Next Article in Journal
Impact of Ficus deltoidea Aqueous Extract on Maternal Hepatic Drug Metabolism and Foetal Development in Rats
Previous Article in Journal
Phytochemical Profile, Toxicity Evaluation and Antinociceptive Effect of the n-Butanolic Fraction from the Leaves of Calotropis procera (Aiton) W.T Aiton (Apocynaceae)
Previous Article in Special Issue
The Bitter Gourd Transcription Factor McNAC087 Confers Cold Resistance in Transgenic Arabidopsis
 
 
Search for Articles:
Title / Keyword
Author / Affiliation / Email
Journal
Article Type
 
 
Advanced Search
 
Section
Special Issue
Volume
Issue
Number
Page
 
Journals
Plants
Volume 14
Issue 23
10.3390/plants14233621
plants-logo
Submit to this Journal Review for this Journal Propose a Special Issue
► Article Menu

Article Menu

share Share announcement Help format_quote Cite question_answer Discuss in SciProfiles
first_page
settings
Order Article Reprints
Font Type:
Arial Georgia Verdana
Font Size:
Aa Aa Aa
Line Spacing:
Column Width:
Background:
Article

Genome-Wide Analysis of the Dof Gene Family in Soybean and Functional Identification of GmDof63 in Response to Phytophthora sojae Infection

by
Sujie Fan
,
Haiyuan Chen
,
Yuhan Huo
,
Yang Song
,
Piwu Wang
,
Zhuo Zhang
* and
Liangyu Jiang
*
Plant Biotechnology Center, College of Agronomy, Jilin Agriculture University, Changchun 130118, China
*
Authors to whom correspondence should be addressed.
Plants 2025, 14(23), 3621; https://doi.org/10.3390/plants14233621
Submission received: 22 October 2025 / Revised: 19 November 2025 / Accepted: 25 November 2025 / Published: 27 November 2025
(This article belongs to the Special Issue Crop Functional Genomics and Biological Breeding—2nd Edition)
Browse Figures
Versions Notes

Abstract

Phytophthora root and stem infection by Phytophthora sojae is a global and devastating disease of soybeans. Selecting disease-resistant varieties is the most economical and effective measure for controlling this disease. Delving into the disease resistance and defense molecular mechanisms can lay a theoretical foundation for solving this problem. Here, we screened the soybean genome and identified 78 GmDof genes distributed on nineteen chromosomes. Subcellular localization analysis revealed that the majority of GmDof proteins were located in the cell nucleus. Phylogenetic analysis categorized these genes into nine subfamilies. Gene structure analysis showed that all GmDofs contained 0 to 2 introns, and most of them did not have introns. Motif and conserved domain analysis showed that all GmDofs contained a common motif (motif-1) and a typical conserved C2-C2 domain. The prediction of cis-acting elements in promoter regions revealed numerous cis-regulatory elements responsible for stress responses, plant growth and development, plant hormone responses, and light responses. RNA-seq and quantitative real-time PCR results showed that GmDof63 (Glyma.16G145000) was specifically expressed at high levels after P. sojae infection. GmDof63 was strongly induced by SA and ETH treatments. The soybean seedlings overexpressing GmDof63 displayed enhanced resistance to P. sojae infection compared with the wild-type soybean seedlings. Further experiments indicated that the expression levels of pathogenesis-related protein genes PR1a, PR4, PR5a, and PR10 were significantly up-regulated in GmDof63-overexpressing transgenic soybean seedlings. Taken together, these findings reveal the mechanism by which GmDof63 directly or indirectly regulates the expression of PR genes to modulate the soybean response to P. sojae infection.

1. Introduction

Soybean [Glycine max (L.) Merr.] is an important food and economic crop worldwide, and the significance and urgency of soybean research have become increasingly prominent [1,2]. Phytophthora root and stem rot of soybean is a devastating disease caused by Phytophthora sojae, leading to stunting, death or premature senescence of seedlings [3]. It can result in crop failure in the severely affected fields, causing billions of dollars of economic losses to the global soybean production each year [3,4]. Although several P. sojae strains and quantitative trait loci (QTLs) related to P. sojae resistance in soybean have been identified, the mechanisms underpinning the functions and regulation of resistance genes remain to be studied [5,6,7].
The DNA binding with one finger (Dof) family is a plant-specific class of transcription factors [8,9,10]. Members of the Dof family have been identified in many species, such as Arabidopsis thaliana [11], Oryza sativa [11], G. max [12], Zea mays [13], Solanum tuberosum [14], Prunus persica [15], and Capsicum annuum [16]. Dof transcription factors (TFs) typically consist of 200–400 amino acid residues and contain oligomerization sites [17]. The highly conserved domain consists of 50–52 residues at the N-terminus, containing a C2-C2 zinc finger domain CX2CX21CX2C [18,19]. Dof TFs recognize and bind to the AAAG-rich sequences or the CTTTT sequence of their target genes [20].
Dof TFs play a crucial role in various biological processes, including plant growth and development, signal transduction, and abiotic stress responses [21,22,23]. In Arabidopsis, the overexpression of OBP4 (a Dof TF) promotes cell proliferation in the differentiation zone and induces the formation of callus [24]. In blueberry plants, VcDof2 and VcDof45 are considered to play significant roles in the flowering and fruit development processes, and VcDof1, VcDof11 and VcDof15 exhibit positive responses and upregulated expression under abiotic stress conditions [25]. The amount of oil contained in the seeds of cotton growing on specific land is related to GhDof1. When GhDof1 is highly expressed, the oil content in cotton seeds will increase, while the protein content will decrease [26]. The cycling Dof factor 2 (CDF2) causes Arabidopsis to become insensitive to photoperiod and to delay flowering by reducing the level of CONSTANS [27]. In apple plants, MdCDOF3 and MdDOF3.6 activate the cytokinin oxidase MdCKX7 in response to sorbitol signals, thereby accelerating the leaf senescence process [28]. The wheat endosperm-specific TF TaDOF6 promotes grain development by regulating the expression of TaSWEET13h and facilitating the transport of sugars and gibberellins [29]. SlDof22 can bind to the promoter of SlSOS1 to down-regulate the expression of this gene, which compromises the salt stress tolerance [30]. In Camellia sinensis, CsDOF51 and CsDOF12 exhibit significant expression changes under drought stress and high-temperature stress, respectively, and CsDOF44 shows significant changes under both conditions [31].
To date, the precise roles of Dof or Dof-like proteins in regulating biotic stress responses remain poorly understood. In tobacco, Sar8.2b can be activated by the Dof TF, which is related to systemic acquired resistance [32]. The atypical Dof TF OsDes1 can specifically recognize the promoter region of the defense-related gene OsPR1b, thereby activating the expression of OsPR1b to enhance the resistance to Xanthomonas oryzae pv. oryzae [33]. However, few studies report the Dof genes responsive to P. sojae infection in soybean. On another note, salicylic acid (SA), Methyl jasmonic acid (MeJA), ethylene (ETH), and abscisic acid (ABA) are all small molecule hormones, and they can coordinate the defense responses of plants through a complex signaling network, thereby regulating plant immunity [34,35,36,37]. Dof TFs have been shown to mediate responses to various hormone signals. SsDOF1-7 in sugarcane have been shown to contain several cis-elements involved in SA, MeJA, and ABA responses [38]. In Arabidopsis, a DOF transcription factor gene, OBP3, is mainly responsive to SA, and DOF5.8 positively regulates the target gene ANAC069 under abiotic stress conditions [39]. FtDof gene was mainly up-regulated under MeJA treatment but down-regulated under SA, ABA, gibberellic acid (GA), and indole-3-acetic acid (IAA) treatment in Tartary buckwheat [40]. The VvDOF3 gene was rapidly induced by exogenous SA, MeJA, and powdery mildew infection [41]. These findings suggest that Dof TFs may play a key role by regulating the levels of plant hormone signals in response to environmental stress.
In this work, we sought to uncover the functional roles of the Dof gene family members in soybean defense against P. sojae infection. In order to do this, we identified 78 Dof family genes in soybean and analyzed their physicochemical properties, chromosomal localization, evolutionary relationships, gene structures, conserved motifs, intraspecies and interspecies collinearity, and promoter elements. We employed RNA sequencing and the quantitative RT-PCR method to screen out the key Dof gene in soybean response to P. sojae infection. Subsequently, GmDof63 (Glyma.16G145000) was chosen for further analysis. Notably, GmDof63 was significantly upregulated under SA and ETH treatments. The soybean seedlings overexpressing GmDof63 displayed enhanced resistance to P. sojae infection compared with the wild-type (WT) soybean seedlings. These findings lay a good framework for further research into the involvement of the soybean Dof gene in defense against P. sojae infection.

2. Results

2.1. Identification and Characterization of the Dof Genes in Soybean

In this study, a total of 78 Dof genes were identified in the soybean genome and renamed as GmDof1 to GmDof78 based on the location order of soybean chromosomes from top to bottom. The GmDofs showed the amino acid sequence lengths ranging from 171 aa (GmDof18) to 503 aa (GmDof35), the molecular weights ranging from 19.65 kDa (GmDof18) to 54.73 kDa (GmDof35), and the theoretical pI ranging from 4.69 to 10.23, with 26 members having the pI less than 7 and 52 members having the pI greater than 7. Except for GmDof37, GmDof47, and GmDof60, the other members were unstable proteins. All the GmDofs were identified as hydrophilic proteins. Subcellular localization prediction indicated that except for GmDof47, located in the cytoplasm, and GmDof19 and GmDof60, located in the chloroplast, the other GmDofs were located in the cell nucleus (Table S1).

