Search
Submit Manuscript
1.9CiteScore
Advanced Search
MAP  >  Fruit Research
2024 Volume 4
Article Contents
Abstract
Introduction
Results
Whole-genome resequencing and variant calling
Phylogenetic analysis
Population structure and principal component analysis (PCA)
Analysis of gene flow among Hainan tea, Assamica, and Sinensis
Kinship analysis
Analysis of genetic diversity of Hainan tea
Discussion
Materials and methods
Sample collection
DNA sample preparation and sequencing
Quality control and filtering
Variation calling and annotation
Population genetics analysis
Relationship inference
Genetic variation and FST calculations
Gene flow analysis
f3 and Patterson’s D statistics
Author contributions
Data availability
Acknowledgments
Conflict of interest
Rights and permissions
References
About this article
Next Previous
ARTICLE   Open Access    

Identification and characterization of grape VAP27 gene family and their roles in disease resistance

  • 1.

    State Key Laboratory of Crop Stress Resistance and High-Efficiency Production, College of Horticulture, Northwest A&F University, Yangling 712100, Shaanxi, China

  • 2.

    Key Laboratory of Horticultural Plant Biology and Germplasm Innovation in Northwest China, Ministry of Agriculture, Northwest A&F University, Yangling 712100, Shaanxi, China

  • # Authors contributed equally: Ruonan Li, Bianbian Wang

More Information
  • Received: 24 January 2024
    Revised: 03 April 2024
    Accepted: 24 April 2024
    Published online: 17 May 2024
    Fruit Research  4,  Article number: e019 (2024)  |  Cite this article
  • Vesicle-associated membrane protein (VAMP)-associated proteins (VAP27s), which are widely expressed in plants and animals, play an important role in metabolism, physiology, growth, and development, disease resistance, and immunity. While the function of this family has been elucidated in model plants like Arabidopsis thaliana and tomato, its role in grapevine remains unclear. In this present study, 12 vesicle-associated protein-membrane protein genes were identified in the grapevine genome by bioinformatics, designated as the VAP27 gene family. A phylogenetic tree, encompassing 53 genes from three model plants, Arabidopsis thaliana, Oryza sativa, and Solanum lycopersicum, revealed the subdivision of the VAP27 gene family into three subfamilies, each presumably serving different functions, besides localizing in endoplasmic reticulum, individual members also localize in nucleus. Additionally, we compared the transcriptional levels and subcellular localizations of the VvVAP27 family members across different plant tissues (flower, leaf, seed, root, fruit, tendril, and stem), indicating site-specific functionalities for different gene members. To investigate the responsiveness of the VAP27 gene family to pathogen infection, particularly Plasmopara viticola on host plants, we analyzed the expression patterns of VAP27 genes post-infection. Our findings revealed divergent expression profiles among different members at different stages of infection. The gene family responded to the infection of downy mildew on grapevine and could inhibit the spread of Phytophthora capsici lesions in Nicotiana benthamiana. These results provide an important basis for further studies delving into the functions of the VAP27 gene family in plant growth and disease resistance.

  • Introduction

    Tea (Camellia sinensis (L.) O. Kuntze) stands as China’s earliest documented tree crop, boasting a domestication history spanning over 3,000 years. Initially employed as a medicinal herb with roots dating back nearly 5,000 years, it later evolved into a beverage widely embraced for consumption[1]. On a global scale, cultivated tea plants are classified into two primary groups: C. sinensis var. sinensis (CSS) and C. sinensis var. assamica (CSA)[2].

    Hainan Island, positioned in the northern part of the South China Sea, has a rich history of tea plant cultivation and extensive planting areas. There were reports of the abundant tea plant resources on Hainan Island at the end of the Qing Dynasty. For instance, the American missionary and botanist Benjamin Couch Henry uncovered a significant number of wild tea trees during his extensive exploration of the Li ethnic group area in Hainan, confirming the abundance of ancient tea tree resources on the island[3]. As the Yunnan-Guizhou Plateau is widely recognized as a potential geographical origin of tea[4−6], most studies on tea plant population genomics encompass samples from southwestern China, particularly CSA varieties[1, 6−8], leaving research on tea plants in Hainan Island relatively sparse. The self-incompatibility of tea trees results in high offspring heterozygosity, and the abundant wild tea plant germplasm on the island provides a wealth of genetic variation, laying the groundwork for cultivating new varieties with desirable traits[7]. Despite Hainan Island’s abundance of tea resources, fully comprehending the genetic resources of tea plants there poses a challenge due to its unique climate and geographical environment. Hence, a genome-wide investigation into the genetic diversity of Hainan tea is imperative for a comprehensive understanding of the genetic resource background of Hainan tea.

    It is noteworthy that the tea plant species on the island closely resembles CSA and is referred to as ‘Hainan dayezhong’[9]. However, evidence is insufficient to conclusively determine whether the Hainan dayezhong belongs to CSA or not. The classification of Hainan tea presents a significant challenge for several reasons: Firstly, C. sinensis plants are prone to hybridization between different species, posing a challenge in accurately classifying various hybrid progenies. Secondly, numerous morphological characteristics of tea plants resemble each other, complicating precise taxonomic delineations[10]. Lastly, traditional classification of tea plants primarily relies on morphological characteristics, which may sometimes conflict with the latest molecular-based classification results[8]. Despite Hainan tea’s identification by the National Crop Variety Approval Committee in 1985, its taxonomic status within the Camellia genus on Hainan Island remains unclear due to the absence of support from modern genomic research data.

    Islands, as an ideal system for studying the effects of geographical isolation and long-distance diffusion, offer valuable insights into species evolution, encompassing phenomena such as adaptive radiation and speciation[11]. Previous studies have documented the discovery of several new plant species on Hainan Island, including Holttumochloa[12], Euphorbia[13], Cycadaceae[14], among others. Moreover, advancements in whole-genome resequencing technology have confirmed the independent evolutionary histories and parallel domestication processes of CSS and CSA[7, 8]. Building upon these findings, our hypothesis suggests that tea trees on Hainan Island may constitute a distinct species separated from CSS and CSA, and that Hainan tea has undergone an independent evolutionary trajectory on the island.

    To furnish molecular evidence regarding the genomic divergence and relationship of Hainan tea with CSS and CSA, and to elucidate the genetic background of Hainan tea on Hainan Island, we procured 500 samples of Hainan tea from the Baisha, Qiongzhong, Wuzhishan, and Ledong regions of Hainan Province, China. Employing whole-genome resequencing technology, we identified SNPs in the Hainan tea samples and constructed a phylogenetic tree that included both cultivated tea and Hainan tea, utilizing the Yunkang 10 as the reference genome. Subsequently, detailed analyses of population structure and kinship relationships were conducted to offer a comprehensive understanding of the population structure and genetic diversity of Hainan tea and to unveil the phylogenetic relationships between Hainan tea and global Camellia sinensis varieties. This study furnished robust genomic data support and further corroborated the independent status of Hainan tea within the taxonomy of Camellia sinensis. Concurrently, these findings furnished a crucial scientific foundation for the conservation of tea germplasm resources and molecular breeding on Hainan Island. Furthermore, the research methodologies and techniques employed herein hold the potential to provide valuable insights into the origin and domestication analyses of other species on Hainan Island, as well as for the investigation of genetic diversity.

    Results

    Whole-genome resequencing and variant calling

    In this study, 500 tea tree samples were collected from four major tea-producing regions in Hainan: Ledong, Qiongzhong, Baisha, and Wuzhishan (Fig. 1). Notably, the samples encompassed a substantial number of ancient tea trees. Detailed sample information is provided in the Methods and Materials section and Supplemental Table S1. Following the sequencing, a total of 6.9 Tb of raw sequencing data were obtained. Subsequently, the original data source underwent filtration and was aligned with the reference genome (Yunkang 10), yielding a final average alignment rate of 98.98%. Notably, A-MCXB3-1D was excluded from the dataset due to its notably low mapping rate of 27.27% (Supplemental Fig. S1; Table S1).

    Figure 1.  Geographical distribution of tea samples collected and analyzed in this study.
    The color of circles denotes the collection area of Hainan tea samples, with circle size indicating the corresponding sample count. Larger circles indicate higher sample counts in the respective areas.
    DownLoad: Full-Size Img PowerPoint

    Based on the results from Supplemental Fig. S2, which contain information about SNPs initially detected using GATK, hard filtering conditions were established initially. Subsequently, SNPs with a minor allele frequency (MAF) of at least 0.05 were retained, resulting in a total of 32,334,340 SNPs (Supplemental Table S2). Among them, 91.3% were located in the intergene region, 4.85% in the intron region, 0.12% in the 5' UTR, and 0.28% in the 3' UTR. In addition, 1.01% of SNPs were located in the upstream region of the gene, 1.06% in the downstream region. Exon SNPs accounted for 1.34% of the total SNPs, of which non-synonymous SNPs and synonymous SNPs accounted for 49.82% and 47.93%, respectively (Tables 1, 2).

    Table 1.  The number of SNPs in different genome structures.
    VariantsTypeCore set
    SNPTotal32,334,340
    Intergenic29,520,274
    Intronic1,566,641
    Exonic433,604
    5' UTR40,383
    3' UTR92,101
    UTR5;UTR3229
    Upstream326,710
    Downstream341,541
    Upstream;downstream7,803
    Splicing4,838
    Exonic;splicing216
     | Show Table
    DownLoad: CSV
    Table 2.  The number of large-effect SNPs.
    VariantsTypeCore set
    SNPTotal (exonic + exonic;splicing)433,820
    Nonsynonymous243,634
    Synonymous179,902
    Nonsyn/Syn ratio1.35
    Stop-gain9,699
    Stop-loss518
    Unknown67
     | Show Table
    DownLoad: CSV

    Phylogenetic analysis

    To explore the phylogenetic relationship between tea trees of CSS and CSA on Hainan Island, genomic data of CSS and CSA were obtained from various global regions from the teabase database (http://teabase.ynau.edu.cn/)[15], as well as from the Genome Sequence Archive project number PRJCA001158[8]. Additionally, KM6 (Camellia cuspidata) was selected as an outgroup. The chosen tea plant materials are listed in Supplemental Table S3. Subsequently, we constructed a phylogenetic tree between the resequenced samples and globally cultivated tea trees using the maximum likelihood method with SNPs extracted from prior results. Phylogenetic analysis revealed distinct categorization of the samples into four main classes: global Aassamica1, global Assamica2, global Sinensis, and tea trees from Hainan Island. Notably, global Assamica and Sinensis clustered together on one side of the tree, while the tea tree samples from Hainan Island formed a separate, distinct cluster, without mingling with global Assamica and Sinensis. Particularly noteworthy is the significant geographic clustering observed in samples collected from the Limu Mountain area (abbreviated as LMS in Fig. 2), forming a subgroup within the Hainan tea sample cluster. Conversely, samples from other regions lacked a discernible pattern of geographic or regional clustering. For simplicity, we collectively refer to Hainan tea samples as ‘Hainan tea’. Based on the phylogenetic tree clustering results, we abbreviated tea samples from the Limu Mountain region as LMS and those from outside the Limu Mountain region as OLMS.

