Purification and Characterization of a New Ribosome Inactivating Protein from Cinchonaglycoside C-treated Tobacco Leaves

A new ribosome-inactivating protein(RIP)with a molecular weight of 31 kDa induced by Cinchonaglycoside C(1)designatedCLP31,was isolated from tobacco leaves.Analysis of this protein sequence indicated that it belongs to the RIP family and itwas distinct from the other plant RIPs reported previously at its N-terminal amino acid sequence.CIP31 can directly impairsynthesis of coat protein(CP)of tobacco mosaic virus(TMV),which resulted in inhibition of TMV long distance movementand multiplication in tobacco plants at concentrations of ng/mL.Furthermore,no toxicity was shown to the growth andfertility of the plants.CIP31 was synthesized only in the presence of Cinchonaglycoside C(1)and was independent of thesalicylic acid(SA)signal pathway.We provided evidence for the SA-independent biological induction of resistance.     

毛头鬼伞真菌具有抗烟草花叶病毒活性的y3基因的克隆及对烟草的转化

【目的】蛋白质Y3具有抗烟草花叶病毒(TMV)活性并由y3基因编码。本文的目的是从真菌毛头鬼伞(Coprinus comatus)中克隆y3基因全长并在植物体中展现其对TMV的抑制活性。【方法】我们利用试剂盒5′-Full RACE Core Set(TaKaRa)扩增了y3基因cDNA5′-端未知序列,通过RT-PCR获得了全长序列,并把该全长序列与CaMV 35 S启动子和NOS终止子一起插入多克隆位点(MCS)构建了植物表达载体pCAMBIA1301-y3,用于农杆菌介导的烟草转化。【结果】y3基因全长534碱基对,包含1个开放阅读框(ORF),编码一条含130个氨基酸残基的肽链(GenBank检索号:GQ859168;EMBL:FN546262)。其cDNA序列和由它推到的氨基酸序列均与已发表的y3基因部分片段有高度相似性(94%)。Northern杂交分析证实了y3基因在转基因烟草中得到表达。接种TMV的转基因植株表现出抗TMV的活性。【结论】我们克隆了y3基因全长并得到了转基因植株。在转基因植株中,由于y3基因的表达改善了植株的抗病毒活性。y3基因的克隆和表达无疑为该基因的进一步研究奠定了基础。     

Expression of recombinant trichosanthin,a ribosome-inactivating protein,in transgenic tobacco

Trichosanthin (TCS) is an antiviral plant defense protein, classified as a type-I ribosome-inactivating protein, found in the root tuber and leaves of the medicinal plant Trichosanthes kirilowii. It is processed from a larger precursor protein, containing a 23 amino acid amino (N)-terminal sequence (pre sequence) and a 19 amino acid carboxy (C)-terminal extension (pro sequence). Various constructs of the TCS gene were expressed in transgenic tobacco plants to determine the effects of the amino- and carboxy-coding gene sequences on TCS expression and host toxicity in plants. The maximum TCS expression levels of 2.7% of total soluble protein (0.05% of total dry weight) were obtained in transgenic tobacco plants carrying the complete prepro-TCS gene sequence under the Cauliflower mosaic virus 35S RNA promoter. The N-terminal sequence matched the native TCS sequence indicating that the T. kirilowii signal sequence was properly processed in tobacco and the protein translation inhibitory activity of purified rTCS was similar to native TCS. One hundred-fold lower expression levels and phenotypic aberrations were evident in plants expressing the gene constructs without the C-terminal coding sequence. Transgenic tobacco plants expressing recombinant TCS exhibited delayed symptoms of systemic infection following exposure to Cucumber mosaic virus and Tobacco mosaic virus (TMV). Local lesion assays using extracts from the infected transgenic plants indicated reduced levels of TMV compared with nontransgenic controls.     

