A two-point mutated gene of acetolactate synthase (ALS) was isolated from rice cells, which was cultured together with an ALS-inhibiting herbicide, bispyribac-sodium (BS). The mutations involved residues of tryptophan at position 548 to leucine (W548L) and serine at position 627 to isoleucine (S627I). The ALS expressed in Escherichia coli from this gene showed resistance to multiple herbicides including pyrimidinylcarboxylate (PC), sulfonylurea and imidazolinone herbicides, and showed stronger resistance to PC herbicides than to other herbicides. BS, a PC herbicide, had almost no effect on the enzyme even at 100 mM, which is an approximately 10,000-fold higher concentration than the concentration required for 50% inhibition of the wild-type. The resistance level of W548L/S627I mutating ALS to BS was stronger than the additive effect predicted from the degree of resistance of each single amino acid mutated ALS. Transformed rice cells carrying this gene and a regenerated rice plant expressed resistance to BS, suggesting that this gene is useful as a selectable marker for introducing foreign traits into rice when used with PC herbicides.

Mutated acetolactate synthase (ALS) genes of rice and Arabidopsis, which confer resistance to ALS-inhibiting herbicides, were generated using PCR and overlap extension. Recombinant ALSs from these mutated genes were prepared as glutathione S-transferase-fused proteins, and sensitivities of the proteins to ALS-inhibiting herbicides were examined. Nine kinds of rice ALSs that have mutations at the P171 position (each amino acid is represented by one letter) showed high resistance to a herbicide, bensulfuron-methyl, but the resistance level to a herbicide, bispyribac-sodium (BS), changed among ALSs. S627I ALS of rice expressed high resistance to herbicides: pyriminobac and pyrithiobac-sodium. P171H/R172S ALS of rice showed greater resistance to a herbicide, chlorsulfuron than the additive effect predicted from the resistance of each single mutated ALS. P171H/W548L ALS and P171H/S627I ALS of rice showed similar synergistic resistance to BS. On the other hand, P197S, W574L, S653I, P197H/R198S and W574L/S653I-mutated ALSs of Arabidopsis expressed similar sensitivities to herbicides as those of rice ALSs with the corresponding mutations. These results proposed that P171-mutated ALSs of rice can be used as model enzymes for resistant weed management to ALS-inhibiting herbicides, and rice-mutated ALS genes encoding mutated ALSs such as S627I, P171H/R172S, P171H/W548L, P171H/S627I, and Arabidopsis-mutated ALS genes encoding mutated ALSs, such as S653I, P197H/R198S and W574L/S653I, are useful as new selectable marker genes for genetic transformation of plants when used together with ALS-inhibiting herbicides to which mutated ALSs express high resistance.

Strategies employed for the production of genetically modified (GM) crops are premised on (1) the avoidance of gene transfer in the field; (2) the use of genes derived from edible organisms such as plants; (3) preventing the appearance of herbicideresistant weeds; and (4) maintaining transgenes without obstructing plant cell propagation. To this end, we developed a novel vector system for chloroplast transformation with acetolactate synthase (ALS). ALS catalyzes the first step in the biosynthesis of the branched amino acids, and its enzymatic activity is inhibited by certain classes of herbicides. We generated a series of Arabidopsis (Arabidopsis thaliana) mutated ALS (mALS) genes and introduced constructs with mALS and the aminoglycoside 3#-adenyltransferase gene (aadA) into the tobacco (Nicotiana tabacum) chloroplast genome by particle bombardment. Transplastomic plants were selected using their resistance to spectinomycin. The effects of herbicides on transplastomic mALS activity were examined by a colorimetric assay using the leaves of transplastomic plants. We found that transplastomic G121A, A122V, and P197S plants were specifically tolerant to pyrimidinylcarboxylate, imidazolinon, and sulfonylurea/pyrimidinylcarboxylate herbicides, respectively. Transplastomic plants possessing mALSs were able to grow in the presence of various herbicides, thus affirming the relationship between mALSs and the associated resistance to herbicides. Our results show that mALS genes integrated into the chloroplast genome are useful sustainable markers that function to exclude plants other than those that are GM while maintaining transplastomic crops. This investigation suggests that the resistance management of weeds in the field amid growing GM crops is possible using (1) a series of mALSs that confer specific resistance to herbicides and (2) a strategy that employs herbicide rotation.

Trichoderma asperellum SKT-1 and Gibberella fujikuroi, known as causal agents of "Bakanae" disease, were both transformed with genes encoding green fluorescent protein (GFP) and hygromycin B (hygB) by restriction enzyme-mediated integration (REMI). Rice seeds inoculated with GFP-tagged G. fujikuroi showed "Bakanae" symptoms. GFP-tagged SKT-1 maintained biocontrol activity against the pathogen by soaking seeds in SKT-1 spore suspension. Then, we monitored in situ interactions between SKT-1 and G. fujikuroi on rice seeds using GFP-tagged transformations under confocal scanning laser stereomicroscopy. G. fujikuroi disappeared from the embryo of rice seeds after treatment with SKT-1, whereas SKT-1 was observed on the embryo 24 hr after initiation of germination. In addition, the hyphae of G. fujikuroi were penetrated by the hyphae of SKT-1, and degradation of the cell walls of G. fujikuroi was observed under SEM in co-culture. The cell wall of G. fujikuroi on the embryo of rice seeds was lysed, suggesting that mycoparasitism is the mode of action of T. asperellum SKT-1.