Skip to main content
Download PDF

Ectopic and transient expression of VvDIR4 gene in Arabidopsis and grapes enhances resistance to anthracnose via affecting hormone signaling pathways and lignin production

BMC Genomics volume 25, Article number: 895 (2024) Cite this article

Abstract

Background

DIR (Dirigent) proteins play important roles in the biosynthesis of lignin and lignans and are involved in various processes such as plant growth, development, and stress responses. However, there is less information about VvDIR proteins in grapevine (Vitis vinifera L).

Results

In this study, we used bioinformatics methods to identify members of the DIR gene family in grapevine and identified 18 VvDIR genes in grapevine. These genes were classified into 5 subfamilies based on phylogenetic analysis. In promoter analysis, various plant hormones, stress, and light-responsive cis-elements were detected. Expression profiling of all genes following Colletotrichum gloeosporioides infection and phytohormones (salicylic acid (SA) and jasmonic acid (JA)) application suggested significant upregulation of 17 and 6 VvDIR genes, respectively. Further, we overexpressed the VvDIR4 gene in Arabidopsis thaliana and grapes for functional analysis. Ectopic expression of VvDIR4 in A. thaliana and transient expression in grapes increased resistance against C. gloeosporioides and C. higginsianum, respectively. Phenotypic observations showed small disease lesions in transgenic plants. Further, the expression patterns of genes having presumed roles in SA and JA signaling pathways were also influenced. Lignin contents were measured before and after C. higginsianum infection; the transgenic A. thaliana lines showed higher lignin content than wild-type, and a significant increase was observed after C. higginsianum infection.

Conclusions

Based on the findings, we surmise that VvDIR4 is involved in hormonal and lignin synthesis pathways which regulate resistance against anthracnose. Our study provides novel insights into the function of VvDIR genes and new candidate genes for grapevine disease resistance breeding programs.

Peer Review reports

Background

During the process of plant evolution, plants have developed complex and effective defense mechanisms to mitigate environmental impacts. These mechanisms include oxidative cleavage, lignin synthesis, lignin deposition, and release of acids as signaling pathways [1,2,3]. Lignin is an important component of plant cell walls and is a randomly polymerized macromolecule consisting of highly substituted phenylpropane units. Lignin is one of the most abundant organic polymeric materials on land and primarily constitutes the secondary cell wall. It enhances cell wall hardness through cross-linking with cellulose and hemicellulose and improves the ability to resist adverse biotic and abiotic stresses [4]. Dirigent (DIR) proteins were initially discovered in Forsythia and Pinus and were found to have roles in lignin synthesis [5]. Subsequent studies revealed the widespread distribution of DIR proteins in vascular plants, including ferns, gymnosperms, and angiosperms [6]. Research has also found that DIR proteins participate in responses to diseases, pests, and certain abiotic stresses [7]. Most DIR genes do not contain introns [8]. Ralph et al. [9] and Sahani et al. [10] conducted phylogenetic analysis of 150 DIR proteins from different species and classified the DIR gene family into six subfamilies: DIR-a, DIR-b/d, DIR-c, DIR-e, DIR-f, and DIR-g, with DIR-c being specific to monocotyledonous plants. Genes from the DIR-a subfamily were primarily involved in lignin-related reactions, while limited information is available about the specific functions of genes within other subfamilies.

The main function of DIR genes is to regulate plant stress responses by participating in the control of lignin and suberin biosynthesis pathways [11]. DIR genes exhibit inducible responses to pathogen attacks, for example PsDIR1 gene in peas being was induced in response to infections by Cladosporium fulvum and Alternaria solani [12]. The DIR gene in Brassica rapa enhanced resistance to Verticillium dahliae and Alternaria brassicicola [13]. BrDIR2 gene in oilseed rape was induced after infection with Fusarium oxysporum f.sp. conglutinans and in cabbage, four homologous genes to BrDIR were found to respond to the same fungus infection [14, 15]. The HfrDIR genes in wheat responded to induced infection by Hessian fly [16]. In strawberry, 33 FvDIR genes were identified and FvDIR13 showed response to Colletotrichum gloeosporioides infection [17]. Eleven SIDIR genes in Solanum lycopersicum enhanced resistance against Pst DC3000 infection [18]. The CsDIR16 gene in Cucumis sativus showed upregulated expression under cucumber mosaic virus stress [19]. In Vigna angularis, the expression of VaDIR14, VaDIR16, and VaDIR33 was significantly induced following fungus infection [20]. Moreover, certain DIR genes may exhibit responsiveness to the induction of disease-related hormones. For example, the expression of the SHDIR11 and SHDIR16 genes in Saccharum officinarum was induced by SA and MeJA [21]. FvDIR1 in Fragaria vesca was induced by MeJA, while FvDIR7 and FvDIR24 were significantly induced by SA [17]. In Populus, PeDIR19 showed significant upregulation following exogenous hormones (MeJA and ABA) application [22]. Overexpression of GmDIR22 in Glycine max plants enhanced resistance to Phytophthora sojae via increasing total lignin accumulation [23]. Overexpression of TaDIR13 in Nicotiana tabacum increased lignin accumulation and enhanced resistance to Pseudomonas syringae [24]. Overexpression of GhDIR1 gene in cotton increased lignin contents, enhanced lignification of the epidermis and vascular bundles, and improved tolerance to Verticillium wilt [25]. Six DIR genes in Pinus species are strongly induced after mechanical damage and bark beetle attack [9]. These all findings suggest the critical roles of DIR genes in plant stress resistance.

Grapes (Vitis vinifera L.) are among the most economically valuable cultivated fruit trees, having a rich cultivation history and diverse range of uses. Most grape production worldwide involves European grapes (V. vinifera ssp.), which are renowned for their high quality and yield, resulting in significant economic benefits. However, European grapes are more susceptible to fungal diseases, especially anthracnose, when compared to V. labrusca L. Anthracnose stands out as a major grape disease, posing a serious threat to the viticulture industry [26]. Anthracnose, also known as late rot, is primarily caused by C. gloeosporioides and is widespread globally. C. gloeosporioides mostly affects mature fruits, causing the growth of black and brown anthracnose spots on the infected fruits and leaves. It also leads to surface ulceration of the fruits, juice extravasation, and mold contamination, all of which severely impact grape yield and quality. The role of DIR genes in disease resistance has been well-studied in many crops [11, 17, 27, 28]. However, there is limited information available on grape DIR genes. The critical roles of DIR genes in disease resistance and the importance of grapes justify the need for DIR genes identification and functional characterization in grapevine.

This study utilized bioinformatics methods to identify the VvDIR gene family members in grapes, and analyzed their encoded protein’s physicochemical properties, gene structure, promoter elements, evolutionary relationships, and subcellular localization predictions. The expression analysis of VvDIR genes was analyzed following inoculation with C. gloeosporioides and hormone (SA and JA) treatments. Further, we investigated the functionality of the VvDIR4 gene in disease resistance mechanisms by overexpressing it in A. thaliana and grape leaf tissues through stable and transient transformation. This research contributes to the further study of the DIR gene family in grapes, laying the foundation for understanding its role in anthracnose disease resistance.

