Structure-function analysis of interallelic complementation in ROOTY transheterozygotes

Auxin is a crucial plant growth regulator. Forward genetic screens for auxin-related mutants have led to the identification of key genes involved in auxin biosynthesis, transport, and signaling. Loss-of-function mutations in the genes involved in indole glucosinolate biosynthesis, a metabolically-related route that produces defense compounds from indolic precursors shared with the auxin pathway, result in auxin overproduction. We identified an allelic series of fertile, hypomorphic mutants for an essential indole glucosinolate route gene ROOTY (RTY) that show a range of high-auxin defects. Genetic characterization of these lines uncovered phenotypic suppression by cyp79b2 b3, wei2, and wei7 mutants and revealed the phenomenon of interallelic complementation in several RTY transheterozygotes. Structural modeling of RTY shed light on the structure-to-function relations in the RTY homo- and heterodimers and unveiled the likely structural basis of interallelic complementation. This work underscores the importance of employing true null mutants in genetic complementation studies.

In this study, we describe an allelic series of hypomorphic mutants for one of the IG 79 pathway genes, RTY. The RTY protein is a C-S lyase that catalyzes the conversion of S-80 (alkylacetohydroximoyl)-L-cysteine to thiohydroximate in the synthesis of IGs (Mikkelsen 81 et al., 2004). Partial inactivation of this enzymatic step due to missense mutations in RTY 82 leads to milder defects than previously reported for the seedling-lethal null mutants of this 83 gene, with the developmentally stunted but fertile new alleles of RTY in our collection 84 ranging in their severity from moderate to mild. Unexpectedly, genetic characterization of 85 these lines uncovered several instances of interallelic complementation between missense 86 mutants of RTY. Computational modeling of the RTY protein structure and mapping of RTY 87 mutations onto the structural model of the RTY protein not only highlighted key residues 88 essential for dimerization and binding of the substrate, but also shed light on structure-to-89 function relationships, revealing the likely structural basis of interallelic complementation. 90 The combination of genetics and modeling employed in this work provided an effective, 91 synergistic strategy to investigating the molecular basis of the complementation 92 phenomenon in F1 transheterozygotes. Given the scarcity of reports describing interallelic 93 complementation despite the ubiquitous use of allelic testing in the genetic 94 characterization of mutants, this study serves as a cautionary tale that underscores the 95 need of utilizing true null alleles of genes of interest in all complementation crosses. 96 97 Results

99
Characterization of a new allele of the RTY locus 100 101 Downward curling (i.e., epinasty) of cotyledons (Fig. 1A) and true leaves (Fig. 1B) is a 102 characteristic feature of auxin biosynthesis mutants that produce excess IAA, such as gain- showed varying degrees of epinasty and dwarfism in adults (see below). To gain insights 114 into the nature of the affected gene(s), we performed phenotypic and genetic 115 characterization of these lines. 116 The first mutant of that subclass characterized was TG8B that showed characteristic 117 cotyledon and leaf epinasty in light-exposed seedlings and soil-grown adults (Fig. 1A, B) 118 and lacked apical hooks in etiolated seedlings (Fig. 1C) Table  140 S1).

141
To determine whether TG8B is, indeed, an allele of RTY, we developed a set of five 142 primer pairs that amplify five overlapping gene fragments and cover the entire RTY gene 143 ( Fig. 2A for both mutants: in fragment 3 for rty and fragment 4 for TG8B ( Fig. 2A) . 2E) and normalized auxin response (Fig. 2F).

