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1.
Reduced toxicity and broad spectrum resistance to viral and fungal infection in transgenic plants expressing pokeweed antiviral protein II 总被引:3,自引:0,他引:3
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. 相似文献
2.
L Barbieri A Bolognesi P Cenini A I Falasca A Minghetti L Garofano A Guicciardi D Lappi S P Miller F Stirpe 《The Biochemical journal》1989,257(3):801-807
1. Ribosome-inactivating proteins were found in high amounts in one line of cells of Phytolacca americana (pokeweed) cultured in vitro and, in less quantity, in lines of Saponaria officinalis (soapwort) and of Zea mays (corn) cells. 2. The main ribosome-inactivating protein from pokeweed cells was purified to homogeneity. It is a protein with Mr 29,000 and basic pI, similar to the 'pokeweed antiviral protein' (PAP), a ribosome-inactivating protein from pokeweed leaves. We propose to call the pokeweed antiviral protein isolated from pokeweed cells PAP-C. 3. PAP-C inactivates ribosomes in a less-than-equimolar ratio, thus inhibiting protein synthesis by a rabbit reticulocyte lysate with an IC50 (concentration causing 50% inhibition) of 0.067 nM (2 ng/ml), and modifies rRNA in a manner apparently identical to that of ricin and other ribosome-inactivating proteins. It inhibits protein synthesis by intact cells with an IC50 of 0.7-3.4 microM, and is toxic to mice with an LD50 of 0.95 mg/kg. 相似文献
3.
Transgenic creeping bentgrass with delayed dollar spot symptoms 总被引:7,自引:0,他引:7
Zhenfei Guo Stacy Bonos William A. Meyer Peter R. Day Faith C. Belanger 《Molecular breeding : new strategies in plant improvement》2003,11(2):95-101
Creeping bentgrass (Agrostis palustris Huds) is animportant turfgrass used on golf course greens and fairways. It is susceptibleto a number of fungal pathogens and requires considerable fungicide use fordisease control. Transgenic approaches may be useful in improving the level ofdisease resistance. We have generated transgenic creeping bentgrass plantsexpressing PR5K from Arabidopsis thaliana (L.) Henyh. PR5Kis a receptor protein kinase whose extracellular domain is homologous to thePR5family of pathogenesis-related proteins. In a field test of plants inoculatedwith the fungal pathogen dollar spot (Sclerotiniahomoeocarpa F.T. Bennett) four of the eight transgenic lines showeddelays in disease expression of 29 to 45 days, relative to the control plants. 相似文献
4.
A non-toxic pokeweed antiviral protein mutant inhibits pathogen infection via a novel salicylic acid-independent pathway 总被引:8,自引:0,他引:8
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. 相似文献
5.
6.
Stacy A. Bonos 《Journal of Phytopathology》2011,159(1):12-18
The dollar spot disease, incited by Sclerotinia homoeocarpa F.T. Bennet, is one of the most important diseases of creeping bentgrass (Agrostis stolonifera L.) on golf courses. An understanding of the inheritance of dollar spot resistance could enhance genetic improvement efforts in creeping bentgrass. The objectives of this study were to evaluate the response of two creeping bentgrass crosses to two different isolates of S. homoeocarpa, determine gene action and identify number of loci involved in resistance to individual fungal isolates. Parental clones, pseudo F2, pseudo F3, BC1 and BC2 progenies from two crosses were established in a field trial in a randomized complete block split‐plot design in the fall of 2002. Progeny of each generation (subplots) were inoculated with each of two isolates of S. homoeocarpa (main plots) applied at a rate of 0.25 g/m2 of prepared inoculum and evaluated for dollar spot disease. Minimum loci calculations averaged 1.0–2.6. Midparent heterosis calculations were not significant. Backcross population means were closest to the recurrent parent. Generation mean analysis supports a simple additive‐dominance model for both crosses and both isolates, although there was also some evidence of epistatic gene action depending on the cross and the isolate. These results confirm previous research that dollar spot disease is quantitatively inherited and indicate that there may be a few genes interacting in a mainly additive fashion to confer dollar spot disease resistance in creeping bentgrass. 相似文献
7.
