Thickening of the cell wall increases the resistance of S. cerevisiae to commercial formulations of glyphosate

The use of glyphosate-based herbicides is widespread and despite its extensive use, its effects are yet to be deciphered completely. The additives in commercial formulations of glyphosate, though labeled as inert when used individually, have adverse effects when used in combination with other additives and the active ingredient. As a species, Saccharomyces cerevisiae has a wide range of resistance to glyphosate-based herbicides. To investigate the underlying genetic differences between sensitive and resistant strains, global changes in gene expression were measured when yeast were exposed to a commercial formulation of glyphosate (CFG). Changes in gene expression involved in numerous pathways such as DNA replication, MAPK signaling, meiosis, and cell wall synthesis. Because so many diverse pathways were affected, these strains were then subjected to in-lab-evolutions (ILE) to select mutations that confer increased resistance. Common fragile sites were found to play a role in adaptation mechanisms used by cells to attain resistance with long-term exposure to CFG. The cell wall structure acts as a protective barrier in alleviating the stress caused by exposure to CFG. The thicker the cell wall, the more resistant the cell is against CFG. Hence, a detailed study of the changes occurring at the genome and transcriptome level is essential to better understand the possible effects of CFG on the cell as a whole. Author Summary We are exposed to various chemicals in the environment on a daily basis. Some of these chemicals are herbicides that come in direct contact with the food we consume. This makes the thorough investigation of these chemicals crucial. Some of the most commonly used herbicides around the world are glyphosate-based. Their mode of action effects a biosynthetic pathway that is absent in mammals and insects and so it is deemed safe for consumption. However, there are many additives to these herbicides that increase its effects. Thorough testing of these commercially available herbicides is essential to decipher all the potentially adverse effects that it could have on a cell. Saccharomyces cerevisiae has a wide range of genetic diversity, making it is suitable to test different chemicals and identify any harmful effects. In this study, we exposed yeast cells to some glyphosate-based herbicides available in the market, to understand what effects it could have on a cell. We found that the additives in the herbicides have an effect on the cell wall and the mode of entry of glyphosate into the cell.

that is representative of a phenotype similar to most commercial formulations, Cr41 was used for all 154 further experiments. 155 A polygenic trait that has a strain and condition-based pattern in response to Cr41 156 There are tens of thousands of SNPs between these four strains. To explore how genomes 157 change to permit adaptation to high levels of Cr41, the yeast were serially passaged in media containing 158 Cr41 by performing In-Lab-Evolutions (ILEs). Three biological replicates were passaged in YM, YM 159 supplemented with WYF and YPD media, with 0.25% glyphosate from the Cr41 in minimal media and 1% 160 in rich media respectively (Fig 2A). The strains were diluted through six passages by transferring 1% 161 culture to fresh media. Once the resistant populations were identified, single colonies were isolated. 162 These strains were then released from the selective pressure for two passages and then the Cr41 163 resistance was confirmed (Fig 2B-E). This step was performed in order to ensure that the resistance 164 wasn't entirely due to epigenetic mechanisms that cannot be detected through whole-genome 165 sequencing. The resistant cells sequenced for each strain along with the condition are listed in S2 Table.  166 The ILEs selected a large number of SNPs as well as duplicated regions. The synonymous mutations were 167 filtered out and only the genes containing non-synonymous mutations were taken into consideration. A 168 total of 148 genes (S3 Table) accumulated at least one non-synonymous SNP within the coding region 169 among all the sequenced strains treated with Cr41. The genes that accumulated more than one SNP 170 and/or indel in any of the samples from different strains were prioritized and focused on for this study. 