Induced broad-spectrum resistance to pathogens in transgenic plants expressing nontoxic mutant forms of pokeweed antiviral protein (Pathogen resistance)

Ribosome inactivating proteins are characterized by their ability to enzymatically remove specific purine residues from a conserved sequence, sarcin/ricin (S/R) loop in the large ribosomal RNA. Pokeweed antiviral protein (PAP) is one of three ribosome inactivating proteins found in pokeweed, Phytolacca americana. PAP has potent antiviral activity against animal and plant viruses. Expression of PAP in transgenic plants confers broad-spectrum resistance to viral infection but is toxic to the host plant.; By utilizing random mutagenesis and positive selection in yeast, Saccharomyces cerevisiae, several nontoxic mutants of PAP were isolated. Mutations that abolish toxicity of PAP were localized either at the active site of PAP, or near its N-terminus, or resulted in premature termination of PAP polypeptide near the C-terminus.; Nontoxic PAP transgenes were expressed in tobacco Nicotiana tabacum cv Samsun and potato Solanum tuberosum cv Russet Burbank. Expression of nontoxic PAP was not deleterious to the host plant and conferred broad-spectrum pathogen resistance, indicating that viral and fungal protection could be dissociated from the toxicity of PAP.; Analysis of the expression of the host genes for pathogenesis related proteins (PRs) revealed that PAP and its nontoxic C-terminal mutant form, PAPc, induced constitutive expression of acidic and basic isoforms of PRs independently of salicylic acid production in transgenic tobacco. Active site mutant form of PAP, PAPx, neither induced PR expression, nor conferred pathogen resistance. Virus and fungus resistant tobacco expressing nontoxic N-terminal form of PAP, PAPn, failed to induce acidic PRs. Instead, constitutive upregulation of wounding inducible protein kinase, WIPK, and proteinase inhibitor PI-II was detected, suggesting that PAPn induced a subset of host defense genes, which are associated with the wounding response.; To confer broad-spectrum pathogen resistance in transgenic plants, the intact active site of the PAP transgenes was required, but depurination and association with host ribosomes was not necessary.