Trichosanthin, alpha-momorcharin and beta-momorcharin: identity of abortifacient and ribosome-inactivating proteins 总被引:4,自引:0,他引:4
H W Yeung W W Li Z Feng L Barbieri F Stirpe 《International journal of peptide and protein research》1988,31(3):265-268
The abortifacient proteins trichosanthin, alpha-momorcharin and beta-momorcharin at nM concentrations inhibit cell-free protein synthesis. The momorcharins and the ribosome-inactivating proteins isolated from Momordica charantia seeds cross-react with the respective antisera. The ribosome-inactivating proteins saporins, pokeweed antiviral protein (PAP) and, to a lesser extent, gelonin have abortifacient activity on pregnant mice. 相似文献
8.
Josh A. Honig Christine Kubik Martin Majewski Christine Poulsen Eric Weibel Keenan Amundsen Scott E. Warnke William A. Meyer Stacy A. Bonos 《Molecular breeding : new strategies in plant improvement》2014,34(1):185-203
Creeping bentgrass (Agrostis stolonifera L.) is the most widely utilized cool-season turf species for intensively managed sports playing surfaces, including bowling greens and golf course putting greens, tees, and fairways. One of the biggest disease problems affecting creeping bentgrass is dollar spot disease caused by Sclerotinia homoeocarpa F.T. Bennett. Relative to traditional food crops, little attention has been paid to applying molecular technology to traditional creeping bentgrass breeding programs. The objective of this study was to develop a PCR-based linkage map of creeping bentgrass and identify quantitative trait loci (QTLs) associated with dollar spot resistance. Mapping populations segregating for dollar spot resistance were created, phenotyped for disease resistance, and genotyped for simple sequence repeat, conserved intron scanning primer, intron length polymorphism, and amplified fragment length polymorphism markers. As expected, 14 linkage groups (LGs) were detected for each parental map, covering a total of 1,424 and 1,374 cM for the 7418-3 and the L93-10 parental maps, respectively. A total of eight QTL regions (23 markers) for dollar spot resistance were observed for three isolates (Crenshaw, PRG, and UMass1) in our creeping bentgrass mapping populations. LGs 1, 4, and 5 contained at least two overlapping QTL regions to different isolates, indicating that these regions may play a significant role in dollar spot resistance. Identification of QTLs associated with disease resistance will help to facilitate marker-assisted selection in traditional creeping bentgrass breeding programs. 相似文献
9.
Pokeweed antiviral protein inactivates pokeweed ribosomes; implications for the antiviral mechanism 总被引:9,自引:0,他引:9
Maureen S. Bonness Michael P. Ready James D. Irvin Tom J. Mabry 《The Plant journal : for cell and molecular biology》1994,5(2):173-183
Pokeweed antiviral protein (PAP) and other ribosome-inactivating proteins (RIPs) had previously been thought to be incapable of attacking conspecific ribosomes, thus having no effect on endogenous processes. This assertion conflicts with a model for PAP's in vivo antiviral mechanism in which PAP (a cell wall protein) selectively enters virus-infected cells and disrupts protein synthesis, thus causing local suicide and preventing virus replication. We show here that pokeweed ( Phytolacca americana ) ribosomes, as well as endod ( Phytolacca dodecandra ) ribosomes, are indeed highly sensitive to inactivation by conspecific RIPs. Ribosomes isolated from RIP-free pokeweed and endod suspension culture cells were found to be highly active in vitro , as measured by poly(U)-directed polyphenylalanine synthesis. Phytolacca ribosomes challenged with conspecific RIPs generated doseresponse curves (IC50 of 1 nM PAP or dodecandrin) very similar to those from wheat germ ribosomes. To determine if Phytolacca cells produce a cytosolic 'anti-RIP' protective element, ribosomes were combined with Phytolacca postribosomal supernatant factors from culture cells, then challenged with conspecific RIPs. Resulting IC50 values of 3–7 nM PAP, PAP-II, PAP-S or dodecandrin indicate that supernatants from these Phytolacca cells lack a ribosomal protective element. This research demonstrates that PAP inactivates pokeweed ribosomes (and is therefore potentially toxic to pokeweed cells) and supports the local suicide model for PAP's in vivo antiviral mechanism. The importance of spatial separation between PAP and ribosomes of cells producing this RIP is emphasized, particularly if crop plants are transformed with the PAP gene to confer antiviral protection. 相似文献
10.
