Efficient Disruption and Replacement of an Effector Gene in the Oomycete Phytophthora sojae using CRISPR/Cas9

Keywords: genome-editing, effector, CRISPR/Cas9, Oomycetes, Phytophthora

21

Phytophthora sojae is a pathogenic oomycete that infects soybean seedlings as 22 well as stems and roots of established plants, costing growers $1-2 billion per 23 year. Due to its economic importance, P. sojae has become a model for the 24 study of oomycete genetics, physiology and pathology. Despite the availability of 25 several genome sequences, the lack of efficient techniques for targeted 26 mutagenesis and gene replacement have long hampered genetic studies of 27 pathogenicity in Phytophthora species. Here, we describe a CRISPR/Cas9 28 system enabling rapid and efficient genome editing in P. sojae. Using the RXLR 29 effector gene Avr4/6 as target, we observed that in the absence of a homologous 30 template, the repair of Cas9-induced double-strand breaks (DSBs) in P. sojae 31 was mediated by non-homologous end joining (NHEJ), primarily resulting in short 32 indels. Most mutants were homozygous, presumably due to gene conversion 33 triggered by Cas9-mediated cleavage of non-mutant alleles. When donor DNA 34 was present, homology directed repair (HDR) was observed, which resulted in 35 the replacement of the target gene with the donor DNA. By testing the specific 36 virulence of several NHEJ mutants and HDR -mediated gene replacements on 37 soybeans, we have validated the contribution of Avr4/6 to recognition by soybean 38 R gene loci, Rps4 and Rps6, but also uncovered additional contributions to 39 resistance by these two loci. Our results establish a powerful tool for studying 40 functional genomics in Phytophthora, which provides new avenues for better 41 control of this pathogen. 
42 43 . 

INTRODUCTION 44

Phytophthora sojae is a destructive pathogen which causes "damping off" of 45 soybean seedlings as well as stem and root rot of established plants (Tyler, gene replacement is an efficient and useful strategy for testing the function of 110 specific genes in P. sojae such as Avr4/6, which should be useful for all (Fang & Tyler, 2015). version has been widely used in a variety of organisms (Cong et al., 2013, Zhang 125 et al., 2014, Peng et al., 2015), and matches P. sojae codon usage relatively well, 126 with a codon adaptation index calculated at 0.776 127 (http://genomes.urv.es/CAIcal/E-CAI/). To test if this protein could be efficiently 128 expressed in P. sojae, we fused GFP to the C-terminus of hSpCas9. Furthermore, 129 to direct hSpCas9-GFP into the P. sojae nucleus, we used a strong synthetic 130 NLS derived from a P. sojae bZIP transcription factor (Fang & Tyler, 2015), which 131 guidelines and web tool, sgRNA Designer (Doench et al., 2014). Then sgRNA 177 candidates rated highly by the web tool were further filtered by off-target analysis. 

178

Finally, two sgRNAs (sgRNA version A and B) were selected in which the 179 respective Cas9 cleavage sites overlapped unique restriction enzymes sites (Bst 180 UI and Tsp 45I respectively) that could be used to rapidly screen for mutations 181 (Fig. 2A). 

182

To examine the activity of the designed sgRNAs, Avr4/6-A and Avr4/6-B, 183 we carried out a sgRNA-mediated in vitro cleavage assay of target DNA. In these 184 assays, the sgRNAs were synthesized using T7 RNA polymerase, and SpCas9 185 protein was purchased from New England Biolabs. Cas9/sgRNA-B completely 186 cleaved the target DNA, whereas Cas9/sgRNA-A showed no activity in vitro (Fig. 187 2B). 

188

In parallel with the in vitro assays, we used transient expression in P. and 10 pure mutant lines respectively (summarized in Table 1). All 27 pure lines 236 showed a homogeneous sequence profile (Fig. S2), indicating that all of them 237 were already homozygous, and carried the same mutation in both alleles. All 10 238 of the T32 lines were homozygous for the same mutation. The lines derived from 239 T18 included 9 lines homozygous for one Avr4/6 mutation and one homozygous 240 for a different mutation. The lines derived from T11 included two homozygous for 241 one mutation (mut1) and five homozygous for a second mutation (mut2). The (Figure 3B and Table 1). 

250

We also tested the inherent stability of the mutants by sub-culturing each 251 of the single zoospore lines for at least three generations on media without G418 

287

The results suggested that HDR had occurred, but the frequency was variable 288 depending on the length of the flanking sequences in the donor DNA plasmids. 

289

The transformant population generated with the 1 kb flanking sequences showed 

294

Next, to characterize HDR events in detail, we generated single P. sojae 295 transformants derived from the 1 kb arm donor. After screening 68 individual 296 G418 resistant transformants for GFP production, we identified 18 transformants 297 bearing the two CRISPR components. Then, using nested primers specific for 298 HDR events (Fig. 4B), we found evidence for gene replacement events in 9 of 299 the transformants (Fig. 4D). Sanger sequencing across the junctions of the 300 flanking sequences and NPT II in the nested PCR products was also consistent 301 with replacement of the Avr4/6 ORF with NPT II gene (data not shown). Three 302 HDR mutants, namely HDR -T12, -T29 and -T39, that readily produced 303 zoospores, were selected for functional tests. After zoospore isolation, the Avr4/6 304 region of each single zoospore line was examined by PCR amplification using 305 primers flanking the two homologous arms and cleavage of the amplicon by the 306 restriction enzyme Nco I. We found that all of the 11 single zoospore lines 307 obtained from HDR-T12, and all 8 obtained from HDR-T39 were homozygotes 308 (Fig. 4E); this was further validated by Sanger sequencing (Fig. 4F). In contrast, 309 all 20 of the single zoospore lines of T29 appeared to be heterozygotes that 310 contained a HDR event in just one of the two Avr4/6 alleles. This was further 311 verified by PCR amplification using primers outside of homology arms and in 312 NPT II gene. More detailed analysis of three of the HDR-T29 lines revealed that 313 the non-HDR alleles possessed the same mutation, an adenine deletion, in every 314 case (Fig. S3), presumably caused by NHEJ. 2). We observed that the frameshifted mutants T32-3 326 and T11-2-5 both showed increased killing of Rps4-or Rps6-containing soybean 327 seedlings in both Williams and Harosoy backgrounds (Table2). The increased 328 killing of both Rps4 and Rps6 plants by T32-3 was statistically significant (p < 329 0.01), while the increased killing by T11-2-5 of Rps4 but not Rps6 plants was 330 significant (p<0.05). On the other hand, the increased killing in every case was 331 still significantly (p < 0.05) less than the killing of rps plants lacking Rps4 or Rps6 332 (Table 2). Thus T32-3 was scored as intermediate on Rps4 and Rps6 plants, 2). 

340

The two homozygous HDR mutants (T12-1 and T39-1), both showed 341 significantly (p < 0.01) more killing of Rps4-or Rps6-containing soybean 342 seedlings in both Williams and Harosoy backgrounds (Table 2 and Fig. 5), but the 343 killing was significantly (p < 0.05) less than on rps plants. Thus, both mutants and even larger numbers are underway (Jiang et al., 2013, Jiang & Tyler, 2012, 350 Kamoun et al., 2014). These genomes contain 15,000 to 25,000 genes, of which 351 approximately half in most species show the rapid sequence divergence 352 expected of infection-related genes (Tyler et al., 2006, Haas et al., 2009, Baxter 353 et al., 2010, Jiang & Tyler, 2012, Jiang et al., 2013). Many of the diverging genes 354 encode large families of effectors, toxins, hydrolytic enzymes, hydrolytic enzyme 355 inhibitors, and ABC transporters, as well as numerous proteins of unknown 356 function (Tyler et al., 2006, Haas et al., 2009, Jiang & Tyler, 2012). To date 357 however, the tools available for assessing the functions of these genes have 358 been limited to RNAi-mediated gene silencing and to expression of ectopic 359 transgenes. RNAi-mediated gene silencing has proven useful (Judelson et al., 360 1993b, Ah-Fong et al., 2008), especially with the introduction of transient 361 silencing (Whisson et al., 2005, Wang et al., 2011). However the level of 362 silencing is unpredictable and often unsatisfactory, and some genes have nuclear proteins to the point that a small highly efficient NLS tag could be 375 constructed (to be published elsewhere). A second hurdle was that no RNA 376 polymerase III promoters had been characterized in oomycetes, and that the U6 377 genes we attempted to use did not appear to be well transcribed under our 378 transformation conditions. This we solved by using a strong RNA polymerase II sojae, presumably due to their highly repetitive nature. The humanized SpCas9 384 protein was readily expressed, but unexpectedly the hSpCas9-GFP fusion 385 protein we used to validate expression was not effective in generating mutations 386 in P. sojae. despite the fact that Cas9-GFP fusions have been used in other 387 organisms such as Toxoplasma gondii (Shen et al., 2014). We were successful 388 only after we used the non-fused hSpCas9. A fourth hurdle was identifying 389 effective sgRNAs. One of the two sgRNAs predicted using the sgRNA Designer 390 web tool proved ineffective both in vitro and in vivo. This sgRNA, sgRNA-A, was 391 found to be strongly self-complementary, potentially preventing hybridization with 392 the target DNA. This observation, which is in agreement with Peng et al., (2015), 393 underlines that sgRNAs with strong secondary structure predictions should be be 394 eliminated. We also observed a discrepancy between in vitro and in vivo assays 395 of Cas9/sgRNA activity, namely that the sgRNA lacking ribozymes was functional 396 in the in vitro assay, whereas only the sgRNA flanked by ribozymes was effective 397 in vivo. There clearly is room for further optimization of sgRNA design. 

398

Using a single sgRNA, we were able to generate small indels at the site of 399 the Cas9 cleavage. While these were useful, future attempts to disrupt genes 400 with large deletions will likely require a pair of sgRNAs, or else the use of and Rps6 loci, consistent with the findings of Dou et al., (2010). However, the 448 mutants did not kill the Rps4-and Rps6-containing plants as completely as they 449 killed rps plants lacking Rps4 and Rps6. The Rps4 and Rps6-containing isolines 450 were produced by introgression, not by transformation with individual R genes 451 (Sandhu et al., 2004). Since the Rps4 and Rps6 loci, which are allelic, both 452 contain many NB-LRR genes (Sandhu et al., 2004), we speculate that additional (Dou et al., 2008b). The -1 frameshift in T11-2-5 also eliminated the dEER motif, 473 but in its place created a highly positively charged sequence (RKKRNARSR). 

474

Thus we speculate that this positively charged sequence may act as a surrogate 475 cell entry sequence (Snyder & Dowdy, 2004, Dou et al., 2008b, Kale et al., 2010, 476 Milletti, 2012) delivering the N-terminal fragment for recognition by Rps4. 

477

Of the three in-frame deletions, the two single amino acid deletion mutants S1 in the supplemental 511 material. A map and sequence file for the plasmid backbones used for 512 expressing Cas9 and the sgRNAs targeting the Avr4/6 locus can be found in Fig. 

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S4 in the supplemental material. To express hSpCas9 and sgRNAs effectively in To test the activity of the designed sgRNAs, an in vitro cleavage assay 554 was carried out as previously described (Gao & Zhao, 2014). Briefly, sgRNA was 555 in vitro transcribed through run-off reactions with T7 RNA polymerase using the 556 MEGAshortscript TM T7 kit (Ambion) according to the manufacturer's manual. 

557

Templates for sgRNA synthesis were generated by PCR amplification from the 558 sgRNA expression plasmid pYF2.2-sgRNAs (Table S1). The target DNA was 559 amplified from pCR2.1-Avr4/6 using primer M13F and M13R (Supplemental 560 material). SpCas9 nuclease was purchased from New England Biolabs, and the 561 cleavage assay was performed according to the product manual. Fisher's exact test (Sokal & Rohlf, 1995) S1). (F) Sanger sequencing traces of junction regions confirming that the Avr4/6 ORF was cleanly replaced by the NPT II ORF in a representative zoospore purified clone (HDR-T12-1). Start and stop codons are in red bold. 



