Expression of the yeast L3 and the pokeweed antiviral protein genes confers resistance to trichothecene mycotoxins

Rong Di and Nilgun Tumer

Biotech Center, Cook College, Rutgers University, New Brunswick, NJ

Trichothecenes are a highly diverse class of toxic, sesquiterpenoid secondary metabolites that are produced mainly by plant pathogenic fungi. The contamination of important agricultural products, such as wheat, barley or maize with the trichothecene mycotoxin, deoxynivalenol (DON) due to infection with Fusarium graminearum and F. culmorum is a worldwide problem. Trichothecene mycotoxins interact with the peptidyltransferase site of eukaryotic ribosomes and inhibit protein synthesis. Ribosomal protein L3 (RPL3) participates in the formation of the peptidyltransferase center. Mutations in the RPL3 gene (called TCM1) were initially identified by conferring resistance to trichodermin, a trichothecene mycotoxin that inhibits the peptidyl transferase reaction. To determine if expression of the yeast RPL3 gene will confer resistance to trichothecene mycotoxins, we generated transgenic tobacco plants expressing either the wild type or mutant forms of the yeast RPL3 alone or together with pokeweed antiviral protein (PAP), a ribosome inactivating protein that inhibits viral and fungal infection. Transgenic plants containing the wild type yeast RPL3 and PAP or a mutant form of the yeast RPL3 (L3D) and PAP were phenotypically normal. Similarly, transgenic lines expressing the yeast RPL3 or L3D alone were indistinguishable from wild type plants. To determine if transgenic tobacco plants expressing the yeast RPL3 genes are resistant to trichothecenes, seeds from transgenic and wild type plants were germinated on MS medium, containing 1 µM DAS (4,15-diacetoxyscirpenol) or 10 µM of DON and their root length was measured at the end of six weeks. Plants from all transgenic lines showed resistance to DAS and DON compared to the wild type plants. However, the highest level of resistance was observed with transgenic plants expressing the yeast RPL3 genes together with PAP. To confirm that yeast RPL3 genes were expressed in these plants, we carried out real-time PCR analysis using primers specific for the yeast RPL3 genes, which do not hybridize to the tobacco L3 genes. The results confirmed the expression of the yeast RPL3 genes in the transgenic lines. These results demonstrate that we can obtain phenotypically normal transgenic plants that show high levels of resistance to DON and DAS by coexpressing the wild type or mutant forms of the yeast RPL3 together with PAP in transgenic tobacco plants.