2.2. Chromosome Localization of GmDofs

According to the soybean genome database, 78 soybean Dof genes were unevenly distributed among 20 chromosomes, with the exception of chromosome 14 (Figure 1). The number of GmDofs in each chromosome differed considerably. Chromosome 13 had the largest number (11) of GmDofs, followed by chromosome 15 (8) and chromosome 7 (6). Chromosomes 4, 6, and 19 had five GmDofs; chromosomes 2, 5, 8, 17, and 18 had four GmDofs; chromosomes 1 and 11 had three GmDofs; and chromosomes 3, 9, 10, 12, 16, and 20 had two GmDofs.

2.3. Phylogenetic Relationships of GmDofs

To ensure the reliability of the analysis, we employed the Maximum likelihood (ML) method to perform multiple sequence alignment of GmDofs and 36 Arabidopsis Dof members and constructed a phylogenetic tree of all Dof proteins (Figure 2). The phylogenetic clustering of GmDofs was associated with the uneven distribution of their conserved domains. The GmDofs were clustered into nine subfamilies (A, B1, B2, C1, C2.1, C2.2, C3, D1, and D2), which showed significant differences in the number of GmDofs. Overall, the evolutionary tree structure of GmDofs is mainly related to the evolutionary relationships between species, and the genes of species with evolutionary relationships tend to cluster on the same branch of the evolutionary tree.

2.4. Gene Structures and Motif Distribution of GmDofs

To examine the structural diversity of GmDofs, we investigated the number of exon-introns and the distribution of conserved domains of GmDofs. This analysis provided evidence for unveiling the evolution of structural diversity in the soybean Dof family. Almost all GmDofs contained very few or no introns (Figure 3). The genes in the A, B2, and D2 subfamilies did not contain introns. The GmDofs classified in the same subfamily showed similar gene structures. To elucidate the evolution of GmDofs, MEME analysis identified 20 conserved motifs. Notably, N-terminal regions contained a highly conserved motif-1, and GmDofs within the same subfamily displayed similar motif composition (Figure 3). Most A subfamily members contained motif-1 and motif-14. B1 subfamily members contained motif-1, motif-10, and motif-13. Most B2 subfamily members contained motif-1, motif-17, and motif-20. Most C1 subfamily members contained motif-1 and motif-9. Most C2.1 subfamily members contained motif-1 and motif-18. C2.2 subfamily members contained motif-1 and motif-6. Most C3 subfamily members contained motif-1 and motif-10. Most D1 subfamily members contained motif-1, motif-2, motif-3, motif-4, motif-5, motif-6, motif-7, motif-8, motif-11, motif-12, motif-16, motif-18, and motif-19. D2 subfamily members only contained motif-1. All GmDofs contained the typical conserved Dof domain, categorized under the zinc finger superfamily, indicating evolutionary conservation of functional domains among GmDofs.

2.5. Collinearity of GmDofs

To further elucidate the potential functions of the GmDofs, we analyzed the duplication events giving rise to GmDofs. The distribution of GmDofs varied among different chromosomes, indicating substantial differences in their chromosomal evolution. The soybean genome carries 110 GmDof gene pairs involved in duplication events, which are located on different chromosomes, with the greatest number of GmDof homologous gene pairs on chromosome 13 (Figure 4). The findings indicate that gene segmental duplication events may have been the main driving force behind the evolution of GmDofs. Furthermore, we calculated the synonymous (Ks) and non-synonymous (Ka) substitution rates (Ka/Ks) of 110 segmentally duplicated pairs (Table S2). The Ka/Ks ratios for segmentally duplicated gene pairs ranged from 0.07 to 0.64, with an average of 0.29, indicating that the GmDofs have undergone strong negative purifying selection pressure. Furthermore, the frequency distribution of the Ka/Ks ratios showed that more than 78% of duplicated gene pairs had ratios ranging from 0.1 and 0.3. These results demonstrate the conserved evolution of soybean Dof genes.

2.6. Interspecies Collinearity of GmDofs

To further investigate the gene duplications in the Dof genes, we analyzed the collinearity of GmDofs with the Dof genes in other species, including A. thaliana, O. sativa, Nicotiana tabacum, and S. tuberosum (Figure 5). The results showed that the collinear gene pairs in rice, a monocot model plant, were significantly fewer than those in dicot genomes. In addition, more genetic overlap was found between soybean and S. tuberosum than between soybean and other plants, signifying a closer evolutionary relationship of soybean to S. tuberosum.

2.7. Predicted Cis-Acting Elements in the Promoters of GmDofs

In this study, PlantCARE was used to analyze the cis-acting elements in the sequence upstream (2 kb) from the start codon of each GmDof. The results showed that the responsive elements were widely present in the Dof family genes of soybean (Figure 6A). All cis-acting elements were classified into four categories according to their functions: stress response, plant growth and development, plant hormone response, and light response (Figure 6B,C). Stress response-related element analysis showed that MYC, a drought-responsive element, was the most numerous (29.35%), and STRE (a stress-responsive element) was the second most numerous element (16.57%). The promoters of 92.30% of GmDofs contained the MYC element, and those of 78.21% of GmDofs contained the STRE element, suggesting that GmDofs played a role in regulating drought and stress responses. Plant growth and development-related element analysis showed that the AAGAA motif, an auxin-responsive element, was the most numerous (52.76%). The promoters of 87.18% of GmDofs contained the AAGAA motif, suggesting that most GmDofs played a role in regulating auxin responses. The analysis of plant hormone response-related elements showed that ABRE was the most numerous element (33.12%), which was the ABA-responsive element. The promoters of 83.33% of GmDofs contained the ABRE element, suggesting that GmDofs played an important role in regulating ABA response. Among the light-responsive elements, the box 4 element was the most numerous (43.69%). The promoters of 97.44% of GmDofs contained the box 4 element, suggesting that GmDofs played an important role in light response.

2.8. Screening of Dof Genes in Soybean After P. sojae Infection

Hypocotyls of JN4507 and JN28 seedlings were inoculated with P. sojae isolate PSR01. At 48 h post-inoculation of P. sojae, it was observed that the JN28 soybean seedlings remained firm, with only slight browning of the stems, while the JN4507 soybean seedlings exhibited extended lesions, with the entire plant wilting and emitting a foul odor (Figure 7A). The seedling incubation assay demonstrated that JN28 showed a strong immune response against P. sojae isolate PSR01, whereas JN4507 was completely susceptible.
To determine whether Dof members control P. sojae infection, we profiled the expression of GmDofs in soybean seedlings during P. sojae infection by RNA sequencing. We collected three replicates of P. sojae-treated JN28 and JN4507 samples at 48 h post-inoculation to capture the transcriptional changes during P. sojae infection. The control samples were inoculated with an agar block without P. sojae. The differentially expressed genes were identified by a pairwise comparison of the transcriptome datasets (JN28-P. sojae vs. JN4507-P. sojae) (Figure 7B). We further identified the significantly upregulated expression of GmDof63 (Glyma.13G329000) through the volcano plot (Figure 7C). Quantitative RT-PCR results indicated that the expression of GmDof63 was upregulated in the highly resistant soybean variety JN28 after P. sojae infection (p < 0.01), and the accumulation of GmDof63 peaked at 48 h post-inoculation (Figure 7D). These results indicated that GmDof63 was a potentially crucial gene in soybean during P. sojae infection.

2.9. Sequence Characteristics and Expression Pattern of GmDof63

To study GmDof63 expression, we first cloned the complete sequence of GmDof63 from JN28 using the RT-PCR technique, which contained a 711 bp open reading frame that encodes a 236-amino acid protein with the zinc finger domain CX2CX21CX2C (Figure 8A). We further explored the expression pattern of GmDof63 in soybean under different stresses. As shown in Figure 8B, the expression of GmDof63 was significantly upregulated at 6 h and reached a peak at 24 h after SA treatment, and the expression of GmDof63 was significantly upregulated at 24 h and reached a peak at 24 h after ETH treatment, but the expression level of the GmDof63 did not show any significant change after treatment with MeJA and ABA. The results suggested that GmDof63 participates in multiple signaling pathways.

2.10. Subcellular Localization of GmDof63

To investigate the subcellular localization of GmDof63, 35S::GFP vector or 35S::GmDof63-GFP vector (Figure 9A) was transformed into Arabidopsis mesophyll protoplasts. H2B-mCherry was chosen as the nuclear marker protein, which can encode histone H2B fused with the red fluorescent protein mCherry. As can be seen from Figure 9B, the transformed cells carrying 35S::GFP showed a strong green fluorescence signal throughout the entire cell, whereas the transformed cells carrying 35S::GmDof63-GFP showed a strong green fluorescence signal only in the nucleus, and this finding was consistent with the location of the nuclear marker protein H2B-mCherry. These results suggested that GmDof63 is a nucleus-localized transcription factor.