  CRISPR/Cas9 system for P. sojae 115 To establish a CRISPR/Cas9 system for P. sojae, several functionalities had to 116 be established, including efficient expression of Cas9 in P. sojae (our attempts to 117 express TALENs in P. sojae were never successful due to strong gene silencing), 118 efficient expression of guide RNAs (no oomycete RNA polymerase III promoters 119 have been characterized), and targeting of the Cas9 enzyme to the P. sojae 120 nucleus (commonly used mammalian nuclear localization signals function poorly 121 in P. sojae) 


122

  For use in P. sojae we selected the Streptococcus pyogenes Cas9 123 encoded by a gene with human-optimized codons (hSpCas9), because this Cas9 




  124 




  189 sojae protoplasts to test whether the hSpCas9 and sgRNAs produced in vivo 190 could modify the endogenous Avr4/6 gene. The two Avr4/6-specific sgRNAs 191 were assembled into the P. sojae expression plasmid under the control of the 192 RPL41 promoter, either flanked with ribozymes (Avr4/6-sgRNA-A R , -B R ) or 193 without ribozymes (Avr4/6-sgRNA-A, -B). The sgRNA constructs were co-194 transformed with the hSpCas9 expression plasmid into P. sojae strain P6497 by 195 PEG-mediated protoplast transformation. Transformants were enriched by G418 196 selection 12 h after transformation when hyphae had regenerated. After 24 h, 197 DNA was extracted from the culture containing the pooled transformants. Avr4/6 198 sequences were amplified from the pool of genomic DNAs and screened for 199 mutants resistant to the relevant restriction enzymes (Bst UI for A and Tsp 45I for 200 B). We found that the Avr4/6 amplicons from the two sgRNA-A transformations 201 (constructs with and without ribozymes) were still fully subject to restriction 202 enzyme cleavage, indicating failure of the Cas9-mediated mutagenesis (Fig. 2C). 203 In contrast, the transformation utilizing the sgRNA version B flanked by 204 ribozymes yielded restriction enzyme resistant amplicons (Fig. 2C). However the 205 transformation utilizing the sgRNA version B without ribozymes did not yield 206 restriction enzyme resistant amplicons (Fig 2C). To validate the enzyme 207 cleavage results, we further sequenced the nested PCR products amplified from 208 the enzyme digestion products from the sgRNA-B R transformants. The sequence 209 chromatograms showed pure sequences proximal to the target site, and mixed 210 sequences distal to the target site (data not shown), suggesting the presence of 211 indel mutations at the target site. These observations indicated that in P. sojae, 212 RNA polymerase II can be successfully used for generating sgRNA, provided 213 that ribozymes are employed to remove the surrounding sequences from the 214 transcripts. In addition, based on the intensity of the restriction enzyme-resistant 215 band on the agarose gel relative to the intensity of the fragments resulting from 216 Tsp 45I cleavage, around 25-30% of the amplicons appeared to carry a mutation 217 in the restriction site (Fig. 2C). The failure of the sgRNA version A may result 218 from strong self-complementarity that we subsequently discovered in its 219 sequence using RNA structure 220 (http://rna.urmc.rochester.edu/RNAstructureWeb/Servers/Predict1/Predict1.html), 221 which could block its binding to target DNA. 222 To characterize CRISPR/Cas9-generated Avr4/6 mutations in detail, the 223 transformation with the ribozyme-containing Avr4/6-sgRNA-B R construct was 224 repeated. Individual G418-resistant transformants were isolated and screened for 225 the presence of GFP indicating the presence of the sgRNA construct. Of 50 226 primary transformants screened, 6 exhibited green fluorescence. Of these, 4 227 yielded Avr4/6 amplicons that were partially or fully resistant to Tsp 45I digestion 228 (Fig. 3A), indicating the presence of Avr4/6 mutations. Since P. sojae protoplasts 229 and hyphae are multinucleate, and hence might be expected to harbor nuclei 230 with a diversity of Avr4/6 mutations, we isolated zoospores (which are 231 mononucleate) from three of the primary transformants (T11, T18 and T32; the 232 fourth, T30, did not produce zoospores). Ten single zoospore lines were isolated 233 from each transformant, and the Avr4/6 amplicons were screened by restriction 234 enzyme digestion and by Sanger sequencing. T11, T18 and T32 yielded 7, 10 235 


242

  three remaining lines were heterozygous and biallelic, containing a third mutation 243 paired with mut1. (Fig. 3B and Fig. S2). No lines retained any wild type alleles, 244 either homozygous or heterozygous. 245 Each of the mutations consisted of a short indel, located specifically at the 246 Cas9 cleavage site, i.e. between the 17 th and 18 th nucleotide of the sgRNA target 247 site. Deletions of one, three and six bp were observed, one contained a one bp 248 insertion, and one combined a three bp deletion with a one bp replacement 249 




  252 selection. All of the mutated sites examined remained the same as the first 253 generation, based on the restriction enzyme cleavage assay and sequencing of 254 the Avr4/6 amplicon. In addition, we noticed that a few of the sub-cultured 255 mutants had lost the ability to grow under G418 selection, showing that mutations 256 were stable regardless of presumptive continued presence of the sgRNA:Cas9 257 construct. Interestingly, one transformant T47 which did not show obvious 258 mutations in the first generation acquired the same single adenine insertion as 259 T32-3 after sub-culturing of the unpurified transformant for one generation (Fig. 260 S3), presumably because the sgRNA:Cas9 constructs were integrated into the 261 genome and continued to actively cleave the target in each generation. 262 Collectively, these results indicate that our CRISPR/Cas9 system can efficiently 263 and specifically trigger the introduction of NHEJ mutations, typically short indels, 264 into the P. sojae genome. mediated repair of sgRNA-guided Cas9 cuts has proven an efficient 269 way to facilitate gene replacements via homology-directed repair (HDR) (Cong et 270 al., 2013, Mali et al., 2013). To determine if sgRNA:Cas9 mediated DSB could 271 stimulate homologous recombination in P. sojae, we co-transformed the CRISPR 272 constructs that were successfully used for mutation of Avr4/6, along with uncut 273 donor DNA plasmids that contained the entire NPT II ORF flanked by different 274 lengths of the sequences surrounding the Avr4/6 gene. An equimolar ratio of the 275 three plasmids was used (Fig. 4A). Since preliminary experiments had shown 276 that expression of the NPT II gene from the Avr4/6 promoter was insufficient for 277 G418 selection, the NPT II gene was included in the Cas9 plasmid for selection 278 of transformants. We used homology arms consisting of three different lengths of 279 5′ and 3′ flanking sequences, namely, 250 bp, 500 bp, and 1 kb, to assess which 280 would enable the highest recombination efficiency (Fig. 4B). The NPT II gene in 281 the Cas9 expression plasmid served as a negative control, because it lacked any 282 Avr4/6 flanking sequences. 283 Following co-transformation and G418 enrichment, the bulk transformants 284 were subjected to genomic DNA extraction and PCR analysis. PCR 285 amplifications using primers located outside the Avr4/6 homology arms and 286 within the NPT II gene were used to detect homologous recombination events. 


290

  the highest frequency of gene replacement. The population generated with the 291 500 bp flanking sequences showed a much lower frequency compared to the 1 292 kb population. The population from the 250 bp flanking sequences showed very 293 low recombination frequencies (Fig. 4C). 




  test the effects of the CRISPR/Cas9-induced Avr4/6 mutations 319 on P. sojae recognition by plants containing the Rps4 and Rps6 loci, the five 320 homozygous NHEJ mutants and two homozygous HDR mutants were inoculated 321 onto hypocotyls of soybean isolines containing Rps4 (L85-2352) or Rps6 (L89-322 1581) in a Williams background, as well as isolines containing Rps4 (HARO4272) 323 or Rps6 (HARO6272) in a Harosoy background. 4 days after inoculation (dpi), 324 the specific virulence of the different mutants was scored and analyzed by 325 Fisher's exact test (Table 




  scored as avirulent and intermediate respectively. The other 334 NHEJ mutants, containing in-frame deletions, also showed increased killing of 335 Rps4-and Rps6-containing plants, but significantly (p < 0.03) less killing than 336 observed with rps plants. The Avr4/6 mutant having a two amino acid deletion 337 (T11-1) showed an intermediate phenotype that was close to fully virulent on 338 Rps4 plants while the two mutants a single amino acid deletion (18-1 and 18-2) 339 showed intermediate to avirulent phenotypes (Fig. 5; Table 


344

  were scored as intermediate to virulent on Rps4 and Rps6. 




  oomycete genomes have now been sequenced, 




  349 


363

  required a major effort to find a few silenced lines. Expression of ectopic 364 transgenes has also proven useful. However major limitations have been the 365 unpredictability of the expression level and the related issue of potential over-366 expression artifacts. The inability to eliminate the contribution of the endogenous 367 gene has also limited the usefulness of ectopic transgenes. 368 Here we have adapted the CRIPSR/Cas9 sequence-specific nuclease 369 technology for use in Phytophthora sojae. This involved overcoming several 370 technical hurdles. One of these was the fact that commonly used mammalian 371 nuclear localization sequences (NLS) do not function in P. sojae and that P. 




  372sojae nuclear proteins use dispersed NLS signals (to be published elsewhere). A 373 very large effort was required to delineate the NLS signals from several P. sojae 374 




  379 promoter (RPL41) in conjunction with self-cleaving ribozymes to release the 380 sgRNA from the RNA polymerase II transcript. A third hurdle was effective 381 expression of the nuclease protein. Out initial attempts to use TALENs were 382 blocked because the TALEN constructs were silenced extremely strongly in P. 




  383 




  401homology-directed repair to introduce specific mutations or to replace the gene 402 entirely, as we did in replacing Avr4/6 with NPT II. Of interest is the fact that from 403 two of our transformant lines we were able to recover two or three different 404 mutations. Several of these mutations were segregated into different single 405 zoospore lines, indicating that the mutations had occurred in different nuclei of 406 the regenerating protoplasts. One set of lines were biallelic indicating that two 407 different mutations had occurred in the same nucleus. The fact that relatively few 408 distinct mutations were recovered in each case might be explained if the 409 mutations occurred very early during protoplast regeneration when there were 410 limited numbers of nuclei present. Alternatively, the recovered mutations may 411 have been the ones that occurred the earliest and therefore the nuclei carrying 412 them came to dominate the heterokaryotic transformant. Also of interest is the 413 observation that most of the mutations were recovered as homozygotes in this 414 diploid organism. We speculate that once a mutation occurred in one allele that 415 made it resistant to further Cas9 cleavage, cleavage of the remaining unmodified 416 allele by Cas9 in most cases led to gene conversion of that allele to match the 417 first allele. 418 It was reported that in some cases over-expression of Cas9 negatively 419 impacts the growth of the organism, e.g. the parasite Trypanosoma cruzi (Peng 420 et al., 2015). However, during sub-culture and infection assays of all our Cas9-421 expressing mutants, we did not observe negative effects on P. sojae growth nor 422 on overall virulence. 423 CRISPR-mediated gene disruptions and gene replacements will find 424 numerous applications in P. sojae and other oomycetes. Gene disruptions will 425 enable genes to be knocked out much more surely than by silencing. With careful 426 design of sgRNAs, single members of closely related gene families could be 427 eliminated, or else tags for transcriptional measurements could be introduced, to 428 determine their individual contributions. Alternatively, entire gene clusters could 429 be eliminated with a pair of sgRNAs. Gene replacements will enable mutations of 430 all kinds, including promoter mutations and epitope and fluorescent protein tags, 431 to be introduced into the endogenous gene where expression and phenotypes 432 will not be confounded by position effects, over-expression artifacts, and 433 contributions from the unmodified native gene. Targeted gene insertions are also 434 expected to solve a longstanding problem in some oomycetes such as P. sojae 435 where ectopic transgenes are invariably poorly expressed, even from the 436 strongest promoter. Gene disruptions will also be useful for creating a much 437 wider choice of selectable markers for transformation, through the creation of 438 auxotrophic mutants. Disruption of the Ku70 and/or Ku80 genes required for non-439 homologous end joining may also result in higher efficiencies of homologous 440 gene replacement (Arentshorst et al., 2012). Gene deletions could also be used 441 to remove integrated transgenes so that selectable markers such as NPT II can 442 be recycled for repeated transformation experiments. 443 Some of the advantages of these CRISPR-enabled approaches are 444 illustrated by insights gained from our manipulation of the Avr4/6 gene. The 445 elimination of the gene by replacement with NPT II confirms that Avr4/6 makes a 446 major contribution to recognition of the pathogen by plants containing the Rps4 




  447 




  453NB-LRR genes at these loci (or even the Rps4 and Rps6 genes themselves) can 454 recognize additional effectors produced by the P. sojae strain used in these 455 studies (P6497).Dou et al (2010) also observed that P. sojae strains silenced for 456 Avr4/6 did not completely kill Rps4 and Rps6-containing lines, but those 457 observations were ascribed to incomplete silencing of Avr4/6. With the availability 458 of complete Avr4/6 deletion mutants, we can now more confidently conclude that 459 the Rps4 and Rps6 loci make additional contributions to resistance other than 460 through recognition of Avr4/6. 461 The two frameshift mutants killed Rps6 plants nearly as well as the HDR 462 mutants (62% combined killing versus 74%) indicating that these mutants were463 no longer recognized by Rps6-containing plants. T32-3 killed Rps4 plants better 464 than the two HDR mutants (83% versus 74% killing), but T11-2-5 was clearly 465 avirulent on the Rps4 plants (40% versus 74%; WT = 31%). The explanation for 466 this difference may lie in the observation by Dou et al (2010) that the N-terminal 467 domain of Avr4/6, up to and including the dEER motif, is sufficient for recognition 468 by Rps4 plants, whereas recognition by Rps6 plants requires the C-terminus. 469 Since the site of the CRISPR-induced NHEJ mutations is immediately upstream 470 of the dEER motif, the +1 frameshift in T32-3 retained the N-terminal domain but 471 eliminated the dEER motif, presumably abolishing effector entry into the plant 472 




  478 scored as intermediate to avirulent on both Rps4 and Rps6 plants, suggesting 479 that recognition was only slightly impaired. The two amino acid deletion mutant 480 (T11-1) however was actually slightly more virulent on Rps4 plants than the HDR 481 mutants (86% versus 74% killing) while slightly less virulent on the Rps6 plants 482 (64%). We speculate that the two residue deletion may have disrupted the 483 structure of Avr4/6 with possibly a stronger effect on the immediate region of the 484 N-terminus required for recognition by Rps4 plants. 485 In summary, the adaptation of CRISPR/Cas9-mediated gene targeting to 486 oomycetes is expected to rapidly advance the functional analysis of these 487 extremely destructive and important plant and animal pathogens. 




