 83.5%were mapped to the host.More 21-nt reads(-7.7E+06 reads)were found than 24-nt(~6.20E+06 reads)or 22-nt(-4.30E+06 reads)reads.De novo assembly of the high-quality contigs revealed the presence of three earlier repoted viruses in the Czech Republic:BYDVs(31.48%),WSMV(24.23%)and WDV(2666%).We also showed the presence of cereal yellow dwarf virus(14.33%;two species CYDV-RPS and CYDV-RPV family Luteoviridae/Polerovirus)and wheat yellow dwarf virus(WYDV,3.30%;Luteoviridae).Phylogenetic analysis showed CYDV and WYDV grouped separately from BYDVs.Furthermore,several recombination breakpoints were found among the groups of yellow dwarf viruses(BYDVs,CYDV,and WYDV).Using RNA deep sequencing,we confirmed the presence of the three known viruses(BYDVs,WSMV,and WDV)and the first record of two species of CYDV and WYDV in wheat in the Czech Republic.
基金the Czech Ministry of Education, Youth and Sports funding programme LH12161the Czech Ministry of Agriculture funding programme MZE RO0417
文摘Wheat dwarf virus （WDV）, an important cereal pathogen, is closely related to Maize streak virus （MSV）, a model virus of the Mastrevirus genus. Based on its similarity to known MSV resistance strategies, a truncated part of the WDV replication- associated （RepA） gene （WDVRepA215） and the WDV RepA gene with a mutated retinoblastoma-related protein （RBR） interaction domain （WDVRepA215RBRre^t） were cloned into the plPKb002 expression vector and transformed into immature embryos of spring barley cv. Golden Promise plants through Agrobacterium-mediated transformation. A detailed study of T1-generation plants infected by leafhoppers （Psammotettix alienus） fed on infection sources of variable strength was performed over a 5-week period encompassing the initial stages of virus infection. A DNA WDV TaqMan qPCR assay normalized using the DNA puroindoline-b SYBR Green qPCR assay for samples on a per week basis revealed an approximately 2-week delay in WDVRepA215RBR^mut plants to WDVRepA215 plants before significant increases in the WDV viral levels occurred. Both WDVRepA215 and WDVRepA215RBR^mut plants showed similar levels of transgenic transcripts over the screened period; however, the transgenic plants also showed increased numbers of infected plants compared to the control plants.