2.11. GmDof63 Enhances Resistance of Transgenic Soybean Seedlings to P. sojae

We next focused on the function of GmDof63. To confirm the functions of GmDof63 in soybean response to P. sojae, we constructed a GmDof63-overexpressing vector 35S::GmDof63 with BAR as the selective marker (Figure 10A). We then used an efficient Agrobacterium-mediated transformation system described by Paz et al. (2004) [42] and Li et al. (2017) [43] to generate GmDof63-overexpressing transgenic soybean seedlings. In the T0 and T2 generations, the transgenic soybean seedlings were verified by the BAR LibertyLink strip (Envirologix, Portland, OR, USA) (Figure S1). Three lines of positive transgenic soybean seedlings were inoculated with P. sojae zoospores in a hydroponic assay. The zoospore suspension was prepared according to the method described by Shrestha et al. (2016) [44] and Yang et al. (2021) [45]. Under standard culture conditions, no significant differences in leaf phenotype were observed between transgenic and WT plants. However, the WT soybean roots exhibited extended lesions, browning, and wilting, while the GmDof63-overexpressing soybean roots remained firm with only slight browning (Figure 10B). Quantitative RT-PCR results showed that the expression of GmDof63 in GmDof63-overexpressing soybean roots was higher than that of WT soybean roots at 48 h post-inoculation (Figure 10C). The relative accumulation of P. sojae was significantly lower in GmDof63-overexpressing soybean roots than in WT soybean roots at 48 h post-inoculation (Figure 10D). The above results indicated that the GmDof63-overexpressing transgenic soybean seedlings displayed enhanced resistance to P. sojae infection compared with the WT soybean seedlings.
Pathogenesis-related (PR) genes are some of the most important genes in the defense response of plants against pathogens [46,47,48,49]. To test whether GmDof63 can regulate the expression of PR genes, we performed quantitative RT-PCR analysis in roots of GmDof63-overexpressing and WT soybean at 48 h post-inoculation. The expression of PR1a, PR4, PR5a, or PR10 in GmDof63-overexpressing soybean roots was higher than that of WT soybean roots (Figure 10E). Previous research has revealed that Dof proteins often recognize the AAAG or TTTC motif in their target promoters [50,51,52], so we analyzed the AAAG or TTTC motif in the promoter sequence of the PR genes. The result showed that a large number of AAAG or TTTC motifs were present in the promoter sequence of PR1a, PR4, PR5a, and PR10 (Figure S2), indicating that GmDof63 may directly or indirectly regulate the expression of PR genes to modulate the soybean response to P. sojae infection.

3. Discussion

The Dof family is a type of plant-specific TFs belonging to the single zinc finger protein superfamily. With the development of bioinformatics, since the first Dof gene was cloned from maize, the Dof gene family has been deeply identified and analyzed in plants such as Arabidopsis, rice, maize, potato and sweet pepper [23,53]. However, the research on Dof genes in soybean is still limited, and there are few reports on the roles of Dof genes in response to P. sojae infection.
In this study, we identified 78 members of the Dof family in soybean and analyzed their physicochemical properties, chromosomal localization, evolutionary relationships, gene structures, intraspecies and interspecies collinearity, and promoter elements. Subcellular localization is an important characteristic of proteins, and protein function is closely related to protein localization [54,55]. For example, proteins in the cell nucleus mainly participate in gene transcription and DNA repair [56]. In this study, the subcellular localization prediction showed that 75, 1, and 2 GmDofs were located in the cell nucleus, cytoplasm, and chloroplast, respectively.
Motif, conserved domain, and gene structure prediction indicated that all GmDofs contained a common motif (motif-1), suggesting that this motif may play a key role. The diversity of motifs among different Dof members may be related to their complex functions. Different gene families have their own conserved domains. All GmDofs had a complete C2-C2 single-finger zinc structure, which was consistent with the research results of Wu et al. (2019) [57] and Luo et al. (2022) [58]. Gene structure analysis showed that all GmDofs had 0 to 2 introns, and most of them did not have introns. This feature indicated that the gene structure of GmDofs was conserved.
The evolution and expansion of gene families are closely related to gene duplication. The occurrence of gene duplication may be due to fragment duplication, tandem duplication or whole-genome duplication [59,60]. Some duplicated genes may retain similar functions and show partial or complete divergence from each other [61]. Many TF families in plants have undergone gene duplication events [62]. In this study, we discovered that GmDofs were unevenly distributed on chromosomes other than chromosome 14. Homology analysis showed that the soybean genome contained 110 pairs of Dof homologous genes, all of which originated from fragment duplication, indicating that fragment replication may have played a dominant role in the evolution and expansion of the GmDof family. In addition, the Ka/Ks values of 110 pairs of Dof homologous genes were all less than 1, indicating that the Dof genes have undergone purifying selection during long-term evolution. Interspecies collinearity showed that the number of co-linear Dof gene pairs of soybean with monocotyledonous model plants such as rice was significantly lower than that with dicotyledonous plants such as A. thaliana, O. sativa, N. tabacum and S. tuberosum, and the gene overlap with S. tuberosum was the most, indicating a closer evolutionary relationship with S. tuberosum.
Dof TFs play a significant role in regulating plant growth and development, stress responses, and plant hormone signal transduction [63,64,65,66,67]. Studies have shown that cis-acting elements are involved in the responses to various environmental stresses [68,69,70,71]. The MYC and STRE elements play an important role in stress responses. The AAGAA-motif element is involved in plant responses to environmental stresses. ABRE played an important role in regulating ABA response. The Box 4 element acts as a photosensitive element, participating in the plant response to light conditions. We can further understand the functions of GmDofs by analyzing the cis-acting elements of the promoters of GmDofs. The most common cis-acting elements predicted in the promoter region of GmDofs contained MYC, STRE, AAGAA-motif, ABRE, and Box 4 elements. Therefore, GmDofs play a crucial role in plant growth and development, stress responses, and plant hormone signal transduction.
Phytophthora root and stem rot, a devastating disease caused by P. sojae, has caused serious losses to soybean production worldwide. Studies have confirmed that there are extensive genetic variations (including those conferring disease resistance) in major crops, providing an opportunity to utilize these variations to enhance the soybean response to P. sojae. For example, miR393 and miR166 have been found to play a crucial role in responding to P. sojae infection [72]. Overexpression of the bHLH transcription factor GmPIB1 can enhance resistance to P. sojae [73]. The zinc finger protein-type TF GmZFP03 boosts the resistance to P. sojae by targeting the promoters of two SOD1 genes and activating their expression [74]. GmCAT1 can slightly cause cell death in Arabidopsis and promote the development of P. sojae, and the interaction between PsAvh113-GmDPB-GmCAT1 may be a potential defense mechanism that is conducive to the infection of P. sojae [75]. However, the natural variations in Dof genes have not been examined in soybeans responding to P. sojae infection. Here, we identified one Dof gene, GmDof63, in soybean via RNA sequencing, which exhibited an upregulated expression in the resistant materials. The function of GmDof63 was verified through genetic transformation. The GmDof63-overexpressing transgenic soybean seedlings demonstrate enhanced resistance to P. sojae. Quantitative RT-PCR results showed that the expression level of GmDof63 significantly increased in GmDof63-overexpressing transgenic soybean after P. sojae infection, and the relative accumulation of P. sojae was significantly lower in GmDof63-overexpressing transgenic soybean than in WT soybean, further indicating that this gene played a role in the response to P. sojae infection.
Much research has shown that Dof TFs play a key role in mediating plant hormone pathways [38,39,40,41,76]. We discovered the similar research result that the transcription level of GmDof63 in soybean seedlings was significantly induced after spraying SA and ETH. Previous studies have shown that transcription factors can specifically bind to the promoters of PR genes to regulate their expression, thereby responding to pathogen infection [77,78]. In this study, the expression levels of PR1a, PR4, PR5a and PR10 were significantly up-regulated in GmDof63-overexpressing transgenic soybean seedlings, and a large number of AAAG or TTTC motifs were present in the promoter sequence of PR1a, PR4, PR5a, and PR10, so we speculated that GmDof63 may directly or indirectly regulate the expression of these PR genes. Furthermore, previous research has also revealed that PRs usually function as effector genes for systemic acquired resistance (SAR), and this resistance is mediated by SA [79,80]. So, we speculate that the high expression levels of PR genes indicate the activation of the SA signaling pathway. Taken together, these findings suggested that GmDof63 may serve as a key regulatory center for the SA and ETH signaling pathways, integrating these pathways to cope with P. sojae infection.
In the follow-up work, the phenotypes of GmDof63-overexpressing transgenic soybean and the expression levels of GmDof63 and PRs will be evaluated at multiple time points after P. sojae infection, which could offer a more detailed understanding of the temporal dynamics of the defense response. Now, the functional verification of GmDofs in response to P. sojae infection can not only reveal the molecular mechanism underpinning the soybean response to pathogen infection but also provide direct molecular markers and candidate genes for the breeding of new varieties with specific disease resistance traits, demonstrating promising application prospects.