Results

Identification and physiochemical properties of VvDIR proteins

18 DIR genes were identified in the grape genome and were unevenly distributed on 8 chromosomes. The genes were named according to their chromosomal positions (VvDIR1 to VvDIR18). The physicochemical properties and basic information including the deduced protein length, Mw, pI, and instability index of all the VvDIR proteins are mentioned in Table 1. The VvDIR proteins have coding sequence lengths ranging from 369 bp (VvDIR11) to 3846 bp (VvDIR17). The pI values range from 4.57 (VvDIR18) to 10.89 (VvDIR11), while the MW ranges from 14.28 kDa (VvDIR12) to 121.32 kDa (VvDIR3). The instability index varied from 21.8 (VvDIR2) to 60.84 (VvDIR17). Most (15 out of 18) of the VvDIR proteins were predicted to be present in chloroplasts and plasmids.

Table 1 Basic details and physicochemical properties of VvDIR proteins
Full size table

Chromosomal distribution and evolutionary history of VvDIR genes

Eighteen VvDIR genes were mapped unequally on eight grape chromosomes (Fig. 1) and Chromosome 6 contained the highest number of VvDIR members (5 genes), followed by chromosome 8 (4 genes) and chromosome (3 genes). The VvDIR genes were clustered at the top of chromosome 6 and the lower half of chromosome 8, suggesting that these chromosomes underwent secondary duplication events (Fig. 1). In plants, tandem duplication and segmental duplication are the two most common causes of gene family expansion [29]. Only one event of segmental duplication (VvDIR5/VvDIR10) was identified (Fig. 2).

Fig. 1
figure 1

Chromosomal distribution of the VvDIR genes. The size of the chromosome is shown using a vertical scale (Mb)

Full size image
Fig. 2
figure 2

Collinearity analysis of VvDIR genes. A red line connects the syntenic relationships of VvDIR genes

Full size image

Phylogenetic relationships and classification of VvDIR proteins

Based on phylogenetic trees constructed among the DIR proteins of grapes, rice, and A. thaliana, the DIR proteins were divided into five subfamilies (DIR-a, b/d, e, f, and g) (Fig. 3). The VvDIR proteins were unevenly distributed in the subfamilies (DIR-a, b/d, e, f, and g) and the DIR-a subfamily contained the highest VvDIR genes (VvDIR2, 4, 6, 9, 13, and 14). The DIR-e subfamily contained five VvDIR (VvDIR1, 3, 7, 8, and 15) genes followed by DIR-g subfamily which has four VvDIR genes (VvDIR5, 11, 17, and 18). The DIR-b/d, DIR-f, DIR-c subfamilies contained 2, 1, and 0 members, respectively. The alignment of clustering patterns of phylogenetic trees with previous classifications indicates the accuracy of the results [9, 30, 31].

Fig. 3
figure 3

Phylogenetic analysis of DIR genes in A. thaliana, O. sativa, and V. vinifera. The full-length amino acid sequences of DIR genes from A. thaliana (At), O. sativa (Os), and V. vinifera (Vv, blue) were aligned by Clustal W. MEGA 7.0 was used to create the phylogenetic tree, using the maximum-likelihood method with 1000 bootstrap repetitions. Different subfamilies are marked with different colors

Full size image

Gene structure architecture of VvDIR members

The conserved motifs, domains, and evolutionary relationships of the 18 VvDIR members were analyzed to get deep insights (Fig. 4). The MEME suite was utilized to detect motifs in VvDIR proteins (Fig. 4B). Details of each subject sequence are listed in Table S2. These conserved motifs annotated by the SMART program. It was found that motif 3 was present in 15 members, followed by motif 4 which was present in 10 members. Motifs 5, 6, and 7 were present in 4, 3, and 4 VvDIR genes, respectively. The lowest number of motifs (2) was found in VvDIR17. The motifs of the same group showed more similarities with each other, indicating that they may have similar functions. For example, motif 9 is only found in members of subfamily DIR-e and subfamily DIR-f, while motif 6 is only found in members of subfamily DIR-b/d and subfamily DIR-a. The presence of different motifs in different DIR genes suggests that they may have diverse biochemical and biological roles.

Fig. 4
figure 4

Gene structure, conserved motifs, and phylogenetic relationship of VvDIR genes. (A) Phylogenetic tree of VvDIR genes. The phylogenetic tree was generated using the maximum-likelihood method with 1000 bootstrap replicates utilizing the full-length protein sequences of VvDIR genes. The VvDIR genes can be divided into five subgroups, which are denoted with different colors, respectively. (B) Motif distribution patterns. (C) Domains of the VvDIR genes

Full size image

Analysis of cis-acting elements

The promoter region of the VvDIR genes contains various regulatory elements, including TGA-element, AuxRE, AuxRR-core, TATC-box, GARE-motif, P-box, ABRE, CGTCA-motif, TGACG-motif, SARE, and TCA-element, which are associated with responses to auxin (IAA), gibberellin (GA), abscisic acid (ABA), methyl jasmonate (MeJA), and salicylic acid (SA) (Fig. 5). Specifically, 15 CGTCA motifs were identified in the promoter regions of 10 members, while 12 members contained 29 TCA-elements. These findings suggest the critical roles of VvDIR genes play in plant stress responses by enhancing the synthesis of lignin and lignans. Moreover, anaerobic-induced elements were present in most of the members, followed by MeJA-responsiveness.

Fig. 5
figure 5

Promoter analysis of grape DIR genes

Full size image

Expression analysis of VvDIR genes following C. gloeosporioides and signaling molecules application

Grape leaves following C. gloeosporioides infection showed different expression patterns of VvDIR genes (Fig. 6). All genes except VvDIR17 showed upregulation upon inoculation with C. gloeosporioides. Most of the genes displayed the same pattern of upregulation and showed peak expression at 48 h post inoculation (hpi). However, there were differences as well, for example, VvDIR 5, 8, 11, 12, 14 initially showed upregulation followed by subsequent downregulation, and VvDIR11, 12, 14 displayed the highest expression at 6 hpi. The VvDIR4 showed the highest increase in expression levels at 48 hpi and VvDIR 5, 8 showed the peak expression at 12 hpi.

Further, we also analyzed the response of six DIR genes (VvDIR2, 4, 6, 7, 11, and 13) to the application of two hormones (SA and MeJA), as shown in Fig. 7. VvDIR2 showed the highest response to SA at 6 hpi. VvDIR6 was upregulated at 3 hpi in response to MeJA, while VvDIR4 and VvDIR11 also displayed a significant response to MeJA and had the highest response at 3 hpi (80.54) and 24 hpi (71.09), respectively. On the other hand, VvDIR7 and VvDIR13 had a significant response to SA, reaching a peak at 3 hpi (6151.26 and 9.50, respectively), but only showed a slight response to MeJA stress. The observed changes in the expression levels of VvDIR4 to C. gloeosporioides infection and hormone application suggest its important roles in the disease resistance mechanism.

