Comparative Transcriptomics Suggests Early Modifications by Vintec® in Grapevine Trunk of Hormonal Signaling and Secondary Metabolism Biosynthesis in Response to Phaeomoniella chlamydospora and Phaeoacremonium minimum.

Given their well-known antifungal abilities, species of the genus Trichoderma are of significant interest in modern agriculture. Recent studies have shown that Trichoderma species can induce plant resistance against different phytopathogens. To further extend this line of investigation, we investigate herein the transcriptomic response of grapevine trunk to Vintec®, which is a Trichoderma atroviride SC1-based commercial formulation for biological control of grapevine trunk diseases and which reduces wood colonization. The aim of the study is to understand whether the biocontrol agent Vintec® modifies the trunk response to Phaeoacremonium minimum and Phaeomoniella chlamydospora, which are two esca-associated fungal pathogens. An analysis of transcriptional regulation identifies clusters of co-regulated genes whose transcriptomic reprogramming in response to infection depends on the absence or presence of Vintec®. On one hand, the results show that Vintec® differentially modulates the expression of putative genes involved in hormonal signaling, especially those involved in auxin signaling. On the other hand, most significant gene expression modifications occur among secondary-metabolism-related genes, especially regarding phenylpropanoid metabolism and stilbene biosynthesis. Taken together, these results suggest that the biocontrol agent Vintec® induces wood responses that counteract disease development.

A novel approach to discriminate the volatilome of Vitis vinifera berries by selected ion flow tube mass Spectrometry analysis and chemometrics.

This study investigated a quick way to discriminate grape varieties based on their composition in volatile compounds through a SIFT-MS scan coupled with simple chemometrics approaches such as analysis of variance (ANOVA), principal component analysis (PCA) and hierarchical ascendant classification (HAC). The 23 studied grape varieties were distinguishable using O2+, H3O+ and NO+ as reagent ions, and the combination of these three ions. For its ability to ionize most compounds, to efficiently fragment them to generate ions with distinct m/z ratio, and to enhance the differentiation of compounds of similar masses, O2+ reagent ion should be preferentially considered. The use of one single ion rather than three enables to limit the time of analysis and the number of variables to be treated. The technique allowed the distinction of high and low aroma compounds producers as confirmed by headspace solid-phase microextraction followed by gas chromatography-mass spectrometry (HS-SPME/GC-MS) analyses. SIFT-MS is a quick and interesting tool with potential application in various fields of viticulture such as phenotyping of grape varieties or non-targeted studies on the impact of environmental factors or viticultural practices on grape aroma composition.

Modification of Early Response of Vitis vinifera to Pathogens Relating to Esca Disease and Biocontrol Agent Vintec® Revealed By Untargeted Metabolomics on Woody Tissues.

Esca disease is one of the most destructive grapevine trunk diseases. Phaeoacremonium minimum and Phaeomoniella chlamydospora are two of the known fungal pathogens associated with this disease. Today, biocontrol agents against Esca are mainly based on the use of the strain of the mycoparasite fungal genus Trichoderma such as the Vintec® product. The aim of this study was to investigate early response of woody tissues to Esca pathogens and identify metabolites that could be correlated with a biocontrol activity within a complex woody matrix. An untargeted liquid chromatography-high-resolution mass spectrometry metabolomic approach coupled to a spectral similarity network was used to highlight clusters of compounds associated with the plant response to pathogens and biocontrol. Dereplication highlighted the possible role of glycerophospholipids and polyphenol compounds, the latest mainly belonging to stilbenoids. Antifungal activity of some relevant biomarkers, evaluated in vitro on Phaeomoniella chlamydospora and Botrytis cinerea, suggests that some of these compounds can play a role to limit the development of Esca pathogens in planta.

Development of screening methods for functional characterization of UGTs from Stevia rebaudiana.