180
Possible genetic mechanisms of RTY complementation in F1 181 182 We next decided to investigate why the F1 generation of the cross between the classical rty 183 mutant (King et al., 1995) and the new allele, TG8B, showed full complementation. We 184 could envision two possibilities. One possible scenario explaining the wild-type 185 morphology of the rty/TG8B transheterozygote is that the rty phenotype might have been 186 suppressed by one copy of the wei2-1 mutation, since a rty wei2 double mutant was utilized 187 for this cross and, therefore, the F1 was genotypically rty/TG8B wei2/+. This is, however, 188 an unlikely possibility, as two copies of the wei2-1 mutant allele are required for the 189 suppression of the high-auxin defects of sur2 and rty (Stepanova et al., 2005) or of TG8B 190 (Fig. 2E, 2F), and the sesquimutants TG8B/TG8B wei2/+, rty/rty wei2/+ (Supplemental Fig.  191 S2A, S2B) and sur2/sur2 wei2/+ (Supplemental Fig. S2C To further explore the basis of mutant phenotype complementation in the RTY 211 transheterozygote, we analyzed several additional putative rty alleles identified in our 212 mutant screen. TD11B, TF5D, and TH5H show varying degree of cotyledon and leaf 213 epinasty in light-grown plants (Fig. 3A, B) and lack apical hooks in dark-grown seedlings 214 (Fig. 3C), as do rty and TG8B. Adult phenotypes range in severity in terms of rosette size 215 and fertility, with TD11B being the weakest of all mutants, and TH5H being the strongest in 216 this subset, but not as severe as rty itself (Fig. 3B, D). It is not surprising that neither of the 217 novel mutants is as strong as the classical rty allele (King et al., 1995), as only fertile 218 mutants have been recovered from our epinastic cotyledon genetic screens. In fact, several 219 additional rty-like lines were initially selected in seedlings but lost during propagation due 220 to their lethality in young adults.

221
Backcrosses of TD11B, TF5D, and TH5H to wild-type Col and Ler revealed that all 222 three mutants are recessive ( Table 1). CEL1 assay, sequencing and/or mapping of these 223 mutants were performed and demonstrated that all of these lines are, indeed, new alleles of 224 rty ( Fig. 2A) Table S1) that is less than 200Kb away from the RTY locus. TF5D shows a CEL1-assay 229 detectable polymorphism in the fragment 3 of RTY ( Fig. 2A). Sequencing of this fragment 230 in the TF5D mutant background has uncovered a G1109A nucleotide substitution in RTY 231 genomic DNA that corresponds to a D243N mutation in the 3 rd exon. Furthermore, another 232 recessive rty-like mutant, TJ12H, that harbors an identical mutation to that of TD11B ( Fig.  233 2A) but has been isolated from a different EMS family and thus has arisen independently 234 from TD11B, is phenotypically indistinguishable from TD11B (Table 1), further suggesting 235 that the high-auxin phenotypes of both of these mutants are a consequence of the D243N 236 amino acid substitution in the RTY protein and not some other mutation. Finally, the TH5H 237 mutant has been found to harbor a G215A missense mutation in the 1 st exon of RTY 238 genomic DNA, resulting in a G72D amino acid substitution ( Fig. 2A). Taken together, these 239 results demonstrate that an allelic series of the RTY locus has been identified. Not 240 surprisingly, the severity of the high-auxin phenotypes in RTY mutants (Fig. 3) correlates 241 with the degree of conservation of the specific amino acid affected by the mutation (Fig.  242  2B).

243
To test the ability of the new rty alleles to form functional heteromers in F1 244 transheterozygotes, we intercrossed all of the available RTY locus mutants, i.e. rty (in the 245 wei2-1 background for fertility reasons), TG8B, TD11B, TF5D, and TH5H, and examined the 246 morphology of the F1 generation (Fig. 4). Remarkably, some but not all interallelelic 247 combinations were able to complement each other, potentially offering insights into the 248 structure-function relations in RTY heteromers. For example, the TG8B amino acid 249 substitution mutant was able to complement all other missense alleles of RTY to wild-type 250 level (Fig. 4), demonstrating full interallelic complementation in F1 transheterozygotes.

