The MYB Transcription Factor GmMYB78 Negatively Regulates Phytophthora sojae Resistance in Soybean

Phytophthora root rot is a devastating disease of soybean caused by Phytophthora sojae. However, the resistance mechanism is not yet clear. Our previous studies have shown that GmAP2 enhances sensitivity to P. sojae in soybean, and GmMYB78 is downregulated in the transcriptome analysis of GmAP2-overexpressing transgenic hairy roots. Here, GmMYB78 was significantly induced by P. sojae in susceptible soybean, and the overexpressing of GmMYB78 enhanced sensitivity to the pathogen, while silencing GmMYB78 enhances resistance to P. sojae, indicating that GmMYB78 is a negative regulator of P. sojae. Moreover, the jasmonic acid (JA) content and JA synthesis gene GmAOS1 was highly upregulated in GmMYB78-silencing roots and highly downregulated in overexpressing ones, suggesting that GmMYB78 could respond to P. sojae through the JA signaling pathway. Furthermore, the expression of several pathogenesis-related genes was significantly lower in GmMYB78-overexpressing roots and higher in GmMYB78-silencing ones. Additionally, we screened and identified the upstream regulator GmbHLH122 and downstream target gene GmbZIP25 of GmMYB78. GmbHLH122 was highly induced by P. sojae and could inhibit GmMYB78 expression in resistant soybean, and GmMYB78 was highly expressed to activate downstream target gene GmbZIP25 transcription in susceptible soybean. In conclusion, our data reveal that GmMYB78 triggers soybean sensitivity to P. sojae by inhibiting the JA signaling pathway and the expression of pathogenesis-related genes or through the effects of the GmbHLH122-GmMYB78-GmbZIP25 cascade pathway.


Introduction
Phytophthora root and stem rot of soybean is a devastating worldwide soybean disease caused by the oomycete Phytophthora sojae, which can result in billions of dollars in economic losses globally each year on average [1,2].Due to the complexity of the virulence changes in the P. sojae population, new pathogenic types are constantly emerging [3].Therefore, it is necessary to study the genes that respond to P. sojae stress in order to better understand the response mechanism of soybeans under P. sojae-induced stress.These genes will provide useful information for genetic engineering and breeding.
The transcription factor (TF) is also known as the trans-acting factor [4]. Various plant transcription factors such as MYB, bHLH, AP2/ERF, WRKY, and NAC have been reported, with most of these transcription factors playing an important role in responding to the tolerance of plants to external environmental stresses [5][6][7][8][9].Among many transcription factors, MYB is one of the most widely distributed and functionally strong members of the plant transcription factor family [10].According to the number of MYB domains, MYB family is divided into four subfamilies: the 1R-MYB, R2R3-MYB, 3R-MYB, and 4R-MYB family [10].The 1R-MYB family has only one MYB domain, which plays an important role in regulating plant transcription and maintaining the chromosome structure [11].The R2R3-MYB family genes contain two conserved R2 and R3 repeat sequences in the MYB binding domain, and also include a regulatory domain (activating or inhibitory function) in the variable region at the C-terminus.This subfamily, with the most members and diverse functions among the four subfamilies, is widely involved in cell differentiation, secondary metabolism, and stress responses [12,13].The 3R-MYB family genes have a conserved domain composed of R1, R2, and R3, mainly regulating cell differentiation and cell cycle control [14].The 4R-MYB subfamily genes have a conserved domain composed of four R1/R2 repeat sequences, and the number of genes in this subfamily discovered in plants is currently very small [10].
Plant hormones are essential substances that regulate various physiological and biochemical reactions in plants, allowing normal life activities to proceed; The MYB gene expression in plants is closely related to plant hormones [15,16].GhMYB18 can activate gene expression in salicylic acid (SA) and phenylpropane signaling pathways, promoting the synthesis of SA and flavonoid compounds [17].Wheat TaMYB391 and TaMYB29 can promote gene expression in the SA signaling pathway, participating in the biosynthetic pathway of SA [18,19].Research has found that AtMYB44 can interact with the Arabidopsis abscisic acid (ABA) receptor RCAR1/PYL9 and regulate the expression of the ABA response gene RAB18, participating in the ABA signaling pathway [20].AcoMYB4 negatively regulates osmotic pressure by participating in ABA biosynthesis and signal transduction pathways [21].FtMYB22 can interact with the ABA receptor proteins RCAR1/2 to form heterodimers and play a role in the ABA signaling pathway [22].MYB7, as a negative regulator of ABA signaling, participates in seed germination by inhibiting the expression of ABI5 (ABA insensitive 5) [23].GmABAS1 directly binds to the promoter of ABI5 to suppress its expression, thereby enhancing soybean sensitivity to ABA [24].RhMYB108 can bind to the promoters of target genes RhNAC053, RhNAC092, and RhSAG113, participating in ethylene and jasmonic acid (JA) signaling pathways in roses (Rosa hybrida) [25].The R2R3-MYB transcription factor GhMYB25 can interact with GhJAZ2 to regulate the JA signaling pathway [26].Tea tree CsMYB46 and CsMYB105 can interact with CsJAZ3, CsJAZ10, and CsJAZ21 in the cell nucleus, thereby participating in the JA biosynthetic pathway [27].
MYB transcription factors play an important role in responding to fungal, bacterial, and oomycete infections [28][29][30][31].For example, MdMYB73 can promote gene expression in the SA synthesis pathway, increase SA content, and enhance resistance to Botryosphaeria dothidea in apple [6].TuMYB46L (Triticum urartu) can bind to the promoter region of the ethylene synthesis-related gene TuACO3 to inhibit its expression, and the overexpression of TuMYB46L can reduce the ethylene content in wheat, leading to the increased susceptibility of wheat to Blumeria graminis f. sp.tritici [30].The overexpression of AtMYB44 in Arabidopsis downregulates the defense response against Alternaria brassicola [31].Rice myb negatively regulates rice resistance to Pyricularia grise by participating in the JA signaling pathway [32].MdMYB30 enhances transgenic apple callus resistance to Botryosphaeria dothidea by regulating wax biosynthesis, and the overexpression of MdMYB30 in Arabidopsis can enhance resistance to Pseudomonas syringae pv.tomato DC3000 [33].Studies have shown that the overexpression of CaMYB78 can increase chickpea resistance to Fusarium solani [34].The MYB transcription factor CaPHL8 can enhance pepper resistance to Ralstonia solanacearum by directly or indirectly regulating the expression of defense-related genes [35].PalbHLH1 and PalMYB90 can activate the expression of genes related to the flavonoid biosynthesis pathway, thereby increasing the accumulation of flavonoids in poplar and enhancing its resistance to fungal or bacterial infections [36].The R2R3-MYB transcription factor CaMYB39 can induce the expression of genes involved in flavonoid biosynthesis to increase the accumulation of flavonoids, and the overexpression of CaMYB39 can induce defense gene expression and enhance chickpea resistance to Ascochyta rabiei [37].TaMYB391 and TaMYB29 can promote the expression of PR genes and SA signaling pathway genes, and then increase the accumulation of SA, thereby regulating the defense response to Puccinia striiformis f. sp.tritici [18].Furthermore, knocking out TaMYB4 weakens the resistance of wheat to Puccinia striiformis f. sp.tritici [38].VdMYB1 (Vitis davidii) enhances the resistance to Erysiphe necator by binding to the promoter of the key regulatory factor of flavonoid metabolism, Stilbene Synthase (STS), and activating STS expression [39].PnMYB2 (Panax notoginseng) positively regulates its resistance to Fusarium solani by modulating JA signaling, photosynthesis, and the expression of the disease-resistant gene [40].
Our previous study identified an AP2/ERF transcription factor, GmAP2 (Phytozome.Glyma.03G136100), which negatively regulates soybean resistance to P. sojae [41].A downregulated MYB transcription factor named GmMYB78 was identified through a transcriptome analysis of GmAP2-overexpressing transgenic hairy roots.In this study, GmMYB78 (GenBank accession no.XM_003536811.5)was isolated from susceptible soybean cultivar 'Dongnong 50', and its involvement in the defense response against P. sojae was investigated.The overexpression and RNA interference analyses indicated that GmMYB78 negatively regulates soybean resistance to P. sojae, possibly through the downregulation of pathogenesis-related gene expression, JA synthesis, and signal transduction pathways.In addition, it may also be through the GmbHLH122 transcriptional inhibition of GmMYB78 and the GmMYB78 transcriptional activation of GmbZIP25 that inhibits resistance to P. sojae.
In order to analyze the expression profiles of GmMYB78, we used RT-qPCR to detect the transcription level of GmMYB78 in the resistant soybean cultivar 'Suinong 10' and the susceptible soybean cultivar 'Dongnong 50'.As shown in Figure 1A, the relative expression levels in roots, leaves, and cotyledons of the susceptible soybean cultivar 'Dongnong 50' were significantly higher than those of the highly resistant soybean cultivar 'Suinong 10' (** p < 0.01).We further analyzed whether GmMYB78 was induced by P. sojae.The results showed that the expression of GmMYB78 in 'Dongnong 50' increased rapidly after inoculation with P. sojae, and reached the peak at 9 h, and then decreased gradually.By contrast, the expression of GmMYB78 in 'Suinong 10' was rapidly downregulated at 6 h post-inoculation (hpi) (Figure 1B).It was concluded that GmMYB78 could be induced by P. sojae, and the expression level of GmMYB78 was different between resistant and susceptible soybean cultivars.The expression level of GmMYB78 in soybean 'Dongnong 50' was significantly higher than that in soybean 'Suinong 10', indicating that GmMYB78 may be a negative regulator in response to P. sojae infection.
post-inoculation (hpi) (Figure 1B).It was concluded that GmMYB78 could be induced by P. sojae, and the expression level of GmMYB78 was different between resistant and susceptible soybean cultivars.The expression level of GmMYB78 in soybean 'Dongnong 50' was significantly higher than that in soybean 'Suinong 10', indicating that GmMYB78 may be a negative regulator in response to P. sojae infection.
In addition, we assessed the subcellular localization of GmMYB78 in Arabidopsis protoplasts that co-transfected the GmMYB78-GFP construct or 35S:GFP control construct (Figure 1C) with the nuclear marker H2B-mCherry (encoding histone H2B fused to the red fluorescent protein mCherry).As shown in Figure 1D, we detected GFP fluorescence throughout the cells expressing the 35S:GFP control plasmid.By contrast, only GmMYB78-GFP and nuclear marker H2B-mCherry fusion protein were observed in the nucleus.These results proved that GmMYB78 is a nucleus-localized transcription factor.In addition, we assessed the subcellular localization of GmMYB78 in Arabidopsis protoplasts that co-transfected the GmMYB78-GFP construct or 35S:GFP control construct (Figure 1C) with the nuclear marker H2B-mCherry (encoding histone H2B fused to the red fluorescent protein mCherry).As shown in Figure 1D, we detected GFP fluorescence throughout the cells expressing the 35S:GFP control plasmid.By contrast, only GmMYB78-GFP and nuclear marker H2B-mCherry fusion protein were observed in the nucleus.These results proved that GmMYB78 is a nucleus-localized transcription factor.

