3.2 Expression of Als1 and Als2 mutations
Results of the cDNA cloning of the Als1 and Als2 mutations are shown in the 1% Tris-borate-EDTA (TBE) agarose gel in Fig. 3 [30 min at 100 V, 1 kb ladder (NEB Cat. No. N0468S)]. The amplified cDNA fragments appear to match the expected fragment sizes of 2066 bp and 1942 bp for Als1 (see Fig. 3, lanes marked ‘a’ and ‘b’ respectively) and 2083 bp and 1957 bp for Als2 (see Fig. 3, lanes marked ‘c’ and ‘d’ respectively). The faint bands in the no-RT control lanes can be attributed to slight genomic contamination, as the original RNA isolation preparation was not DNase treated. The remaining RNA prep was subsequently DNase treated before cDNA synthesis and RT-qPCR. The RT-qPCR results are shown in quantification cycle (Cq) plots in Fig. 4, where Cq is the terminology adopted in the MIQE guidelines. Lower Cq values indicate higher levels of the specific mRNA being analyzed. The Cq values are also listed in Table 5. The Cq values for Als1 and Als2 were higher than the Cq value for the eIF-4A control in the W4-4 sample. This suggests that the expression of Als1 and Als2 was relatively low compared with eIF-4A. The no-RT control did not register Cq values for Als1 and Als2. Although the eIF-4A/no-RT combination had an average Cq value of 34.93, this value is significantly higher than the average Cq value of 21.41 for eIF-4A in the W4-4 sample, and the genomic contamination can be regarded as insignificant. The ratio of Als2 to Als1 is calculated using the formula
Figure 3. Als1 and Als2 alleles were amplified from W4-4 cDNA with primers from Table 3: a – ALS1F and ALS3′UTRR; b – ALS1F and ALSR; c – ALS2F and ALS3'UTRR; d – ALS2F and ALSR. Faint bands in no-RT control lanes are slight genomic contamination in the RNA preparation used for cDNA synthesis.
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Figure 4. Reverse transcriptase quantitative PCR results for Als1, Als2 and eIF-4A genes: (a) with W4-4 cDNA only; (b) with no-RT control only (all done in triplicate).
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Table 5. Cq values for RT-qPCR results
|Sample||Gene||Efficiency||Cq||Average Cq||Maximum Cq||Minimum Cq||Cq SD|
where the average of the efficiencies for Als1 and Als2 was used, as well as the averages of the Cq values. This resulted in a ratio of 1.30, which suggests that the level of expression of Als1 and Als2 is essentially the same.
Together, the cDNA cloning and the RT-qPCR results confirm the expression of both of the Als1 (P178S) and Als2 (W560L) genes, suggesting that the mutations in both of these genes contribute to the high levels of sulfonylurea tolerance in the W4-4 soy line.
3.3 Evaluation of herbicide tolerance
Als1, Als2 and the Als1 + Als2 combination dramatically reduced soybean response to post-emergence applications of herbicides inhibiting the acetolactate synthase enzyme (Fig. 5). Based on visual analysis of dose–response curves, when compared with the wild type, Als1 significantly improved soybean tolerance to chlorimuron, nicosulfuron, rimsulfuron, sulfometuron, thifensulfuron, tribenuron and flucarbazone. When compared with the wild type, Als2 improved soybean tolerance to imazapyr, chlorimuron, nicosulfuron, rimsulfuron, sulfometuron, thifensulfuron, tribenuron and flucarbazone. When compared with the wild type, inclusion of the combination of Als1 + Als2 improved soybean tolerance to imazapyr, pyrithiobac sodium, chlorimuron, nicosulfuron, rimsulfuron, sulfometuron, thifensulfuron, tribenuron and flucarbazone. When compared with soybean containing Als1 only or Als2 only, inclusion of the combination of Als1 + Als2 improved soybean tolerance to imazapyr, pyrithiobac sodium, nicosulfuron, rimsulfuron, sulfometuron and flucarbazone. These test results confirm that the inclusion of Als1, Als2 and the combination of Als1 + Als2 improve the tolerance of soybean to at least four of the five chemical families active on ALS. Wild-type soybean showed little phytotoxic response to imazethapyr or cloransulam methyl. Both of these herbicides are inherently selective for soybean tolerance and are labeled for weed control in soybean. Test results for imazethapyr and cloransulam methyl show that Als1, Als2 and the combination of Als1 + Als2 do not increase the sensitivity of soybean to either herbicide.
Data presented in Fig. 5 were used to estimate doses of herbicides that elicit either 10% or 50% phytotoxicity response of ‘wild-type’ soybean and soybean containing the Als1, Als2 or Als1 + Als2 genes. These EC10 and EC50 values, and their confidence intervals, were subsequently used for isobole analysis to test for zero interaction, antagonism or synergism among the various ALS genotypes. For example, in Fig. 6, the predicted dose–response curves based on log-logistic analysis are compared with the observed data for pyrithiobac sodium for the three soybean biotypes. Amounts of pyrithiobac sodium to cause 50% phytotoxicity were estimated to be 74 (62–89) g AI ha−1, 49 (46–53) g AI ha−1 and 450 (330 to >560) g AI ha−1 [mean (95% confidence interval)] for soybean containing Als1 only, Als2 only or Als1 + Als2 genes respectively. These EC50 values were then compared to test for interactions between the Als1 and Als2 genes.
Figure 6. Soybean response to post-emergence applications of pyrithiobac sodium: (a) Als1 only; (b) Als2 only; (c) Als1 + Als2; (d) isobole plot based on EC50 for Als1 + Als2, illustrating synergism versus the zero interaction line.
