Plant resistance to fungal infection induced by nontoxic pokeweed antiviral protein mutants

Zoubenko, Oleg; Uckun, Fatih M.; Hur, Yoonkang; Chet, I.; Tumer, Nilgun E.

doi:10.1038/nbt1097-992

Cited by 77 publications

(53 citation statements)

References 26 publications

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“…Genes coding for defense proteins have already been employed to enhance plant resistance against fungal and bacterial phytopathogens (38,39,51). For instance, transgenic orange plants expressing a tomato thaumatin-like protein exhibited increased tolerance toward Phytophtora citrophthora (13); constitutive overexpression of an antimicrobial protein gene Ace-AMP1 from Allium cepa in Oryza sativa subsp.…”

Section: Discussionmentioning

confidence: 99%

Expression of a Novel Small Antimicrobial Protein from the Seeds of Motherwort ( Leonurus japonicus ) Confers Disease Resistance in Tobacco

Yang

Xiao

Wang

et al. 2007

Appl Environ Microbiol

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Medicinal plants are valuable resources of natural antimicrobial materials. A novel small protein with antimicrobial activities, designated LJAMP1, was purified from the seeds of a medicinal herb, motherwort (Leonurus japonicus Houtt). LJAMP1 is a heat-stable protein with a molecular mass of 7.8 kDa and a determined isoelectric point of 8.2. In vitro assays showed that LJAMP1 inhibits the growth of an array of fungi and bacteria. The hyphal growth inhibition by LJAMP1 was more evident against hyphomycete fungi, such as Alternaria alternata, Cercospora personata, and Aspergillus niger. The N-terminal amino acid sequence of LJAMP1 was determined, and its coding gene was consequently cloned by the rapid amplification of cDNA ends. The gene LJAMP1 has no intron and encodes a polypeptide of 95 amino acids, in which the first 27 residues was deduced as a signal peptide. The mature LJAMP1 shows relatively low identity to plant napin-like storage proteins. Northern blot assays revealed that LJAMP1 is expressed preferentially in seeds. Bioassays in transgenic tobacco demonstrated that that overexpression of LJAMP1 significantly enhanced the resistance of tobacco against not only the fungal pathogen A. alternata but also the bacterial pathogen Ralstonia solanacearum, while no visible alteration in plant growth and development was observed.

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Section: Discussionmentioning

confidence: 99%

Expression of a Novel Small Antimicrobial Protein from the Seeds of Motherwort ( Leonurus japonicus ) Confers Disease Resistance in Tobacco

Yang

Xiao

Wang

et al. 2007

Appl Environ Microbiol

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“…Transgenic plants expressing nontoxic mutated PAP forms such as PAPx (active site mutant), PAPn (N-terminal mutant), and PAPc (C-terminal mutant) have shown that Glu-176, Gly-75, and 25 C-terminal amino acids are critical amino acid residues involved in ribosome depurination (Smirnov et al, 1997;Tumer et al, 1997;Zoubenko et al, 1997Zoubenko et al, , 2000Hudak et al, 2000). Comparison of cDNA sequences of PAP-H and PAP shows that PAP-H also contains those amino acid residues involved in ribosome depurination.…”

Section: Discussionmentioning

confidence: 99%

“…Accordingly, PAP-S cross-reacts with PAP antibodies, but PAP II does not react with PAP antibodies (Barbieri et al, 1982). PAP is thought to play a defense role because it depurinates ribosomes from all organisms tested, and because its expression in transgenic tobacco (Nicotiana tabacum cv Samsan and Nicotiana benthamiana) plants leads to resistance against viral and fungal infection (Lodge et al, 1993;Zoubenko et al, 1997). However, a clear understanding of the role of PAP in pokeweed has not been achieved.…”

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confidence: 99%

Isolation and Characterization of a Novel Ribosome-Inactivating Protein from Root Cultures of Pokeweed and Its Mechanism of Secretion from Roots

Park

Lawrence²,

Linden

et al. 2002

Plant Physiology

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Ribosome-inactivating proteins are N-glycosidases that remove a specific adenine from the sarcin/ricin loop of the large rRNA, thus arresting protein synthesis at the translocation step. In the present study, a novel type I ribosome-inactivating protein, termed PAP-H, was purified from Agrobacterium rhizogenes-transformed hairy roots of pokeweed (Phytolacca americana). The protein was purified by anion- and cation-exchange chromatography. PAP-H has a molecular mass of 29.5 kD as detected by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and its isoelectric point was determined to be 7.8. Yeast (Saccharomyces cerevisiae) ribosomes incubated with PAP-H released the 360-nucleotide diagnostic fragment from the 26S rRNA upon aniline treatment, an indication of its ribosome-inactivating activity. Using immunofluorescence microscopy, PAP-H was found to be located in the cell walls of hairy roots and root border cells. PAP-H was determined to be constitutively secreted as part of the root exudates, with its secretion enhanced by a mechanism mediated by ethylene induction. Purified PAP-H did not show in vitro antifungal activity against soil-borne fungi. In contrast, root exudates containing PAP-H as well as additional chitinase, β-1,3-glucanase, and protease activities did inhibit the growth of soil-borne fungi. We found that PAP-H depurinates fungal ribosomes in vitro and in vivo, suggesting an additive mechanism that enables PAP-H to penetrate fungal cells.

