Rice ragged stunt virus（RRSV） is a serious rice disease in Asia, causing serious yield losses on rice. The capsid protein（CP） gene of the major outer capsid protein of RRSV was expressed in Escherichia coli BL21（...Rice ragged stunt virus（RRSV） is a serious rice disease in Asia, causing serious yield losses on rice. The capsid protein（CP） gene of the major outer capsid protein of RRSV was expressed in Escherichia coli BL21（DE3） using the pMAL-C2 X expression vector. The recombinant protein was used as the immunogen to immunize BALB/c mice. A hybridoma cell line 8A12 secreting monoclonal antibody（MAb） against RRSV was obtained by fusing mouse myeloma cells（Sp 2/0） with spleen cells from the immunized BALB/c mice. Western blot analysis showed that the MAb 8A12 can specifically react with RRSV CP. Using the MAb, an antigen-coated-plate enzyme-linked immunosorbent assay（ACP-ELISA）, a dot enzyme-linked immunosorbent assay（dot-ELISA）, and immunocapture-RT-PCR（IC-RT-PCR） assay were developed to detect RRSV. The established ACP-ELISA, dot-blot ELISA and IC-RT-PCR methods could detect RRSV in infected rice tissue crude extracts with dilutions of 1：40 960, 1：1 280 and 1：655 360（w/v, g mL-1）, respectively. The ACP-ELISA and dot-blot ELISA methods could detect RRSV in infected insect vector crude extracts with dilutions of 1：12 800 and 1：1 600（an individual planthopper μL-1）, respectively. The field survey revealed that Rice ragged stunt disease occurs on rice in Hainan, Yunnan, Guangxi, Sichuan, Guizhou, Fujian, Hunan, Jiangxi and Zhejiang in China.展开更多
Intracellular polyamines(putrescine,spermidine,and spermine)have emerged as important molecules for viral infection;however,how viruses activate polyamines biosynthesis to promote viral infection remains unclear.Ornit...Intracellular polyamines(putrescine,spermidine,and spermine)have emerged as important molecules for viral infection;however,how viruses activate polyamines biosynthesis to promote viral infection remains unclear.Ornithine decarboxylase 1(ODC1)and its antienzyme 1(OAZ1)are major regulators of polyamine biosynthesis in animal cells.Here,we report that rice yellow stunt virus(RYSV),a plant rhabdovirus,could activate putrescine biosynthesis in leafhoppers to promote viral propagation by inhibiting OAZ1 expression.We observed that the reduction of putrescine biosynthesis by treatment with difluormethylornithine(DFMO),a specific nontoxic inhibitor of ODC1,or with in vitro synthesized dsRNAs targeting ODC1 mRNA could inhibit viral infection.In contrast,the supplement of putrescine or the increase of putrescine biosynthesis by treatment with ds RNAs targeting OAZ1 mRNA could facilitate viral infection.We further determined that both RYSV matrix protein M and ODC1 directly bind to the ODC-binding domain at the C-terminus of OAZ1.Thus,viral propagation in leafhoppers would decrease the ability of OAZ1 to target and mediate the degradation of ODC1,which finally activates putrescine production to benefit viral propagation.This work reveals that polyamine-metabolizing enzymes are directly exploited by a vector-borne virus to increase polyamine production,thereby facilitating viral infection in insect vectors.展开更多
