The PLCP gene family of grapevine (Vitis vinifera L.) : Characterization and differential expression in response to Plasmopara Viticola
ABSTRACT: In order to investigate the putative roles of the VvPLCP genes in grapevine resistance, the leaves-specific expression patterns of VvPLCPs were analyzed according to transcriptome data in two cultivars including V. vinifera cv. ‘Zitian Seedless’ and Vitis rootstocks ‘Kober 5BB’ when infected with P. viticola Overall design: Expression data for VvPLCP genes was obtained from this transcriptome data. The expression levels of V. vinifera cv.‘Zitian Seedless’and Vitis rootstocks‘Kober 5BB’leaves at 0, 24, and 72 h after P. viticola BS-4-MW infection
Project description:The Eurasian grapevine (Vitis vinifera), an Old World species now cultivated worldwide for high-quality wine production, is extremely susceptible to the agent of downy mildew, Plasmopara viticola. The cultivation of resistant V. vinifera varieties would be a sustainable way to reduce the damage caused by the pathogen and the impact of disease management, which involves the economic, health and environmental costs of frequent fungicide application. We report the finding of unique downy mildew resistance traits in a winemaking cultivar from the domestication center of V. vinifera, and characterize the expression of a range of genes associated with the resistance mechanism. Based on comparative experimental inoculations, confocal microscopy and transcriptomics analyses, our study shows that V. vinifera cv. Mgaloblishvili, native to Georgia (South Caucasus), exhibits unique resistance traits against P. viticola. Its defense response, leading to a limitation of P. viticola growth and sporulation, is determined by the overexpression of genes related to pathogen recognition, the ethylene signaling pathway, synthesis of antimicrobial compounds and enzymes, and the development of structural barriers. The unique resistant traits found in Mgaloblishvili highlight the presence of a rare defense system in V. vinifera against P. viticola which promises fresh opportunities for grapevine genetic improvement.
Project description:Downy mildew of grapevine (Vitis vinifera L.), caused by the oomycete pathogen Plasmopara viticola, is one of the most serious concerns for grape production worldwide. It has been widely reported that the pathogenesis-related 4 (PR4) protein plays important roles in plant resistance to diseases. However, little is known about the role of PR4 in the defense of grapevine against P. viticola. In this study, we engineered loss-of-function mutations in the VvPR4b gene from the cultivar "Thompson Seedless" using the CRISPR/Cas9 system and evaluated the consequences for downy mildew resistance. Sequencing results showed that deletions were the main type of mutation introduced and that no off-target events occurred. Infection assays using leaf discs showed that, compared to wild-type plants, the VvPR4b knockout lines had increased susceptibility to P. viticola. This was accompanied by reduced accumulation of reactive oxygen species around stomata. Measurement of the relative genomic abundance of P. viticola in VvPR4b knockout lines also demonstrated that the mutants had increased susceptibility to the pathogen. Our results confirm that VvPR4b plays an active role in the defense of grapevine against downy mildew.
Project description:Cryopreservation is used for the long-term conservation of plant genetic resources. This technique very often induces lethal injury or tissue damage. In this study, we measured indicators of viability and cell damage following cryopreservation and vitrification-cryopreservation in Vitis vinifera L. axillary buds cv. "Flame seedless" stored in liquid nitrogen (LN) for: three seconds, one hour, one day, one week and one month; after LN thawed at 38 °C for three minutes. The enzymatic activity of catalase (CAT) and superoxide dismutase (SOD), as well as the amount of malondialdehyde (MDA), total protein and viability were assayed.