Induction of Benzoic Acid 2-Hydroxylase in Virus-Inoculated Tobacco

Salicylic acid (SA) plays an important role in the induction of plant resistance to pathogens. An accompanying article (N. Yalpani, J. Leon, M.A. Lawton, I. Raskin [1993] Plant Physiol 103: 315-321) shows that SA is synthesized via the decarboxylation of cinnamic acid to benzoic acid (BA), which is, in turn, hydroxylated to SA. Leaf extracts of tobacco (Nicotiana tabacum L. cv Xanthi-nc) catalyze the 2-hydroxylation of BA to SA. The monooxygenase catalyzing this reaction, benzoic acid 2-hydroxylase (BA2H), required NAD(P)H or reduced methyl viologen as an electron donor. BA2H activity was detected in healthy tobacco leaf extracts (1-2 nmol h-1 g-1 fresh weight) and was significantly increased upon inoculation with tobacco mosaic virus (TMV). This increase paralleled the levels of free SA in the leaves. Induction of BA2H activity was restricted to tissue expressing a hypersensitive response at 24[deg]C. TMV induction of BA2H activity and SA accumulation were inhibited when inoculated tobacco plants were incubated at 32[deg]C. However, when inoculated plants were incubated for 4 d at 32[deg]C and then transferred to 24[deg]C, they showed a 15-fold increase in BA2H activity and a 65-fold increase in free SA content compared with healthy plants incubated at 24[deg]C. Treatment of leaf tissue with the protein synthesis inhibitor cycloheximide blocked the induction of BA2H activity by TMV. The effect of TMV inoculation on BA2H could be duplicated by infiltrating leaf discs of healthy plants with BA. This response was observed even when applied levels of BA were much lower than the levels observed in vivo after virus inoculation. Feeding tobacco leaves with phenylalanine, cinnamic acid, or o-coumaric acid (putative precursors of SA) failed to trigger the induction of BA2H activity. BA2H appears to be a pathogen-inducible protein with an important regulatory role in SA accumulation during the development of induced resistance to TMV in tobacco.     

The type-1 and type-2 ribosome-inactivating proteins from<Emphasis Type="Italic"> Iris</Emphasis> confer transgenic tobacco plants local but not systemic protection against viruses

The antiviral activity of the type-2 ribosome-inactivating protein (RIP) IRAb from Iris was analyzed by expressing IRAb in tobacco (Nicotiana tabacum L. cv. Samsun NN) plants and challenging the transgenic plants with tobacco mosaic virus (TMV). Although constitutive expression of IRAb resulted in an aberrant phenotype, the plants were fertile. Transgenic tobacco lines expressing IRAb showed a dose-dependent enhanced resistance against TMV infection but the level of protection was markedly lower than in plants expressing IRIP, the type-1 RIP from Iris that closely resembles the A-chain of IRAb. To verify whether IRIP or IRAb can also confer systemic protection against viruses, transgenic RIP-expressing scions were grafted onto control rootstocks and leaves of the rootstocks challenged with tobacco etch virus (TEV). In spite of the strong local antiviral effect of IRIP and IRAb the RIPs could not provide systemic protection against TEV. Hence our results demonstrate that expression of the type-1 and type-2 RIPs from Iris confers tobacco plants local protection against two unrelated viruses. The antiviral activity of both RIPs was not accompanied by an induction of pathogenesis-related proteins. It is suggested that the observed antiviral activity of both Iris RIPs relies on their RNA N-glycohydrolase activity towards TMV RNA and plant rRNA.Abbreviations GUS -Glucuronidase - IRAb Iris agglutinin b - IRIP Iris type-1 RIP - PAG Polynucleotide:adenosine glycosylase - PAP Phytolacca americana antiviral protein - PR Pathogenesis-related - RIP Ribosome-inactivating protein - TCS Trichosanthin - TEV Tobacco etch virus - TMV Tobacco mosaic virus     

Cloning and expression of antiviral/ribosome-inactivating protein from <Emphasis Type="Italic">Bougainvillea</Emphasis> x<Emphasis Type="Italic">buttiana</Emphasis>

A full-length cDNA encoding ribosome-inactivating/antiviral protein (RIP/AVP)from the leaves of Bougainvillea x buttiana was isolated.The cDNA consisted of 1364 nucleotides with an open reading frame (ORF)of 960 nucleotides encoding a 35.49 kDa protein of 319 amino acids.The deduced amino acid sequence has a putative active domain conserved in RIPs/AVPs and shows a varying phylogenetic relationship to the RIPs from other plant species.The deduced protein has been designated BBAP1 (Bougainvillea x buttiana antiviral protein1).The ORF was cloned into an expression vector and expressed in E.coli as a fusion protein of approximately 78 kDa.The cleaved and purified recombinant BBAP1 exhibited ribosome-inhibiting rRNA N-glycosidase activity,and imparted a high level of resistance against the tobacco mosaic virus (TMV).     

