Map-based cloning of a novel rice cytochrome P450 gene CYP81A6 that confers resistance to two different classes of herbicides

Abstract

Development of hybrid rice has greatly contributed to increased yields during the past three decades. Two bentazon-lethal mutants 8077S and Norin8m are being utilized in developing new hybrid rice systems. When the male sterile lines are developed in such a mutant background, the problem of F1 seed contamination by self-seeds from the sterile lines can be solved by spraying bentazon at seedling stage. We first determined the sensitivity of the mutant plants to bentazon. Both mutants showed symptoms to bentazon starting from 100 mg/l, which was about 60-fold, lower than the sensitivity threshold of their wild-type controls. In addition, both mutants were sensitive to sulfonylurea-type herbicides. The locus for the mutant phenotype is bel for 8077S and bsl for Norin8m. Tests showed that the two loci are allelic to each other. The two genes were cloned by map-based cloning. Interestingly, both mutant alleles had a single-base deletion, which was confirmed by PCR-RFLP. The two loci are renamed bel ( a ) (for bel) and bel ( b ) (for bsl). The wild-type Bel gene encodes a novel cytochrome P450 monooxgenase, named CYP81A6. Analysis of the mutant protein sequence also revealed the reason for bel ( a ) being slightly tolerant than bel ( b ). Introduction of the wild-type Bel gene rescued the bentazon- and sulfonylurea-sensitive phenotype of bel ( a ) mutant. On the other hand, expression of antisense Bel in W6154S induced a mutant phenotype. Based on these results we conclude that the novel cytochrome P450 monooxygenase CYP81A6 encoded by Bel confers resistance to two different classes of herbicides.

A Novel Rice Cytochrome P450 Gene, CYP72A31, Confers Tolerance to Acetolactate Synthase-Inhibiting Herbicides in Rice and Arabidopsis

“…BSM), which have different chemical structures from pyrimidinyl carboxylate herbicides (e.g. BS; Zhang et al, 2002;Pan et al, 2006;Wang et al, 2012). It was also reported that japonica rice varieties Kamenoo-4 and Joshu tended to be more sensitive to BSM than indica rice varieties IR-8 and IR-26 (Kobayashi et al, 1995), suggesting the possibility that, in addition to the CYP81A6 gene, the CYP72A31 gene is involved in BSM tolerance.…”

“…This mutant showed 60-fold higher sensitivity to bensulfuronmethyl (BSM), a sulfonylurea herbicide, compared with wild-type rice (Pan et al, 2006). Genetic mapping and complementation tests revealed that a cytochrome P450, CYP81A6, is involved in BSM tolerance (Pan et al, 2006). As far as we know, this is the only example of the isolation and characterization of a cytochrome P450 gene involved in nontarget herbicide tolerance in rice.…”

“…In rice (Oryza sativa), an herbicide-sensitive mutant has been produced by g-ray irradiation (Zhang et al, 2002). This mutant showed 60-fold higher sensitivity to bensulfuronmethyl (BSM), a sulfonylurea herbicide, compared with wild-type rice (Pan et al, 2006). Genetic mapping and complementation tests revealed that a cytochrome P450, CYP81A6, is involved in BSM tolerance (Pan et al, 2006).…”

Cytochrome P450 CYP81A12 and CYP81A21 Are Associated with Resistance to Two Acetolactate Synthase Inhibitors in Echinochloa phyllopogon

“…In rice, BSM is mainly metabolized through O-demethylation of the methoxy group at position 4 of the pyrimidine ring (Takeda et al, 1986). Purified rice microsomes catalyzed this reaction (Deng and Hatzios, 2002), and a CYP81A6 knockout mutant of rice was susceptible to BSM (Pan et al, 2006). Therefore, P450 activity of CYP81A6 appears to catalyze the O-demethylation of BSM, although direct evidence for the reaction has yet to be reported.…”

CRISPR‐S: an active interference element for a rapid and inexpensive selection of genome‐edited, transgene‐free rice plants

“…Silencing CYP81A6 may render rice plants susceptible to the herbicide (Pan et al ., 2006). We speculated that a CYP81A6‐ hpRNAi may be a suitable marker for CRISPR/Cas9 expression.…”