The jasmonate-induced 60 kDa protein of barley exhibits N-glycosidase activity in vivo.

Upon jasmonate treatment barley leaf segments express a putative ribosome-inactivating protein (JIP60). The influence of this protein on translation in planta has been analysed by using barley plants and tobacco plants transformed with a barley cDNA encoding JIP60. In both plant systems JIP60 exhibited N-glycosidase activity in vivo. The depurination of the 25S rRNA of tobacco and barley ribosomes led to accumulation of translationally inactive polysomes.     

Expression of the ribosome-inactivating protein JIP60 from barley in transgenic tobacco leads to an abnormal phenotype and alterations on the level of translation

In this paper we report the in-planta activity of the ribosome-inactivating protein JIP60, a 60-kDa jasmonate-induced protein from barley (Hordeum vulgare L.), in transgenic tobacco (Nicotiana tabacum L.) plants. All plants expressing the complete JIP60 cDNA under the control of the cauliflower mosaic virus (CaMV) 35S promoter exhibited conspicuous and similar phenotypic alterations, such as slower growth, shorter internodes, lanceolate leaves, reduced root development, and premature senescence of leaves. Microscopic inspection of developing leaves showed a loss of residual meristems and higher degree of vacuolation of mesophyll cells as compared to the wild type. When probed with an antiserum which was immunoreactive against both the N- and the C-terminal half of JIP60, a polypeptide with a molecular mass of about 30 kDa, most probably a processed JIP60 product, could be detected. Phenotypic alterations could be correlated with the differences in the detectable amount of the JIP60 mRNA and processed JIP60 protein. The protein biosynthesis of the transformants was characterized by an increased polysome/monosome ratio but a decreased in-vivo translation activity. These findings suggest that JIP60 perturbs the translation machinery in planta. An immunohistological analysis using the JIP60 antiserum indicated that the immunoreactive polypeptide(s) are located mainly in the nucleus of transgenic tobacco leaf cells and to a minor extent in the cytoplasm. Received: 31 July 1996 / Accepted: 18 February 1997     

A non-toxic pokeweed antiviral protein mutant inhibits pathogen infection via a novel salicylic acid-independent pathway

Pokeweed antiviral protein (PAP), a ribosome-inactivating protein isolated from Phytolacca americana, is characterized by its ability to depurinate the sarcin/ricin (S/R) loop of the large rRNA of prokaryotic and eukaryotic ribosomes. In this study, we present evidence that PAP is associated with ribosomes and depurinates tobacco ribosomes in vivo by removing more than one adenine and a guanine. A mutant of pokeweed antiviral protein, PAPn, which has a single amino acid substitution (G75D), did not bind ribosomes efficiently, indicating that Gly-75 in the N-terminal domain is critical for the binding of PAP to ribosomes. PAPn did not depurinate ribosomes and was non-toxic when expressed in transgenic tobacco plants. Unlike wild-type PAP and a C-terminal deletion mutant, transgenic plants expressing PAPn did not have elevated levels of acidic pathogenesis-related (PR) proteins. PAPn, like other forms of PAP, did not trigger production of salicylic acid (SA) in transgenic plants. Expression of the basic PR proteins, the wound-inducible protein kinase and protease inhibitor II, was induced in PAPn-expressing transgenic plants and these plants were resistant to viral and fungal infection. These results demonstrate that PAPn activates a particular SA-independent, stress-associated signal transduction pathway and confers pathogen resistance in the absence of ribosome binding, rRNA depurination and acidic PR protein production.     

Expression of Pokeweed Antiviral Protein in Transgenic Plants Induces Virus Resistance in Grafted Wild-Type Plants Independently of Salicylic Acid Accumulation and Pathogenesis-Related Protein Synthesis

Pokeweed antiviral protein (PAP), a 29-kD protein isolated from Phytolacca americana, inhibits translation by catalytically removing a specific adenine residue from the large rRNA of the 60S subunit of eukaryotic ribosomes. Transgenic tobacco (Nicotiana tabacum) plants expressing PAP or a variant (PAP-v) were shown to be resistant to a broad spectrum of plant viruses. Expression of PAP-v in transgenic plants induces synthesis of pathogenesis-related proteins and a very weak (<2-fold) increase in salicylic acid levels. Using reciprocal grafting experiments, we demonstrate here that transgenic tobacco rootstocks expressing PAP-v induce resistance to tobacco mosaic virus infection in both N. tabacum NN and nn scions. Increased resistance to potato virus X was also observed in N. tabacum nn scions grafted on transgenic rootstocks. PAP expression was not detected in the wild-type scions or rootstocks that showed virus resistance, nor was there any increase in salicylic acid levels or pathogenesis-related protein synthesis. Grafting experiments with transgenic plants expressing an inactive PAP mutant demonstrated that an intact active site of PAP is necessary for induction of virus resistance in wild-type scions. These results indicate that enzymatic activity of PAP is responsible for generating a signal that renders wild-type scions resistant to virus infection in the absence of increased salicylic acid levels and pathogenesis-related protein synthesis.     