  P. sojae isolate P6497 (Race 2) used in this study was routinely 493 grown and maintained in cleared V8 medium at 25 °C in the dark. Zoospores 494 were induced and isolated from ~ 1-week old cultures grown on clarified V8 agar, 495 as previously described(Judelson et al., 1993a). Single P. sojae transformants 496 were incubated in 12-well plates containing V8 media supplemented with 50 497 µg/mL G418 (Geneticin) for 2~3 days before genomic DNA was isolated by were selected according to the web-tool sgRNA Designer 501 (http://www.broadinstitute.org/rnai/public/analysis-tools/sgrna-design; (Doench et 502 al., 2014). Potential off-target sites were checked using the FungiDB 503 (www.fungidb.org) alignment search tool (BLASTN) against the P. sojae genome 504 and visual inspection of the results. Sequences that perfectly matched the final 505 12 nt of the target sequence and NGG PAM sequence were discarded (Cong et 506 al., 2013). Ribozymes were designed according to Gao, et al. (2014). The first six 507 nucleotides of the Hammerhead (HH) ribozyme were designed to be the reverse 508 complement of the first six nucleotides of the sgRNA target sequences. 509 Plasmid construction 510 All the primers used in this study are listed in the Table 




  514P. sojae, we first created a new Phytophthora expression plasmid backbone 515 pYF2 by combining elements from pHamT34(Judelson et al., 1991), pUN(Dou 516 et al., 2008a) and pGFPN(Ah-Fong & Judelson, 2011) as follows. (i) The HSP70 517 terminator was PCR amplified from pGFPN by using primers BlHSP70T_AgeI_F 518 and BlHSP70T_NsiI_EcoRI_R which added an Eco RI site, and was then 519 inserted into pUN using Kpn I and Nsi I sites, placing the NPT II gene under the 520 control of the RPL41 promoter and HSP70 terminator. (ii) The entire NPT II 521 cassette from (i) was then extracted by digestion with Eco RI and inserted into 522 the Eco RI site of pHAMT34, resulting in pYF1. (iii) A synthetic multiple cloning 523 site fragment containing the restriction enzyme sites Xma I-Cla I-Stu I-Sac II-Spe 524 I-Bsi WI-Afl II-Kpn I was introduced between the Xma I and Kpn I sites of pYF1 525 by oligo annealing, creating pYF2. The plasmid pYF2-GFP was generated by 526 adding an eGFP fragment amplified from pGFPN using primers GFP_AflII_F and 527 GFP_ApaI_R into the restriction enzyme sites Afl II and Apa I of pYF2. The 528 PsNLS, reported in Fang & Tyler (2015), was tested using the plasmid pYF2-529 PsNLS-2XGFP, which was constructed by two steps. (i) An extra GFP was 530 amplified from the same plasmid pGFPN using primers GFP_SpeI_F and 531 GFP_Afl II_R and inserted into the restriction sites Spe I and Afl II of the plasmid 532 pYF2-GFP, generating pYF2-2XGFP. (ii) The PsNLS was inserted by annealing 533 of two oligonucleotides encoding the NLS 534 (MHKRKREDDTKVRRRMHKRKREDDTKVRRRMHKRKREDDTKVRRR). 535 To generate hSpCas9-expression plasmids pYF2-PsNLS-hSpCas9-GFP 536 and pYF2-PsNLS-hSpCas9, the coding fragment of hSpCas9 nuclease from the 537 plasmid pSpCas9 (BB)-2A-GFP (PX458, Addgene plasmid # 48138) were 538 subcloned into the Spe I and Afl II sites or Spe I and Apa I sites (there is an Apa I 539 site in hSpCas9, so the two digested fragments were cloned sequentially), 540 respectively, of plasmid pYF2-PsNLS-2XGFP containing the P. sojae NLS. 541 To generate plasmids pYF2.2-GFP-sgRNA and pYF2.2-GFP-Ribo-sgRNA, 542 the plasmid pYF2-GFP was first modified to pYF2.2-GFP by mutating the 543 unexpected Age I site in the junction of eGFP and the Ham34 terminator to a Bsp 544 EI site through QuikChange Lightning Multi Site-Directed Mutagenesis (Agilent 545 Technologies), and the Kpn I site was also removed. Then sgRNAs with or 546 without ribozyme were PCR amplified from the same plasmid, PX458, and 547 inserted into the sites Age I and Nhe I of pYF2.2-GFP. 548 To generate the construct for replacing the entire ORF of Avr4/6, the NPT 549 II coding region together with 250 bp, 500 bp or 1 kb of 5′ and 3′ flanking regions 550 outside the Avr4/6 coding region were PCR amplified and cloned into the plasmid 551 pBluescript II KS+ by In-Fusion ® HD Cloning Kit (Clontech). 


552sgRNA:

  Cas9 in vitro activity assay 553 


562

  Improved transformation of P. sojae 563Polyethylene glycol (PEG) mediated protoplast transformations were conducted 564 to introduce DNA into P. sojae. To achieve a higher transformation rate, we 565 improved the previously described transformation methods(Mcleod et al., 2008, 566Dou et al., 2008a) as follows. 2-4 days old P. sojae mycelial mats, cultured in 567 nutrient pea broth, were harvested and pre-treated with 0.8 M mannitol for 10 min, 568 then digested in 20 mL enzyme solution (0.4 M mannitol, 20 mM KCl, 20mM 569 MES, pH 5.7, 10 mM CaCl 2 , 0.5% Lysing Enzymes from Trichoderma harzianum 570 [Sigma L1412], and 0.5% CELLULYSIN ® Cellulase [Calbiochem 219466]) for ~40 571 min at room temperature with gentle shaking. The mixture was filtered through a 572 Falcon™ Nylon Mesh Cell Strainer (BD Biosciences) and protoplasts were 573 pelleted by centrifugation at 1,200g for 2 min in a Beckman Coulter benchtop 574 centrifuge with swing buckets. After washing with 30 mL W5 solution (5 mM KCl, 575 125 mM CaCl 2 , 154 mM NaCl, and 177 mM glucose), protoplasts were 576 resuspended in 10 mL W5 solution and left on ice for 30 min. Protoplasts were 577 collected by centrifugation at 1,200 g for 2 min in the Beckman centrifuge and 578 resuspended at 10 6 /mL in MMg solution (0.4 M mannitol, 15 mM MgCl 2 and 4 579 mM MES, pH 5.7). DNA transformation was conducted in a 50 mL Falcon tube, 580 where 1 mL protoplasts were well mixed with 20-30 µg DNA for single plasmid 581 transformation. For co-transformation experiments, 20-30 µg of the plasmid 582 carrying the NPT II selectable marker gene was used, together with an equimolar 583 ratio of any other DNAs included. Then, three successive aliquots of 580 µl each 584 of freshly made polyethylene glycol (PEG) solution (40% PEG 4000 v/v, 0.2 M 585 mannitol and 0.1 M CaCl 2 ) were slowly pipetted into the protoplast suspension 586 and gently mixed. After 20 min incubation on ice, 10 mL pea broth containing 0.5 587 M mannitol were added, and the protoplasts were regenerated overnight at 18℃ 588 in the dark. For production of stable transformants, the regenerated protoplasts 589 were collected by centrifugation at 2,000 g for 2 min in the Beckman centrifuge, 590 and then resuspended and evenly divided into three Falcon tubes containing 50 591 mL liquid pea broth containing 1% agar (42 ºC), 0.5 M mannitol and 50 µg/mL 592 G418 (AG Scientific). The resuspended protoplasts were then poured into empty 593 60 mm × 15 mm petri dishes. Mycelial colonies could be observed after 2 d 594 incubation at 25℃ in the dark. The visible transformants were transferred to V8 595 liquid media containing 50 µg/mL G418 and propagated for 2~3d at 25 ºC prior to 596 analysis. For transient expression, 50 µg/mL G418 was usually added into the 597 regeneration medium after overnight recovery, to enrich the positive 598 transformants. After 1 d incubation at 25 ºC in the dark, hyphae were collected 599 for genomic DNA extraction. 600 Detection and quantification of targeted mutagenesis 601 To detect the results of targeted mutagenesis in transformants, total genomic 602 DNA (gDNA) was extracted from pooled or individual P. sojae transformants. For 603 pooled transformants, 48 h after transformation 1 mL of the mycelial culture was 604 pelleted, resuspended in 500 mL lysis buffer (200 mM Tris, pH 8.0, 200 mM NaCl, 605 25 mM EDTA, pH 8.0, 2% SDS, plus 0.1 mg/mL RNase added prior to use) and 606 broken by vortexing with 0.5 mm glass beads. For individual transformants, 607 approximately a 7 mm diameter clump of P. sojae hyphae were blotted dry on 608 Kimwipe™ paper, then frozen in liquid nitrogen. Frozen mycelia was transferred 609 into a 1.5 mL Eppendorf tube and ground to a powder using a polypropylene 610 pestle, then resuspended in 500 µL lysis buffer. Hyphal lysates were incubated at 611 37 ºC, 30 min for RNA digestion. gDNA mixture was extracted with an equal 612 volume of phenol-chloroform-isoamyl alcohol (25:24:1, pH 8.3), and then with an 613 equal volume of chloroform. The aqueous phase was recovered and precipitated 614 by adding 0.5 volume of isopropanol. After being pelleted and washed with 70% 615 ethanol, the gDNA was dried under vacuum and dissolved in 40 µL water. 616 All PCR amplifications were conducted using Phusion ® high-fidelity DNA 617 polymerase (New England Biolabs) in order to exclude the possibility of 618 mutations causing during PCR amplification. Generally ~10 ng gDNA was used 619 as PCR template. Nested PCR was conducted if necessary, using 1:1000 diluted 620 PCR products as a DNA template for the second round. 621 To detect NHEJ mutations, the entire 372 bp Avr4/6 ORF was amplified 622 and examined by digestion with the relevant restriction enzyme. For pooled 623 transformants, a nested PCR was performed to enrich the mutated target before 624 sequencing; this step was not needed for individual transformants, including 625 single zoospore lines. To detect HDR events in pooled and individual 626 transformants (other than zoospore lines), primers located outside the Avr4/6 627 homology arms and in NPT II gene were used. For screening single zoospore 628 lines, PCR was performed by only using primers outside the homology arms. In 629 both cases, nested PCR was carried out for efficient amplification of the targets. 630 PCR products were sequenced directly by the Sanger dideoxy method in the 631 Oregon State University Center for Genome Research and Biocomputing. microscopy (Zeiss LSM 780 NLO) was used to examine 635 the expression and subcellular localization of hSPCas9 fused to the NLS and to 636 GFP. Living hyphae were picked from liquid cultures after 2-3 days growth and 637 regeneration of transformants. Samples were stained with DAPI (4', 6-diamidino-638 2-phenylindole) for 20 min in the dark (Talbot, 2001) before microscopy 639 examination. Images were captured using a 63X oil objective with 640 excitation/emission settings (in nm) 405/410-490 for DAPI, and 488/510~535 for Avr4/6 mutants to infect soybean plants carrying Rps4 and Rps6 645 was evaluated by hypocotyl inoculation as previously described (Dou et al., 646 2010). The wild type and mutant P.sojae strains were grown on V8 plates without 647 G418 selection for ~5 days. Seeds of soybean cultivars HARO(1-7)1 (rps), 648 HARO4272 (Harosoy background, Rps4, Rps7), HARO6272 (Harosoy 649 background, Rps6, Rps7), Williams (rps), L85-2352 (Williams background, Rps4), 650 and L89-1581 (Williams background, Rps6) were provided by M. A. Saghai-651 Maroof (Virginia Tech). Each pathogenicity test was performed in triplicate, each 652 replicate consisting of at least 19 seedlings. A strain was considered avirulent if 653 the number of inoculated Rps4 or Rps6 seedlings surviving was significantly 654 higher than among the seedlings lacking the Rps gene, as determined by 655 


Fig. 1 .

 1 Fig. 1. Cas9 and guide RNA constructs for P. sojae genome editing. (A) Top: Schematic of the plasmid for expression of hSpCas9 fused to eGFP and an NLS in P. sojae. eGFP, enhanced green fluorescent protein. Bottom: P. sojae hyphae expressing PsNLS-hSpCas9-GFP from pYF2, counter-stained with DAPI; scale bars, 10 µm. (B) Top: Schematic of the plasmids used for expression of CRISPR constructs in P. sojae. Cas9 expression is driven by the Ham34 promoter, and cloned together with the selectable marker NPTII (encoding G418 resistance) driven by the RPL41 promoter (P. sojae ribosomal protein L41) in one plasmid. Transcription of sgRNA (including flanking ribozymes) is driven by the RPL41 promoter cloned into the same plasmid with an eGFP expression cassette (used as a screening marker). Bottom: Design of double ribozyme construct for release of sgRNAs from the primary RNA polymerase II transcript. 


Fig. 2 .

 2 Fig. 2. Design and evaluation of sgRNAs for targeting of Avr4/6. (A) Two sgRNA target sites within the Avr4/6 ORF. Target sites of sgRNA A (sgRNA-A) and B (sgRNA-B) are highlighted in blue and yellow, respectively. sgRNAs A and B target Avr4/6 on the negative (-) and positive DNA strand (+), respectively. The sgRNA-A site overlaps with a Bst UI restriction enzyme site (CGCG) and sgRNA-B with a Tsp 45I site (GT G / C AC). PAM, Protospacer Adjacent Motif is in bold red. (B) In vitro cleavage assay indicating Avr4/6 sgRNA-B can direct Cas9 cleavage of target PCR products but sgRNA-A cannot. DNA template was amplified from pBS-Avr4/6 by using M13F and M13R (Supplemental material). The colors of the original gel are inverted for clarity. (C) PCR and restriction enzyme analysis of P. sojae pooled transient expression transformants indicating that only sgRNA-B flanked by ribozymes (sgRNA-B R ) produced amplicons resistant to restriction enzyme cleavage (arrowhead). Approximately 25-30% of the amplicon was resistant to Tsp 45I digestion. Experiment was performed in triplicate. sgRNA -A and -B, sgRNA lacking ribozymes; gRNA-A R , -B R , gRNAs flanked by ribozymes. Mock, P. sojae transformants only receiving Cas9 plasmid. In (B) and (C), all gel panels placed together were from the same gel; white dividers indicate lanes not adjacent in those gels. 


Fig. 3 .

 3 Fig. 3. Characterization of individual P. sojae NHEJ-mediated mutants. (A) Tsp 45 I screening of Avr4/6 amplicons from six individual P. sojae transformants carrying hSpCas9 and sgRNA-B R plasmids. Arrowhead indicates Tsp 45 I-resistant amplicons. Transformants are expected to carry a mixture of modified and non-modified Avr4/6 genes. (B) Left: Sequences of Avr4/6 mutant amplicons from single zoospore lines derived from transformants T11, T18 and T32 (T30 produced no zoospores). Target sites are highlighted in yellow and the PAM sequences are in bold red. (Right) Summary of virulence assays of Avr4/6 mutants on Rps4-and Rps6-containing cultivars. V, virulent; A, avirulent; I, intermediate; I/A intermediate to avirulent; I/V intermediate to virulent. 


Fig. 4 .