Fig. 6
figure 6

VvDIR genes expression profiles in response to C. gloeosporioides inoculation. qRT-PCR was used for expression analysis and a heatmap was generated using TBtools. The red and green color scale indicate high and low expression levels, respectively

Full size image
Fig. 7
figure 7

VvDIR genes expression profiles in response to SA and MeJA treatment by qRT-PCR. Error bars represent the standard deviation (SD) of three biological replicates

Full size image

Ectopic expression of VvDIR4 inA. thaliana enhances the resistance to C. higginsianum

Expression profiling revealed that the VvDIR4 gene showed significant change in expression in strawberry following C. gloeosporioides infection, and the expression was upregulated with external SA and JA treatment. Therefore, the VvDIR4 gene was selected for further investigation and ectopically expressed in A. thaliana. Among the 27 transgenic T2 lines, three independent transgenic lines (T3-10, T3-14, and T3-25) having the highest expression levels of VvDIR4 were selected for subsequent experiments (Fig. 8C). The leaves of the selected transgenic and wild-type lines were inoculated with C. higginsianum. For phenotypic observations, leaf samples were taken after 3 days (Fig. 8A). Both transgenic and wild-type plants responded to C. higginsianum, displaying black lesions around the inoculation sites. However, the disease symptoms in wild-type plants were more severe than those in transgenic plants, with the largest lesion diameter observed in the wild-type plants and the smallest in T3-10 (Fig. 8A and B).

To further investigate the molecular mechanism of VvDIR4 transgenic plants following C. higginsianum inoculation, we measured the transcription levels of important genes having presumed roles in the SA (AtPR1 and AtICS1) and JA (AtPDF1.2) signaling pathways (Fig. 8D-F). In the T3-10 line, the AtPR1 gene was significantly upregulated at 12 hpi and reached its highest expression at 48 hpi (Fig. 8E). At 12 hpi, the expression levels of the AtICS1 gene in all three transgenic lines reached their maximum, significantly higher than that in the wild-type line (Fig. 8D). The expression levels of the AtPDF1.2 gene in all transgenic lines were higher than those in the wild-type line at all time points, with the highest expression levels occurring at 48 hpi in T3-14 and at 12 hpi in the other two lines (Fig. 8F).

Fig. 8
figure 8

Ectopic expression of VvDIR4 gene in A. thaliana and expression levels at 0, 12, 24, 48, and 72 hpi. Data are the mean values and SDs of three replications and asterisks denote a statistically significant difference (* p < 0.05, ** p < 0.01, Student’s t-test) between wild-type and transgenic lines. (A) Symptoms of disease in WT and transgenic A. thaliana leaves after 72 hpi. (B) The average lesion diameter on leaves after three days of inoculation. (C) The transcript level of VvDIR4 in leaves of WT and transgenic lines. (D) Expression analysis of AtPR1 gene. (E) Expression analysis of AtICS1 gene. (F) Expression analysis of AtPDF1.2 gene. (G) Lignin content in WT and transgenic A. thaliana (whole plants) after C. higginsianum inoculation

Full size image

Overexpression of VvDIR4 increases acid-soluble lignin content in A. thaliana

Members of the DIR-a subfamily are involved in lignin synthesis [17, 32]. To verify whether the VvDIR4 gene is also involved in lignin synthesis, we measured the lignin content in wild-type and transgenic A. thaliana plants after C. gloeosporioides inoculation. The lignin content in transgenic A. thaliana started to increase at 24 hpi and reached its highest level at 120 hpi (Fig. 9G). In contrast, the lignin content in wild-type plants did not show significant changes and remained lower than that in transgenic plants at all time points (Fig. 9G), indicating VvDIR4 gene may enhance disease resistance in A. thaliana by regulating lignin synthesis.

The overexpression of VvDIR4 gene has been shown to enhance the resistance of grapevine against C. gloeosporioides

For further functional verification of the VvDIR4 gene, a pCAMBIA2300-VvDIR4-GFP overexpression vector was constructed and transiently transformed into the C. gloeosporioides susceptible grape cultivar ‘Thompson Seedless’ leaves using the Agrobacterium-mediated transient transformation method. Transient overexpression of the empty vector pCAMBIA2300-GFP in leaves served as the control. After 24 h of transient transformation, the leaves were inoculated with agar plates containing C. gloeosporioides mycelium. The expression level of VvDIR4 in transiently transformed grape leaves was significantly higher than that in the control (Fig. 9C). Phenotypic observations revealed that overexpression of VvDIR4 enhanced grape resistance against C. gloeosporioides, as the damage severity of the control leaves was significantly greater than that of the transiently overexpressed VvDIR4 leaves (Fig. 9A and B).

Mittler et al. [33] reported that plants can restrict or even kill pathogens by inducing the production of reactive oxygen species (ROS) and have ROS scavenging systems that maintain a dynamic balance with ROS production. Furthermore, superoxide dismutase (SOD), peroxidase (POD), and catalase (CAT) are important antioxidant enzymes in plants [34]. Therefore, the activities of CAT, SOD, and POD in grape leaves after C. gloeosporioides infection were also estimated. CAT, SOD, and POD activities in the leaves overexpressing VvDIR4 were higher than those in the control group during the infection process and reached their peaks at 72 h (Fig. 9D-F). These results suggest the involvement of VvDIR4 in the C. gloeosporioides infection resistance mechanism.

Fig. 9
figure 9

The VvDIR4 gene was transiently overexpressed in grapes cultivars Thompson seedless. These leaves were inoculated with a PDA disk containing C. gloeosporioides mycelium and harvested at 0, 24, 72, and 120 h. Data are the mean values and SDs of three replications. Asterisks denote a statistically significant difference (* p < 0.05, ** p < 0.01, Student’s t test) between the empty vector control and transgenic lines. (A) Disease symptoms of the control and transiently transformed leaves at 120 hpi. (B) Average lesion area on the leaves at 120 hpi. (C) Expression of VvDIR4 in the control and transiently transformed grape leaves. (D-F) CAT activity (D), POD activity (E), and SOD activity (F) after inoculation with C. gloeosporioides

Full size image

Discussion

The DIR genes have key roles in the biosynthesis of plant lignin and phenylpropanoids and serve as key regulatory factors that can be activated in response to biotic and abiotic stresses [9, 11, 35]. The activation makes them significantly involved in stress resistance, particularly in the defense against pathogens. The involvement of DIR genes in the biotic stress resistance mechanism has been studied in various plant species, including rice [31], soybean [36], and pepper [37]. However, there is less information available about their roles in the grapevine. Here, 18 VvDIR family genes were identified in the grapevine genome using bioinformatics methods and were analyzed in terms of their physicochemical properties, gene structures, phylogenetic relationships, motif distribution, expansion patterns, and expression profiling. Moreover, the role of the VvDIR4 gene in anthracnose resistance was validated by overexpressing A. thaliana and grapevine. Our results provide new candidate genes for disease resistance breeding and basic information about the roles of VvDIR genes in anthracnose resistance.