251
Good complementation was also observed between two stronger RTY alleles, the classical 252 rty and TH5H, whereas no or only very weak complementation was seen between rty or 253 TH5H and the moderate allele TF5D (Fig. 4). Several RTY allele intercrosses (e.g., 254 rty/TD11B, TH5H/TD11B, and TF5D/TD11B F1 transheterozygotes) displayed phenotypes 255 less severe than that seen in the parents (in this case, TD11B) ( Fig. 4, Supplemental Fig.  256 S3), yet the morphology of these F1s was also distinct from that of wild-type plants, 257 suggesting partial complementation. 258 In summary, we found that the phenomenon of interallelic complementation is not 259 uncommon in the RTY gene, with all missense mutations examined complementable by at 260 least one other missense allele. More importantly, although we found that the strength of We next employed molecular modeling to provide structural reasoning for the ability of 273 transheterozygous mutations in RTY to restore the RTY activity and thus lead to a 274 phenotype that is milder than either of the homozygous parents. A three-dimensional (3D) 275 structural model of dimeric RTY was generated using the crystal structure of the closest 276 RTY homolog from Arabidopsis thaliana, the aforementioned aminotransferase MEE17 277 (PDB code: 5WMH) (Holland et al., 2018) (Supplemental Fig. S4). The mutations described 278 above were then mapped onto the wild-type dimeric RTY model (Fig. 5, Table 2), landing 279 in varied locations throughout the dimeric structures. In particular, the D315 residue 280 (mutated in TG8B) is located directly in the dimerization interface, suggesting that an 281 amino acid substitution at this site may affect the efficiency of RTY protein dimerization. 282 This site also falls within 8Å from the PLP cofactor and substrate binding pocket of the 283 other monomer (across the dimerization interface) and lines the binding pocket entrance. 284 To experimentally test the possible involvement of the D315N in the dimer formation, wild-285 type RTY (WT) and the D315N (TG8B) mutant were expressed in a heterologous system as 286 part of bait and prey hybrid proteins in yeast, one fused with a transcriptional activation 287 domain (AD) and another with a DNA binding domain of GAL4 (BD) (Supplemental Fig. S5).

288
We observed that the activity of the LacZ reporter in plates was dramatically reduced when 289 both the bait and the prey carry the D315N mutation (TG8B + TG8B), whereas a nearly 290 wild-type level of the reporter activity was seen for the interallelic combination with WT type morphology suggests that those other alleles of RTY do not interfere with the 301 dimerization process. These results also imply that the D315N mutation does not directly 302 affect the catalytic activity of the protein besides preventing the formation of functionally 303 active RTY homodimers. Therefore, if a heterodimer forms between D315N and a 304 catalytically impaired monomer such as D243N (see next), the resulting dimer should still 305 be able to carry out the C-S lyase reaction (due to the catalytic functionality of D315N). 306 The second RTY mutation for which the structural model provides a likely 307 explanation is D243N (defective in TF5D), for which the side-chain of D243 falls in the 308 binding pocket of the pyridoxal phosphate (PLP) cofactor (between 1-2 Å) ( Fig. 5 inactive.

315
Other RTY mutations, G72D (TH5H), P213S (rty), and G260E (TD11B/TJ12H), map 316 away from the catalytic pocket or the dimerization interface and therefore are harder to 317 interpret with respect to the mutant defects they trigger (Fig. 5, Table 2). In these cases, 318 we wondered whether the phenotypes of different interallelic combinations could provide 319 some clues on the structure-function relationship of these mutations. The wild-type 320 phenotype of the interallelic combination between G72D and the dimerization mutant 321 D315N suggests that the G72D is unlikely to directly affect dimerization. On the other hand, 322 the strong transheterozygous mutant defects of the G72D and the PLP-binding-impaired 323 D243N combination would suggest that these two mutants may affect the same function of 324 the protein, in other words, the PLP binding and/or substrate recruitment. In fact, this is 325 not so far-fetched based on the structure analysis, as the residue G72 is solvent exposed 326 and lines the entrance to the PLP binding channel in our structural model (Fig. 5). 327 The positions of the two other missense mutations, P213S (that is found in our strongest 328 allele, rty) and G260E (seen in the weakest allele in the series, TD11B), in the 3D model do 329 not provide strong clues on the possible structure-function alterations in these mutants, as 330 they are located distal to the PLP binding pocket. P213 and G260 occupy a center of mass 331 (COM -a point representing the mean position of the matter in a protein), space ~7 and 332 13Å away from D243 (the residue in the substrate binding pocket likely implicated in PLP 333 binding), respectively (Fig. 5).