The barley 60 kDa jasmonate-induced protein (JIP60) is a novel ribosome-inactivating protein

The N-terminal region of a 60 kDa, jasmonate-induced protein of barley leaves (JIP60) is shown to be homologous to the catalytic domains of plant ribosome-inactivating proteins (RIP). Western blotting of leaf extracts and in vitro reconstitution experiments indicate that JIP60 is synthesized as a precursor which is processed in vivo. This is in keeping with in vitro translation experiments indicating that a deletion derivative of the N-terminal region, but not the putative precursor, strongly inhibits protein synthesis on reticulocyte ribosomes. The inhibition of ribosome function is associated with depurination of 26S rRNA, characteristic of plant RIPs. This indicates that JIP60 is a novel ribosome-inactivating protein requiring at least two processing events for full activation. JIP60 derivatives do not significantly inhibit in vitro protein synthesis on wheat germ ribosomes. These and other results suggest that JIP60 may be involved in plant defence.     

Type 1 ribosome-inactivating proteins depurinate plant 25S rRNA without species specificity.

Four different type 1 ribosome-inactivating proteins (RIPs) with RNA N-glycosidase activity were tested for their ability to attack the large rRNA of plant ribosomes derived from tobacco plants, as well as from the plant species from which the particular RIP had been isolated. Incubation of tobacco ribosomes with RIPs isolated from either Phytolacca americana L. (pokeweed), Dianthus barbatus L. (carnation), Spinacia oleracea L. (spinach) or Chenopodium amaranthicolor Coste and Reyn. (chenopodium) rendered the 25S rRNA sensitive to aniline-catalyzed hydrolysis, generating a single rRNA-fragment of about 350 nucleotides. The same fragment was generated when rRNAs from pokeweed, carnation, spinach or chenopodium ribosomes were aniline-treated without any deliberate treatment of the ribosomes with the respective RIP. This indicated that ribosomes from all RIP-producing plants were already inactivated by their own RIPs during preparation. These results demonstrate that plant ribosomes are generally susceptible to RIP attack, including modification by their own RIPs. Direct sequencing of the newly generated fragments revealed that a single N-glycosidic bond at an adenosine residue within the highly conserved sequence 5'-AGUACGAGAGGA-3' was cleaved by all of the RIPs investigated, a situation also found in animal, yeast and Escherichia coli ribosomes.     

Expression of a truncated form of ribosomal protein L3 confers resistance to pokeweed antiviral protein and the Fusarium mycotoxin deoxynivalenol

The contamination of important agricultural products such as wheat, barley, or maize with the trichothecene mycotoxin deoxynivalenol (DON) due to infection with Fusarium species is a worldwide problem. Trichothecenes inhibit protein synthesis by targeting ribosomal protein L3. Pokeweed antiviral protein (PAP), a ribosome-inactivating protein binds to L3 to depurinate the alpha-sarcin/loop of the large rRNA. Plants transformed with the wild-type PAP show lesions and express very low levels of PAP because PAP autoregulates its expression by destabilizing its own mRNA. We show here that transgenic tobacco plants expressing both the wild-type PAP and a truncated form of yeast L3 (L3delta) are phenotypically normal. PAP mRNA and protein levels are very high in these plants, indicating that L3delta suppresses the autoregulation of PAP mRNA expression. Ribosomes are not depurinated in the transgenic plants expressing PAP and L3delta, even though PAP is associated with ribosomes. The expression of the endogenous tobacco ribosomal protein L3 is up-regulated in these plants and they are resistant to the Fusarium mycotoxin DON. These results demonstrate that expression of an N-terminal fragment of yeast L3 leads to trans-dominant resistance to PAP and the trichothecene mycotoxin DON, providing evidence that both toxins target L3 by a common mechanism.     

Analysis of the in planta antiviral activity of elderberry ribosome-inactivating proteins.