All genes were unevenly distributed on eight chromosomes including three pairs of tandemly duplicated genes and one pair of segmentally duplicated genes (Figs. 1 and 2). These results suggest that both tandem and segmental duplication have played a role in the expansion of DIR gene family in grapes. There are 25, 45, 34, 22, and 18 DIR genes in A. thaliana, Vigna radiata, Medicago sativa, Brassica napus, and V. vinifera respectively [9, 14, 36, 38]. These diverse numbers of DIR genes in different plants suggest the DIR family changed extensively during genome evolution. There are 6 subfamilies in most of the plants, but grapes contained only five of six DIR subfamilies, and perhaps DIR-c was lost during grape evolution (Fig. 3).

Motif distribution patterns can provide some clues about gene functions. Most members of the same subfamily have almost similar motif distribution patterns with few exceptions suggesting close evolutionary relationships among genes of the same family [39]. Promoter analysis suggested that most of the genes are responsive to phytohormones (GA, SA, and MeJA) suggesting their involvement in hormone signaling pathways. These results are consistent with the findings of Cheng et al. [39]. We conducted a study on the changes in gene expression levels in in detached grape leaves following C. gloeosporioides inoculation, nearly all genes exhibited significant alterations in expression. 12 genes showed significant upregulation at 48 hpi, suggesting their key roles in anthracnose disease resistance mechanism (Fig. 6).

Plant hormone signal transduction pathways play an important role in systemic acquired resistance (SAR) in plants [40]. SA and MeJA are endogenous plant hormones and have important roles in plant immune responses. SA is primarily involved in the basic defense against biotrophic pathogens, whereas MeJA controls the response to necrotrophic pathogens [41, 42]. In our study, following SA and MeJA treatments, certain VvDIR genes showed differential expression patterns. Specifically, VvDIR2, 4, 7, and 13 genes showed upregulation against SA application, and VvDIR4 and 6 displayed upregulation to MeJA treatment. Interestingly VvDIR4 showed a positive response to both hormones, suggesting a role in hormonal signaling pathways of JA and MeJA (Fig. 7). These results further validate our predication made on cis-elements analysis that DIR genes have a role in hormone signaling pathways, especially SA and MeJA. In conclusion, VvDIR genes may play important roles in biotic and abiotic stress resistance in grapes.

To get more insights, VvDIR4 was ectopically and transiently expressed in A. thaliana and grapevine leaves, respectively. The transgenic plants of A. thaliana exhibited smaller lesions and higher disease resistance compared to wild-type plants (Fig. 8A). These results further support the role of VvDIR4 in disease resistance. Moreover, to further justify the role of VvDIR4 the expression patterns of genes having presumed roles in hormone signaling pathways were studied. Following C. higginsianum infection AtPR1 and AtICS1 showed significantly differential expression patterns in transgenic A. thaliana plants as compared to wild type (Fig. 8D, E). These findings are in line with previous results that AtPR1 and AtICS1 are involved in the SA pathway in A. thaliana [43, 44]. Moreover, AtPDF1.2 also showed higher expression in transgenic plants, suggesting the role of VvDIR4 in JA signaling pathways. The high expression of PDF1.2 indicates activation of the JA hormone pathway [44]. These are also consistent with the findings of Shi et al. [17].

Transient overexpression of the VvDIR4 gene in susceptible grapevine cultivar ‘Thompson Seedless’ improved its resistance against C. gloeosporioides. After 120 h of inoculation with C. gloeosporioides, the transiently overexpressed leaves showed significantly smaller lesion areas compared to the control (empty vector) (Fig. 9A-B). ROS network also plays an important role in plant defense signaling [33]. Upon pathogen infection, ROS is rapidly generated to prevent pathogen entry or induce resistance genes inhibiting pathogen growth [45]. However, high levels of ROS can be toxic and negatively impact subcellular components and metabolism in plants [46]. To cope with this, plants have ROS scavenging systems that maintain a dynamic balance with ROS production. SOD, POD, and CAT are important antioxidant enzymes in plants [34]. In our study, after C. gloeosporioides infection, the transient overexpression of VvDIR4 in leaves increased CAT, SOD, and POD activities compared to the control (Fig. 9D-F). These findings suggest that VvDIR4 may enhance plant resistance to C. gloeosporioides by increasing the activity of antioxidant enzymes to suppress the production of ROS.

Conclusions

DIR proteins, particularly those in the DIR-A subfamily, play important roles in lignin biosynthesis. During pathogen infections, they contribute to enhanced plant resistance by promoting lignin accumulation [11, 47]. VvDIR4, belonging to the DIR-A subfamily, was investigated for its involvement in lignin synthesis. We measured lignin content in both wild-type and transgenic A. thaliana plants using the method described by Syros et al. [48], both before and after inoculation with C. higginsianum. The transgenic lines exhibited higher lignin content than the wild type, with a significant upregulation observed after C. higginsianum infection. These results endorse that VvDIR4 may play critical roles in grape lignin formation, thereby enhancing resistance to C. gloeosporioides infection through increased lignin biosynthesis. Our study offers fundamental insights into VvDIR genes, with a detailed functional analysis of VvDIR4. We surmise that VvDIR4 contributes to anthracnose resistance in grapes by influencing lignin synthesis and influencing JA and SA signaling pathways. These findings are valuable for grape disease resistance breeding programs.

Methods

Plant materials and seedlings treatment

The grape varieties (V. vinifera L.), “Red Globe” and “Thompson Seedless,” planted at the research experimental base of Fujian Agriculture and Forestry University were used. A. thaliana plants were grown for 4 weeks in a growth chamber at 21 °C, 70% relative humidity, and a 12-hour light/12-hour dark cycle. The pathogens were sprayed on A. thaliana and grape plants, respectively. The concentration of inoculated pathogens (C. gloeosporioides and C. higginsianum) was 1 × 106 conidia/mL. Grape plants were kept in a growth chamber at 28 °C and 85% humidity after inoculation, while A. thaliana was placed in a growth chamber with a 16-hour light (23 °C)/8-hour dark (21 °C) cycle. To evaluate the biological activity of VvDIR4 against anthracnose, 28 A. thaliana transgenic lines and Col-0 wild-type plants were infected with C. higginsianum, following the recommendations of Casado-Diaz et al. [49] and Wen et al. [50]. Samples were collected at 0, 12, 24, 48, and 72 h post-inoculation (hpi) from transgenic and wild-type A. thaliana plants. Grape leaves were sprayed with a 5 mM salicylic acid (SA) or 50 mM methyl jasmonate (MeJA) solution, while control plants were sprayed with distilled water. Each grape leaf was sprayed with 1.5 mL of each exogenous hormone using a hand sprayer. Leaf samples were collected at 0, 3, 6, 12, and 24 h after spraying. To extract RNA, all collected samples were immediately frozen in liquid nitrogen and stored at -80 °C. Each experiment was conducted with three biological and technical replicates.