The Sambucus nigra type-2 ribosome-inactivating protein SNA-I' exhibits in planta antiviral activity in transgenic tobacco 总被引:4,自引:0,他引:4
Transgenic tobacco (Samsun NN) plants transformed with a cDNA clone encoding SNA-I' from Sambucus nigra synthesize, and correctly process and assemble, a fully active type-2 ribosome-inactivating protein. Expression of SNA-I' under the control of the 35S cauliflower mosaic virus promoter enhances the plant's resistance against infection with tobacco mosaic virus. In contrast to type-1 ribosome-inactivating proteins, the expression of SNA-I' does not affect the growth and fertility of the transgenic plants and is not accompanied by an increased expression of pathogenesis-related proteins indicating that its antiviral activity most probably differs from that of pokeweed antiviral protein. 相似文献
11.
Summary Creeping bentgrass is a very important turfgrass species used extensively on golf course greens, fairways, and tees. One of
the challenges of creeping bentgrass management is the control of grassy weeds, most of which respond to herbicides in a similar
manner to that of creeping bentgrass. As part of a weed management program for golf courses, Roundup?-tolerant creeping bentgrass will be simple to employ and more effective in controlling problem weeds than currently available
methods. The goal of this research was to evaluate fitness-related reproductive traits in four transgenic creeping bentgrass
events modified to express a Roundup?-tolerant gene, cp4 epsps, to determine if these creeping bentgrass events had gained an unexpected reproductive fitness advantage. We compared transgenic
events ASR 333, ASR801 with their nontransformed tissue culture line, C99056L and transgenic events ASR365, ASR368 with their
non-transformed tissue culture line, B99061R. Populations of plants from three conventional cultivars were also included for
comparison to determine whether significant variations, if present in transgenic events, were novel to the non-transformed
organism, Agrostis stolonifera L. Our results showed that none of the four transgenic events surveyed were significantly different from the respective non-transformed
tissue culture line plants for the following characteristics: first heading date, anthesis duration, inflorescence length,
number of florets per inflorescence, pollen size, and seed-set capacity through open-pollination. One of the transgenic events,
ASR333, needed significantly more days for anthesis initiation than the nontransformed tissue culture line, C99056L; while
another transgenic event, ASR801, exhibited significantly shorter pollen longevity than plants of the tissue culture line,
C99056L. However, ASR801 was not significantly different from the conventional cultivars ‘Penn A-4’ and ‘Penncross’ for pollen
longevity. Plants of both transgenic events ASR365 and ASR368 did not differ significantly from plants of the tissue culture
line, B99061R, for all characters measured. 相似文献
12.
An Arabidopsis β-glucosidase, AtBG1 is known to hydrolyze glucose-conjugated, biologically inactive abscisic acid (ABA) to produce active ABA, which increases the level of ABA in plants. Since an increase of ABA in plants confers tolerance against abiotic stress such as drought, we introduced the pCAMBIA3301 vector harboring the AtBG1 gene into creeping bentgrass through Agrobacterium-mediated transformation. After transformation, putative transgenic plants were selected using the BASTA resistance assay at a concentration of 0.8?%. Genomic integration of the AtBG1 gene was confirmed by genomic PCR and Southern blot analysis, and gene expression was validated by Northern blot and Western blot analyses. Interestingly, the transgenic bentgrass plants overexpressing AtBG1 had a dwarf phenotype with reduced growth rates when compared to wild-type creeping bentgrass. In addition, the transgenic plants accumulated higher ABA levels and displayed enhanced drought tolerance. These results suggest that the expression of AtBG1 in plants induces the accumulation of higher ABA levels, which results in the formation of dwarf creeping bentgrass and enhances the survival in water-limiting environments. Key message We used an Arabidopsis β-glucosidase AtBG1 to engineer a crop with elevated active ABA levels, and developed transgenic creeping bentgrass with enhanced drought tolerance and dwarf phenotype. 相似文献
13.
