- Open Access
Identification of Vitis vinifera MYB transcription factors and their response against grapevine berry inner necrosis virus
BMC Plant Biology volume 23, Article number: 279 (2023)
The myeloblastosis (MYB) superfamily is the largest transcription factor family in plants that play diverse roles during stress responses. However, the biotic stress-responsive MYB transcription factors of the grapevine have not been systematically studied. In China, grapevine berries are often infected with the grapevine berry inner necrosis virus (GINV), which eventually reduces the nutritional quality and commodity value.
The present study identified and characterized 265 VvMYB or VvMYB-related genes of the “Crimson seedless” grapevine. Based on DNA-binding domain analysis, these VvMYB proteins were classified into four subfamilies, including MYB-related, 2R-MYB, 3R-MYB, and 4R-MYB. Phylogenetic analysis divided the MYB transcription factors into 26 subgroups. Overexpression of VvMYB58 suppressed GINV abundance in the grapevine. Further qPCR indicated that among 41 randomly selected VvMYB genes, 12 were induced during GINV infection, while 28 were downregulated. These findings suggest that VvMYB genes actively regulate defense response in the grapevine.
A deeper understanding of the MYB TFs engaged in GINV defense response will help devise better management strategies. The present study also provides a foundation for further research on the functions of the MYB transcription factors.
The MYB transcription factors (TF) form the largest family of TFs in all eukaryotes and the second largest TF superfamily in flowering plants [1,2,3,4,5]. The first MYB gene identified was the V-myb avian myeloblastosis viral oncogene homolog. Currently, they were found widely distributed in higher plants and play multiple roles in various processes associated with plant growth and development [2,3,4]. MYB proteins are characterized by a highly conserved N-terminal DNA-binding domain repeat (R), consisting of at least 1–4 imperfect tandem repeats (R) of 50–53 amino acids [1, 3]. In addition, this highly conserved domain comprises regularly spread triplet tryptophan residues that group together to make a hydrophobic core; sometimes, aromatic or hydrophobic amino acid residues replace the tryptophan residues . The N-terminal DNA-binding domains mainly associated with DNA-binding and protein–protein interactions [7, 8]. The MYB domain is highly conserved among high plants, according to the MYB repeat number and the identified of the MYB repeats, MYB proteins are generally classified as MYB-related, 2R-MYB (R2R3-MYB), 3R-MYB (R1R2R3-MYB), 4R-MYB [3, 9]. In plants, the MYB gene was first identified in Zea mays in 1987 as involved in anthocyanin biosynthesis in aleurone tissues . Subsequently, research has identified the MYB gene family members in numerous plant genomes, including 199 in Arabidopsis thaliana , 492 in upland cotton , 299 in cassava , 244 in soybean , and 192 in Populus . Plant MYB proteins play crucial roles in various biological processes, including growth and development , cell metabolism [17, 18], cell fate, and stress responses . Recently, numerous researchers have found that MYB TFs play vital roles in signal transduction, hormone synthesis, primary and secondary metabolism, and against pathogen infection [20,21,22]. In pepper, MYB TF positively regulates the host defense against Ralstonia solanacearum infection . In rice, the MYB TF participates in broad-spectrum blast resistance . In apple, MdMYB30 plays a vital role in regulating cuticular wax accumulation and enhances Botryosphaeria dothidea resistance . Meanwhile, MdMYB73 regulates the salicylic acid pathway and confers resistance against the fungal pathogen Botryosphaeria dothidea . In the Chinese wild grapevine (Vitis davidii), MYB TF activates the expression of the stilbene synthase gene and positively regulates the defense response against invading pathogens . These earlier findings indicated the role of MYB TFs in modulating plant resistance against pathogens and the underlying mechanisms; however, little is known about the role of MYB TFs in regulating defense responses against plant viruses.
Grapevine (Vitis vinifera L.) is among the earliest domesticated fruit species and the most economically important fruit crop worldwide . More than 71 different viruses infect grapes, eventually reducing nutritional quality and commodity value, resulting in heavy losses . Grapevine berry inner necrosis virus (GINV) is a major pathogen that causes severe damage; it belongs to the genus Trichovirus and the family Betaflexiviridae. GINV was first identified in 1984 in Japan. Recently, a new variant of GINV has been identified from the “Beta” and “Thompson seedless” grapevines in China . The GINV is widely distributed in China and is highly associated with the ring spot symptom . The GINV-infected grapevine plantlets exhibit leaf chlorotic mottling and ringspot, and the infected berries show discoloration on the fruit surface and necrosis of the flesh [29, 31]. In China, over a third of grapevine plants have been infected with the GINV . GINV infections are closely related to ring spot symptoms. In the field, GINV is transmitted by grapevine erineum mites [29,30,31]. Therefore, there is the need to identify host factors associated with defensive responses in grapevine plants. The former research results indicated that MYB proteins regulates the host defense against invading pathogens [22,23,24,25,26]. In grapevine, VvMYB TFs were identified only in R2R3 subfamily [32, 33]. In this study, we undertook a comprehensive genome-wide characterization and expression analysis of four distinct subfamilies MYB TFs during GINV infection. The study's findings will provide a valuable resource for subsequent research on the functions and regulatory mechanisms of VvMYB proteins, potentially crucial for antiviral defense response in the grapevine.