Project description:Vacuolar processing enzymes (VPEs) have received considerable attention due to their caspase-1-like activity and ability to regulate programmed cell death (PCD), which plays an essential role in the development of stenospermocarpic seedless grapes ovules. To characterize VPEs and the relationship between stenospermocarpic grapes and the VPE gene family, we identified 3 Vitis vinifera VPE genes (Vv?VPE, Vv?VPE, and Vv?VPE) from the PN40024 grape genome and cloned the full-length complementary DNAs (cDNAs) from the 'Vitis vinifera cv. Pinot Noir' and 'Vitis vinifera cv. Thompson Seedless' varietals. Each of the VPEs contained a typical catalytic dyad [His (177), Cys (219)] and substrate binding pocket [Arg (112), Arg (389), Ser (395)], except that Ser (395) in the Vv?VPE protein sequence was replaced with alanine. Phylogenetic analysis of 4 Arabidopsis thaliana and 6 Vitis vinifera VPEs revealed that the 10 VPEs form 3 major branches. Furthermore, the 6 grapevine VPEs share a similar gene structure, with 9 exons and 8 introns. The 6 grapevine VPEs are located on 3 different chromosomes. We also tested the enzymatic activity of recombinant VPEs expressed in the Pichia Pastoris expression system and found that the VvVPEs exhibit cysteine peptidase activity. Tissue-specific expression analysis showed that Vv?VPE is only expressed in flowers, buds and ovules, that Vv?VPE is expressed in various tissues, and that Vv?VPE was expressed in roots, flowers, buds and ovules. The results of quantitative real-time PCR (qRT-PCR) suggested that Vv?VPE in seeded grapes increased significantly at 30 days after full-bloom (DAF), close to the timing of endosperm abortion at 32 DAF. These results suggested that Vv?VPE is related to ovule abortion in seedless grapes. Our experiments provide a new perspective for understanding the mechanism of stenospermocarpic seedlessness and represent a useful reference for the further study of VPEs.
Project description:BACKGROUND: Grapevine protection against diseases needs alternative strategies to the use of phytochemicals, implying a thorough knowledge of innate defense mechanisms. However, signalling pathways and regulatory elements leading to induction of defense responses have yet to be characterized in this species. In order to study defense response signalling to pathogens in Vitis vinifera, we took advantage of its recently completed genome sequence to characterize two putative orthologs of NPR1, a key player in salicylic acid (SA)-mediated resistance to biotrophic pathogens in Arabidopsis thaliana. RESULTS: Two cDNAs named VvNPR1.1 and VvNPR1.2 were isolated from Vitis vinifera cv chardonnay, encoding proteins showing 55% and 40% identity to Arabidopsis NPR1 respectively. Constitutive expression of VvNPR1.1 and VvNPR1.2 monitored in leaves of V. vinifera cv chardonnay was found to be enhanced by treatment with benzothiadiazole, a SA analog. In contrast, VvNPR1.1 and VvNPR1.2 transcript levels were not affected during infection of resistant Vitis riparia or susceptible V. vinifera with Plasmopara viticola, the causal agent of downy mildew, suggesting regulation of VvNPR1 activity at the protein level. VvNPR1.1-GFP and VvNPR1.2-GFP fusion proteins were transiently expressed by agroinfiltration in Nicotiana benthamiana leaves, where they localized predominantly to the nucleus. In this system, VvNPR1.1 and VvNPR1.2 expression was sufficient to trigger the accumulation of acidic SA-dependent pathogenesis-related proteins PR1 and PR2, but not of basic chitinases (PR3) in the absence of pathogen infection. Interestingly, when VvNPR1.1 or AtNPR1 were transiently overexpressed in Vitis vinifera leaves, the induction of grapevine PR1 was significantly enhanced in response to P. viticola. CONCLUSION: In conclusion, our data identified grapevine homologs of NPR1, and their functional analysis showed that VvNPR1.1 and VvNPR1.2 likely control the expression of SA-dependent defense genes. Overexpression of VvNPR1 has thus the potential to enhance grapevine defensive capabilities upon fungal infection. As a consequence, manipulating VvNPR1 and other signalling elements could open ways to strengthen disease resistance mechanisms in this crop species.