Identification of DIR genes in grapevine

The genome, proteome, and annotation information for A. thaliana, Oryza sativa, and V. vinifera L. were obtained from the TAIR (https://www.arabidopsis.org), PlantTFDB (http://planttfdb.gao-lab.org), and Phytozome (https://phytozome-next.jgi.doe.gov). The Hidden Markov Model (HMM) profile for the Dirigent domain (PFAM 03018) was obtained from the Pfam database (http://pfam.xfam.org/) and used to search grape protein databases [51]. The local Blast analysis was performed using TBtools [52]. Sequences with an E-value less than 1e− 5 were selected as candidate sequences and redundant sequences were removed. The presence of a complete DIR domain in all putative DIR genes was checked using the SMART (http://smart.embl.de/) and CDD (https://www.ncbi.nlm.nih.gov/Structure/bwrpsb/bwrpsb.cgi) websites. Based on this analysis 18 VvDIR genes were selected for further study [53, 54]. The ProtParam tool provided by the ExPASy Server (https://web.expasy.org/protparam/) was utilized to calculate the physicochemical properties of VvDIR genes, including protein molecular weight (Mw), amino acid counts, and isoelectric point (pI) values. The subcellular location of proteins was predicted using the Plant-mPLoc Server (http://www.csbio.sjtu.edu.cn/bioinf/plant-multi/) [55].

Phylogenetic studies and multiple sequence alignment

One phylogenetic tree was generated among DIR protein sequences of A. thaliana, O. sativa, and V vinifera L. and another phylogenetic tree was generated among VvDIR proteins. The trees were generated with MEGA 7 (http://www.megasoftware.net.) using the neighbor-joining (NJ) and maximum likelihood (ML) methods, respectively. Bootstrap values were set at 1000 replications to evaluate the statistical reliability of the phylogenetic trees [56]. The resulting trees were visualized using Evolgenius online software (https://evolgenius.info//evolview-v2/#login).

Distribution of the VvDIR genes on chromosomes and duplication events

The chromosomal locations of VvDIR gene family members were identified according to physical location information from the Phytozome (https://phytozome-next.jgi.doe.gov). The chromosomal distribution map of the VvDIR gene family members were performed using TBtools [52]. Tandemly duplicated VvDIR genes were analyzed using MCScanX software [57], Duplication and collinearity analysis of VvDIR genes according to Basic Circos function module in TBtools.

Conserved motif distribution and gene structure analysis

For cis-regulatory elements analysis, the 2 kb upstream sequence of each VvDIR gene was extracted using the “GXF Sequences Extract” tool in TBtools software [52] and the obtained sequences were analyzed using the online PlantCare database (https://bioinformatics.psb.ugent.be/webtools/plantcare/html/). ‘TBtools Basic BIO sequence View’ tool was utilized for visualization purposes.

RNA extraction and PCR analysis

RNA was extracted from “Red Globe " grape leaves using the RNApre Pure Plant Total RNA Extraction Kit (Tiangen, Beijing, China) following company guidelines. cDNA was synthesized using the PrimeScript™ RT Reagent Kit with gDNA Eraser (Takara Bio, Dalian, China). For qPCR analysis cDNA was diluted 6 times. Gene-specific primers were designed using Primer Premier 5.0 software (Table S1). Quantitative real-time PCR (qRT-PCR) was performed using the SYBR Premix Ex Taq (2×) kit and the Applied Biosystems StepOne Plus instrument. The reaction mixture (20 µL) contained 10µL of SYBR Pre-mix Ex Taq (2×), 0.4 µL of each primer (10 µmol/L), 2 µL cDNA template, and 7.2 µL ddH2O. The PCR was carried out under the following conditions: pre-denaturation at 95 °C for 2 min; denaturation at 95 °C for 5 s, annealing at 60 °C for 34 S, for 40 cycles; melt curve analysis: 95 °C for 15 S, 60 °C for 20 S, 95 °C for 15 S. Each reaction was performed three times and grape Actin (Accession no. XM_002282480) was used as an internal reference. The gene expression profiles were visualized using TBtools software and the relative expression levels were calculated using the 2−ΔΔCt method [58].

Plasmid construction and transformation

The VvDIR4 ORF sequence (582 bp) was amplified using LA Taq (TaKaRa Bio, Dalian, China) and then cloned into pMD20-T (TaKaRa Bio, Dalian, China). The resulting sequence was verified by sequencing (FuZhou ShangYa BioInc., Fuzhou, China) and submitted to GenBank (Acc. NO. OR903152). To generate the transgenic plants, the sequence was cloned into the binary vector pCAMBIA1300-HA (CAMBIA company) using specific primers VvDIR4-clone-F and VvDIR4-clone-R containing BamH I and Kpn I restriction sites (Table S1). The recombinant plasmid was transformed into Agrobacterium tumefaciens GV3101 using a freeze-thaw technique [59] and then transferred into wild A. thaliana using the floral dip method [60]. The transgenic plants were screened on a solid MS medium containing 50 mg/L hygromycin [61].

Measurement of the acid-soluble lignin content after C. higginsianum inoculation

Samples of leaves and stems were collected from both wild-type and transgenic plants of A. thaliana to analyze the lignin content using the acetyl bromide method [62]. After inoculating A. thaliana with C. higginsianum, samples were collected at three different time points: 0, 12, and 24 hpi, for a total of 128 plants. The extraction of lignin was performed following the instructions provided in the lignin extraction kit from Herui Bioscience (Herui Bio, Fujian, China), and the amount of lignin was measured and calculated accordingly.

Transient expression of VvDIR4 in grapes

The VvDIR4 gene was cloned into the pCAMBIA2300-HA vector (CAMBIA company) using specific primers VvDIR4-GFP-F and VvDIR4-GFP-R containing Kpn I and Xba I sites (Table S1). The recombinant plasmid was transformed into A. tumefaciens GV3101 using the freeze-thaw technique. The transformed Agrobacterium was then transiently transformed into “Thompson Seedless” grape leaves using a vacuum infiltration method [63]. The transformed “Thompson Seedless” grape leaves were incubated in the dark for 2 days and used for pathogen infection experiments. Leaf samples were collected at 0, 24, 72, and 120 h for antioxidant enzyme activity detection. Herui Bio Kit (Fujian, China) was used according to the manufacturer’s instructions for enzyme activity detection. Each experiment used 10 transiently transformed leaf samples as biological replicates, and each experiment was conducted thrice.

Statistical analysis

The averages and standard deviations of at least three independent replicates were used in all the experiments. Statistical analysis was performed using SPSS version 21.0. Student’s t-test was used to detect the differences between the mean of the expression level, and the overall statistical significance of the data was determined at *p < 0.05 and **p < 0.01 levels.

Data availability

All data generated or analyzed during this study are included in this published article and its additional files.