GmMYB78 Increases Susceptibility to P. sojae in Transgenic Soybean Hairy Roots
To analyze the function of GmMYB78, the plant overexpressing vector GmMYB78-OE (35S:GmMYB78-4myc overexpression) and RNA interference vector GmMYB78-RNAi were constructed and transformed into susceptible soybean cultivar 'Dongnong 50' to produce transgenic soybean hairy roots by the high-efficiency A. rhizogenes-mediated transformation (Figure 2A).We characterized the GmMYB78-OE transgenic hairy roots using RT-qPCR analysis (Figure 2B) and western blotting (Figure S2A), and the GmMYB78-RNAi trans-genic hairy roots using qRT-PCR analysis and Quickstix Kits for Liberty Link (bar) strips (Figures 2B and S2B).After 72 hpi, the GmMYB78-OE transgenic lines displayed more serious disease symptoms than roots infected with the empty vector (EV), with the inoculation site and its surroundings being dark brown and having obvious soft rot (Figure 2A).In contrast, the GmMYB78-RNAi transgenic lines had no obvious symptoms compared with the EV (Figure 2A).The total area of lesions and the relative biomass content of P. sojae were significantly higher in the roots of the GmMYB78-OE lines compared with those infected with the EV (Figure 2C-E).By contrast, the area of lesions and relative biomass of P. sojae were significantly lower in the roots of the GmMYB78-RNAi lines compared with the EV control (Figure 2C-E).Taken together, these results indicated that GmMYB78 is a negative regulator of the response to P. sojae infection.GmEF1β and GmTUB4 were used as the reference genes and expression is relative to that of the EV controls, the values of which were set as 1. (C) Lesion size in GmMYB78-OE, GmMYB78-RNAi, and EV transgenic soybean hairy roots at 72 hpi.(D,E) Relative biomass accumulation of P. sojae in the GmMYB78-OE, GmMYB78-RNAi, and EV roots based on transcript levels of (D) the fungal TEF1 gene and (E) the PSEL1 (P.sojae elicitin gene 1) gene.Fungal biomass is relative to that of the plant.The experiment was performed on three biological replicates, each with three technical replicates, and statistical significance was analyzed using Student's t-test (** p < 0.01).Bars indicate the standard error of the mean.
2.3.GmMYB78 Inhibits Pathogenesis-Related (PR) Gene Expression in Response to P. sojae Infection The PR gene is an important part of the plant defense response to invasive pathogens, and MYB transcription factors can participate in the plant stress response by regulating the expression of PR genes [19,38].To investigate the potential defense mechanism of the GmMYB78-regulated response to P. sojae, we examined the expression of some candidate PR genes GmPR1 (GenBank accession no.AF136636), GmPR2 (GenBank accession no.M37753), GmPR3 (GenBank accession no.AF202731), and GmPR10 (GenBank accession no.FJ960440).The results showed that the expression levels of GmPR2, GmPR3, and GmPR10 were significantly lower than those of EV in GmMYB78-OE hairy roots, while the expression level of GmPR1 was not significantly different from that of EV.In GmMYB78-RNAi hairy roots, the expression levels of GmPR2, GmPR3, and GmPR10 showed the opposite results, and the expression level of GmPR1 was not significantly different from that of EV (Figure 3A-D).The above results showed that GmMYB78 responds to P. sojae by negatively regulating the expression of GmPR2, GmPR3, and GmPR10.
M37753), GmPR3 (GenBank accession no.AF202731), and GmPR10 (GenBank accession no.FJ960440).The results showed that the expression levels of GmPR2, GmPR3, and GmPR10 were significantly lower than those of EV in GmMYB78-OE hairy roots, while the expression level of GmPR1 was not significantly different from that of EV.In GmMYB78-RNAi hairy roots, the expression levels of GmPR2, GmPR3, and GmPR10 showed the opposite results, and the expression level of GmPR1 was not significantly different from that of EV (Figure 3A-D).The above results showed that GmMYB78 responds to P. sojae by negatively regulating the expression of GmPR2, GmPR3, and GmPR10.