Background
Turfgrass species are agriculturally and economically important perennial crops. Turfgrass species are highly susceptible to a wide range of fungal pathogens. Dollar spot and brown patch, two important diseases caused by fungal pathogens Sclerotinia homoecarpa and Rhizoctonia solani, respectively, are among the most severe turfgrass diseases. Currently, turf fungal disease control mainly relies on fungicide treatments, which raises many concerns for human health and the environment. Antimicrobial peptides found in various organisms play an important role in innate immune response.Methodology/Principal Findings
The antimicrobial peptide - Penaeidin4-1 (Pen4-1) from the shrimp, Litopenaeus setiferus has been reported to possess in vitro antifungal and antibacterial activities against various economically important fungal and bacterial pathogens. In this study, we have studied the feasibility of using this novel peptide for engineering enhanced disease resistance into creeping bentgrass plants (Agrostis stolonifera L., cv. Penn A-4). Two DNA constructs were prepared containing either the coding sequence of a single peptide, Pen4-1 or the DNA sequence coding for the transit signal peptide of the secreted tobacco AP24 protein translationally fused to the Pen4-1 coding sequence. A maize ubiquitin promoter was used in both constructs to drive gene expression. Transgenic turfgrass plants containing different DNA constructs were generated by Agrobacterium-mediated transformation and analyzed for transgene insertion and expression. In replicated in vitro and in vivo experiments under controlled environments, transgenic plants exhibited significantly enhanced resistance to dollar spot and brown patch, the two major fungal diseases in turfgrass. The targeting of Pen4-1 to endoplasmic reticulum by the transit peptide of AP24 protein did not significantly impact disease resistance in transgenic plants.Conclusion/Significance
Our results demonstrate the effectiveness of Pen4-1 in a perennial species against fungal pathogens and suggest a potential strategy for engineering broad-spectrum fungal disease resistance in crop species. 相似文献14.
Chakraborty N Curley J Warnke S Casler MD Jung G 《TAG. Theoretical and applied genetics. Theoretische und angewandte Genetik》2006,113(8):1421-1435
Dollar spot caused by Sclerotinia homoeocarpa F. T. Bennett is the most economically important turf disease on golf courses in North America. Dollar spot resistance in a creeping bentgrass cultivar would greatly reduce the frequency, costs, and environmental impacts of fungicide application. Little work has been done to understand the genetics of resistance to dollar spot in creeping bentgrass. Therefore, QTL analysis was used to determine the location, number and effects of genomic regions associated with dollar spot resistance in the field. To meet this objective, field inoculations using a single isolate were performed over 2 years and multiple locations using progeny of a full sib mapping population ‘549 × 372’. Dollar spot resistance seems to be inherited quantitatively and broad sense heritability for resistance was estimated to be 0.88. We have detected one QTL with large effect on linkage group 7.1 with LOD values ranging from 3.4 to 8.6 and explaining 14–36% of the phenotypic variance. Several smaller effect QTL specific to rating dates, locations and years were also detected. The association of the tightly linked markers with the LG 7.1 QTL based on 106 progeny was further examined by single marker analysis on all 697 progeny. The high significance of the QTL on LG 7.1 at a sample size of 697 (P < 0.0001), along with its consistency across locations, years and ratings dates, indicated that it was stable over environments. Markers tightly linked to the QTL can be utilized for marker-assisted selection in future bentgrass breeding programs. 相似文献
15.
X-ray crystallographic analysis of the structural basis for the interactions of pokeweed antiviral protein with its active site inhibitor and ribosomal RNA substrate analogs.
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I. V. Kurinov D. E. Myers J. D. Irvin F. M. Uckun 《Protein science : a publication of the Protein Society》1999,8(9):1765-1772
The pokeweed antiviral protein (PAP) belongs to a family of ribosome-inactivating proteins (RIP), which depurinate ribosomal RNA through their site-specific N-glycosidase activity. We report low temperature, three-dimensional structures of PAP co-crystallized with adenyl-guanosine (ApG) and adenyl-cytosine-cytosine (ApCpC). Crystal structures of 2.0-2.1 A resolution revealed that both ApG or ApCpC nucleotides are cleaved by PAP, leaving only the adenine base clearly visible in the active site pocket of PAP. ApCpC does not resemble any known natural substrate for any ribosome-inactivating proteins and its cleavage by PAP provides unprecedented evidence for a broad spectrum N-glycosidase activity of PAP toward adenine-containing single stranded RNA. We also report the analysis of a 2.1 A crystal structure of PAP complexed with the RIP inhibitor pteoric acid. The pterin ring is strongly bound in the active site, forming four hydrogen bonds with active site residues and one hydrogen bond with the coordinated water molecule. The second 180 degrees rotation conformation of pterin ring can form only three hydrogen bonds in the active site and is less energetically favorable. The benzoate moiety is parallel to the protein surface of PAP and forms only one hydrogen bond with the guanido group of Arg135. 相似文献
16.