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The isobole method of analysis for gene interaction is based on the dose response of biologically active agents in combination and uses ‘isoeffective’ or equally effective doses for each of the components to build an isobole graph. In this study, the agents being evaluated were the Als1 and Als2 gene mutations. Soybean responses to increasing doses of herbicides were used to assess the effectiveness of each gene to improve tolerance to ALS-inhibiting herbicides. If there is no interaction between the genes, an isoeffective amount of one gene can be substituted for the other gene. The zero interaction line in the isobole is a straight line connecting isoeffective rates (e.g. EC50) for each of the two genes (Fig. 6d). An EC50 line with confidence intervals was estimated for the observed response of soybean containing the combination of Als1 + Als2. These values were then plotted on the isobole graph by parsing out the contribution to the (Als1 + Als2) combination's EC50 from its Als1 and Als2 components using a 1:1 ratio assumption. A 1:1 ratio is assumed because Als1 + Als2 consists of a single mutation of Als1 on one ALS gene and a single mutation of Als2 on an independent ALS gene. If the zero interaction line is below the lower confidence interval for the combination, synergy is indicated. In addition, if the combination of the Als1 and Als2 genes is synergistic in their ability to improve soybean tolerance to ALS herbicides, substantially more herbicide is required to produce the same level of phytotoxic response in soybean containing both ALS genes than in soybean containing only one of the ALS genes. For example, substantially more pyrithiobac sodium (450 g AI ha−1) was required to cause 50% phytotoxicity in the Als1 + Als2 biotype versus the amount of pyrithiobac sodium necessary to cause a 50% phytotoxic response in either the Als1 only (74 g AI ha−1) or the Als2 only (49 g AI ha−1) biotypes (Fig. 6). Synergistic activity of the combination of Als1 plus Als2 genes to improve soybean tolerance to ALS herbicides was observed for imazapyr, pyrithiobac sodium, rimsulfuron, sulfometuron, tribenuron and flucarbazone (Table 6). These active ingredients represent four of the five chemical families with herbicidal activity on the acetolactate synthase enzyme, suggesting that the combination of Als1 + Als2 provides improved tolerance of soybean to all chemistries with activity on this site of action. Isobole analysis was not conducted for imazethapyr, chlorimuron, thifensulfuron or cloransulam methyl because insufficient herbicide was applied to produce substantial phytotoxicity in soybean containing either the Als1 or the Als2 gene and in soybean containing both genes. Wild-type soybean can tolerate these specific herbicides through independent (non-ALS-based) metabolic mechanisms. Although there was insufficient soybean response to test for synergism for imazethapyr, chlorimuron, thifensulfuron or cloransulam methyl, data from Fig. 5 show that inclusion of the Als1, Als2 and Als1 + Als2 mutations at least maintains and can dramatically improve soybean tolerance to these herbicides.
Table 6. Dose response and isobole analysis data of soybeans treated with ALS herbicides
|Chemical family||Active ingredient||EC isobole||Biotypea||Geneinteraction|
|Wild type (als1 + als2)||Als1 only||Als2 only||Als1 + Als2|
|(g AI ha−1)||(g AI ha−1)||(g AI ha−1)||(g AI ha−1)|
|Imidazolinone||Imazapyr||EC50||22 (20–23)||39 (34–44)||94 (71–140)||110 (87 to >140)b||Synergistic|
|Pyrimidinylthiobenzoate||Pyrithiobac sodium||EC50||<35c||74 (62–89)||49 (46–53)||450 (330 to >560)||Synergistic|
|Sulfonylurea||Nicosulfuron||EC10||38 (26–49)||190 (90–220)||110 (60–140)||>280||Zero interaction|
|Sulfonylurea||Rimsulfuron||EC10||10 (8–12)||29 (18–37)||24 (18–29)||120 (80 to >140)||Synergistic|
|Sulfonylurea||Sulfometuron||EC10||<4.4||15 (9–19)||<4.4||>70 (17 to >70)||Synergistic|
|Sulfonylurea||Tribenuron||EC10||4 (<4–5)||70 (70)||5 (<4–8)||>70||Synergistic|
|Triazolinone||Flucarbazone||EC10||<17.5||230 (120 to >280)||<17.5||>280||Synergistic|
ALS genes in other crops, including maize (Zea mays L.), wheat (Triticum aestivum L.), oilseed rape (Brassica napus L.), rice (Oryza sativa L.) and sunflower (Helianthus annuus L.), have been modified to improve the tolerance of these crops primarily to the imidazolinone herbicides, one of the five chemical families with activity on ALS. Tolerance to imidazolinone herbicides improved dramatically in maize, oilseed rape, rice and wheat when serine was replaced with asparagine at position 653 (positions in this section reference A. thaliana). In maize, a mutation from tryptophan to leucine at position 574 improved maize crop tolerance to the imidazolinone, sulfonylurea, triazolopyrimidine and pyrimidinylthiobenzoate chemical families. Consistent with these results in maize, in the present studies, a mutation from tryptophan to leucine at position 574 improved soybean tolerance to all five chemical families with ALS activity. For the imidazolinone-tolerant crops, oilseed rape and spring wheat, two mutations of ALS loci were required to produce crop hybrids or varieties with sufficient tolerance to be treated commercially with an imidazolinone herbicide for weed control. In oilseed rape, the two loci were unlinked and additive for improved tolerance to the herbicide. No information is given regarding how the two loci in spring wheat interact. In these studies with soybean, mutations at the two independent loci (positions 197 and 574) acted synergistically for improved crop tolerance to several of the ALS herbicides from different chemical families.