Materials and methods
Plant materials and growth conditions
The Vitis vinifera cultivar “Crimson seedless” and Nicotiana benthamiana (N. benthamiana) were used for this study. Berries were collected from 10-year-old grapevine trees grown in a grapevine orchard at the Henan Institute of Science and Technology, Xinxiang City, Henan Province, China (E 113°55', N 35°18'). All grapevine plants were assessed for viruses twice annually, and those tested negative for GINV were chosen for the study. The grapevine berries collected from the same tree were immediately infiltrated with agrobacterium (Agrobacterium tumefaciens) cells. The tissues and organs (roots, phloem, leaf blades, fruits) also collected from the same tree of “Crimson seedless” grapevines using tissue-specific expression analysis. We cloned GFP into GINV infection clone plasmid after CaMV 35S promoter using seamless cloning and assembly kit (Mei5 Biotechnology, Co., Ltd, China). Then pGINV-GFP plasmid was transferred into the agrobacterium strain GV3101. N. benthamiana was grown under controlled conditions at 25 °C with a 16 h light/8 h dark regime in illumination incubator.
Identification and sequence analysis of the MYB genes
All annotated grapevine proteins were downloaded from the Ensembl Plants database (https://plants.ensembl.org/Vitis_vinifera/Info/Index) and the National Centre for Biotechnology Information database (http://0-www-ncbi-nlm-nih-gov.brum.beds.ac.uk/) to identify the complete list of grapevine MYB genes. Arabidopsis thaliana MYB protein sequences were also downloaded from Ensembl Plants and NCBI. A Hidden Markov Model search (HMMsearch) was conducted using the HMM profile of the MYB-like DNA-binding domain (PF00249), which was obtained using the Pfam program (https://pfam.xfam.org/). The preliminary screening of members was performed using HMMER3.1, with P < 0.001. All the searched sequences were then submitted to the SMART tool (http://smart.embl.de/) and NCBI CDD (https://0-www-ncbi-nlm-nih-gov.brum.beds.ac.uk/Structure/bwrpsb/bwrpsb.cgi) to detect the MYB-like DNA-binding domain and to remove the sequences without this domain.
The amino acid sequences of the VvMYBs were analyzed with ExPASy ProtParam (http://www.expasy.org/tools/protparam.html) to obtain the number of amino acids, theoretical isoelectric point (pI), molecular weight, and instability index. Furthermore, VvMYB candidate genes were examined using domain analysis programs of Pfam and SMART.
Chromosomal location and phylogenetic analysis of VvMYBs
All VvMYB genes were mapped onto the grapevine chromosomes based on the information available in Ensembl Plants and NCBI databases. The annotation data, including information on the gene's position on the chromosome, chromosome number, and length, were uploaded in the TXT format file. Finally, the genes were mapped to the chromosomes using MapChart software (version 2.3).
A phylogenetic tree was constructed using the amino acid sequences of the MYB proteins of Arabidopsis and V. vinifera in MEGA 6.0 software (http://www.megasoftware.net/) following the neighbor-joining (NJ) method (Parameter setting: Bootstrap method-1000 replicates, Poisson model, Pairwisedeletion). We used the trimAl software to remove the redundant sequences information, and used the FigTree software to embellish phylogenetic tree.
Agrobacterium-mediated infection, RNA extraction, and cDNA synthesis
The agrobacterium (with infectious clone of GINV-GFP, pGD-VvMYB58-Flag, pGD-VvMYB11-Flag, or pGD-GUS recombinant plasmid) were independently transformed into the grapevine berries. The agrobacterium cell with infiltration buffer (10 mM MES/NaOH pH 5.6, 10 mM MgCl2, 150 μM acetosyringone) was injected into the “Crimson seedless” grapevine berries with a 1 mL syringe, using mock cells in the infiltration buffer as control. Agrobacterium-mediated transformation method as described in a previous work . Grapevine berries were collected 3, 5, 7, and 9 days after infection. Relative expression levels of coat protein (CP) and VvMYB58 genes were examined using qRT-PCR. Meanwhile, agrobacterium-mediated transient expression of the proteins (VvMYB20, VvMYB58, VvMYB100, VvMYB170, VvMYB191, VvMYB205, VvMYB251, VvMYB258, and VvMYB263) were carried out in N. benthamiana leaves, respectively as described previously . The grapevine berries and the N. benthamiana leaves were ground individually in liquid nitrogen. Total RNA was isolated from the samples using the Quick RNA Isolation Kit (Waryong, RNAzol, China) according to the manufacturer’s instructions and reverse transcribed into complementary DNA (cDNA) using random pentadecamer primers according to a previously described method .
RT-PCR and quantitative real-time PCR (qRT-PCR)
RT-PCR was performed to analyze the expression levels of VvMYB genes (VvMYB1-VvMYB265), using primers (150–500 bp amplicon) listed in Supplementary Table S5 and the fast PCR mix (Mei5bio, HiPer Taq HiFi PCR mix, China) on a thermal cycler (MyCycler; Bio-Rad). Total RNA was independently extracted from different tissues and organs (roots, phloem, leaf blades, fruits) using Quick RNA Isolation Kit (Waryong, RNAzol, China). The program used was as follows: 3 min at 95 °C, followed by 32 cycles of 95 °C for 25 s, 55–64 °C for 25 s, and 72 °C for 30 s, and a final extension for 5 min at 72 °C. Grapevine β-actin gene was used as a housekeeping gene to normalize the cDNA concentrations. Each PCR was replicated (n = 3) using the cDNA samples obtained from independent experiments. Agarose gel (1.5%) electrophoresis was used to check the amplicons (150–500 bp).qRT-PCR was performed with the SYBR® PrimeScript™ RT-PCR Kit (Takara) according to the manufacturer’s instructions on an ABI 7500 thermocycler (Applied Biosystems, USA). Grapevine berries were collected at 1, 3, 5, 7, and 9 days after agroinfiltrated. Total RNA was independently extracted from frozen sample also using Quick RNA Isolation Kit (Waryong, RNAzol, China).The relative expression levels of the genes were determined using the comparative ΔΔCT method. Grapevine β-actin gene was used as a reference gene.
