Expression of disease resistance in genetically modified grapevines correlates with the contents of viral sequences in the T-DNA and global genome methylation

Increased tolerance to pathogens is an important goal in conventional and biotechnology-assisted grapevine breeding programs worldwide. Fungal and viral pathogens cause direct losses in berry production, but also affect the quality of the final products. Precision breeding strategies allow the introduction of resistance characters in elite cultivars, although the factors determining the plant’s overall performance are not fully characterized. Grapevine plants expressing defense proteins, from fungal or plant origins, or of the coat protein gene of grapevine leafroll-associated virus 3 (GLRaV-3) were generated by Agrobacterium-mediated transformation of somatic embryos and shoot apical meristems. The responses of the transformed lines to pathogen challenges were investigated by biochemical, phytopathological and molecular methods. The expression of a Metarhizium anisopliae chitinase gene delayed pathogenesis and disease progression against the necrotrophic pathogen Botrytis cinerea. Modified lines expressing a Solanum nigrum osmotin-like protein also exhibited slower disease progression, but to a smaller extent. Grapevine lines carrying two hairpin-inducing constructs had lower GLRaV-3 titers when challenged by grafting, although disease symptoms and viral multiplication were detected. The levels of global genome methylation were determined for the genetically engineered lines, and correlation analyses demonstrated the association between higher levels of methylated DNA and larger portions of virus-derived sequences. Resistance expression was also negatively correlated with the contents of introduced viral sequences and genome methylation, indicating that the effectiveness of resistance strategies employing sequences of viral origin is subject to epigenetic regulation in grapevine.     

Secreted proteins produced by fungi associated with <Emphasis Type="Italic">Botryosphaeria</Emphasis> dieback trigger distinct defense responses in <Emphasis Type="Italic">Vitis vinifera</Emphasis> and <Emphasis Type="Italic">Vitis rupestris</Emphasis> cells

Grapevine trunk diseases (Eutypa dieback, esca and Botryosphaeria dieback) are caused by a complex of xylem-inhabiting fungi, which severely reduce yields in vineyards. Botryosphaeria dieback is associated with Botryosphaeriaceae. In order to develop effective strategies against Botryosphaeria dieback, we investigated the molecular basis of grapevine interactions with a virulent species, Neofusicoccum parvum, and a weak pathogen, Diplodia seriata. We investigated defenses induced by purified secreted fungal proteins within suspension cells of Vitis (Vitis rupestris and Vitis vinifera cv. Gewurztraminer) with putative different susceptibility to Botryosphaeria dieback. Our results show that Vitis cells are able to detect secreted proteins produced by Botryosphaeriaceae, resulting in a rapid alkalinization of the extracellular medium and the production of reactive oxygen species. Concerning early defense responses, N. parvum proteins induced a more intense response compared to D. seriata. Early and late defense responses, i.e., extracellular medium alkalinization, cell death, and expression of PR defense genes were stronger in V. rupestris compared to V. vinifera, except for stilbene production. Secreted Botryosphaeriaceae proteins triggered a high accumulation of δ-viniferin in V. vinifera suspension cells. Artificial inoculation assays on detached canes with N. parvum and D. seriata showed that the development of necrosis is reduced in V. rupestris compared to V. vinifera cv. Gewurztraminer. This may be related to a more efficient induction of defense responses in V. rupestris, although not sufficient to completely inhibit fungal colonization. Overall, our work shows a specific signature of defense responses depending on the grapevine genotype and the fungal species.     

