The transcription factors VaERF16 and VaMYB306 interact to enhance resistance of grapevine to Botrytis cinerea infection.

Botrytis cinerea is a fungus that infects cultivated grape (Vitis vinifera); the identification and characterization of resistance mechanisms in the host is of great importance for the grape industry. Here, we report that a transcription factor in the ethylene-responsive factor (ERF) family (VaERF16) from Chinese wild grape (Vitis amurensis 'Shuang You') is expressed during B. cinerea infection and in response to treatments with the hormones ethylene and methyl jasmonate. Heterologous overexpression of VaERF16 in Arabidopsis thaliana substantially enhanced resistance to B. cinerea and the bacterium Pseudomonas syringae DC3000 via the salicylic acid and jasmonate/ethylene signalling pathways. Yeast two-hybrid, bimolecular fluorescence complementation, and co-immunoprecipitation assays indicated that VaERF16 interacts with the MYB family transcription factor VaMYB306. Overexpression of VaERF16 or VaMYB306 in grape leaves increased resistance to B. cinerea and caused an up-regulation of the defence-related gene PDF1.2, which encodes a defensin-like protein. Conversely, silencing of either gene resulted in increased susceptibility to B. cinerea. Yeast one-hybrid and dual-luciferase assays indicated that VaERF16 increased the transcript levels of VaPDF1.2 by binding directly to the GCC box in its promoter. Notably, VaMYB306 alone did not bind to the VaPDF1.2 promoter, but the VaERF16-VaMYB306 transcriptional complex resulted in higher transcript levels of VaPDF1.2, suggesting that the proteins function through their mutual interaction. Elucidation of this regulatory module may be of value in enhancing resistance of grapevine to B. cinerea infection.

Comparative transcriptomic analysis highlights contrasting levels of resistance of Vitis vinifera and Vitis amurensis to Botrytis cinerea.

Botrytis cinerea is a major grapevine (Vitis spp.) pathogen, but some genotypes differ in their degree of resistance. For example, the Vitis vinifera cultivar Red Globe (RG) is highly susceptible, but V. amurensis Rupr Shuangyou (SY) is highly resistant. Here, we used RNA sequencing analysis to characterize the transcriptome responses of these two genotypes to B. cinerea inoculation at an early infection stage. Approximately a quarter of the genes in RG presented significant changes in transcript levels during infection, the number of which was greater than that in the SY leaves. The genes differentially expressed between infected leaves of SY and RG included those associated with cell surface structure, oxidation, cell death and C/N metabolism. We found evidence that an imbalance in the levels of reactive oxygen species (ROS) and redox homeostasis probably contributed to the susceptibility of RG to B. cinerea. SY leaves had strong antioxidant capacities and improved ROS homeostasis following infection. Regulatory network prediction suggested that WRKY and MYB transcription factors are associated with the abscisic acid pathway. Weighted gene correlation network analysis highlighted preinfection features of SY that might contribute to its increased resistance. Moreover, overexpression of VaWRKY10 in Arabidopsis thaliana and V. vinifera Thompson Seedless enhanced resistance to B. cinerea. Collectively, our study provides a high-resolution view of the transcriptional changes of grapevine in response to B. cinerea infection and novel insights into the underlying resistance mechanisms.

Genome-wide identification and expression analysis of the B-box transcription factor gene family in grapevine (Vitis vinifera L.).

BACKGROUND: B-box (BBX) zinc-finger transcription factors play important roles in plant growth, development, and stress response. Although these proteins have been studied in model plants such as Arabidopsis thaliana or Oryza sativa, little is known about the evolutionary history or expression patterns of BBX proteins in grapevine (Vitis vinifera L.). RESULTS: We identified a total of 25 VviBBX genes in the grapevine genome and named them according to the homology with Arabidopsis. These proteins were classified into five groups on the basis of their phylogenetic relationships, number of B-box domains, and presence or absence of a CCT domain or VP motif. BBX proteins within the same group showed similar exon-intron structures and were unevenly distributed in grapevine chromosomes. Synteny analyses suggested that only segmental duplication events contributed to the expansion of the VviBBX gene family in grapevine. The observed syntenic relationships between some BBX genes from grapevine and Arabidopsis suggest that they evolved from a common ancestor. Transcriptional analyses showed that the grapevine BBX genes were regulated distinctly in response to powdery mildew infection and various phytohormones. Moreover, the expression levels of a subset of BBX genes in ovules were much higher in seedless grapevine cultivars compared with seeded cultivars during ovule development, implying a potential role in seed abortion. Additionally, VviBBX8, VquBBX15a and VquBBX29b were all located in the nucleus and had transcriptional activity except for VquBBX29b. CONCLUSIONS: The results of this study establish the genome-wide analysis of the grapevine BBX family and provide a framework for understanding the biological roles of BBX genes in grapevine.