The virus disease threatening wheat production in Hulunbcir District of Inner - Mongolia was identified as NCMV (Northern Cereal Mosaic Virus) or WRSV (Wheat Rosette Stunt Virus) by tests of host range, transmission, ...The virus disease threatening wheat production in Hulunbcir District of Inner - Mongolia was identified as NCMV (Northern Cereal Mosaic Virus) or WRSV (Wheat Rosette Stunt Virus) by tests of host range, transmission, virus particle and scrology. The main vector for spreading the virus is planthopper, Laodelphax striatellus (Fallen), which overwinters in the stubbles of wheat underground. The overwintered virulifcrous nymphs emerged in late April is responsible for the early infection of the disease. Agropyron repens, an important perennial weed in cultivated regions, is also an important wild host of the virus and its vector. Severe loss is induced when wheat is infected before 3 - leaf stage. The disease incidence could be predicted by a model with the population and virulifcrous rate of overwintered vectors as independent variables.展开更多
为建立可同时检测甘薯褪绿矮化病毒（SPCSV）、甘薯G病毒（SPVG）和甘薯羽状斑驳病毒（SPFMV）多重RT-PCR检测方法,本文根据SPCSV热激蛋白基因（hsp70）及SPVG、SPFMV外壳蛋白基因（CP）基因核苷酸序列的保守区域设计特异性引物,通过引...为建立可同时检测甘薯褪绿矮化病毒（SPCSV）、甘薯G病毒（SPVG）和甘薯羽状斑驳病毒（SPFMV）多重RT-PCR检测方法,本文根据SPCSV热激蛋白基因（hsp70）及SPVG、SPFMV外壳蛋白基因（CP）基因核苷酸序列的保守区域设计特异性引物,通过引物筛选,优化多重RT-PCR反应条件,建立了能同时检测SPCSV、SPVG和SPFMV 3种病毒的多重RT-PCR检测方法。该体系能有效扩增出大小为304、433、601 bp 3个特异性片段。测序结果表明3种病毒与参考序列的一致性达94%-99%。应用建立的多重RT-PCR检测方法可稳定、准确、灵敏地同时检测单一或复合侵染3种甘薯病毒,为甘薯脱毒和病毒病诊断奠定基础。展开更多
MicroRNAs （miRNAs） are pivotal modulators of plant development and host-virus interactions. However, the roles and action modes of specific miRNAs involved in viral infection and host susceptibility remain largely u...MicroRNAs （miRNAs） are pivotal modulators of plant development and host-virus interactions. However, the roles and action modes of specific miRNAs involved in viral infection and host susceptibility remain largely unclear. In this study, we show that Rice ragged stunt virus （RRSV） infection caused increased accumulation of miR319 but decreased expression of miR319-regulated TCP （TEOSINTE BRANCHED/ CYCLOIDEA/PCF） genes, especially TCP21, in rice plants. Transgenic rice plants overexpressing miP,319 or downregulating TCP21 exhibited disease-like phenotypes and showed significantly higher susceptibility to RRSV in comparison with the wild-type plants. In contrast, only mild disease symptoms were observed in RRSV-infected lines overexpressing TCP21 and especially in the transgenic plants overexpressing miR319- resistant TCP21. Both RRSV infection and overexpression of miR319 caused the decreased endogenous jasmonic acid （JA） levels along with downregulated expression of JA biosynthesis and signaling-related genes in rice. However, treatment of rice plants with methyl jasmonate alleviated disease symptoms caused by RRSV and reduced virus accumulation. Taken together, our results suggest that the induction of miR319 by RRSV infection in rice suppresses JA-mediated defense to facilitate virus infection and symp- tom development.展开更多
The ubiquitin-proteasome system(UPS)is an important post-translational regulatory mechanism that controls many cellular functions in eukaryotes.Here,we show that stable expression of P3 protein encoded by Rice grassy ...The ubiquitin-proteasome system(UPS)is an important post-translational regulatory mechanism that controls many cellular functions in eukaryotes.Here,we show that stable expression of P3 protein encoded by Rice grassy stunt virus(RGSV),a negative-strand RNA virus in the Bunyavirales,causes developmental abnormities similar to the disease symptoms caused by RGSV,such as dwarfing and excess tillering,in transgenic rice plants.We found that both transgenic expression of P3 and RGSV infection induce ubiquitination and UPS-dependent degradation of rice NUCLEAR RNA POLYMERASE D1a(OsNRPD1a),one of two orthologs of the largest subunit of plant-specific RNA polymerase IV(Pol IV),which is required for RNA-directed DNA methylation(RdDM).Furthermore,we identified a P3-inducible U-box type E3 ubiquitin ligase,designated as P3-inducible protein 1(P3IP1),which interacts with OsNRPD1a and mediates its ubiquitination and UPS-dependent degradation in vitro and in vivo.Notably,both knockdown of OsNRPD1 and overexpression of P3IP1 in rice plants induced developmental phenotypes similar to RGSV disease symptomss.Taken together,our findings reveal a novel virulence mechanism whereby plant pathogens target host RNA Pol IV for UPS-dependent degradation to induce disease symptoms.Our study also identified an E3 ubiquitin ligase,which targets the RdDM compotent NRPD1 for UPS-mediated degradation in rice.展开更多