335
Experimental testing of the 3D structural model of RTY 336 337 To test the potential capacity of the computational model of RTY to explain the phenotypes 338 (complemented or not complemented) of F1 transheterozygotes, we have mapped the 339 amino acid substitution of another novel RTY allele, IK6G12, which has been recently 340 identified in our laboratory from another genetic screen in the wei8-1 mutant background.

341
From prior studies we knew that wei8-1 is deficient in the tryptophan aminotransferase 342 activity in the parallel IPyA branch of auxin biosynthesis (Supplemental Fig. S1) and this 343 mutation does not suppress the epinastic cotyledon and leaf phenotypes of rty and sur2 344 (Stepanova et al., 2008). IK6G12 (wei8-1) is a moderately strong recessive allele of RTY 345 (Fig. 3) that harbors a C1890T missense mutation in the sixth exon that alters proline 404 346 to serine ( Fig. 2A, B). Based on our 3D model, P404 is located ~19 Å from the PLP binding 347 pocket, ~40 Å from the PLP binding pocket within the other monomer, and ~16 Å from the 348 nearest residue in the dimerization interface (Y104) (Fig. 5, Table 2). Thus, the structural 349 effects of P404S are likely to be indirect through creating conformational changes to the 350 entrance of the PLP binding pocket or alteration to the dimerization interface and, 351 therefore, should be functionally different than the structural consequences of all other 352 missense mutants characterized in our study. Accordingly, we predicted that the IK6G12 353 allele should be able to, at least in part, complement all of the available missense mutants. 354 In fact, when we performed crosses between IK6G12 and other alleles of RTY to 355 experimentally test our predictions, we found that indeed IK6G12 was able to fully 356 complement TG8B and partially all other missense mutants in the F1 transheterozygotes 357 (Fig. 4). These observations support the idea that interallelic complementation is possible 358 for mutant combinations that disable functionally non-overlapping protein domains. Our 359 findings are also consistent with the notion that lack of complementation may be indicative 360 of the two allelic mutations affecting domains of similar functions. 361 As the genetic analyses and computational modeling of RTY described above 362 strongly indicate that the use of missense mutations in complementation tests can be 363 misleading, we decided to also utilize a true null allele of RTY. Our expectation was that a 364 mutant that makes no full-length RTY protein should be unable to complement any of the 365 amino acid substitution alleles of rty. We identified a previously uncharacterized 366 insertional allele of RTY, Sail_1164_G12 (CS878294), that harbors a T-DNA at the beginning 367 of the second exon of RTY immediately upstream of the nucleotide 638 in the RTY genomic 368 DNA ( Fig. 2A). This T-DNA line can still make partial 3' RTY mRNA, as detected by qRT-369 PCR with primers that anneal downstream of the insertion site (Supplemental Fig. S6), but 370 the levels of the 3' product are reduced more than three fold. The corresponding truncated 371 protein, if produced, should be non-functional as it is expected to lack at least the first 207 372 amino acids, since the first available methionine downstream of the T-DNA insertion site in 373 frame with the RTY protein is M208. Based on our mutant analysis, even a single amino 374 acid substitution in the N-terminus, G72D (TH5H), leads to severe functional consequences, 375 and the deletion of the entire N-terminal part of the protein would remove significant 376 portions of the dimerization interface (86%) as well as nearly half of the PLP binding 377 pocket. It is highly unlikely that this protein can fold correctly, let alone be able to dimerize 378 and function. 379 Consistent with the notion that the Sail_1164_G12 allele is null, the mutant was 380 unable to rescue any of the missense RTY alleles in interallelic combinations (Fig. 4). This 381 lack of complementation is not simply due to a more severe phenotype of Sail_1164_G12 382 compared to the other alleles of RTY employed in this study, as the strength of this T-DNA 383 mutant is similar to that of the classical missense allele, rty, utilized herein (Fig. 3). Since 384 Sail_1164_G12 is an exonic insertional mutant that cannot produce full-length RTY RNA, in 385 F1 transheterozygotes this mutant should not be able to form RTY protein heteromers and 386 should only produce homomeric mutant RTY protein complexes. Consistent with that 387 expectation, for the majority of RTY mutants, the severity of the phenotypes in the F1 388 generation of inter-allelic crosses with the T-DNA allele of RTY was found to be enhanced 389 with respect to that of the homozygous missense mutant parents (Fig. 4), suggesting that 390 these interallelic RTY mutants may be haploinsufficient. Surprisingly, the 391 TG8B/Sail_1164_G12 F1 transheterozygotes were phenotypically indistinguishable from 392 TG8B itself (Fig. 4) In contrast with the problems encountered when employing missense RTY mutants 420 in complementation testing, utilization of the true null T-DNA allele of RTY, Sail_1164_G12, 421 was very helpful at establishing (or, in this case, confirming) the allelic relationships 422 between all RTY gene mutants. This discovery emphasizes the critical importance of using 423 true (i.e., full-length RNA) null alleles for all complementation tests, at least for the genes 424 whose products may form dimers or higher order complexes and, therefore, interallelic 425 complementation is possible. 