GmMYB78 Is a Negative Regulator of JA-Dependent Signaling during the Response to P. sojae
In order to further explore the susceptible mechanism of GmMYB78 to P. sojae and explore its potential downstream target genes, in this study, the transcriptome (RNA-seq) and metabolome analysis were performed on GmMYB78-OE and EV transgenic soybean hairy roots.There were 617 differentially expressed genes (false-discovery rate set at <0.01, fold-change set at ≥2) identified between GmMYB78-OE and EV transgenic hairy roots, including 199 upregulated differentially expressed genes and 418 downregulated differ-  In order to further explore the susceptible mechanism of GmMYB78 to P. sojae and explore its potential downstream target genes, in this study, the transcriptome (RNA-seq) and metabolome analysis were performed on GmMYB78-OE and EV transgenic soybean hairy roots.There were 617 differentially expressed genes (false-discovery rate set at <0.01, fold-change set at ≥2) identified between GmMYB78-OE and EV transgenic hairy roots, including 199 upregulated differentially expressed genes and 418 downregulated differentially expressed genes (Figure 4A,B).The GO functional analysis and KEGG enrichment indicated that these differentially expressed genes are involved in multiple life processes such as the plant hormone transduction pathway and stress response (Figure 4C,D).A metabolomics analysis showed that there were 96 differential metabolites (VIP ≥ 1, fold-change set at ≥2) identified between GmMYB78-OE and EV transgenic hairy roots, of which 17 were upregulated, and 79 were downregulated, including the jasmonic acid transduction pathway (Figure S3A-C).The combined analysis of the transcriptome and metabolome of GmMYB78-OE transgenic hairy roots showed that jasmonic acid (JA) was downregulated in the JA signal transduction pathway, and the expression of the key gene GmAOS1 (Allene oxide synthase (A) Volcano plots of significantly differentially expressed genes in GmMYB78-OE vs. EV transgenic soybean hairy roots after the RNA-seq analysis.(B) Heat map of significantly differentially expressed genes between the EV and GmMYB78-OE transgenic soybean hairy roots, as determined using an RNA-seq analysis.Using a false discovery rate < 0.05 and a fold change ≥ 1 as the screening criteria, a total of 617 differentially expressed genes (DEGs) were identified.The scale bar indicates the fold changes (log2 values).(C) Gene annotation of KEGG classification of differentially expressed genes.The differentially expressed genes are mainly involved in multiple biological processes, including plant-pathogen interaction, and plant hormone signal transduction.(D) Gene Ontology functional classification of the differentially expressed genes.The differentially expressed genes were placed into the three main GO categories: biological process, cellular component, and molecular function.
The combined analysis of the transcriptome and metabolome of GmMYB78-OE transgenic hairy roots showed that jasmonic acid (JA) was downregulated in the JA signal transduction pathway, and the expression of the key gene GmAOS1 (Allene oxide synthase 1) (GenBank accession no.NP_001236432.1) in the JA synthesis pathway was also downregulated (Figure S3D,E).MYB transcription factors play an important role in plant defense responses by regulating JA signaling [40, 42,43].In order to analyze the regulatory role of GmMYB78 in the JA signaling pathway, we measured the JA content in GmMYB78-OE, EV, and GmMYB78-RNAi transgenic soybean hairy roots.The results showed that the JA content in GmMYB78-OE transgenic hairy roots was significantly lower than that in EV, while the JA content in GmMYB78-RNAi was significantly higher than that in EV hairy roots (Figure 5A).In addition, we also analyzed the transcriptional levels of the JA synthesis gene GmAOS1, and two negative regulators of the JA signaling pathway GmJAZ1 (Phytozome.Glyma.07g041400) and GmJAZ2 (Phytozome.Glyma16g010000) [44] in GmMYB78 transgenic hairy roots by qRT-PCR.The results showed that the expression of GmAOS1 was significantly lower in GmMYB78-OE transgenic hairy roots than that in EV, and significantly higher in GmMYB78-RNAi than that in EV roots (Figure 5B), while the expression of GmJAZ1 and GmJAZ2 was significantly higher in GmMYB78-OE transgenic hairy roots than that in EV, and significantly lower in GmMYB78-RNAi transgenic hairy roots than that in EV (Figure 5C,D).In summary, GmMYB78 can inhibit the JA signaling pathway by negatively regulating the JA synthesis gene GmAOS1 expression and reducing the biosynthesis of JA, thereby negatively regulating soybean resistance to P. sojae.GmMYB78 transgenic hairy roots by qRT-PCR.The results showed that the expression of GmAOS1 was significantly lower in GmMYB78-OE transgenic hairy roots than that in EV, and significantly higher in GmMYB78-RNAi than that in EV roots (Figure 5B), while the expression of GmJAZ1 and GmJAZ2 was significantly higher in GmMYB78-OE transgenic hairy roots than that in EV, and significantly lower in GmMYB78-RNAi transgenic hairy roots than that in EV (Figure 5C,D).In summary, GmMYB78 can inhibit the JA signaling pathway by negatively regulating the JA synthesis gene GmAOS1 expression and reducing the biosynthesis of JA, thereby negatively regulating soybean resistance to P. sojae.
Previous studies have shown that JA and salicylic acid (SA) signaling pathways sometimes have antagonistic effects in plants [45,46].In this study, GmMYB78 inhibits the JA signaling pathway (Figure 5).In order to further clarify whether GmMYB78 is involved in the SA signaling pathway, we analyzed the transcriptional level of the SA synthesis gene GmICS (GenBank no.XM_003522145) and the salicylic acid signaling pathway gene GmPAL (GenBank no.NM_001250027) in GmMYB78 transgenic hairy roots.The results showed that the expression levels of GmICS and GmPAL displayed no significant differences between GmMYB78-OE/GmMYB78-RNAi transgenic hairy roots and EV roots (Figure S4), suggesting that GmMYB78 is not involved in the SA signaling pathway.

GmMYB78 Regulates the Transcription of GmbZIP25
To test the activation of the transcription function of GmMYB78, we performed a transient expression assay in yeast cells using a GAL4-responsive reporter system.Transformed yeast cells harbouring DBD-P53 + T-antigen (pGBKT7-53 + pGADT7-T, positive control), BD-GmMYB78 (pGBKT7-GmMYB78), and DBD-GmWRKY31 (pGBKT7-GmWRKY31) [47], which exhibited a transcriptional activation ability in our previous Previous studies have shown that JA and salicylic acid (SA) signaling pathways sometimes have antagonistic effects in plants [45,46].In this study, GmMYB78 inhibits the JA signaling pathway (Figure 5).In order to further clarify whether GmMYB78 is involved in the SA signaling pathway, we analyzed the transcriptional level of the SA synthesis gene GmICS (GenBank no.XM_003522145) and the salicylic acid signaling pathway gene GmPAL (GenBank no.NM_001250027) in GmMYB78 transgenic hairy roots.The results showed that the expression levels of GmICS and GmPAL displayed no significant differences between GmMYB78-OE/GmMYB78-RNAi transgenic hairy roots and EV roots (Figure S4), suggesting that GmMYB78 is not involved in the SA signaling pathway.

