Glyceollin Transcription Factor GmMYB29A2 Regulates Soybean 3 Resistance to Phytophthora sojae

Glyceollin isomers I, II, and III the major secondary metabolites of that, collectively with other 5- deoxyisoflavonoids, provide race-specific resistance to Phytophthora sojae. The NAC- 60 family transcription factor (TF) GmNAC42-1 is an essential regulator of some but not all 61 glyceollin biosynthesis genes, indicating other essential TF(s) of the glyceollin gene 62 regulatory network remain to be identified. Here, we conducted comparative 63 transcriptomics on soybean hairy roots of the variety Williams 82 (W82) and imbibing 64 seeds of Harosoy 63 (H63) upon treatment with wall glucan elicitor (WGE) from P. sojae 65 and identified two homologous R2R3-type MYB TF genes, GmMYB29A1 and 66 GmMYB29A2 , upregulated during the times of peak glyceollin biosynthesis. Overexpression and RNAi silencing of GmMYB29A2 increased and decreased expression of GmNAC42-1 , GmMYB29A1 , and glyceollin biosynthesis genes and metabolites, 69 respectively, in response to WGE. By contrast, overexpressing or silencing GmMYB29A1 70 decreased glyceollin I accumulation with marginal or no effects on the expressions of glyceollin synthesis genes, suggesting a preferential role in promoting glyceollin turnover and/or competing biosynthetic pathways. GmMYB29A2 interacted with the promoters of 73 two glyceollin I biosynthesis genes in vitro and in vivo . Silencing GmMYB29A2 in W82, a soybean variety that encodes the resistance gene Rps1k , rendered it compatible with


Introduction
7 bicolor) (Höll et al., 2013;Frerigmann et al., 2015;Ibraheem et al., 2015). By comparing 142 the transcriptomes of soybean plants responding to a novel glyceollin biosynthesis 143 elicitor. namely acidity stress, and the suppressor dehydration, we identified the NAC 144 (NAM/ATAF1/2/CUC2)-family TF gene GmNAC42-1. GmNAC42-1 is up-and down- worldwide, respectively. The major source of resistance relied upon by breeders has been 160 the resistance to P. sojae (Rps) genes that primarily encode nucleotide binding site 161 leucine-rich repeat (NBS-LRR) receptor proteins that recognize on a gene-for-gene basis 162 the effector proteins encoded by P. sojae avirulence (Avr) genes (Gao et al., 2005;Song 163 et al., 2013). Rps1k has been the most widely relied upon Rps gene by breeders in the 164 major soybean-producing regions of the U.S. for the last two decades because it has 165 provided resistance to several of the most commonly encountered races of P. sojae (Gao 166 et al., 2005). Soybean genotypes encoding Rps1k , such as Williams 82 (W82) and 8 Rps1k (Yoshikawa et al., 1978;Hahn et al., 1985). RNAi silencing of the CHALCONE 170 REDUCTASE (CHR) gene ( Fig. 1) has provided genetic evidence that daidzein and/or the 171 glyceollins mediate Rps1k and Rps1c resistance to race 1 P. sojae (Graham et al., 2007). 172 Further, overexpressing the biosynthesis genes 4-COUMARIC ACID: COENZYME A 173 LIGASE (4CL) or ISOFLAVONE REDUCTASE (IFR) that increased the levels of 5-174 deoxyisoflavonoids enhanced resistance to P. sojae (Cheng et al., 2015;Chen et al., 175 2019). However, similar to many other plant-pathogen interactions, the exact 176 metabolite(s) responsible for providing resistance have remained unknown. Here, we 177 have identified two TF genes that have roles in regulating the biosynthesis of glyceollin I. 178 We also provide evidence that glyceollin I is the 5-deoxyisoflavonoid responsible for 179 providing resistance to race 1 P. sojae. 12 found that 'Sequence-specific DNA binding TF activity' was the major gene ontology, 218 comprising 14.3% (Fig. 3B). The myeloblastosis (MYB) family of TFs was the most 219 prevalent in this ontology (14.0%), followed by WRKY family genes (11.3%) (Fig. 3C). 220 Among the ten most highly upregulated TFs were three ETHYLENE RESPONSE 221 FACTOR (ERF) and three MYB family genes ( Table 2). Since evidence suggested that 222 ethylene is not a regulator of glyceollin biosynthesis (Paradies et al., 1980), we chose to 223 investigate further the MYB genes. The two most highly upregulated MYB genes were GmMYB29A1 and GmMYB29A2 228 ( Table 2). WGE increased their expressions 3.33-and 2.81-fold in H63 seeds and 2.48-229 and 0.87-fold in W82 hairy roots, respectively. RT-qPCR confirmed their upregulation by 230 WGE treatment in both tissues (Fig. 4A). The G. max RNA-seq Atlas (SoyBase.org) 231 demonstrated that the expression of both genes was not developmentally regulated, like 232 G4DT, and unlike the isoflavone glycoside biosynthesis and TF genes HIDH and 233 GmMYB176 (Fig. 4B). During the course of our study, a closely related soybean MYB 234 gene GmMYB29 was implicated by GWAS in positively regulating isoflavone glycoside 235 biosynthesis in developing soybean organs (Chu et al., 2017). The RNA-seq Atlas 236 revealed that GmMYB29 expression was developmentally regulated like HIDH and 237 GmMYB176 (Fig. 4B). 238 A phylogenetic analysis of GmMYB29A1 and GmMYB29A2 with representative 239 proteins from each of the MYB subgroups indicated that they were most closely related 240 to the R2R3-type MYBs AtMYB14 and VvMYB14 (Fig. 4C) (Chen et al., 2013;Höll et al., 2013). Other more distantly related proteins in this cluster 14 of camalexin phytoalexins in Arabidopsis (Frerigmann et al., 2015), AtPAP1/AtMYB75 246 for anthocyanin biosynthesis (Borevitz et al., 2000), and AtPFG1/AtMYB12, 247 AtPFG2/AtMYB11, and AtPFG3/AtMYB111 for flavonol glycoside biosynthesis 248 (Stracke et al., 2001). An outlier was GmMYB363, whose gene was also highly 249 upregulated by WGE in both H63 seeds and W82 hairy roots ( Table 2). The isoflavonoid 250 regulator GmMYB176 was not found in this cluster.