    Figure 2.  Phylogenetic relationship between Hainan tea and cultivated tea.
    Branch color indicates the sample source. Blue, purple, and green branches in the upper section of the phylogenetic tree denote cultivated tea across global regions. CSA is divided into Assamac1 (blue) and Assamac2 (purple). The lower section illustrates the phylogenetic relationships among Hainan tea samples from distinct regions.
    DownLoad: Full-Size Img PowerPoint

    Further population rooted tree analysis (Supplemental Fig. S3) indicated that Hainan tea exhibited significant genetic divergence from CSS and CSA, making it challenging to classify them within the same category. This suggested a considerable evolutionary divergence of Hainan tea from CSS and CSA, although additional evidence is required to bolster this hypothesis. Notably, a discernible genetic distance exists between OLMS and LMS in the evolutionary tree, and they do not form a distinct category, consistent with the findings of phylogenetic analysis. In comparison to the outgroups, Hainan tea clustered more closely with global Assamica and Sinensis, prompting speculation that Hainan tea may belong to a Camellia species distinct from CSS and CSA.

    Population structure and principal component analysis (PCA)

    Utilizing the data from Fig. 2 as the foundation, sequencing data from 21 bloom Camellia, 24 oilseed Camellia, 41 wild Camellia and 15 other Camellia groups were extracted from the teabase database (Supplemental Table S3), followed by population structure analysis and principal component analysis. K values ranging from 1 to 9 and their corresponding cross-validation (CV) error values were examined (Fig. 3a; Supplemental Fig. S4). Notably, at K = 2, Hainan tea diverged from other Camellia species, manifesting distinct ancestral compositions. With K = 3, a subsequent separation occurred with Sinensis exhibiting novel ancestral components. By K = 4, Assamica segregated from the genetic makeup of the added Camellia plant samples. At this juncture, Hainan tea demonstrated a distinctive population genetic background compared to the global Assamica and Sinensis, corroborating the findings depicted in Fig. 2. Furthermore, at K = 8, the cross-validation error minimized (Fig. 3a), indicating the optimal model where multiple species within Hainan tea and Camellia were delineated into eight genetically discrete populations. Within these populations, the LMS group displayed an autonomous genetic composition (depicted in purple). Particularly noteworthy, populations from the LD-JFL and QZ-BML, originating from two rainforest reserves, exhibited a considerable blend of purple genetic backgrounds. Additionally, the NS-JM and SM-SM populations shared a common genetic makeup (depicted in dark brown). It’s of significance to observe that certain SM-MN populations encompassed a genetic background akin to global Assamica1, plausibly due to the historical introduction of Assamica in Hainan Province.

    Figure 3.  The population structure and principal component analysis results for various species of Hainan tea and the Camellia genus are presented, alongside gene flow maps illustrating interactions between cultivated tea and Hainan tea.
    (a) Population structure of Hainan tea and global Assamica, global Sinensis, 21 bloom Camellia, 24 oilseed Camellia, 41 wild Camellia, and 15 other Camellia groups. The picture shows the population structure of K value from 2 to 8 analyses. (b) Principal component analysis. These samples can be divided into six different groups, represented by six circles. Among them, Other, Bloom Camellia and Oilseed Camellia are grouped together, while Hainan Tea, global Assamica1, global Assamica2, global Sinensis, and wild Camellia are grouped separately. (c) Graph illustrating the results of the optimal gene flow analysis conducted using Treemix. This figure shows the history of gene flow among three populations of Assamica, Sinensis and Hainan tea. Arrows are used to mark possible population migration events in the figure, the length of the arrow indicates the intensity of migration, and the direction of the arrow indicates the direction of migration.
    DownLoad: Full-Size Img PowerPoint

    In the principal component analysis of Hainan tea and Camellia species (Fig. 3b; Supplemental Fig. S5), PC1, PC2, and PC3 carried weights of 6.25%, 5.20%, and 4.53% respectively. PC1 distinctly segregated Hainan tea from other Camellia species, suggesting a unique genetic background for Hainan tea. This finding aligned with the outcomes of the population structure analysis (K = 2). PC2 separated global Assamica1 from Assamica2, while PC3 separated global Assamica from Sinensis. Consistent with the population structure results, PCA also indicated the inclusion of SM-SM samples within global Assamica1, further supporting the hypothesis that certain samples clustered within global Assamica was due to the introduction of CSA from Yunnan Province to Hainan Province. It’s noteworthy that Hainan tea displayed a closer clustering with Assamica and Sinensis in the PCA plot compared to the subsequently added Camellia genus. The findings from population structure and PCA complemented those of Fig. 2, demonstrating that Hainan tea not only stands distinct from Assamica and Sinensis but also lacks any shared genetic components with the newly added Camellia genus population.

    Analysis of gene flow among Hainan tea, Assamica, and Sinensis

    To provide additional evidence for our hypothesis proposing that Hainan tea belongs to a distinct species within the Camellia genus, separate from Assamica and Sinensis, we conducted f3 statistical analysis and examined the genetic relationships among Hainan tea, Assamica, and Sinensis using Treemix and D statistics.

    During the f3 statistical analysis, we observed that the f3 values between Hainan tea and cultivated tea were highly similar, with a difference of only 0.00347, indicating a close genetic relationship between them. Specifically, the f3 values of OLMS and LMS exceeded those of Hainan tea and cultivated tea, implying a higher genetic similarity between OLMS and LMS. This discovery enhanced our understanding of the potential genetic connection between Hainan tea and LMS, suggesting a potential sharing of deeper genetic characteristics.

    In the gene flow analysis, OptM determined the optimal migration model to be m = 1, implying the possibility of a migration event among the studied populations (Supplemental Fig. S6a, S6b). Employing a model with m = 1, we identified gene flow from Sinensis to Assamica1 (Fig. 3c), supported by a significant D value in the D statistics (Supplemental Table S5). In summary, these findings indicated that Hainan tea exhibits limited similarity to Assamica and Sinensis, with no significant historical gene flow between Hainan tea and Assamica or Sinensis.

    Kinship analysis

    In order to assess the genetic relationship between Hainan tea samples, KING software was used to analyze the genetic relationship of Hainan tea, Assamica and Sinensis, and the results are shown in Fig. 4a. In the genetic process, individuals accumulate their mutations, which can be shared among different individuals, so two individuals with the same mutations do not necessarily have the same ancestors. This similarity of mutations is called Identical by state (IBS). The King software can calculate the IBS value based on the SNPs, and reflect the composition and reliability of the kinship relationship among groups through the ratio of the kinship coefficient. The abscissa in the figure represented the proportion when IBS is 0, closer to 0, and higher the reliability of the result. The kinship coefficient of the vertical axis was mainly divided into three levels. A coefficient lower than 0.0442 indicated that the kinship relationship among individuals was far away, while a negative kinship coefficient indicated that there may be a large difference in the population structure between two individuals.

    Figure 4.  The genetic relationships between samples and the genetic differentiation coefficient (FST) between different populations, as well as the corresponding nucleotide diversity (π).
    (a) The family analysis results are based on KING software. The X-axis is the IBS value, and the Y-axis is the kinship coefficient. The data comprise pairs of samples exhibiting close kinship, which were selected from Global Assamica, Global Sinensis, and Hainan tea. Sample pairs from Hainan tea are highlighted in red. The three intervals, separated by dotted lines from top to bottom, represent primary, secondary, and tertiary degrees of kinship, respectively. (b) The FST heat map llustraties the genetic differences among the five populations of OLMS, LMS, Assamica1, Assamica2 and Sinensis. (c) π values of OLMS, LMS, Assamica and Sinensis populations.
    DownLoad: Full-Size Img PowerPoint

    Highlighted in red in Fig. 4a was the sample pair with close genetic relationship screened out of Hainan tea samples, that was, kinship greater than 0.0442 (Supplemental Table S6). The closely related samples primarily belonged to the WDB group in the SM-MN area (refer to Fig. 4a; Supplemental Table S6 for specifics). All Hainan tea samples in this region were sourced from artificially managed tea gardens, with human activities influencing the reproduction of tea trees. Moreover, the area has a history of large-scale tea plant cultivation, suggesting that the close relationship between these samples may stem from the expansion of tea tree cultivation areas.

    Analysis of genetic diversity of Hainan tea

    Pairwise FST values were computed for five populations: Assamica1, Assamica2, Sinensis, Hainan tea, and LMS, revealing a range between 0.036 and 0.328 (Fig. 4b; Table 3). Notably, the minimal group distinction was observed between Hainan tea and LMS, resulting in an FST value of 0.036. Importantly, when compared to Assamica, the distinction between tea and Sinensis populations in the Hainan and LMS regions was less pronounced. This observation aligned with the findings from the phylogenetic analysis of f3. Genetic diversity levels for these five populations were additionally assessed through the analysis of π values. As illustrated in Fig. 4c, tea populations in the Hainan and LMS regions exhibited greater genetic diversity compared to Assamica and Sinensis. It is notable that teas from Hainan and LMS regions display heightened genetic diversity levels. The level of genetic diversity among tea populations is relatively consistent.

    Table 3.  Genetic differentiation coefficient (FST) among groups. FST values range from 0 and 1, with higher values indicating greater genetic differences among populations.
    FSTAssamica1Assamica2OLMSLMSSinensis
    Assamica10.2360.2390.2360.321
    Assamica20.2810.2820.328
    OLMS0.0360.209
    LMS0.212
    Sinensis
     | Show Table
    DownLoad: CSV

    Discussion

    Although Hainan Island is rich in wild tea tree resources and possesses vast plantation areas of rainforest tea trees, tea tree resources have not yet been comprehensively investigated and fully developed. In this study, we selected a large number of ancient tea tree samples from the rainforest area, and analyzed them by whole genome resequencing, obtaining 32,334,340 SNPs. This dataset is the most extensive resequencing dataset of Hainan tea samples reported so far.