GmMYB78 Regulates the Transcription of GmbZIP25
To test the activation of the transcription function of GmMYB78, we performed a transient expression assay in yeast cells using a GAL4-responsive reporter system.Transformed yeast cells harbouring DBD-P53 + T-antigen (pGBKT7-53 + pGADT7-T, positive control), BD-GmMYB78 (pGBKT7-GmMYB78), and DBD-GmWRKY31 (pGBKT7-GmWRKY31) [47], which exhibited a transcriptional activation ability in our previous studies, grew well in synthetic dropout medium without tryptophan, histidine, and adenine [SD (-Trp/-His/-Ade)] and showed α-galactosidase (α-gal) activity, whereas yeast cells that were empty (pGBKT7, negative control) exhibited no α-gal activity (Figure 6A).These data confirm that GmMYB78 has transcriptional activation activity.In order to further analyze the downstream genes activated by GmMYB78, we selected eight stress-related upregulated differentially expressed genes based on the RNA-seq for validation by RT-qPCR in GmMYB78-OE and GmMYB78-RNAi transgenic hairy roots.Of the eight genes tested, the expression levels of GmPRR13, GmbZIP25, GmMYB177, GmLOX9, GmCHS13, and GmARM were upregulated (Figure 6B).On the contrary, the expression levels of GmPRR13, GmbZIP25, GmMYB177, GmLOX9, GmCHS13, and GmARM were downregulated in GmMYB78-RNAi transgenic hairy roots (Figure 6C).In addition, GmbZIP25 transcript levels increased the most in the GmMYB78-OE hairy roots compared to EV (Figure 6B) and showed the strongest decrease in GmMYB78-RNAi (Figure 6C).The above results suggest that GmbZIP25 may be a downstream target of GmMYB78 in defense responses.
MYB transcription factors can specifically bind to MBS (MYB-binding sites) cis-acting elements (T/C) AAC (G/T) G (A/C/T) (A/C/T), (C/T) NGTT (A/G), ACC (A/T) A (A/C) (T/C), and ACC (A/T) (A/C/T) (A/C/T) (A/C/T) in the target gene promoter, and then participate in plant growth and metabolic pathways and respond to various biotic and abiotic stress defense responses [48][49][50][51].We analyzed the cis-acting elements in the promoter of GmbZIP25 and found that pGmbZIP25 contained two MYB transcription factor-specific binding motifs, i.e., CAACGGAT and ACCAAC.Therefore, we further analyzed the binding ability of GmMYB78 to the GmbZIP25 promoter.Firstly, the 2000 bp upstream promoter of GmbZIP25 was cloned, and a 387 bp-797 bp interval, which contained the MYB transcription factor-specific binding motifs CAACGGAT and ACCAAC, was isolated.The recombinant vector pGmbZIP25-pBait-AbAi was constructed according to the 387 bp-797 bp interval sequence.The recombinant vector was transformed into yeast Y1H Gold and the concentration of AbA inhibiting its yeast growth strain was screened.The results showed that, when the AbA concentration was 400 ng/mL, the growth of the bait strain Y1H [pBait-AbAi-pGmbZIP25] was completely inhibited (Figure S5), so the yeast one-hybrid test was carried out at this concentration.
Next, the recombined vector GmMYB78-AD was transformed into the Y1H Gold [pBait-AbAi-pGmbZIP25] yeast strain, with pGADT7-53 or pGADT7 transformed into the yeast cells Y1H Gold [p53-AbAi] as positive and negative controls, respectively.They were then sequentially spread on SD/-Leu and SD/-Leu + AbA 400 agar plates and cultured upside down at 30 • C for 3-7 days.As shown in Figure 6D, all yeast cells grew normally on the SD/-Leu agar plates.The positive control and the GmMYB78-AD-fused Y1H Gold [pBait-AbAi-pGmbZIP25] yeast cells grew normally, while the negative control did not grow on the SD/-Leu + AbA 400 agar plates.The results indicated that GmMYB78 can directly bind to the promoter of GmbZIP25.
Finally, the mechanism of GmMYB78 regulating GmbZIP25 expression was explored by the luciferase reporter system.We cloned the full-length (2000 bp) promoter of Gm-bZIP25 and constructed the reporter vectors p35S:Rluc-pGmbZIP25:LUC.The effector construct harboring GmMYB78 was expressed under the control of the 35S promoter (p35S: GmMYB78-Myc), and an EV control (Figure 6E).The following combinations were each co-transformed into N. benthamiana leaves: p35S:Rluc-pGmbZIP25:LUC + EV and p35S:Rluc-pGmbZIP25:LUC + p35S: GmMYB78-Myc.After 3 d, 1 mM D-Luciferin was sprayed onto the transformed leaves and imaged using a chemiluminescence system.Compared with the leaves transfected with p35S:Rluc-pGmbZIP25:LUC + EV, p35S:Rluc-pGmbZIP25:LUC + p35S: GmMYB78-Myc displayed a significantly enhanced chemiluminescence signal (Figure 6F).The results of the LUC/Rluc relative activity assay further indicated that GmMYB78 could directly enhance GmbZIP25 expression (Figure 6G).The above results indicated that GmMYB78 can directly bind to the promoter of GmbZIP25 to enhance its expression.

GmbHLH122 Regulates the Transcription of GmMYB78
In our previous study, GmMYB78 was downregulated in GmAP2-OE hairy roots according to the transcriptome sequencing analysis [41].To determine whether GmMYB78 is a downstream target gene directly regulated by GmAP2, the binding of GmAP2 to the Gm-MYB78 promoter was analyzed by the yeast one-hybrid.Firstly, the AbA concentration that inhibited Y1H [pBait-AbAi-pGmMYB78] was determined to be 500 ng/mL (Figure S6A).Further yeast one-hybrid experiments showed that the positive control yeast cells grew normally, while Y1H Gold [pBait-AbAi-pGmMYB78] fused with GmAP2-AD and the negative control did not grow on SD/-Leu + AbA 500 agar plates, indicating that GmMYB78 is not a direct downstream target gene of GmAP2 (Figure S6B).In addition, the luciferase reporter system analysis results indicated that, compared with the co-transfection of N. benthamiana leaves with the reporter vector p35S:Rluc-pGmMYB78:LUC and EV, there was no significant difference in the chemiluminescence signal of N. benthamiana leaves co-transfected with the reporter vector p35S:Rluc-pGmMYB78:LUC and the effector vector p35S:GmAP2-Myc (Figure S6C).Furthermore, the relative activity of LUC/REN further proves that GmMYB78 is not a direct downstream target gene regulated by GmAP2 (Figure S6D).
In order to further determine the upstream regulatory factors of GmMYB78, we used a yeast one-hybrid (Y1H) screen of a cDNA library from the soybean disease-resistant cultivar 'Suinong 10' infected with P. sojae based on the promoter (113 bp-670 bp) of GmMYB78.The ESTs from four candidate genes encoding proteins that might be regulatory factors of GmMYB78 are listed in Table S1, of which a bHLH transcription factor GmbHLH122 (XM-003543501.5) was appraised.In this study, we further analyzed the transcriptional regulation of GmMYB78 by GmbHLH122 by the yeast one-hybrid.The recombined vector GmbHLH122-AD was transformed into the Y1H Gold [pBait-AbAi-pGmMYB78] yeast strain, with pGADT7-53 or pGADT7 transformed into the yeast cells Y1H Gold [p53-AbAi] as positive and negative controls, respectively.They were then sequentially spread on SD/-Leu and SD/-Leu + AbA 500 agar plates and cultured upside down at 30 • C for 3-7 days.As shown in Figure 7A, all yeast cells grew normally on the SD/-Leu agar plates.The positive control and the GmbHLH122-AD-fused Y1H Gold [pBait-AbAi-pGmMYB78] yeast cells grew normally, while the negative control did not grow on the SD/-Leu + AbA 500 agar plates.The results indicated that GmbHLH122 can directly bind to the promoter of GmMYB78.

Expression Analysis of GmbLHL122, GmMYB78, and GmbZIP25 in Response to P. sojae
In order to further elucidate the transcriptional regulatory mechanism of the GmbLHL122-GmMYB78-GmbZIP25 module in response to P. sojae, we measured the expression levels of GmbHLH122, GmMYB78, and GmbZIP25 in the susceptible soybean cultivar 'Dongnong 50' and the resistant soybean cultivar 'Suinong 10' upon P. sojae infection.In 'Dongnong 50', GmbHLH122 expression was not obviously activated by P. sojae infection, while GmMYB78 expression was quickly upregulated, reaching a peak at 9 h post-inoculation (hpi); GmbZIP25 was also upregulated and peaked at 12 hpi (Figure 8A).In 'Suinong 10', infection by P. sojae quickly induced the upregulation of GmbHLH122 expression, which reached a peak at 12 hpi, while the expression of GmMYB78 and GmbZIP25 were not obviously activated by P. sojae infection (Figure 8B).These results suggest that GmbLHL122 is highly expressed in resistant soybean cultivars, while GmMYB78 and GmbZIP25 are more highly expressed in susceptible soybean cultivars following P. sojae infection.

Expression Analysis of GmbLHL122, GmMYB78, and GmbZIP25 in Response to P. sojae
In order to further elucidate the transcriptional regulatory mechanism of t GmbLHL122-GmMYB78-GmbZIP25 module in response to P. sojae, we measured the e pression levels of GmbHLH122, GmMYB78, and GmbZIP25 in the susceptible soybean c tivar 'Dongnong 50' and the resistant soybean cultivar 'Suinong 10' upon P. sojae infectio In 'Dongnong 50', GmbHLH122 expression was not obviously activated by P. sojae inf tion, while GmMYB78 expression was quickly upregulated, reaching a peak at 9 h po inoculation (hpi); GmbZIP25 was also upregulated and peaked at 12 hpi (Figure 8A).'Suinong 10', infection by P. sojae quickly induced the upregulation of GmbHLH122 e pression, which reached a peak at 12 hpi, while the expression of GmMYB78 a GmbZIP25 were not obviously activated by P. sojae infection (Figure 8B).These resu suggest that GmbLHL122 is highly expressed in resistant soybean cultivars, wh GmMYB78 and GmbZIP25 are more highly expressed in susceptible soybean cultivars f lowing P. sojae infection.