基金supported by the National Basic Research Program of China(2010CB126203)the special fund for Agro-scientific Research in the Public Interest,China(201003031)+1 种基金Earmarked Funds for Modern Agro-industry Technology Research SystemZhejiang Provincial Natural Science Foundation of China(Z3090039)
文摘Rice ragged stunt virus（RRSV） is a serious rice disease in Asia, causing serious yield losses on rice. The capsid protein（CP） gene of the major outer capsid protein of RRSV was expressed in Escherichia coli BL21（DE3） using the pMAL-C2 X expression vector. The recombinant protein was used as the immunogen to immunize BALB/c mice. A hybridoma cell line 8A12 secreting monoclonal antibody（MAb） against RRSV was obtained by fusing mouse myeloma cells（Sp 2/0） with spleen cells from the immunized BALB/c mice. Western blot analysis showed that the MAb 8A12 can specifically react with RRSV CP. Using the MAb, an antigen-coated-plate enzyme-linked immunosorbent assay（ACP-ELISA）, a dot enzyme-linked immunosorbent assay（dot-ELISA）, and immunocapture-RT-PCR（IC-RT-PCR） assay were developed to detect RRSV. The established ACP-ELISA, dot-blot ELISA and IC-RT-PCR methods could detect RRSV in infected rice tissue crude extracts with dilutions of 1：40 960, 1：1 280 and 1：655 360（w/v, g mL-1）, respectively. The ACP-ELISA and dot-blot ELISA methods could detect RRSV in infected insect vector crude extracts with dilutions of 1：12 800 and 1：1 600（an individual planthopper μL-1）, respectively. The field survey revealed that Rice ragged stunt disease occurs on rice in Hainan, Yunnan, Guangxi, Sichuan, Guizhou, Fujian, Hunan, Jiangxi and Zhejiang in China.
基金supported by the National Natural Science Foundation of China(31871931)the Fujian Agriculture and Forestry University Outstanding Young Scientists Project(xjq201705)National Key Research and Development Project of China(2018YFD0200306)。
文摘Intracellular polyamines(putrescine,spermidine,and spermine)have emerged as important molecules for viral infection;however,how viruses activate polyamines biosynthesis to promote viral infection remains unclear.Ornithine decarboxylase 1(ODC1)and its antienzyme 1(OAZ1)are major regulators of polyamine biosynthesis in animal cells.Here,we report that rice yellow stunt virus(RYSV),a plant rhabdovirus,could activate putrescine biosynthesis in leafhoppers to promote viral propagation by inhibiting OAZ1 expression.We observed that the reduction of putrescine biosynthesis by treatment with difluormethylornithine(DFMO),a specific nontoxic inhibitor of ODC1,or with in vitro synthesized dsRNAs targeting ODC1 mRNA could inhibit viral infection.In contrast,the supplement of putrescine or the increase of putrescine biosynthesis by treatment with ds RNAs targeting OAZ1 mRNA could facilitate viral infection.We further determined that both RYSV matrix protein M and ODC1 directly bind to the ODC-binding domain at the C-terminus of OAZ1.Thus,viral propagation in leafhoppers would decrease the ability of OAZ1 to target and mediate the degradation of ODC1,which finally activates putrescine production to benefit viral propagation.This work reveals that polyamine-metabolizing enzymes are directly exploited by a vector-borne virus to increase polyamine production,thereby facilitating viral infection in insect vectors.