171 Dip5 in part transports glyphosate into the cell as shown in our previous study [10], and contained SNPs 172 in three of the sequenced samples ( Fig S1). 173 Principal Component Analysis (PCA plots) were plotted for each sample taking all the SNPs into 174 consideration in order to identify the major sources of variation. This was done to ensure that the main 175 sources of variation corresponds to the biological conditions and was not a mere effect of experimental bias. An R-package, pcadapt (Luu et al. 2017) visualized the patterns that naturally exist between the 177 strains. Hence, resistant strains clustered together and so did the sensitive strains when the data from 178 all the conditions were pooled together ( Fig 3A). The underlying background genetic differences 179 dominated the genome comparisons. Further analysis of the strain-specific PCA plots showed that the 180 clustering of the controls vs. the Cr41 treated samples in S288c was based on PC2 (Fig 3B), and PC1 did 181 the same in the case of RM11 (Fig 3C). 182 Sections of the genome containing as few as two to as many as a hundred genes increased in 183 copy number (CN) as shown by copy number variation analysis (CNV). All the strains in this study were 184 haploid and having a CN of less than one can only occur at nonessential genes. In this analysis, any genes 185 that were partially duplicated were excluded under the assumption that the change in copy number is 186 associated with synteny or the other genes present in their immediate surroundings (S4 Table). Any 187 gene that underwent a CNV in a treated cell but also had an increase in CN in any control sample, was 188 filtered out. This made it apparent that there is an underlying connection between the strain and the 189 condition (i.e. YM, YM with WYF or YPD) it was evolved under. As in the case of YJM789, CNVs occurred 190 only in cells that were evolved in minimal media with WYF. S288c had the most genes (81 genes) that 191 underwent CNV while RM11 did not have any, which was one of the most CFG resistant strain. Some of 192 the S288c genes that underwent CNV were found in all the conditions, but many of which were found in 193 cells evolved in minimal media supplemented with WYF. Though these genes underwent duplication in a 194 specific condition (i.e., WYF), they did not belong to a single pathway. Their functionality ranged from 195 mitochondrial maintenance as in the case of MRX14, MRLP1 to biosynthesis of secondary metabolites 196 such as INO2. This led to the hypothesis that the route used to attain resistance was dependent on the 197 media type. Hence, if a certain strain was evolved in YM, it may not be resistant to the CFG in YPD. To 198 confirm this hypothesis, semi-quantitative growth assays of the evolved strains was carried out in 199 different combinations of media conditions. The strains evolved in rich media did not confer cross-200 resistance in minimal media. However, those evolved in minimal media and WYF concur cross-resistance 201 ( Fig S2). 202 Many regions that underwent CNV were flanked by fragile sites, mainly consisting of 203 transposable elements [39] and one long regulatory ncRNA, ICR1 (Table 1). Fragile sites are defined as 204 regions of the genome that make it difficult for the cell to undergo replication and sometimes result in 205 chromosome breakage. Studies in other organisms such as C. elegans and human cells have shown 206 evidence of DNA damage in these sites [15,40]. Past studies lack conclusive evidence to declare that Ty 207 elements are involved in fragile site rearrangements resulting in CNV of certain regions. However, there 208 is evidence to prove that these elements are involved in processes such as translocations, deletions, etc. 209 (Dunham et al. 2002;Roeder & Fink, 1980). Most of the regions undergoing an increase in copy number 210 and being flanked by Ty elements were found in S288c cells, evolved in the presence of Cr41. All the 211 genes that contained non-synonymous SNPs or underwent CNV (S4 Table) were referred to as affected 212 genes because they were affected by the Cr41 treatment ( Fig 3D) To determine if there was a correlation between the genes that underwent changes in the ILEs 220 and those that are differentially expressed on exposure to Cr41, an RNAseq experiment was carried out. 221 The RNA was extracted and sequenced from cells exposed to Cr41 in minimal media and media 222 