 4 Fig. 4. HDR-mediated replacement of the Avr4/6 ORF with an NPT II ORF. (A) Strategy used for gene replacement. Plasmids containing a homologous donor DNA (NPT II with Avr4/6 flanking sequences) were co-transformed with the Avr4/6 sgRNA-B R and hSpCas9 constructs. (B) Three different sizes of homology arms, 250 bp, 500 bp and 1 kb, flanking the Avr4/6 locus were used. The NPT II gene in the hSpCas9 expression plasmid served as the control (0 bp homologous arm, Mock). Primers used to screen the HDR mutants and validate the replaced region are shown as arrows. Primer pairs F1/R1, and nested primer pairs F2/R3, F3/R2 and F2/R2 were used for HDR mutant screening. Red arrow indicates the CRISPR/Cas9 cleavage site (between 232 bp and 233 bp of Avr4/6 ORF). (C) Analysis of genomic DNA from pooled transformants produced using the four sizes of flanking sequences, using nested PCR. Arrowheads indicate sizes expected if HDR has occurred. ACTIN = actin control for DNA quality. (D) Screening of individual HDR transformants generated with the 1 kb flanking sequence plasmid. The 9 positive HDR mutants are highlighted in bold. (E) PCR analysis of representative zoospore-purified lines of HDR mutants, demonstrating that T12 and T39 are homozygotes while T29 is a heterozygote. DNA sizes (bp) before restriction enzyme cleavage: WT, 2936; HDR mutants, 3327; after Nco I digestion: WT, 2273 + 663; HDR mutant, 1945 + 751 +631. The arrowhead indicates the fragment amplified from the NPT II-replaced allele. Primer F4= Avr4/6_up500bp_PhusF (TableS1). (F) Sanger sequencing traces of junction regions confirming that the Avr4/6 ORF was cleanly replaced by the NPT II ORF in a representative zoospore purified clone (HDR-T12-1). Start and stop codons are in red bold. 


Fig. 5 .

 5 Fig. 5. Infection phenotypes of Avr4/6 mutants Representative photos showing soybean seedlings inoculated on the hypocotyls with P. sojae Avr4/6 HDR mutants. L85-2352 and HARO4272 contain Rps4 while L89-1581 and HARO6272 contain Rps6. Photographs were taken 4 days post-inoculation. 




  Fig. S1 Phytophthora U6 promoter evaluation. (A) Alignment of selected oomycete U6 genes, showing that U6 transcripts are highly conserved. The numbers of U6 genes were variable in different oomycete species, 8 in P. sojae P6497 (PsojP6497), 127 in P. infestans (PinfT30-4), 5 in capsici LT1534 (PcapLT1534), 1 in P. parasitica (Ppar INRA-310), and 1 in H. arabidopsidis (HaraEmoy2) respectively. Genome data are obtained from the fungidb.org website. (B) Alignment of the 8 annotated P. sojae U6 genes. PsU6-1 was used to test promoter activity. The red lines indicate the border of the upstream and downstream tRNAs repectively. (C) One of the 127 P. infestans U6 genes cloned to test U6 promoter activity. (Top) Position of the PiU6 gene on Phytophthora infestans T30-4 Supercontig 65. (Bottom) PiU6 sequence used for promoter activity test. The putative U6 coding region is underlined; a putative TATA-box is in red. (D) The plasmid used for testing the functions of the PsU6-1 and PiU6 promoters in P. sojae. Residues 1-150 bp of eGFP was used as a transcription detection maker. Arrows indicate the primer pair U6GFP_F and U6GFP_R used for cloning the eGFP fragment and also for detection of U6 transcripts by RT-PCR. The Eco NI restriction enzyme site used for inserting the GFP detection marker is underlined in (A) and (B) and double underlined in (C). 


Fig. S2

 S2 Fig. S2 Representative sequencing chromatograms of the Avr4/6 mutations in the single zoospore-purified mutants. Regions in box showing the sgRNA target sites within the Avr4/6 gene. The unambiguous sequencing profiles indicate that these mutant lines are all homozygous. 


Fig. S3

 S3 Fig. S3Sanger sequencing profiles revealing that the sub-cultured Cas9:sgRNA transformant T47 (NHEJ-T47) and HDR mutant T29 (HDR-T29-2) had NHEJ mutations (one bp insertion and deletion respectively). Red triangles indicate the differences between wild type (WT) and mutants. 




  


Table 1 . Avr4/6 CRISPR/Cas-induced NHEJ mutations

 1 and the number was not significantly Rps gene, and the number was also not significantly different than the 664 number of surviving seedlings inoculated with the unmodified control strain 665 P6497. A strain was also considered intermediate if the number of inoculated 666 Rps4 or Rps6 seedlings surviving was significantly higher than among the 667 seedlings lacking the Rps gene, and the number was also significantly lower than 668 the number of surviving seedlings inoculated with the unmodified control strain 669 P6497. Intermediate phenotypes were further designated intermediate/virulent or 


	656	
	657	different than the number of surviving seedlings inoculated with the unmodified
	658	control strain P6497. A strain was considered virulent if the number of inoculated
	659	Rps4 or Rps6 seedlings surviving was not significantly different than among the
	660	seedlings lacking the Rps gene, and the number was significantly fewer than the
		number of surviving seedlings inoculated with the unmodified control strain

661 P6497. A strain was considered intermediate if the number of inoculated Rps4 or 662 Rps6 seedlings surviving was not significantly different than among the seedlings 663 lacking the 
Table 2 . Characterization of the virulence of P. sojae Avr4/6 NHEJ-and 822 HDR-mutants on soybeans

 2 
	823												
	824												
		Williams		Ws (rps)		L85-2352 (Rps4)			L89-1581 (Rps6)
		Strains		sv	a	vir	b	sv	p-value1	c	p-value2	d	vir	sv	p-value1 p-value2 vir
		WT		0/60	V		39/58	-		-		A 30/46	-	-	A
		T11-1		0/40	V		7/47	0.014		<0.0001 I/V 12/35 <0.0001	0.0074	I/A
		T11-2-5 fs	e	0/52	V		41/70 <0.0001	0.36		A 18/53 <0.0001	0.034	I/A
		T18-1		1/40	V		18/53	0.0001		0.0006		I	15/32 <0.0001	0.16	A
		T18-2		0/39	V		16/45 <0.0001	0.0016		I/A 12/26 <0.0001	0.21	A
		T32-3 fs		0/54	V		6/60	0.029		<0.0001 I/V 18/51 <0.0001	0.0044	I/A
		HDR-T12-1 0/20	V		9/28	0.0063		0.0027		I	8/32	0.0174	0.0006	I/V
		HDR-T39-1 0/19	V		11/31	0.0035		0.0067		I	7/33	0.0390	0.0002	I/V
	825												
	826												
		Harosoy		(1-7)1 (rps)		4272 (Rps4)			6272 (Rps6)
		Strains		sv		vir	sv	p-value1 p-value2 vir	sv	p-value1 p-value2 vir
		WT		0/40	V	60/85	-		-		A	53/76	-	-	A
		T11-1		0/38	V	5/37	0.025	<0.0001 I/V 15/39 <0.0001	0.0024	I/A
		T11-2-5 fs 0/40	V	47/76 <0.0001	0.25		A	34/79 <0.0001	0.0011	I/A
		T18-1		0/40	V	19/36 <0.0001	0.094		A	15/28 <0.0001	0.16	A
		T18-2		0/39	V	20/35 <0.0001	0.20		A	16/40 <0.0001	0.0027	I/A
		T32-3 fs		0/42	V	15/64	0.0004	<0.0001	I	25/64 <0.0002	0.0003	I
		HDR-12-1 0/20	V	6/28	0.034	<0.0001 I/V 10/35 0.0090	<0.0001 I/V
		HDR-39-1 0/21	V	10/33	0.0043	0.0001		I/V	9/31	0.0073	0.0002	I/V
	827												
	828 829	a sv, soybean seedlings surviving after infection/total seedlings b Vir, virulence of WT and mutants, V, virulent; A, avirulent; I, intermediate; I/A
	830 831 832 833	intermediate to avirulent; I/V intermediate to virulent c p-value1, the difference between Rps4/Rps6 and the rps control d p-value2, the difference between mutants and WT P. sojae e fs = frameshift mutant
	834												

Table 1 .

 1 The sequences of oligonucleotides used in this study 


	Supplemental Primer name	Sequence	Usage
	BlHSP70T_AgeI_F	5'	
		GGCCACCGGTGTCGACTAGA	
		ATACTTAGCAATTGGTTAG 3'	PCR BIHSP70
	BlHSP70T_NsiI_Eco	5'	terminator
	RI_R	TGCATGCATGCAGGAATTCCC	
		CCAATTCCCCGGATCGTC 3'	
	ClaI-AflII instert_F	CGATAGGCCTCCGCGGACTA	
		GTCGTACGC	Oligo annealing, to
	ClaI-AflII instert_R	TTAAGCGTACGACTAGTCCGC	create pYF2
		GGAGGCCTAT	
	GFP_AflII_F	CGGCTTAAGATGGGCAAGGG	
		CGAGGAAC	PCR eGFP, to create
	GFP_ApaI_R	TAGGGCCCTCACTTGTAGAGT	pYF2-GFP
		TCATCCATGCCATG	
	a548t_t553a_F a548t_t553a_R	5'-TGAGGGCCCGGTTCCGGAGC CTACTTCTTG-3' 5'-CAAGAAGTAGGCTCCGGAAC CGGGCCCTCA-3'	Mutate Age I in the junction of eGFP and the Ham34 terminator to Bsp EI, to create pYF2.2-GFP
	GFP_SpeI_F	5'	
		GGACTAGTATGGGCAAGGGC	
		GAGGAAC 3'	PCR eGFP, to create
	GFP_AflII_R	5'	pYF2.2-2XGFP
		GACTTAAGCTTGTAGAGTTCA	
		TCCATGCCATGC 3'	
	PsU6 _HindIII_F	5'	
		CCCAAGCTTCTCCTGCCTACA	
	PsU6_SpeI_R	ACAAACACCAACG 3' 5'	Clone P. sojae U6
		CGGACTAGTCCACTACTCCCT	
		CGCGCCAATC 3'	
	PiU6_ HindIII_F	5'	
		CCCAAGCTTCCTCAGCAGACT	
	PiU6_SpeI_R	CGCGGTAGC 3' 5'	Clone P. infestans U6
		CGGACTAGTGGTTACAATGCT	
		CACCATCAGCC 3'	
	U6GFP_F	5'	Insert 150 bp GFP
		GAGCCTGCGCAAGGATGGGC	fragment into U6 gene
		AAGGGCGAGGAAC 3'	and detect the
	U6GFP_R	5'	transcription of the
		GTGCCTTGCGCAGGGGTAGT	GFP fragment



  Spe I and Afl II sites or Spe I and Apa I sites (there is an Apa I site in hSpCas9, so the two digested fragments were cloned sequentially), respectively, of plasmid pYF2-PsNLS-2XGFP containing the P. sojae NLS.To generate the construct for replacing the entire ORF of Avr4/6, the NPT II coding region together with 250 bp, 500 bp or 1 kb of 5′ and 3′ flanking regions outside the Avr4/6 coding region were PCR amplified and cloned into the plasmid pBluescript II KS+ by In-Fusion ® HD Cloning Kit (Clontech).NPT II gene is in gray background. Nucleotides in green, pink are the border of, 250 bp, 500 bp and 1 kb homologous arms. 