文摘The virus disease threatening wheat production in Hulunbcir District of Inner - Mongolia was identified as NCMV (Northern Cereal Mosaic Virus) or WRSV (Wheat Rosette Stunt Virus) by tests of host range, transmission, virus particle and scrology. The main vector for spreading the virus is planthopper, Laodelphax striatellus (Fallen), which overwinters in the stubbles of wheat underground. The overwintered virulifcrous nymphs emerged in late April is responsible for the early infection of the disease. Agropyron repens, an important perennial weed in cultivated regions, is also an important wild host of the virus and its vector. Severe loss is induced when wheat is infected before 3 - leaf stage. The disease incidence could be predicted by a model with the population and virulifcrous rate of overwintered vectors as independent variables.
文摘为建立可同时检测甘薯褪绿矮化病毒（SPCSV）、甘薯G病毒（SPVG）和甘薯羽状斑驳病毒（SPFMV）多重RT-PCR检测方法,本文根据SPCSV热激蛋白基因（hsp70）及SPVG、SPFMV外壳蛋白基因（CP）基因核苷酸序列的保守区域设计特异性引物,通过引物筛选,优化多重RT-PCR反应条件,建立了能同时检测SPCSV、SPVG和SPFMV 3种病毒的多重RT-PCR检测方法。该体系能有效扩增出大小为304、433、601 bp 3个特异性片段。测序结果表明3种病毒与参考序列的一致性达94%-99%。应用建立的多重RT-PCR检测方法可稳定、准确、灵敏地同时检测单一或复合侵染3种甘薯病毒,为甘薯脱毒和病毒病诊断奠定基础。
文摘MicroRNAs （miRNAs） are pivotal modulators of plant development and host-virus interactions. However, the roles and action modes of specific miRNAs involved in viral infection and host susceptibility remain largely unclear. In this study, we show that Rice ragged stunt virus （RRSV） infection caused increased accumulation of miR319 but decreased expression of miR319-regulated TCP （TEOSINTE BRANCHED/ CYCLOIDEA/PCF） genes, especially TCP21, in rice plants. Transgenic rice plants overexpressing miP,319 or downregulating TCP21 exhibited disease-like phenotypes and showed significantly higher susceptibility to RRSV in comparison with the wild-type plants. In contrast, only mild disease symptoms were observed in RRSV-infected lines overexpressing TCP21 and especially in the transgenic plants overexpressing miR319- resistant TCP21. Both RRSV infection and overexpression of miR319 caused the decreased endogenous jasmonic acid （JA） levels along with downregulated expression of JA biosynthesis and signaling-related genes in rice. However, treatment of rice plants with methyl jasmonate alleviated disease symptoms caused by RRSV and reduced virus accumulation. Taken together, our results suggest that the induction of miR319 by RRSV infection in rice suppresses JA-mediated defense to facilitate virus infection and symp- tom development.
基金This work was supported by the National Natural Science Foundation of China(no.31772128,31722045,U1905203,and 31701757)the Fok Ying Tung Education Foundation(161024).
文摘The ubiquitin-proteasome system(UPS)is an important post-translational regulatory mechanism that controls many cellular functions in eukaryotes.Here,we show that stable expression of P3 protein encoded by Rice grassy stunt virus(RGSV),a negative-strand RNA virus in the Bunyavirales,causes developmental abnormities similar to the disease symptoms caused by RGSV,such as dwarfing and excess tillering,in transgenic rice plants.We found that both transgenic expression of P3 and RGSV infection induce ubiquitination and UPS-dependent degradation of rice NUCLEAR RNA POLYMERASE D1a(OsNRPD1a),one of two orthologs of the largest subunit of plant-specific RNA polymerase IV(Pol IV),which is required for RNA-directed DNA methylation(RdDM).Furthermore,we identified a P3-inducible U-box type E3 ubiquitin ligase,designated as P3-inducible protein 1(P3IP1),which interacts with OsNRPD1a and mediates its ubiquitination and UPS-dependent degradation in vitro and in vivo.Notably,both knockdown of OsNRPD1 and overexpression of P3IP1 in rice plants induced developmental phenotypes similar to RGSV disease symptomss.Taken together,our findings reveal a novel virulence mechanism whereby plant pathogens target host RNA Pol IV for UPS-dependent degradation to induce disease symptoms.Our study also identified an E3 ubiquitin ligase,which targets the RdDM compotent NRPD1 for UPS-mediated degradation in rice.