Cryopreservation of grapevine (<Emphasis Type="Italic">Vitis</Emphasis> spp.)—a review

Grapevine (Vitis genus) is one of the economically most important fruits worldwide. Some species and cultivars are rare and have only a few vines, but represent national heritages with a strong need for preservation. Field collections are labor intensive, and expensive to maintain, and are exposed to natural disasters. In addition, infection with pathogens, especially viruses, is common in grapevine because of vegetative propagation, which is conventionally used for this genus. Cryopreservation provides an alternative and ideal means for the long-term preservation of Vitis germplasm, which can be used as a backup to field collections for important autochthonous cultivars or only as cryo-banks for rare, native cultivars that are worthy of preservation. Cryotherapy, based on cryopreservation protocols, provides an efficient method for the eradication of grapevine viruses. This review provides comprehensive and updated information on cryopreservation for long-term preservation of genetic resources and cryotherapy for virus eradication in Vitis. Additional research in grapevine cryopreservation and cryotherapy is needed.     

<Emphasis Type="Italic">Muscadinia rotundifolia</Emphasis> ‘Noble’ defense response to <Emphasis Type="Italic">Plasmopara viticola</Emphasis> inoculation by inducing phytohormone-mediated stilbene accumulation

Downy mildew (DM), one of the most devastating grape diseases worldwide, is caused by the biotrophic oomycete Plasmopara viticola (Pv). In general, grapevine responds to Pv infection with the accumulation of phytoalexins as part of the innate immune system, and diverse phytoalexins are induced on grapevines with different DM-resistance levels in response to Pv invasion. However, the regulation of phytoalexin biosynthesis during grapevine against Pv is still unclear. Herein, we detected stilbenes by UPLC-ESI-MS/MS and found that resveratrol was accumulated to higher level and earlier in the DM-immune Muscadinia rotundifolia ‘Noble’ than that in the DM-susceptible Vitis vinifera ‘Thompson Seedless’ after Pv inoculation. Additionally, a considerable amount of pterostilbene and ε-viniferin was found in ‘Noble’, while a little was detected in ‘Thompson Seedless’. Resveratrol was glycosylated into piceid both in ‘Noble’ and ‘Thompson Seedless’ after Pv inoculation. The qPCR analysis of gene expression indicated that the resveratrol-synthesis gene (STS) was induced by Pv inoculation earlier in ‘Noble’ than that in ‘Thompson Seedless’, while the pterostilbene-synthesis gene (ROMT) was induced in ‘Noble’ but not in ‘Thompson Seedless’ at all. The piceid-synthesis gene (GT) was generally up-regulated in both cultivars. Sequence analysis of STS, ROMT, and GT promoters revealed that they contained cis-regulatory elements responsive to phytohormones and pathogens. Following Pv inoculation, the level of SA, MeJA, and ABA was found to be consistently higher in ‘Noble’ than those in ‘Thompson Seedless’. The results of exogenous hormone elicitation further demonstrated that the accumulation of stilbenes was regulated by phytohormones. The earlier and higher accumulation of phytohormones and consequent induction of stilbene synthesis may play an important role in grapevine defense against downy mildew disease.     

Effect of potential biocontrol agents selected among grapevine endophytes and commercial products on crown gall disease

The current strategies for the control of Agrobacterium vitis crown gall in grape are generally unsuccessful once the pathogen has established in vineyards. Experimental trials were conducted to evaluate the effectiveness of treatments based on non-pathogenic endophytes isolated from asymptomatic grapevines growing in vineyards with high incidence of crown gall and on microorganisms isolated from commercial products. Two-year in planta trials conducted on rootstocks treated with endophytic isolates showed the effectiveness of two bacterial endophytes, both in the genus Curtobacterium, and one fungal isolate in the genus Acremonium in reducing crown gall development. For the commercial biological control agents, Bacillus subtilis SR63 and Trichoderma asperellum T1 were the most effective strains against A. vitis, indicating commercial products could be reserves to draw upon to identify useful biocontrol agents. Based on the combination of data in this work, microorganisms, both endophytes and those formulated in commercial products, were identified that can potentially be exploited for the control of grapevine crown gall disease.     