Glycosylation is a key modification that contributes to determine bioactivity and bioavailability of plant natural products, including that of terpenoids and steviol glycosides (SVglys). It is mediated by uridine-diphosphate glycosyltransferases (UGTs), that achieve their activity by transferring sugars on small molecules. Thus, the diversity of SVglys is due to the number, the position and the nature of glycosylations on the hydroxyl groups in C-13 and C-19 of steviol. Despite the intense sweetener property of SVglys and the numerous studies conducted, the SVglys biosynthetic pathway remains largely unknown. More than 60 SVglys and 68 putative UGTs have been identified in Stevia rebaudiana. This study aims to provide methods to characterize UGTs putatively involved in SVglys biosynthesis. After agroinfiltration-based transient gene expression in Nicotiana benthamiana, functionality of the recombinant UGT can be tested simply and directly in plants expressing it or from a crude extract. The combined use of binary vectors from pGWBs series to produce expression vectors containing the stevia's UGT, enables functionality testing with many substrates as well as other applications for further analysis, including subcellular localization.

Dynamic virulence-related regions of the plant pathogenic fungus Verticillium dahliae display enhanced sequence conservation.

Plant pathogens continuously evolve to evade host immune responses. During host colonization, many fungal pathogens secrete effectors to perturb such responses, but these in turn may become recognized by host immune receptors. To facilitate the evolution of effector repertoires, such as the elimination of recognized effectors, effector genes often reside in genomic regions that display increased plasticity, a phenomenon that is captured in the two-speed genome hypothesis. The genome of the vascular wilt fungus Verticillium dahliae displays regions with extensive presence/absence polymorphisms, so-called lineage-specific regions, that are enriched in in planta-induced putative effector genes. As expected, comparative genomics reveals differential degrees of sequence divergence between lineage-specific regions and the core genome. Unanticipated, lineage-specific regions display markedly higher sequence conservation in coding as well as noncoding regions than the core genome. We provide evidence that disqualifies horizontal transfer to explain the observed sequence conservation and conclude that sequence divergence occurs at a slower pace in lineage-specific regions of the V. dahliae genome. We hypothesize that differences in chromatin organisation may explain lower nucleotide substitution rates in the plastic, lineage-specific regions of V. dahliae.

UGT76G1 polymorphism in Stevia rebaudiana: New variants for steviol glycosides conjugation.

Steviol glycosides (SVglys) are secondary metabolites derived from terpenoids exhibiting high-sweetening properties produced in Stevia rebaudiana leaves. Their great diversity is due to the number, the position and the nature of glycosylations on the steviol aglycone. Steviol conjugation is mediated by uridine-diphosphate glycosyltransferases (UGTs). Four UGTs have been clearly identified as involved in SVglys metabolism: UGT74G1, UGT85C2, UGT76G1 and UGT73E1. Natural non-functional mutants with nonsense codon have yet been observed for UGT76G1. To investigate the variability of UGT76G1 functionality, natural mutants with low or no content of rebaudioside A and C were identified in a germplasm collection of Stevia rebaudiana. These compounds are known to be the direct products of UGT76G1 and their biosynthesis is governed by a single gene at the locus Rae (Rebaudioside A enablement). Crosses were done with remarkable accessions including phenotypes with low (0-3%) and high proportions (70%) of rebaudioside A and C, to investigate the functionality of the Rae locus in the parents. Seven variants of UGT76G1 were found, among them 4 lead to a functional protein and 3 lead to non-functional isoforms. Five of these variants are new. We found that non-functionality of UGT76G1 towards SVglys is not due to a premature nonsense codon, which appears to be an extreme case to explain the loss of functionality of an UGT. Variations in steviol glycoside profile in stevia leaves is partly due to UGT76G1 polymorphism: amino acid substitutions in parts of the protein involved in the substrate specificity can be found by sequence comparison.

Accounting for biotic spatial variability in fields: Case of resistance screening against sunflower Verticillium wilt.

