Identification And Functional Analysis Of Genes Involved In Metabolism-Based Mesosulfuron-Methyl Resistance In Alopecurus Aequalis

Wheat(Triticum aestivum L.)is one of the most important crops worldwide,and it is the the second largest food crop in the China.Shortawn foxtail(Alopecurus aequalis Sobol.)is an invasive grass weed that widely spreads in the east,south-central,and southwest regions and parts of the Yellow River basin,seriously disturbing wheat production.Mesosulfuron-methyl is a highly efficient acetolactate synthase(ALS)-inhibiting herbicide widely used for control of this species.However,in recent years,some A.aequalis populations have evolved resistance to mesosulfuron-methyl due to its single site of action and long-term reuse.As documented,many A.aequalis populations located in the wheat fields across provinces of Jiangsu,Anhui,etc.,can not be effectively controlled by mesosulfuron-methyl applied at its field-recommanded rate.Moreover,some populations even evolved multiple-resistance to herbicides with different modes of action,severely threatening the agricultural sustainable development.In this study,we collected a total of 80 populations of A.aequalis from the crop fields across provinces of Jiangsu,Anhui,Henan,and Shandong,after which we investigated the resistance mechanisms of the AH-18 population harboring metabolic resistance to mesosulfuron-methyl.1.A whole-plant single-dose testing was performed to determine the sensitivity of all the80 populations to mesosulfuron-methyl.In total,36 populations such as JS-03,JS-08,JS-10,etc.were susceptible to mesosulfuron-methyl,while the other 44 populations such as JS-01,JS-02,JS-06,etc.were resistant to it.Meanwhile,the resistance levels of some of the resistant populations were characterized.The results showed that all of them had evolved high-level resistance to mesosulfuron-methyl with their resistance indexes ranged from 12.75 to 225.90.2.The full-length ALS genes were amplied and compared between susceptible and resistant populations.The results showed that the ALS genes of all the resistance populations mutated.Of them,an ALS1 gene Trp-574-Leu mutation occurred in 28 populations such as JS-01,JS-02,AH-04,etc.;an ALS1 gene Pro-197-Tyr mutation occurred in the populations AH-12and AH-36;an ALS1 gene Pro-197-Arg mutation occurred in the populations AH-18,JS-04,and JS-05;an Pro-197-Ser mutation occurred in the populations AH-27,AH-29,and AH-52;and an ALS1 gene Pro-197-Thr mutation occurred in seven populations including JS-06,JS-07,JS-09,AH-15,AH-46,AH-51,and SD-03.In the population AH-29,some individuals have an ALS1 gene Trp-574-Leu mutation while the others have an ALS1 gene Pro-197-Ser mutation.Notably,no mutation was found in the ALS1 genes of the populations AH-14 and AH-32,while their ALS2 genes occurred Trp-574-Leu and Pro-197-Thr mutations,respectively.This indicates that all the resistant populations have evolved target-site-based resistance to mesosulfuron-methyl.3.Whole-plant dose-response experiments were conducted to determine the resistance pattern of the population AH-36 to other herbicides.The results showed that AH-36 was highly resistant to mesosulfuron-methyl,pyroxsulam,and flucarbazone-sodium,yet only moderately resistant to imazethapyr and pyrithiobac sodium salt.Based on the RI values,the GR₅₀ values of AH-36 were 7.8 to 28.5 times greater for all tested ALS inhibitors relative to AH-20.Additionally,AH-36 was sensitive to the PSII inhibitor isoproturon given its RI value of 1.3.4.A real-time quantification PCR assay was conducted to compare the relative expression levels of the ALS genes between susceptible population SD-01 and resistant population AH-18.The results revealed that the expression of ALS genes can be induced by mesosulfuron-methyl treatment.Also,the expression of ALS was significantly up-regulated in AH-18 compared with SD-01 before and after mesosulfuron-methyl treatment.5.A whole-plant single-dose testing was performed to determine the effects of the P450s inhibitor malathion on the control efficacy of mesosulfuron-methyl to the resistant A.aequalis populations.The results showed that malathion significantly increased the sensitivity of five populations including JS-05,AH-15,AH-18,AH-29,and AH-36 to mesosulfuron-methyl,of which the population AH-18 exhibited the most significant P450-inhibitory phenotype.This implys the P450s may participate in the mesosulfuron-methyl metabolism in A.aequalis.6.The P450s activity was investigated by determining