Transcriptome Analysis of the Grape-Elsinoë ampelina Pathosystem Reveals Novel Effectors and a Robust Defense Response.

Elsinoë ampelina is an ascomycetous fungus that causes grape anthracnose, a potentially devastating disease worldwide. In this study, a dual RNA-seq analysis was used to simultaneously monitor the fungal genes related to pathogenesis and grape genes related to defense during the interaction at 2, 3, 4, and 5 days postinoculation. Consistent with their potential roles in pathogenicity, genes for carbohydrate-active enzymes, secondary metabolite synthesis, pathogen-host interaction, and those encoding secreted proteins are upregulated during infection. Based on Agrobacterium tumefaciens-mediated transient assays in Nicotiana benthamiana, we further showed that eight and nine candidate effectors, respectively, suppressed BAX- and INF1-mediated programmed cell death. The host response was characterized by the induction of multiple defense systems against E. ampelina, including synthesis of phenylpropanoids, stilbenes, and terpenoid biosynthesis, cell-wall modifications, regulation by phytohormones, and expression of defense-related genes. Together, these findings offer new insights into molecular mechanisms underlying the grape-E. ampelina interaction.[Formula: see text] Copyright © 2021 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

The jasmonate-ZIM domain gene VqJAZ4 from the Chinese wild grape Vitis quinquangularis improves resistance to powdery mildew in Arabidopsis thaliana.

Grape (Vitis vinifera L.) is one of the most widely cultivated and economically important fruits. Most cultivated varieties of grape are highly susceptible to fungal diseases, and one of the most pervasive is powdery mildew, caused by Uncinula necator. The jasmonate-ZIM domain (JAZ) family proteins are critical for plant responses to environmental stresses. Here, we report the characterization of VqJAZ4, a jasmonate-ZIM domain gene isolated from Vitis quinquangularis, a Chinese wild Vitis species that exhibits high tolerance to several kinds of fungi. Subcellular localization assay indicated that the VqJAZ4 protein is targeted to the nucleus. The VqJAZ4 gene was strongly induced by U. necator inoculation, as well as by the defense-related hormones methyl jasmonate (MeJA) and salicylic acid (SA). The upregulation of VqJAZ4 after inoculation was dependent on its promoter sequences. Expression of VqJAZ4 in Arabidopsis thaliana improved resistance to powdery mildew. Histochemical staining assays indicated that plants expressing VqJAZ4 displayed a larger number of dead cells and stronger reactive oxygen species (ROS) burst than non-transgenic control (NTC) plants. Expression analysis of several disease-related genes suggested that VqJAZ4 expression enhanced defense responses though SA and/or JA signaling pathways. We also found that VqJAZ4-expressing Arabidopsis showed increased susceptibility to Botrytis cinerea. Taken together, these results provide evidence that VqJAZ4 may play an important role in response to fungal pathogens in grape, and may represent a candidate for future grape molecular breeding for disease resistance.

Genomic Organization of the B3-Domain Transcription Factor Family in Grapevine (Vitis vinifera L.) and Expression during Seed Development in Seedless and Seeded Cultivars.

Members of the plant-specific B3-domain transcription factor family have important and varied functions, especially with respect to vegetative and reproductive growth. Although B3 genes have been studied in many other plants, there is limited information on the genomic organization and expression of B3 genes in grapevine (Vitis vinifera L.). In this study, we identified 50 B3 genes in the grapevine genome and analyzed these genes in terms of chromosomal location and syntenic relationships, intron-exon organization, and promoter cis-element content. We also analyzed the presumed proteins in terms of domain structure and phylogenetic relationships. Based on the results, we classified these genes into five subfamilies. The syntenic relationships suggest that approximately half of the genes resulted from genome duplication, contributing to the expansion of the B3 family in grapevine. The analysis of cis-element composition suggested that most of these genes may function in response to hormones, light, and stress. We also analyzed expression of members of the B3 family in various structures of grapevine plants, including the seed during seed development. Many B3 genes were expressed preferentially in one or more structures of the developed plant, suggesting specific roles in growth and development. Furthermore, several of the genes were expressed differentially in early developing seeds from representative seeded and seedless cultivars, suggesting a role in seed development or abortion. The results of this study provide a foundation for functional analysis of B3 genes and new resources for future molecular breeding of grapevine.