Subcellular localization of VvMYB proteins
The full-length coding sequences of VvMYB20, VvMYB58, VvMYB100, VvMYB170, VvMYB191, VvMYB205, VvMYB251, VvMYB258, and VvMYB263 without a stop codon were amplified from the cDNA obtained from “Crimson seedless” grapevine berries using gene-specific primers. The fragments were identified by sequencing and fused to the green fluorescent protein (GFP) under the control of the double CaMV35S promoter in the modified plant expression vector pCam35s-GFP to produce pVvMYB20-GFP, pVvMYB58-GFP, pVvMYB100-GFP, pVvMYB170-GFP, pVvMYB191-GFP, pVvMYB205-GFP, pVvMYB251-GFP, pVvMYB258-GFP, and pVvMYB263-GFP plasmids, respectively. All recombinant plasmids were independently transferred into the agrobacterium strain GV3101 and infiltrated in N. benthamiana leaves as described in a previous work . Fluorescence signals in the cells were visualized 72 h post-agroinfiltration using an Olympus FluoView 3000 confocal microscope equipped with an Olympus FluoView FV10-ASW 4.0 Viewer Software. Fluorescence images were captured at an excitation wavelength of 488 nm.
Protein extraction and Western blotting
Protein extraction and Western blotting were performed as described previously . Grapevine berries were ground in liquid nitrogen and mixed with a 2 × SDS sample buffer containing 10% (v/v) β-mercaptoethanol. Proteins were separated by SDS-PAGE (12%), and Western blotting was performed by probing with the rabbit anti-Flag or anti-GFP antiserum (diluted 1:5000).
Identification and characterization of the MYB gene family in V. vinifera
With reference to Ensembl Plants, and transcriptome databases , members of the VvMYB family were searched using HMMsearch with a HMM profile of PF00249.
After SMART tool and NCBI CDD verification, a total of 265 VvMYB or VvMYB-related genes were isolated in the grapevine as candidate VvMYB genes across these two databases. We named these sequences according to the corresponding gene identifiers in the genome browsers (Table S1). The gene identifiers (e.g. VIT_00s0194g00130) assigned to genes may help to avoid the confusion that often results when multiple names are used for the same gene in such a large gene family [9, 13]. We distinguished each of the VvMYBs using a provisional nomenclature system and named them from VvMYB1 to VvMYB265 (Table S1). The chemical and physical properties of these VvMYB proteins are shown in Table S1. The length of the VvMYB genes varied from 165 bp (VvMYB4) to 9075 bp (VvMYB103) (Table S1). Sequence analysis showed that these VvMYB proteins or polypeptides had a length ranging from 54 to 1738 amino acids (aa), with a predicted molecular weight ranging from 6.27 to 190.21 kDa (Table S1) and a theoretical isoelectric point (pI) ranging from 3.85 (VvMYB27) to 10.79 (VvMYB75) (Table S1). The grand average of hydropathicity index (GRAVY) of the VvMYB proteins was negative (− 1.371 to − 0.149), except for seven MYBs (VvMYB34, VvMYB46, VvMYB154, VvMYB155, VvMYB201, VvMYB202, VvMYB259), indicating hydrophilicity of proteins (Table S1). The aliphatic index of these proteins ranged from 36.4 (VvMYB97) to 123.75 (VvMYB155) (Table S1), while the instability index was more than 40, indicating an unstable protein structure . These observations showed that most VvMYB proteins were unstable, except for twenty-nine (Table S1).
Classification of VvMYB transcription factors
To verify the reliability of our results, we also performed SMART analysis to identify all of the putative VvMYB protein sequences in Ensembl Plants, and transcriptome databases. The results were consistent with the Pfam outcome. The VvMYB proteins were classified into four sub-families are based on the number and location of MYB-like DNA-binding domain (PF00249). Our study classified the VvMYB or VvMYB-related genes into four distinct subfamilies MYB-related, 2R-MYB (R2R3-MYB), 3R-MYB (R1R2R3-MYB), 4R-MYB (Table 1, Table S2), consistent with the member grouping in Arabidopsis , cassava , and apple , but there are differences in the quantity distribution. Among them, the R2R3-MYB subfamily accounted for more than one-third of the total VvMYB proteins, while R1-MYB and R1R2R3-MYB subgroups accounted for 29.43% and 26.42%, respectively; the R1R2R3R4-MYB subgroup accounted for 6.04% of VvMYBs only (Table 1).
Chromosomal distribution of VvMYBs
Analysis of the chromosomal distribution of 265 possible members of the VvMYB or VvMYB-related genes based on Ensembl Plants and NCBI annotation. Genome chromosomal location analysis revealed that 209 VvMYB genes were unevenly distributed on all 19 chromosomes (Fig. 1). The TFs VvMYB1 to VvMYB13 were distributed on scaffolds (Chr0). Meanwhile, the chromosomal location information was unavailable for forty-three of the VvMYBs. We also found that the density and distribution pattern of the VvMYBs on the chromosomes were not uniform. Some chromosomal regions and single chromosomes (chromosomes 4, 7, 8, 11, 14, and 17) showed a high density of VvMYBs, while others did not (chromosomes 1, 10, 16, and 19) (Fig. 1, Table S3). The highest density of VvMYB genes was observed on chromosomes 8 and 14, with 19 and 21 VvMYB genes, and the lowest density was observed on chromosome 10, with only 4 VvMYB genes (Fig. 1, Table S3).
