Transcriptionally linked simultaneous overexpression of P450 genes for broad-spectrum herbicide resistance.

Broad-spectrum herbicide resistance (BSHR), often linked to weeds with metabolism-based herbicide resistance, poses a threat to food production. Past studies have revealed that overexpression of catalytically promiscuous enzymes explains BSHR in some weeds; however, the mechanism of BSHR expression remains poorly understood. Here, we investigated the molecular basis of high-level resistance to diclofop-methyl in BSHR late watergrass (Echinochloa phyllopogon) found in the United States, which cannot be solely explained by the overexpression of promiscuous cytochrome P450 monooxygenases CYP81A12/21. The BSHR late watergrass line rapidly produced 2 distinct hydroxylated diclofop acids, only 1 of which was the major metabolite produced by CYP81A12/21. RNA-seq and subsequent reverse transcription quantitative PCR (RT-qPCR)-based segregation screening identified the transcriptionally linked overexpression of a gene, CYP709C69, with CYP81A12/21 in the BSHR line. The gene conferred diclofop-methyl resistance in plants and produced another hydroxylated diclofop acid in yeast (Saccharomyces cerevisiae). Unlike CYP81A12/21, CYP709C69 showed no other herbicide-metabolizing function except for a presumed clomazone-activating function. The overexpression of the 3 herbicide-metabolizing genes was also identified in another BSHR late watergrass in Japan, suggesting a convergence of BSHR evolution at the molecular level. Synteny analysis of the P450 genes implied that they are located at mutually independent loci, which supports the idea that a single trans-element regulates the 3 genes. We propose that transcriptionally linked simultaneous overexpression of herbicide-metabolizing genes enhances and broadens the metabolic resistance in weeds. The convergence of the complex mechanism in BSHR late watergrass from 2 countries suggests that BSHR evolved through co-opting a conserved gene regulatory system in late watergrass.

Gene expression shapes the patterns of parallel evolution of herbicide resistance in the agricultural weed Monochoria vaginalis.

The evolution of herbicide resistance in weeds is an example of parallel evolution, through which genes encoding herbicide target proteins are repeatedly represented as evolutionary targets. The number of herbicide target-site genes differs among species, and little is known regarding the effects of duplicate gene copies on the evolution of herbicide resistance. We investigated the evolution of herbicide resistance in Monochoria vaginalis, which carries five copies of sulfonylurea target-site acetolactate synthase (ALS) genes. Suspected resistant populations collected across Japan were investigated for herbicide sensitivity and ALS gene sequences, followed by functional characterization and ALS gene expression analysis. We identified over 60 resistant populations, all of which carried resistance-conferring amino acid substitutions exclusively in MvALS1 or MvALS3. All MvALS4 alleles carried a loss-of-function mutation. Although the enzymatic properties of ALS encoded by these genes were not markedly different, the expression of MvALS1 and MvALS3 was prominently higher among all ALS genes. The higher expression of MvALS1 and MvALS3 is the driving force of the biased representation of genes during the evolution of herbicide resistance in M. vaginalis. Our findings highlight that gene expression is a key factor in creating evolutionary hotspots.

Characterization of the acetolactate synthase gene family in sensitive and resistant biotypes of two tetraploid Monochoria weeds, M. vaginalis and M. korsakowii.

Monochoria vaginalis and M. korsakowii are allotetraploid noxious weeds in rice cultivation. Occurrences of resistance to acetolactate synthase (ALS)-inhibiting herbicides have been reported in these weeds in Japan since the 1990s. The existence of multiple copies of ALS genes in both species has hindered and complicated the detailed study of molecular mechanisms in them. To determine the copy number and full-length of ALS genes in both species, we first amplified partial sequences of ALS genes and separated them by cloning. Five and three distinct sequences were identified in M. vaginalis and M. korsakowii, respectively. RACE and TAIL PCR successfully isolated full-length ALS genes, revealing that one copy of ALS genes in both species is a pseudogene formed by a frameshift mutation. Interestingly, one of the four putative functional ALS genes in M. vaginalis contains an intron in the 3'-untranslated region. Amplification and sequencing of the full-length ALS genes in sensitive and suspected resistant lines revealed a non-synonymous point mutation at codon Pro197, resulting in amino acid substitutions (Leu, Ser, or Ala) well known to endow ALS inhibitor resistance. Importantly, codon Pro197 of the M. korsakowii pseudogene encodes leucine (Leu) both in resistant and sensitive plants, which is also known to confer ALS inhibitor resistance when ALS genes are functional. Dose responses to imazosulfuron of the lines analyzed for ALS genes were in agreement with the existence of the mutations. These results suggest that some caution is needed when diagnosing molecular resistance in M. korsakowii. The information of copy number and full-length sequences will help diagnose ALS resistance and make a basis for the study of the evolution of ALS resistance in Monochoria spp.

Functional characterization of cytochrome P450 CYP81A subfamily to disclose the pattern of cross-resistance in Echinochloa phyllopogon.