GmMYB78 Negatively Regulates P. sojae Infection by Inhibiting the Expression of Pathogenesis-Related Genes
Transcription factors MYB play important roles in plants responding to fungal, bacterial, and oomycete infections [28,31,32,[52][53][54].For example, the overexpression of At-MYB44 weakens the Arabidopsis defense response against Pseudomonas syringae pv.tomato DC3000 [31].VaMYB306 can form a transcription complex with VaERF16 to enhance the transcription of the defense-related gene VaPDF1.2,thereby positively regulating resistance to Botrytis cinerea [55].Our previous studies demonstrated that GmBTB/POZ can enhance soybean resistance to P. sojae [56], and an AP2/ERF transcription factor GmAP2 was identified by the yeast two-hybrid, which negatively regulates soybean resistance to P. sojae [41].In this study, RNA-seq analysis of GmAP2 identified a downregulated MYB family transcription factor, GmMYB78.In order to explore whether GmMYB78 is a potential downstream target gene of GmAP2, we used the yeast one-hybrid and dual luciferase reporter system to verify it.The results showed that GmAP2 does not directly regulate the expression of GmMYB78 (Figure S6B-D), suggesting GmMYB78 may indirectly participate in the defense response of soybean to P. sojae through GmAP2.To further explore whether GmMYB78 is involved in the response to P. sojae infection in soybean, transgenic soybean hairy roots of GmMYB78 were analyzed for disease resistance, indicating that GmMYB78 is a negative regulator in response to P. sojae infection (Figure 2).Multiple homologous genes of GmMYB78 were also identified in other species [57][58][59][60], among which JAMYB in Medicago truncatula was identified as a candidate gene related to Erysiphe pisi resistance [59].
PR genes are some of the most important genes in the plant defense response to pathogens [61][62][63][64].Transcription factors can respond to pathogens by regulating the expression of PR genes [65,66].Studies have found that soybean GmERF113 can enhance transgenic soybean resistance to P. sojae by positively regulating the expression of GmPR1 and GmPR10-1 [67].GmNPR1 enhances soybean resistance to P. sojae by activating the expression of GmPR1a, GmPR2, GmPR3, and GmPR10 [47].To investigate whether GmMYB78 can also participate in the response of soybeans to P. sojae by affecting the expression of PR genes, we analyzed the expression levels of PR genes in GmMYB78 transgenic hairy roots.The results indicate that GmMYB78 can downregulate the expression of GmPR2, GmPR3, and GmPR10, rather than GmPR1, reducing soybean resistance to P. sojae.PR1 usually act as effector genes for systemic acquired resistance (SAR), a process mediated by salicylic acid (SA); high expression levels of these genes indicate the activation of SA signaling [68,69].Since GmMYB78 is not involved in the SA signaling pathway, we speculate that one of the reasons why GmMYB78 cannot regulate GmPR1 expression is its inability to respond to SA signaling.These findings suggested that GmMYB78 can reduce the resistance to P. sojae through the negative regulation of pathogenesis-related gene expression.

GmMYB78 Is Involved in Soybean Defense Response to P. sojae through JA Signaling Pathway
The JA signaling pathway plays an important role in plant immunity and plants can respond to biotic stresses through the JA signaling pathway [40, 44,70].For example, Avh94 can interact with the JA signaling repressor JAZ1/2, stabilizing JAZ1/2 to inhibit JA signal transduction, thereby negatively regulating soybean resistance to P. sojae [44].The overexpression of OsMPK15 can lead to the significant downregulation of SA and JA-related genes, thereby negatively regulating rice resistance to Magnaporthe oryzae [71].Cotton GhJAZ2 can interact with GhbHLH171 and inhibit its transcriptional activity, thereby inhibiting JA synthesis, reducing plant tolerance to Verticillium dahliae [72].Rice OsMYC2 activates its expression by binding to the G-box motif in the OsJAZ10 promoter, enhancing sensitivity to JA and positively regulating rice resistance to Xanthomonas oryzae pv.oryzae (Xoo) [73].
It has been reported that MYB transcription factors can participate in plant responses to biotic stress through the JA signaling pathway [40, 74,75].For example, silencing the MYB transcription factor GhODO1 impairs JA-mediated defense signals, inhibits the expression of genes in the JA synthesis pathway, thereby reducing JA accumulation, and negatively regulates the resistance of cotton (Gossypium hirsutum) to Verticillium dahlia [43].RcMYB84 interacts with the key repressor of JA signaling, JAZ1, and their complex binds to the RcMYB123 promoter through the CAACTG motif to inhibit its transcription, thereby reducing rose resistance to Botrytis cinerea [76].In this study, we found that JA was downregulated in the metabolome of GmMYB78 and the key gene for JA synthesis, GmAOS1, showed a downregulated expression in the RNA-seq assay of GmMYB78.Further research found that the JA content was decreased and the expression of GmAOS1 was downregulated in GmMYB78-OE transgenic hairy roots.The JA content and JA pathway-related gene expression in GmMYB78 transgenic hairy roots confirmed that GmMYB78 can reduce soybean resistance to P. sojae by negatively regulating JA synthesis and signal transduction.Research has shown that the JA and salicylic acid (SA) signaling pathways can sometimes have antagonistic effects within plant bodies [45,46].Therefore, the expression levels of genes related to the SA signaling pathway were analyzed in GmMYB78 transgenic soybean hairy roots.The results demonstrate that GmMYB78 does not affect the SA synthesis or gene expression involved in its signaling transduction pathway (Figure S4C,D).Furthermore, SA signaling pathway genes and related metabolites were not found in the transcriptome and metabolome analysis of GmMYB78, leading us to speculate that GmMYB78 is not involved in the SA signaling transduction pathway.These results indicating that GmMYB78 can reduce resistance to P. sojae, potentially functioning via effects on the expression of JA-related genes and reduced accumulation of JA.