    The classification of Camellia species basing on traditional taxonomy is very challenging[8], and Hainan dayezhong, as a unique Camellia species in Hainan, lacks the support of genomics data so far, and its status in taxonomy is always unknown so that it is often not yet a CSA. We analyzed the population relationship between Hainan tea and globally cultivated tea trees based on resequencing data to clarify the status of Hainan tea in Camellia from a genomic perspective. By constructing a phylogenetic tree between Hainan tea and globally cultivated tea trees, it can be observed that Hainan tea does not belong to either CSS or CSA, but rather forms an independent branch and clusters into a single taxon. It is important to note that in this cluster of Hainan tea, the samples from the LMS group formed distinct geographic subgroups, whereas the samples from the OLMS group did not appear to be geographically clustered (Fig. 2). This may be attributed to the fact that samples from the LMS population were collected in the Limu Mountain Rainforest Reserve, which is relatively undisturbed by human activities. In contrast, other areas have more human activities, which may lead to the mixing of genetic backgrounds of Hainan tea in multiple regions[16].

    Although the Wuzhishan region is located in a tropical rainforest reserve, according to the Qiongzhong County Record, the state actively promoted tea planting in the region in the mid-1990s and introduced CSA varieties for breeding and cultivation. Therefore, the samples from the Wuzhishan region did not show obvious geographical clustering (Fig. 2). Additional results of population structure and principal component analysis further confirmed this observation. The population structure analysis revealed that Hainan tea has an independent genetic background, whereas LMS differs from OLMS in genetic background. It is particularly noteworthy that, except for LMS, OLMS presented a mixture of genetic backgrounds, which coincided with the results of phylogenetic trees (Figs 2, 3a). The results of principal component analysis also clearly showed the independent group status of Hainan tea with CSS and CSA (Fig. 3b; Supplemental Fig. S5). Despite the presence of several Hainan tea samples in the global Assamica1 cluster, this is consistent with the historical context of the introduction of CSA from Yunnan in the mid-1990s.

    Geographic isolation is one of the main causes of species formation[17]. When populations of the same breeding stock separate, they face independent evolutionary histories defined by natural selection, genetic drift, adaptation, and colonization to local conditions[18]. Hainan, as a tropical island, has extensive rainforests that provide high-quality growing environments for plants, and the island’s geography provides the necessary geographic isolation for new species to arise. The results of the population structure analysis, which incorporated data from additional Camellia plants, clearly indicated that Hainan tea possesses a distinct genetic background compared to other Camellia species. Moreover, Hainan tea clustered closer to CSS and CSA in the principal component analysis while remaining distant from other Camellia (Fig. 3a, b). Therefore, we cautiously proposed that Hainan tea represents a novel variety of Camellia sinensis distinct from CSS and CSA. Notably, samples from the LMS region form a distinct subgroup cluster in the phylogenetic tree depicted in Fig. 2 and demonstrated an independent genetic component in the population structure analysis, akin to the scenario observed with G. hirsutum L. purpurascens on Hainan Island[19]. Thus, it was deduced that Hainan tea from the LMS region constitutes a unique endemic variety within the Hainan tea species.

    Genetic drift is one of the important mechanisms for maintaining genetic diversity among biological populations. High levels of genetic drift help to reduce genetic differences and increase the homogeneity between two populations[20]. However, when physical barriers prevent genetic drift, different populations may form or experience physical isolation that prevents the exchange of genetic materials. These physical barriers are usually, although not always, caused by natural factors[21].

    Hainan Island, once connected to the mainland, has undergone a long period of rotation and movement, rotating counter-clockwise from its original position in the Beibu Gulf to its current position. The initial separation occurred in the Paleocene (ca. 65 Mya), while the major part of the rotational drift occurred in the Eocene[22]. During the Quaternary, ice ages and interglacial periods alternated, the most recent major ice age occurring about 15 Kya ago. The onset of the Ice Age led to a drop in global temperatures and a steady decline in global sea levels, which led to the formation of natural land bridges between sea islands and continents. During the ~8,000-year-long Ice Age (15 Kya-7 Kya ago), genetic exchange of species between Hainan Island and neighboring continents may have occurred. For example, a literature survey study found the existence of gene flow between Hainan’s native Painted Lady and the Chinese Painted Lady in South China[23]. However, the cold global climate during the Ice Age reduced the population size of the species, especially for the cold-intolerant CSA, and the likelihood of genetic exchange diminished[24]. After the Ice Age ended, the rise of the sea level led to the emergence of Qiongzhong Strait, which switched Hainan Island once again to the island mode. This geological event may have hindered genetic exchange between Hainan Island tea trees and those on the mainland, leading to their gradual and independent evolution in response to the tropical island climate. As a result, a new variant emerged, possibly falling under the categorization of Camellia sinensis[25].

    Considering the potential existence of land bridges facilitating gene flow between Hainan tea and mainland tea plants, we intensively investigated the gene flow between Hainan tea and cultivated tea. First, we performed f3 statistical analysis (Supplemental Table S4) and found that the genetic relationship between LMS and OLMS was closer comparing to cultivated tea, which is consistent with the results in Fig. 2 and 3a. Especially noteworthy is that Hainan tea was closer to Sinensis comparing to Assamica. The results of the Treemix analysis visually demonstrated how geographic isolation significantly impeded gene flow between cultivated and Hainan teas (Fig. 3c). In addition, the Dsuite program was applied to perform ABBA-BABA analysis, and this result further supported our view (Supplemental Table S5). These findings strongly suggested that the geographic separation of Hainan tea has prevented the exchange of genetic material between it and cultivated tea, thus contributing to its possible independent evolution as a new variant of Camellia sinensis.

    Groups that are highly segregated and lack genetic drift are usually prone to inbreeding[26]. However, the current analyses showed (Fig. 4a) that Hainan tea do not show excessive kinship among each other, and the concentration of samples with high kinship was overwhelmingly from samples from the WDB group (Supplemental Table S6), a group whose tea trees came from an artificially managed tea plantation. This phenomenon may be caused by anthropogenic factors. Genetic diversity, species diversity, and ecosystem diversity are the three pillars of biodiversity. Tea plants are typically propagated asexually via cuttings. If individuals propagated through this method are presented in the study samples, a significant portion of sample pairs will exhibit an affinity coefficient exceeding 0.354. Nevertheless, the present findings do not corroborate this hypothesis (Fig. 4a). Based on the principles of population genetics, the conservation of biodiversity is ultimately the conservation of genetic diversity[27]. Nucleotide diversity is an important indicator for assessing the diversity of DNA sequences in a species or population[28]. The processes of domestication and breeding have reduced the genetic diversity of crops, and the widespread cultivation of monoculture crop varieties has led to an increase in genetic vulnerability[29,30]. Wild ancient tea trees, as a precious natural resource with high genetic diversity, are of great value for the study of the evolutionary mechanisms and diversity of the tea trees[31]. Interestingly, the Hainan tea and LMS have higher genetic diversity than CSS and CSA (Fig. 4c), even though Hainan tea is affected by geographic isolation, resulting in restricted gene flow (Fig. 3c). This can be partially attributed to the unique climatic conditions of the tropical island, which are very favorable for the growth of tea trees. Combined with minimal anthropogenic disturbance, this has resulted in less natural pressure on tea tree population expansion, thus helping to maintain genetic diversity[16]. Furthermore, the genetic relationships between tea plants in Hainan and LMS were closer to those of Sinensis than to Assamica, and the genetic relationships between tea trees in Hainan and LMS were closer to each other (Fig. 4b; Table 3). This is consistent with the results obtained in the f3 statistical analysis (Supplemental Table S4), suggesting that Hainan tea and Assamica taxa diverged earlier than Hainan tea and Sinensis taxa.

    In summary, the whole-genome resequencing of 500 Hainan tea samples from major tea-producing regions of Hainan Island was performed in this study, and 32,334,340 SNPs were successfully identified. The results of this study strongly support the existence of Hainan tea as a new variant of Camellia sinensis, which is genetically distinct from CSS and CSA, and also reveal the existence of Hainan tea in the LMS region as an independently evolved local variety. Although Hainan tea did not show significant gene flow between Hainan tea and cultivated tea trees due to the geographic barrier of the strait, it still maintained high genetic diversity, which manifested itself in high π values. The results of this study help to clarify the position of Hainan tea in the taxonomy of Camellia sinensis from a genomic perspective. Additionally, they provide reliable data support for an in-depth understanding of the genetic background and diversity of Hainan tea on the island. Furthermore, they offer an important scientific basis for the conservation of tea germplasm resources and molecular breeding on Hainan Island. In addition, our research methods and techniques can also provide lessons and references for the analyses of the origin and domestication of other species on Hainan Island, as well as for genetic diversity studies.

    Materials and methods

    Sample collection

    Systematically, 500 samples of Hainan tea from Hainan Province, China were collected. These included Jianfengling (Ledong, 28 samples), Limu Mountain (Qiongzhong, 160 samples), Gaofeng Fangtong Village (Nankai, Baisha, 41 samples), Miao Village Junior Class (Nankai, Baisha, 53 samples), Mengya Village (Nankai, Baisha, 24 samples), Shifu Village Junior Class (Nankai, Baisha, 26 samples), Yaxing (Nankai, Baisha, 15 samples), Junmin Village (Nansheng, Wuzhishan, 13 samples), Maoxiang Village (Nansheng, Wuzhishan, 32 samples), Baimaling (Qiongzhong, 13 samples), Fanglong Village (Shuiman, Wuzhishan, 14 samples), Maona Village (Shuiman, Wuzhishan, 47 samples), and Shuiman Village (Shuiman, Wuzhishan, 34 samples). Additionally, the teabase database[15] and data from various Camellia species in the genome sequence archive with project number PRJCA001158 from Genome Sequence Archive[8] were utilized for analysis. Notably, the KM6 strain (Cuspidata Camellia) was selected as an outgroup for our study and subsequent analyses. Detailed information on the study samples can be found in Supplemental Tables S1, S3, and Fig. 1.

    DNA sample preparation and sequencing

    Five hundred tea accessions were acquired exclusively from Hainan province in China. Young leaves were harvested from these plants and rapidly frozen in liquid nitrogen. Total DNA extraction was performed using the DNAsecure plant kit (Tiangen, Beijing). Subsequently, 2 µg of genomic DNA from each accession was utilized to prepare sequencing libraries according to the manufacturer’s protocol using the NEBNext Ultra DNA Library Prep Kit (NEB Inc., America). Sequencing was carried out on an Illumina NovaSeq 6000 sequencer, generating paired-end sequencing libraries with an approximate insert size of 400 bp.

    Quality control and filtering

    The paired-end resequencing reads underwent filtering utilizing fastp (Version: 0.12.2)[32]. This process eliminated reads containing adapter sequences or poly-N sequences, as well as low-quality reads (defined as reads with more than 40% bases having Phred quality scores ≤ 20) from the raw data. The outcome of this step was the production of clean data, which were then utilized for subsequent downstream analyses.