the the mesosulfuron-methyl dose resulting in 50%growth inhibition(GR₅₀)in the absence and presence of the P450s inhibitor malathion.The results showed that the pretreatment of malathion greatly reduced the resistance level of the AH-18 population by 41%to mesosulfuron-methyl,while it did not lead to a significant change in the susceptibility of the SD-01 population.This indicates that P450s play a key role in the metabolic resistance to mesosulfuron-methyl in AH-18.7.The GSTs activity toward CDNB was compared between SD-01 and AH-18.The results revealed that the GSTs activity of A.aequalis could be induced by mesosulfuron-methyl treatment.Moreover,GSTs activities in AH-18 were significantly higher than those in SD-01before and after mesosulfuron-methyl treatment.This indicates that GSTs may also participate in the mesosulfuron-methyl metabolism in A.aequalis,and the GSTs activity significantly enhanced in AH-18.8.LC-MS/MS analysis was conducted to determine the amounts of herbicide residues in the populations SD-01 and AH-18 at 1,3,5,and 7 d after mesosulfuron-methyl treatment.We found that in terms of herbicide metabolic rate,AH-18 plants metabolized mesosulfuron-methyl significantly faster than did the SD-01 plants at every sampling time.This further demonstrates that AH-18 has evolved significant metabolic resistance to mesosulfuron-methyl.9.A RNA-Seq transcriptome analysis was performed to identify candidate genes that may confer metabolic resistance to mesosulfuron-methyl in the population AH-18.The expression level of the selected genes was verified by q RT-PCR,and the genes with an identical expression between RNA-Seq and q RT-PCR were identified as the final candidates.The results showed that a total of 17 contigs could serve as major candidates for contributing to metabolic mesosulfuron-methyl resistance,including four P450s,two GSTs,two GTs,two ABC transporters,and seven additional contigs with functional annotations related to oxidation,hydrolysis,and plant stress physiology.10.An i TRAQ assay was carried out to identify candidate proteins that may confer metabolic resistance to mesosulfuron-methyl in the population AH-18.The abundance of the selected proteins was verified by PRM,and the proteins with an identical expression between i TRAQ and PRM were identified as the final candidates.The results showed that a total of 22proteins could serve as major candidates for contributing to metabolic mesosulfuron-methyl resistance,including one esterase,four GSTs,two GTs,three ABC transporters,and 12additional proteins with functional annotations related to oxidation,peroxidation,and reduction.11.By performing an associating analysis between RNA-Seq and i TRAQ,and considering also the sequencing depths of different omics and the expression levels and functional significances of the candidates,a total of eight genes were selected for further functional verification.Transgenic Arabidopsis over-expressing CYP709C56,GSTF1,and GSTU1exhibited significant resistant phenotypes to mesosulfuron-methyl.Also,their sensitivities to herbicide was related to the transcript accumulation levels of the transgenes.12.The recombinant GSTU1 proteins were obtained by using a prokaryotic expression system,and its activity towards mesosulfuron-methyl was investigated.The results indicate that GSTU1 showed catalytic activity towards mesosulfuron-methyl.This indicates the GSTU1indeed involved in the metabolic resistance of AH-18 to mesosulfuron-methyl.In summary,the present study investigated the occurrence and development of mesosulfuron-methyl-resistant A.aequalis populations in major rice-wheat rotation regions of the China.The studies on the resistance mechanisms revealed that all the resistant populations harbored target-site based resistance to mesosulfuron-methyl,in which some populations also evolved metabolic resistance to it.We found that the metabolic resistance to mesosulfuron-methyl of the population AH-18 was resulted from enhanced activities of P450s,GSTs,etc.By combining the RNA-Seq,q RT-PCR,i TRAQ,PRM,and transgene,we found that three genes may play key roles in mesosulfuron-methyl metabolism.We also obtained the recombinant proteins of one of the key genes and comfirmed its capacity on herbicide detoxification.This study may provide potential gene resource for herbicide resistance diagnosis and the improvement of crop quality,and provide a reference on investigation on the metabolic resistance metabolisms of other weed species to specific herbicides.