Project description:The genus Vitis is represented by several coexisting species in Europe. Our study focuses on naturalised rootstocks that originate in viticulture. The consequences of their presence to the landscape and to native European species (Vitis vinifera ssp. silvestris) are evaluated. This study compares ecological traits (seven qualitative and quantitative descriptors) and the genetic diversity (10 SSR markers) of populations of naturalised rootstocks and native wild grapevines. 18 large naturalised rootstock populations were studied in the Rhône watershed. Wild European grapevines are present in four main habitats (screes, alluvial forests, hedges, and streamside hedges). In contrast, naturalised rootstock populations are mainly located in alluvial forests, but they clearly take advantage of alluvial system dynamics and connectivity at the landscape level. These latter populations appear to reproduce sexually, and show a higher genetic diversity than Vitis vinifera ssp. silvestris. The regrouping of naturalised rootstocks in interconnected populations tends to create active hybrid swarms of rootstocks. The rootstocks show characters of invasive plants. The spread of naturalised rootstocks in the environment, the acceleration of the decline of the European wild grapevine, and the propagation of genes of viticultural interest in natural populations are potential consequences that should be kept in mind when undertaking appropriate management measures.
Project description:The reduction of synthetic fungicides in agriculture is necessary to guarantee a sustainable production that protects the environment and consumers' health. Downy mildew caused by the oomycete Plasmopara viticola is the major pathogen in viticulture worldwide and responsible for up to 60% of pesticide treatments. Alternatives to reduce fungicides are thus utterly needed to ensure sustainable vineyard-ecosystems, consumer health and public acceptance. Essential oils (EOs) are amongst the most promising natural plant protection alternatives and have shown their antibacterial, antiviral and antifungal properties on several agricultural crops. However, the efficiency of EOs highly depends on timing, application method and the molecular interactions between the host, the pathogen and EO. Despite proven EO efficiency, the underlying processes are still not understood and remain a black box. The objectives of the present study were: a) to evaluate whether a continuous fumigation of a particular EO can control downy mildew in order to circumvent the drawbacks of direct application, b) to decipher molecular mechanisms that could be triggered in the host and the pathogen by EO application and c) to try to differentiate whether essential oils directly repress the oomycete or act as plant resistance primers. To achieve this a custom-made climatic chamber was constructed that enabled a continuous fumigation of potted vines with different EOs during long-term experiments. The grapevine (Vitis vinifera) cv Chasselas was chosen in reason of its high susceptibility to Plasmopara viticola. Grapevine cuttings were infected with P. viticola and subsequently exposed to continuous fumigation of different EOs at different concentrations, during 2 application time spans (24 hours and 10 days). Experiments were stopped when infection symptoms were clearly observed on the leaves of the control plants. Plant physiology (photosynthesis and growth rate parameters) were recorded and leaves were sampled at different time points for subsequent RNA extraction and transcriptomics analysis. Strikingly, the Oregano vulgare EO vapour treatment during 24h post-infection proved to be sufficient to reduce downy mildew development by 95%. Total RNA was extracted from leaves of 24h and 10d treatments and used for whole transcriptome shotgun sequencing (RNA-seq). Sequenced reads were then mapped onto the V. vinifera and P. viticola genomes. Less than 1% of reads could be mapped onto the P. viticola genome from treated samples, whereas up to 30% reads from the controls mapped onto the P. viticola genome, thereby confirming the visual observation of P. viticola absence in the treated plants. On average, 80% of reads could be mapped onto the V. vinifera genome for differential expression analysis, which yielded 4800 modulated genes. Transcriptomic data clearly showed that the treatment triggered the plant's innate immune system with genes involved in salicylic, jasmonic acid and ethylene synthesis and signaling, activating Pathogenesis-Related-proteins as well as phytoalexin synthesis. These results elucidate EO-host-pathogen interactions for the first time and indicate that the antifungal efficiency of EO is mainly due to the triggering of resistance pathways inside the host plants. This is of major importance for the production and research on biopesticides, plant stimulation products and for resistance-breeding strategies.