Abbreviations

aa:

Amino acid

Chr:

Chromosome numbers

qPCR:

Quantitative real-time polymerase chain reaction

HMM:

Hidden Markov Models

Hpi:

Hours post inoculation

hpt:

Hours post treatment

ICS1:

Isochorismate synthase

ICS1:

Isochorismate synthase 1

LOX3:

Lipoxygenase 3

MEME:

Multiple Em for Motif Elicitation

MeJA:

Methyl jasmonate

SA:

Salicylic acid

MW:

Molecular weight

ORF:

Open reading frame

PDF1.2:

Plant defensing 1.2

pI:

Isoelectric point

PR1:

Pathogenesis-related gene 1

References

  1. Barbara N, Kunkel, Brooks M. Cross talk between signaling pathways in pathogen defense. Curr Opin Plant Biol. 2002;5(4):325–31.

    Article  Google Scholar 

  2. Jones JDG, Dangl JL. The plant immune system. Nature. 2006;444(7117):323–9.

    Article  CAS  PubMed  Google Scholar 

  3. Singh A, Jha SK, Bagri J, Pandey GK. ABA inducible rice protein phosphatase 2 C confers ABA insensitivity and abiotic stress tolerance in Arabidopsis. PLoS ONE. 2015;10(4):e0125168.

    Article  PubMed  PubMed Central  Google Scholar 

  4. Tiburzy R, Reisener HJ. Resistance of wheat to Puccinia graminis f. sp. tritici: association of the hypersensitive reaction with the cellular accumulation of lignin-like material and callose. Physiol Mol Plant Pathol. 1990;36:109–20.

    Article  CAS  Google Scholar 

  5. Gavin LB, Wang HB, Davin LB. Stereoselective biomolecular phenoxy radical coupling by an auxiliary (dirigent) protein without an active center. Science. 1997;275(5298):362–6.

    Article  Google Scholar 

  6. Laurence B, Davin M, Jourden A, Patten A. Dissection of lignin macromolecular configuration and assembly: comparison to related biochemical processes in allyl/propenyl phenol and lignan biosynthesis. Nat Prod Rep. 2008;25(6):1015–90.

    Article  Google Scholar 

  7. Hosmani PS, Kamiya T, Danku J, Naseer S, Geldner N, Guerinot ML. Dirigent domain-containing protein is part of the machinery required for formation of the lignin-based casparian strip in the root. Proc Nat Acad Sci. 2013;110(35):14498–14503.

  8. Chen JL, Zhang ZJ, Liu XY, Zhu XF. Genome identification and analysis of Phyllostachys edulis Dirigent gene family. Plant Physiol J. 2019;55(9):1406–17.

    Google Scholar 

  9. Ralph SG, Park JY, Bohlmann J, Mansfield SD. Dirigent proteins in conifer defense: gene discovery, phylogeny, and differential wound- and insect-induced expression of a family of DIR and DIR-like genes in spruce (Picea spp). Plant Mol Biol. 2006;60:21–40.

    Article  CAS  PubMed  Google Scholar 

  10. Sahani PA, Ujinwal M, Singh S. Genome wide in silico characterization of dirigent protein family in flax (Linum usitatissimum L). Plant Archives. 2018;18:61–8.

    Google Scholar 

  11. Paniagua C, Bilkova A, Jackson P, Dabravolski S, Riber W, Didi V, Houser J, Gigli-Bisceglia N, Wimmerova M, Budínská E, Hamann T, Hejatko J. Dirigent proteins in plants: modulating cell wall metabolism during abiotic and biotic stress exposure. J Exp Bot. 2017;68:3287–301.

    Article  CAS  PubMed  Google Scholar 

  12. Hadwiger LA, Chiang CC, Horovitz D. Expression of disease resistance response genes in near-isogenic pea cultivars following challenge by Fusarium oxysporum race 1. Mol Plant Pathol. 1992;40:259–69.

    Article  Google Scholar 

  13. Wang YP, Fristensky B. Transgenic canola lines expressing pea defense gene: DRR206 have resistance to aggressive blackleg isolates and to Rhizoctonia solani. Mol Plant Breed. 2001;8:263–71.

    Article  CAS  Google Scholar 

  14. Thamil Arasan SK, Park J, Ahmed NU, Jung HJ, Hur Y, Kang KK, Lim YP, Nou IS. Characterization and expression analysis of dirigent family genes related to stresses in Brassica. Plant Physiol Biochem. 2013;67:144–53.

    Article  CAS  PubMed  Google Scholar 

  15. Kumar TAS, Park JI, Ahmed NU. Analysis of Dirigent Family Genes against Stresses in Brassica sp. In: International Plant and Animal Genome Conference Asia; 2013.

  16. Subramanyam S, Smith DF, Clemens JC, Webb MA, Sardesai N, Williams CE. Functional characterization of hfr1, a high-mannose N-glycan-specific wheat lectin induced by hessian fly larvae. Plant Physiol. 2008;147(3):1412–26.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  17. Shi Y, Shen Y, Ahmad B, Yao L, He T, Fan J, Liu Y, Chen Q, Wen Z. Genome-wide identification and expression analysis of dirigent gene family in strawberry (Fragaria vesca) and functional characterization of FvDIR13. Sci Hort. 2022;297.

  18. Sun HR, Zhang JN, Ren M, Wang YF. Genome-wide identification and expression analysis of tomato DIR gene family. Agric Biotechnol. 2023;31(1):36–49.

    Google Scholar 

  19. Liu CH, Qin ZW, Zhou XY, Xin M, Wang CH, Liu D, Lis N. Expression and functional analysis of the propamocarb-related gene CsDIR16 in cucumbers. BMC Plant Biol. 2018;18:16.

    Article  PubMed  PubMed Central  Google Scholar 

  20. Ke X, Yuan MQ, Xu XD, Yin LH, Guo YX, Zuo YH. Identification of the dirigent gene family in adzuki bean and the effect of rust infection on the expression of different members. Acta Agron Sinica. 2022;48(11):2774–85.

    Google Scholar 

  21. Damaj MB, Kumpatla SP, Emani C, Beremand PD, Avutu S, Reddy AS, Rathore KS, Buenrostro-Nava MT, Curtis LS, Thomas TL, Mirkov TE. Sugarcane DIRIGENT and O-METHYLTRANSFERASE promoters confer stem-regulated gene expression in diverse monocots. Planta. 2010;231:1439–58.

    Article  CAS  PubMed  Google Scholar 

  22. Li L, Wang Y, Zhou P, Yang X, Tan W, Sun W, Zhu G. Cloning and expression analysis of PeDIR19 gene in Populus. Mol Plant Breed. 2022;09:2899–907.

    Google Scholar 

  23. Ninghui L, Ming Z, Tengfei L, Lidong D, Qun C, Junjiang W. A novel soybean dirigent gene GmDIR22 contributes to promotion of lignan biosynthesis and enhances resistance to phytophthora sojae. Front Plant Sci. 2017;8:1185.

    Article  Google Scholar 

  24. Ma Q, Liu Y. TaDlR13, a dirigent protein from wheat, promotes lignan biosynthesis and enhances pathogen resistance. Plant Mol Biology Report. 2015;33(1):143–52.