4. Materials and Methods

4.1. Identification of Dof Members in Soybean Genome

The soybean Wm82.a4.v1 genome was downloaded from the Soybase database (https://www.soybase.org/ (accessed on 12 April 2023)) for the identification of Dof genes. The nucleotide and protein sequences of Dof family members of A. thaliana were downloaded from TAIR (https://www.arabidopsis.org/ (accessed on 12 April 2023)), and the nucleotide and protein sequences of Dof family members of O. sativa were downloaded from the Rice Genome Annotation Project (http://rice.uga.edu/ (accessed on 12 April 2023)). The Hidden Markov Model file corresponding to the Dof domain (PF02701) was downloaded from the Pfam database (http://pfam.xfam.org/ (accessed on 13 April 2023)). Dof members were searched from the soybean database via HMMER 3.0 (http://hmmer.org/ (accessed on 15 April 2023)). The BLASTp v2.12.0 tool was used to compare these sequences against the acquired Dof protein sequences of O. sativa and A. thaliana, and the genes with E-values ≤ 1 × 10−5 were retained. These collected putative Dof members were confirmed by the Pfam (http://pfam.sanger.ac.uk/ (accessed on 15 April 2023)) and InterPro (https://www.ebi.ac.uk/interpro/ (accessed on 15 April 2023)) databases. The longest CDS transcript in the same gene was selected as the representative sequence.

4.2. Physicochemical Characterization of Soybean Dof Members

The physicochemical properties of the predicted proteins of soybean Dof genes were calculated via the ProtParam tool (https://web.expasy.org/protparam/ (accessed on 26 April 2023)), including amino acid sequence length, theoretical isoelectric point (pI), molecular weight, instability index, aliphatic index, and grand average of hydropathicity. The subcellular localization of the soybean Dof members was predicted by Wolf Psort online (https://wolfpsort.hgc.jp/ (accessed on 18 May 2023)), and the top predicted position was selected as the result.

4.3. Chromosomal Location Analysis of Soybean Dof Members

Gene Location Visualize from GTF/GFF module in Tbtools v2.310 software was used to map all non-redundant soybean Dof genes on the 20 soybean chromosomes on the basis of the information in the soybean database.

4.4. Phylogenetic Analysis of Soybean Dof Members

The amino acid sequences of Dof members from soybean and Arabidopsis were selected for phylogenetic analysis. ClustalW and ClustalX with default parameters were used for multiple sequence alignment. The phylogenetic tree was constructed by the Maximum likelihood (ML) method in MEGA12 software, with the Bootstrap value set at 1000. Subsequently, iTOL (https://itol.embl.de/ (accessed on 23 April 2023)) was used for visualization and beautification of the phylogenetic tree.

4.5. Gene Structure and Motif Analysis of Soybean Dof Members

The structure of soybean Dof genes, including cDNA sequences and the corresponding genomic DNA sequences, was extracted from the soybean genome annotation file. Finally, TBtools v2.310 was used for visualization analysis. Online MEME (http://www.OMIcsclass.com/article/67 (accessed on 19 May 2023)) was employed to analyze the motif structure of soybean Dof members, with the maximum number, minimum width, and maximum width of motifs set at 20, 6, and 50, respectively.

4.6. Analysis of Collinearity and KaKs of Soybean Dof Members

The genome sequences and annotation files of Arabidopsis (TAIR10.55) and rice (v7.0) were downloaded from the Phytozome v13 website (https://phytozome-next.jgi.doe.gov/ (accessed on 23 May 2023)). The genome sequences and annotation files of tobacco (Niben261) were downloaded from the Sol Genomics Network database (https://solgenomics.net/ (accessed on 23 May 2023)). The genome sequences and annotation files of potato (SolTub_3.0) were downloaded from the Ensemble Plants database (https://plants.ensembl.org/index.html (accessed on 23 May 2023)). The MCScanX program was used to analyze the collinearity of Dof members in soybean with those in Arabidopsis, rice, tobacco, and potato. The syntenic relationships within Dof members of soybean were visualized using the TBtools software. The non-synonymous/synonymous substitution rate (Ka/Ks) is an important indicator for measuring the selection pressure on gene evolution. TBtools software was used to visualize and analyze the Ka/Ks results (Ka/Ks < 1 indicates purifying selection, Ka/Ks = 1 indicates neutral evolution, and Ka/Ks > 1 indicates positive selection).

4.7. Prediction of Cis-Acting Elements of Soybean Dof Members

The 2000 bp sequence upstream of each soybean Dof member was downloaded from the Phytozome v13 website. The sequences were analyzed via the CARE search tool in the PlantCARE database (http://bioinformatics.psb.ugent.be/webtools/plantcare/html (accessed on 26 May 2023)). The cis-acting elements were visualized and summarized by the Basic Biosequence View and HeatMap modules in TBtools.

4.8. Plant Materials and Pathogen Strain

Jinong 4507 (JN4507) is a soybean variety susceptible to P. sojae infection. The high-yield variety Jinong 28 (JN28), developed by the Plant Biotechnology Center of the Jilin Agricultural University, is resistant to P. sojae infection. The special variety Dongnong 50 (DN50), developed by the College of Agriculture of the Northeast Agricultural University, is susceptible to P. sojae infection. In this study, JN28 was used for gene isolation, and DN50 was used for soybean transformation. Soybean seedlings were grown in a greenhouse with vermiculite as the growth medium. The seedlings were cultivated under a photoperiod of 16L/8D, 25 °C, and the relative humidity of 70%.
P. sojae race 1 (PSR01), a dominant race in Jilin Province, was kindly provided by Professor Shuzhen Zhang from Northeast Agricultural University. PSR01 was cultured on V8 juice agar in a polystyrene dish and activated by incubation at 22–25 °C.

4.9. Resistance Identification, RNA Extraction, and Transcriptome Sequencing

To examine the phenotypes of JN4507 and JN28 in response to P. sojae infection, we used a sterile scalpel to inoculate the agar plugs covered by mycelia (2 cm × 2 cm) on wounded hypocotyls of 14-day-old soybean seedlings. The inoculum was prepared from 7-day-old fungal cultures grown on V8 juice agar. We extracted total RNA from the inoculation point of soybean seedlings with Trizol reagent (Sangon Biotech, Shanghai, China). RNA quality was determined by the Qubit2.0 RNA test kit (Thermo-Life, Waltham, MA, USA). A high-quality RNA sample (OD260/280 of 1.8 to 2.2, OD260/230 ≥ 2.0, RIN ≥ 6.5, 28S:18S ≥ 1.0, >10 μg) was used to construct the sequencing library. The transcriptome sequencing work was carried out by Sangon Biotech.

4.10. Quantitative RT-PCR

One Step RT-PCR Kit (Code No. PCR-311, TOYOBO, Tokyo, Japan) was used for standard expression determination, and the specific primers for the target genes and housekeeping genes were designed with Primer 5 software. The reaction procedure was described as follows: 95 °C for 30 s; followed by 40 cycles of 95 °C for 5 s, 60 °C for 15 s, and 72 °C for 45 s. The relative expression levels were calculated by the 2−ΔΔCT method. GmActin (Glyma.18G290800.1) was used as the internal control.

4.11. Primer Sequences Used in the Present Study

The specific primers used for all assays are listed in Table S3.

5. Conclusions

A total of 78 soybean Dof genes were identified and phylogenetically divided into 9 subfamilies. Gene structure analysis showed that all GmDofs contained 0 to 2 introns, and most of them did not have introns. Motif and conserved domain analysis showed that all GmDofs contained a common motif (motif-1) and a typical conserved C2-C2 domain. Investigations into CREs indicated the presence of various stress-responsive, plant growth and development, hormone-responsive, and light-responsive regulatory elements in the promoter region of GmDofs. Subsequently, RNA-seq and qRT-PCR results showed that GmDof63 was specifically expressed at high levels after P. sojae infection. GmDof63 was strongly induced by SA and ETH treatments. GmDof63 enhanced resistance to P. sojae infection in GmDof63-overexpressing transgenic soybean seedlings. Furthermore, the expression levels of PR genes PR1a, PR4, PR5a, and PR10 were significantly up-regulated in GmDof63-overexpressing transgenic soybean seedlings. Our study provides a basis for further research on the functions of soybean Dof family members in biotic stress tolerance.

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/plants14233621/s1, Table S1: Detailed information of all identified soybean Dof members; Table S2: Segmental duplication of GmDofs among Glycine max chromosomes; Table S3: Oligonucleotide primers used in this study; Table S4: Source data underlying the graphs presented in the main figures. Figure S1: Detection of the T0 and T2 transgenic plants with PAT/Bar LibertyLink strips. Figure S2: The AAAG or TTTC motif in the promoter sequence of PR1a, PR4, PR5a, and PR10.

Author Contributions

Conceptualization, S.F.; Data curation, H.C.; Formal analysis, H.C.; Funding acquisition, Z.Z. and S.F.; Investigation, Y.H.; Methodology, H.C.; Project administration, S.F.; Resources, Y.H.; Software, H.C.; Supervision, P.W.; Validation, Z.Z.; Visualization, Y.S.; Writing—original draft, S.F.; Writing—review and editing, L.J. All authors have read and agreed to the published version of the manuscript.

Funding

This research was funded by Key Agricultural Technology Research and Development Program of Jilin Province (20240303011NC), Science and Technology Research Program of the Jilin Provincial Department of Education (JJKH20240437KJ) and Science and Technology Development Plan Project of Jilin Province (20220101340JC).

Data Availability Statement

Data are contained within the article and Supplementary Materials.