supplemented with WYF. The transcriptome analysis was performed on two strains, one of which was 223 sensitive (S288c) to Cr41 exposure and the other resistant (RM11). To decide on which two strains to 224 consider for this study, we also factored in that the two strains had the most variation in the ILE study in 225 terms of number of SNPs and the genes that underwent CNV. S288c, the sensitive strain treated with 226 Cr41, had a much higher number of differentially expressed genes from the RNAseq (Fig 4A). In YM, 227 1100 genes (S5 Table) and 438 genes (S6 Table) in media supplemented with WYF were differentially 228 expressed in S288c when exposed to Cr41 with a final concentration of 0.25% glyphosate. In RM11, only 229 58 (S7 Table) and 53 (S8 Table) genes were differentially expressed in YM and media supplemented with 230 WYF, respectively. RM11 differentially expressed few genes in both conditions i.e., minimal media and 231 media supplemented with WYF ( Fig 4B) and had a higher correlation with the conditions under which 232 the cells were exposed to Cr41. KEGG pathway enrichment analysis of the differentially expressed genes 233 in S288c revealed that a large number of these genes corresponded to pathways involved in the cell 234 cycle, meiosis, DNA replication, and MAPK signaling pathways, especially in WYF (Fig 4C and 4D). Among 235 many others, SED1 is one of the cell wall genes that are differentially regulated along with a few 236 transposable elements in S288c, but no SNPs were found in the ILEs. The downregulated genes mainly 237 associated with biosynthesis of secondary metabolites and amino acids in both minimal media and 238 media supplemented with WYF. 239 S288c cells arrested in G1 on exposure to Cr41 but not pure glyphosate 240 The RNAseq data showed upregulation of the expression of cell cycle regulator genes encoding 241 proteins that are G1 phase regulators such as Rad53, Cdc28, Nrm1 and Swi4 (Bertoli et al. 2013). 242 Increased expression of these genes suggested that cells were arrested in G1 on exposure to Cr41. To 243 ascertain if it was the glyphosate itself or a cumulative effect of all the additives in CFG that caused cell 244 cycle arrest, cells treated with Cr41 and pure glyphosate were subjected to flow cytometry. RM11 and 245 S288c were grown to log phase and then the asynchronous populations were exposed to 0.25% pure 246 glyphosate and Cr41 ( Fig S4). Within 30 minutes of Cr41 exposure, 70% of the population arrested in G1 247 and remained the same over the course of 6 hours while the culture density was maintained 248 consistently ( Fig 5). This was not the case in the untreated and pure-glyphosate treated cells. In these 249 two cases, the populations were evenly distributed across the G1, G2, and S phases. RM11 cells did not 250 show any changes in the cell cycle with respect to the two treatments. It was only the strain that was 251 sensitive to Cr41 exposure, S288c that underwent G1 arrest almost immediately on exposure to Cr41. 252 sed1∆ mutants are highly sensitive to Cr41 exposure 253 The genes commonly affected in the ILE study and differentially expressed in the RNAseq 254 analysis are involved in various pathways, including functions ranging from cell wall proteins, 255 mitochondrial proteins, MAPK related proteins to meiosis (S4 Table). The BY4741 (also an S288c derived 256 strain) knockout collection was used to test the growth phenotype of the different genes as 257 representatives from some of these pathways. Five genes were selected for further characterization, all 258 of which encoded proteins in the cell wall or cell membrane. Det1 plays an integral role in intracellular 259 sterol transport [44]. Pst1 encodes a GPI protein that works with Ecm33 to maintain cell wall integrity 260 [45]. The presence of Emc33 could be the cause for lack of sensitivity in pst1∆ mutants on exposure to 261 Cr41. SED1 is a gene that is expressed as a major stress-induced cell wall glycoprotein [25]. FLO11 262 encodes a GPI-anchored cell wall protein, whose transcription is regulated by the MAPK pathway [46]. 