	>pYF2.2-GFP AAGCTTGGCGTAATCATGGTCATAGCTGTTTCCTGTGTGAAATTGTTATCCGCTCAC AATTCCACACAACATACGAGCCGGAAGCATAAAGTGTAAAGCCTGGGGTGCCTAAT GAGTGAGCTAACTCACATTAATTGCGTTGCGCTCACTGCCCGCTTTCCAGTCGGGA AACCTGTCGTGCCAGCTGCATTAATGAATCGGCCAACGCGCGGGGAGAGGCGGTT TGCGTATTGGGCGCTCTTCCGCTTCCTCGCTCACTGACTCGCTGCGCTCGGTCGTT CGGCTGCGGCGAGCGGTATCAGCTCACTCAAAGGCGGTAATACGGTTATCCACAG AATCAGGGGATAACGCAGGAAAGAACATGTGAGCAAAAGGCCAGCAAAAGGCCAG GAACCGTAAAAAGGCCGCGTTGCTGGCGTTTTTCCATAGGCTCCGCCCCCCTGAC GAGCATCACAAAAATCGACGCTCAAGTCAGAGGTGGCGAAACCCGACAGGACTAT AAAGATACCAGGCGTTTCCCCCTGGAAGCTCCCTCGTGCGCTCTCCTGTTCCGACC CTGCCGCTTACCGGATACCTGTCCGCCTTTCTCCCTTCGGGAAGCGTGGCGCTTTC TCAATGCTCACGCTGTAGGTATCTCAGTTCGGTGTAGGTCGTTCGCTCCAAGCTGG GCTGTGTGCACGAACCCCCCGTTCAGCCCGACCGCTGCGCCTTATCCGGTAACTA TCGTCTTGAGTCCAACCCGGTAAGACACGACTTATCGCCACTGGCAGCAGCCACTG GTAACAGGATTAGCAGAGCGAGGTATGTAGGCGGTGCTACAGAGTTCTTGAAGTG GTGGCCTAACTACGGCTACACTAGAAGGACAGTATTTGGTATCTGCGCTCTGCTGA AGCCAGTTACCTTCGGAAAAAGAGTTGGTAGCTCTTGATCCGGCAAACAAACCACC GCTGGTAGCGGTGGTTTTTTTGTTTGCAAGCAGCAGATTACGCGCAGAAAAAAAGG ATCTCAAGAAGATCCTTTGATCTTTTCTACGGGGTCTGACGCTCAGTGGAACGAAAA CTCACGTTAAGGGATTTTGGTCATGAGATTATCAAAAAGGATCTTCACCTAGATCCT TTTAAATTAAAAATGAAGTTTTAAATCAATCTAAAGTATATATGAGTAAACTTGGTCTG ACAGTTACCAATGCTTAATCAGTGAGGCACCTATCTCAGCGATCTGTCTATTTCGTT CATCCATAGTTGCCTGACTCCCCGTCGTGTAGATAACTACGATACGGGAGGGCTTA CCATCTGGCCCCAGTGCTGCAATGATACCGCGAGACCCACGCTCACCGGCTCCAG ATTTATCAGCAATAAACCAGCCAGCCGGAAGGGCCGAGCGCAGAAGTGGTCCTGC AACTTTATCCGCCTCCATCCAGTCTATTAATTGTTGCCGGGAAGCTAGAGTAAGTAG TTCGCCAGTTAATAGTTTGCGCAACGTTGTTGCCATTGCTACAGGCATCGTGGTGT CACGCTCGTCGTTTGGTATGGCTTCATTCAGCTCCGGTTCCCAACGATCAAGGCGA GTTACATGATCCCCCATGTTGTGCAAAAAAGCGGTTAGCTCCTTCGGTCCTCCGAT CGTTGTCAGAAGTAAGTTGGCCGCAGTGTTATCACTCATGGTTATGGCAGCACTGC ATAATTCTCTTACTGTCATGCCATCCGTAAGATGCTTTTCTGTGACTGGTGAGTACT CAACCAAGTCATTCTGAGAATAGTGTATGCGGCGACCGAGTTGCTCTTGCCCGGCG TCAATACGGGATAATACCGCGCCACATAGCAGAACTTTAAAAGTGCTCATCATTGGA AAACGTTCTTCGGGGCGAAAACTCTCAAGGATCTTACCGCTGTTGAGATCCAGTTC GATGTAACCCACTCGTGCACCCAACTGATCTTCAGCATCTTTTACTTTCACCAGCGT TTCTGGGTGAGCAAAAACAGGAAGGCAAAATGCCGCAAAAAAGGGAATAAGGGCG ACACGGAAATGTTGAATACTCATACTCTTCCTTTTTCAATATTATTGAAGCATTTATC AGGGTTATTGTCTCATGAGCGGATACATATTTGAATGTATTTAGAAAAATAAACAAAT AGGGGTTCCGCGCACATTTCCCCGAAAAGTGCCACCTGACGTCTAAGAAACCATTA TTATCATGACATTAACCTATAAAAATAGGCGTATCACGAGGCCCTTTCGTCTCGCGC GTTTCGGTGATGACGGTGAAAACCTCTGACACATGCAGCTCCCGGAGACGGTCAC AGCTTGTCTGTAAGCGGATGCCGGGAGCAGACAAGCCCGTCAGGGCGCGTCAGC GGGTGTTGGCGGGTGTCGGGGCTGGCTTAACTATGCGGCATCAGAGCAGATTGTA CTGAGAGTGCACCATATGCGGTGTGAAATACCGCACAGATGCGTAAGGAGAAAATA CCGCATCAGGCGCCATTCGCCATTCAGGCTGCGCAACTGTTGGGAAGGGCGATCG GTGCGGGCCTCTTCGCTATTACGCCAGCTGGCGAAAGGGGGATGTGCTGCAAGGC GATTAAGTTGGGTAACGCCAGGGTTTTCCCAGTCACGACGTTGTAAAACGACGGCC AGTGAATTCCCCCAATTCCCCGGATCGTCTCATAGCTGCTCAAGACGTTTAGCAGA TTGAGGATGCAGATCTTCTACTAATGAAACTACTGCAAACTACCAACAAAGAGGCGA CCACAAAATGGACAGCCTATGCCGAGATTTGTTTAAGATACTCGATTTGCCGTCCAA TTTTCATCAAGACGAAACACAGTGCATCGAACTGTGTTGTAAAAATTCATTTATTCTT GCAATTGTGACTCTCAAAGATTGATTAAACTTGAAAGCTGGTGAAAGCTTTCCTTCT TTTGGCATGTTTGTTACTAAGGCGGCGTAAGCACAATAGGCCCAGACTCATGCAAA CCAGCCATAGTGACCTTCTCACTATGTTTCGCTTGAGATTTACTAGACTCTCGGGCA TCTTCACAAAAGATGACAACCGAATCAACAAATTAAGTCAACGCTAAATCAAATATTT AGAAAATAAATTTTATTGTCGAAAAAACTCTGCTTTGACAATTTTCAAACTAACCAAT TGCTAAGTATTCTAGTCGACACCGGTACCTATCAGAAGAACTCGTCAAGAAGGCGA TAGAAGGCGATGCGCTGCGAATCGGGAGCGGCGATACCGTAAAGCACGAGGAAG CGGTCAGCCCATTCGCCGCCAAGCTCTTCAGCAATATCACGGGTAGCCAACGCTAT GTCCTGATAGCGGTCCGCCACACCCAGCCGGCCACAGTCGATGAATCCAGAAAAG CGGCCATTTTCCACCATGATATTCGGCAAGCAGGCATCGCCATGGGTCACGACGA GATCCTCGCCGTCGGGCATGCGCGCCTTGAGCCTGGCGAACAGTTCGGCTGGCG CGAGCCCCTGATGCTCTTCGTCCAGATCATCCTGATCGACAAGACCGGCTTCCATC CGAGTACGTGCTCGCTCGATGCGATGTTTCGCTTGGTGGTCGAATGGGCAGGTAG CCGGATCAAGCGTATGCAGCCGCCGCATTGCATCAGCCATGATGGATACTTTCTCG GCAGGAGCAAGGTGAGATGACAGGAGATCCTGCCCCGGCACTTCGCCCAATAGCA GCCAGTCCCTTCCCGCTTCAGTGACAACGTCGAGCACAGCTGCGCAAGGAACGCC CGTCGTGGCCAGCCACGATAGCCGCGCTGCCTCGTCCTGCAGTTCATTCAGGGCA CCGGACAGGTCGGTCTTGACAAAAAGAACCGGGCGCCCCTGCGCTGACAGCCGG AACACGGCGGCATCAGAGCAGCCGATTGTCTGTTGTGCCCAGTCATAGCCGAATA GCCTCTCCACCCAAGCGGCCGGAGAACCTGCGTGCAATCCATCTTGTTCAATCATT GTTGCCCGGGTGGATGCTCAGATGCTAGCGTCACCGAACGAATTAAAACTATAGTA AGTAAAGACTGTCGATGTAGCCATGTGGGGCCAATAGCTGCGAGGCTCAGAGAGA GACAAGGGGGTCGCCACTTACCCCGAACGATGTTCCAAGCAGAGCAGTCAGCAGA AAGTGAGGCTTGTTGCTGTCGGCGAGCGCAGCCTTGGGATCAACATGCGAGAGGA TAGTTGGCTGCGAGCTTGCCAGCGTGGAGAGACTTGGTTATTGTAGGCAACAAGTC CCGCATGAAGGGCGTTGTATGGTGCAAAAAAGAGGAGATGGAATCTTCAAACACTT TGATTGGCTGGACAGCGGGCGCGGACTACTGGCCAACCAAAATGTGACCCCACTT GAGTGACACTTTCCCCACTATAGTGTCTTCAACTTGCACTGCAAGTGAAGTTGTTCT TGTTATTGCACTCGTTCTTACGTTATGGCTAGTACGCGCAGATGCAGAGCCTCCTC GGTGGTATTGCACGCCAACGAACCTCCATTCTCTGCGACATAAGGCTGTGGCAGTG CGCCCAGGAACTCGTAGAGAATGGTCAACTGCGCCGTCACCACTCCCGTGTTCGA GTCTTCCGTCGTGACGAGGGCCGACTTCACCGTCGCCACATTCGACAGCGCAGTG CGGTACATCTCCATCAGCATCGTGTCCAGTTGGGCTGTCACTTCCGTCGCGTTCGC CGTGAAGTCCTCGACAAAACTGTACGCCCAGTTGCCGAGCTCGAAGCTGACGCTG GCGAATCCGCTCGCCAGGTCGAAGGTCAAGATCTGCTCTCCAGGACTGTAGTCCA GCACGATCTGCCTCCCTATTGTGGTGCTGTATGCCAGTGGACAGCTCTTCGCCCAC AGACAAGCGTAGCTGGACTCGAGCAGCTCACGCACTGTACTCGACCATTCCGTGC CGTCCGATGGACTCAAGCAGTCCTGCAGCAGCCAAGTTGCATCAACGCTGCTGTC GATATCGTCCTTCTCCAGCATCGTAGCTATCAGTGTCAAGTCTGCCTCAAGCAGAA CACCCATACACGCTGAGAACGCTCGGCACTCGGGCGAGCTGCTGCAATCCTCCAA TAACGTCGTGCACTCGCTGCAAGTTGGGGCTACATCATCCAGTTGTGAACCTGAAT ACAGTCCACCAAGTCGAGGCTGCTCCAAATCCAATGTTATGGTGATACCAGTGACT TCGTCTCCATCGTCGATCAGATCAGGAATCTCAGAGGTGATAAATGCTGGTTCAGG CTGGCCAGTGGTGTCTACCACCGCGAGGTCGCCAAACTGCCGACGTCGCACAATG GGGATGACAGTTGGGTCGTCAGGAACAAACTCCAGGTCTATGGTGAAGGGTGTGG TAGTGGTCATATCTTGCATCGAGTTTACAGCTTCGAACGTGAGACGGAAGCTCTGG GCATTGCCGCTTGATGACGACCCGCCTACGAAGAAGGACCACGAAGTATCCCCAC CGAGACTCTGAGCACCAACCAGCACCCACGGAGCCGGATAAGTAGAAGTTGAACC AGCAACACTTGATCCGGGAGGAAGACCAATTGACGAAGATAGTGCAGCGTGAATG CTCTGCAGTTTACGAAGAAATATTGGAGCCAACTGATCGGCGCCTGTGAACCGGAG CTTGATACACAGTCTAGAAGTGGTGGAAGAGCACACGTCATCAGCATCTACCAATG CTCCAGATGATGCCGTCGACTCAGGGTTATCGAAGCCGTAGATAAAACTTGTTTGA TGTTCCAGCAATGATTCCGACGAGGTAATGAAGCCGTCGCTGACCATGTCTATCTC TGGTACGGGATCTAACAGTCCAGGCAAATATTGCGGCCAAACGGGTGATCCATCG GCGTGGACGTCCATCTCTCGCTCAATCAGCTCGAGGCTGCCGAGTGAATACAAAG AAATCGTGGAAGCGAGTTGCTGCGAGGAAATGACGGAACGAATTCTTGCCATTGTT ATGGTTGGTTTACGATAAATAAAGAATGATTGTCAGCTGGCTTAAACATAGAAATTT CTACCATGTTAAGTACACACAGTTGCCTCAAAATAGTCACGGTTTTAGCAAGAATCG AAATATTGACAAAAAGGGATCACTCCATACGACTATAGCGGTCATCAACCAAAGAGA AACAATTCTAAGTAGCGTGGGGGATGGTTTCTTTGCATTAGAAGTCACGTAGTTTTG TTTCAAGCCGCCATCAGTCTACTCTACTTTGGTCTTTATTCTCTGTTATAAAATAAAG CTGTCACTGCGCTTGTTCAGCGTTATATTTCATTTGTAAAGCGCCATCACCGATTCG TCACACTCGAATACCAAATGCTAGTATAAAAAAAAATTGTTCTACAAACGGCCTTCTT TTGAAAACAATCGTTCTTTCACAAAAAAAAACAACGAAAATGGAAAAAAGGAAATTG TATTAAATGCATAGACACAAAATCTGCAACTTCGCACTCAGTGCACACAGCTCGACC TTCGGCTTACAAGAAGTAGGCACCGGTACCGGGCCCTCACTTGTAGAGTTCATCCA TGCCATGCGTGATCCCAGCAGCCGTCACGAACTCCAGCAGGACCATGTGGTCCCT CTTCTCGTTGGGGTCCTTGGAGAGGGCAGATTGCGTGGACAGGTAATGGTTGTCC GGCAGCAGGACAGGGCCATCGCCGATGGGCGTGTTCTGCTGGTAGTGGTCCGCC AGTTGCACGCTTCCATCTTCGATGTTGTGCCTGATCTTGAAGTTCACCTTGATGCCG TTCTTCTGCTTGTCGGCCATGATGTATACGTTGTGGGAGTTGTAGTTGTACTCCAGC TTGTGTCCGAGGATGTTTCCATCTTCCTTGAAATCGATGCCTTTCAGCTCGATGCGG TTCACCAGGGTATCACCTTCGAACTTGACTTCGGCACGTGTCTTGTAGTTCCCGTC ATCCTTGAAGAAGATAGTCCTTTCTTGCACGTAGCCTTCGGGCATGGCGCTCTTGA AGAAGTCATGCTGCTTCATGTGATCTGGGTACCGGGAGAAGCACTGAACACCGTAG GTGAAAGTGGTGACGAGGGTTGGCCACGGAACAGGGAGCTTTCCGGTAGTGCAGA TGAACTTCAGGGTGAGCTTTCCGTAGGTGGCATCACCTTCACCCTCTCCGCTGACG GAGAACTTGTGCCCGTTCACATCACCATCCAGTTCCACCAGGATTGGGACCACGCC AGTGAACAGTTCCTCGCCCTTGCCCATCTTAAGCTTGTAGAGTTCATCCATGCCATG CGTGATCCCAGCAGCCGTCACGAACTCCAGCAGGACCATGTGGTCCCTCTTCTCG TTGGGGTCCTTGGAGAGGGCAGATTGCGTGGACAGGTAATGGTTGTCCGGCAGCA GGACAGGGCCATCGCCGATGGGCGTGTTCTGCTGGTAGTGGTCCGCCAGTTGCAC GCTTCCATCTTCGATGTTGTGCCTGATCTTGAAGTTCACCTTGATGCCGTTCTTCTG CTTGTCGGCCATGATGTATACGTTGTGGGAGTTGTAGTTGTACTCCAGCTTGTGTC CGAGGATGTTTCCATCTTCCTTGAAATCGATGCCTTTCAGCTCGATGCGGTTCACCA GGGTATCACCTTCGAACTTGACTTCGGCACGTGTCTTGTAGTTCCCGTCATCCTTG AAGAAGATAGTCCTTTCTTGCACGTAGCCTTCGGGCATGGCGCTCTTGAAGAAGTC ATGCTGCTTCATGTGATCTGGGTACCGGGAGAAGCACTGAACACCGTAGGTGAAA GTGGTGACGAGGGTTGGCCACGGAACAGGGAGCTTTCCGGTAGTGCAGATGAACT TCAGGGTGAGCTTTCCGTAGGTGGCATCACCTTCACCCTCTCCGCTGACGGAGAA CTTGTGCCCGTTCACATCACCATCCAGTTCCACCAGGATTGGGACCACGCCAGTGA ACAGTTCCTCGCCCTTGCCCATACTAGTCCGCGGAGGCCTATCGATAAGCTTGATA TCGAATTAATTCCTGCAGCCCGGGTTGTCGGTGAGAGTGAAGGTCGAAAAAGAGTC GGTTGGGACTTGGGCACGTGAGAGTGAAAAGGAGAATGAGCCTTCCGATCGTGGG CGAGTCGGGCGAGGCTTTTTGAGCGTCCGGTTGTGGCTTCACAACCCCACAAACA GGTGGTTTTCGGATGTAAAAGAGGAAGTGAAATGGCCAATCAGAAACGATCCCTGT GCAAGAGATGTCTTTAATGGGGCGCAAAAAAGACGATCAAAAAGAGCGGATAAGAG AGTTCCGGCGAGACATGTGGTGGTGGTGCTATAATTAACGGATGGTAGGACGCAA AAGCGCTCTGCACGGGTCAGGTTGAGAAGGTTCTCCAATCTTCTCTCTCTCTGTGA CATGTGGTATTTCTCAGAAACCTTCGTGTAAACGCGAGGGAATGGACACGGAAATT AAGGTCTGAAAAAGGGTACTTGACTCGAGGTTTGTGGTTACGCCGGCTGAAGACG CCTAACCTCGCGTGCGATATGCCAGCTGTCGACATTGGAAAAGCCGAGATTGTCGC GGGTACGTAGAGGCGACCCTTTGTCCATCAGAGGGAGAGGAACCGACCAGTGGAT AGGAGGGATAGACTTTATTTTGAGGGCGGTATAATGGTTATGGTAGCGACATTTTG ATTTGACTTGAAAAAGATAAGAAAAAAAAAGGAAAAACTCGACTGCTTTGATGGGAT AGCGATTTTTCCGTGGGTAGTCAGTTGGCCCTTGATTTTTACCATCAAGTACGTCCA TATGACGGGAATAGGGACGCGGGTGGATATTAGGAGCGGCAGCTGACGTGGCAG CTCATAGACAAGGGGGTGCTTGAAAGGCTGTCTATGGTGGTGACGCCGTCTTGCG GTGTGGCAATGAGTTGTATCATTTAATCGAATACGAGGTATAA >pYF2.2-GFP