Acknowledgments

We would like to thank Shuzhen Zhang and her team (Soybean Research Institute, Key Laboratory of Soybean Biology of Chinese Education Ministry, Northeast Agricultural University, Harbin, China) for providing the Phytophthora sojae race 1 strain used in this study.

Conflicts of Interest

The authors declare no conflicts of interest.

References

  1. Schmitthenner, A.F. Problems and progress in control of Phytophthora root rot of soybean. Plant Dis. 1985, 69, 362–368. [Google Scholar] [CrossRef]
  2. Anderson, T.R.; Buzzell, R.I. Diversity and frequency of races of Phytophthora megasperma f. sp. glycinea in soybean fields in Essex County, Ontario, 1980–1989. Plant Dis. 1992, 76, 587–589. [Google Scholar] [CrossRef]
  3. Tyler, B.M. Phytophthora sojae: Root rot pathogen of soybean and model oomycete. Mol. Plant Pathol. 2007, 8, 1–8. [Google Scholar] [CrossRef]
  4. Chang, K.F.; Hwang, S.F.; Ahmed, H.U.; Zhou, Q.; Strelkov, S.E.; Conner, R.L.; McLaren, D.L.; Henriquez, M.A.; Harding, M.W.; Turnbull, G.D. First report of Phytophthora sojae causing root rot in soybean [Glycine max (L.) Merr.] in Alberta, Canada. Crop Prot. 2017, 91, 49–56. [Google Scholar] [CrossRef]
  5. Demirbas, A.; Rector, B.G.; Lohnes, D.G.; Fioritto, R.J.; Graef, G.L.; Cregan, P.B.; Shoemarker, R.C.; Specht, J.E. Simple sequence repeat markers linked to the soybean Rps genes for Phytophthora resistance. Crop Sci. 2001, 41, 1220–1227. [Google Scholar] [CrossRef]
  6. Burnham, K.D.; Dorrance, A.E.; Francis, D.M. Rps8, a new locus in soybean for resistance to Phytophthora sojae. Crop Sci. 2003, 43, 101–110. [Google Scholar] [CrossRef]
  7. Ping, J.; Fitzgerald, J.C.; Zhang, C.; Lin, F.; Bai, Y.; Wang, D.; Aggarwal, R.; Rehman, M.; Crasta, O.; Ma, J. Identification and molecular mapping of Rps11, a novel gene conferring resistance to Phytophthora sojae in soybean. Theor. Appl. Genet. 2016, 129, 445–451. [Google Scholar] [CrossRef]
  8. Zou, X.; Sun, H. DOF transcription factors: Specific regulators of plant biological processes. Front. Plant Sci. 2023, 14, 1044918. [Google Scholar] [CrossRef]
  9. Liu, J.; Jin, Y.; Wu, Y.S.; Liu, Y.; Wang, W.b.; Ren, S.S.; Diao, S.F.; Chen, Y.L. Advances on the structural characteristics and function of Dof gene in plant. Biotechnol. Bull. 2020, 36, 180–219. [Google Scholar] [CrossRef]
  10. Wang, T.; Yue, J.J.; Wang, X.J.; Xu, L.; Li, L.B.; Gu, X.P. Genome-wide identification and characterization of the Dof gene family in moso bamboo (Phyllostachys heterocycla var. Pubescens). Genes Genom. 2016, 38, 733–745. [Google Scholar] [CrossRef]
  11. Lijavetzky, D.; Carbonero, P.; Vicente-Carbajosa, J. Genome-wide comparative phylogenetic analysis of the rice and Arabidopsis Dof gene families. BMC Evol. Biol. 2003, 3, 17. [Google Scholar] [CrossRef]
  12. Gou, C.; Zhang, G.; Deng, Z.; Lin, C.; Li, H.; Liu, H.; Fang, X. Genome-wide analysis of the DNA-binding with one finger gene family reveals soybean expression pattern and functional analysis. Int. J. Mol. Sci. 2025, 26, 6192. [Google Scholar] [CrossRef]
  13. Yanagisawa, S.; Izui, K. Molecular cloning of two DNA-binding proteins of maize that are structurally different but interact with the same sequence motif. J. Biol. Chem. 1993, 268, 16028–16036. [Google Scholar] [CrossRef] [PubMed]
  14. Venkatesh, J.; Park, S.W. Genome-wide analysis and expression profiling of DNA-binding with one zinc finger (Dof) transcription factor family in potato. Plant Physiol. Biochem. 2015, 94, 73–85. [Google Scholar] [CrossRef]
  15. Chen, M.; Liu, X.; Huan, L.; Sun, M.; Liu, L.; Chen, X.; Gao, D.; Li, L. Genome-wide analysis of Dof family genes and their expression during bud dormancy in peach (Prunus persica). Sci. Hortic. 2017, 214, 18–26. [Google Scholar] [CrossRef]
  16. Wu, Z.; Cheng, J.; Cui, J.; Xu, X.; Liang, G.; Luo, X.; Chen, X.; Tang, X.; Hu, K.; Qin, C. Genome-wide identification and expression profile of Dof transcription factor gene family in pepper (Capsicum annuum L.). Front. Plant Sci. 2016, 7, 574. [Google Scholar] [CrossRef] [PubMed]
  17. Gupta, S.; Malviya, N.; Kushwaha, H.; Nasim, J.; Bisht, N.C.; Singh, V.K.; Yadav, D. Insights into structural and functional diversity of Dof (DNA binding with one finger) transcription factor. Planta 2015, 241, 549–562. [Google Scholar] [CrossRef]
  18. Li, S.; Zhang, W.; Si, C.; Chen, J.; Huang, Y.; Li, M.; Liang, H.; Duan, J.; He, C. Genome-Wide identification and functional characterization of the Dof family in Dendrobium officinale. Int. J. Mol. Sci. 2025, 26, 2671. [Google Scholar] [CrossRef]
  19. Wang, Y.; Wang, H.; Li, W.; Dai, G.; Chen, J. Genome-wide identification and expression analysis of the LbDof transcription factor family genes in Lycium barbarum. Plants 2025, 14, 1567. [Google Scholar] [CrossRef]
  20. Wang, P.; Wang, D.; Li, Y.; Li, J.; Liu, B.; Wang, Y.; Gao, C. The transcription factor ThDOF8 binds to a novel cis-element and mediates molecular responses to salt stress in Tamarix hispida. J. Exp. Bot. 2024, 75, 3171–3187. [Google Scholar] [CrossRef]
  21. Zhao, C.; Bai, H.; Li, C.; Pang, Z.; Xuan, L.; Lv, D.; Niu, S. Genome-wide identification of the Dof gene family in Kiwifruit (Actinidia chinensis) and functional validation of AcDOF22 in response to drought stress. Int. J. Mol. Sci. 2024, 25, 9103. [Google Scholar] [CrossRef]
  22. Dai, J.; Wu, Y.; Liu, J.; Wei, L.; Zeng, Y.; Liu, S.; Zhang, J.; Liu, G.; Huang, G. Genome-wide identification and expression analysis of Dof gene family members in mulberry trees (Morus notabilis L.) under drought stress. BMC Genomics 2025, 26, 744. [Google Scholar] [CrossRef]
  23. Liu, Y.; Liu, N.; Deng, X.; Liu, D.; Li, M.; Cui, D.; Hu, Y.; Yan, Y. Genome-wide analysis of wheat DNA-binding with one finger (Dof) transcription factor genes: Evolutionary characteristics and diverse abiotic stress responses. BMC Genom. 2020, 21, 276. [Google Scholar] [CrossRef] [PubMed]
  24. Ramirez-Parra, E.; Perianez-Rodriguez, J.; Navarro-Neila, S.; Gude, I.; Moreno-Risueno, M.A.; del Pozo, J.C. The transcription factor OBP4 controls root growth and promotes callus formation. New Phytol. 2017, 213, 1787–1801. [Google Scholar] [CrossRef]
  25. Li, T.; Wang, X.; Elango, D.; Zhang, W.; Li, M.; Zhang, F.; Pan, Q.; Wu, Y. Genome-wide identification, phylogenetic and expression pattern analysis of Dof transcription factors in blueberry (Vaccinium corymbosum L.). PeerJ 2022, 10, e14087. [Google Scholar] [CrossRef]
  26. Su, Y.; Liang, W.; Liu, Z.; Wang, Y.; Zhao, Y.; Ijaz, B.; Hua, J. Overexpression of GhDof1 improved salt and cold tolerance and seed oil content in Gossypium hirsutum. J. Plant Physiol. 2017, 218, 222–234. [Google Scholar] [CrossRef] [PubMed]
  27. Fornara, F.; Panigrahi, K.C.; Gissot, L.; Sauerbrunn, N.; Rühl, M.; Jarillo, J.A.; Coupland, G. Arabidopsis Dof transcription factors act redundantly to reduce CONSTANS expression and are essential for a photoperiodic flowering response. Dev. Cell 2009, 17, 75–86. [Google Scholar] [CrossRef] [PubMed]
  28. Zhang, W.J.; Ma, C.N.; Du, L.D.; Xiang, Y.; Xiao, F.; Liu, Y.T.; Wang, C.K.; Li, W.K.; Zhao, T.T.; Hu, D.G. Two DNA-binding one zinc finger transcription factors, MdCDOF3 and MdDOF3.6, accelerate leaf senescence by activating cytokinin oxidase MdCKX7 in response to sorbitol signaling in apple. Hortic. Res. 2025, 12, uhaf120. [Google Scholar] [CrossRef]
  29. Ding, R.; Xiao, T.; Li, S.; Qiang, J.; Zhang, H.; Chang, H.; Yan, Y.; Li, X. Wheat endosperm-specific transcription factor TaDOF6 enhances grain development by regulating TaSWEET13h expression and facilitating sugar and gibberellin transport. Front. Plant Sci. 2025, 16, 1608090. [Google Scholar] [CrossRef]
  30. Cai, X.; Zhang, C.; Shu, W.; Ye, Z.; Li, H.; Zhang, Y. The transcription factor SlDof22 involved in ascorbate accumulation and salinity stress in tomato. Biochem. Biophys. Res. Commun. 2016, 474, 736–741. [Google Scholar] [CrossRef]
  31. Wen, Y.; Tan, C.; Zhang, Y.; Wu, H.; Chen, D.; Yue, H.; Ding, Z.; Cao, S.; Zheng, K. Genome-wide characterization and functional analysis of CsDOF transcription factors in Camellia sinensis cv. Tieguanyin under combined heat-drought stress. Plants 2025, 14, 1829. [Google Scholar] [CrossRef]
  32. Song, F.; Goodman, R.M. Cloning and identification of the promoter of the tobacco Sar8.2b gene, a gene involved in systemic acquired resistance. Gene 2002, 290, 115–124. [Google Scholar] [CrossRef]
  33. Qiu, T.; Wei, S.; Fang, K.; Zhang, M.; Li, Y.; Feng, Y.; Cheng, Y.; Zhang, S.; Tian, J.; Gao, A.; et al. The atypical Dof transcriptional factor OsDes1 contributes to stay-green, grain yield, and disease resistance in rice. Sci. Adv. 2024, 10, eadp0345. [Google Scholar] [CrossRef] [PubMed]
  34. Trusov, Y.; Sewelam, N.; Rookes, J.E.; Kunkel, M.; Nowak, E.; Schenk, P.M.; Botella, J.R. Heterotrimeric G proteins-mediated resistance to necrotrophic pathogens includes mechanisms independent of salicylic acid-, jasmonic acid/ethylene- and abscisic acid-mediated defense signaling. Plant J. 2009, 58, 69–81. [Google Scholar] [CrossRef] [PubMed]
  35. Campos, M.L.; Kang, J.H.; Howe, G.A. Jasmonate-triggered plant immunity. J. Chem. Ecol. 2014, 40, 657–675. [Google Scholar] [CrossRef]
  36. Mersmann, S.; Bourdais, G.; Rietz, S.; Robatzek, S. Ethylene signaling regulates accumulation of the FLS2 receptor and is required for the oxidative burst contributing to plant immunity. Plant Physiol. 2010, 154, 391–400. [Google Scholar] [CrossRef] [PubMed]
  37. Charitha, P.A.J.; Emilee, R.M.S.; Jocelyn, A.O.; Stephen, E.S. The complex roles of plant hormones during clubroot disease development in the Brassicaceae. J. Plant Growth Regul. 2025, 44, 5692–5712. [Google Scholar] [CrossRef]