    Article  CAS  Google Scholar 

  25. Shi H, Liu Z, Zhu L, Zhang C, Chen Y, Zhou Y, Li F, Li X. Overexpression of cotton (Gossypium hirsutum) dirigent l gene enhances lignification that blocks the spread of Verticillium Dahliae. Acta Biochim Biophys Sin. 2012;44(7):555–64.

    Article  CAS  PubMed  Google Scholar 

  26. Armijo G, Schlechter R, Agurto M, Muñoz D, Nuñez C, Arce-Johnson P. Grapevine pathogenic microorganisms: understanding infection strategies and host response scenarios. Front Plant Sci. 2016;7:382.

    Article  PubMed  PubMed Central  Google Scholar 

  27. Singh DK, Mehra S, Chatterjee S, Purty RS. In silico identification and validation of miRNA and their DIR specific targets in Oryza sativa indica under abiotic stress. Non-coding RNA Res. 2020;5(4):167–77.

    Article  CAS  Google Scholar 

  28. Li L, Sun W, Zhou P, Wei H, Wang P, Li H. Genome-wide characterization of dirigent proteins in Populus: gene expression variation and expression pattern in response to Marssonina brunnea and phytohormones. Forests. 2021;12(4):507–507.

  29. Cannon SB, Mitra A, Baumgarten A, Young ND, May G. The roles of segmental and tandem gene duplication in the evolution of large gene families in Arabidopsis thaliana. BMC Plant Biol. 2004;4:10.

    Article  PubMed  PubMed Central  Google Scholar 

  30. Li Q, Chen JF, Xiao Y, Di P, Zhang L, Chen WS. The dirigent multigene family in Isatis Indigotica: gene discovery and differential transcript abundance. BMC Genomics. 2014;15:388.

    Article  PubMed  PubMed Central  Google Scholar 

  31. Liao Y, Liu S, Jiang Y, Hu CQ, Zhang XW, Cao XF, Xu ZJ, Gao XL, Li LH, Zhu JQ. Genome-wide analysis and environmental response profiling of dirigent family genes in rice (Oryza sativa). Genes Genomics. 2017;39:47–62.

    Article  CAS  Google Scholar 

  32. Burlat V, Kwon M, Lunde BM, Lewis NG. Dirigent proteins and dirigent sites in lignifying tissues. Phytochemistry. 2001;57(6):883–97.

    Article  CAS  PubMed  Google Scholar 

  33. Mittler R, Vanderauwera S, Gollery M, Breusegem FV. Reactive oxygen gene network of plants. Trends Plant Sci. 2004;9(10):490–8.

    Article  CAS  PubMed  Google Scholar 

  34. Ma P, Su S, Li Y, Zhong P, Hou Y, Wei B. Effects of exogenous proline on osmotic regulation and antioxidant enzyme activity in Bai Ci (Ziziphus Jujube Mill.) Leaves under natural drought conditions. J Gansu Agricultural Univ. 2020;(04):121–127.

  35. Vanholme R, Moreel K, Darrah C. Metabolic engineering of novel lignin in biomass crops. New Phytol. 2012;196(4):978–1000.

    Article  CAS  PubMed  Google Scholar 

  36. Ma XF, Xu WY, Liu T, Chen RY, Zhu H, Zhang HY, Cai CM. Functional characterization of soybean (Glycine max) DIRIGENT genes reveals an important role of GmDIR27 in the regulation of pod dehiscence. Genomics. 2021;113(1 Pt 2):979–90.

    Article  CAS  PubMed  Google Scholar 

  37. Khan A, Li RJ, Sun JT, Zhang HJ, Li MA, Wang JE, Gong Z. Genome-wide analysis of dirigent gene family in pepper (Capsicum annuum L.) and characterization of DIRCa7 in biotic and abiotic stresses. Sci Rep. 2018;8(1):5500.

    Article  PubMed  PubMed Central  Google Scholar 

  38. Song M, Peng X. Genome-wide identification and characterization of DIR genes in Medicago truncatula. Biochem Genet. 2019;57(4):487–506.

    Article  CAS  PubMed  Google Scholar 

  39. Cheng X, Su X, Muhammad A, Li M, Zhang J, Sun Y, Li G, Jin Q, Cai Y, Lin Y. Molecular characterization, evolution, and expression profiling of the Dirigent (DIR) family genes in Chinese white pear (Pyrus Bretschneideri). Front Genet. 2018;136.

  40. Betsuyaku S, Katou S, Takebayashi Y, Sakakibara H, Nomura N, Fukuda H. Salicylic acid and jasmonic acid pathways are activated in spatially different domains around the infection site during effector-triggered immunity in Arabidopsis thaliana. Plant Cell Physiol. 2018;59(1):8–16.

    Article  CAS  PubMed  Google Scholar 

  41. Amil-Ruiz F, Garrido-Gala J, Gadea J, Blanco-Portales R, Muñoz-Mérida A, Trelles O, de Los Santos B, Arroyo FT, Aguado-Puig A, Romero F, Mercado JÁ, Pliego-Alfaro F, Muñoz-Blanco J, Caballero JL. Partial activation of SA- and JA-defensive pathways in strawberry upon Colletotrichum acutatum interaction. Front Plant Sci. 2016;7:1036.

    Article  PubMed  PubMed Central  Google Scholar 

  42. Li JL, Han GL, Sun CF. Research advances of MYB transcription factors in plant stress resistance and breeding. Plant Signal Behav. 2019;14(8):1613131.

    Article  PubMed  PubMed Central  Google Scholar 

  43. Van Loon LC, Van Strien EA. The families of pathogenesis-related proteins, their activities, and comparative analysis of PR-1 type proteins. Physiol Mol Plant Pathol. 1999;55(2):85–97.

    Article  Google Scholar 

  44. Zhao XT, Song LY, Jiang LW, Zhu YT, Gao QH, Wang DD, Xie J, Lv M, Liu P, Li M. The integration of transcriptomic and transgenic analyses reveals the involvement of the SA response pathway in the defense of chrysanthemum against the necrotrophic fungus Alternaria spp. Hortic Res. 2020;7(1):80.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  45. Nandini P, Shetty HJ, Lyngs. Roles of reactive oxygen species in interactions between plants and pathogens. Eur J Plant Pathol. 2008;121(3):267–80.

    Article  Google Scholar 

  46. Guofu H, Yiming L, Tianqi D, Bingyu Z, Guowen C, Jing J. Antioxidant metabolism variation associated with alkali-salt tolerance in thirty switch grass (Panicum virgatum) lines. PLoS ONE. 2018;13(6):e0199681.

    Article  Google Scholar 

  47. Davin LB, Wang HB, Crowell AL, Bedgar DL, Martin DM, Sarkanen S, Lewis NG. Stereoselective bimolecular phenoxy radical coupling by an auxiliary (dirigent) protein without an active center. Science. 1997;275:362–6.

    Article  CAS  PubMed  Google Scholar 

  48. Syros T, Yupsanis T, Zafiriadis H, Economou A. Activity and isoforms of peroxidases, lignin and anatomy, during adventitious rooting in cuttings of Ebenus Cretica L. Plant Physiol. 2004;161(1):69–77.