426 Interallelic (also known as heteroallelic) complementation in transheterozygotes 427 has been observed in many species, from yeast to humans, but is not a very commonly Celenza and colleagues stated lack of complementation in three crosses between alf1-1, rty, 495 and hls3 mutant, again suggesting that these mutations affect the same gene (Celenza et al., 496 1995). Finally, the seven sur1 alleles have each been crossed to one other sur1 allele and 497 also found to be non-complementing (Boerjan et al., 1995). One obvious difference 498 between these previously reported alleles of RTY and our new allelic series is the strength 499 of the mutations. The EMS mutants described herein are phenotypically milder relative to 500 the rty allele and are partially fertile (Fig. 3D), as these were derived from large M2 501 families from which all strong mutants (that were initially selected but failed to set seeds) 502 have been simply lost. Unlike our missense alleles, many (but not all) of the previously 503 published RTY alleles may be null. Nonetheless, given that the phenotypically null rty 504 mutant could complement several of our weaker alleles of RTY, the cautionary take-home 505 message of this study stays the same: allelism tests require the use of a full-length RNA-null 506 mutant as a reference in complementation crosses or can otherwise provide misleading 507 results. In our case, in light of the complementation observed in rty crosses, we chose to 508 invest time into the characterization of these mutants because initially we erroneously 509 concluded that these mutants were novel. 510 511 Molecular modeling of dimeric RTY 512 513 The computational modeling of the three-dimensional RTY structure provided 514 important clues to explain the F1 generation transheterozygote complementation in the 515 RTY heterodimers and also demonstrated potential predictive power of the model. To our 516 knowledge, this is the first time that these types of approaches have been used to Discerning structure-to-function relationships requires quality structural data (e.g., 526 from nuclear magnetic resonance, X-ray crystallography, or cryo-electron microscopy) for 527 the protein of interest or a related protein. To our knowledge, no protein structure is 528 currently available for RTY from Arabidopsis thaliana, but several related proteins have 529 been structurally characterized through crystallization. Although the RTY protein sequence 530 aligns with tyrosine and other aromatic amino acid aminotransferases from mouse, human, 531 and bacterial proteomes, we chose the sequence of MEE17 (PDB 5WMH) from Arabidopsis 532 thaliana, a bifunctional aspartate aminotransferase and glutamate/aspartate-prephenate 533 aminotransferase (Holland et al., 2018), as the template for our analysis. MEE17 is a far 534 better template for this molecular modeling study, as it is derived from the same species as 535 RTY, harbors PLP in the binding pocket, includes an extra alpha helical structure that is 536 lacking in the related human tyrosine aminotransferase (PDB 3DYD) (Supplemental Fig.  537 S4), contains coordinates for 399 out of 475 residues, and aligns well with the RTY 538 sequence, with an E-value of 3.8E-17. 539 The structural interrogation of the RTY molecular model presented here allowed us 540 to infer possible structure-to-function relationships for the mutations characterized in this 541 study. We worked under the premise that mutations that affect the same functional domain 542 should not complement each other in the transheterozygous plants. This basic assumption, 543 together with the structural models, provides possible mechanism(s) for the disruption of 544 the dimerization interface, cofactor binding pocket, or alteration of the catalytic domain. 545 For example, D243 is likely directly involved in PLP/substrate binding and any disruption 546 in this region is likely to interfere with RTY's ability to recruit substrates. When examining 547 the phenotypes of the transheterozygous F1 plants, we observed that G72D has the 548 strongest phenotype when combined with the D243N mutation, suggesting that both affect 549 the same functional domain, in this case, the PLP binding pocket. This is further supported 550 by the fact that although G72 is not directly located in the cofactor binding pocket, it is a 551 solvent-exposed residue that lines the channel entrance for this cofactor. Similarly, while 552 the very different phenotypes of the P213 (strong) and G260 (weak) mutants do not 553 suggest that these two mutations affect the same functional domain, their location 554 immediately adjacent to the substrate-binding pocket and the fact that the strongest 555 phenotype of any of the G260E-containing interallelic combinations is the one with P213S 556 insinuates that these two mutations may disable the same protein functionality. 557 Residue D315 is positioned directly in the dimerization interface and any disruption 558 here could lead to alteration of the interface and a decrease in the dimer's ability to form. In 559 addition, this mutation is also in close proximity to the substrate binding pocket of the 560 adjacent monomer (~8Å away). Of these two non-mutually exclusive possibilities, our 561 analysis suggests that the D315N mutations predominantly affects dimerization and is less 562 likely to affect substrate binding. This possibility is supported by the yeast two-hybrid data 563 that revealed reduced binding of the mutant monomers to one another, as well as by the 564 fact that D315N complemented all other missense mutations examined, including those 565 predicted to affect the cofactor and substrate binding, thus disfavoring the possibility of 566 D315N interfering with substrate recruitment. Conversely, the position of P404 in the 3D 567 model failed to provide convincing support for a specific functional alteration in this 568 mutant. Again, the combination of structural data analysis and transheterozygous 569 phenotypes provided a reasonable hypothesis for the structure-function relationship of 570 these mutations. In fact, distinct location of the P404S in the 3D model far away from the 571 dimerization interface and the substrate binding pocket suggested that this mutation may 572 disable a functional domain distinct from those affected by the other missense mutations 573 analyzed. Consistent with this line of thought, we discovered partial complementation in all 574 transheterozygotes containing the P404S mutations, suggesting that it impairs an activity 575 not critically disturbed in any of the other alleles.