263 The flo11∆ mutants showed growth defects in minimal media. VBA5 is a paralog of VBA3, and it codes 264 for a plasma membrane protein that plays a role in amino acid uptake [47]. Not all the knockout genes 265 changed growth in response to Cr41 exposure as many of the genes may work in unison to have an 266 overall effect on resistance to the treatment. Another possibility is the specific mutation could have a 267 gain of function effect, which cannot be mimicked by using a knockout collection. One of the cell wall 268 genes that had a definitive response was SED1. SED1 was found in both, the ILE data with its copy 269 number doubled, and expression decreased by 1.475 log2 fold in the transcriptome analysis. The sed1 270 mutant was extremely sensitive to Cr41 exposure in both rich and minimal media ( Fig 6A). In the S288c 271 ILE strain that developed resistance to Cr41, SED1 underwent duplication in all media conditions, i.e., 272 minimal media with and without WYF, and rich media. Downregulation of SED1 gene in YM in S288c 273 (sensitive strain) could contribute to the sensitivity of the cells to Cr41. No mutations were found in 274 SED1 from the ILE sequencing but there are several insertions and deletions found in RM11, YJM789,275 and AWRI1631 in reference to S288c (Fig 6B). Sed1 is a stress-induced structural GPI 276 (glycosylphosphatidylinositol) cell wall glycoprotein (Shimoi et al.1998), hence its sensitivity to Cr41 due 277 to the additives could be affecting the integrity of the cell. 278

Calcofluor white (CFW) rescues growth inhibition of S288c caused by Cr41 exposure 279
The analysis of affected genes in the evolved strains and the differentially expressed genes gave 280 rise to a significant number of genes that are associated with the cell wall. To test if the cell wall played 281 an important role in the effectiveness of Cr41, one sensitive and one resistant strain that was treated 282 with Cr41 was exposed to Calcofluor white (CFW). CFW is known for its property of inducing cell wall 283 stress, as it is a chitin antagonist and results in increased deposition of chitin making the cell walls 284 thicker (Liesche, et al. 2015;Roncero and Duran 1985). A quantitative liquid growth assay was carried 285 out using S288c and RM11. The growth of RM11 was inhibited by pure glyphosate and to a greater 286 extent by Cr41. Treating the RM11 samples with CFW did not have a significant effect in YM for Cr41 or 287 PG (p value=0.19 and 0.503). In WYF, it was significant with Cr41 (p-value=0.011) but not PG (p-288 value=0.08). All strains closely related to S288c showed a higher sensitivity to pure glyphosate compared 289 to Cr41 over the first 50 hours ( Fig S5). Treating S288c cells in YM exposed to pure glyphosate with CFW 290 did not have a significant effect (p-value = 0.378) (Fig 6C-E). However, CFW significantly alleviated 291 growth inhibition caused by Cr41 (p-value = 0.024). Cells treated with CFW have increased cell wall 292 volume by about 30% and the wall/ cell ratio also increases significantly (Liesche et al. 2015). The 293 increase in cell wall thickness could be the main contributor to the rescue of cells treated with Cr41. This 294 indicated that the additives in Cr41 along with the glyphosate have a cumulative effect on the cell wall 295 of sensitive strains such as S288c, which was alleviated by CFW treatment. The increase in cell wall 296 thickness on CFW treatment helps the cells withstand the effects of Cr41, also it may reduce the amount 297 of Cr41 entering into the cell. However, treating cells exposed to only pure glyphosate with CFW does 298 not result in alleviation of growth inhibition. This implies that CFW is not rescuing cells from the 299 glyphosate in Cr41 but the alleviating the effects of the additives on the cell wall. 300 Discussion 301 Glyphosate-based herbicides are most commonly used around the world because of the 302 specificity of glyphosate, in acting solely on the aromatic amino acid pathway, that is absent in humans 303 and other eukaryotes. The additives and surfactants present in the commercial formulations are chosen 304 due to their intrinsic inert and non-toxic properties. There are variations from one CFG to the other in 305 terms of these additives which results in different extents of the effectiveness of the herbicide [13]. The 306 supposed non-activeingredients that are added in CFG , enhance the potency of glyphosate and were 307 not inert as seen by changes in the transcriptome. A crucial part is the study of all the changes occurring 308 within the cell to make the herbicide more effective due to the presence of additives and surfactants. 