Introduction of a Modified Ribosomal Protein L3 Gene as a Strategy to Increase Trichothecene Toxin Resistance in Plants

Fusarium graminearum, the causative agent of wheat head blight, produces the trichothecene deoxynivalenol (DON). DON acts as a protein synthesis inhibitor and is a significant factor contributing to the disease. However, acetylation of the C-3 hydroxyl group of a number of different trichothecenes reduces the toxicity to yeast as well as to the singlecelled plant, Chlamydomonas, suggesting that the C-3 OH site is critical for toxicity. Since the TRI101 gene in F. graminearum encodes a 3-O-acetyltransferase, this gene may function for self-protection against DON and its intermediates. Disruption of the FgTRI101 was done to test if, and to what extent, this gene is involved in self-protection. The degree of virulence on wheat by mutants deficient in TRI101 activity has also been evaluated. FgTRI101 was cloned from Gz3639 into the vector pT7Blue3. The selectable marker, hygromycin B, as well as its promoter, was inserted into an EcoRI site, located approximately in the middle of the 1.4 kb DNA sequence encoding FgTRI101. Disruption was verified by both PCR and Southern hybridization. Fungal disruptants were tested on toxincontaining medium to determine if the mutants were more sensitive than the wild type strain. They were also analyzed to determine what intermediates in the pathway they synthesized. Previous work in our labs has shown that F. sporotrichioides mutants carrying a disrupted FsTRI101 gene accumulate isotrichodermol, a compound shown by us to be toxic to Chlamydomonas. However, TRI101-disrupted mutants in F. graminearum do not accumulate the C-3 hydroxylated compound and instead accumulate the C-3,8 hydroxylated compound, which has been shown to be less toxic in our Chlamydomonas toxicity tests. To test whether FgTRI101 disruptants were less virulent than wild type, wheat virulence testing in the greenhouse showed that the fungal TRI101 disruptants produced far less disease than the wild type. Our results support the conclusion, that in F. graminearum, FgTRI101 plays a role in self-protection but is not the sole mechanism. Disruption mutants are weakened in their ability to infect and cause disease in wheat, most likely because they do not produce large quantities of a C-3 OH toxic byproduct. 2000 National Fusarium Head Blight Forum 5 Biotechnology CHARACTERIZATION OF WHEAT PR-PROTEINS CDNA’S FOR TRANSFORMATION OF WHEAT TO ENHANCE RESISTANCE TO SCAB A. Anand, W. Li, N. Sakthivel, S. Krishnaveni, S. Muthukrishnan*, B.S. Gill2, J.S.Essig, R.E.Adams V.Janakiraman, H.N.Trick Department of Biochemistry; 2 Department of Plant Pathology, Kansas State University, Manhattan, KS, 66506 *Corresponding author: PH: (785) 532-6939, E-mail: [email protected]