In breeding for disease resistance, the magnitude of the genetic response is difficult to appreciate because of environmental stresses that interact with the plant genotype. We discuss herein the fundamental problems in breeding for disease resistance with the aim being to better understand the interactions between plant, pathogen, and spatial patterns. The goal of this study is to fine tune breeding decisions by incorporating spatial patterns of such biotic factors into the definition of disease-occurrence probability. We use a preexisting statistics method based on geostatistics for a descriptive analysis of biotic factors for trial quality control. The plant-population structure used for spatial-pattern analysis consists of two F1-hybrid cultivars, defined as symptomatic and asymptomatic controls with respect to the studied pathogen. The controls are inserted at specific locations to establish a grid arrangement over the field that include the F1-hybrid cultivars under evaluation. We characterize the spatial structure of the pathogen population and of the general plant environment-with undetermined but present abiotic constraints-not by using direct notation such as flower time or rainfall but by using plant behavior (i.e., leaf symptom severity, indirect notation). The analysis indicates areas with higher or lower risk of disease and reveals a correlation between the symptomatic control and the effective level of disease for sunflowers. This result suggests that the pathogen and/or abiotic components are major factors in determining the probability that a plant develops the disease, which could lead to a misinterpretation of plant resistance.

A Method to Detect and Quantify Eutypa lata and Diplodia seriata-Complex DNA in Grapevine Pruning Wounds.

Trunk diseases are factors that limit sustainability of vineyards worldwide. Botryosphaeria and Eutypa diebacks are caused by several fungi belonging to the Botryosphaeriaceae and Diatrypaceae, respectively, with Diplodia seriata and Eutypa lata being two of the most common species. Previous information indicated that the traditional isolation method used to detect these pathogens from plant samples could underestimate their incidence levels. In the present study, we designed two sets of primers that target the ß-tubulin gene and that are amenable for quantitative real-time PCR (qPCR) Sybr-Green assays for the detection and quantification of D. seriata-complex (DseCQF/R) and E. lata (ElQF/R) DNA. The design of a species-specific assay was achieved for E. lata. For D. seriata, a species-specific assay could not be designed. The low interspecific diversity across ß-tubulin genes resulted in an assay that could not discriminate D. seriata from some closely related species either not yet reported or presenting a low prevalence on grapevine, such as D. intermedia. We validated our technique on grapevine spur samples naturally and artificially infected with D. seriata and E. lata during the dormant season. Experimental grapevines were located in two counties of northern California where the incidence of both pathogens was previously reported. The qPCR assays revealed that a high frequency of pruning wound infections (65%) was achieved naturally by E. lata, while low infection frequency (less than 5%) was observed using the reisolation method. For D. seriata-complex, low (5%) to no natural infection frequencies were observed by the qPCR and the reisolation method, respectively. These results also provided evidence that our qPCR detection methods were more sensitive to assess the incidence of E. lata and D. seriata-complex in plant samples, than traditional isolation techniques. Benefits of molecular methods for the detection of canker pathogens in the field under natural conditions are discussed.

Differing Alterations of Two Esca Associated Fungi, Phaeoacremonium aleophilum and Phaeomoniella chlamydospora on Transcriptomic Level, to Co-Cultured Vitis vinifera L. calli.

The filamentous fungi Phaeoacremonium aleophilum (P.al, Teleomorph: Togninia minima) and Phaeomoniella chlamydospora (P.ch) are believed to be causal agents of wood symptoms associated with the Esca associated young vine decline. The occurrence of these diseases is dramatically increasing in vineyards all over the world whereas efficient therapeutic strategies are lacking. Both fungi occupy the same ecological niche within the grapevine trunk. We found them predominantly within the xylem vessels and surrounding cell walls which raises the question whether the transcriptional response towards plant cell secreted metabolites is comparable. In order to address this question we co-inoculated grapevine callus culture cells with the respective fungi and analyzed their transcriptomes by RNA sequencing. This experimental setup appears suitable since we aimed to investigate the effects caused by the plant thereby excluding all effects caused by other microorganisms omnipresent in planta and nutrient depletion. Bioinformatics analysis of the sequencing data revealed that 837 homologous genes were found to have comparable expression pattern whereas none of which was found to be differentially expressed in both strains upon exposure to the plant cells. Despite the fact that both fungi induced the transcription of oxido- reductases, likely to cope with reactive oxygen species produced by plant cells, the transcriptomics response of both fungi compared to each other is rather different in other domains. Within the transcriptome of P.ch beside increased transcript levels for oxido- reductases, plant cell wall degrading enzymes and detoxifying enzymes were found. On the other hand in P.al the transcription of some oxido- reductases was increased whereas others appeared to be repressed. In this fungus the confrontation to plant cells results in higher transcript levels of heat shock and chaperon-like proteins as well as genes encoding proteins involved in primary metabolism.

