Transgenic rice plants expressing trichothecene 3-O-acetyltransferase show resistance to the Fusarium phytotoxin deoxynivalenol.

Fusarium head blight (FHB) is a devastating disease of small grain cereal crops caused by the necrotrophic pathogen Fusarium graminearum and Fusarium culmorum. These fungi produce the trichothecene mycotoxin deoxynivalenol (DON) and its derivatives, which enhance the disease development during their interactions with host plants. For the self-protection, the trichothecene producer Fusarium species have Tri101 encoding trichothecene 3-O-acetyltransferase. Although transgenic expression of Tri101 significantly reduced inhibitory action of DON on tobacco plants, there are several conflicting observations regarding the phytotoxicity of 3-acetyldeoxynivalenol (3-ADON) to cereal plants; 3-ADON was reported to be highly phytotoxic to wheat at low concentrations. To examine whether cereal plants show sufficient resistance to 3-ADON, we generated transgenic rice plants with stable expression and inheritance of Tri101. While root growth of wild-type rice plants was severely inhibited by DON in the medium, this fungal toxin was not phytotoxic to the transgenic lines that showed trichothecene 3-O-acetylation activity. This is the first report demonstrating the DON acetylase activity and DON-resistant phenotype of cereal plants expressing the fungal gene.

A fluorescent antibiotic resistance marker for rapid production of transgenic rice plants.

Blasticidin S (BS) is an aminoacylnucleoside antibiotic used for the control of rice blast disease. To establish a new cereal transformation system, we constructed a visual marker gene designated gfbsd, encoding an enhanced green fluorescent protein (EGFP) fused to the N-terminus of BS deaminase (BSD). It was cloned into a monocot expression vector and introduced into rice (Oryza sativa L. cv. Nipponbare) calluses by microprojectile bombardment. Three to five weeks after the bombardment, multicellular clusters emitting bright-green EGFP fluorescence were obtained with 10 microg/ml BS, which is not sufficient to completely inhibit the growth of non-transformed tissues. Fluorescent sectors (approximately 2mm in diameter) excised from the calluses regenerated into transgenic plantlets (approximately 10 cm in height) as early as 51 (average 77+/-11) days after the bombardment. The visual antibiotic selection was more efficient and required less time than the bialaphos selection with bar. In addition, the small size (1.1 kb) of gfbsd is preferable for construction of transformation vectors. This new marker gene will make a significant contribution in molecular genetic studies of rice plants.

New TAXI-type xylanase inhibitor genes are inducible by pathogens and wounding in hexaploid wheat.

TAXI-I (Triticum aestivum xylanase inhibitor I) is a wheat grain protein that inhibits arabinoxylan fragmentation by microbial endo-beta-1,4-xylanases used in the food industry. Although TAXI was speculated to be involved in counterattack against pathogens, there is actually no evidence to support this hypothesis. We have now demonstrated the presence of TAXI family members with isolation of two mRNA species, Taxi-III and Taxi-IV. At the nucleotide sequence level, Taxi-III and Taxi-IV were 91.7% and 92.0% identical, respectively, to Taxi-I, and Taxi-III and Taxi-IV were 96.8% identical. Accumulation of Taxi-III/IV transcripts was most evident in roots and older leaves where transcripts of Taxi-I were negligible. When challenged by fungal pathogens Fusarium graminearum and Erysiphe graminis, the concentrations of Taxi-III/IV transcripts increased significantly. In contrast, the increases in Taxi-I transcripts in response to these pathogens were rather limited. Both Taxi-I and Taxi-III/IV were strongly expressed in wounded leaves. The upstream region of Taxi-III contained W boxes and GCC boxes, which are sufficient to confer pathogen and wound inducibility on promoters. Recombinant TAXI-III protein inhibited Aspergillus niger and Trichoderma sp. xylanases: it was also active against some spelt xylan-induced xylanases of F. graminearum. These features suggest that some, but not all, TAXI-type xylanase inhibitors have a role in plant defense.

Expression in cereal plants of genes that inactivate Fusarium mycotoxins.

Trichothecene 3-O-acetyltransferase (encoded by Tri101) inactivates the virulence factor of the cereal pathogen Fusarium graminearum. Zearalenone hydrolase (encoded by zhd101) detoxifies the oestrogenic mycotoxin produced by the same pathogen. These genes were introduced into a model monocotyledon rice plant to evaluate their usefulness for decontamination of mycotoxins. The strong and constitutive rice Act1 promoter did not cause accumulation of TRI101 protein in transgenic rice plants. In contrast, the same promoter was suitable for transgenic production of ZHD101 protein; so far, five promising T0 plants have been generated. Low transgenic expression of Tri101 was suggested to be increased by addition of an omega enhancer sequence upstream of the start codon.