    Variation calling and annotation

    The paired-end resequencing reads were aligned to our tea reference genome using BWA (Version: 0.7.17-r1188)[33], employing default parameters. The mapping results were converted into the BAM format and unmapped as well as non-unique reads were filtered using SAMtools (Version: 1.3.1)[34]. Additionally, duplicated reads were removed using the Picard package (picard.sourceforge.net, Version: 2.1.1).

    Following BWA alignment, we performed realignment of reads around indels using GATK in a two-step process. Initially, the RealignerTargetCreator package was utilized to identify regions necessitating realignment. Subsequently, the identified regions were realigned using IndelRealigner, resulting in a realigned BAM file for each accession. Variant detection was conducted following the recommended best practice workflow by GATK[35]. Specifically, variants were called for each accession using the GATK HaplotypeCaller[35]. A joint genotyping step was carried out to merge variations comprehensively from the gVCF files. During the filtering step, the SNP filter expression was set as ‘QD < 2.0 || MQ < 40.0 || FS > 60.0 || SOR > 5.0 || MQRankSum < −12.5 || ReadPosRankSum < −8.0 || QUAL < 30’. SNPs that were not bi-allelic were excluded, resulting in the creation of the basic set. Subsequently, SNPs with more than 20% missing calls and MAF less than 0.05 were further eliminated to generate the core set, which was used for phylogenetic tree construction, PCA, and population structure analysis.

    SNPs were annotated according to the tea genome using the ANNOVAR package (Version: 2015-12-14)[36]. Based on the genome annotation, SNPs were classified into various genomic regions, including exonic regions (overlapping with coding exons), splicing sites (within 2 bp of a splicing junction), 5' UTRs, 3' UTRs, intronic regions (overlapping with introns), upstream and downstream regions (within a 1 kb region upstream or downstream from the transcription start site), and intergenic regions. SNPs located in coding exons were further categorized into synonymous SNPs (which did not cause amino acid changes), nonsynonymous SNPs (which caused amino acid changes), stop gain mutations (mutations resulting in the gain of a stop codon), and stop-loss mutations (mutations resulting in the loss of a stop codon). Indels within exonic regions were classified based on whether they caused frame-shift mutations (3 bp insertion or deletion) and whether they resulted in the gain or loss of a stop codon.

    Population genetics analysis

    Whole-genome SNPs were utilized to construct the maximum likelihood (ML) phylogenetic tree with 100 bootstrap replicates using SNPhylo (Version: 20140701)[37]. Camellia cuspidata (KM6) served as an outgroup to provide corresponding positional information. The phylogenetic tree was visualized and color-coded using iTOL (http://itol.embl.de).

    Chromosomal SNPs were filtered by removing SNPs in linkage disequilibrium with PLINK (Version v1.90b3.38)[38] , employing a window size of 50 SNPs (advancing 1 SNP at a time) and an r2 threshold of 0.5. Principal component analysis was conducted using Genome-wide Complex Trait Analysis (GCTA, version: 1.25.3) software[39] , and the first three eigenvectors were plotted. Population structure analysis was performed using the ADMIXTURE program (Version: 1.3)[40] with a block-relaxation algorithm. The number of genetic clusters (K) was predefined from 2 to 9, and the cross-validation error (CV) procedure was run to explore convergence of individuals. Default methods and settings were applied in all analyses.

    Relationship inference

    The relationship between each accession was examined using KING (Version: 2.2.5)[41] , utilizing the basic set SNPs with the option ‘--kinship’. This option employed the KING-Robust algorithm to estimate pair-wise kinship coefficients. Close relatives were reliably inferred based on the estimated kinship coefficients using the following simple algorithm: an estimated kinship coefficient range greater than 0.354 indicates a duplicate relationship, while ranges of [0.177, 0.354], [0.0884, 0.177], and [0.0442, 0.0884] correspond to 1st-degree, 2nd-degree, and 3rd-degree relationships, respectively.

    Genetic variation and FST calculations

    The calculation of average pairwise diversity within each population (π) was conducted using 100 kb sliding windows. Population differentiation (FST) was assessed through pairwise FST comparisons among populations.

    Gene flow analysis

    Admixture graphs of geographically defined Hainan tea populations were inferred using TreeMix[42], employing a Maximum Likelihood (ML) approach based on a Gaussian model of allele frequency change. The topology of the ML trees varies depending on the number of migration events (m) permitted in the model, ranging from m = 0 to m = 5. Bootstrap values on the tree were derived from 1,000 replicates. Admixture events among different tea populations were indicated by arrows on the graph, with KM6 serving as the root. To ensure robustness, each migration event was iterated 10 times with a random seed. The optimal number of migration edges was determined using the R package ‘OptM’ (Version: v0.1.6)[43].

    f3 and Patterson’s D statistics

    The f3 statistics were computed using the R package ‘admixr’ (Version: 0.9.1)[44] for all conceivable combinations of tea groups, with KM6 serving as the outgroup. SNPs exhibiting missing data and monomorphism were excluded from the analysis.

    To assess the presence of introgression signals among tea groups, Patterson’s D (also known as the ABBA-BABA test) and f4 admixture ratio statistics for all possible trios of tea groups were calculated using Dtrios in Dsuite (Version: 0.4 r42)[45], with KM6 designated as the outgroup. SNPs with missing data and monomorphism were removed from consideration.

    To investigate the species-level relationships among tea groups, we explored the backbone of the phylogeny using the PoMo model[46] within IQ-Tree[47]. This analysis included 1,000 bootstrap replicates, employing the ultrafast bootstrap approximation method. The tree was rooted using KM6 as the outgroup.

    Author contributions

    The authors confirm contribution to the paper as follows: study conception and design: Duan S; draft manuscript preparation: Guo D, Li D; manuscript revision and editing: Huang Y, Duan S; tea samples collection: Wang Z, Li D, Zhou Y, Xiang G, Zhang W, Wang W, Fang Z, Hao T, Zheng D, Lei Y, Yang L, Zhang W, Tang S, Zheng L, Cao Y. All authors reviewed the results and approved the final version of the manuscript.

    Data availability

    The data that supported the findings of this study have been deposited into the CNGB Sequence Archive (CNSA) of China National GeneBank DataBase (CNGBdb) with accession number CNP0005405. In addition, the sequencing data are also accessible from the tea database (http://teabase.ynau.edu.cn/index/download/index) and the BIG Data Center under the accession number PRJCA001158.

    Acknowledgments

    This work was supported by the Hainan Academy of Agricultural Sciences Research Project (HAAS2022KJCX03), Research and Demonstration on Key Technologies of Germplasm Resource Bank Construction and Resource Innovation Utilization of Wuzhishan Big Leaf Tea (ZDYF2024XDNY245) and Monitoring and Analysis of Key Quality Components of Hainan Big Leaf Black Tea and Development and Demonstration of New Standardized Processing Technology (WZSKTPXM202202). We express our sincere gratitude to the People’s Government of Wuzhishan City, Hainan Province, and the Wuzhishan Scenic Area of Hainan Tropical Rainforest National Park for their generous support and assistance in this project.

  • The authors declare that they have no conflict of interest.

  • [1]

    Wang P, Richardson C, Hawkins TJ, Sparkes I, Hawes C, et al. 2016. Plant VAP27 proteins: domain characterization, intracellular localization and role in plant development. New Phytologist 210:1311−26

    doi: 10.1111/nph.13857

    CrossRef   Google Scholar

    [2]

    Nishimura Y, Hayashi M, Inada H, Tanaka T. 1999. Molecular cloning and characterization of mammalian homologues of vesicle-associated membrane protein-associated (VAMP-associated) proteins. Biochemical and Biophysical Research Communications 254:21−26

    doi: 10.1006/bbrc.1998.9876

    CrossRef   Google Scholar

    [3]

    Skehel PA, Martin KC, Kandel ER, Bartsch D. 1995. A VAMP-binding protein from Aplysia required for neurotransmitter release. Science 269:1580−83

    doi: 10.1126/science.7667638

    CrossRef   Google Scholar

    [4]

    Laurent F, Labesse G, de Wit P. 2000. Molecular cloning and partial characterization of a plant VAP33 homologue with a major sperm protein domain. Biochemical and Biophysical Research Communications 270:286−92

    doi: 10.1006/bbrc.2000.2387

    CrossRef   Google Scholar

    [5]

    Wang P, Hawkins TJ, Richardson C, Cummins I, Deeks MJ, et al. 2014. The plant cytoskeleton, NET3C, and VAP27 mediate the link between the plasma membrane and endoplasmic reticulum. Current Biology 24:1397−405

    doi: 10.1016/j.cub.2014.05.003

    CrossRef   Google Scholar

    [6]

    Zhang L, Zhang H, Liu P, Hao H, Jin J, et al. 2011. Arabidopsis R-SNARE Proteins VAMP721 and VAMP722 are required for cell plate formation. PLoS ONE 6:e26129

    doi: 10.1371/journal.pone.0026129

    CrossRef   Google Scholar

    [7]

    Zhang T, Li Y, Li C, Zang J, Gao E, et al. 2023. Exo84c interacts with VAP27 to regulate exocytotic compartment degradation and stigma senescence. Nature Communications 14:4888

    doi: 10.1038/s41467-023-40729-5

    CrossRef   Google Scholar

    [8]

    Stefano G, Renna L, Wormsbaecher C, Gamble J, Zienkiewicz K, et al. 2018. Plant endocytosis requires the ER membrane-anchored proteins VAP27-1 and VAP27-3. Cell Reports 23:2299−307

    doi: 10.1016/j.celrep.2018.04.091

    CrossRef   Google Scholar

    [9]

    Sparkes IA, Ketelaar T, De Ruijter NCA, Hawes C. 2009. Grab a Golgi: laser trapping of Golgi bodies reveals in vivo interactions with the endoplasmic reticulum. Traffic 10:567−71

    doi: 10.1111/j.1600-0854.2009.00891.x

    CrossRef   Google Scholar

    [10]

    Kamemura K, Chihara T. 2019. Multiple functions of the ER-resident VAP and its extracellular role in neural development and disease. The Journal of Biochemistry 165:391−400

    doi: 10.1093/jb/mvz011

    CrossRef   Google Scholar

    [11]

    Kuster A, Nola S, Dingli F, Vacca B, Gauchy C, et al. 2015. The Q-soluble N-ethylmaleimide-sensitive factor attachment protein receptor (Q-SNARE) SNAP-47 regulates trafficking of selected vesicle-associated membrane proteins (VAMPs). Journal of Biological Chemistry 290:28056−69

    doi: 10.1074/jbc.M115.666362

    CrossRef   Google Scholar

    [12]

    Codjoe JM, Richardson RA, McLoughlin F, Vierstra RD, Haswell ES. 2022. Unbiased proteomic and forward genetic screens reveal that mechanosensitive ion channel MSL10 functions at ER–plasma membrane contact sites in Arabidopsis thaliana. eLife 11:e80501

    doi: 10.7554/eLife.80501

    CrossRef   Google Scholar

    [13]