Affinity purification of streptavidin using tobacco mosaic virus particles as purification tags

A new protein affinity purification system has been developed. Recombinant tobacco mosaic virus (TMV) was used as an affinity matrix for isolation and purification of the given protein of interest. In model experiments, streptavidin-specific heptapeptide sequence TLIAHPQ was inserted into TMV coat protein near the C end. This oligopeptide did not interfere significantly with viral replication, assembly, and movement. Recombinant TMV functioned as an epitope tag recognizing streptavidin in plant protein extracts. Plant protein extracts containing streptavidin were incubated with recombinant TMV virions. Affinity complexes of viral particles with the protein of interest were collected by centrifugation. Recombinant TMV-streptavidin complex was dissociated with 0.2M acetic acid, pH 4.6, and was passed through membrane filter Nanosep 300K by centrifugation. The filtrate contained pure streptavidin. Recombinant TMV was left on the filter. TMV particles collected from the filter could be used for at least two more purification cycles. The streptavidin-specific recombinant TMV system was applied successfully for purification of streptavidin from Streptomyces avidinii. The authors believe that the TMV-based affinity system can also be used for the purification of other proteins.     

Systemic induction of a Phytolacca insularis antiviral protein gene by mechanical wounding, jasmonic acid, and abscisic acid

We have isolated a gene encoding a ribosome-inactivating protein (RIP) from Phytolacca insularis, designated as P. insularis antiviral protein 2 (PIP2). The PIP2 gene contained an open reading frame encoding a polypeptide of 315 amino acids. The deduced amino acid sequence of PIP2 was similar to those of other RIPs from Phytolacca plants. Recombinant PIP2 was expressed in Escherichia coli and was used to investigate its biological activities. Recombinant PIP2 inhibited protein synthesis in rabbit reticulocyte lysate by inactivating ribosomes through N-glycosidase activity. It also exhibited antiviral activity against tobacco mosaic virus (TMV). Expression of the PIP2 gene was developmentally regulated in leaves and roots of P. insularis. Furthermore, expression of the PIP2 gene was induced in leaves by mechanical wounding. The wound induction of the PIP2 gene was systemic. Expression of the PIP2 gene also increased in leaves in a systemic manner after treatment with jasmonic acid (JA) and abscisic acid (ABA), but not with salicylic acid (SA). These results imply that plants have employed the systemic synthesis of the defensive proteins to protect themselves more efficiently from infecting viruses.     

Isolation and characterization of cDNA clones encoding pathogenesis-related proteins from tobacco mosaic virus infected tobacco plants.

Infection of the tobacco cultivar Samsun NN by tobacco mosaic virus (TMV) results in a hypersensitive response. During this defense reaction several host encoded proteins, known as pathogenesis-related proteins (PR-proteins), are induced. Poly(A)+ RNA from TMV infected tobacco plants was used to construct a cDNA library. Thirty two cDNA clones were isolated and after digestion with different restriction endonucleases, twenty clones were found to code for PR-1a, six clones for PR-1b, and four clones for PR-1c. Two independent cDNA clones of each class were further characterized by DNA sequence analysis. All clones analyzed contained the 138 amino acid coding regions of their respective mature proteins, but only partial sequences of the signal peptides. Minor differences between the nucleotide sequences for clones belonging to the same class were detected. Comparison of the amino acid sequence for PR-1a deduced from its nucleotide sequence with published data obtained by Edman degradation of the protein showed four differences. Analysis of the 3' ends of the cDNA clones indicates that various alternate poly(dA) addition sites are used. Southern blot analysis using these cDNAs as probes suggests the presence of multiple PR-protein genes in the genomes of tobacco and tomato plants.     