GmbHLH122-GmMYB78-GmbZIP25 Regulatory Module Is Involved in the Response to P. sojae in Soybean
Research has shown that MYB transcription factors can be regulated as downstream target genes of defense-related genes, thereby responding to environmental stress [77,78].The wheat stripe rust resistance transcription factor TaWRKY10 may bind to the W-Box elements on the promoters of MYB transcription factors TaLHYa, TaLHYb, and TaLHYd to regulate the expression of TaLHY [79].In order to identify the upstream protein that directly regulates GmMYB78, this study used the GmMYB78 promoter region as bait protein to screen for candidate upstream regulatory genes from a yeast one-hybrid cDNA library induced by P. sojae, and preliminary evidence through the yeast one-hybrid and dual-luciferase reporter systems demonstrated that GmbHLH122 can directly bind to the GmMYB78 promoter and suppress its expression (Figure 7).bHLH transcription factors are widely involved in the response to biotic stress in plants [8,41].For example, the bHLH transcription factor GmPIB1 can significantly reduce the accumulation of ROS (reactive oxygen species) and directly bind to the cis-acting elements in the promoter of GmSPOD1 to inhibit its expression, positively regulating soybean resistance to P. sojae [8].The overexpression of the bHLH transcription factor GhbHLH171 can activate JA synthesis signaling pathways, thereby enhancing the tolerance to Verticillium dahliae in cotton [72].In this study, GmMYB78 is a negative regulator in soybean response to P. sojae infection, and GmbHLH122, as a direct regulator of GmMYB78, was induced by P. sojae infection in resistant cultivar 'Suinong 10' (Figure 8B) and may be involved in the resistance to P. sojae infection in soybean, which will be the focus of future research.
MYB transcription factors activate or inhibit the expression of downstream defenserelated genes by specifically binding to the promoters, thus participating in the regulation of plant defense responses [48][49][50].For example, TuMYB46L can bind to the promoter region of the ethylene synthesis-related gene TuACO3, inhibiting ethylene biosynthesis and negatively regulating wheat resistance to powdery mildew [30].BjMYB1 can bind to chitinase BjCHI1 promoter, activating BjCHI1 expression to enhance transgenic Arabidopsis resistance to Botrytis cinerea [80].CsMYB96 directly binds to the CsCBP60g (calmodulin binding protein 60 g) promoter, activating SA signal transduction to enhance resistance of citrus and Arabidopsis to fungal pathogens [81].This study demonstrates that GmMYB78 is located in the nucleus (Figure 1B) and can function as a transcriptional activation factor to activate the transcription of reporter genes (Figure 6A).In addition, the transcriptome se-quencing of GmMYB78 transgenic soybean hairy roots revealed that GmMYB78 can activate the expression of multiple stress-responsive genes including GmbZIP25 (Figure 6B,C), with GmbZIP25 showing the most significant change in expression in GmMYB78 transgenic hairy roots, thus further validating GmbZIP25 as a downstream target gene directly regulated by GmMYB78.An analysis of the GmbZIP25 promoter revealed the presence of two MYB binding motifs in its promoter region.Further yeast one-hybrid and dual-luciferase reporter system assays confirmed that GmMYB78 can directly bind to the GmbZIP25 promoter and activate its expression (Figure 6D-F).It has been reported that bZIP transcription factors, as important defense signaling genes, are widely involved in plant defense responses to biotic stresses [82].CsAtf1, bZIP transcription factor, can enhance the virulence of Colletotrichum siamense by regulating the expression of CsPbs2 and CsHog1 in the MAPK pathway, thereby reducing rubber tree resistance to anthracnose [83].Knocking out TabZIP74 increased the susceptibility of wheat seedlings to stripe rust [84].Silencing RcbZIP17 can reduce the resistance of roses to Botrytis [85].In this study, GmMYB78 is a negative regulator in soybean response to P. sojae infection, and GmbZIP25, as a target gene directly activated by GmMYB78 (Figure 6D-G), was induced by P. sojae infection in susceptible cultivar 'Dongnong 50' (Figure 8A) and may also be involved in the defense response of soybean to P. sojae.We will further verify the binding of GmMYB78 to the promoter region of GmbZIP25 and analyze the function of GmbZIP25 in the future works of research.This will reveal the role of the GmbHLH122-GmMYB78-GmbZIP25 regulatory pathway in the soybean response to P. sojae infection, providing a theoretical basis for unraveling the molecular mechanisms of GmMYB78 in response to P. sojae.
Based on these results, we propose a model that explains the mechanisms of GmMYB78 response to P. sojae infection (Figure 9).According to our model, in the susceptible cultivar 'Dongnong 50', GmbHLH122 is not activated as a result of P. sojae infection, while GmMYB78 is upregulated and accumulated, and represses downstream GmbZIP25 expression, downregulates the expression of GmPR genes and JA-synthesis-pathway-related genes, and reduces JA content, thereby inhibiting the plant defense responses (left); in the resistant cultivar 'Suinong 10', GmbHLH122 transcription is induced by P. sojae infection.The high levels of GmbHLH122 inhibits the transcription of GmMYB78, which reduce its promotive effect on GmbZIP25 expression, upregulates GmPR genes and JA-synthesis-pathway-related genes, and promotes the accumulation of JA content, thereby enhancing the defense response to P. sojae (right).Thus, our study provides novel insights into the mechanisms by which the GmbHLH122-GmMYB78-GmbZIP25 pathway modulates the defense response of soybean during infection by P. sojae. .Knocking out TabZIP74 increased the susceptibility of wheat seedlings to stripe rust [84].Silencing RcbZIP17 can reduce the resistance of roses to Botrytis [85].In this study, GmMYB78 is a negative regulator in soybean response to P. sojae infection, and GmbZIP25, as a target gene directly activated by GmMYB78 (Figure 6D-G), was induced by P. sojae infection in susceptible cultivar 'Dongnong 50' (Figure 8A) and may also be involved in the defense response of soybean to P. sojae.We will further verify the binding of GmMYB78 to the promoter region of GmbZIP25 and analyze the function of GmbZIP25 in the future works of research.This will reveal the role of the GmbHLH122-GmMYB78-GmbZIP25 regulatory pathway in the soybean response to P. sojae infection, providing a theoretical basis for unraveling the molecular mechanisms of GmMYB78 in response to P. sojae.
Based on these results, we propose a model that explains the mechanisms of GmMYB78 response to P. sojae infection (Figure 9).According to our model, in the susceptible cultivar 'Dongnong 50', GmbHLH122 is not activated as a result of P. sojae infection, while GmMYB78 is upregulated and accumulated, and represses downstream GmbZIP25 expression, downregulates the expression of GmPR genes and JA-synthesis-pathway-related genes, and reduces JA content, thereby inhibiting the plant defense responses (left); in the resistant cultivar 'Suinong 10', GmbHLH122 transcription is induced by P. sojae infection.The high levels of GmbHLH122 inhibits the transcription of GmMYB78, which reduce its promotive effect on GmbZIP25 expression, upregulates GmPR genes and JAsynthesis-pathway-related genes, and promotes the accumulation of JA content, thereby enhancing the defense response to P. sojae (right).Thus, our study provides novel insights into the mechanisms by which the GmbHLH122-GmMYB78-GmbZIP25 pathway modulates the defense response of soybean during infection by P. sojae.Figure 9.A proposed model for the molecular mechanism of the GmMYB78 in soybeans responding to P. sojae infection.In the susceptible soybean cultivar 'Dongnong 50', GmbHLH122 is not activated by infection, while GmMYB78 is upregulated.The high levels of GmMYB78 suppresses the expression of GmbZIP25 by binding to its promoter, and downregulates the expression of GmPR genes and JA-synthesis-pathway-related genes, and reduces JA content, thereby inhibiting the defense responses (left).In the resistant cultivar 'Suinong 10', GmbHLH122 is activated by P. sojae infection.The high levels of GmbHLH122 inhibits GmMYB78 transcription, which reduce its promotive effect on GmbZIP25 expression, upregulates GmPR genes and JA synthesis pathway related genes, and Figure 9.A proposed model for the molecular mechanism of the GmMYB78 in soybeans responding to P. sojae infection.In the susceptible soybean cultivar 'Dongnong 50', GmbHLH122 is not activated by infection, while GmMYB78 is upregulated.The high levels of GmMYB78 suppresses the expression of GmbZIP25 by binding to its promoter, and downregulates the expression of GmPR genes and JA-synthesis-pathway-related genes, and reduces JA content, thereby inhibiting the defense responses (left).In the resistant cultivar 'Suinong 10', GmbHLH122 is activated by P. sojae infection.The high levels of GmbHLH122 inhibits GmMYB78 transcription, which reduce its promotive effect on GmbZIP25 expression, upregulates GmPR genes and JA synthesis pathway related genes, and promotes the accumulation of JA content, thereby enhancing the defense response to P. sojae (right).

Plant Materials and Growth Conditions
This study used 'Dongnong 50' (highly susceptible to Phytophthora sojae race 1, the predominant race in Heilongjiang, China), and 'Suinong 10', carrying resistance against P. sojae [86].'Dongnong 50' and 'Suinong 10' seeds were sown in soil and grown in a growth chamber at 28 • C and 70% relative humidity under a 16 h light/8 h dark photoperiod.These plants were used for gene isolation and analysis of relative transcript levels [87,88] when the first true leaf was about to unfold (V1) [89].Phytophthora sojae No.1 physiological race was preserved in the Key Laboratory of Soybean Biology, Ministry of Education for identification of pathogen inoculation.Arabidopsis thaliana seeds were grown in a growth chamber at 23 • C under an 8 h light/16 h dark photoperiod.Arabidopsis plant used for subcellular localization assay before bolting.Nicotiana benthamiana seeds were grown in a growth chamber at 28 • C under a 16 h light/8 h dark photoperiod.N. benthamiana plants at 6 weeks old were used for dual-luciferase assays.

RT-qPCR
Total RNA was isolated from leaves or hairy roots of soybean using Trizol reagent (Invitrogen, Shanghai, China), and RT-qPCR analysis was performed on a LightCycler96 instrument (Roche, Switzerland) with a real-time PCR kit (TOYOBO, Osaka, Japan).The gene expression levels were calculated by the 2 −∆∆Ct method with GmEF1β (GenBank accession no.NM_001248778) and GmTUB4 (GenBank accession no.EV263740) as the internal control.The reaction conditions consisted of an initial 5 min pre-incubation at 94 • C, and 40 cycles at 94 • C for 30 s, 59 • C for 30 s, and 72 • C for 40 s, followed by a melting-curve analysis from 55 • C to 100 • C with a final cooling for 10 min at 72 • C. The relative transcript abundance of each target gene was calculated using the 2 −∆∆CT method.The primers used for expression analysis are shown in Table S2.