    Reyes-Impellizzeri S, Moreno AA. 2021. The endoplasmic reticulum role in the plant response to abiotic stress. Frontiers in Plant Science 12:755447

    doi: 10.3389/fpls.2021.755447

    CrossRef   Google Scholar

    [14]

    Ung KL, Schulz L, Kleine-Vehn J, Pedersen BP, Hammes UZ, et al. 2023. Auxin transport at the endoplasmic reticulum: roles and structural similarity of PIN-FORMED and PIN-LIKES. Journal of Experimental Botany 74:6893−903

    doi: 10.1093/jxb/erad192

    CrossRef   Google Scholar

    [15]

    Sparkes I, Hawes C, Frigerio L. 2011. FrontiERs: movers and shapers of the higher plant cortical endoplasmic reticulum. Current Opinion in Plant Biology 14:658−65

    doi: 10.1016/j.pbi.2011.07.006

    CrossRef   Google Scholar

    [16]

    Saravanan RS, Slabaugh E, Singh VR, Lapidus LJ, Haas T, et al. 2009. The targeting of the oxysterol-binding protein ORP3a to the endoplasmic reticulum relies on the plant VAP33 homolog PVA12. The Plant Journal 58:817−30

    doi: 10.1111/j.1365-313X.2009.03815.x

    CrossRef   Google Scholar

    [17]

    Sanderfoot AA, Raikhel NV. 1999. The specificity of vesicle trafficking: coat proteins and SNAREs. The Plant Cell 11:629−41

    doi: 10.1105/tpc.11.4.629

    CrossRef   Google Scholar

    [18]

    El Kasmi F, Krause C, Hiller U, Stierhof YD, Mayer U, et al. 2013. SNARE complexes of different composition jointly mediate membrane fusion in Arabidopsis cytokinesis. Molecular Biology of the Cell 24:1593−601

    doi: 10.1091/mbc.e13-02-0074

    CrossRef   Google Scholar

    [19]

    Ichikawa M, Hirano T, Enami K, Fuselier T, Kato N, et al. 2014. Syntaxin of plant proteins SYP123 and SYP132 mediate root hair tip growth in Arabidopsis thaliana. Plant and Cell Physiology 55:790−800

    doi: 10.1093/pcp/pcu048

    CrossRef   Google Scholar

    [20]

    Kim S, Choi Y, Kwon C, Yun HS. 2019. Endoplasmic reticulum stress-induced accumulation of VAMP721/722 requires CALRETICULIN 1 and CALRETICULIN 2 in Arabidopsis. Journal of Integrative Plant Biology 61:974−80

    doi: 10.1111/jipb.12728

    CrossRef   Google Scholar

    [21]

    Kwon C, Neu C, Pajonk S, Yun HS, Lipka U, et al. 2008. Co-option of a default secretory pathway for plant immune responses. Nature 451:835−40

    doi: 10.1038/nature06545

    CrossRef   Google Scholar

    [22]

    Yun HS, Kwaaitaal M, Kato N, Yi C, Park S, et al. 2013. Requirement of vesicle-associated membrane protein 721 and 722 for sustained growth during immune responses in Arabidopsis. Molecules and Cells 35:481−88

    doi: 10.1007/s10059-013-2130-2

    CrossRef   Google Scholar

    [23]

    Tao K, Waletich JR, Arredondo F, Tyler BM. 2019. Manipulating endoplasmic reticulum-plasma membrane tethering in plants through fluorescent protein complementation. Frontiers in Plant Science 10:635

    doi: 10.3389/fpls.2019.00635

    CrossRef   Google Scholar

    [24]

    Siao W, Wang P, Voigt B, Hussey PJ, Baluska F. 2016. Arabidopsis SYT1 maintains stability of cortical endoplasmic reticulum networks and VAP27-1-enriched endoplasmic reticulum–plasma membrane contact sites. Journal of Experimental Botany 67:6161−71

    doi: 10.1093/jxb/erw381

    CrossRef   Google Scholar

    [25]

    Huang G, Liu Z, Gu B, Zhao H, Jia J, et al. 2019. An RXLR effector secreted by Phytophthora parasitica is a virulence factor and triggers cell death in various plants. Molecular Plant Pathology 20:356−71

    doi: 10.1111/mpp.12760

    CrossRef   Google Scholar

    [26]

    Lan X, Liu Y, Song S, Yin L, Xiang J, et al. 2019. Plasmopara viticola effector PvRXLR131 suppresses plant immunity by targeting plant receptor-like kinase inhibitor BKI1. Molecular Plant Pathology 20:765−83

    doi: 10.1111/mpp.12790

    CrossRef   Google Scholar

    [27]

    Gessler C, Pertot I, Perazzolli M. 2011. Plasmopara viticola: a review of knowledge on downy mildew of grapevine and effective disease management. Phytopathologia Mediterranea 50:3−44

    Google Scholar

    [28]

    Fasoli M, Dal Santo S, Zenoni S, Tornielli GB, Farina L, et al. 2012. The grapevine expression atlas reveals a deep transcriptome shift driving the entire plant into a maturation program. The Plant Cell 24:3489−505

    doi: 10.1105/tpc.112.100230

    CrossRef   Google Scholar

    [29]

    Li M, Jiao Y, Wang Y, Zhang N, Wang B, et al. 2020. CRISPR/Cas9-mediated VvPR4b editing decreases downy mildew resistance in grapevine (Vitis vinifera L.). Horticulture Research 7:149

    doi: 10.1038/s41438-020-00371-4

    CrossRef   Google Scholar

    [30]

    Liu G, Wang B, Lecourieux D, Li M, Liu M, et al. 2021. Proteomic analysis of early-stage incompatible and compatible interactions between grapevine and P. viticola. Horticulture Research 8:100

    doi: 10.1038/s41438-021-00533-y

    CrossRef   Google Scholar

    [31]

    Liu R, Chen T, Yin X, Xiang G, Peng J, et al. 2021. A Plasmopara viticola RXLR effector targets a chloroplast protein PsbP to inhibit ROS production in grapevine. The Plant Journal 106:1557−70

    doi: 10.1111/tpj.15252

    CrossRef   Google Scholar

    [32]

    Toledo-Ortiz G, Huq E, Quail PH. 2003. The Arabidopsis basic/helix-loop-helix transcription factor family. The Plant Cell 15:1749−70

    doi: 10.1105/tpc.013839

    CrossRef   Google Scholar

    [33]

    Heim MA, Jakoby M, Werber M, Martin C, Weisshaar B, et al. 2003. The basic helix-loop-helix transcription factor family in plants: a genome-wide study of protein structure and functional diversity. Molecular Biology and Evolution 20:735−47

    doi: 10.1093/molbev/msg088

    CrossRef   Google Scholar

    [34]

    Zhang L, Ma J, Liu H, Yi Q, Wang Y, et al. 2021. SNARE proteins VAMP721 and VAMP722 mediate the post-Golgi trafficking required for auxin-mediated development in Arabidopsis. The Plant Journal 108:426−40

    doi: 10.1111/tpj.15450

    CrossRef   Google Scholar

    [35]

    Baena G, Xia L, Waghmare S, Karnik R. 2022. SNARE SYP132 mediates divergent traffic of plasma membrane H+-ATPase AHA1 and antimicrobial PR1 during bacterial pathogenesis. Plant Physiology 189:1639−61

    doi: 10.1093/plphys/kiac149

    CrossRef   Google Scholar

    [36]

    Yi C, Park S, Yun HS, Kwon C. 2013. Vesicle-associated membrane proteins 721 and 722 are required for unimpeded growth of Arabidopsis under ABA application. Journal of Plant Physiology 170:529−33

    doi: 10.1016/j.jplph.2012.11.001

    CrossRef   Google Scholar

    [37]

    Liu Y, Lan X, Song S, Yin L, Dry IB, et al. 2018. In planta functional analysis and subcellular localization of the oomycete pathogen Plasmopara viticola candidate RXLR effector repertoire. Frontiers in Plant Science 9:286

    doi: 10.3389/fpls.2018.00286

    CrossRef   Google Scholar

  • Cite this article

    Li R, Wang B, Zha M, Zhang K, Li M, et al. 2024. Identification and characterization of grape VAP27 gene family and their roles in disease resistance. Fruit Research 4: e019 doi: 10.48130/frures-0024-0019
    Li R, Wang B, Zha M, Zhang K, Li M, et al. 2024. Identification and characterization of grape VAP27 gene family and their roles in disease resistance. Fruit Research 4: e019 doi: 10.48130/frures-0024-0019
    shu

Figures(6)  /  Tables(1)

Download PDF

Article Metrics

Article views(2952) PDF downloads(379)

Other Articles By Authors

ARTICLE   Open Access    

Identification and characterization of grape VAP27 gene family and their roles in disease resistance

  • 1.

    State Key Laboratory of Crop Stress Resistance and High-Efficiency Production, College of Horticulture, Northwest A&F University, Yangling 712100, Shaanxi, China

  • 2.

    Key Laboratory of Horticultural Plant Biology and Germplasm Innovation in Northwest China, Ministry of Agriculture, Northwest A&F University, Yangling 712100, Shaanxi, China

  • Received: 24 January 2024
  • Revised: 03 April 2024
  • Accepted: 24 April 2024
  • Published online: 17 May 2024
Fruit Research  4,  Article number: e019  (2024)  |  Cite this article

Abstract: Vesicle-associated membrane protein (VAMP)-associated proteins (VAP27s), which are widely expressed in plants and animals, play an important role in metabolism, physiology, growth, and development, disease resistance, and immunity. While the function of this family has been elucidated in model plants like Arabidopsis thaliana and tomato, its role in grapevine remains unclear. In this present study, 12 vesicle-associated protein-membrane protein genes were identified in the grapevine genome by bioinformatics, designated as the VAP27 gene family. A phylogenetic tree, encompassing 53 genes from three model plants, Arabidopsis thaliana, Oryza sativa, and Solanum lycopersicum, revealed the subdivision of the VAP27 gene family into three subfamilies, each presumably serving different functions, besides localizing in endoplasmic reticulum, individual members also localize in nucleus. Additionally, we compared the transcriptional levels and subcellular localizations of the VvVAP27 family members across different plant tissues (flower, leaf, seed, root, fruit, tendril, and stem), indicating site-specific functionalities for different gene members. To investigate the responsiveness of the VAP27 gene family to pathogen infection, particularly Plasmopara viticola on host plants, we analyzed the expression patterns of VAP27 genes post-infection. Our findings revealed divergent expression profiles among different members at different stages of infection. The gene family responded to the infection of downy mildew on grapevine and could inhibit the spread of Phytophthora capsici lesions in Nicotiana benthamiana. These results provide an important basis for further studies delving into the functions of the VAP27 gene family in plant growth and disease resistance.