Phylogenetic analysis of VvMYBs
Furthermore, to elucidate the potential evolutionary relationships between the various VvMYB TFs, we constructed an NJ phylogenetic tree using the amino acid sequences of the grapevine and Arabidopsis MYBs. We used the amino acid sequences of 199 AtMYB genes (AtMYB1 to AtMYB199) obtained from the Ensembl Plants and NCBI database for the analysis. The AtMYB grouping was consistent with the provisional nomenclature of the VvMYB subfamilies. Analysis of the phylogeny and the protein sequences categorized the 199 AtMYB genes and 265 VvMYB, respectively. As shown in Fig. 2, 265 VvMYB proteins were distributed among 26 subfamilies, which were designated Ia through Xd (Fig. 2). Most MYB subgroups contained more grapevine members than Arabidopsis . The largest subfamily was Ia, with 56 VvMYB proteins, and the smallest was X, with only two VvMYB proteins (Fig. 2, Table S4). The VvMYB domain is highly conserved as other species, according to the number of MYB-like DNA-binding domain (PF00249).
Tissue-specific expression profiles of VvMYB genes
Generally, the MYB genes exhibit different expression patterns under biotic or abiotic stress responses and physiological and developmental stimuli. Studies have also demonstrated the variable expression of MYB or MYB-related genes in different varieties or tissues or under various developmental phases . In this study, we performed a RT-PCR to investigate the expression profiles of VvMYB genes in roots, phloem, leaf blades, and fruits (berries) of grapevine. Among the 265 VvMYB or VvMYB-related genes, 236 VvMYB genes exhibited various expression patterns in different grapevine tissues. However, we did not detect any transcripts of 29 VvMYB genes (11.33%) in these four grapevine tissues. Thirty- VvMYB genes (11.32%) were expressed in two tissues (leaf blade and fruit), and most VvMYB genes were expressed in the leaf blade (85.66%) and fruit tissues (87.55%) of the grapevine (Fig. 3, S1).
Subcellular localization analysis of VvMYBs
Most TFs are localized in the nucleus. To investigate the subcellular localization of VvMYB proteins, we randomly selected nine VvMYB genes using transient expression in N. benthamiana. We cloned the full-length cDNA of VvMYB20 (NM_001281204.1), VvMYB58 (XM_010650081.2), VvMYB100 (XM_002280991.3), VvMYB170 (XM_002272670.4), VvMYB191 (XM_002283539.3), VvMYB205 (NM_001281231.1), VvMYB251 (XM_002284201.3), VvMYB258 (XM_002284212.4), and VvMYB263 (XM_010646850.2) from “Crimson seedless” grapevine by RT-PCR and fused to the C-terminal GFP fusion protein to analyze their subcellular localization. Nine VvMYB:GFP fusion proteins and pCam35s-GFP vector separately transferred into the agrobacterium strain GV3101 and infiltrated in N. benthamiana leaves. We observed GFP fluorescence of nine fusion proteins in transiently transformed N. benthamiana leaf epidermis at 3 days post-inoculation (dpi) only in the nucleus. Among them, three fusion proteins (VvMYB58:GFP, VvMYB251:GFP, and VvMYB263:GFP) were highly expressed in N. benthamiana leaf (Fig. 4).
Expression analysis of VvMYBs genes in response to GINV infection
Further, to examine the role of VvMYB or VvMYB-related genes in grapevine under GINV infection, we compared 41 VvMYB genes expression between GINV infection and mock using qRT-PCR. These were selected based on the transcriptome data sets . In previous studies, we only selected one time point (7 dpi) using transcriptome sequencing. To investigate their expression profile in grapevine berries at various time intervals, we selected five time points at 1, 3, 5, 7, and 9 dpi. Here, 12 VvMYB genes were upregulated, while 28 VvMYB genes were downregulated during GINV infection; VvMYB59 showed no significant difference during the entire process (Fig. 5, Table S6). Among them, VvMYB11 and VvMYB58 were markedly induced at five time points during GINV infection. VvMYB11 and VvMYB58 were continually induced during GINV infection (Fig. 5, Table S6). Fourteen VvMYB genes were significantly downregulated during GINV infection. These genes mainly distributed in three subfamily VII, VIII, and X (3/14, 4/14, 3/14) (Figs. 2 and 5). Taken together, these results demonstrated that VvMYB genes actively regulate defense response against GINV in the grapevine.
VvMYB58 suppresses GINV accumulation
GINV infection significantly increased the expression of mainly VvMYB11 and VvMYB58 (Fig. 5, Table S6). Therefore, to investigate whether VvMYB11 or VvMYB58 is involved in defense responses in grapevine, we cloned the full-length cDNA of VvMYB11 (XM_002264114.4) and VvMYB58 (XM_010650081.2) by RT-PCR from “Crimson seedless” grapevine. We independently cloned the two genes into pGD-Flag vector, and generated pGD-VvMYB11-Flag and pGD-VvMYB58-Flag recombinant plasmids. The agrobacterium with infectious clone of GINV-GFP and pGD-VvMYB58-Flag were co-infiltrated into the grapevine berries. And GINV-GFP and pGD-GUS were co-infiltrated served as a negative control. qRT-PCR assays indicated that the transcript levels of VvMYB58 at 5, 7, and 9 dpi were 326%, 413% and 478% of that in controls (Fig. 6A), and the relative levels of GINV RNA were about 43%, 26%, and 16% of that in controls (Fig. 6B). Western blot analysis showed that transient expression of VvMYB58-Flag leads to an obvious decrease in viral accumulation (Fig. 6C). “Crimson seedless” berries infiltrated with GINV showed necrotic symptoms at 7 dpi (Fig. S2D), which is consistent with our previous research . Overexpression of VvMYB58 suppressed GINV replication but does not affect symptoms (Fig. S2E). In addition, the GINV agroinoculated N. benthamiana plants behaved chlorotic mottle at 7 dpi (Fig. S2E). Nevertheless, overexpression of VvMYB11 did not affect GINV accumulation (Fig. S2A-B). The results demonstrated that the overexpression of VvMYB58 decrease GINV abundance in the grapevine berries (Fig. 6).