Gene Cloning, Sequence Analyses, and Plasmid Construction
The full-length GmMYB78, GmbHLH122, and GmbZIP25 genes were amplified by PCR using the gene-specific primers (Table S2).Sequence alignments were performed using DNAMAN (http://www.lynnon.com/,accessed on 3 December 2021).Molecular Evolutionary Genetics Analysis (MEGA) software 5.1 were used to analyze phylogenetic of GmMYB78 and other MYBs.The ORF of GmMYB78 was cloned into the vector pCAM-BIA3301 with the bar gene and 4 × Myc tag as the selectable marker under the control of the cauliflower mosaic virus 35S (CaMV35S) promoter to overexpress the GmMYB78 gene.GmMYB78-RNAi constructs were created based on pFGC5941 vector following the methods described by Kerschen et al. [90].

Subcellular Localization Assays of the GmMYB78 Protein
The full-length coding sequence of GmMYB78 was cloned into pCAMBIA1302 (GFP) vector.The coding sequence was placed under the control of the CaMV 35S promoter and cloned in-frame and upstream of the sequence encoding green fluorescent protein (GFP).The resulting 35S:GmMYB78-GFP expression plasmid and 35S:GFP control were individually co-transfected with nuclear Marker H2B-mCherry into Arabidopsis protoplasts using PEG-mediated transfection, as described by Yoo et al. [91].The transformed protoplasts were observed on a confocal spectral microscope imaging system (Leica TCS SP8, Wetzlar, Germany).

Agrobacterium Rhizogenes-Mediated Transformation of Soybean Hairy Roots
The recombinant constructs of GmMYB78-OE and GmMYB78-RNAi were introduced into Agrobacterium rhizogenes K599 strain and transformed into susceptible soybean cultivar 'Dongnong 50' to induce transgenic soybean hairy roots according to a modified method described by Graham et al. [92] and Kereszt et al. [93].Briefly, we removed the growth point of soybean cotyledon and cut the wound on the surface of cotyledons.Agrobacterium rhizogenes bacteria carrying the target gene plasmid were cultured to OD 600 = 0.6 and the harvested cells were resuspended.A. rhizogenes suspension was applied to the cut surface of hypocotyls.The treated soybean seedlings were put on root-inducing medium in dishes and placed in an incubator at 25 • C for 3 weeks.The empty vectors were used as controls.The transgenic hairy taproots will be used for P. sojae inoculation.The lateral roots grown on the taproots were tested to confirm the positive taproots.The transgenic hairy taproots with overexpression-target gene were tested via immunoblots, and RNAi transgenic hairy taproots were verified by QuickStix Kit for LibertyLink (bar) strip detection.The expression level of the overexpression and interference genes were also detected by RT-qPCR.The healthy taproots with similar size were selected for P. sojae infection.

Assessment of Soybean Disease Responses
The pathogen resistances of the GmMYB78-OE, GmMYB78-RNAi, and empty vector (EV) transgenic soybean hairy root lines were assessed using artificial inoculation assays with P. sojae, as described by Ward et al. [87] and Dou et al. [94], with minor modifications.When the hairy roots generated at the infection site grew approximately for 3 weeks, which grew to around 7 cm and were detected as positive ones, the tap roots were incubated with P. sojae zoospores (approximately 1 × 10 5 spores mL −1 ) in a mist chamber at 25 • C with 100% relative humidity for 72 h.EV soybean hairy roots were used as controls.Disease symptoms were imaged using a Nikon B7000 camera at 72 h after inoculation, and the total area of lesions was determined using ImageJ software, https://imagej.net/ij/index.html,accessed on 8 January 2024).

Determination of JA Levels
Jasmonic acid (JA) was extracted from GmMYB78 or EV transgenic soybean hairy roots and quantified using HPLC-mass spectrometry, as described by Zhu et al. [95].

RNA-Seq and Metabolome Analysis
The hairy roots of GmMYB78-OE and the EV control grown under non-stress conditions were used for RNA-seq or metabolites analysis.For RNA-seq assay, the cDNA libraries were sequenced on the Illumina sequencing platform by Metware Biotechnology Co., Ltd.(Wuhan, China).We used HISATv2.1.0to construct the index, and compared clean reads to the reference genome (https://phytozome.jgi.doe.gov/,accessed on 12 January 2022).DESeq2 v1.22.1 was used to analyze the differential expression between GmMYB78-OE and EV transgenic soybean hairy roots.During differential expression gene detection, fold change ≥1, FDR < 0.05 was used as a screening standard.The enrichment analysis is performed based on the hypergeometric test.For KEGG, the hypergeometric distribution test is performed with the unit of pathway; for GO, it is performed based on the GO term.
For metabolome assay, significantly regulated metabolites between groups were determined by VIP ≥ 1 and absolute log2FC (fold change) ≥ 1. Identified metabolites were annotated using KEGG compound database (http://www.kegg.jp/kegg/compound/,accessed on 4 February 2022); annotated metabolites were then mapped to KEGG pathway database (http://www.kegg.jp/kegg/pathway.html,accessed on 4 February 2022).Pathways with significantly regulated metabolites mapped to them were then fed into MSEA (metabolite sets enrichment analysis); their significance was determined by hypergeometric test's p-values.

Yeast One-Hybrid (Y1H) Assays
Yeast one-hybrid assays were used to examine the binding of GmbHLH122 to the GmMYB78 promoter and GmMYB78 to the GmbZIP25 promoter.The coding sequence of GmbHLH122 or GmMYB78 was cloned into the pGADT7 plasmid, while the promoter of GmMYB78 or GmbZIP25 was inserted into the pBait-AbAi plasmid.The background leakiness of the pBait-AbAi-pGmMYB78 or pBait-AbAi-pGmbZIP25 recombinant plasmid was suppressed by adding AbA to the synthetic defined (SD) medium lacking -Ura (SD/-Ura) medium.The recombinant vector GmbHLH122-AD or GmMYB78-AD was transformed into Y1H [pBait-AbAi-pGmMYB78] or Y1H [pBait-AbAi-pGmbZIP25] yeast cells and spotted onto synthetic defined (SD) medium lacking -Leu (SD-Leu) or SD/-Leu/AbA to determine the interactions.The pGADT7-P53 and pGADT7 plasmids were used as positive and negative controls, respectively.

Transient Transcription Dual-Luciferase Assays
The promoter region of GmMYB78 or GmbZIP25 was amplified, cloned into the pGreenII 0800-LUC vector, and used as the reporter.The 35S:GmbHLH122-Myc or 35S:GmMYB78-Myc construct was used as effector.The effector and reporter constructs were introduced into Agrobacterium (Agrobacterium tumefaciens strain GV3101).Mixtures of agrobacteria resuspended in infiltration buffer in a 1:1 ratio were infiltrated into healthy leaves of 28-day-old N. benthamiana leaves [96].The N. benthamiana were incubated under continuous dark light for 2 days and white light for 1 day after infiltration, sprayed with luciferin (1 mM luciferin and 0.01% [v/v] Triton X-100), and photographed using a chemiluminescence imaging system (Tanon 5200).Leaf samples were collected to measure firefly luciferase (LUC) and Renilla luciferase (Rluc) activities with a commercial dual-luciferase assay kit (Promega; PR-E1910).The Rluc in the pGreenII 0800-LUC vector was used as an internal control.LUC activity was normalized to Rluc activity.

Statistical Analysis
All experiments were performed at least three times and significant differences between means were determined using Student's t-test.