309 The additives in the CFG along with other functions play a key role in the entry of the active ingredient 310 into the cell. This is done by first compromising the first protective physical barrier that they come in 311 contact with, the cell wall. The effectiveness varies based on the structure and composition of the cell 312 wall along with the alleles of the different mannoproteins that the cell contains. When the 313 concentrations and combinations of additives change, this also changes their cumulative effect on the 314 cell. When multiple CFG were tested on different strains of yeast, this resulted in variation in their 315 growth phenotype. The strains chosen for this study have been isolated from different environments as 316 prior exposure was suspected to have an effect of the extent of tolerance to CFG. The agricultural 317 isolates (RM11, AWRI1631) were more resistant to CFGs and glyphosate compared to the laboratory 318 strain and the clinical isolate (YJM789). The agricultural isolates may have come in contact with 319 glyphosate or similar herbicides in the past, which could have led to the development of resistance 320 mechanisms based on past exposure. The lab strain and clinical isolate are not in environments 321 commonly exposed to CFG, making it less likely for them to have been exposed in the past, contributing 322 to their sensitivity. 323 The genetic variation between these strains was used to gain a better understanding of the 324 effects of the CFGs as a whole at the genome and transcriptome level. In case of some stressors, 325 adaptation can occur through a specific route depending on the regions of the genome most effected by 326 the stressor. Response to CFG is a polygenic trait as the different components of the herbicide act together to affect many genes that contribute to various pathways. Hence adaptation can result from 328 small modifications that occur in many different genes as a cumulative effect, as well as one particular induced fragile site expression, in turn resulting in duplication of genes present between two Ty 338 elements. Having the Ty elements in these regions provided the opportunity for these genes to undergo 339 duplication, and those with a beneficial effect were retained as the population progressed. Over the 340 course of serial passaging of these strains, the duplicated regions along with the effects of SNPs in some 341 of the genes may have provided the advantage that the cells needed to overcome sensitivity to Cr41. To 342 assess if there was an overarching contribution by a particular gene that underwent modification or it 343 was the cumulative effect of many of the changes, some of the genes were tested using the knockout 344 collection. This led to the observation that Sed1 is an important contributor to the cell's resistance of 345

CFG. 346
The most drastic effects of glyphosate-based herbicides on the cell apart from the aromatic 347 amino acid pathway was on the cell wall. The cell wall is the mode of entry into the cell and a vital 348 structure in maintaining the osmotic integrity of the cell. Changing the combinations or concentrations 349 of additives in an herbicide alters the mode of entry of the active component into the cell. In our 350 previous study, we have shown that proteins such as Dip5, the aspartic and glutamic acid permease [10] 351 are involved in the import of some glyphosate into the cell. Dip5 was found by comparing S288c and 352 YJM789 genomes with QTL analysis. Therefore only genes that had genetic variation between these 353 strains would be detected and there could be permeases/ transports that also regulate glyphosate that 354 are genetically identical between strains that would not be identified in QTL analysis. The effect of 355 pleiotropic drug response genes is more evident in rich media. PDR5 is the most polymorphic gene in 356 yeast [53]. Pdr5 shares 96% amino acid similarity between S288c and YJM789. Whereas Pdr5 is 99.7% 357 similar between S288c and RM11, which may have led to the masking of its contribution to this study. 