263
GmMYB29A2 encoded a serine at this residue like MYB14 and MYB15 TFs from 264 Arabidopsis, grapevine, and Lotus japonicus, whereas GmMYB29A1 encoded a proline 265 (see Supplementary Fig. S1). 266 Overall, we selected GmMYB29A1 and GmMYB29A2 for functional analysis since they 267 had similar MYB DNA binding domains as GmMYB29 and thus could potentially 268 regulate some of the same isoflavonoid genes. To evaluate whether GmMYB29A1 and/or GmMYB29A2 regulate glyceollin biosynthesis, 272 we first silenced their gene expression in WGE-treated W82 hairy roots. RNAi triggers 273 were designed to target ~250 bp of their respective 3'-UTRs (Fig. 5A). A 3.2-fold 274 silencing of GmMYB29A1 did not affect the expression of GmMYB29A2, 275 or glyceollin biosynthesis genes with the exception of a 1.5-fold reduction in the 276 expression of G4DT (Fig. 5B). This was accompanied by a 1.6-fold reduction in the 277 levels of glyceollin I metabolites (Fig. 5C). By contrast, a 2.2-fold silencing of 278 GmMYB29A2 reduced the expressions of GmMYB29A1, IFS2,I2'H and 279 G4DT from 1.7-to 15.2-fold (Fig. 5D). It also resulted in a 3.4-fold reduction in the 280 amounts of glyceollin I and a 1.5-to 4.9-fold increase in the amounts of 6''-O-281 malonyldaidzin, daidzein, and 6''-O-malonylgenistin (Fig. 5E).