Candidate genes within a 143 kb region of the flower sex locus in <Emphasis Type="Italic">Vitis</Emphasis>

Wild Vitis species are dioecious plants, while the cultivated counterpart, Vitis vinifera subspec. vinifera, generally shows hermaphroditic flowers. In Vitis the genetic determinants of flower sex have previously been mapped to a region on chromosome 2. In a combined strategy of map-based cloning and the use of the publicly available grapevine reference genome sequence, the structure of the grapevine flower sex locus has been elucidated with the subsequent identification of candidate genes which might be involved in the development of the different flower sex types. In a fine mapping approach, the sex locus in grapevine was narrowed down using a population derived from a cross of a genotype with a Vitis vinifera background (‘Schiava Grossa’ × ‘Riesling’) with the male rootstock cv. ‘Börner’ (V. riparia × V. cinerea). A physical map of 143 kb was established from BAC clones spanning the 0.5 cM region defined by the closest flanking recombination break points. Sequencing and gene annotation of the entire region revealed several candidate genes with a potential impact on flower sex formation. One of the presumed candidate genes, an adenine phosphoribosyltransferase, was analysed in more detail. The results led to the development of a marker for the presence or absence of the female alleles, while the male and hermaphroditic alleles are still to be differentiated. The impact of other candidate genes is discussed, especially with regard to plant hormone actions. The markers developed will permit the selection of female breeding lines which do not require laborious emasculation thus considerably simplifying grapevine breeding. The genetic finger prints displayed that our cultivated grapevines frequently carry a female allele while homozygous hermaphrodites are rare.     

Development of immunodiagnostics for the detection of <Emphasis Type="Italic">Grapevine leafroll</Emphasis>-<Emphasis Type="Italic">associated virus</Emphasis> 3 (GLRaV-3) in grapevine using in vitro expression and purification of its coat protein gene

A previously cloned coat protein (CP) gene of Grapevine leafroll-associated virus 3 (GLRaV-3) from cultivar Cabernet Souvignon was over-expressed in Escherichia coli strain BL21 expression system as ~ 43 kDa fusion protein containing polyhistidine tag (6His) at its N terminal. The protein was purified from insoluble fraction and reacted positively in western blotting with commercial anti GLRaV-3 polyclonal antiserum (Bioreba, Switzerland) and hence, used as immunogen for the production of polyclonal antisera in New Zealand white rabbits. Polyclonal antiserum specific to GLRaV-3 detected the virus by double antibody sandwich enzyme linked immunosorbent assay using commercial alkaline phosphatase (ALP) conjugated globulin fraction (Bioreba, Switzerland) in GLRaV-3 positive grapevine samples. The immunoreactivity of the antiserum was confirmed through western blotting. The purified antiserum was conjugated with ALP. The primary antiserum along with ALP conjugate successfully detected the GLRaV-3 from the infected sample at 1:8000 and 1:10,000 dilutions, respectively. To the best of our knowledge, it is the first global study wherein the CP of GLRaV-3 was cloned in pET28a(+) expression vector having many advantages over the earlier used expression vectors. The cloned CP gene was expressed, purified and subjected to the production of immunoreagents. The developed immunoreagents will be useful for certification programmes as well as for large scale virus screening to produce GLRaV-3 free grapevines. The indigenously developed immunereagents will provide a cost-effective way of managing grapevine leafroll disease in Indian sub-continent.     

In silico identification and computational characterization of endogenous small interfering RNAs from diverse grapevine tissues and stages

Small interfering RNAs (siRNAs) are effectors of regulatory pathways underlying plant development, metabolism, and stress- and nutrient-signaling regulatory networks. The endogenous siRNAs are generally not conserved between plants; consequently, it is necessary and important to identify and characterize siRNAs from various plants. To address the nature and functions of siRNAs, and understand the biological roles of the huge siRNA population in grapevine (Vitis vinifera L.). The high-throughput sequencing technology was used to identify a large set of putative endogenous siRNAs from six grapevine tissues/organs. Subsequently, Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis was performed to classify the target genes of siRNA. In total, 520,519 candidate siRNAs were identified and their expression profiles exhibited typical temporal characters during grapevine development. In addition, we identified two grapevine trans-acting siRNA (TAS) gene homologs (VvTAS3 and VvTAS4) and the derived trans-acting siRNAs (tasiRNAs) that could target grapevine auxin response factor (ARF) and myeloblastosis (MYB) genes. Furthermore, the GO and KEGG analysis of target genes showed that most of them covered a broad range of functional categories, especially involving in disease-resistance process. The large-scale and completely genome-wide level identification and characterization of grapevine endogenous siRNAs from the diverse tissues by high throughput technology revealed the nature and functions of siRNAs in grapevine.     