358 Sed1 is a GPI-cell wall protein that is highly expressed when the cells are in stationary phase and is a 359 required protein for cells in this phase when they are under stress (Shimoi et al. 1998). SED1 is not very 360 highly conserved across strains and a deletion leading to a similarity of only 87.87% between the S288c 361 and RM11 alleles. Like Pdr5, the role of Sed1 appears to be primarily aimed at dealing with the effects of 362 the additives in Cr41. Like the pdr5 mutant, the sed1 was very sensitive on YPD which has higher levels 363 of Cr41 to induce growth inhibition. The MAPK pathway along with Hog1 genes coregulate genes 364 involved in maintaining the integrity of the cell and cell wall. Several Hog1 MAP kinase genes (FUS3, 365 STE5, BMH2, and AFR1) contained gene duplications in the evolved strains. This pathway is activated 366 under conditions of hyperosmotic stress and is usually accompanied by differential expression of various 367 GPI-cell wall proteins. The CFG as a whole could be inducing hyperosmotic stress resulting in the 368 activation of the Hog1 MAP kinase pathway which could also contribute to arresting in G1 phase 369 (Escoté, et al. 2004). 370 In this study, we highlight the importance of studying different CFG to gain a better 371 understanding of all the pathways affected on exposure to Cr41 and to recognize the various non-target 372 adaptation mechanisms. Humans have proteins that have structure and functional similarity to those 373 found in yeast. As this herbicide is used so extensively on produce that is used for human consumption, 374 it is important that we understand the effects of the chemicals we are being exposed to. Recent studies have shown the presence of glyphosate and its metabolites in urine samples in humans [55,56] through 376 the levels of glyphosate humans are exposed to through food consumption is much lower than those 377 used in this study to treat yeast cells. Without analyzing the effects at the molecular level, it is difficult to 378 predict if there will be any long-term effects. Not seeing any effects immediately is not necessarily an 379 indication that there would not be any effects after long-term exposure. Hence, it is crucial to study the 380 effects of different CFG to be able to regulate the use of additives and surfactants. 381

Variations in growth phenotype 383
A semi-quantitative growth assay was carried out on exposure of AWRI1631, RM11, YJM789, 384 and S288c, to different formulations of glyphosate-based herbicides. The media used for the study was 385 nutrient-rich (YPD) and nutrient minimal (YM) solid media. The rich media is comprised of yeast extract, 386 peptone, and dextrose; as for the minimal media, it consists of yeast-nitrogen base and 2% dextrose. 387 The minimal media was supplemented with aromatic amino acids, namely 20 g/ml tryptophan (W), 30 388 g/ml tyrosine (Y) and 50 g/ml phenylalanine (F). It was also supplemented with 100 g/ml aspartic (D) 389 acid for certain conditions. This assay was performed as previously described in [10], as follows. These 390 were exposed to the glyphosate-based herbicides and the concentration of glyphosate was standardized 391 to 1.0% in rich and 0.15% in minimal media respectively. The different formulations used were, Compare 392 and Save (CAS), WeedPro (WP), Super Cncentrated (SC), Credit 41 (Cr41) and pure glyphosate as a 393 control. 394 Quantitative growth analysis was carried out using a TECAN M200, automatic plate reader [57]. 395 The growth of cells in liquid media was measured every 1 hour at 600nm under shaking conditions. 396 Along with Credit41 and pure glyphosate, the cells were treated with 5mM calcofluor white, to measure 397 the progression of their growth over 50 hours.

Gene expression analysis 399
RNA was extracted from S288c and RM11 cells grown in minimal media with and without 400 supplementing WYF. The cells were treated with 0.25% Credit41 for 90 minutes with 5 replicates of each 401 condition. The samples were then washed, and the total RNA was extracted using hot phenol method 402 [58]. Paired-end cDNA libraries were built using the RNA extracted using Epicenre's ScriptSeq Yeast kit. 403 The sequencing was performed using 76bp paired-end reads on the Illumina HiSeq platform resulting in 404 5-8.5 million read pairs. 405 Salmon v0.13.1 was used to estimate the transcript level abundance from the RNA seq-read 406 data [59]. To do so, indexing of the S288c reference transcriptome was first carried out. This index was 407 used by Salmon against each sample to generate quant.sf files containing the length, abundance in 408 terms of Transcripts Per Million (TPM) and the estimated number of reads for each 409 transcript(https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE135473 secure token: 410 arkxmokwdlufjir). The differential expression of genes between the two strains and each condition was 411 calculated using DESeq2 v1.8.1. DESeq2 was also used to generate a sample to sample distance map (Fig  412   S3). The program clusterProfiler v3.10.0 was used to carry out KEGG Pathway Enrichment Analysis, to 413 identify the pathways involved, based on the clustering of genes. 414