AAGCTTGGCGTAATCATGGTCATAGCTGTTTCCTGTGTGAAATTGTTATCCGCTCAC AATTCCACACAACATACGAGCCGGAAGCATAAAGTGTAAAGCCTGGGGTGCCTAAT GAGTGAGCTAACTCACATTAATTGCGTTGCGCTCACTGCCCGCTTTCCAGTCGGGA AACCTGTCGTGCCAGCTGCATTAATGAATCGGCCAACGCGCGGGGAGAGGCGGTT TGCGTATTGGGCGCTCTTCCGCTTCCTCGCTCACTGACTCGCTGCGCTCGGTCGTT CGGCTGCGGCGAGCGGTATCAGCTCACTCAAAGGCGGTAATACGGTTATCCACAG AATCAGGGGATAACGCAGGAAAGAACATGTGAGCAAAAGGCCAGCAAAAGGCCAG GAACCGTAAAAAGGCCGCGTTGCTGGCGTTTTTCCATAGGCTCCGCCCCCCTGAC GAGCATCACAAAAATCGACGCTCAAGTCAGAGGTGGCGAAACCCGACAGGACTAT AAAGATACCAGGCGTTTCCCCCTGGAAGCTCCCTCGTGCGCTCTCCTGTTCCGACC CTGCCGCTTACCGGATACCTGTCCGCCTTTCTCCCTTCGGGAAGCGTGGCGCTTTC TCAATGCTCACGCTGTAGGTATCTCAGTTCGGTGTAGGTCGTTCGCTCCAAGCTGG GCTGTGTGCACGAACCCCCCGTTCAGCCCGACCGCTGCGCCTTATCCGGTAACTA TCGTCTTGAGTCCAACCCGGTAAGACACGACTTATCGCCACTGGCAGCAGCCACTG GTAACAGGATTAGCAGAGCGAGGTATGTAGGCGGTGCTACAGAGTTCTTGAAGTG GTGGCCTAACTACGGCTACACTAGAAGGACAGTATTTGGTATCTGCGCTCTGCTGA AGCCAGTTACCTTCGGAAAAAGAGTTGGTAGCTCTTGATCCGGCAAACAAACCACC GCTGGTAGCGGTGGTTTTTTTGTTTGCAAGCAGCAGATTACGCGCAGAAAAAAAGG ATCTCAAGAAGATCCTTTGATCTTTTCTACGGGGTCTGACGCTCAGTGGAACGAAAA CTCACGTTAAGGGATTTTGGTCATGAGATTATCAAAAAGGATCTTCACCTAGATCCT TTTAAATTAAAAATGAAGTTTTAAATCAATCTAAAGTATATATGAGTAAACTTGGTCTG ACAGTTACCAATGCTTAATCAGTGAGGCACCTATCTCAGCGATCTGTCTATTTCGTT CATCCATAGTTGCCTGACTCCCCGTCGTGTAGATAACTACGATACGGGAGGGCTTA CCATCTGGCCCCAGTGCTGCAATGATACCGCGAGACCCACGCTCACCGGCTCCAG ATTTATCAGCAATAAACCAGCCAGCCGGAAGGGCCGAGCGCAGAAGTGGTCCTGC AACTTTATCCGCCTCCATCCAGTCTATTAATTGTTGCCGGGAAGCTAGAGTAAGTAG TTCGCCAGTTAATAGTTTGCGCAACGTTGTTGCCATTGCTACAGGCATCGTGGTGT CACGCTCGTCGTTTGGTATGGCTTCATTCAGCTCCGGTTCCCAACGATCAAGGCGA GTTACATGATCCCCCATGTTGTGCAAAAAAGCGGTTAGCTCCTTCGGTCCTCCGAT CGTTGTCAGAAGTAAGTTGGCCGCAGTGTTATCACTCATGGTTATGGCAGCACTGC ATAATTCTCTTACTGTCATGCCATCCGTAAGATGCTTTTCTGTGACTGGTGAGTACT CAACCAAGTCATTCTGAGAATAGTGTATGCGGCGACCGAGTTGCTCTTGCCCGGCG TCAATACGGGATAATACCGCGCCACATAGCAGAACTTTAAAAGTGCTCATCATTGGA AAACGTTCTTCGGGGCGAAAACTCTCAAGGATCTTACCGCTGTTGAGATCCAGTTC GATGTAACCCACTCGTGCACCCAACTGATCTTCAGCATCTTTTACTTTCACCAGCGT TTCTGGGTGAGCAAAAACAGGAAGGCAAAATGCCGCAAAAAAGGGAATAAGGGCG ACACGGAAATGTTGAATACTCATACTCTTCCTTTTTCAATATTATTGAAGCATTTATC AGGGTTATTGTCTCATGAGCGGATACATATTTGAATGTATTTAGAAAAATAAACAAAT AGGGGTTCCGCGCACATTTCCCCGAAAAGTGCCACCTGACGTCTAAGAAACCATTA TTATCATGACATTAACCTATAAAAATAGGCGTATCACGAGGCCCTTTCGTCTCGCGC GTTTCGGTGATGACGGTGAAAACCTCTGACACATGCAGCTCCCGGAGACGGTCAC AGCTTGTCTGTAAGCGGATGCCGGGAGCAGACAAGCCCGTCAGGGCGCGTCAGC GGGTGTTGGCGGGTGTCGGGGCTGGCTTAACTATGCGGCATCAGAGCAGATTGTA CTGAGAGTGCACCATATGCGGTGTGAAATACCGCACAGATGCGTAAGGAGAAAATA CCGCATCAGGCGCCATTCGCCATTCAGGCTGCGCAACTGTTGGGAAGGGCGATCG GTGCGGGCCTCTTCGCTATTACGCCAGCTGGCGAAAGGGGGATGTGCTGCAAGGC GATTAAGTTGGGTAACGCCAGGGTTTTCCCAGTCACGACGTTGTAAAACGACGGCC AGTGAATTCCCCCAATTCCCCGGATCGTCTCATAGCTGCTCAAGACGTTTAGCAGA TTGAGGATGCAGATCTTCTACTAATGAAACTACTGCAAACTACCAACAAAGAGGCGA CCACAAAATGGACAGCCTATGCCGAGATTTGTTTAAGATACTCGATTTGCCGTCCAA TTTTCATCAAGACGAAACACAGTGCATCGAACTGTGTTGTAAAAATTCATTTATTCTT GCAATTGTGACTCTCAAAGATTGATTAAACTTGAAAGCTGGTGAAAGCTTTCCTTCT TTTGGCATGTTTGTTACTAAGGCGGCGTAAGCACAATAGGCCCAGACTCATGCAAA CCAGCCATAGTGACCTTCTCACTATGTTTCGCTTGAGATTTACTAGACTCTCGGGCA TCTTCACAAAAGATGACAACCGAATCAACAAATTAAGTCAACGCTAAATCAAATATTT AGAAAATAAATTTTATTGTCGAAAAAACTCTGCTTTGACAATTTTCAAACTAACCAAT TGCTAAGTATTCTAGTCGACACCGGTACCTATCAGAAGAACTCGTCAAGAAGGCGA TAGAAGGCGATGCGCTGCGAATCGGGAGCGGCGATACCGTAAAGCACGAGGAAG CGGTCAGCCCATTCGCCGCCAAGCTCTTCAGCAATATCACGGGTAGCCAACGCTAT GTCCTGATAGCGGTCCGCCACACCCAGCCGGCCACAGTCGATGAATCCAGAAAAG CGGCCATTTTCCACCATGATATTCGGCAAGCAGGCATCGCCATGGGTCACGACGA GATCCTCGCCGTCGGGCATGCGCGCCTTGAGCCTGGCGAACAGTTCGGCTGGCG CGAGCCCCTGATGCTCTTCGTCCAGATCATCCTGATCGACAAGACCGGCTTCCATC CGAGTACGTGCTCGCTCGATGCGATGTTTCGCTTGGTGGTCGAATGGGCAGGTAG CCGGATCAAGCGTATGCAGCCGCCGCATTGCATCAGCCATGATGGATACTTTCTCG GCAGGAGCAAGGTGAGATGACAGGAGATCCTGCCCCGGCACTTCGCCCAATAGCA GCCAGTCCCTTCCCGCTTCAGTGACAACGTCGAGCACAGCTGCGCAAGGAACGCC CGTCGTGGCCAGCCACGATAGCCGCGCTGCCTCGTCCTGCAGTTCATTCAGGGCA CCGGACAGGTCGGTCTTGACAAAAAGAACCGGGCGCCCCTGCGCTGACAGCCGG AACACGGCGGCATCAGAGCAGCCGATTGTCTGTTGTGCCCAGTCATAGCCGAATA GCCTCTCCACCCAAGCGGCCGGAGAACCTGCGTGCAATCCATCTTGTTCAATCATT GTTGCCCGGGTGGATGCTCAGATGCTAGCGTCACCGAACGAATTAAAACTATAGTA AGTAAAGACTGTCGATGTAGCCATGTGGGGCCAATAGCTGCGAGGCTCAGAGAGA GACAAGGGGGTCGCCACTTACCCCGAACGATGTTCCAAGCAGAGCAGTCAGCAGA AAGTGAGGCTTGTTGCTGTCGGCGAGCGCAGCCTTGGGATCAACATGCGAGAGGA TAGTTGGCTGCGAGCTTGCCAGCGTGGAGAGACTTGGTTATTGTAGGCAACAAGTC CCGCATGAAGGGCGTTGTATGGTGCAAAAAAGAGGAGATGGAATCTTCAAACACTT TGATTGGCTGGACAGCGGGCGCGGACTACTGGCCAACCAAAATGTGACCCCACTT GAGTGACACTTTCCCCACTATAGTGTCTTCAACTTGCACTGCAAGTGAAGTTGTTCT TGTTATTGCACTCGTTCTTACGTTATGGCTAGTACGCGCAGATGCAGAGCCTCCTC GGTGGTATTGCACGCCAACGAACCTCCATTCTCTGCGACATAAGGCTGTGGCAGTG CGCCCAGGAACTCGTAGAGAATGGTCAACTGCGCCGTCACCACTCCCGTGTTCGA GTCTTCCGTCGTGACGAGGGCCGACTTCACCGTCGCCACATTCGACAGCGCAGTG CGGTACATCTCCATCAGCATCGTGTCCAGTTGGGCTGTCACTTCCGTCGCGTTCGC CGTGAAGTCCTCGACAAAACTGTACGCCCAGTTGCCGAGCTCGAAGCTGACGCTG GCGAATCCGCTCGCCAGGTCGAAGGTCAAGATCTGCTCTCCAGGACTGTAGTCCA GCACGATCTGCCTCCCTATTGTGGTGCTGTATGCCAGTGGACAGCTCTTCGCCCAC AGACAAGCGTAGCTGGACTCGAGCAGCTCACGCACTGTACTCGACCATTCCGTGC CGTCCGATGGACTCAAGCAGTCCTGCAGCAGCCAAGTTGCATCAACGCTGCTGTC GATATCGTCCTTCTCCAGCATCGTAGCTATCAGTGTCAAGTCTGCCTCAAGCAGAA CACCCATACACGCTGAGAACGCTCGGCACTCGGGCGAGCTGCTGCAATCCTCCAA TAACGTCGTGCACTCGCTGCAAGTTGGGGCTACATCATCCAGTTGTGAACCTGAAT ACAGTCCACCAAGTCGAGGCTGCTCCAAATCCAATGTTATGGTGATACCAGTGACT TCGTCTCCATCGTCGATCAGATCAGGAATCTCAGAGGTGATAAATGCTGGTTCAGG CTGGCCAGTGGTGTCTACCACCGCGAGGTCGCCAAACTGCCGACGTCGCACAATG GGGATGACAGTTGGGTCGTCAGGAACAAACTCCAGGTCTATGGTGAAGGGTGTGG TAGTGGTCATATCTTGCATCGAGTTTACAGCTTCGAACGTGAGACGGAAGCTCTGG GCATTGCCGCTTGATGACGACCCGCCTACGAAGAAGGACCACGAAGTATCCCCAC CGAGACTCTGAGCACCAACCAGCACCCACGGAGCCGGATAAGTAGAAGTTGAACC AGCAACACTTGATCCGGGAGGAAGACCAATTGACGAAGATAGTGCAGCGTGAATG CTCTGCAGTTTACGAAGAAATATTGGAGCCAACTGATCGGCGCCTGTGAACCGGAG CTTGATACACAGTCTAGAAGTGGTGGAAGAGCACACGTCATCAGCATCTACCAATG CTCCAGATGATGCCGTCGACTCAGGGTTATCGAAGCCGTAGATAAAACTTGTTTGA TGTTCCAGCAATGATTCCGACGAGGTAATGAAGCCGTCGCTGACCATGTCTATCTC TGGTACGGGATCTAACAGTCCAGGCAAATATTGCGGCCAAACGGGTGATCCATCG GCGTGGACGTCCATCTCTCGCTCAATCAGCTCGAGGCTGCCGAGTGAATACAAAG AAATCGTGGAAGCGAGTTGCTGCGAGGAAATGACGGAACGAATTCTTGCCATTGTT ATGGTTGGTTTACGATAAATAAAGAATGATTGTCAGCTGGCTTAAACATAGAAATTT CTACCATGTTAAGTACACACAGTTGCCTCAAAATAGTCACGGTTTTAGCAAGAATCG AAATATTGACAAAAAGGGATCACTCCATACGACTATAGCGGTCATCAACCAAAGAGA AACAATTCTAAGTAGCGTGGGGGATGGTTTCTTTGCATTAGAAGTCACGTAGTTTTG TTTCAAGCCGCCATCAGTCTACTCTACTTTGGTCTTTATTCTCTGTTATAAAATAAAG CTGTCACTGCGCTTGTTCAGCGTTATATTTCATTTGTAAAGCGCCATCACCGATTCG TCACACTCGAATACCAAATGCTAGTATAAAAAAAAATTGTTCTACAAACGGCCTTCTT TTGAAAACAATCGTTCTTTCACAAAAAAAAACAACGAAAATGGAAAAAAGGAAATTG TATTAAATGCATAGACACAAAATCTGCAACTTCGCACTCAGTGCACACAGCTCGACC TTCGGCTTACAAGAAGTAGGCTCCGGAACCGGGCCCTCACTTGTAGAGTTCATCCA TGCCATGCGTGATCCCAGCAGCCGTCACGAACTCCAGCAGGACCATGTGGTCCCT CTTCTCGTTGGGGTCCTTGGAGAGGGCAGATTGCGTGGACAGGTAATGGTTGTCC GGCAGCAGGACAGGGCCATCGCCGATGGGCGTGTTCTGCTGGTAGTGGTCCGCC AGTTGCACGCTTCCATCTTCGATGTTGTGCCTGATCTTGAAGTTCACCTTGATGCCG TTCTTCTGCTTGTCGGCCATGATGTATACGTTGTGGGAGTTGTAGTTGTACTCCAGC TTGTGTCCGAGGATGTTTCCATCTTCCTTGAAATCGATGCCTTTCAGCTCGATGCGG TTCACCAGGGTATCACCTTCGAACTTGACTTCGGCACGTGTCTTGTAGTTCCCGTC ATCCTTGAAGAAGATAGTCCTTTCTTGCACGTAGCCTTCGGGCATGGCGCTCTTGA AGAAGTCATGCTGCTTCATGTGATCTGGGTACCGGGAGAAGCACTGAACACCGTAG GTGAAAGTGGTGACGAGGGTTGGCCACGGAACAGGGAGCTTTCCGGTAGTGCAGA TGAACTTCAGGGTGAGCTTTCCGTAGGTGGCATCACCTTCACCCTCTCCGCTGACG GAGAACTTGTGCCCGTTCACATCACCATCCAGTTCCACCAGGATTGGGACCACGCC AGTGAACAGTTCCTCGCCCTTGCCCATCTTAAGCGTACGACTAGTCCGCGGAGGC CTATCGATAAGCTTGATATCGAATTAATTCCTGCAGCCCGGGTTGTCGGTGAGAGT GAAGGTCGAAAAAGAGTCGGTTGGGACTTGGGCACGTGAGAGTGAAAAGGAGAAT GAGCCTTCCGATCGTGGGCGAGTCGGGCGAGGCTTTTTGAGCGTCCGGTTGTGGC TTCACAACCCCACAAACAGGTGGTTTTCGGATGTAAAAGAGGAAGTGAAATGGCCA ATCAGAAACGATCCCTGTGCAAGAGATGTCTTTAATGGGGCGCAAAAAAGACGATC AAAAAGAGCGGATAAGAGAGTTCCGGCGAGACATGTGGTGGTGGTGCTATAATTAA CGGATGGTAGGACGCAAAAGCGCTCTGCACGGGTCAGGTTGAGAAGGTTCTCCAA TCTTCTCTCTCTCTGTGACATGTGGTATTTCTCAGAAACCTTCGTGTAAACGCGAGG GAATGGACACGGAAATTAAGGTCTGAAAAAGGGTACTTGACTCGAGGTTTGTGGTT ACGCCGGCTGAAGACGCCTAACCTCGCGTGCGATATGCCAGCTGTCGACATTGGA AAAGCCGAGATTGTCGCGGGTACGTAGAGGCGACCCTTTGTCCATCAGAGGGAGA GGAACCGACCAGTGGATAGGAGGGATAGACTTTATTTTGAGGGCGGTATAATGGTT ATGGTAGCGACATTTTGATTTGACTTGAAAAAGATAAGAAAAAAAAAGGAAAAACTC GACTGCTTTGATGGGATAGCGATTTTTCCGTGGGTAGTCAGTTGGCCCTTGATTTTT ACCATCAAGTACGTCCATATGACGGGAATAGGGACGCGGGTGGATATTAGGAGCG GCAGCTGACGTGGCAGCTCATAGACAAGGGGGTGCTTGAAAGGCTGTCTATGGTG GTGACGCCGTCTTGCGGTGTGGCAATGAGTTGTATCATTTAATCGAATACGAGGTA TAA encoding the NLS (MHKRKREDDTKVRRRMHKRKREDDTKVRRRMHKRKREDDTKVRRR). To generate hSpCas9-expression plasmids pYF2-PsNLS-hSpCas9-GFP and pYF2-PsNLS-hSpCas9, the coding fragment of hSpCas9 nuclease from the plasmid pSpCas9 (BB)-2A-GFP (PX458, Addgene plasmid # 48138) were catggtcccagcctcctcgctggcgccggctgggcaacatgcttcggcatggcgaatgggac >PsNLS-Cas9 P. sojae NLS in red, enzymes used for cloning are underscored. Start and stop codon are in bold. ATGCACAAGCGCAAGCGCGAGGACGACACCAAGGTCCGTCGTCGCATGCACAAGC GCAAGCGCGAGGACGACACCAAGGTCCGTCGTCGCATGCACAAGCGCAAGCGCG AGGACGACACCAAGGTCCGTCGTCGCACTAGTATGGCCCCAAAGAAGAAGCGGAA GGTCGGTATCCACGGAGTCCCAGCAGCCGACAAGAAGTACAGCATCGGCCTGGAC ATCGGCACCAACTCTGTGGGCTGGGCCGTGATCACCGACGAGTACAAGGTGCCCA GCAAGAAATTCAAGGTGCTGGGCAACACCGACCGGCACAGCATCAAGAAGAACCT