Since the initial identification in 1982 , the MYB family of TFs has been a research topic. Currently, the MYB genes have been evaluated in about 74 species with sequenced genomes , such as Arabidopsis , upland cotton , cassava , soybean , apple , and populus . Over the past few decades, plant MYBs have emerged as key regulators of responses to diverse abiotic stresses, such as salinity stress , high- and low-temperature stress , drought , and phosphate starvation . The MYB TFs also play crucial roles in regulating the responses against infection by pathogens, such as bacteria [23, 24] and fungi [25, 26]. However, little is known about the MYB-mediated transcriptional regulation during grapevine response to viruses. The previous research have been identified VvMYB genes only in R2R3 subfamily, which explored wine quality-related MYB  and regulation of stilbene accumulation . This study conducted a comprehensive genome-wide analysis of the VvMYB gene superfamily in the grapevine plants. The study identified 265 VvMYB or VvMYB-related genes from the grapevine plant genome and transcriptome database . These findings were to refine and expand the member of grapevine MYB superfamily [32, 33]. Our results provide a robust theoretical foundation for further antiviral defense response studies in grapevine.
The present study found similarities in the conserved sequences among the VvMYB proteins within the same subfamilies; however, differences were detected in their chemical and physical characteristics, such as pI (VvMYB75 and VvMYB157 or VvMYB167) and the instability index (VvMYB218 and VvMYB72) (Table S1). These differences may be due to the discrepancies in the amino acids in the non-conserved regions of the VvMYB members, suggesting that each VvMYB protein may act differently in its microenvironment [4, 38]. Further sequence analysis showed that these VvMYB proteins or polypeptides vary widely in length (54 to 1738 amino acids), and most VvMYBs are hydrophilic (Table S1). These chemical and physical properties have the similar characterization to the Arabidopsis MYBs [12, 13]. Analysis of the physiological and biochemical properties suggested similar pI, GRAVY, instability index, and aliphatic index for most VvMYB proteins (Table S1), indicating a close evolutionary relationship. Moreover, the study found an uneven distribution of 209 VvMYB genes on the 19 chromosomes of the grapevine (Fig. 1). The VvMYB genes appeared concentrated in the homologous regions in chromosomes (Fig. 1). Our results showed ten VvMYB genes within a 2–6 Mb region in Chr4, eleven VvMYB genes within a 3–10 Mb region in Chr7, sixteen VvMYB genes within a 10–23 Mb region in Chr8, eleven VvMYB genes within 20–28 Mb region in Chr14, and thirteen VvMYB genes within a 4–13 Mb region in Chr17 (Fig. 1). These results suggest that the segmental and tandem duplications contributed to the expansion of VvMYB genes in the grapevine. The MYB domain is classified as VvMYB-related, R2, R3, or R4 type based on the number of repeats [43, 44]. Similarly, the present study classified the 265 VvMYB or VvMYB-related genes into four distinct subfamilies (Table 1, Table S2). However, our observations on the distribution are not simlar to the reports by Salih et al. on upland cotton MYB . Grapevine has an almost similar number of VvMYB R1, R2, and R3 members, but most upland cotton MYBs belong to the R2-MYB subfamily, and differences in the number of R2R3-MYB subfamily members, reflecting species differences (Table 1). We identified 26 subgroups containing 265 VvMYB genes (Fig. 2). The largest subfamily VIIIa, only had seven VvMYB proteins. Meanwhile, the VvMYBs were widely distributed in Ic, III(a + b), and VIIIb subfamilies, and Ic and VIIIb groups had two main branches (Fig. 2). The VvMYBs were not clustered into the AtMYB subfamilies VIIIa and Ib. These results indicated that the VvMYBs and the AtMYBs separated during the evolutionary process.