Genetic identification of SNP markers linked to a new grape phylloxera resistant locus in <Emphasis Type="Italic">Vitis cinerea</Emphasis> for marker-assisted selection

Background

Grape phylloxera (Daktulosphaira vitifoliae Fitch) is a major insect pest that negatively impacts commercial grapevine performance worldwide. Consequently, the use of phylloxera resistant rootstocks is an essential component of vineyard management. However, the majority of commercially available rootstocks used in viticulture production provide limited levels of grape phylloxera resistance, in part due to the adaptation of phylloxera biotypes to different Vitis species. Therefore, there is pressing need to develop new rootstocks better adapted to specific grape growing regions with complete resistance to grape phylloxera biotypes.

Results

Grapevine rootstock breeding material, including an accession of Vitis cinerea and V. aestivalis, DRX55 ([M. rotundifolia x V. vinifera] x open pollinated) and MS27-31 (M. rotundifolia specific hybrid), provided complete resistance to grape phylloxera in potted plant assays. To map the genetic factor(s) of grape phylloxera resistance, a F₁ V. cinerea x V. vinifera Riesling population was screened for resistance. Heritability analysis indicates that the V. cinerea accession contained a single allele referred as RESISTANCE TO DAKTULOSPHAIRA VITIFOLIAE 2 (RDV2) that confers grape phylloxera resistance. Using genetic maps constructed with pseudo-testcross markers for V. cinerea and Riesling, a single phylloxera resistance locus was identified in V. cinerea. After validating SNPs at the RDV2 locus, interval and linkage mapping showed that grape phylloxera resistance mapped to linkage group 14 at position 16.7 cM.

Conclusion

The mapping of RDV2 and the validation of markers linked to grape phylloxera resistance provides the basis to breed new rootstocks via marker-assisted selection that improve vineyard performance.

     

Development of SSR markers linked to QTL reducing leaf hair density and grapevine downy mildew resistance in <Emphasis Type="Italic">Vitis vinifera</Emphasis>

Dense leaf hairs of grapevines have been known to act as a preexisting defense structure for preventing the incidence of grapevine downy mildew, because the pathogen, Plasmopara viticola, needs water to invade grapevines, and water is repelled by a hydrophobic surface due to dense leaf hairs. In the present study, we performed regression analyses of downy mildew resistance with leaf hair density using hybrids of Vitis labrusca origin and confirmed the effect of leaf hairs. Reducing the repelling effect of leaf hairs by detergent application canceled the effect of leaf hair, which confirmed the hypothesis. Thereafter, based on QTL analyses in the population of V. vinifera ‘Muscat of Alexandria’ × the interspecific hybrid ‘Campbell Early,’ we identified a major locus in linkage group (LG) 5 of ‘Muscat of Alexandria’ controlling leaf hair density. This locus was previously reported as a small effect QTL for downy mildew resistance, however we found that the locus had high LOD scores explaining 71.9%–78.5% of the phenotypic variance of leaf hairs. Moreover, this locus was detected as a QTL for downy mildew resistance. The effect of this locus was confirmed in two other hybrid populations. Finally, we could successfully identify three traditional V. vinifera table grapes ‘Muscat of Alexandria,’ ‘Katta Kurgan,’ and ‘Parkent’ as the origin of the allele linked to hairlessness by defining the SSR haplotypes. The use of this locus for marker-assisted selections would be a promising strategy for producing grapevines with resistance by preexisting defense structure.     