  38. Cai, M.; Lin, J.; Li, Z.; Lin, Z.; Ma, Y.; Wang, Y.; Ming, R. Allele specific expression of Dof genes responding to hormones and abiotic stresses in sugarcane. PLoS ONE 2020, 15, e0227716. [Google Scholar] [CrossRef]
  39. Virág, E.; Nagy, Á.; Tóth, B.B.; Kutasy, B.; Pallos, J.P.; Szigeti, Z.M.; Máthé, C.; Kardos, G.; Hegedűs, G. Master regulatory transcription factors in β-aminobutyric acid-induced resistance (BABA-IR): A perspective on phytohormone biosynthesis and signaling in Arabidopsis thaliana and Hordeum vulgare. Int. J. Mol. Sci. 2024, 25, 9179. [Google Scholar] [CrossRef]
  40. Li, J.; Zhang, Y.; Xu, L.; Wang, C.; Luo, Y.; Feng, S.; Yuan, Y.; Yang, Q.; Feng, B. Genome-wide identification of DNA binding with one finger (dof) gene family in tartary buckwheat (Fagopyrum tataricum) and analysis of its expression pattern after exogenous hormone stimulation. Biology 2022, 11, 173. [Google Scholar] [CrossRef]
  41. Yu, Y.H.; Bian, L.; Wan, Y.T.; Jiao, Z.L.; Yu, K.K.; Zhang, G.H.; Guo, D.L. Grape (Vitis vinifera) VvDOF3 functions as a transcription activator and enhances powdery mildew resistance. Plant Physiol. Biochem. 2019, 143, 183–189. [Google Scholar] [CrossRef]
  42. Paz, M.M.; Shou, H.; Guo, Z.; Zhang, Z.; Banerjee, A.K.; Wang, K. Assessment of conditions affecting Agrobacterium-mediated soybean transformation using the cotyledonary node explants. Euphytica 2004, 136, 167–179. [Google Scholar] [CrossRef]
  43. Li, S.; Cong, Y.; Liu, Y.; Wang, T.; Shuai, Q.; Chen, N.; Gai, J.; Li, Y. Optimization of Agrobacterium-mediated transformation in soybean. Front. Plant Sci. 2017, 8, 246. [Google Scholar] [CrossRef]
  44. Shrestha, S.D.; Chapman, P.; Zhang, Y.; Gijzen, M. Strain specific factors control effector gene silencing in Phytophthora sojae. PLoS ONE 2016, 11, e0150530. [Google Scholar] [CrossRef]
  45. Yang, X.; Jiang, X.; Yan, W.; Huang, Q.; Sun, H.; Zhang, X.; Zhang, Z.; Ye, W.; Wu, Y.; Govers, F.; et al. The mevalonate pathway is important for growth, spore production, and the virulence of Phytophthora sojae. Front. Microbiol. 2021, 12, 772994. [Google Scholar] [CrossRef]
  46. Alexander, D.; Goodman, R.M.; Gut-Rella, M.; Glascock, C.; Weymann, K.; Friedrich, L.; Maddox, D.; Ahl-Goy, P.; Luntz, T.; Ward, E. Increased tolerance to two oomycete pathogens in transgenic tobacco expressing pathogenesis-related protein 1a. Proc. Natl. Acad. Sci. USA 1993, 90, 7327–7331. [Google Scholar] [CrossRef]
  47. Sessa, G.; Yang, X.Q.; Raz, V.; Eyal, Y.; Fluhr, R. Dark induction and subcellular localization of the pathogenesis-related PRB-1b protein. Plant Mol. Biol. 1995, 28, 537–547. [Google Scholar] [CrossRef] [PubMed]
  48. Xu, P.F.; Jiang, L.Y.; Wu, J.J.; Li, W.B.; Fan, S.J.; Zhang, S.Z. Isolation and characterization of a pathogenesis-related protein 10 gene (GmPR10) with induced expression in soybean (Glycine max) during infection with Phytophthora sojae. Mol. Biol. Rep. 2014, 41, 4899–4909. [Google Scholar] [CrossRef] [PubMed]
  49. Breen, S.; Williams, S.J.; Outram, M.; Kobe, B.; Solomon, P.S. Emerging insights into the functions of pathogenesis-related protein 1. Trends Plant Sci. 2017, 22, 871–879. [Google Scholar] [CrossRef]
  50. Umemura, Y.; Ishiduka, T.; Yamamoto, R.; Esaka, M. The Dof domain, a zinc finger DNA-binding domain conserved only in higher plants, truly functions as a Cys2/Cys2 Zn finger domain. Plant J. 2004, 37, 741–749. [Google Scholar] [CrossRef] [PubMed]
  51. Yanagisawa, S. The Dof family of plant transcription factors. Trends Plant Sci. 2002, 7, 555–560. [Google Scholar] [CrossRef]
  52. Chen, P.; Yan, M.; Li, L.; He, J.; Zhou, S.; Li, Z.; Niu, C.; Bao, C.; Zhi, F.; Ma, F.; et al. The apple DNA-binding one zinc-finger protein MdDof54 promotes drought resistance. Hortic. Res. 2020, 7, 195. [Google Scholar] [CrossRef]
  53. Khan, I.; Khan, S.; Zhang, Y.; Zhou, J.P. Genome-wide analysis and functional characterization of the Dof transcription factor family in rice (Oryza sativa L.). Planta 2021, 253, 101. [Google Scholar] [CrossRef]
  54. Yang, C.; Lai, Y.M.; Yao, N. Plant sphingolipids: Subcellular distributions and functions. Curr. Opin. Plant Biol. 2025, 85, 102704. [Google Scholar] [CrossRef]
  55. Marques, A.C.; Vinckenbosch, N.; Brawand, D.; Kaessmann, H. Functional diversification of duplicate genes through subcellular adaptation of encoded proteins. Genome Biol. 2008, 9, R54. [Google Scholar] [CrossRef]
  56. Yanagisawa, S.; Schmidt, R.J. Diversity and similarity among recognition sequences of Dof transcription factors. Plant J. 1999, 17, 209–214. [Google Scholar] [CrossRef] [PubMed]
  57. Wu, J.; Chen, L.; Chen, M.; Zhou, W.; Dong, Q.; Jiang, H.; Cheng, B. The Dof-domain transcription factor ZmDOF36 positively regulates starch synthesis in transgenic maize. Front. Plant Sci. 2019, 10, 465. [Google Scholar] [CrossRef]
  58. Luo, T.; Song, Y.; Gao, H.; Wang, M.; Cui, H.; Ji, C.; Wang, J.; Yuan, L.; Li, R. Genome-wide identification and functional analysis of Dof transcription factor family in Camelina sativa. BMC Genom. 2022, 23, 812. [Google Scholar] [CrossRef]
  59. Magadum, S.; Banerjee, U.; Murugan, P.; Gangapur, D.; Ravikesavan, R. Gene duplication as a major force in evolution. J. Genet. 2013, 92, 155–161. [Google Scholar] [CrossRef] [PubMed]
  60. Panchy, N.; Lehti-Shiu, M.; Shiu, S.H. Evolution of gene duplication in plants. Plant Physiol. 2016, 171, 2294–2316. [Google Scholar] [CrossRef] [PubMed]
  61. Qiao, X.; Li, Q.; Yin, H.; Qi, K.; Li, L.; Wang, R.; Zhang, S.; Paterson, A.H. Gene duplication and evolution in recurring polyploidization-diploidization cycles in plants. Genome Biol. 2019, 20, 38. [Google Scholar] [CrossRef]
  62. Kondrashov, F.A. Gene duplication as a mechanism of genomic adaptation to a changing environment. Proc. Biol. Sci. 2012, 279, 5048–5057. [Google Scholar] [CrossRef]
  63. Wu, Q.; Liu, X.; Yin, D.D.; Yuan, H.; Xie, Q.; Zhao, X.F.; Li, X.B.; Zhu, L.H.; Li, S.G.; Li, D.Y. Constitutive expression of OsDof4, encoding a C2-C2 zinc finger transcription factor, confesses its distinct flowering effects under long- and short-day photoperiods in rice (Oryza sativa L.). BMC Plant Biol. 2017, 17, 166. [Google Scholar] [CrossRef] [PubMed]
  64. Wu, Y.F.; Lee, S.K.; Yoo, Y.; Wei, J.; Kwon, S.Y.; Lee, S.W.; Jeon, J.S.; An, G. Rice transcription factor OsDOF11 modulates sugar transport by promoting expression of sucrose transporter and SWEET genes. Mol. Plant 2018, 11, 833–845. [Google Scholar] [CrossRef] [PubMed]
  65. Qin, H.; Wang, J.; Chen, X.B.; Wang, F.F.; Peng, P.; Zhou, Y.; Miao, Y.C.; Zhang, Y.O.; Gao, Y.D.; Qi, Y.D.; et al. Rice OsDOF15 contributes to ethylene-inhibited primary root elongation under salt stress. New Phytol. 2019, 223, 798–813. [Google Scholar] [CrossRef] [PubMed]
  66. Shim, Y.; Kang, K.; An, G.; Paek, N.C. Rice DNA-binding one zinc finger 24 (OsDOF24) delays leaf senescence in a jasmonate-mediated pathway. Plant Cell Physiol. 2019, 60, 2065–2076. [Google Scholar] [CrossRef]
  67. Zhang, Y.; Verhoeff, N.I.; Chen, Z.; Chen, S.; Wang, M.; Zhu, Z.; Ouwerkerk, P.B.F. Functions of OsDof25 in regulation of OsC4PPDK. Plant Mol. Biol. 2015, 89, 229–242. [Google Scholar] [CrossRef]
  68. Zuo, Z.F.; Kang, H.G.; Park, M.Y.; Jeong, H.; Sun, H.J.; Song, P.S.; Lee, H.Y. Zoysia japonica MYC type transcription factor ZjICE1 regulates cold tolerance in transgenic Arabidopsis. Plant Sci. 2019, 289, 110254. [Google Scholar] [CrossRef]
  69. Mayordomo, I.; Estruch, F.; Sanz, P. Convergence of the target of rapamycin and the Snf1 protein kinase pathways in the regulation of the subcellular localization of Msn2, a transcriptional activator of STRE (Stress Response Element)-regulated genes. J. Biol. Chem. 2002, 277, 35650–35656. [Google Scholar] [CrossRef]
  70. Lohani, N.; Babaei, S.; Singh, M.B.; Bhalla, P.L. Genome-wide in silico identification and comparative analysis of Dof gene family in Brassica napus. Plants 2021, 10, 709. [Google Scholar] [CrossRef]
  71. Liu, J.; Meng, Q.L.; Xiang, H.T.; Shi, F.M.; Ma, L.G.; Li, Y.C.; Liu, C.L.; Liu, Y.; Su, B.H. Genome-wide analysis of Dof transcription factors and their response to cold stress in rice (Oryza sativa L.). BMC Genom. 2021, 22, 800. [Google Scholar] [CrossRef]
  72. Wong, J.; Gao, L.; Yang, Y.; Zhai, J.X.; Arikit, S.; Yu, Y.; Duan, S.Y.; Chan, V.; Xiong, Q.; Yan, J.; et al. Roles of small RNAs in soybean defense against Phytophthora sojae infection. Plant J. 2014, 79, 928–940. [Google Scholar] [CrossRef]
  73. Cheng, Q.; Dong, L.D.; Gao, T.J.; Liu, T.F.; Li, N.H.; Wang, L.; Chang, X.; Wu, J.J.; Xu, P.F.; Zhang, S.Z. The bHLH transcription factor GmPIB1 facilitates resistance to Phytophthora sojae in Glycine max. J. Exp. Bot. 2018, 69, 2527–2541. [Google Scholar] [CrossRef]
  74. Li, W.; Zheng, X.; Cheng, R.; Zhong, C.; Zhao, J.; Liu, T.H.; Yi, T.; Zhu, Z.; Xu, J.; Meksem, K.; et al. Soybean ZINC FINGER PROTEIN03 targets two SUPEROXIDE DISMUTASE1s and confers resistance to Phytophthora sojae. Plant Physiol. 2023, 69, 2527–2541. [Google Scholar] [CrossRef]
  75. Zhu, X.; Guo, L.; Zhu, R.; Zhou, X.; Zhang, J.; Li, D.; He, S.; Qiao, Y. Phytophthora sojae effector PsAvh113 associates with the soybean transcription factor GmDPB to inhibit catalase-mediated immunity. Plant Biotechnol. J. 2023, 21, 1393–1407. [Google Scholar] [CrossRef] [PubMed]
  76. Kang, H.G.; Singh, K.B. Characterization of salicylic acid-responsive, Arabidopsis Dof domain proteins: Overexpression of OBP3 leads to growth defects. Plant J. 2000, 21, 329–339. [Google Scholar] [CrossRef]