17.
Association of the ribosome-inactivating proteins (RIPs): pokeweed antiviral protein (PAP), gelonin, Momordica charantia inhibitor (MCI), with reconstituted Sendai virus envelopes (RSVE) was obtained without detectable loss of activities either of RIPs or of viral envelope glycoproteins. RIPs are inactive towards intact cells, but, once encapsulated in RSVE, they become cytotoxic. The concentration of RSVE-associated PAP, which causes 50% inhibition of protein synthesis by Friend erythroleukemic cells, is 0.5 ng/ml. Substances capable to inhibit the viral activities block the acquired cytotoxicity of RIPs associated to RSVE. 相似文献
18.
Agricultural crops worldwide suffer from a vast array of fungal diseases which cause severe yield losses. Upon interaction
with a pathogen, plants initiate a complex network of defense mechanisms, among which is a dramatic increase in chitinase
activity. Chitinases are capable of hydrolyzing chitin-containing fungal cell walls and are therefore thought to play a major
role in the plant’s response. One of the strategies to increase plant tolerance to fungal pathogens is the constitutive
overexpression of proteins involved in plant-defense mechanisms. The level of protection observed in transgenic plants harboring
heterologous chitinase genes varies, depending on the particular combination of enzyme, plant and pathogen tested. Nevertheless,
most of these transgenic plants exhibit increased tolerance to fungal diseases relative to their non-transgenic counterparts.
The combined expression of chitinases with other plant-defense proteins such as glucanases and ribosome-inactivating proteins
further enhances the plant’s resistance to fungal attack.
Received 29 January 1997/ Accepted in revised form 01 July 1997 相似文献
19.
A minimal gene cassette comprised of the ubiquitin (Ubi) promoter + green fluorescent protein (Gfp) gene + Nos terminator DNA sequences, derived from the plasmid vector pPZP201-Gfp was utilized for transformation of creeping bentgrass using particle bombardment. Bentgrass calli bombarded individually
with equivalent amounts of the cassette or whole plasmid DNA were compared for Gfp expression and the GFP-positive calli were subsequently regenerated into plants. Percentage of GFP expressing calli and the
number of GFP spots/calli were significantly higher in calli that were bombarded with the minimal gene cassette when compared
to the whole plasmid. The Gfp expression was stable up to the T2 generation in minimal gene cassette transformants and there was a lower degree of gene silencing. Southern blot analysis
of transgenic plants derived from minimum gene cassette bombardment revealed the presence of single or few copy of the transgene
and fairly simple integration patterns. In comparison, whole plasmid transformants had multiple copies and complex integration
patterns of the transgene. These results illustrate the advantages of using simple gene cassette for stable plant transformation
in bentgrass with possible applications to other plant species. 相似文献
20.
Production of 6-methylsalicylic acid by expression of a fungal polyketide synthase activates disease resistance in tobacco 总被引:4,自引:0,他引:4
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Salicylic acid (SA) has been shown to act as a signal molecule that is produced by many plants subsequent to the recognition of potentially pathogenic microbes. Increases in levels of SA often trigger the activation of plant defenses and can result in increased resistance to subsequent challenge by pathogens. We observed that the polyketide 6-methylsalicylic acid (6-MeSA), a compound that apparently is not endogenous to tobacco, can mimic SA. Tobacco leaves treated with 6-MeSA show enhanced accumulation of the pathogenesis-related (PR) proteins PR1, beta-1,3-glucanase, and chitinase and also develop increased resistance to tobacco mosaic virus. We transformed tobacco with 6msas, the 6-methylsalicylic acid synthase (6MSAS) gene from Penicillium patulum, to generate plants that constitutively accumulate 6-MeSA. Analysis of primary transformants and the first generation progeny of 6MSAS tobacco revealed that plants can be engineered to accumulate significant amounts of 6-MeSA as a conjugate. Levels of total 6-MeSA increased with plant age. Increased 6-MeSA accumulation correlated with increased levels of PR1 and chitinase proteins and resulted in enhanced resistance of NN genotype 6MSAS tobacco to tobacco mosaic virus. Our results demonstrate that a multistep biosynthetic pathway can be engineered into plants using a single fungal polyketide synthase gene. The functional expression of 6msas can be used to activate disease resistance pathways that normally are induced by SA. 相似文献