Variations in Early Response of Grapevine Wood Depending on Wound and Inoculation Combinations with Phaeoacremonium aleophilum and Phaeomoniella chlamydospora.

Defense mechanisms in woody tissue are poorly understood, especially in vine colonized by trunk pathogens. However, several investigations suggest that molecular mechanisms in the central tissue of Vitis vinifera L. may be involved in trunk-defense reactions. In this work, the perception of Phaeoacremonium aleophilum and Phaeomoniella chlamydospora alone or together were investigated in cuttings of Cabernet Sauvignon trunks. Plant responses were analyzed at the tissue level via optical microscopy and at the cellular level via plant-gene expression. The microscopy results revealed that, 6 weeks after pathogen inoculation, newly formed vascular tissue is less developed in plants inoculated with P. chlamydospora than in plants inoculated with P. aleophilum. Co-inoculation with both pathogens resulted in an intermediate phenotype. Further analysis showed the relative expression of the following grapevine genes: PAL, PR10.3, TL, TLb, Vv17.3, STS, STS8, CWinv, PIN, CAM, LOX at 10, 24, 48, and 120 h post-inoculation (hpi). The gene set was induced by wounding before inoculation with the different pathogens, except for the genes CAM and LOX. This response generated significant noise, but the expression of the grapevine genes (PAL, PR10.3, TL, TLb, Vv17.3, STS, STS8, CWinv, and PIN) still differed due to perception of mycelium by the plant. Furthermore, at 48 hpi, the induction of PAL and STS8 differs depending on the pathogen, and a specific pattern emerges from the different inductions associated with the different treatments. Based on these results, we conclude that V. vinifera L. trunk perceives the presence of pathogens differently depending on the inoculated pathogen or even on the combination of co-inoculated pathogens, suggesting a defense orchestration in the perennial organs of woody plants.

NtRING1, putative RING-finger E3 ligase protein, is a positive regulator of the early stages of elicitin-induced HR in tobacco.

KEY MESSAGE: NtRING1 is a RING-finger protein with a putative E3 ligase activity. NtRING1 regulates HR establishment against different pathogens. Loss-/gain-of-function of NtRING1 altered early stages of HR phenotype establishment. Plant defence responses against pathogens often involve the restriction of pathogens by inducing a hypersensitive response (HR). cDNA clones DD11-39, DD38-11 and DD34-26 were previously obtained from a differential screen aimed at characterising tobacco genes with an elicitin-induced HR-specific pattern of expression. Our precedent observations suggested that DD11-39, DD38-11 and DD34-26 might play roles in the HR establishment. Only for DD11-39 a full-length cDNA sequence was obtained and the corresponding protein encoded for a type-HC RING-finger/putative E3 ligase protein which we termed NtRING1. The expression of NtRING1 was upregulated upon HR induction by elicitin, Ralstonia solanacearum, or tobacco mosaic virus (TMV) in tobacco. Silencing of NtRING1 remarkably delayed the establishment of elicitin-induced HR in tobacco as well as the expression of different early induction genes in tissues undergoing HR. Accordingly, transient overexpression of NtRING1 accelerated the HR launching upon elicitin treatment. Taking together, our data suggests that NtRING1 plays a functional role in the early establishment of HR.

Deciphering the Niches of Colonisation of Vitis vinifera L. by the Esca-Associated Fungus Phaeoacremonium aleophilum Using a gfp Marked Strain and Cutting Systems.