GATCGGAGCCCTGCTGTTCGACAGCGGCGAAACAGCCGAGGCCACCCGGCTGAA GAGAACCGCCAGAAGAAGATACACCAGACGGAAGAACCGGATCTGCTATCTGCAA GAGATCTTCAGCAACGAGATGGCCAAGGTGGACGACAGCTTCTTCCACAGACTGG AAGAGTCCTTCCTGGTGGAAGAGGATAAGAAGCACGAGCGGCACCCCATCTTCGG CAACATCGTGGACGAGGTGGCCTACCACGAGAAGTACCCCACCATCTACCACCTG AGAAAGAAACTGGTGGACAGCACCGACAAGGCCGACCTGCGGCTGATCTATCTGG CCCTGGCCCACATGATCAAGTTCCGGGGCCACTTCCTGATCGAGGGCGACCTGAA CCCCGACAACAGCGACGTGGACAAGCTGTTCATCCAGCTGGTGCAGACCTACAAC CAGCTGTTCGAGGAAAACCCCATCAACGCCAGCGGCGTGGACGCCAAGGCCATCC TGTCTGCCAGACTGAGCAAGAGCAGACGGCTGGAAAATCTGATCGCCCAGCTGCC CGGCGAGAAGAAGAATGGCCTGTTCGGAAACCTGATTGCCCTGAGCCTGGGCCTG ACCCCCAACTTCAAGAGCAACTTCGACCTGGCCGAGGATGCCAAACTGCAGCTGA GCAAGGACACCTACGACGACGACCTGGACAACCTGCTGGCCCAGATCGGCGACCA GTACGCCGACCTGTTTCTGGCCGCCAAGAACCTGTCCGACGCCATCCTGCTGAGC GACATCCTGAGAGTGAACACCGAGATCACCAAGGCCCCCCTGAGCGCCTCTATGA TCAAGAGATACGACGAGCACCACCAGGACCTGACCCTGCTGAAAGCTCTCGTGCG GCAGCAGCTGCCTGAGAAGTACAAAGAGATTTTCTTCGACCAGAGCAAGAACGGCT ACGCCGGCTACATTGACGGCGGAGCCAGCCAGGAAGAGTTCTACAAGTTCATCAA GCCCATCCTGGAAAAGATGGACGGCACCGAGGAACTGCTCGTGAAGCTGAACAGA GAGGACCTGCTGCGGAAGCAGCGGACCTTCGACAACGGCAGCATCCCCCACCAG ATCCACCTGGGAGAGCTGCACGCCATTCTGCGGCGGCAGGAAGATTTTTACCCATT CCTGAAGGACAACCGGGAAAAGATCGAGAAGATCCTGACCTTCCGCATCCCCTACT ACGTGGGCCCTCTGGCCAGGGGAAACAGCAGATTCGCCTGGATGACCAGAAAGAG CGAGGAAACCATCACCCCCTGGAACTTCGAGGAAGTGGTGGACAAGGGCGCTTCC GCCCAGAGCTTCATCGAGCGGATGACCAACTTCGATAAGAACCTGCCCAACGAGA AGGTGCTGCCCAAGCACAGCCTGCTGTACGAGTACTTCACCGTGTATAACGAGCTG ACCAAAGTGAAATACGTGACCGAGGGAATGAGAAAGCCCGCCTTCCTGAGCGGCG AGCAGAAAAAGGCCATCGTGGACCTGCTGTTCAAGACCAACCGGAAAGTGACCGT GAAGCAGCTGAAAGAGGACTACTTCAAGAAAATCGAGTGCTTCGACTCCGTGGAAA TCTCCGGCGTGGAAGATCGGTTCAACGCCTCCCTGGGCACATACCACGATCTGCT AAGATATCGTGCTGACCCTGACACTGTTTGAGGACAGAGAGATGATCGAGGAACG GCTGAAAACCTATGCCCACCTGTTCGACGACAAAGTGATGAAGCAGCTGAAGCGG CGGAGATACACCGGCTGGGGCAGGCTGAGCCGGAAGCTGATCAACGGCATCCGG GACAAGCAGTCCGGCAAGACAATCCTGGATTTCCTGAAGTCCGACGGCTTCGCCA ACAGAAACTTCATGCAGCTGATCCACGACGACAGCCTGACCTTTAAAGAGGACATC CAGAAAGCCCAGGTGTCCGGCCAGGGCGATAGCCTGCACGAGCACATTGCCAATC TGGCCGGCAGCCCCGCCATTAAGAAGGGCATCCTGCAGACAGTGAAGGTGGTGGA CGAGCTCGTGAAAGTGATGGGCCGGCACAAGCCCGAGAACATCGTGATCGAAATG GCCAGAGAGAACCAGACCACCCAGAAGGGACAGAAGAACAGCCGCGAGAGAATG AAGCGGATCGAAGAGGGCATCAAAGAGCTGGGCAGCCAGATCCTGAAAGAACACC CCGTGGAAAACACCCAGCTGCAGAACGAGAAGCTGTACCTGTACTACCTGCAGAAT GGGCGGGATATGTACGTGGACCAGGAACTGGACATCAACCGGCTGTCCGACTACG ATGTGGACCATATCGTGCCTCAGAGCTTTCTGAAGGACGACTCCATCGACAACAAG GTGCTGACCAGAAGCGACAAGAACCGGGGCAAGAGCGACAACGTGCCCTCCGAA GAGGTCGTGAAGAAGATGAAGAACTACTGGCGGCAGCTGCTGAACGCCAAGCTGA TTACCCAGAGAAAGTTCGACAATCTGACCAAGGCCGAGAGAGGCGGCCTGAGCGA ACTGGATAAGGCCGGCTTCATCAAGAGACAGCTGGTGGAAACCCGGCAGATCACA AAGCACGTGGCACAGATCCTGGACTCCCGGATGAACACTAAGTACGACGAGAATG ACAAGCTGATCCGGGAAGTGAAAGTGATCACCCTGAAGTCCAAGCTGGTGTCCGAT TTCCGGAAGGATTTCCAGTTTTACAAAGTGCGCGAGATCAACAACTACCACCACGC CCACGACGCCTACCTGAACGCCGTCGTGGGAACCGCCCTGATCAAAAAGTACCCT AAGCTGGAAAGCGAGTTCGTGTACGGCGACTACAAGGTGTACGACGTGCGGAAGA TGATCGCCAAGAGCGAGCAGGAAATCGGCAAGGCTACCGCCAAGTACTTCTTCTAC AGCAACATCATGAACTTTTTCAAGACCGAGATTACCCTGGCCAACGGCGAGATCCG GAAGCGGCCTCTGATCGAGACAAACGGCGAAACCGGGGAGATCGTGTGGGATAAG GGCCGGGATTTTGCCACCGTGCGGAAAGTGCTGAGCATGCCCCAAGTGAATATCG TGAAAAAGACCGAGGTGCAGACAGGCGGCTTCAGCAAAGAGTCTATCCTGCCCAA GAGGAACAGCGATAAGCTGATCGCCAGAAAGAAGGACTGGGACCCTAAGAAGTAC GGCGGCTTCGACAGCCCCACCGTGGCCTATTCTGTGCTGGTGGTGGCCAAAGTGG AAAAGGGCAAGTCCAAGAAACTGAAGAGTGTGAAAGAGCTGCTGGGGATCACCAT CATGGAAAGAAGCAGCTTCGAGAAGAATCCCATCGACTTTCTGGAAGCCAAGGGCT ACAAAGAAGTGAAAAAGGACCTGATCATCAAGCTGCCTAAGTACTCCCTGTTCGAG CTGGAAAACGGCCGGAAGAGAATGCTGGCCTCTGCCGGCGAACTGCAGAAGGGA AACGAACTGGCCCTGCCCTCCAAATATGTGAACTTCCTGTACCTGGCCAGCCACTA TGAGAAGCTGAAGGGCTCCCCCGAGGATAATGAGCAGAAACAGCTGTTTGTGGAA CAGCACAAGCACTACCTGGACGAGATCATCGAGCAGATCAGCGAGTTCTCCAAGA GAGTGATCCTGGCCGACGCTAATCTGGACAAAGTGCTGTCCGCCTACAACAAGCA CCGGGATAAGCCCATCAGAGAGCAGGCCGAGAATATCATCCACCTGTTTACCCTGA CCAATCTGGGAGCCCCTGCCGCCTTCAAGTACTTTGACACCACCATCGACCGGAA GAGGTACACCAGCACCAAAGAGGTGCTGGACGCCACCCTGATCCACCAGAGCATC ACCGGCCTGTACGAGACACGGATCGACCTGTCTCAGCTGGGAGGCGACCTTAAGC CTCGTGCTGGGACACTGGAACCCACTAGAACAGGCTGATGAGCATAAGGTTCACG CGGGGCCCTAA Sequence of plasmid having donor DNA >pBS_KS_Avr4/6-1k CTAAATTGTAAGCGTTAATATTTTGTTAAAATTCGCGTTAAATTTTTGTTAAATCAGCT CATTTTTTAACCAATAGGCCGAAATCGGCAAAATCCCTTATAAATCAAAAGAATAGA AAGAATCGAACCTTCATTTAGCCTTTCTCTTTGAAAAAAGCGCCGGAGTCTGTGTGG CCTTTTTCAATATTATTGAAGCATTTATCAGGGTTATTGTCTCATGAGCGGATACATA GCCGCTAAAACAACTTCATAGCACCACATTTCTATAGAATTGTAAACCCTGAGAGCA AATGCCGCAAAAAAGGGAATAAGGGCGACACGGAAATGTTGAATACTCATACTCTT CGGGAAAGCACTCGTATTTGTATCCATAAGCATATTCCTGACTCGATCAGTATCTGA TCTTCAGCATCTTTTACTTTCACCAGCGTTTCTGGGTGAGCAAAAACAGGAAGGCAA ttcttgacgagttcttctgaGTTTCACAGGCGTTCTGGCTGGAAATCCCGCAATCGTGCCAATA GGATCTTACCGCTGTTGAGATCCAGTTCGATGTAACCCACTCGTGCACCCAACTGA agcttggcggcgaatgggctgaccgcttcctcgtgctttacggtatcgccgctcccgattcgcagcgcatcgccttctatcgcc GCAGAACTTTAAAAGTGCTCATCATTGGAAAACGTTCTTCGGGGCGAAAACTCTCAA tttctggattcatcgactgtggccggctgggtgtggcggaccgctatcaggacatagcgttggctacccgtgatattgctgaag CGGCGACCGAGTTGCTCTTGCCCGGCGTCAATACGGGATAATACCGCGCCACATA cgcgcatgcccgacggcgaggatctcgtcgtgacccatggcgatgcctgcttgccgaatatcatggtggaaaatggccgct AGATGCTTTTCTGTGACTGGTGAGTACTCAACCAAGTCATTCTGAGAATAGTGTATG gccggtcttgtcgatcaggatgatctggacgaagagcatcaggggctcgcgccagccgaactgttcgccaggctcaagg TTATCACTCATGGTTATGGCAGCACTGCATAATTCTCTTACTGTCATGCCATCCGTA gcatacgcttgatccggctacctgcccattcgaccaccaagcgaaacatcgcatcgagcgagcacgtactcggatggaa AAGCGGTTAGCTCCTTCGGTCCTCCGATCGTTGTCAGAAGTAAGTTGGCCGCAGTG aagtgccggggcaggatctcctgtcatctcaccttgctcctgccgagaaagtatccatcatggctgatgcaatgcggcggct CAGCTCCGGTTCCCAACGATCAAGGCGAGTTACATGATCCCCCATGTTGTGCAAAA ggctggccacgacgggcgttccttgcgcagctgtgctcgacgttgtcactgaagcgggaagggactggctgctattgggcg GTTGCCATTGCTACAGGCATCGTGGTGTCACGCTCGTCGTTTGGTATGGCTTCATT aggggcgcccggttctttttgtcaagaccgacctgtccggtgccctgaatgaactgcaggacgaggcagcgcggctatcgt ATTGTTGCCGGGAAGCTAGAGTAAGTAGTTCGCCAGTTAATAGTTTGCGCAACGTT gggtggagaggctattcggctatgactgggcacaacagacaatcggctgctctgatgccgccgtgttccggctgtcagcgc AAGGGCCGAGCGCAGAAGTGGTCCTGCAACTTTATCCGCCTCCATCCAGTCTATTA AAGCGCCCTGCAATTCCAAGTCCAACGatgattgaacaagatggattgcacgcaggttctccggccgctt CGCGAGACCCACGCTCACCGGCTCCAGATTTATCAGCAATAAACCAGCCAGCCGG ACAGCGTACCATTGACCTGTAGAGCCCACCCAGATCATAATTAATCTTCCAATAAGC GTAGATAACTACGATACGGGAGGGCTTACCATCTGGCCCCAGTGCTGCAATGATAC ACATGCGTAATGCGATACGGCGCGGTGTCTCAGAACCACCCGCCGATTTTGTACTG ACCTATCTCAGCGATCTGTCTATTTCGTTCATCCATAGTTGCCTGACTCCCCGTCGT ATTCCTTACTCCGTTTTCTGGTGTGTACAGTATCGGGTCGTAAACACATGCGTAAAC CTAAAGTATATATGAGTAAACTTGGTCTGACAGTTACCAATGCTTAATCAGTGAGGC ATCCGTACGTTTTGGAGTTGGCTCGCCTGTTCTCATCAATGCCGTCGATCGTAGCA TATCAAAAAGGATCTTCACCTAGATCCTTTTAAATTAAAAATGAAGTTTTAAATCAAT AAATACGGTAAATGTTATAGGTGCCCTGCAGCTTGTCCGACATGTACATCGTTCATC GGGGTCTGACGCTCAGTGGAACGAAAACTCACGTTAAGGGATTTTGGTCATGAGAT CAAAATGGCTCCTTTTTCGGCAACCCCATTATAACTAAACCCCCACAACAACTAAAT GCAGCAGATTACGCGCAGAAAAAAAGGATCTCAAGAAGATCCTTTGATCTTTTCTAC TCCTACCTCGATCATCAATCACTTCATACAAACTTTTCATAAAGACTTCGCCCGTCTC AGCTCTTGATCCGGCAAACAAACCACCGCTGGTAGCGGTGGTTTTTTTGTTTGCAA GGTATACCTACCATATTTAGTTATAATGGGGGTGGTACAAAATGGGAATGAGTACTT CAGTATTTGGTATCTGCGCTCTGCTGAAGCCAGTTACCTTCGGAAAAAGAGTTGGT CTCGAGGTCGACGGTATCGATAAGCTTGATATCGAATTCGTAGGAAATCTCAAATTC GGCGGTGCTACAGAGTTCTTGAAGTGGTGGCCTAACTACGGCTACACTAGAAGGA CAGTGAGCGCGCGTAATACGACTCACTATAGGGCGAATTGGGTACCGGGCCCCCC CTTATCGCCACTGGCAGCAGCCACTGGTAACAGGATTAGCAGAGCGAGGTATGTA CGATTAAGTTGGGTAACGCCAGGGTTTTCCCAGTCACGACGTTGTAAAACGACGGC GACCGCTGCGCCTTATCCGGTAACTATCGTCTTGAGTCCAACCCGGTAAGACACGA GGTGCGGGCCTCTTCGCTATTACGCCAGCTGGCGAAAGGGGGATGTGCTGCAAGG GGTGTAGGTCGTTCGCTCCAAGCTGGGCTGTGTGCACGAACCCCCCGTTCAGCCC TACAGGGCGCGTCCCATTCGCCATTCAGGCTGCGCAACTGTTGGGAAGGGCGATC CTCCCTTCGGGAAGCGTGGCGCTTTCTCATAGCTCACGCTGTAGGTATCTCAGTTC AGTGTAGCGGTCACGCTGCGCGTAACCACCACACCCGCCGCGCTTAATGCGCCGC CCCTCGTGCGCTCTCCTGTTCCGACCCTGCCGCTTACCGGATACCTGTCCGCCTTT GTGGCGAGAAAGGAAGGGAAGAAAGCGAAAGGAGCGGGCGCTAGGGCGCTGGCA AGGTGGCGAAACCCGACAGGACTATAAAGATACCAGGCGTTTCCCCCTGGAAGCT CGGAACCCTAAAGGGAGCCCCCGATTTAGAGCTTGACGGGGAAAGCCGGCGAAC TTTTCCATAGGCTCCGCCCCCCTGACGAGCATCACAAAAATCGACGCTCAAGTCAG GTGAACCATCACCCTAATCAAGTTTTTTGGGGTCGAGGTGCCGTAAAGCACTAAAT TGAGCAAAAGGCCAGCAAAAGGCCAGGAACCGTAAAAAGGCCGCGTTGCTGGCGT GTGGACTCCAACGTCAAAGGGCGAAAAACCGTCTATCAGGGCGATGGCCCACTAC CAAAGGCGGTAATACGGTTATCCACAGAATCAGGGGATAACGCAGGAAAGAACATG CCGAGATAGGGTTGAGTGTTGTTCCAGTTTGGAACAAGAGTCCACTATTAAAGAAC CCACTCAGTATGACCGAGATATCCGGTAAACCCACTTGCAGCTTTTTCAAAACCCTT ATATCCCTCCCTCTTCGTCGACGCCTAGCGAAGTGTGCATCAGCTGGCTGACGGAT GCCTTGGTCGAGTCGTCACTCCGGAGTTGTCTCGCATCGGGACTAAGTAAGCGAG GCGCAGCGGATCCACTAGTTCTAGAGCGGCCGCCACCGCGGTGGAGCTCCAGCTT TTGTTCCCTTTAGTGAGGGTTAATTGCGCGCTTGGCGTAATCATGGTCATAGCTGTT TCCTGTGTGAAATTGTTATCCGCTCACAATTCCACACAACATACGAGCCGGAAGCAT AAAGTGTAAAGCCTGGGGTGCCTAATGAGTGAGCTAACTCACATTAATTGCGTTGC GCTCACTGCCCGCTTTCCAGTCGGGAAACCTGTCGTGCCAGCTGCATTAATGAATC GGCCAACGCGCGGGGAGAGGCGGTTTGCGTATTGGGCGCTCTTCCGCTTCCTCG CTCACTGACTCGCTGCGCTCGGTCGTTCGGCTGCGGCGAGCGGTATCAGCTCACT TTTGAATGTATTTAGAAAAATAAACAAATAGGGGTTCCGCGCACATTTCCCCGAAAA subcloned into the gcgatcctgatgagtccgtgaggacgaaacgagtaagctcgtc↓GATCGCATCGTCTGTCACCAgtttta gagctagaaatagcaagttaaaataaggctagtccgttatcaacttgaaaaagtggcaccgagtcggtgctttt↓ggccgg GAAAATTATCAAGGACAAGGACTTCCTGGACAATGAGGAAAACGAGGACATTCTGG TTTAAGGCGCTCGGGGACCCTTTCGGCCTCTGGAGCAGCTTGCCAAGACCGCTCG GTGCCAC