576
The 3D molecular model of RTY described above has provided structure-to-function 577 reasons for the observed phenotypes for a majority of the RTY mutant alleles characterized 578 in this study. We cannot rule out more nuanced structural reasons for the phenotypes 579 observed, such as complementary effects of the mutations stabilizing either the substrate-580 binding pocket, dimerization interface, and/or interactions with potential partner proteins 581 in some of the transheterozygous mutant combinations. Future experimental high-582 resolution structural data or molecular dynamic simulations could provide greater insight 583 into the structural effects of these mutations, such as altered conformations within the 584 dimerization interface leading to weaker dimers or within the substrate binding pocket 585 reducing or abolishing substrate recruitment. In a meantime, a combination of genetics and 586 3D computational models enabled us to explain the molecular consequences of several RTY 587 missense mutations and their interallelic combinations. reported. New mutant combinations were generated by crossing various mutants to each 601 other and phenotyping/genotyping the lines in the F2 and F3 generation (see below). 602 Reporter introgression was also done by crosses and phenotyping of progeny in F2 and F3. 603 Seeds were germinated in plates in AT plates (1x Murashige and Skoog salts, 1% Sucrose, 604 pH 6.0 with 1 M KOH, 0.7% Bacto-Agar) in the dark for 3 days and then moved to constant 605 light, as indicated. For propagation, 10-to 14-day-old seedlings were transplanted to soil 606 and grown under 16-hour light/8-hour dark cycle in 1:1 mix of Fafard superfine 607 germinating mix and Fafard 4P mix. 608 A RTY cDNA complementation construct driven by the 35S promoter and fused to a 609 C-terminal TAP-tag in LIC6 vector was ordered from ABRC (DKLAT2g20610) and 610 transformed into TG8B via floral dip method (Clough and Bent, 1998). T1s were selected in 611 gentamycin and propagated, with images of complemented lines taken in the T2 612 generation.