Figure 2 .
Figure 2. GmMYB78 is a negative regulator in response to P. sojae.(A) Symptoms of GmMYB78-OE, GmMYB78-RNAi, and empty vector (EV) soybean hairy roots inoculated by P. sojae at 72 hpi.Bars = 0.5 cm.(B) Relative expression of GmMYB78 in three GmMYB78-OE, GmMYB78-RNAi, and EV transgenic soybean hairy root as determined by qRT-PCR.GmEF1β and GmTUB4 were used as the reference genes and expression is relative to that of the EV controls, the values of which were set as 1. (C) Lesion size in GmMYB78-OE, GmMYB78-RNAi, and EV transgenic soybean hairy roots at 72 hpi.(D,E) Relative biomass accumulation of P. sojae in the GmMYB78-OE, GmMYB78-RNAi, and EV roots based on transcript levels of (D) the fungal TEF1 gene and (E) the PSEL1 (P.sojae elicitin gene 1) gene.Fungal biomass is relative to that of the plant.The experiment was performed on three biological replicates, each with three technical replicates, and statistical significance was analyzed using Student's t-test (** p < 0.01).Bars indicate the standard error of the mean.2.3.GmMYB78 Inhibits Pathogenesis-Related (PR) Gene Expression in Response to P. sojae

Figure 2 .
Figure 2. GmMYB78 is a negative regulator in response to P. sojae.(A) Symptoms of GmMYB78-OE, GmMYB78-RNAi, and empty vector (EV) soybean hairy roots inoculated by P. sojae at 72 hpi.Bars = 0.5 cm.(B) Relative expression of GmMYB78 in three GmMYB78-OE, GmMYB78-RNAi, and EV transgenic soybean hairy root as determined by qRT-PCR.GmEF1β and GmTUB4 were used as the reference genes and expression is relative to that of the EV controls, the values of which were

Figure 3 .
Figure 3. Relative expression levels of pathogenesis-related genes GmPR1 (A), GmPR2 (B), GmPR3 (C), and GmPR10 (D) in GmMYB78 transgenic soybean hairy roots.The experiment was performed on three biological replicates, each with three technical replicates, and statistical significance was analyzed using Student's t-test (* p < 0.05, ** p < 0.01).Bars indicate the standard error of the mean.

Figure 3 .
Figure 3. Relative expression levels of pathogenesis-related genes GmPR1 (A), GmPR2 (B), GmPR3 (C), and GmPR10 (D) in GmMYB78 transgenic soybean hairy roots.The experiment was performed on three biological replicates, each with three technical replicates, and statistical significance was analyzed using Student's t-test (* p < 0.05, ** p < 0.01).Bars indicate the standard error of the mean.

2. 4 .
GmMYB78 Is a Negative Regulator of JA-Dependent Signaling during the Response to P. sojae Int. J. Mol.Sci.2024, 25, x FOR PEER REVIEW 8 of 24

Figure 4 .
Figure 4. Transcriptomic analysis of gene expression profiles in response to GmMYB78 overexpression.(A) Volcano plots of significantly differentially expressed genes in GmMYB78-OE vs.EV transgenic soybean hairy roots after the RNA-seq analysis.(B) Heat map of significantly differentially expressed genes between the EV and GmMYB78-OE transgenic soybean hairy roots, as determined using an RNA-seq analysis.Using a false discovery rate < 0.05 and a fold change ≥ 1 as the screening criteria, a total of 617 differentially expressed genes (DEGs) were identified.The scale bar indicates the fold changes (log2 values).(C) Gene annotation of KEGG classification of differentially expressed genes.The differentially expressed genes are mainly involved in multiple biological processes, including plant-pathogen interaction, and plant hormone signal transduction.(D) Gene Ontology functional classification of the differentially expressed genes.The differentially expressed genes were placed into the three main GO categories: biological process, cellular component, and molecular function.

Figure 4 .
Figure 4. Transcriptomic analysis of gene expression profiles in response to GmMYB78 overexpression.(A)Volcano plots of significantly differentially expressed genes in GmMYB78-OE vs. EV transgenic soybean hairy roots after the RNA-seq analysis.(B) Heat map of significantly differentially expressed genes between the EV and GmMYB78-OE transgenic soybean hairy roots, as determined using an RNA-seq analysis.Using a false discovery rate < 0.05 and a fold change ≥ 1 as the screening criteria, a total of 617 differentially expressed genes (DEGs) were identified.The scale bar indicates the fold changes (log2 values).(C) Gene annotation of KEGG classification of differentially expressed genes.The differentially expressed genes are mainly involved in multiple biological processes, including plant-pathogen interaction, and plant hormone signal transduction.(D) Gene Ontology functional classification of the differentially expressed genes.The differentially expressed genes were placed into the three main GO categories: biological process, cellular component, and molecular function.

Figure 5 .
Figure 5. Investigation of the relationship between GmMYB78 and the jasmonic acid (JA) pathway in soybean.(A) JA contents in GmMYB78-OE, GmMYB78-RNAi, and EV transgenic soybean hairy roots.(B-D) Relative transcript level of GmAOS1 (B), GmJAZ1 (C), or GmJAZ2 (D) in GmMYB78-OE, GmMYB78-RNAi, and EV transgenic soybean hairy roots.The level of the control sample (EV lines) was set to unity.The experiment was statistically analyzed using Student's t-test (** p < 0.01).Bars indicate the standard error of the mean.

Figure 5 .
Figure 5. Investigation of the relationship between GmMYB78 and the jasmonic acid (JA) pathway in soybean.(A) JA contents in GmMYB78-OE, GmMYB78-RNAi, and EV transgenic soybean hairy roots.(B-D) Relative transcript level of GmAOS1 (B), GmJAZ1 (C), or GmJAZ2 (D) in GmMYB78-OE, GmMYB78-RNAi, and EV transgenic soybean hairy roots.The level of the control sample (EV lines) was set to unity.The experiment was statistically analyzed using Student's t-test (** p < 0.01).Bars indicate the standard error of the mean.

Figure 7 .
Figure 7. GmbLHL122 directly regulates GmMYB78.(A) Verification of GmbHLH122 regulating GmMYB78 in yeast.(B) Schematic diagram of the reporter vector and effector vector.(C) Dualluciferase assay in N. benthamiana leaves showing that GmbLHL122 promotes the expression of GmMYB78.Representative photographs are shown.(D) Detection of LUC/Rluc activity to verify that GmbLHL122 promotes the expression of GmMYB78.The combination of the reporter construct (pGmMYB78: LUC) and EV was used as a control.The experiment was performed on three biological replicates, each with three technical replicates, and the results were statistically analyzed using Student's t-test (** p < 0.01).Bars indicate the standard deviation of the mean (n = 3).

Figure 7 .
Figure 7. GmbLHL122 directly regulates GmMYB78.(A) Verification of GmbHLH122 regulat GmMYB78 in yeast.(B) Schematic diagram of the reporter vector and effector vector.(C) Dual-lu erase assay in N. benthamiana leaves showing that GmbLHL122 promotes the expression GmMYB78.Representative photographs are shown.(D) Detection of LUC/Rluc activity to verify t GmbLHL122 promotes the expression of GmMYB78.The combination of the reporter constr (pGmMYB78: LUC) and EV was used as a control.The experiment was performed on three biologi replicates, each with three technical replicates, and the results were statistically analyzed using S dent's t-test (** p < 0.01).Bars indicate the standard deviation of the mean (n = 3).

Figure 8 .
Figure 8. Temporal expression patterns of GmbHLH122, GmMYB78, and GmbZIP25 in response to sojae infection in (A) the susceptible cultivar 'Dongnong 50' and (B) the resistant soybean culti 'Suinong 10'.The expression was determined by qRT-PCR using GmEF1β and GmTUB4 as the r erence genes.Bars indicate the standard deviation of the mean (n = 3).

Figure 8 .
Figure 8. Temporal expression patterns of GmbHLH122, GmMYB78, and GmbZIP25 in response to P. sojae infection in (A) the susceptible cultivar 'Dongnong 50' and (B) the resistant soybean cultivar 'Suinong 10'.The expression was determined by qRT-PCR using GmEF1β and GmTUB4 as the reference genes.Bars indicate the standard deviation of the mean (n = 3).
Int. J. Mol.Sci.2024, 25, x FOR PEER REVIEW 16 of 24 transcription factors, as important defense signaling genes, are widely involved in plant defense responses to biotic stresses [82].CsAtf1, bZIP transcription factor, can enhance the virulence of Colletotrichum siamense by regulating the expression of CsPbs2 and CsHog1 in the MAPK pathway, thereby reducing rubber tree resistance to anthracnose [83]