ACKNOWLEDGEMENTS 675

We thank F. Arredondo, S. Taylor and D. Wellappili (Oregon State University) for


This work was supported in part by grant 2011-68004-30104 from the Agriculture 681 and Food Research Initiative of the USDA National Institute for Food and 682


SUPPLEMENTARY INFORMATION

Supplementary Table 1 The sequences of oligonucleotides used in this study Supplementary Fig. S1 Phytophthora U6 promoter evaluation Supplementary Fig. S2 Phytophthora U6 promoter evaluation Supplementary Fig. S3 Representative sequencing chromatograms of the Avr4/6 mutations in the single zoospore-purified mutants Supplementary Fig. S4 Plasmid backbones used for expression of hSpCas9 and sgRNA in P. sojae 

Supplementary Methods


Supplementary Sequence Information


Supplemental methods


Generation of P. sojae CRISPR/Cas9 plasmids.

To express hSpCas9 and sgRNAs effectively in P. sojae, we first created a new Phytophthora expression plasmid backbone pYF2 by combining elements from pHamT34 (Judelson et al., 1991), pUN (Dou et al., 2008)
Yufeng Fang, Brett M Tyler
Efficient Disruption and Replacement of an Effector Gene in the Oomycete Phytophthora sojae using CRISPR/Cas9