    HTML

    Introduction

    • The vesicle-associated membrane protein (VAMP)-associated protein (VAPs) family has been identified as a highly conserved group of proteins identified both in plants[1] and animals[2]. It was first identified in animals participating in the transportation of related substances by forming SNARE protein complexes[3]. Their plant homologues are named VAP27 because the first member identified had a molecular weight of 27 kDa[4]. The structure of VAPs usually contains an N-terminal major sperm domain (aa 1–129), a coiled-coil domain (aa 178–234), and a C-terminal transmembrane domain (aa 234–253)[4]. It is reported that the N-terminal sperm domain is crucial for the interaction between VAP27-1 and NET3C, and the fixation of VAP27-1 in the ER-PM contact site[5]. In Arabidopsis, 10 VAP homologs have been identified[1]. To date, numerous research on VAPs have been reported in the plant kingdom, and increasing proteins relevant to VAPs have also been discovered[5−8]. For example, NET3C and VAP27 form homo-dimers or homo-oligomers that function in mediating the communication of PM and ER via interacting with actin and microtubules for lipid transport, calcium influx, and other vital biological processes[5]. VAP27-1 and VAP27-3 have been revealed to interact with clathrin and play a central role in maintaining clathrin homeostatic dynamics at endocytic membranes and regulating endocytosis[8].

      According to existing studies, VAP proteins family localize to the endoplasmic reticulum (ER) and ER/plasma membrane (PM) contact sites and are tightly linked to the cytoskeleton that plays a supporting role[9−12]. The endoplasmic reticulum (ER), as one of the important components of the intimal system, plays an indispensable role in protein synthesis, folding and quality control, protein secretion, lipid biosynthesis, and calcium storage[13,14]. The ER is also actively involved in endocytosis with mechanisms that have not been clearly defined[15]. The transportation and translocation of various proteins, steroids, lipids, and other synthesized molecules typically depend on ER-centered traditional vesicular trafficking pathways[16,17]. The close association between the ER and the PM, facilitated by proteins like VAPs, is essential for vesicle trafficking from the ER. Given the intimate connection between the VAP family and the plasma membrane, researchers propose VAP interactions with proteins involved in plant development and maintaining structural stability. This explains the requirement of VAPs for growth, cell division, and abiotic stress responses[18−22]. Recent research in Arabidopsis thaliana also supports this idea, revealing that SYT1, an ER-resident protein[23], plays a vital role in stabilizing the ER network and connection between VAP27-1-enriched ER and plasma membrane[24]. VAMP721/722 are components of the default secretory pathway and can transport substances required for cell growth, suggesting a potential role in plant autoimmune regulation.

      There have been studies demonstrating that the plasma membrane participates in the secretion of immune protein for the activation of plant immune defense against pathogen invasion. For instance, the antimicrobial proteins secreted through vesicle trafficking was targeted and destroyed by the RxLR effector of Phytophthora brassicae by working together with host RABA-type GTPase, subsequently compromising the immune system[25]. There are also reports indicating that the VAP protein family could influence the development of various plants and defense networks. The immune mechanism of the VAP protein family in Arabidopsis thaliana and tomato has been confirmed[20,22].

      Grapevine (Vitis vinifera L.) is distinguished as one economically valuable fruit, appreciated both for fresh consumption and the production of various processed items such as wine and grape juice. Environmental stresses can seriously affect grapevine growth and development in cropland. For instance, high humidity on prolonged rainy days during critical maturation stages can compromise the quality of grapevine, while drought conditions can drastically reduce fruit yields. Additionally, biotic stresses, such as downy mildew, powdery mildew, anthracnose, and others[26,27], pose threats by impeding normal leaf growth and causing yield losses. Given its substantial economic importance, grapevine cultivation is widespread across various countries. The identification of significant functional genes becomes of utmost interest.

      At present, research on the VAP27 protein is limited, particularly in the context of grapevine. Therefore, in this study, we identified and analyzed the VAP27 protein family through bioinformatic analysis of genomic and transcriptomic data. The structure and function of the VAP27 gene were preliminarily analyzed, laying the foundation for further study of gene functionality.

    Materials and methods

      Identification and annotation of VAP27s in grape

    • The grape genome sequences of Vitis vinifera cv. 'Pinot Noir' (PN40024.v4) were downloaded from Ensemble Plants (https://plants.ensembl.org/Vitis_vinifera/Info/Index). Initial identification involved querying the grape genome database using the Arabidopsis VAPs protein sequences through BLAST. Next, an HMM file was constructed using the seed alignment file for the VAP domain (PF00635) obtained from the Pfam database, utilizing the HMMER3 software package. HMM searches were then performed against local protein databases of grape sequencing using HMMER3. To ensure accuracy, the physical localizations of all candidate Vitis vinifera VAP27s (VvVAP27s) on chromosomes were examined, and redundant sequences with identical chromosome locations were excluded. All obtained VAP27 protein sequences were subjected to Pfam analysis (http://pfam.xfam.org/) to verify the DBD domain. The presence of DBD domains and coiled-coil structures was confirmed using SMART (http://toolkit.tuebingen.mpg.de/marcoil) and MARCOIL (http://toolkit.tuebingen.mpg.de/marcoil). Sequences lacking the DBD domain or a coiled-coil structure were eliminated from further analysis.

      Phylogenetic, exon-intron structure, and motif analysis of VAP27 Genes

    • Genome sequences, CDS sequences, and protein sequences of the VAP27 family were downloaded for analysis. An unrooted phylogenetic tree was constructed for sequences from grapes, Arabidopsis thaliana, tomatoes, and rice using the Neighbor-Joining (NJ) method with the bootstrap test replicated 1,000 times. The software used for creating these phylogenetic trees was MEGA5. The exons and introns of grape VAP27 genes were determined based on alignments of transcribed sequences and corresponding genomic sequences, and the visualization of VAP27 gene structures was performed with the online Gene Structure Display Server 2.0. Conserved motifs and domains of grape VAP27 genes were identified using MEME 4.11.2 (http://meme-suite.org/tools/meme) and SMART (http://toolkit.tuebingen.mpg.de/marcoil) software.

      Transcriptome analysis of VvVAP27 gene

    • Using published data[28], the expression patterns of VvVAP27 gene family at 54 stages of grape plant development were analyzed using the average expression values of three biological replicates. Clustering analysis plots from RNA-seq datasets were created using FPKM (fragments mapped per kilobase read per million times) values. The expression heatmap of the grape VvVAP27 gene family was drawn using TBtools.

      Plant materials and treatments

    • The grapevine materials used in this study are Vitis vinifera 'Pinot Noir' and Vitis piasezkii 'Liuba-8', cultured in the Grape Repository of Northwest A&F University, Yangling, Shaanxi, China. The P. viticola population was collected from the susceptible Vitis plants as per previous studies[29−31]. Briefly, infected leaves were collected and washed in sterile distilled water three times. The leaves were positioned with the abaxial side facing up on sterile moist filter papers in trays and incubated overnight at room temperature to allow P. viticola sporulation. Leaves on which fresh sporangia developed were transferred into a large Petri dish and washed gently with sterile distilled water. The sporangium suspension was filtered with three-layer sterilized gauze. The concentration was adjusted to 5 × 104 sporangia/mL using a hemocytometer under a light microscope.

      For inoculation, the third to fifth fully expanded leaves from the top were detached and washed three times in sterile distilled water, and inoculated with 10 μL drops of the sporangia suspension on the abaxial leaf surface. The inoculated leaves were placed on sterile Petri dishes (90 mm in diameter) containing three-layer sterile moist filter papers and incubated in an incubator at 23 ± 1 °C, 90% relative humidity, and a photoperiod of 16 h light and 8 h dark. Samples were collected at 0, 6, 12, 24, 48, 96, and 120 h post-inoculation (hpi), with 0 hpi as the control samples. The collected samples were promptly frozen in liquid nitrogen and stored at −80 °C. Each biological replicate was a pool of three independent leaves. The tobacco plant material, N. benthamiana, was routinely grown at 25/20 °C in a greenhouse under white light (18 h light/6 h dark).

      RNA extraction and RT–qPCR

    • Total RNA was extracted from grapevine leaves using the RNA Mini Kit (Omega, USA) following the manufacturer's instructions. The EasyScript® One-Step gDNA Removal and cDNA Synthesis SuperMix kit (TransGen Biotech, China) was used to perform reverse transcription and synthesize double-stranded cDNAs. In the reverse transcription, 500 ng RNA was used with the Anchored Oligo (dT) 18 as the primer. The remaining reaction components included 10 μL of 2× ES Reaction Mix, 1.0 μL of gDNA Remover, 1.0 μL of EasyScript® RT Enzyme Mix, and sterile distilled H2O were added to reach a final volume of 20 μL. The reaction was carried out at 42 °C for 15 min, 85 °C for 10 s. Quantitative PCR was performed on an Applied Biosystems QuantStudio 6 (Thermo Fisher Scientific, USA) with PerfectStart® Green qPCR SuperMix (TransGen Biotech, China), according to the recommended protocol. In brief, each reaction mixture contained 10 μL of 2× TransStart Top Green qPCR SuperMix, 2.0 μL of cDNA template, 0.5 μL of each primer, and 8.0 μL of sterile distilled H2O. Cycling parameters included an initial step at 50 °C for 2 min and 94 °C for 30 s, followed by 45 cycles at 95 °C for 5 s, 59 °C for 15 s, and 72 °C for 30 s. Melt-curve analyses were performed with a program starting at 95 °C for 15 s and then a constant increase from 60 to 95 °C. Data were analyzed by the 2−ΔΔCT method for calculating gene relative expression levels with three biological replicates. Gene-specific primers were designed using Primer Premier 5.0 software and gene transcripts were normalized to VvActin as internal standards.

      Transient transformation of Agrobacterium tumefaciens

    • The pCambia2300-VAP27s-GFP construct was introduced into the A. tumefaciens GV3101 strain, and recombinant colonies were verified through growth on a selective medium and PCR analysis. For Agrobacterium-mediated transient transformation assays, bacterial cells were collected by centrifugation and then resuspended in infiltration buffer (10 mM MgCl2, 10 mM MES, 200 μM acetosyringone). The bacterial suspension was diluted to a concentration of OD600 = 0.6 and incubated for 3 h at 28 °C before infiltration. Infiltration on tobacco leaves was carried out using a syringe without a needle.