Transgenic tobacco plants with ribosome inactivating protein gene cassin from Cassia occidentalis and their resistance to tobacco mosaic virus]

Cassin, the new gene of ribosome-inactivating protein (RIP) isolated from Cassia occidentalis, was inserted into expression vector pBI121 to produce plant expression vector pBI121-cassin (Figs.1, 2). pBI121-cassin was introduced into tobacco cultivar 'K326' by the Agrobacteriurm tumefaciens transformation method and more than 100 independent transformants were obtained. Southern blot hybridization analysis showed that a single gene locus was inserted into the chromosome of the transgenic tobacco lines (Fig.5) and PCR analysis of segregation population of progeny indicated that the inheritance of transgene was dominant in transgenic lines (Fig.4, Table 1). Results of RT-PCR and Northern blot hybridization analysis showed that transgene could be transcribed correctly (Figs.5, 6) . Three self-pollination lines of transgenic T(1) and T(2) were challenged with TMV at different concentration titers by mechanical inoculation. The transgenic lines exhibited different levels of resistance to TMV with the nontransgenic plants. After both titers of TMV concentration were inoculated, transgenic lines were considered as the highly resistant type with a delay of 4-13 d in development of symptoms and 10%-25% of test plants were infected, while nontransgenic control plants were susceptible typical symptoms on the newly emerged leaves (Table 2). One T(2) line, T(2)-8-2-1, was regarded as an immune type because it did not show any symptoms during 70 d and all plants were shown to be virus free by ELISA tests.     

杆状病毒p35基因诱导烟草产生广谱抗病机理分析

来源于昆虫病毒和动物的抗细胞凋亡基因能够诱导植物对生物或者非生物胁迫产生抗性.但其抗性机理有不同甚至相反的报道.本研究将来源于苜蓿银纹夜蛾核多角体病毒的p35基因转化烟草,T1代转化烟草Western blotting检测P35蛋白的表达,转化烟草接种烟草花叶病毒(Tobacco mosaic virus,TMV)抗病效果增强.进一步的抗病机理研究表明,转化和野生型烟草感染TMV后诱导过氧化氢积累无明显区别,野生型烟草感染24 h后出现DNA Laddering而转化烟草则没有;Western blotting结果显示PR-1蛋白表达没有显著差异.但接种另外一种病原真菌核盘茵(Sclerotiniasclerotiorum)后的RT-PCR分析结果表明,表达P35蛋白的烟草可增强感染核盘菌后PR-1基因的转录.而且表达时间提前.以上结果说明p35基因介导的广谱抗病反应的机理与接种的不同病原有关,对不同病原物的抗病机理存在差异,除抑制细胞凋亡外,还可能通过激活PR基因的表达提高对病原物的抗病能力.     

Salicylic acid has cell-specific effects on tobacco mosaic virus replication and cell-to-cell movement

Tobacco mosaic virus (TMV) and Cucumber mosaic virus expressing green fluorescent protein (GFP) were used to probe the effects of salicylic acid (SA) on the cell biology of viral infection. Treatment of tobacco with SA restricted TMV.GFP to single-epidermal cell infection sites for at least 6 d post inoculation but did not affect infection sites of Cucumber mosaic virus expressing GFP. Microinjection experiments, using size-specific dextrans, showed that SA cannot inhibit TMV movement by decreasing the plasmodesmatal size exclusion limit. In SA-treated transgenic plants expressing TMV movement protein, TMV.GFP infection sites were larger, but they still consisted overwhelmingly of epidermal cells. TMV replication was strongly inhibited in mesophyll protoplasts isolated from SA-treated nontransgenic tobacco plants. Therefore, it appears that SA has distinct cell type-specific effects on virus replication and movement in the mesophyll and epidermal cell layers, respectively. Thus, SA can have fundamentally different effects on the same pathogen in different cell types.     

Salicylic acid is a systemic signal and an inducer of pathogenesis-related proteins in virus-infected tobacco.