In plants, MYB TFs are involved in various processes, such as cell fate and identity [38, 45], primary and secondary metabolism , development , and biotic and abiotic stress responses . The complex function of the MYB protein family is reflected in the tissue-specific expression patterns of the various members. Nearly half of VvMYB TFs exhibited differences in the expression patterns in grapevine plants (Fig. 3, S1), similar to cotton  and cassava MYBs . Regulation of the specific stress genes temporal and spatial expression patterns is essential to plant stress responses . Studies have confirmed the role of MYB TFs of Arabidopsis in defense responses . In Arabidopsis, Botrytis cinerea induced the expression of BOTRYTIS SUSCEPTIBLE1 gene encodes the R2R3-type MYB TF, which increased tolerance to necrotrophic pathogens and abiotic stresses . Meanwhile, the R2R3-type MYB30 of Arabidopsis positively regulated programmed cell death associated with hypersensitive response . However, there is little research on the functions of MYB TFs in defense responses in the grapevine. Therefore, the present study analyzed the temporal and spatial expression patterns of VvMYBs during GINV infection to understand their roles in defense responses in the grapevine. The qRT-PCR results indicated that among the 41 randomly selected VvMYB genes, 12 were induced during GINV infection, while 28 were downregulated (Fig. 5, Table S6), indicating an alteration in VvMYBs during GINV infection. Subsequently, to verify whether VvMYB family members are involved in antiviral defense responses in grapevine, we overexpressed VvMYB11 and VvMYB58, which were significantly induced during GINV infection (Fig. 5). Our results demonstrated that the overexpression of VvMYB58 suppressed GINV abundance in grapevine (Fig. 6), confirming its role in antiviral defense response in grapevine. Moreover, in present study overexpression of VvMYB11 gene did not directly affect GINV-enriched in grapevine. However, VvMYB11 was dramatically induced during GINV infection (Fig. 5). VvMYB11 TF may not works alone in regulation antiviral defense response gene in grapevine. This result showed that differences in functions among the various members of VvMYB. Taken together, our results indicate part of VvMYB gene family members play an crucial role in protecting against GINV infection. VvMYB58 was functionally characterized as a target gene for genetic engineering approaches to improve the disease resistance of fruit trees and other crops to multiple biotic stresses. Thus, a model was proposed to aid in understanding of the VvMYB genes regulate defense response against GINV in grapevine.
The present study performed the first comprehensive and systematic analysis of the MYB gene superfamily in the grapevine. The study identified 265 VvMYB or VvMYB-related genes and divided them into 26 subfamilies according to their evolutionary characteristics. Additionally, our results revealed that VvMYB genes were distributed across the entire grapevine genome. In grapevine berries, 12 VvMYBs were induced, while 28 were downregulated during GINV infection. This means that part of MYB TFs play vital roles in against GINV infection. In the subsequent research, we will deeply explore the molecular mechanism about VvMYB58 restricts GINV enrichment. And VvMYB58 whether also suppresses other viruses or pathogens. Simultaneously, we now have rich resources for subsequent studies of gene cloning and functional characterization of members in this VvMYB family.
Availability of data and materials
All annotated grapevine gene sequences were downloaded from the Ensembl Plants database (https://plants.ensembl.org/Vitis_vinifera/Info/Index) and the National Centre for Biotechnology Information database (http://0-www-ncbi-nlm-nih-gov.brum.beds.ac.uk/). All relevant data presented in this study are provided either in the manuscript or additional files.
Klempnauer KH, Gonda TJ, Bishop JM. Nucleotide sequence of the retroviral leukemia gene v-MYB and its cellular progenitor c-MYB: The architecture of a transduced oncogene. Cell. 1982;31:453–63.
Kranz H, Scholz K, Weisshaar B. c-MYB oncogene-like genes encoding three MYB repeats occur in all major plant lineages. Plant J. 2000;21:231–5.
Dubos C, Stracke R, Grotewold E, Weisshaar B, Martin C, et al. MYB transcription factors in Arabidopsis. Trends Plant Sci. 2010;15:573–81.
Jiang CK, Rao GY. Insights into the diversification and evolution of R2R3-MYB transcription factors in plants. Plant Physiol. 2020;183:637–55.
Jin J, Zhang H, Kong L, Gao G, Luo J. PlantTFDB 3.0: a portal for the functional and evolutionary study of plant transcription factors. Nucleic Acids Res. 2014;42:1182–7.
Saikumar P, Murali R, Reddy EP. Role of tryptophan repeats and flanking amino acids in Myb-DNA interactions. Proc Natl Acad Sci U S A. 1990;87:8452–6.
Wang Z, Tang J, Hu R, Wu P, Hou XL, et al. Genome-wide analysis of the R2R3-MYB transcription factor genes in Chinese cabbage (Brassica rapa ssp. pekinensis) reveals their stress and hormone responsive patterns. BMC Genomics. 2015;16:17.
Ptashne M. How eukaryotic transcriptional activators work. Nature. 1988;335:683–9.
Stracke R, Werber M, Weisshaar B. The R2R3-MYB gene family in Arabidopsis thaliana. Curr Opin Plant Biol. 2001;4:447–56.
Paz-Ares J, Ghosal D, Wienand U, Peterson PA, Saedler H. The regulatory c1 locus of Zea mays encodes a protein with homology to myb proto-oncogene products and with structural similarities to transcriptional activators. EMBO J. 1987;6:3553–8.
Riechmann JL, Heard J, Martin G, Reuber L, Jiang C, et al. Arabidopsis transcription factors: genome-wide comparative analysis among eukaryotes. Science. 2000;290:2105–10.
Salih H, Gong W, He S, Sun G, Sun J, Du X. Genome-wide characterization and expression analysis of MYB transcription factors in Gossypium hirsutum. BMC Genet. 2016;17:129.
Ruan MB, Guo X, Wang B, Yang YL, Li WQ, et al. Genome-wide characterization and expression analysis enables identification of abiotic stress-responsive MYB transcription factors in cassava (Manihot esculenta). J Exp Bot. 2017;68:3657–72.
Du H, Yang SS, Liang Z, Feng BR, Liu L, et al. Genome-wide analysis of the MYB transcription factor superfamily in soybean. BMC Plant Biol. 2012;12:106.
Wilkins O, Nahal H, Foong J, Provart NJ, Campbell MM. Expansion and diversification of the populus R2R3-MYB family of transcription factors. Plant Physiol. 2009;149:981–93.