Although the type-2 ribosome-inactivating proteins (SNA-I, SNA-V, SNLRP) from elderberry (Sambucus nigra L.) are all devoid of rRNA N-glycosylase activity towards plant ribosomes, some of them clearly show polynucleotide-adenosine glycosylase activity towards tobacco mosaic virus RNA. This particular substrate specificity was exploited to further unravel the mechanism underlying the in planta antiviral activity of ribosome-inactivating proteins. Transgenic tobacco (Nicotiana tabacum L. cv Samsun NN) plants expressing the elderberry ribosome-inactivating proteins were generated and challenged with tobacco mosaic virus in order to analyze their antiviral properties. Although some transgenic plants clearly showed antiviral activity, no clear correlation was observed between in planta antiviral activity of transgenic tobacco lines expressing the different ribosome-inactivating proteins and the in vitro polynucleotide-adenosine glycosylase activity of the respective proteins towards tobacco mosaic virus genomic RNA. However, our results suggest that the in planta antiviral activity of some ribosome-inactivating proteins may rely on a direct mechanism on the virus. In addition, it is evident that the working mechanism proposed for pokeweed antiviral protein cannot be extrapolated to elderberry ribosome-inactivating proteins because the expression of SNA-V is not accompanied by induction of pathogenesis-related proteins.     

Reduced toxicity and broad spectrum resistance to viral and fungal infection in transgenic plants expressing pokeweed antiviral protein II

Pokeweed antiviral protein II (PAPII), a 30 kDa protein isolated from leaves of Phytolacca americana, inhibits translation by catalytically removing a specific adenine residue from the large rRNA of the 60S subunit of eukaryotic ribosomes. The protein sequence of PAPII shows only 41% identity to PAP and PAP-S, two other antiviral proteins isolated from pokeweed. We isolated a cDNA corresponding to PAPII and introduced it into tobacco plants. PAPII expressed in transgenic tobacco was correctly processed to the mature form as in pokeweed and accumulated to at least 10-fold higher levels than wild-type PAP. We had previously observed a significant decrease in transformation frequency with PAP and recovered only two transgenic lines expressing 1–2 ng per mg protein. In contrast, eight different transgenic lines expressing up to 250 ng/mg PAPII were recovered, indicating that PAPII is less toxic than PAP. Two symptomless transgenic lines expressing PAPII were resistant to tobacco mosaic virus, potato virus X and the fungal pathogen Rhizoctonia solani. The level of viral and fungal resistance observed correlated well with the amount of PAPII protein accumulated. Pathogenesis-related protein PR1 was constitutively expressed in transgenic lines expressing PAPII. Although PR1 was constitutively expressed, no increase in salicylic acid levels was detected, indicating that PAPII may elicit a salicylic acid-independent signal transduction pathway.     

菠菜甜菜碱醛脱氢酶基因在烟草中的表达

质粒pLS9含有1．5kb的编码菠菜甜菜碱醛脱氢酶(BADH)基因。经限制酶切后克隆到植物表达载体的35S启动子和PolyA终止子之间。经农杆菌介导转化烟草,获得90多株抗卡那霉素再生植株。经PCR检测证明60％以上再生植株含有BADH基因。转基因植株经Western blot,BADH酶活性测定,BADH酶活性特异性染色法检查和耐盐性分析,证明菠菜BADH基因在烟草正常表达。在叶绿体和胞液中均有BADH酶存在。转基因植株能耐较高浓度盐。     

Analysis of stress-induced or salicylic acid-induced expression of the pathogenesis-related 1a protein gene in transgenic tobacco.

The cis-acting elements for regulating gene expression of the tobacco pathogenesis-related 1a protein gene were analyzed in transgenic plants. The 5'-flanking 2.4-kilobase fragment from the pathogenesis-related 1a protein gene was joined to the bacterial beta-glucuronidase gene and introduced into tobacco cells by Agrobacterium-mediated gene transfer. Promoter activity was monitored by quantitative and histochemical assay of beta-glucuronidase activity in leaves of regenerated transgenic plants. The level of beta-glucuronidase activity was clearly increased by treatment with salicylic acid, by cutting stress, and by local lesion formation caused by tobacco mosaic virus infection. Cytochemical studies of the induced beta-glucuronidase activity revealed tissue-specific and developmentally regulated expression of the pathogenesis-related 1a gene after stress or chemical treatment and after pathogen attack. To identify the cis-acting element more precisely, a series of 5'-deleted chimeric genes was constructed and transformed into tobacco plants. Transgenic plants with a 0.3-kilobase fragment of the 5'-flanking region of the pathogenesis-related 1a gene had the same qualitative response as those with the 2.4-kilobase fragment upon treatment with salicylic acid or infection with TMV. Thus, the 0.3-kilobase DNA sequence fragment was sufficient to allow the regulated expression of the pathogenesis-related 1a gene.     