613
EMS mutagenesis was performed on imbibed Columbia seeds as described (Guzmán 614 and Ecker, 1990). The M1 generation was propagated in families of approximately 1000 615 plants each. A total of 27 M1 families were generated and screened in the M2 generation.

616
M2 seeds were plated at 1000-2000 plants per 15cm Petri plate filled with 50ml of AT 617 media (two plates per family), stratified in the cold for 2-4 days, germinated in the dark for 618 3 days and then transferred to light for additional 5-10 days. The plates were periodically 619 screened visually for cotyledon epinasty. Putative mutants were picked, propagated in soil 620 and re-tested in the M3 generation. One additional RTY allele, IK6G12, was derived from an 621 equivalent EMS mutagenesis carried out in the wei8-1 mutant background. 622 623 PCR-based mutant mapping 624 Physical mapping of new mutants was performed in the F2 generation of crosses to Ler. 625 Epinastic F2 seedlings were picked from plates and transplanted to soil. A single leaf per 626 plant was harvested, its genomic DNA extracted, and short genomic fragments that vary in 627 length between Col and Ler were PCR-amplified in 10uL reactions (1uL genomic DNA, 1 uL 628 10xPCR buffer, 0.25uL 2 mM dNTPs, 0.25uL homemade Taq polymerase, 0.25 mL 20uM 629 forward primer, 0.25uL 20uM reverse primer, and 7uL H2O) using 40 cycles of the 630 following PCR program: 30 s at 94°C, 30 s at 56°C, 1 min at 72°C. PCR products were 631 separated on 3-4% agarose 1xTAE gels, photographed, and scored. The indel PCR marker 632 primers F26H11-1-F1 and F26H11-1-R1 utilized in mapping of RTY mutants are listed in 633  Table S1.

635
Mutant genotyping 636 Plant genotyping was performed by CEL1 assay and/or Sanger sequencing (for the EMS 637 mutants) and by PCR (for T-DNA mutants). PCR primers are listed in Supplemental Table  638 S1. PCR conditions were the same as described for mapping, except longer amplification 639 times were allowed (1min per Kb). 640 For the CEL1 assay, gene-specific primers were used to amplify overlapping gene 641 fragments from the mutant and wild-type Columbia samples in 20ul PCR reactions (2uL 642 genomic DNA, 2 uL 10xPCR buffer, 0.5uL 2 mM dNTPs, 0.5uL homemade Taq polymerase, 643 0.5 mL 20uM forward primer, 0.5uL 20uM reverse primer, and 14uL H2O) using 40 cycles of 644 the following PCR program: 30 s at 94°C, 30 s at 56°C, 1-2 min [1min per kb] at 72°C. 5ul of 645 each mutant and 5ul of the wild-type sample for each fragment were combined, and then 646 denatured and annealed to each other in a Thermocycler by heating the mixture for 10 min 647 at 99°C, reducing the temperature to 70°C, and running 70 steps of the program reducing the 648 temperature 0.3°C per step, keeping the samples for 20 seconds at each temperature, going 649 from 70°C to 49°C. Annealed 10ul samples were combined with 10ul CEL1 mix (2ul 10xCEL1 650 buffer, 1ul CEL1, 7ul diH2O), incubated at 45°C for 15-20 minutes, and then immediately 651 moved to ice. Digested chilled samples were resolved on a 1% agarose 1xTAE gel by gel-652 electrophoresis, photographed and scored. 653 654 GUS staining and basic GFP microscopy 655 Samples harboring the DR5:GUS transgene were harvested in ice-cold 90% acetone and 656 stored at -20C overnight or longer. To stain for GUS, samples were washed once with Rinse 657 Buffer (50mM Sodium Phosphate buffer, pH7, 0.5mM K3Fe(CN)6, 0.5mM K4Fe(CN)6) and 658 stained overnight in Rinse Buffer supplemented with 1ug/ml X-gluc (with X-gluc dissolved 659 in DMSO at 5mg per 100ul and diluted with Rinse Buffer to 1ug/ml final concentration). 660 Staining reactions were stopped with 15% ethanol. To remove chlorophyll, GUS-stained 661 seedlings were incubated for 2-3 days in 70% EtOH at 37-50°C. Samples were 662 photographed using Q Capture software with a Q Imaging digital camera hooked to a Leica 663 dissection scope. To visualize expression of DR5:GFP, a Zeiss Axioplan epifluorescence 664 microscope was utilized. Images were captured with a Diagnostic Instruments Color 665 Mosaic camera using Spot Insight software.