Perez-Rodriguez M, Jaffe FW, Butelli E, Glover BJ, Martin C. Development of three different cell types is associated with the activity of a specific MYB transcription factor in the ventral petal of Antirrhinum majus flowers. Development. 2005;132:359–70.
Bedon F, Grima-Pettenati J, Mackay J. Conifer R2R3-MYB transcription factors: sequence analyses and gene expression in wood-forming tissues of white spruce (Picea glauca). BMC Plant Biol. 2007;7:17.
Stracke R, Ishihara H, Huep G, Barsch A, Mehrtens F, et al. Differential regulation of closely related R2R3-MYB transcription factors controls flavonol accumulation in different parts of the Arabidopsis thaliana seedling. Plant J. 2007;50:660–77.
Deluc L, Bogs J, Walker AR, Ferrier T, Decendit A, et al. The transcription factor VvMYB5b contributes to the regulation of anthocyanin and proanthocyanidin biosynthesis in developing grape berries. Plant Physiol. 2008;147:2041–53.
Chaonan L, Ng KY, Fan L. MYB transcription factors, active players in abiotic stress signaling. Environ Exp Bot. 2015;114:80–91.
Ullah C, Unsicker SB, Fellenberg C, Constabel CP, Schmidt A, et al. Flavan-3-ols are an effective chemical defense against rust infection. Plant Physiol. 2017;175:1560–78.
Yu Y, Guo D, Li G, Yang Y, Zhang G, Li S, Liang Z. The grapevine R2R3-type MYB transcription factor VdMYB1 positively regulates defense responses by activating the stilbene synthase gene 2 (VdSTS2). BMC Plant Biol. 2019;19:478.
Noman A, Hussain A, Adnan M, Khan MI, He S. A novel MYB transcription factor CaPHL8 provide clues about evolution of pepper immunity againstsoil borne pathogen. Microb Pathog. 2019;137:103758.
Li W, Zhu Z, Chern M, Yin J, Yang C, et al. A natural allele of a transcription factor in rice confers broad-spectrum blast resistance. Cell. 2017;170:114–26.
Zhang YL, Zhang CL, Wang GL, Wang YX, Qi CH, et al. The R2R3 MYB transcription factor MdMYB30 modulates plant resistance against pathogens by regulating cuticular wax biosynthesis. BMC Plant Biol. 2019;19:362.
Gu KD, Zhang QY, Yu JQ, Wang JH, Zhang FJ, et al. R2R3-MYB transcription factor MdMYB73 confers increased resistance to the fungal pathogen Botryosphaeria dothidea in apples via the salicylic acid pathway. J Agric Food Chem. 2021;69:447–58.
Guo D, Zhao H, Li Q, Zhang G, Jiang J, et al. Genome-wide association study of berry-related traits in grape based on genotyping-by-sequencing markers. Hortic Res. 2019;6:11.
Wang XY, Zhang CW, Huang WT, Yue J, Dou JJ, Wang LY, et al. Crude garlic extract significantly inhibits replication of grapevine viruses. Plant Pathol. 2020;69:149–58.
Fan XD, Hong N, Zhang ZP, et al. Identification of a divergent variant of grapevine berry inner necrosis virus in grapevines showing chlorotic mottling and ring spot symptoms. Arch Virol. 2016;161:2025–7.
Fan XD, Zhang ZP, Ren GJ, et al. Occurrence and genetic diversity of grapevine berry inner necrosis virus from grapevines in China. Plant Dis. 2017;161:144–9.
Fan X, Zhang P, Ren F, et al. Development of a full-length infectious Cdna clone of the grapevine berry inner necrosis virus. Plants. 2020;9:1340.
Matus JT, Aquea F, Arce-Johnson P. Analysis of the grape MYB R2R3 subfamily reveals expanded wine quality-related clades and conserved gene structure organization across Vitis and Arabidopsis genomes. BMC Plant Biol. 2008;8:83.
Wong DCJ, Schlechter R, Vannozzi A, Höll J, Hmmam I, Bogs J, et al. A systems-oriented analysis of the grapevine R2R3-MYB transcription factor family uncovers new insights into the regulation of stilbene accumulation. DNA Res. 2016;23(5):451–66.
Wang X, Liu Y, Guo L, Shen J, Hu H, Zhou R. Transcriptome analysis of Crimson seedless grapevine (Vitis vinifera L.) infected by grapevine berry inner necrosis virus. Current Research in Virological Science. 2022;3:100024.
Zhang C, Wang X, Li H, Wang J, Zeng Q, et al. GLRaV-2 protein p24 suppresses host defenses by interaction with a RAV transcription factor from grapevine. Plant Physiol. 2022;189:1848–65.
Merugu R, Upadhyay VP, Manda S. Bioinformatics analysis and modelling of mycotoxin patulin induced proteins. Intl J Bioinformatics Biological Sci. 2016;4:5–10.
Cao ZH, Zhang SZ, Wang RK, Zhang RF, Hao YJ. Genome wide analysis of the apple MYB transcription factor family allows the identification of MdoMYB121 gene confering abiotic stress tolerance in plants. PLoS ONE. 2013;8:e69955.
Wu Y, Wen J, Xia Y, Zhang L, Du H. Evolution and functional diversification of R2R3-MYB transcription factors in plants. Horticulture Res. 2022;9:uhac058.
Gong Q, Li S, Zheng Y, Duan H, Xiao F, et al. SUMOylation of MYB30 enhances salt tolerance by elevating alternative respiration via transcriptionally upregulating AOX1a in Arabidopsis. Plant J. 2020;102:1157–71.