Systemic induction of pathogenesis-related (PR) proteins in tobacco, which occurs during the hypersensitive response to tobacco mosaic virus (TMV), may be caused by a minimum 10-fold systemic increase in endogenous levels of salicylic acid (SA). This rise in SA parallels PR-1 protein induction and occurs in TMV-resistant Xanthi-nc tobacco carrying the N gene, but not in TMV-susceptible (nn) tobacco. By feeding SA to excised leaves of Xanthi-nc (NN) tobacco, we have shown that the observed increase in endogenous SA levels is sufficient for the systemic induction of PR-1 proteins. TMV infection became systemic and Xanthi-nc plants failed to accumulate PR-1 proteins at 32 degrees C. This loss of hypersensitive response at high temperature was associated with an inability to accumulate SA. However, spraying leaves with SA induced PR-1 proteins at both 24 and 32 degrees C. SA is most likely exported from the primary site of infection to the uninfected tissues. A computer model predicts that SA should move rapidly in phloem. When leaves of Xanthi-nc tobacco were excised 24 hr after TMV inoculation and exudates from the cut petioles were collected, the increase in endogenous SA in TMV-inoculated leaves paralleled SA levels in exudates. Exudation and leaf accumulation of SA were proportional to TMV concentration and were higher in light than in darkness. Different components of TMV were compared for their ability to induce SA accumulation and exudation: three different aggregation states of coat protein failed to induce SA, but unencapsidated viral RNA elicited SA accumulation in leaves and phloem. These results further support the hypothesis that SA acts as an endogenous signal that triggers local and systemic induction of PR-1 proteins and, possibly, some components of systemic acquired resistance in NN tobacco.     

烟草花叶病毒运动蛋白基因的cDNA克隆、序列测定及植物转化

植物病毒侵染宿主植物的一个重要过程是通过它在宿主体内的转移和传播,产生病害。植物病毒在宿主体内的转移主要有两种方式,一种是通过植物维管组织进行的系统转移,另一种是植物病毒在宿主细胞之间的转移,这种转移是通过植物细胞的胞间连丝实现的。实验表明,病毒自身编码的一种蛋白参与了这个转移过程,对烟草花叶病毒(TMV)而言,这种蛋白就是分子量为30kDa的运动蛋白。     

PeaT1-induced systemic acquired resistance in tobacco follows salicylic acid-dependent pathway

Systemic acquired resistance (SAR) is an inducible defense mechanism which plays a central role in protecting plants from pathogen attack. A new elicitor, PeaT1 from Alternaria tenuissima, was expressed in Escherichia coil and characterized with systemic acquired resistance to tobacco mosaic virus (TMV). PeaT1-treated plants exhibited enhanced systemic resistance with a significant reduction in number and size of TMV lesions on wild tobacco leaves as compared with control. The quantitative analysis of TMV CP gene expression with real-time quantitative PCR showed there was reduction in TMV virus concentration after PeaT1 treatment. Similarly, peroxidase (POD) activity and lignin increased significantly after PeaT1 treatment. The real-time quantitative PCR revealed that PeaT1 also induced the systemic accumulation of pathogenesis-related gene, PR-1a and PR-1b which are the markers of systemic acquired resistance (SAR), NPR1 gene for salicylic acid (SA) signal transduction pathway and PAL gene for SA synthesis. The accumulation of SA and the failure in development of similar level of resistance as in wild type tobacco plants in PeaT1 treated nahG transgenic tobacco plants indicated that PeaT1-induced resistance depended on SA accumulation. The present work suggested that the molecular mechanism of PeaT1 inducing disease resistance in tobacco was likely through the systemic acquired resistance pathway mediated by salicylic acid and the NPR1 gene.     

Expression of active hBMP2 in transgenic tobacco plants

Bone morphogenetic protein 2 (BMP2) is important for bone tissue repair. The goal of this research is to construct a high level human BMP2 (hBMP2) expression system using transgenic tobacco plants as a bioreactor. Cauliflower mosaic virus (CaMV) 35S promoter, alfalfa mosaic virus (AMV) enhancer, tobacco mosaic virus (TMV) enhancer, matrix attachment regions (MARs) sequence, and “Kozak” sequence were used to construct recombinant expression vectors and the high-expression vectors were screened out through GUS-fusions assay. The promoter is the most important factor; double-CaMV 35S promoter is more effective than single promoter. The AMV or TMV enhancer is able to promote the foreign protein expression. After four-step purification, the activated hBMP2 (0.02% total soluble protein) was obtained. Our results suggested that the transgenic tobacco has great potential to be used as a bioreactor to produce hBMP2.