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To explore further the functions of GmMYB29A1 and GmMYB29A2, we overexpressed 283 each of their coding sequences (CDSs) in W82 hairy roots using the 35S viral promoter 284 (p35S) to drive transcription. Overexpressing GmMYB29A1 marginally downregulated 285 GmMYB29A2 and G4DT (1.33-to 1.45-fold) with no other changes in glyceollin gene 286 expressions (Fig. 5F). It also caused a 1.9-fold reduction in glyceollin I metabolites (Fig. 287 5G). By contrast, the overexpression of GmMYB29A2 led to upregulation of GmNAC42-288 1, GmMYB29A1, and all the tested biosynthesis genes in WGE-treated roots (Fig. 5H). In 289 the mock (H 2 O)-treated roots, it also upregulated all genes except for IFS1 and IFS2 (Fig. 290 5H). It did not result in glyceollins accumulating in H 2 O-treated roots, but it enhanced the 291 levels of all glyceollin metabolites 2.0-to 4.2-fold in WGE-treated roots (Fig. 5I). Taken 292 together, these results identify GmMYB29A2 as a positive regulator of GmNAC42-1 that 293 is essential for the full activation of glyceollin I biosynthesis.

295
GmMYB29A2 binds the promoters of glyceollin biosynthesis genes IFS2 and G4DT 296 To determine if the putative TF GmMYB29A2 is located in nuclei, we cloned its CDS 297 downstream of an N-terminal synthetic green fluorescent protein (GFP) tag. We CaMV-35S promoter (p35S). nGFP-GmMYB29A2 localized predominantly to the 300 nucleus but was also found in the cytosol as shown by co-localization with propidium 301 iodide fluorescence (see Supplementary Fig. S2). By contrast, GFP expressed by the 302 empty vector was localized to the cytosol and other extra-nuclear compartments. To 303 determine whether the GmMYB29A2 protein directly binds the promoters of glyceollin 304 biosynthesis genes, we conducted electrophoretic mobility shift assays (EMSAs) using 305 N-terminal human influenza hemagglutinin (HA)-GmMYB29A2 translational fusion 306 proteins purified from Escherichia coli and two labeled oligonucleotide probes encoding 307 highly similar predicted MYB elements from G4DTpro2 and IFS2pro2 promoter regions 308 (Fig. 6A). The HA-GmMYB29A2 protein caused a gel shift for both probes that was  Table S5).

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Yeast containing GAL4 AD -GmMYB29A2 exhibited more growth than GAL4 AD -vector 319 for both G4DT and IFS2 promoter segments (Fig. 6D). Most growth was observed for 320 promoter segments that had the greatest number of predicted MYB REs (compare  GmMYB29A2 mediates resistance to race 1 P. sojae 325 Since 5-deoxyisoflavonoids provide race-specific resistance to P. sojae (Subramanian et 326 al., 2005;Graham et al., 2007), we set out to determine whether the glyceollin regulator 327 GmMYB29A2 has a role in regulating resistance. To do this, we silenced GmMYB29A2 328 using RNAi in hairy roots of variety W82. W82 has Rps1k-mediated race-specific 329 resistance to race 1 P. sojae. We also overexpressed the GmMYB29A2 CDS in the roots  lesions that spread from the point of inoculation with race 1 P. sojae (Fig. 7A). Water-335 soaked lesions and mycelium growing out of infected tissues were highly visible in 336 20 accumulated 81.8 µg gt -1 (Fig. 7C), which is slightly below the ED 50 (Yoshikawa et al., 341 1978). No significant reductions in the levels of other 5-deoxyisoflavonoids or genistein-342 derived isoflavonoids were observed. In dramatic contrast, Williams overexpressing 343 GmMYB29A2 4.1-fold caused a 4.0-to 5.8-fold decrease in disease progression compared 344 to the empty vector control (Fig. 7D). Water-soaked lesions and mycelium growth out of 345 the tissues were readily visible for Williams empty vector roots, but were dramatically 346 reduced in the Williams p35S::GmMYB29A2 roots (Fig. 7E). The amount of glyceollin I 347 in p35S::GmMYB29A2 at 24 h preceding the infection measurements was 110 µg gt -1 , 348 which was 2.0-fold higher than the empty vector control (Fig. 7F)