Yang A, Dai X, Zhang WH. A R2R3-type MYB gene, OsMYB2, is involved in salt, cold, and dehydration tolerance in rice. J Exp Bot. 2012;63:2541–56.
Zhao Y, Cheng X, Liu X, Wu H, Bi H, Xu H. The wheat MYB transcription factor TaMYB31is involved in drought stress responses in Arabidopsis. Front Plant Sci. 2018;9:1426–1426.
Baek D, Kim MC, Chun HJ, Kang S, Park J, et al. Regulation of miR399f transcription by AtMYB2 affects phosphate starvation responses in Arabidopsis. Plant Physiol. 2013;161:362–73.
Jin H, Martin C. Multifunctionality and diversity within the plant MYB-gene family. Plant Mol Biol. 1999;41:577–85.
Tombuloglu H. Genome-wide identification and expression analysis of R2R3, 3R-and 4R-MYB transcription factors during lignin biosynthesis in flax (Linum usitatissimum). Genomics. 2020;112:782–95.
Liu J, Osbourn A, Ma P. MYB transcription factors as regulators of phenylpropanoid metabolism in plants. Mol Plant. 2015;8:689–708.
Aya K, Ueguchi-Tanaka M, Kondo M, Hamada K, Yano K, et al. Gibberellin modulates anther development in rice via the transcriptional regulation of GAMYB. Plant Cell. 2009;21:1453–72.
Fernández-Marcos M, Desvoyes B, Manzano C, Liberman LM, Benfey P, et al. Control of Arabidopsis lateral root primordium boundaries by MYB36. New Phytol. 2017;213:105–12.
Zhang Z, Liu X, Wang X, Zhou M, Zhou X, et al. An R2R3 MYB transcription factor in wheat, TaPIMP1, mediates host resistance to Bipolaris sorokiniana and drought stresses through regulation of defenseand stress-related genes. New Phytol. 2012;196:1155–70.
Ahuja I, de Vos RC, Bones AM, Hall RD. Plant molecular stress responses face climate change. Trends Plant Sci. 2010;15:664–74.
Mengiste T, Chen X, Salmeron J, Dietrich R. The BOTRYTIS SUSCEPTIBLE1 gene encodes an R2R3MYB transcription factor protein that is required for biotic and abiotic stress responses in Arabidopsis. Plant Cell. 2003;15:2551–65.
Marino D, Froidure S, Canonne J, Khaled SB, Khafif M, et al. Arabidopsis ubiquitin ligase MIEL1 mediates degradation of the transcription factor MYB30 weakening plant defence. Nat Commun. 2013;4:1476.
We thank Prof. Yafeng Dong (Chinese Academy of Agricultural Sciences,
China) for providing the GINV infectious clone. We appreciate the linguistic assistance provided by TopEdit (www.topeditsci.com) during the preparation of this manuscript.
This work was supported by the National Natural Science Foundation of China (NSFC, 32002022), the Science and Technology Program of Henan Province (232102110029).
Ethics approval and consent to participate
The Vitis vinifera cultivar “Crimson seedless” and N. benthamiana were collected from experimental field at the Henan Institute of Science and Technology, Xinxiang City, Henan Province, China. The experimental research on plants, including collection of plant material, was complied with institutional, national, or international guidelines. And studies were conducted in accordance with local legislation. And also comply with the Convention on the Trade in Endangered Species of Wild Fauna and Flora.
Consent for publication
The authors declare no competing interests.
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Additional file 1: Table S1.
Summary information of physiological and biochemical properties of the VvMYB proteins.
Additional file 2: Table S2.
The VvMYB transcription factors distributed on different subgroups.
Additional file 3: Table S3.
Distribution of VvMYB genes on grapevine chromosomes.
Additional file 4: Table S4.
Summary information of VvMYBs and AtMYBs amino acid sequence FASTA file.
Additional file 5: Table S5.
The primers sequences used in this study.
Additional file 6: Table S6.
Expression patterns of 41 VvMYB genes during the process of GINV infection by qRT-PCR.
Additional file 7: Fig. S1.
Tissue-specific expression patterns of VvMYB genes in “Crimson seedless” grapevine. R, roots; P, phloem; L, leaf blades; F, fruit.
Additional file 8: Fig. S2.
Overexpression of VvMYB11 did not affect viral replication. (A) and (B) pGD-VvMYB11-Flag and GINV-GFP were co-infiltrated into grapevine berries by Agrobacterium-mediated transient transformation; pGD-GUS and GINV-GFP served as the negative control. Error bars represent the SD of three independent biological replicates. *P < 0.05, **P < 0.01. Overexpression of VvMYB58 did not affect the symptoms of virus-infected. Comparison of symptoms of GINV virus-infected GINV virus-infected Nicotiana benthamiana (C) and “Crimson seedless” grapevine fruits with mock at 7 dpi (E). (D) shows symptoms of solitary infection GINV of “Crimson seedless” grapevine fruits.
Additional file 9: Fig. S3.
The original image blots in Fig. 6C.
Additional file 10: Fig. S4.
Part of the original image of Fig. 3.
Rights and permissions
Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, 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 changes were made. 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/4.0/. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data.
About this article
Cite this article
Wang, X., Zhao, S., Zhou, R. et al. Identification of Vitis vinifera MYB transcription factors and their response against grapevine berry inner necrosis virus. BMC Plant Biol 23, 279 (2023). https://0-doi-org.brum.beds.ac.uk/10.1186/s12870-023-04296-7
- MYB transcription factor
- Expression pattern analysis
- Biotic stress
- Grapevine berry inner necrosis virus