INTRODUCTION: Esca disease has become a major threat for viticulture. Phaeoacremonium aleophilum is considered a pioneer of the esca complex pathosystem, but its colonisation behaviour inside plants remains poorly investigated. MATERIAL AND METHODS: In this study, P. aleophilum::gfp7 colonisation was assessed six and twelve weeks post-inoculation in two different types of tissues: in the node and the internode of one year-old rooted cuttings of Cabernet Sauvignon. These processes of colonisation were compared with the colonisation by the wild-type strain using a non-specific lectin probe Alexa Fluor 488-WGA. RESULTS: Data showed that six weeks post-inoculation of the internode, the fungus had colonised the inoculation point, the bark and xylem fibres. Bark, pith and xylem fibres were strongly colonised by the fungus twelve weeks post-inoculation and it can progress up to 8 mm from the point of inoculation using pith, bark and fibres. P. aleophilum was additionally detected in the lumen of xylem vessels in which tyloses blocked its progression. Different plant responses in specific tissues were additionally visualised. Inoculation of nodes led to restricted colonisation of P. aleophilum and this colonisation was associated with a plant response six weeks post-inoculation. The fungus was however detected in xylem vessels, bark and inside the pith twelve weeks post-inoculation. CONCLUSIONS: These results demonstrate that P. aleophilum colonisation can vary according to the type of tissues and the type of spread using pith, bark and fibres. Woody tissues can respond to the injury and to the presence of this fungus, and xylem fibres play a key role in the early colonisation of the internode by P. aleophilum before the fungus can colonise xylem vessels.

A method to detect and quantify Phaeomoniella chlamydospora and Phaeoacremonium aleophilum DNA in grapevine-wood samples.

Grapevines are sensitive to a wide range of fungal pathogens, including agents such as Phaeomoniella chlamydospora and Phaeoacremonium aleophilum that cause tracheomycosis. In the present study, a procedure for DNA extraction from grapevine woody tissue is first evaluated and shown to be suitable for quantitative analysis. Next, a multiplex real-time PCR method targeting the ß-tubulin gene of the pathogens and the actin gene of plant material is developed and its quantitative capability is verified. This protocol was evaluated in inoculated grapevine-wood samples and in young vines from a nursery and was found to be reliable and highly specific. Results obtained from inoculated cuttings show that the fungal colonization process must be considered regardless of the wood phenotype. An analysis of samples of young vines from the nursery shows that a high rate of contamination occurs at the basis of plants and that this contamination is associated with low quantitative values. This finding provides evidence that in vine nurseries, these fungi may be efficient soil-borne pathogens.

Differential regulation of SERK, LEC1-like and pathogenesis-related genes during indirect secondary somatic embryogenesis in grapevine.

A culture model was developed in Vitis vinifera L., cultivar 'Chardonnay' for studying SE (Somatic Embryogenesis). The auxin 2,4-D (2,4-Dichlorophenoxyacetic acid) was used to induce indirect secondary embryogenesis at a high rate, starting from embryos derived from embryogenic cultures previously obtained. Cotyledonary embryos were shown to be more responsive to SE induction than embryos at the torpedo-stage and were used for molecular analyses. The expression of SERK (Somatic Embryogenesis Receptor Kinase), L1L (Leafy Cotyledon1 Like) and a set of PR (Pathogenesis-Related) genes was monitored during the whole SE process. VvSERK1, VvSERK2 and VvL1L were down-regulated by the 2,4-D treatment but expressed in embryonic tissues. On the contrary, VvPR1, VvPR8, VvPR10.1 and VvPR10.3 were strongly up-regulated by the 2,4-D treatment, and their transcripts were not or only weakly detected in clusters of secondary embryos. VvSERK3, VvPR3 and VvPR10.2 were more stably expressed in all tissues examined. The discussion deals with the putative role of the different genes in grapevine SE.

Characterization of VvSERK1, VvSERK2, VvSERK3 and VvL1L genes and their expression during somatic embryogenesis of grapevine (Vitis vinifera L.).