Grapevine leaf tissue colonization by the fungal entomopathogen <Emphasis Type="Italic">Beauveria bassiana</Emphasis><Emphasis Type="Italic">s.l.</Emphasis> and its effect against downy mildew

A study was conducted to examine whether Beauveria bassiana (Balsamo) Vuillemin (Ascomycota: Hypocreales) can colonize grapevine leaf tissues and subsequently confer protection against downy mildew caused by Plasmopara viticola (Berk. and Curt.) Berl. and de Toni. Following the foliar inoculation of plants with conidial suspensions of selected B. bassiana strains, colonization of leaves by the fungus was determined using culture-based and PCR techniques at different time intervals. Seven days following B. bassiana inoculation, grapevine plants were challenged with P. viticola and symptoms were assessed by calculating the disease incidence and severity. Although all tested strains were able to colonize grapevine plants, percent colonization differed significantly among strains. Disease incidence and severity were, on the other hand, significantly reduced in B. bassiana-inoculated plants compared to control plants irrespective of strain. This study is one of very few studies investigating the promising role B. bassiana could play as a plant disease antagonist.     

Efficient elimination of virus complex from garlic (<Emphasis Type="Italic">Allium sativum</Emphasis> L.) by cryotherapy of shoot tips

Vegetative propagation of plants, such as garlic (Allium sativum L.), is known to facilitate the transmission of several virus species throughout the plant cycles. This process favors the onset of complex diseases by accumulation of different species in the same plant, resulting in decreased productivity and production quality. Studies have reported the use of cryotherapy of shoot tips, or meristematic clusters, as an efficient tool for obtaining virus-free plants. This study aimed to evaluate the ability of cryotherapy to eradicate virus complex in garlic plants. Bulbils naturally infected with Onion Yellow Dwarf Virus (OYDV), Leek Yellow Strip Virus (LYSV) and Garlic Common Latent Virus (GCLV) were employed as explants for different virus-cleaning treatments tested. Dot-ELISA and RT-PCR analysis were used to demonstrate the presence/absence of virus complex, and histological analysis was also performed to confirm these results. Five days after cryotherapy, structural analysis revealed that cooling had caused cell damage, as indicated by the increased vacuolization of cells after cryotherapy, as well as slight plasmolysis after thermotherapy. Immunolocalization analysis indicated the subcellular distribution of OYDV in garlic shoot tips in association with the development of plasmodesmata, while no OYDV was detected in the first cell layers of the meristematic dome. Cryotherapy successfully removed virus complex, resulting in virus-free plants with enhanced efficiency, compared to conventional meristem culture-based techniques. Moreover, the synergistic effects of cryotherapy and thermotherapy resulted in a 40 % survival rate of shoot tips and the regeneration of 90, 100 and 80 % OYDV-, LYSV- and GCLV-free plants, respectively.     

Genome-wide identification and analysis of the U-box family of E3 ligases in grapevine

E3 ubiquitin ligases play essential roles in determining the specificity of ubiquitination and subsequent protein degradation. The plant U-box (PUB) family of E3 ligases has been implicated in biotic and abiotic stress signaling and developmental events in various species. A comprehensive bioinformatics analysis identified 56 PUB genes in the grapevine (Vitis vinifera L.) genome. Based on conserved motifs, the VvPUB family was classified into seven subclasses. Expansion of this family was driven by chromosomal, segmental, and tandem duplications. Microarray expression profiling revealed that three PUB genes were preferentially expressed in pollen, four in leaf, and five in root. Moreover, a large number of PUB genes were differentially expressed under abiotic stresses and eight PUB genes likely participated in defense against powdery mildew. The microarray expression data were verified by RT-qPCR. Genome-wide identification of VvPUB genes and examination of their expression will give insights into the functions of U-box genes in grapevine.