The site of action of six different ribosome-inactivating proteins from plants on eukaryotic ribosomes: the RNA N-glycosidase activity of the proteins

The site of action of six different ribosome-inactivating proteins from plants on eukaryotic ribosomes was studied. Treatment of ribosomes with any one of these proteins caused the 28S rRNA extracted from the inactivated ribosomes to become sensitive to treatment with aniline. A fragment containing about 450 nucleotides was released from the 28S rRNA. Further analysis of the nucleotide sequences of the 450-nucleotide fragments revealed that the aniline-sensitive phosphodiester bond was between A-4324 and G-4325 of the 28S rRNA. These results indicate that all six ribosome-inactivating proteins damage eukaryotic ribosomes by cleaving the N-glycosidic bond at A-4324 of the 28S rRNA of the ribosomes, as does ricin A-chain.     

麻疯树curcin启动子的分离及其在转基因烟草中的功能分析

核糖体失活蛋白是一类可以使核糖体28S rRNA的保守α茎环结构域脱嘌呤的蛋白质。麻疯树毒蛋白（curcin）前体基因编码麻疯树胚乳I型核糖体失活蛋白。从麻疯树基因组中克隆得到其5＇侧翼区0.6 kb长的片段,将该片段插入pBI121载体置换其中的CaMV 35S启动子并在相应的转基因烟草中检测报告基因GUS的表达情况。经过GUS活性检测分析发现,该0.6 kb长的片段能够启动报告基因在种子中的表达,并且其在种子不同发育阶段的表达活性存在差异。同时,GUS组织化学染色定位分析表明,在双子叶植物中该启动子片段是胚乳特异性表达的,它从心形胚时期开始持续地在烟草的胚乳中发挥启动活性。     

The mechanism of action of the cytotoxic lectin from Phoradendron californicum: the RNA N-glycosidase activity of the protein

A toxic lectin from Phoradendron californicum (PCL) was found to inactivate catalytically 60 S ribosomal subunits of rabbit reticulocytes, resulting in the inhibition of protein synthesis. To study the mechanism of action of PCL, rat liver ribosomes were treated with the toxin and the extracted rRNA was treated with aniline. A fragment containing about 450 nucleotides was released from the 28 S rRNA. Analysis of the nucleotide sequence of the fragment revealed that the aniline-sensitive phosphodiester bond was between A4324 and G4325 of the 28 S rRNA. These results indicate that PCL inactivates the ribosomes by cleaving an N-glycosidic bond at A4324 of 28 S rRNA in the ribosomes as does ricin A-chain.     

麻疯树curcin 启动子的分离及其在转基因烟草中的功能分析

核糖体失活蛋白是一类可以使核糖体28S rRNA的保守a茎环结构域脱嘌呤的蛋白质。麻疯树毒蛋白(curcin)前体基因编码麻疯树胚乳I 型核糖体失活蛋白。从麻疯树基因组中克隆得到其5'侧翼区0.6 kb长的片段, 将该片段插入pBI121载体置换其中的CaMV 35S启动子并在相应的转基因烟草中检测报告基因GUS的表达情况。经过GUS活性检测分析发现, 该0.6 kb长的片段能够启动报告基因在种子中的表达, 并且其在种子不同发育阶段的表达活性存在差异。同时, GUS组织化学染色定位分析表明, 在双子叶植物中该启动子片段是胚乳特异性表达的, 它从心形胚时期开始持续地在烟草的胚乳中发挥启动活性。     