667
Yeast two-hybrid assay 668 To test the level of interaction between RTY monomers, we employed the yeast two-hybrid 669 assay. Binding between wild-type monomers, TG8B mutant monomers, and wild type with 670 TG8B monomers was evaluated. The cDNA clone G10872 (ABRC) was employed as RTY-671 WT and used as template for the PCR-based approach to generate the TG8B mutant ORF. 672 To obtain TG8B, three PCR reactions (iProof, BioRad) were performed: PCR 1 using 673 primers SUR1_For and sur1_RevTG8B_Internal; PCR 2 with sur1_ForTG8B_Internal and 674 SUR1_Rev (No STOP); and the fusion PCR 3 to join together both fragments harboring the 675 mutation was conducted using the external primers SUR1_For and SUR1_Rev (No STOP) 676 (see Supplemental Table S1 for primer information). The final PCR product was subcloned 677 into pENTR/D-Topo. Both WT and TG8B RTY were transferred to pACT-GW (GAL4-678 Activation tested for lack of trans-activation and then, transformed with the "prey" constructs 682 containing the GAL4-AD. Interaction between "bait" and "prey" was inferred from the 683 activity of the LacZ reporter (Deplancke et al., 2006). Qualitative colony-lift assays were 684 performed to assess the activity of β-galactosidase by visual scoring of the levels of the 685 resulting blue staining in the presence of X-gal (Goldbio) substrate (Deplancke et al., 2006). 686 To quantify the β-galactosidase activity, lysed yeast cells were resuspended in Z-buffer 687 (Deplancke et al., 2006), supplemented with O-nitrophenyl-beta-D-galactopyranoside, 688 ONPG (Sigma), and the amount of yellow product obtained from the ONPG catalysis was 689 can suppress the high-auxin phenotypes of TG8B. 8-day-old seedlings grown in plates for 3 956 days in the dark followed by 5 days in the light and soil-grown 4-week-old adults are 957 shown. (F) GUS staining in the TG8B wei2 and TG8B wei7 double mutants is reduced 958 relative to TG8B itself. 959     Col  CS878294 rty  TH5H   IK6G12  wei8-1  TF5D  TG8B TD11B   Col  CS878294 rty  TH5H  IK6F12 wei8-1 TF5D TG8B TD11B   Figure 4. Interallelic combinations of RTY mutants. Phenotypes of 4-week-old soil-grown F1 and parental plants are shown. "X" marks the lines that failed to set seeds in two independent F1 propagation experiments. Note that the rty wei2-1 and TH5H parental lines are able to set a limited amount of seeds in some trials.  Supplemental Figure S2 A B  Figure S2. Lack of suppression of the TG8B, rty, and sur2 defects by one copy of wei2-1. 8-day-old seedlings grown in plates for 3 days in the dark followed by 5 days in the light are shown in upper panels (A, B, C). 4-week-old soil-grown (A, C) and 3-week-old plate-grown (B) plants are displayed in lower panels. Scale bars in the lower panels represent 5 mm.