      Location analysis

    • Laser confocal microscopy was employed to determine the localization of the VAP27 gene family in plants. The VAP27 gene was inserted into the pCambia2300-GFP vector and the recombinant plasmid was confirmed by Sanger sequencing. Then the recombinant plasmid was transformed into the Agrobacterium strain GV3101. The monoclonal plaque was amplified in liquid culture and confirmed by PCR analysis. For visualization, the ER-RK marker was co-injected with VAP27-GFP into the N. benthamiana leaves. The transformed N. benthamiana leaves were observed using confocal microscopy (TCS SP8 of Leica). The excitation wavelength for green fluorescent protein was set to 488 nm.

      Phytophthora capsici inoculation

    • The mycelium of P. capsici was first cultivated on 10% V8 juice agar medium and then was transferred into 10% liquid V8 medium and cultured in 25 °C darkness for 5 d. The developed hyphae were collected and resuspended in sterile water at 4 °C for 30 min, followed by incubation at room temperature for 30 min to allow the release of zoospores from sporangia. The resulting sporangial suspension was adjusted to 1.0 × 104 sporangia/mL.

      For the P. capsica infection experiment, the A. tumefaciens containing certain plasmids were injected into N. benthamiana. At 48 h post-infection, the inoculated leaves were detached, and their petioles were first wrapped in sterile cotton and then wrapped in two layers of sterile wet filter paper. Then the leaves were first treated using 0.1% Tween-20, followed by inoculation with 30 μL zoospore suspension of P. capsica. The infected leaf samples were kept in the dark at 25 °C to allow the P. capsica development. The lesion area was statistically analyzed.

    Results

      Identification and genomic distribution of the VAP27 family in the grape genome

    • A total of 12 VAP27 genes in the grape genome were identified, designated as Vitis vinifera VAP27 (VvVAP27)1−12 according to their chromosomal positions (Table 1). The genomic distribution revealed an uneven mapping of VAP27s on eight out of the 19 grape chromosomes. Specifically, VvVAP27-1, VvVAP27-2, VvVAP27-3, VvVAP27-4, VvVAP27-7, and VvVAP27-10 were located on Chromosome 2, Chromosome 5, Chromosome 8, Chromosome 12, Chromosome 14, and Chromosome 19, respectively. VvVAP27-5 and VvVAP27-6 were situated on Chromosome 13, while VvVAP27-8 and VvVAP27-9 were positioned on Chromosome 15. VvVAP27-11 and VvVAP27-12 were putatively located on the 'Chromosome Unknown'. Further study will delve into unraveling the biological functions of these 12 VAP27 genes.

      Table 1.  Chromosome distribution of identified 12 grapevine VAP27 genes. Detailed information, including gene locus, gene symbol, length, chromosome, and site is available in the Ensembl Plants Database.

      Protein nameGene IDChrLength (aa)Annotation
      VvVAP27-1Vitvi02g005452238PREDICTED: vesicle-associated protein 1-1
      VvVAP27-2Vitvi05g003605293PREDICTED: vesicle-associated protein 1-2
      VvVAP27-3Vitvi08g001378532PREDICTED: ankyrin-1
      VvVAP27-4Vitvi12g0063812259PREDICTED: vesicle-associated protein 4-1
      VvVAP27-5Vitvi13g0009913470PREDICTED: ankyrin repeat, PH and SEC7 domain containing protein secG
      VvVAP27-6Vitvi13g0085013136PREDICTED: hypothetical protein VITISV_015240
      VvVAP27-7Vitvi14g0034714336PREDICTED: vesicle-associated protein 2-2
      VvVAP27-8Vitvi15g0067715239PREDICTED: vesicle-associated protein 1-2
      VvVAP27-9Vitvi15g0071315239PREDICTED: vesicle-associated protein 1-3
      VvVAP27-10Vitvi19g0030419264PREDICTED: vesicle-associated protein 4-1
      VvVAP27-11Vitvi10g04245Un264PREDICTED: vesicle-associated protein 4-2
      VvVAP27-12Vitvi00g04146Un348PREDICTED: LOW QUALITY PROTEIN: vesicle-associated protein

      Phylogenetic analysis of the VAP27 gene family

    • To elucidate the evolutionary relationships within the VAP27 gene family, we conducted a comprehensive analysis involving 53 VAPs, including 10 from Arabidopsis, 17 from rice, 15 from tomato, and 12 from grape, and the result was visualized by constructing a phylogenetic tree (Fig. 1). The 53 VAP27 members across these four species fell into three distinct groups (Fig. 1 Clade I−III). Clade I, consisted of VvVAP27-1, VvVAP27-6, VvVAP27-8 and VvVAP27-9 gene. Clade II contained 3 VvVAP27 members: VvVAP27-2, VvVAP27-7 and VvVAP27-12. Clade III emerged as the most populated, encompassing five VvVAP27 members: VvVAP27-3, VvVAP27-4, VvVAP27-5, VvVAP27-10, and VvVAP27-11.

      Figure 1. 

      Unrooted phylogenetic tree of VAP27s in grape, Arabidopsis, rice, and tomato. Vv: Vitis vinifera L. grape species; AT: Arabidopsis thaliana; OS: Oryza sativa; Soly: Solanum lycopersicum.

      The clustering patterns suggest a closer evolutionary proximity of the VAP family in grapes to that of dicotyledon tomatoes compared to rice. This supports the reliability of the analysis results.

      Structural analysis of grape VAP27 genes

    • The gene structures of the 12 grapevine VAP27s were explored through a comprehensive examination of exon/intron boundaries. The varying length and splicing patterns observed among the 12 VAP27s are depicted in Fig. 2a. The structural analysis showed a range of intron numbers from 1 to 7. Notably, VvVAP27-3, VvVAP27-6, and VvVAP27-7 were absent of introns, while VvVAP27-5 exhibited a singular intron. The remaining VAP27 member's genes had between six and seven introns (Fig. 2a). The results revealed significant diversity within the VAP27 family.

      Figure 2. 

      (a) Intron-exon structure and (b) conserved motifs of VvVAP27.

      According to previous studies, motifs recognized as playing an important role in interaction and signal transduction within the transcriptional complex[32,33] were analyzed using MEME for the 12 conservative VAP27 genes. This was conducted also because these motifs are closely related to gene classification. Among these VAP27s, a total of 10 motifs were identified (Fig. 2b), with Motif 1 present in all 11 members except VAP27-12, which indicates its high conservation within the VAP27 gene family. Motif 8 and Motif 9 were the least conserved, found only in VvVAP27-4, VvVAP27-10, and VvVAP27-11 (Motif 8), and VvVAP27-1, VvVAP27-8, and VvVAP27-9 (Motif 9). The high sequence similarity among genes within the same branch suggests shared functions and roles in plants. The analysis of motif and gene structure analysis enrich our understanding of the VAP27 family's classification, providing a robust theoretical foundation.

      Expression profiles of VAP27s in different tissues

    • VAP27 RNAi induces various defects in plant morphology, pollen, seed, and root development in Arabidopsis[1], we anticipated a similar involvement of the VvVAP27 gene in grape growth and development. To investigate this, we examined the expression profiles of the 12 VAP27 genes across different tissues of grapevine (Fig. 3). These tissues represented distinct growth and development stages of grapevine, including root, young stem, leaf, inflorescence, skin, veraison berry, and tendril. Examination of transcriptome data from the VvVAP27 family revealed significant variations across different tissues. The majority of family members (VvVAP27-1 to VvVAP27-10) exhibited comparable expression levels in tissues including flowers, berries, leaves, stems, seeds, and shoots, suggesting their involvement throughout various stages of plant growth and development. Only a subset of genes (VvVAP27-11 and VvVAP27-12) showed significant differences in expression among tissues. Expression of the VvVAP27-11 gene was higher in the berries than in the other tissues that maintained relatively consistent levels. In contrast, VvVAP27-12 showed transcriptional peaks exclusively in seeds and flowers, suggesting a potential association of VvVAP27-12 with flowering and fruit development.

      Figure 3. 

      Expression profiles of the grape VvVAP27s gene. Hierarchical clustering of expression profiles of grape VAP27 genes across different tissues.

      Expression patterns of VAP27 genes post-plasmopara viticola inoculation

    • It has been documented that the VAP27 gene family is involved in regulating plant disease resistance against external pathogen infection[6,18−22]. Therefore, we explored whether the VAP27 family exhibits similar functionality in grapevine downy mildew resistance. Our investigation focused on the expression levels of 12 VAP27 gene members at eight time points post-downy mildew inoculation (0, 6, 12, 24, 48, 72, 96, and 120 hpi). Utilizing RT-qPCR, we assessed whether the VAP27 gene responded to the induction of Grape downy mildew (Fig. 4). Vvactin1 was used as a grapevine internal reference gene for normalization[25].

      Figure 4. 

      Heat map showing the expression profiles of VvVAP27 genes at different time points post-downy mildew infection. The color scale represents expression levels, with red indicating high expression level and blue indicating low expression level. The expression was normalized and the data are displayed as log2 values.

      We discovered that genes in the VvVAP27 family genes exhibited specificity in responses to downy mildew induction (Fig. 4). There were a few members significantly overexpressed at the early stage of downy mildew infection in 'Liuba-8'. Only VvVAP27-6 had the highest expression level at 48 hpi compared to other time points. VvVAP27-2 was highly expressed at 120 hpi, with no difference found at other time points. VvVAP27-3, VvVAP27-6, VvVAP27-11, and VvVAP27-12 genes were highly expressed throughout the downy mildew infection period in 'Pinot Noir'. This indicated that these four genes were positively responsive to the induction of downy mildew, suggesting an important role in the grapevine's defense against downy mildew invasion. The expression levels of the other eight genes remained unchanged across different infection periods. We postulate that the varying expression patterns among different members may be related to the regulation of VAP27-mediated plant disease resistance, possibly involving distinct mechanisms of immunity. However, further experimental verification is needed to substantiate these hypotheses.

      Subcellular localization of VvVAP27 genes

    • To better explore the function of the VvVAP27 gene family, subcellular localization analysis was conducted on some selected VvVAP27 genes. By detecting GFP-tagged proteins, we found that most of the genes were localized to the endoplasmic reticulum (ER). This localization aligns with previous literature reports indicating that membrane proteins of vesicle-associated proteins function by participating in the formation and regulation of plant cell membranes. The endoplasmic reticulum participates in the formation of cell membranes and is closely related to secretory vesicles that function in transporting secretory proteins to various parts of the plant to contribute to plant growth, development, and disease resistance[34,35]. VvVAP27-2, VvVAP27-4, VvVAP27-6, and VvVAP27-9 are all mapped to the endoplasmic reticulum. However, VvVAP27-2, VvVAP27-4, and VvVAP27-6 are also detected in the nucleus in addition to the ER (Fig. 5). Based on the analysis of the expression pattern induced by downy mildew and the Phytophthora capsici infection experiment, we speculate that the subcellular localization may affect the gene expression.