利用转基因烟草确定AtELHYPRP2蛋白对赤霉菌的抗性及其亚细胞定位特征

采用遗传转化技术获得了整合有拟南芥AtELHYPRP2(EARLI1-LIKE HYBRID PROLINE-RICH PROTEIN 2,AT4G12500)基因的转基因烟草株系,研究了该基因编码蛋白对真菌病原体赤霉菌的抗性及其亚细胞定位特征。以拟南芥Col-0生态型基因组DNA为模板,通过聚合酶链反应扩增AtELHYPRP2基因编码序列,经限制性酶切后连接至pCAMBIA1302载体,构建产生pCAMBIA1302-AtELHYPRP2-GFP融合表达载体。进一步采用农杆菌LBA4404转化烟草叶片外植体,筛选得到转基因烟草植株。RT-PCR、Western blotting印迹分析结果显示,AtELHYPRP2基因在转化体中可以有效表达。激光共聚焦显微观察发现AtELHYPRP2-GFP融合蛋白产生的绿色荧光与碘化丙啶染色后产生的红色荧光能够重合,说明AtELHYPRP2蛋白定位于细胞表面。真菌侵染实验结果显示,组成性表达AtELHYPRP2基因能够增强烟草对赤霉菌的抗性,被侵染部位有明显的H2O2积累。转基因烟草植株中PR1基因的本底表达水平比野生型高,PR1和PR5基因的系统表达水平比野生型高,说明AtELHYPRP2基因可能在SAR反应中具有一定的作用。     

Synthesis and accumulation of pea plastocyanin in transgenic tobacco plants

The pea plastocyanin gene in a 3.5 kbp Eco RI fragment of pea nuclear DNA was introduced into tobacco by Agrobacterium-mediated transformation. Regenerated plants contained pea plastocyanin located within the chloroplast thylakoid membrane system. Analysis of seedlings from a self-pollinated transgenic plant containing a single copy of the pea plastocyanin gene indicated that seedlings homozygous for the pea gene contained almost twice as much pea plastocyanin as seedlings hemizygous for the pea gene. Homozygous seedlings contained approximately equal amounts of pea and tobacco plastocyanins. The amount of tobacco plastocyanin in leaves of transgenic plants was unaffected by the expression of the pea plastocyanin gene. The mRNA from the pea gene in tobacco was indistinguishable by northern blotting and S1 nuclease protection from the mRNA found in pea. In both pea and transgenic tobacco, expression of the pea plastocyanin gene was induced by light in leaves but was suppressed in roots. Pea plastocyanin free of contaminating tobacco plastocyanin was purified from transgenic tobacco plants and shown to be indistinguishable from natural pea plastocyanin by N-terminal protein sequencing and ¹H NMR spectroscopy.     

A promoter from sugarcane bacilliform badnavirus drives transgene expression in banana and other monocot and dicot plants

A 1369 bp DNA fragment (Sc) was isolated from a full-length clone of sugarcane bacilliform badnavirus (ScBV) and was shown to have promoter activity in transient expression assays using monocot (banana, maize, millet and sorghum) and dicot plant species (tobacco, sunflower, canola and Nicotiana benthamiana). This promoter was also tested for stable expression in transgenic banana and tobacco plants. These experiments showed that this promoter could drive high-level expression of the -glucuronidase (GUS) reporter gene in most plant cells. The expression level was comparable to the maize ubiquitin promoter in standardised transient assays in maize. In transgenic banana plants the expression levels were variable for different transgenic lines but was generally comparable with the activities of both the maize ubiquitin promoter and the enhanced cauliflower mosaic virus (CaMV) 35S promoter. The Sc promoter appears to express in a near-constitutive manner in transgenic banana and tobacco plants. The promoter from sugarcane bacilliform virus represents a useful tool for the high-level expression of foreign genes in both monocot and dicot transgenic plants that could be used similarly to the CaMV 35S or maize polyubiquitin promoter.     

Green-tissue-specific expression of a reconstructed cry1C gene encoding the active fragment of Bacillus thuringiensis delta-endotoxin in haploid tobacco plants conferring resistance to Spodoptera litura.

The DNA sequence of a truncated cry1C gene encoding the active fragment of Bacillus thuringiensis (Bt) delta-endotoxin was fully reconstructed by introduction of silent mutations. Each of the truncated wild type and the synthetic genes encoding the active fragment of the protoxin was introduced into haploid tobacco plants under the control of the rbcS promoter. To facilitate selection of transgenic tobacco plants with high insecticidal activity, a fusion gene encoding both rat CYP1A1 cytochrome P450 and yeast NADPH-P450 oxidoreductase was cotransformed with the wild type cry1C gene. The synthetic gene elevated the levels of Cry1C protein and the mRNA in transgenic tobacco plants as well as mortality in Spodoptera litura larvae. The Cry1C protein was accumulated mainly in the leaf tissues of the transgenic tobacco plants. The results reported here imply that the green-tissue-specific expression of the synthetic cry1C gene is useful for the control of S. litura which was rather resistant to the other types of Bt toxins.