    Article  CAS  Google Scholar 

  49. Casado-Diaz A, Encinas-Villarejo S, Santos B, Schiliro E, Yubero-Serrano EM, Amil-Ruiz F, Pocovi MI, Pliego-Alfaro F, Dorado G, Rey M, Romero F, Muñoz-Blanco J, Caballero JL. Analysis of strawberry genes differentially expressed in response to Colletotrichum infection. Physiol Plant. 2006;128:633–50.

    Article  CAS  Google Scholar 

  50. Wen ZF, Bai JH, Wang L, Yao LP, Ahmad B, Hanif M, Chen QX. Overexpression of a chitinase 2 gene from Chinese wild strawberry improves resistance to anthracnose disease in transgenic Arabidopsis thaliana. Plant Biotechnol Rep. 2020;14(6):725–36.

    Article  Google Scholar 

  51. Chepyshko H, Lai CP, Huang LM, Liu JH, Shaw JF. Multifunctionality and diversity of GDSL esterase/lipase gene family in rice (Oryza sativa L. Japonica) genome: new insights from bioinformatics analysis. BMC Genomics. 2012;13:309.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  52. Chen CJ, Chen H, Zhang Y, Thomas HR, Frank MH, He YH, Xia R. TBtools: an integrative toolkit developed for interactive analyses of big biological data. Mol Plant. 2020;13(8):1194–202.

    Article  CAS  PubMed  Google Scholar 

  53. Marchler-Bauer A, Derbyshire MK, Gonzales NR, Lu S, Chitsaz F, Geer LY, Geer RC, He J, Gwadz M, Hurwitz DI, Lanczycki CJ, Lu F, Marchler GH, Song JS, Thanki N, Wang Z, Yamashita RA, Zhang D, Zheng C, Bryant SH. CDD: NCBI’s conserved domain database. Nucleic Acids Res. 2015;43:D222–6.

    Article  CAS  PubMed  Google Scholar 

  54. Letunic I, Doerks T, Bork P. Recent updates, new developments and status in 2015. Nucleic Acids Res. 2015;43:257–60.

    Article  Google Scholar 

  55. Chou KC, Shen HB. Plant-mPLoc: a top-down strategy to augment the power for predicting plant protein subcellular localization. PLoS ONE. 2010;5(6):e11335.

    Article  PubMed  PubMed Central  Google Scholar 

  56. Kumar S, Stecher G, Tamura K. MEGA7: molecular evolutionary genetics analysis version 7.0 for bigger datasets. Mol Biol Evol. 2016;33(7):1870–4.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  57. Wang Y, Tang H, DeBarry JD, Tan X, Li J, Wang X, Guo H. MCScanX: a toolkit for detection and evolutionary analysis of gene synteny and collinearity. Nucleic Acids Res. 2012;40(7):e49.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  58. Schmittgen TD, Livak KJ. Analyzing real-time PCR data by the comparative C(T) method. Nat Protoc. 2008;3(6):1101–8.

    Article  CAS  PubMed  Google Scholar 

  59. Yu Y, Du J, Wang G, Ji J. Research on the introduction of recombinant plasmids into Agrobacterium tumefaciens by freeze-thaw method. Jilin Agricultural Univ J. 2003;(03):257–259.

  60. Clough SJ, Bent AF. Floral dip: a simplified method for Agrobacterium-mediated transformation of Arabidopsis thaliana. Plant J. 1998;16(6):735–43.

    Article  CAS  PubMed  Google Scholar 

  61. Wen ZJ, Bing Y, Donald P, Efficient CRISPR. /Cas9-mediated gene editing in Arabidopsis thaliana and inheritance of modified genes in the T2 and T3 generations. PLoS ONE. 2014;9(6):e99225.

    Article  Google Scholar 

  62. Barnes W, Anderson C. Acetyl Bromide Soluble Lignin (ABSL) Assay for Total Lignin Quantification from Plant Biomass. Bio-protocol. 2017;7(5):e2149.

    Article  PubMed  PubMed Central  Google Scholar 

  63. Jiang C, Wang D, Zhang J, Xu Y, Zhang C, Zhang J, Wang X, Wang Y. VqMYB154 promotes polygene expression and enhances resistance to pathogens in Chinese wild grapevine. Hortic Res. 2021;8:151.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

Download references

Acknowledgements

We thank reviewers for their helpful comments and the editors for their careful work on the manuscript.

Funding

This study was supported by the National Natural Science Foundation of China (32072526) and Fundamental Research Project of Provincial Public Welfare Research Institutes (2020R10280013).

Author information

Authors and Affiliations

  1. College of Horticulture, Fujian Agriculture and Forestry University, Fuzhou, 350002, China

    Qimeng Zhang, Ning Luo, Xicheng Dai, Qingxi Chen & Zhifeng Wen

  2. Fruit Research Institute, Fujian Academy of Agricultural Sciences, Fuzhou, Fujian, 350013, China

    Jinhui Lin & Yan Lei

  3. National Key Laboratory of Tropical Crop Breeding, Shenzhen Branch, Guangdong Laboratory of Lingnan Modern Agriculture, Key Laboratory of Synthetic Biology, Ministry of Agriculture and Rural Afairs, Agricultural Genomics Institute at Shenzhen, Chinese Academy of Agricultural Sciences, Shenzhen, 518120, China

    Bilal Ahmad

Authors
  1. Qimeng Zhang
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Ning Luo
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Xicheng Dai
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Jinhui Lin
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Bilal Ahmad
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Qingxi Chen
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Yan Lei
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. Zhifeng Wen
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

Z.Q.M and W.Z.F planned, carried out the experiments, analyzed the data, interpreted the results and wrote the manuscript, performed the experiments. L.N and B.A were involved in revision of the manuscript. D.X.C and L.J.H were involved in the laboratory experiments. C.Q.X contributed to acquisition and analysis. L.Y and W.Z.F projected administration, supervision, visualization, and writing review & editing. All authors have read and approved the manuscript.

Corresponding authors

Correspondence to Yan Lei or Zhifeng Wen.

Ethics declarations

Ethics approval and consent to participate

Not applicable.

Consent for publication

Not applicable.

Competing interests

The authors declare no competing interests.

Additional information

Publisher’s note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Electronic supplementary material

Below is the link to the electronic supplementary material.

Supplementary Material 1

Rights and permissions

Open Access This article is licensed under a Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International License, which permits any non-commercial use, sharing, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if you modified the licensed material. You do not have permission under this licence to share adapted material derived from this article or parts of it. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by-nc-nd/4.0/.

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Zhang, Q., Luo, N., Dai, X. et al. Ectopic and transient expression of VvDIR4 gene in Arabidopsis and grapes enhances resistance to anthracnose via affecting hormone signaling pathways and lignin production. BMC Genomics 25, 895 (2024). https://doi.org/10.1186/s12864-024-10830-0

Download citation

  • Received:

  • Accepted:

  • Published:

  • DOI: https://doi.org/10.1186/s12864-024-10830-0

Keywords

BMC Genomics

ISSN: 1471-2164

Contact us