Project description:Study of gene expression during Plasmopara viticola infection in the resistant Vitis vinifera cultivar 'Regent'. The oomycete fungus Plasmopara viticola (Berk. et Curt.) Berl. et de Toni is responsible for grapevine downy mildew disease. Most of the cultivated grapevines are sensitive to this pathogen, thus requiring intensive fungicide treatments. The molecular basis of resistance to this pathogen is poorly understood. We have carried out a cDNA microarray transcriptome analysis to identify grapevine genes associated with resistance traits. Early transcriptional changes associated with downy mildew infection in the resistant Vitis vinifera cultivar ‘Regent’, when compared to the susceptible cultivar ‘Trincadeira’, were analyzed. Transcript levels were measured at three time-points: 0, 6 and 12 hours post inoculation (hpi). Our data indicate that resistance in V. vinifera ‘Regent’ is induced after infection. This study provides the identification of several candidate genes that may be related to ‘Regent’ defense mechanisms, allowing a better understanding of this cultivar's resistance traits. Overall design: 3 time points: 0, 6 and 12 hours post inoculation by P. viticola. Two cultivars: control (Trinacedira) and test (Regent). Two biological replicates were performed at 0 hpi, and 3 biological replicates at 6 and 12hpi. At 12hpi, three technical replicates also were performed.
Project description:Study of gene expression during Plasmopara viticola infection in the resistant Vitis vinifera cultivar 'Regent'. The oomycete fungus Plasmopara viticola (Berk. et Curt.) Berl. et de Toni is responsible for grapevine downy mildew disease. Most of the cultivated grapevines are sensitive to this pathogen, thus requiring intensive fungicide treatments. The molecular basis of resistance to this pathogen is poorly understood. We have carried out a cDNA microarray transcriptome analysis to identify grapevine genes associated with resistance traits. Early transcriptional changes associated with downy mildew infection in the resistant Vitis vinifera cultivar ‘Regent’, when compared to the susceptible cultivar ‘Trincadeira’, were analyzed. Transcript levels were measured at three time-points: 0, 6 and 12 hours post inoculation (hpi). Our data indicate that resistance in V. vinifera ‘Regent’ is induced after infection. This study provides the identification of several candidate genes that may be related to ‘Regent’ defense mechanisms, allowing a better understanding of this cultivar's resistance traits. 3 time points: 0, 6 and 12 hours post inoculation by P. viticola. Two cultivars: control (Trinacedira) and test (Regent). Two biological replicates were performed at 0 hpi, and 3 biological replicates at 6 and 12hpi. At 12hpi, three technical replicates also were performed.
Project description:Plants belonging to Vitis vinifera varieties are usually grafted on different rootstocks to enhance the plant defenses against pathogens and increase productivity under harsh environmental conditions. Particularly, in Emilia-Romagna region (Italy), Vitis vinifera cultivar Lambrusco can be grafted on a hybrid of V. berlandieri × V. riparia (5BB) or V. berlandieri × V. rupestris (1103P). However, the latter shows potassium absorption problems, with a consequent reduction in grapevine production. Since it has recently been demonstrated that the rootstock has the potential to select for different microorganisms at the root-soil interface, here we hypothesized that the potassium deficiency of 1103P could be partially accounted for by the peculiarities of the rootstock microbiome. We thus employed 16S rRNA sequencing to compare root and rhizosphere microbiomes in plants of V. vinifera cultivar Lambrusco grafted on the two aforementioned rootstocks. According to our findings, 1103P shows a reduced diversity in root and rhizosphere microbiomes, including members of potassium-solubilizing microorganisms, possibly explaining the inadequate potassium absorption of this hybrid. Besides confirming the importance of the rootstock as a determinant of the composition of plant microbiomes, our data indicate the relevance of rootstock-selected microbiomes as possible regulators of potassium absorption by V. vinifera.