  77. van Verk, M.C.; Pappaioannou, D.; Neeleman, L.; Bol, J.F.; Linthorst, H.J.M. A novel WRKY transcription factor is required for induction of PR-1a gene expression by salicylic acid and bacterial elicitors. Plant Physiol. 2008, 146, 1983–1995. [Google Scholar] [CrossRef] [PubMed]
  78. Zhang, Y.; Fan, W.; Kinkema, M.; Li, X.; Dong, X. Interaction of NPR1 with basic leucine zipper protein transcription factors that bind sequences required for salicylic acid induction of the PR-1 gene. Proc. Natl. Acad. Sci. USA 1999, 96, 6523–6528. [Google Scholar] [CrossRef] [PubMed]
  79. He, K.; Gou, X.P.; Yuan, T.; Lin, H.H.; Asami, T.; Yoshida, S.; Russell, S.D.; Li, J. BAK1 and BKK1 regulate Brassinosteroid-dependent growth and Brassinosteroidindependent cell-death pathways. Curr. Biol. 2007, 17, 1109–1115. [Google Scholar] [CrossRef]
  80. Ward, E.R.; Uknes, S.J.; Williams, S.C.; Dincher, S.S.; Wiederhold, D.L.; Alexander, D.C.; Ahl-Goy, P.; Metraux, J.P.; Ryals, J.A. Coordinate gene activity in response to agents that induce systemic acquired resistance. Plant Cell 1991, 3, 1085–1094. [Google Scholar] [CrossRef]
Plants 14 03621 g001
Figure 1. Genomic localization of soybean Dof members on chromosomes. Gene spacing are set at 200 kilobases, which is used to calculate the gene density on each chromosome. The color will gradually change from blue (low gene density) to red (high gene density) to represent this difference.
Figure 1. Genomic localization of soybean Dof members on chromosomes. Gene spacing are set at 200 kilobases, which is used to calculate the gene density on each chromosome. The color will gradually change from blue (low gene density) to red (high gene density) to represent this difference.
Plants 14 03621 g001
Plants 14 03621 g002
Figure 2. Phylogenetic tree of the Dof proteins in soybean and Arabidopsis.
Figure 2. Phylogenetic tree of the Dof proteins in soybean and Arabidopsis.
Plants 14 03621 g002
Plants 14 03621 g003
Figure 3. Visualization of motifs, domains, and gene structures of soybean Dof members.
Figure 3. Visualization of motifs, domains, and gene structures of soybean Dof members.
Plants 14 03621 g003
Plants 14 03621 g004
Figure 4. Synteny analysis of interchromosomal relationships of soybean Dof members. This curved structure represents chromosomes, and the different colored lines indicate segmental gene pairs.
Figure 4. Synteny analysis of interchromosomal relationships of soybean Dof members. This curved structure represents chromosomes, and the different colored lines indicate segmental gene pairs.
Plants 14 03621 g004
Plants 14 03621 g005
Figure 5. Synteny analysis of Dof genes. Each horizontal line represents a chromosome, and the red lines indicate the Dof homologous gene pairs.
Figure 5. Synteny analysis of Dof genes. Each horizontal line represents a chromosome, and the red lines indicate the Dof homologous gene pairs.
Plants 14 03621 g005
Plants 14 03621 g006
Figure 6. Cis-acting elements in the promoters of soybean Dof members. (A) Variations in different types of cis-acting elements. (B) Number of cis-acting elements. (C) Pie charts show the proportion of different cis-acting elements in each category.
Figure 6. Cis-acting elements in the promoters of soybean Dof members. (A) Variations in different types of cis-acting elements. (B) Number of cis-acting elements. (C) Pie charts show the proportion of different cis-acting elements in each category.
Plants 14 03621 g006
Plants 14 03621 g007
Figure 7. Screening of Dof genes in soybean after P. sojae infection. (A) The phenotypic characteristics of the resistant variety and the susceptible variety after P. sojae infection. (B) Overlapping between JN28-P. sojae, JN4507-P. sojae and Dof genes. (C) Volcano plot of soybean Dof members. (D) Expression levels of GmDof63 in JN28 seedlings during P. sojae infection. Three biological replicates were used for each sample, and Student’s t-test (* p < 0.05, ** p < 0.01) was conducted to determine statistical significance. Error bars represent ±SD.
Figure 7. Screening of Dof genes in soybean after P. sojae infection. (A) The phenotypic characteristics of the resistant variety and the susceptible variety after P. sojae infection. (B) Overlapping between JN28-P. sojae, JN4507-P. sojae and Dof genes. (C) Volcano plot of soybean Dof members. (D) Expression levels of GmDof63 in JN28 seedlings during P. sojae infection. Three biological replicates were used for each sample, and Student’s t-test (* p < 0.05, ** p < 0.01) was conducted to determine statistical significance. Error bars represent ±SD.
Plants 14 03621 g007
Plants 14 03621 g008
Figure 8. Sequence characteristics and expression pattern of GmDof63. (A) The GmDof63 gene sequence and the GmDof63 protein sequence. The zinc finger domain CX2CX21CX2C are underlined in red. (B) Expression pattern of GmDof63 in soybean under different stresses. The various treatments were the 0.5 mM SA treatment, 100 µM MeJA treatment, 200 mg/L ETH treatment and 50 µM ABA treatment. Three biological replicates were used for each sample, and Student’s t-test (* p < 0.05, ** p < 0.01) was performed to determine statistical significance. Error bars represent ±SD.
Figure 8. Sequence characteristics and expression pattern of GmDof63. (A) The GmDof63 gene sequence and the GmDof63 protein sequence. The zinc finger domain CX2CX21CX2C are underlined in red. (B) Expression pattern of GmDof63 in soybean under different stresses. The various treatments were the 0.5 mM SA treatment, 100 µM MeJA treatment, 200 mg/L ETH treatment and 50 µM ABA treatment. Three biological replicates were used for each sample, and Student’s t-test (* p < 0.05, ** p < 0.01) was performed to determine statistical significance. Error bars represent ±SD.
Plants 14 03621 g008
Plants 14 03621 g009
Figure 9. Subcellular location of GmDof63 in the Arabidopsis mesophyll protoplasts. (A) Construction of 35S::GFP vector and 35S::GmDof63-GFP vector. (B) 35S::GFP vector or 35S::GmDof63-GFP vector was transiently expressed in the Arabidopsis mesophyll protoplasts, respectively. The green fluorescence signal indicates the 35S::GmDof63-GFP or 35S::GFP protein, and the red fluorescence signal indicates the nuclear marker protein H2B-mCherry, and the fluorescence signals were observed under a confocal microscope. Scale bars = 10 µm.
Figure 9. Subcellular location of GmDof63 in the Arabidopsis mesophyll protoplasts. (A) Construction of 35S::GFP vector and 35S::GmDof63-GFP vector. (B) 35S::GFP vector or 35S::GmDof63-GFP vector was transiently expressed in the Arabidopsis mesophyll protoplasts, respectively. The green fluorescence signal indicates the 35S::GmDof63-GFP or 35S::GFP protein, and the red fluorescence signal indicates the nuclear marker protein H2B-mCherry, and the fluorescence signals were observed under a confocal microscope. Scale bars = 10 µm.
Plants 14 03621 g009
Plants 14 03621 g010
Figure 10. GmDof63 enhances soybean resistance to P. sojae. (A) Construction of GmDof63-overexpressing vector 35S::GmDof63. (B) Phenotypes of WT and GmDof63-overexpressing transgenic soybean seedlings treated with P. sojae for 48 h in the soybean root hydroponic assay. (C) Relative expression levels of GmDof63 in WT and GmDof63-overexpressing transgenic soybean seedlings at 48 h post-inoculation. (D) Accumulation of P. sojae in WT and GmDof63-overexpressing transgenic soybean seedlings at 48 h post-inoculation. The relative accumulation of P. sojae in roots was measured based on the relative expression of P. sojae housekeeping gene PsACT (XM_009530461.1) to soybean housekeeping gene GmActin (Glyma.18G290800.1) (ΔCt = CtHK of P. sojae − CtHK of soybean). (E) Relative expression levels of PR1a (AF136636), PR2 (M37753), PR3 (AF202731), PR4 (BT090788), PR5a (M21297), PR10 (FJ960440) in GmDof63-overexpressing transgenic soybean seedlings were compared with those in WT. WT, wild type. Three biological replicates were used for each sample, and Student’s t-test (* p < 0.05, ** p < 0.01) was performed to determine statistical significance. Error bars represent ±SD.
Figure 10. GmDof63 enhances soybean resistance to P. sojae. (A) Construction of GmDof63-overexpressing vector 35S::GmDof63. (B) Phenotypes of WT and GmDof63-overexpressing transgenic soybean seedlings treated with P. sojae for 48 h in the soybean root hydroponic assay. (C) Relative expression levels of GmDof63 in WT and GmDof63-overexpressing transgenic soybean seedlings at 48 h post-inoculation. (D) Accumulation of P. sojae in WT and GmDof63-overexpressing transgenic soybean seedlings at 48 h post-inoculation. The relative accumulation of P. sojae in roots was measured based on the relative expression of P. sojae housekeeping gene PsACT (XM_009530461.1) to soybean housekeeping gene GmActin (Glyma.18G290800.1) (ΔCt = CtHK of P. sojae − CtHK of soybean). (E) Relative expression levels of PR1a (AF136636), PR2 (M37753), PR3 (AF202731), PR4 (BT090788), PR5a (M21297), PR10 (FJ960440) in GmDof63-overexpressing transgenic soybean seedlings were compared with those in WT. WT, wild type. Three biological replicates were used for each sample, and Student’s t-test (* p < 0.05, ** p < 0.01) was performed to determine statistical significance. Error bars represent ±SD.
Plants 14 03621 g010
Disclaimer/Publisher’s Note: The statements, opinions and data contained in all publications are solely those of the individual author(s) and contributor(s) and not of MDPI and/or the editor(s). MDPI and/or the editor(s) disclaim responsibility for any injury to people or property resulting from any ideas, methods, instructions or products referred to in the content.