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“…In contrast, PAP mRNA was detected in transgenic lines expressing the inactive form, PAPx (or PAP E176V ), which contains the point mutation, E176V, at its active site (13). In the present study, we examined the effect of PAP on the stability of its own mRNA and cellular mRNAs in the yeast, Saccharomyces cerevisiae where PAP expression can be tightly controlled.…”

mentioning

confidence: 99%

“…However, a nontoxic C-terminal deletion mutant of PAP inhibited viral infection without depurinating host ribosomes, indicating that antiviral activity could be separated from rRNA depurination (12). Furthermore, expression of nontoxic forms of PAP in transgenic plants induced a stress-associated signal transduction pathway and provided resistance to viral and fungal infection (13,14). PAP is very active against both animal and plant ribosomes.…”

mentioning

confidence: 99%

Pokeweed Antiviral Protein Regulates the Stability of Its Own mRNA by a Mechanism That Requires Depurination but Can Be Separated from Depurination of the α-Sarcin/Ricin Loop of rRNA

Parikh¹,

Coetzer²,

Tumer³

2002

Journal of Biological Chemistry

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Pokeweed antiviral protein (PAP), a single chain ribosome-inactivating protein (RIP) isolated from pokeweed plants (Phytolacca americana), removes specific adenine and guanine residues from the highly conserved, ␣-sarcin/ ricin loop in the large rRNA, resulting in inhibition of protein synthesis. We recently demonstrated that PAP could also inhibit translation of mRNAs and viral RNAs that are capped by binding to the cap structure and depurinating the RNAs downstream of the cap. Cell growth is inhibited when PAP cDNA is expressed in the yeast Saccharomyces cerevisiae under the control of the galactose-inducible GAL1 promoter. Here, we show that overexpression of wild type PAP in yeast leads to a decrease in PAP mRNA abundance. The decrease in mRNA levels is not observed with an active site mutant, indicating that it is due to the N-glycosidase activity of the protein. PAP expression had no effect on steady state levels of mRNA from four different endogenous yeast genes examined, indicating specificity. We demonstrate that PAP can depurinate the rRNA in trans in a translation-independent manner. When rRNA is depurinated and translation is inhibited, the steady state levels of PAP mRNA increase dramatically relative to the U3 snoRNA. Using a PAP variant which depurinates rRNA, inhibits translation but does not destabilize its mRNA, we demonstrate that PAP mRNA is destabilized after its levels are up-regulated by a mechanism that occurs independently of rRNA depurination and translation. We quantify the extent of rRNA depurination in vivo using a novel primer extension assay and show that the temporal pattern of rRNA depurination is similar to the pattern of PAP mRNA destabilization, suggesting that they may occur by a common mechanism. These results provide the first in vivo evidence that a single chain RIP targets not only the large rRNA but also its own mRNA. These findings have implications for understanding the biological function of RIPs.Pokeweed antiviral protein (PAP), 1 a single chain ribosomeinactivating protein (RIP) isolated from the leaves of pokeweed plants (Phytolacca americana), removes specific adenine and guanine residues from the highly conserved, ␣-sarcin/ricin (S/R) loop in the large rRNA (1-3). The enzymatic removal of specific purines from the S/R loop has been reported to interfere with the binding of eEF-2 (elongation factor 2) and inhibit protein synthesis at the translocation step (4, 5). RIPs are protein toxins produced by organisms ranging from bacteria to plants. Because of their selective toxicity, they have been used as biological weapons, to protect plants against pathogens, and as therapies against cancer. Their biological function in the organisms that produce them is unknown. PAP is thought to be a defense protein because it is a potent inhibitor of animal and plant viral pathogens, including human immunodeficiency virus, poliovirus, herpes simplex virus, influenza, potato virus X, and brome mosaic virus (6 -10). Because of its cytotoxicity to dividing cells, PAP is currently und...

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Plant resistance to fungal infection induced by nontoxic pokeweed antiviral protein mutants

Cited by 77 publications

References 26 publications

Expression of a Novel Small Antimicrobial Protein from the Seeds of Motherwort ( Leonurus japonicus ) Confers Disease Resistance in Tobacco

Expression of a Novel Small Antimicrobial Protein from the Seeds of Motherwort ( Leonurus japonicus ) Confers Disease Resistance in Tobacco

Isolation and Characterization of a Novel Ribosome-Inactivating Protein from Root Cultures of Pokeweed and Its Mechanism of Secretion from Roots

Pokeweed Antiviral Protein Regulates the Stability of Its Own mRNA by a Mechanism That Requires Depurination but Can Be Separated from Depurination of the α-Sarcin/Ricin Loop of rRNA

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