355
GmMYB29A2 is essential for the full elicitation of glyceollin biosynthesis 356 Using RNA-seq we found that transcripts of the R2R3 MYB TF genes GmMYB29A2 and 357 GmMYB29A1 were upregulated at the time of peak glyceollin biosynthesis upon 358 elicitation with P. sojae WGE in two soybean genotypes and tissues, namely the hairy 359 roots of W82 and in imbibing seeds of H63 (see Supplemental Tables S1, S2). Their  Table S3). GmNAC42-1 is a TF that is essential but insufficient to fully activate were almost undetectable in the absence of an elicitor but were highly upregulated with 372 glyceollin biosynthesis by WGE (Fig. 4A, B). Second, overexpressing or silencing its 373 gene expressions in W82 hairy roots increased and decreased, respectively, the levels of 374 glyceollin I metabolites and transcripts of G4DT, a gene that is specific to glyceollin I 375 biosynthesis (Fig. 5D, H). Third, the GmMYB29A2 protein bound the G4DT promoter in 376 Y1H and EMSA systems (Fig. 6). Finally, overexpressing and silencing GmMYB29A2 377 in soybean roots inoculated with P. sojae increased and decreased glyceollin I 378 metabolites, respectively, and had no effect on the amounts of isoflavone glycosides ( Fig.   379

380
GmMYB29A2 was only highly expressed upon treatment with an elicitor, however 381 ectopically overexpressing it in the absence of an elicitor provided some insight into its function. Since p35S::GmMYB29A2 upregulated G4DT in the absence of WGE treatment 383 but did not result in glyceollin biosynthesis, this suggested that it alone cannot upregulate 384 all genes required to convert daidzein to glyceollins. Genes that may not be upregulated Grisebach, 1988). Our future work will aim to identify all genes that are directly 396 regulated by GmMYB29A2.

397
Two independent experiments overexpressing GmMYB29A1 in W82 hairy roots 398 demonstrated that GmMYB29A1 slightly downregulated GmMYB29A2 and G4DT but not 399 other glyceollin biosynthesis genes (Fig. 5). It also reduced glyceollin I metabolite levels. 400 P. sojae elicitation was previously shown by pulse-chase experiments to enhance not 401 only glyceollin I biosynthesis but also its turnover (Bhattacharyya and Ward, 1987). The 402 study suggested that glyceollin I, like several other phytoalexins, was not a pathway 'end 403 product'. Since overexpressing GmMYB29A1 marginally affected the expressions of most 404 glyceollin biosynthetic genes, we suggest that it reduced glyceollin I levels by inducing 405 the expressions of metabolizing enzymes. Thus, the role of GmMYB29A1 may be to 406 limit glyceollin I levels. This could at least in part explain the transient accumulation of 407 glyceollin I upon elicitation (Fig. 2). By contrast, GmMYB29A2 would be required for 408 stimulating glyceollin biosynthesis in part by upregulating GmNAC42-1. bind DNA in Y1H and EMSA systems without a bHLH (Fig. 6). 423 In contrast to their identical N-terminal regions, the C-terminal regions of GmMYB29A1 GmMYB29A1 had only one amino acid difference in their SG2 motif, specifically at 434 amino acid 192 (Fig. 4D). GmMYB29A2 encoded a serine at this residue like MYB14 435 and MYB15 TFs from Arabidopsis, grapevine, and L. japonicus, whereas GmMYB29A1 436 encoded a proline. The two proteins also had major differences in the charges of 10 437 amino acids in the C-terminal region outside of the SG2 motif (Fig. 4D). Thus, it is differences in amino acid residues in their C-terminal regions that somehow distinguishes Here, we found that silencing the R2R3 MYB TF gene GmMYB29A2 in W82 hairy roots 473 reduced only the amounts of glyceollin I and not the other 5-deoxyisoflavonoids and 474 caused a breakdown of race-specific resistance to P. sojae ( Fig. 7A-C). Conversely,     Supplemental Table S6.  Bait strains of YM4271 (MATa,ade5,697 leu2-3, 112, tyr1-501, gal4D, gal80D, ade5::hisG) containing G4DT and IFS2 promoter 698 regions that are integrated into the yeast genome were made as described in (Jahan et al.,    One representative biological experiment out of two is shown.