Share and Cite

MDPI and ACS Style

Fan, S.; Chen, H.; Huo, Y.; Song, Y.; Wang, P.; Zhang, Z.; Jiang, L. Genome-Wide Analysis of the Dof Gene Family in Soybean and Functional Identification of GmDof63 in Response to Phytophthora sojae Infection. Plants 2025, 14, 3621. https://doi.org/10.3390/plants14233621

AMA Style

Fan S, Chen H, Huo Y, Song Y, Wang P, Zhang Z, Jiang L. Genome-Wide Analysis of the Dof Gene Family in Soybean and Functional Identification of GmDof63 in Response to Phytophthora sojae Infection. Plants. 2025; 14(23):3621. https://doi.org/10.3390/plants14233621

Chicago/Turabian Style

Fan, Sujie, Haiyuan Chen, Yuhan Huo, Yang Song, Piwu Wang, Zhuo Zhang, and Liangyu Jiang. 2025. "Genome-Wide Analysis of the Dof Gene Family in Soybean and Functional Identification of GmDof63 in Response to Phytophthora sojae Infection" Plants 14, no. 23: 3621. https://doi.org/10.3390/plants14233621

APA Style

Fan, S., Chen, H., Huo, Y., Song, Y., Wang, P., Zhang, Z., & Jiang, L. (2025). Genome-Wide Analysis of the Dof Gene Family in Soybean and Functional Identification of GmDof63 in Response to Phytophthora sojae Infection. Plants, 14(23), 3621. https://doi.org/10.3390/plants14233621

Note that from the first issue of 2016, this journal uses article numbers instead of page numbers. See further details here.

Article Metrics

Back to TopTop