Little is known about the genes expressed during grapevine somatic embryogenesis. Both groups of Somatic Embryogenesis Receptor Kinase (SERK) and Leafy Cotyledon (LEC and L1L) genes seem to play key roles during somatic embryogenesis in various plant species. Therefore, we identified and analysed the sequences of VvSERK and VvL1L (Leafy cotyledon1-Like) genes. The deduced amino acid sequences of VvSERK1, VvSERK2 and VvSERK3 are very similar to that of registered SERK proteins, with highest homologies for the kinase domain in the C-terminal region. The amino acid sequence of VvL1L presents all the domains that are characteristic for LEC1 and L1L proteins, particularly, the 16 amino acid residues that serve as signature of the B-domain. Phylogenetic analysis distinguishes members of subclass LEC1 and subclass L1L, and VvL1L is closely related to L1L proteins. Using semi-quantitative RT-PCR, we studied gene expression of VvSERK1, VvSERK2, VvSERK3 and VvL1L in calli and somatic embryos obtained from anther culture of Vitis vinifera L. cv Chardonnay. Expression of VvSERK2 is relatively stable during in vitro culture. In contrast, VvSERK1, VvSERK3 and VvL1L are expressed more 4 to 6 weeks after transfer of the calli onto embryo induction medium, before the visible appearance of embryos on the calli as seen by environmental scanning electron microscopy. Later on (8 weeks after transfer) VvSERK1 expression is maintained in the embryogenic calli and VvSERK3 in the embryos, whereas VvL1L expression is very low. All together, these data suggest the involvement of VvSERK and VvL1L genes in grapevine somatic embryogenesis.

NtLRP1, a tobacco leucine-rich repeat gene with a possible role as a modulator of the hypersensitive response.

Plant defense responses against pathogens often involve the restriction of the pathogen to its site of penetration achieved through the combined effects of the hypersensitive response (HR) and its tightly connected localized acquired resistance (LAR). The tobacco DD9-3 expressed sequence tag was previously isolated from a screen designed to isolate genes induced early during the HR, thus potentially involved in the induction/regulation of the HR or LAR. Translation of the open reading frame of DD9-3 revealed a leucine-rich repeat (LRR) domain highly homologous with the receptor domain of a receptor kinase, suggesting a potential function in signaling pathways. The full-length cDNA was cloned. It encodes a small (232 amino acids) LRR protein, designated Nicotiana tabacum leucine-rich protein 1 (NtLRP1), containing a signal peptide, four leucine zipper repeats, five LRR repeats, and a C-terminal domain rich in proline. NtLRP1 expression is induced early during the HR initiated by elicitins, Ralstonia solanacearum, or Tobacco mosaic virus. NtLRP1 coupled with the green fluorescent protein localizes to the endoplasmic reticulum (ER). Loss-of-function through virus-induced gene silencing or through RNA interference did not modify the elicitin-induced HR or LAR. Gain-of-function experiments through transient Agrobacterium tumefaciens-mediated NtLRP1 expression in tobacco leaves caused the suppression of the HR induced by 2 nM elicitin and delayed the HR when the elicitin was applied at higher concentrations. The results suggest that NtLRP1 acts as a modulator of the HR and that retention in the ER is essential for its function.

Identification of tobacco ESTs with a hypersensitive response (HR)-specific pattern of expression and likely involved in the induction of the HR and/or localized acquired resistance (LAR).

Plant defense responses against pathogens often involve the restriction of the pathogen to its site of penetration. Restriction is achieved through the combined effects of the hypersensitive response (HR) and its tightly connected localized acquired resistance (LAR). As LAR is induced by unknown signals released by the cells undergoing the HR, LAR inducing/regulating genes must show a HR-specific pattern of expression. Here, we describe a differential display reverse-transcript polymerase chain reaction (DDRT-PCR) strategy to isolate tobacco expressed sequence tags (ESTs) characterized by such an expression profile, which also characterizes genes involved in the induction/execution of the HR. We compared the DDRT-PCR profile of tobacco cell suspensions treated with beta-megaspermin inducing the HR with that of untreated cells and cells treated with alpha-megaspermin inducing a Defense No Death (DND) phenotype. The expression profile of the selected ESTs was analyzed in tobacco plants expressing a beta-megaspermin-induced HR or a DND phenotype, including LAR, induced by three different elicitors. This comprehensive analysis allowed to identify 24 HR-specific ESTs, half of them shows no or non-significant homology with ESTs and genes in the databases. The other half exhibits homology with genes encoding a receptor-like kinase protein, proteins involved in the regulation of plasma membrane structure, proteins of the ubiquitin/26S proteasome proteolytic system, RNA binding proteins, and a protein hypothesized to be a true regulator of the HR.