KEY MESSAGE: CYP81A P450s armor Echinochloa phyllopogon against diverse and several herbicide chemistries. CYP81A substrate preferences can be a basis for cross-resistance prediction and management in E. phyllopogon and other related species. Metabolism-based herbicide resistance is a major threat to agriculture, as it is unpredictable and could extend resistance to different chemical groups and modes of action, encompassing existing, novel and to-be-discovered herbicides. Limited information on the enzymes involved in herbicide metabolism has hindered the prediction of cross-resistance in weeds. Members of CYP81A subfamily in multiple herbicide resistant (MHR) Echinochloa phyllopogon were previously identified for conferring cross-resistance to six unrelated herbicide classes. This suggests a critical role of CYP81As in endowing unpredictable cross-resistances in E. phyllopogon, thus the functions of all its nine putative functional CYP81A genes to 33 herbicides from 24 chemical groups were characterized. Ectopic expression in Arabidopsis thaliana identified the CYP81As that can confer resistance to multiple and diverse herbicides. The CYP81As were further characterized for their enzymatic functions in Escherichia coli. CYP81A expression in E. coli was optimized via modification of the N-terminus, co-expression with HemA gene and culture at optimal temperature. CYP81As metabolized its herbicide substrates into hydroxylated, N-/O-demethylated or both products. The cross-resistance pattern conferred by CYP81As is geared towards all chemical groups of acetolactate synthase inhibitors and is expanded to herbicides inhibiting photosystem II, phytoene desaturase, protoporphyrinogen oxidase, 4-hydroxyphenylpyruvate dioxygenase, and 1-deoxy-D-xylulose 5-phosphate synthase. Cross-resistance to herbicides pyrimisulfan, propyrisulfuron, and mesotrione was predicted and confirmed in MHR E. phyllopogon. This study demonstrated that the functional characterization of the key enzymes for herbicide metabolism could disclose the cross-resistance pattern and identify appropriate chemical options to manage the existing and unexpected cross-resistances in E. phyllopogon.

CYP81A P450s are involved in concomitant cross-resistance to acetolactate synthase and acetyl-CoA carboxylase herbicides in Echinochloa phyllopogon.

Californian populations of Echinochloa phyllopogon have evolved multiple-herbicide resistance (MHR), posing a threat to rice production in California. Previously, we identified two CYP81A cytochrome P450 genes whose overexpression is associated with resistance to acetolactate synthase (ALS) inhibitors from two chemical groups. Resistance mechanisms to other herbicides remain unknown. We analyzed the sensitivity of an MHR line to acetyl-CoA carboxylase (ACCase) inhibitors from three chemical groups, followed by an analysis of herbicide metabolism and segregation of resistance of the progenies in sensitive (S) and MHR lines. ACCase herbicide metabolizing function was investigated in the two previously identified P450s. MHR plants exhibited resistance to all the ACCase inhibitors by enhanced herbicide metabolism. Resistance to the ACCase inhibitors segregated in a 3 : 1 ratio in the F2 generation and completely co-segregated with ALS inhibitor resistance in F6 lines. Expression of the respective P450 genes conferred resistance to the three herbicides in rice, which is in line with the detection of hydroxylated herbicide metabolites in vivo in transformed yeast. CYP81As are super P450s that metabolize multiple herbicides from five chemical classes, and concurrent overexpression of the P450s induces metabolism-based resistance to the three ACCase inhibitors in MHR E. phyllopogon, as it does to ALS inhibitors.

Copy Number Variation in Acetolactate Synthase Genes of Thifensulfuron-Methyl Resistant Alopecurus aequalis (Shortawn Foxtail) Accessions in Japan.

Severe infestations of Alopecurus aequalis (shortawn foxtail), a noxious weed in wheat and barley cropping systems in Japan, can occur even after application of thifensulfuron-methyl, a sulfonylurea (SU) herbicide. In the present study, nine accessions of A. aequalis growing in a single wheat field were tested for sensitivity to thifensulfuron-methyl. Seven of the nine accessions survived application of standard field rates of thifensulfuron-methyl, indicating that severe infestations likely result from herbicide resistance. Acetolactate synthase (ALS) is the target enzyme of SU herbicides. Full-length genes encoding ALS were therefore isolated to determine the mechanism of SU resistance. As a result, differences in ALS gene copy numbers among accessions were revealed. Two copies, ALS1 and ALS2, were conserved in all accessions, while some carried two additional copies, ALS3 and ALS4. A single-base deletion in ALS3 and ALS4 further indicated that they represent pseudogenes. No differences in ploidy level were observed between accessions with two or four copies of the ALS gene, suggesting that copy number varies. Resistant plants were found to carry a mutation in either the ALS1 or ALS2 gene, with all mutations causing an amino acid substitution at the Pro197 residue, which is known to confer SU resistance. Transcription of each ALS gene copy was confirmed by reverse transcription PCR, supporting involvement of these mutations in SU resistance. The information on the copy number and full-length sequences of ALS genes in A. aequalis will aid future analysis of the mechanism of resistance.

Nucleotide substitutions in the acetolactate synthase genes of sulfonylurea-resistant biotypes of Monochoria vaginalis (Pontederiaceae).

Some point mutations in acetolactate synthase (ALS) confer resistance to ALS-inhibiting herbicides in weeds. To clarify the evolution of the herbicide resistance of Monochoria vaginalis, a weed in rice fields in Japan, the nucleotide sequences of four genes encoding ALS were surveyed in five sulfonylurea-resistant (SU-R) and five sulfonylurea-susceptible (SU-S) biotypes. In the ALS1 gene, two SU-R biotypes showed nucleotide substitutions changing Pro197 to Ser and Leu, respectively. In a different gene, ALS3, three other SU-R biotypes showed either of the two nonsynonymous nucleotide substitutions seen in ALS1. Only two biotypes geographically located distantly from each other shared the same mutation conferring SU resistance in the same gene. These patterns of nucleotide substitutions indicate that the SU-R phenotype was acquired independently by different biotypes. Nucleotide diversity values of the genes showing SU-R mutations were higher than those of ALS2 lacking any SU-R mutation and of a putative pseudogene, ALS4. This result suggests that the maintenance of nucleotide variability within target genes provides an opportunity for the evolution of SU-R phenotypes by herbicide-driven selection for mutations conferring resistance.