Flow cytometry 415
To measure the cell cycle arrest on Credit41 and pure glyphosate exposure, S288c and RM11 416 cells were subjected to flow cytometry after exposure. The cultures were started and allowed to grow to 417 mid-log phase from an overnight culture. Once the cells reached mid-log phase, they were treated with 418 0.25% Credit41 and pure glyphosate, in triplicate with and without supplementing WYF in minimal 419 media. Cells were collected at multiple time points (0 mins, 30 mins, 90 mins, 3 hrs, and 6 hrs) over the 420 next 6 hours. The cells were maintained in log-phase throughout the collections, by replenishing with fresh media as needed. The cells were harvested and fixed in 70% ethanol for 2 days [60]. The cells were 422 then washed and treated with RNAse solution for 8-12 hours at 37⁰C. The RNAse solution comprised of a 423 mixture of 2mg/ml RNAse A, 50mM Tris pH 8.0, and 15mM sodium chloride that had been boiled for 15 424 minutes and cooled to room temperature. The cells were then treated with protease solution (5 mg/ml 425 pepsin and 4.5 l/ml concentrated HCl) for 20 minutes at 37⁰C. The cells were then stored in 50 mM Tris 426 pH 7.5 at 4⁰C, until the day of the analysis. Right before flow cytometry analysis, the cells were 427 sonicated at low levels to separate cells. 50ul of the sonicated mixture was transferred into 1ml of 1uM 428 Sytox Green in 50ml Tris pH 7.5. The cells were analyzed on an LSRFortessa, using the FITC channel. The 429 results were analyzed, and the percentage of cells in each stage of the cell cycle was estimated using FCS 430 Express 6.0. The analysis was carried out using the multi-cycle DNA histogram and '+S order=1 model' 431 was selected based on the lowest Chi-square value. 432

Whole-genome sequencing of In-Lab evolutions (ILEs) 433
In-lab evolutions were carried out by exposing cells to Credit41 over a long period of time until a 434 resistant population was isolated. Two sensitive (S288c and YJM789), and two resistant (RM11 and 435 AWRI1631) strains were used for this study. The cells were evolved in minimal media with 0.25% Cr41, 436 with and without supplementing WYF. The cells evolved in rich media were treated with 1.0% Cr41. A 437 control group was evolved in media in the absence of Credit41 to account for mutations that occur due 438 to the procedure, rather than the Credit41 treatment. A single colony was used to start a saturated 439 overnight culture, that was then used as a starter culture to inoculate the different conditions in 440 triplicate. Each passage was grown 2-3 days in media till saturation. After which 1% of the culture was 441 transferred into fresh media. The cells were subjected to 6 passages and tested for resistant populations 442 by plating 10-fold dilutions on solid media. The resistant populations were streaked on plates to isolate 443 single colonies. The resistant colonies were selected based on size and shape. They were then passaged 444 for 2 passages in media lacking Credit41, to ensure the resistance did not rely solely on epigenetic mechanisms. The genomic DNA was then extracted using the 96 well genomic DNA extraction kit, and 446 the phenol-chloroform extraction technique was also used [61]. 447 The whole-genome sequencing data for all 4 strains were aligned to S288c reference sequence 448 (release R64-2-1) by creating an index and using gatk-4.1.1.0 to carry out the alignment. Samtools was 449 used to convert the SAM files to BAM files, from which duplicates were removed. Using gatk 450 HaplotypeCaller, variant calling was carried out to identify all the SNPs and generate vcf files. Using the 451 generated vcf files, PCA variants were identified using pcadapt as described in ( The authors declare that they have no competing financial interests. 462  The sequences for each TM of Dip5 S288c are as follows . 508     Cell cycle distribution of S288c shows arrest in case of treatment with Cr41 but no arrest when treated with pure glyphosate B. Cell cycle distribution of RM11 shows no effect on treatment with Cr41.