VlbZIP30 of grapevine functions in dehydration tolerance via the abscisic acid core signaling pathway.

Drought stress limits the growth and development of grapevines, thereby reducing productivity, but the mechanisms by which grapevines respond to drought stress remain largely uncharacterized. Here, we characterized a group A bZIP gene from "Kyoho" grapevine, VlbZIP30, which was shown to be induced by abscisic acid (ABA) and dehydration stress. Overexpression of VlbZIP30 in transgenic Arabidopsis thaliana enhanced dehydration tolerance. Transcriptome analysis revealed that a major proportion of ABA-responsive and/or drought-responsive genes are transcriptionally regulated by VlbZIP30 during ABA or mannitol treatment at the cotyledon greening stage. We identified an A. thaliana G-box motif (CACGTG) and a potential grapevine G-box motif (MCACGTGK) in the promoters of the 39 selected A. thaliana genes upregulated in the transgenic plants and in the 35 grapevine homologs, respectively. Subsequently, using two grapevine-related databases, we found that 74% (23/31) and 84% (21/25) of the detected grapevine genes were significantly upregulated by ABA and drought stress, respectively, suggesting that these genes are involved in ABA or dehydration stress and may be regulated by VlbZIP30 in grapevine. We propose that VlbZIP30 functions as a positive regulator of dehydration-responsive signaling in the ABA core signaling pathway.

Expression of Vitis amurensis VaERF20 in Arabidopsis thaliana Improves Resistance to Botrytis cinerea and Pseudomonas syringae pv. Tomato DC3000.

Ethylene response factor (ERF) transcription factors play important roles in regulating immune responses in plants. In our study, we characterized a member of the ERF transcription factor family, VaERF20, from the Chinese wild Vitis genotype, V. amurensis Rupr "Shuangyou". Phylogenetic analysis indicated that VaERF20 belongs to group IXc of the ERF family, in which many members are known to contribute to fighting pathogen infection. Consistent with this, expression of VaERF20 was induced by treatment with the necrotrophic fungal pathogen Botrytis cinerea (B. cinerea) in "Shuangyou" and V. vinifera "Red Globe". Arabidopsis thaliana plants over-expressing VaERF20 displayed enhanced resistance to B. cinerea and the bacterium Pseudomonas syringae pv. tomato (Pst) DC3000. Patterns of pathogen-induced reactive oxygen species (ROS) accumulation were entirely distinct in B. cinerea and PstDC3000 inoculated plants. Examples of both salicylic acid (SA) and jasmonic acid/ethylene (JA/ET) responsive defense genes were up-regulated after B. cinerea and PstDC3000 inoculation of the VaERF20-overexpressing transgenic A. thaliana plants. Evidence of pattern-triggered immunity (PTI), callose accumulation and stomatal defense, together with increased expression of PTI genes, was also greater in the transgenic lines. These data indicate that VaERF20 participates in various signal transduction pathways and acts as an inducer of immune responses.

Publisher Correction: Genome-wide identification and expression analyses of the homeobox transcription factor family during ovule development in seedless and seeded grapes.

A correction to this article has been published and is linked from the HTML version of this paper. The error has been fixed in the paper.

Genome-wide identification and expression analyses of the homeobox transcription factor family during ovule development in seedless and seeded grapes.

Seedless grapes are of considerable importance for the raisin and table grape industries. Previous transcriptome analyses of seed development in grape revealed that genes encoding homeobox transcription factors were differentially regulated in seedless compared with seeded grape during seed development. In the present study, we identified a total of 73 homeobox-like genes in the grapevine genome and analyzed the genomic content and expression profiles of these genes. Based on domain architecture and phylogenetic analyses grape homeobox genes can be classified into eleven subfamilies. An analysis of the exon-intron structures and conserved motifs provided further insight into the evolutionary relationships between these genes. Evaluation of synteny indicated that segmental and tandem duplications have contributed greatly to the expansion of the grape homeobox gene superfamily. Synteny analysis between the grape and Arabidopsis genomes provided a potential functional relevance for these genes. The tissue-specific expression patterns of homeobox genes suggested roles in both vegetative and reproductive tissues. Expression profiling of these genes during the course of ovule development in seeded and seedless cultivars suggested a potential role in ovule abortion associated with seedlessness. This study will facilitate the functional analysis of these genes and provide new resources for molecular breeding of seedless grapes.