      Figure 5. 

      Subcellular localization analysis of VvVAP27 members.

      Response of VAP27 gene to P. capsici inoculation

    • To further study the role of VAP27 genes in disease resistance, the VvVAP27 genes that were induced by grape downy mildew in tobacco leaves were screened. These tobacco leaves were transiently transformed by A. tumefaciens that carry a high-level expression vector with an individual VvVAP27 gene insert before being inoculated with P.capsici spore suspension. The findings revealed that VAP27 gene members inhibited the occurrence of the pathogenicity and significantly enhanced the resistance of tobacco leaves to the pathogen. However, the efficacy of pathogen inhibition varied among different VAP27 members. This result is consistent with previous studies on gene responses to downy mildew infection.

      Specifically, both VvVAP27-6 and VvVAP27-9 exhibited a consistent phenotype, inhibiting infection by pathogens (Fig. 6a). Leaf lesion areas were smaller in VvVAP27-6 and VvVAP27-9 expressing leaves compared with controls, suggesting that they effectively promoted plant immunity (Fig. 6b) and that VvVAP27-6 had a higher inhibitory capacity than VvVAP27-9. VvVAP27-2 and VvVAP27-4, did not differ significantly in the size of the lesion area compared with empty-carrier controls. The heterogeneous functions within this family underscore the need for further experimental studies to elucidate the roles of the remaining genes.

      Figure 6. 

      Responses of VvVAP27 induced by Phytophthora capsici infection. The inoculation of Phytophthora capsici was conducted on Nicotiana benthamiana leaves transiently transformed with VvVAP27. (a) Observation of the phenotypes of VvVAP27 family members and the control after inoculation with Phytophthora capsici, visualized by trypan blue staining. (b) Statistical analysis of the lesion areas caused by Phytophthora capsici infection on Nicotiana benthamiana leaves transiently transformed by VvVAP27. (c) The expression of VvVAP27 family members and GFP protein was detected by Western blot. The experiment was repeated three times and asterisks represent the level of significant differences (* p < 0.05, ** p < 0.01).

    Discussion

    • Vesicle-associated membrane proteins (VAMP-associated proteins) (VAPs) are a family of proteins widely expressed in plants, which play a key role in plant defense against both biotic and abiotic stresses. In this study, a family of 12 VAP27 genes were identified in grapes using bioinformatic methods. Consistent with previous studies, the VAMP gene family has demonstrated multifaceted involvement in diverse defense processes across different plant tissues. VAP27-1 and VAP27-3, as non-plant VAP homologs[5], have been localized extensively to the ER and EPCS[1,5,16]. These proteins were identified to promote plant endocytosis and play a role in endocytosis. In Arabidopsis thaliana, VAMP721/722 have been identified as essential factors for growth, cell division, and responses to abiotic stress[18−22]. The PEN1-SNAP33-VAMP721/722 pathway in Arabidopsis thaliana facilitates the transport of vital materials for cell viability to the endoplasmic reticulum[36,37]; VAMP721 and VAMP722 are involved in the secretory transport of substances to endosomal compartments of the plasma membrane to promote the formation of cell plates during plant cytokinesis[6]. The phylogenetic tree analysis revealed a notable similarity in the quantity and gene structure of the VAP27 gene family between grape, Arabidopsis thaliana, and tomato. Hence, it can be inferred that the VAP27 gene family in grape shares similar functions with those in Arabidopsis thaliana[36], and plays comparable roles in growth, development, and immune resistance mechanisms. Additionally, we analyzed the gene structure of 12 identified VvVAP27 genes using MEME. Results (Fig. 2) showed that most of the VvVAP27 genes (VvVAP27-1, VvVAP27-2, VvVAP27-3, VvVAP27-6, VvVAP27-7, VvVAP27-8, VvVAP27-9) contain conserved domains, especially VvVAP27-3 and VvVAP27-5; VvVAP27-1, VvVAP27-8 and VvVAP27-9; VvVAP27-4, VvVAP27-10 and VvVAP27-11 placed within the same group in the phylogenetic tree classification (Fig. 1). This observation may explain the specific biological functions associated with each subfamily. The analysis of motif and gene structure provides a further theoretical basis for the classification of the VvVAP27 subfamily, guiding subsequent in-depth functional studies within these identified groups.

      To understand the regulatory mechanisms of the VAP27 gene family on the growth and development of grapevine, we analyzed the regulatory effect of the VvVAP27 gene family on the growth and development of grapevine using transcriptome content assay (Fig. 3) across different grape tissues (flowers, seeds, leaves, buds, berries, tendrils, stems, roots). Our transcriptome data analysis showed the highest expression of VvVAP27-11 in berries than in other tissues, implying its potential role in regulating fruit development and quality and promoting fruit setting. Similarly, the expression levels of VvVAP27-12 were higher in flower and seed than in other tissues, indicating that VvVAP27-12 plays an important role in flower induction, seed setting, and growth regulation. This study provides a theoretical basis for further understanding the function of the VAP27 family members in the process of grape growth and development.

      It has been found that the VAP gene family can induce plant cell autonomous immunity, acting on the cell surface or post-pathogen entry, thereby impeding pathogenesis. This phenomenon has been well-documented in various plant species, including tomato, Arabidopsis, and tobacco. In tobacco, VAPB proteins interact with proteins in the intestine of tobacco whitefly (Bemisia tabaci) during the transmission of tomato yellow leaf curl virus (TYLCV), and silencing VAPB results in an increase in virus number and transmission rate, demonstrating that VAPB can play a key role in resistance to TYLCV[4]. Moreover, in Arabidopsis, the SNARE proteins VAMP 721/722 direct secretory vesicles to pathogen-attack sites during immune responses, indicating that these vesicles deliver immune molecules and function in immune responses[36]. Additionally, SYP132, an essential protein for defense against bacterial pathogens, specifically interacts with VAMP721/722 in response to the immune control of P. syringae[21]. Given these findings, we were intrigued by the possibility that the VAP27 gene family might play a similar role in disease resistance in grapes. Grapevine downy mildew, caused by the oomycete P. viticola, is one of the most serious diseases in grape production. P. viticola was originally endemic to North America, but it has now spread to all major grape-producing regions worldwide[37]. This study revealed that the majority of VvVAP27 members exhibited responses to downy mildew infection. Notably, VvVAP27-1, VvVAP27-2, VvVAP27-4, and VvVAP27-6 were significantly up-regulated at 0, 6, and 12 h after infection, and VvVAP27-12 was highly expressed at 24, 48, and 72 h after infection with P. viticola, while other genes were highly expressed only at specific stages. These findings suggest that members of the VAP27 gene family are likely to respond to downy mildew infection. Our results provide a new idea to study the effect of the VAP27 gene on grapevine downy mildew, but further research is needed to study the mechanisms of VAP27 gene action.

      In this study, we identified a new grape gene family, named the VAP27 gene family. Through bioinformatic analysis and transcriptome sequencing, we uncovered striking structural and functional similarities between the VAP27 gene family in grape and those in Arabidopsis thaliana and tomato. The current findings suggest a potentially significant role for this gene family in the growth and development of the grape, as well as in orchestrating immune responses against downy mildew.

    Conclusions

    • The vesicle-associated protein-membrane protein gene family (VAP27) in the grape genome was identified, which consists of 12 gene members. Within this family, some members exhibit localization on the endoplasmic reticulum, and a minority reside within the nucleus. The present results demonstrate the induction of the gene family in response to downy mildew in grape and their ability to inhibit the infection of P. capsici, thus playing an important role in plant disease resistance.

    Author contributions

    • The authors confirm contribution to the paper as follows: conceptualization, writing – review & editing: Xu Y, Liu G; validation, visualization, writing – original draft: Li R, Wang B; investigation: Li R, Wang B, Zha M, Zhang K, Li M, Xie L, Chen X, Liu G; funding acquisition: Liu G. All authors reviewed the results and approved the final version of the manuscript.

    Data availability

    • All data generated or analyzed during this study are included in this published article.

      Acknowledgments

      • This work was supported by grants from the National Natural Science Foundation of China (31972374, 32372660, and 31601716).

      Conflict of interest

      • The authors declare that they have no conflict of interest. Yan Xu is the Editorial Board member of Vegetable Research who was blinded from reviewing or making decisions on the manuscript. The article was subject to the journal's standard procedures, with peer-review handled independently of this Editorial Board member and the research groups.

      • # Authors contributed equally: Ruonan Li, Bianbian Wang

      Rights and permissions
      • Copyright: © 2024 by the author(s). Published by Maximum Academic Press, Fayetteville, GA. This article is an open access article distributed under Creative Commons Attribution License (CC BY 4.0), visit https://creativecommons.org/licenses/by/4.0/.
    Figure (6)  Table (1) References (37)
  • About this article
    Cite this article
    Li R, Wang B, Zha M, Zhang K, Li M, et al. 2024. Identification and characterization of grape VAP27 gene family and their roles in disease resistance. Fruit Research 4: e019 doi: 10.48130/frures-0024-0019
    Li R, Wang B, Zha M, Zhang K, Li M, et al. 2024. Identification and characterization of grape VAP27 gene family and their roles in disease resistance. Fruit Research 4: e019 doi: 10.48130/frures-0024-0019
    shu

Catalog

  • HTML

  • Introduction
  • Materials and methods

  • Identification and annotation of VAP27s in grape

  • Phylogenetic, exon-intron structure, and motif analysis of VAP27 Genes

  • Transcriptome analysis of VvVAP27 gene

  • Plant materials and treatments

  • RNA extraction and RT–qPCR

  • Transient transformation of Agrobacterium tumefaciens

  • Location analysis

  • Phytophthora capsici inoculation

  • Results

  • Identification and genomic distribution of the VAP27 family in the grape genome

  • Phylogenetic analysis of the VAP27 gene family

  • Structural analysis of grape VAP27 genes

  • Expression profiles of VAP27s in different tissues

  • Expression patterns of VAP27 genes post-plasmopara viticola inoculation

  • Subcellular localization of VvVAP27 genes

  • Response of VAP27 gene to P. capsici inoculation

  • Discussion
  • Conclusions
  • Author contributions
  • Data availability
  • Acknowledgments
  • Conflict of interest
  • Rights and permissions
  • About this article
logo

Contents

Policies

Services

Partnerships

© 2025 Maximum Academic Press, except Open Access articles. MAP is a member of ALPSP, COPE, CrossRef, cOAlition S, OASPA and STM.

Privacy Policy  |  Site Map  |  Terms and Conditions

/

DownLoad:  Full-Size Img  PowerPoint
Return
Return