BROAD-SPECTRUM RESISTANCE TO VIRAL AND FUNGAL INFECTION BY EXPRESSION OF POKEWEED ANTIVIRAL PROTEIN
N TUMER, K HUDAK, D RONG, O ZOUBENKO, C COETZER, P WANG, B PARIKH, D-J HWANG, M BONNESS and S. SMIRNOV
Biotech Center for Agriculture and the Environment, Rutgers University, New Brunswick, NJ, USA
Background and objectives
Our research focuses on characterizing the mechanism of cytotoxicity and antiviral activity of pokeweed antiviral protein (PAP). PAP has potent antiviral activity against many plant and animal viruses, including HIV. We have expressed PAP cDNA in the biochemically and genetically malleable yeast Saccharomyces cerevisiae. Expression of this cDNA under the control of the galactose-inducible GAL1 promoter showed that yeast cells do not grow in the presence of PAP. We have taken advantage of this inducible PAP expression system and the toxicity of PAP to yeast to isolate PAP mutants that permit yeast growth subsequent to galactose induction.
Results and conclusions
We expressed two different non-toxic PAP mutants, NT123-2, which has a point mutation (E176V) in the active site that abolishes enzymatic activity and NT124-3, which has a nonsense mutation that results in deletion of the C-terminal 25 amino acids (W237Stop), in transgenic tobacco and showed that, unlike wild-type PAP, neither mutant is toxic to transgenic plants . In vivo depurination of rRNA was detected in transgenic tobacco expressing PAP, but not in transgenic plants expressing either the active-site mutant or the C-terminal deletion mutant PAP. When extracts from transgenic plants containing the mutant PAPs were applied exogenously to tobacco leaves in the presence of potato virus X (PVX), the C-terminal deletion mutant had antiviral activity, while the active-site mutant had no antiviral activity. Furthermore, transgenic plants expressing low levels of the C-terminal deletion mutant showed resistance to PVX infection, while transgenic plants expressing very high levels of the active-site mutant PAP were not resistant to PVX. These results demonstrated that an intact active site of PAP is necessary for antiviral activity, toxicity and in vivo depurination of tobacco ribosomes. An intact C-terminus is also required for toxicity, and depurination of tobacco ribosomes, but not for antiviral activity, suggesting that antiviral activity of PAP can be dissociated from its toxicity.
Both class I (basic) and class II (acidic) isoforms of PR proteins are overexpressed in transgenic plants expressing PAP and the nontoxic PAP mutants . Although PR-proteins are constitutively expressed, no increase in salicylic acid levels was detected. Homozygous progeny of transgenic plants expressing either PAP or the nontoxic PAP mutants displayed resistance to the fungal pathogen Rhizoctonia solani. These results showed, for the first time, that expression of PAP or the nontoxic PAP mutants activates multiple plant defence pathways independently of salicylic acid and confers resistance to fungal infection. We carried out reciprocal grafting experiments and demonstrated that transgenic tobacco rootstocks expressing PAP induce resistance to tobacco mosaic virus infection in wild-type Nicotiana tabacum NN and nn genotype scions . 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 PR 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 indicated 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 PR protein synthesis.
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