The ripening disorder berry shrivel affects anthocyanin biosynthesis and sugar metabolism in Zweigelt grape berries.

MAIN CONCLUSION: Timescale analyses suggest the berry shrivel (BS) disorder is induced before veraison with strong effects on anthocyanin biosynthesis, and minor effects on sugar transport and metabolism. Berry shrivel (BS)-affected grapes have low sugar contents, high acidity, less anthocyanins and flaccid berries. To date no pathogenic causes are known, and studies to elucidate the molecular basis leading to symptom induction and development are limited. Here we present a study on pre-symptomatic as well as symptomatic BS berries to characterize early metabolic changes, with focus on anthocyanin biosynthesis and sugars metabolism. Healthy and BS berries from six sampling time points were used (BBCH79-BBCH89). Our objectives are (1) to search for the beginning of BS-related physiological processes; (2) to search for key enzymes and sugar transporters involved in BS induction and development and (3) to understand the consequences on polyphenol biosynthesis. We employed high performance anion exchange chromatography coupled with pulsed amperometric detection (HPAEC-PAD) and liquid chromatography-tandem mass spectrometry (LC-MS/MS) technology for sugar and polyphenol analyses, respectively. Additionally we conducted expression analyses (qPCR) of key genes and enzymatic activity assays. Our results show that BS-related processes start before veraison, as determined by slightly reduced hexose contents and reduced expression levels of a vacuolar invertase (VviGIN1), two monosaccharide transporters (VviTMT2, VviTMT3) and the anthocyanin biosynthesis (VviUFGT, VviMYBA1/2) genes. Lower amounts of delphinidin and cyanidin glycosidic forms were determined, while caftaric acid, quercetin-3-O-glucuronide and (+)-catechin were increased in BS berries. Although not all results were conclusive, especially for the sugar metabolism, our data provide important knowledge to improve the understanding of the highly complex berry shrivel ripening disorder.

Comparative Transcriptome Analysis of Two Root-Feeding Grape Phylloxera (D. vitifoliae) Lineages Feeding on a Rootstock and V. vinifera.

Grape phylloxera is one of the most dangerous insect pests for worldwide viticulture. The leaf- and root-galling phylloxerid has been managed by grafting European grapevines onto American rootstock hybrids. Recent reports pinpoint the appearance of host-adapted biotypes, but information about the biomolecular characteristics underlying grape phylloxera biotypisation and its role in host performance is scarce. Using RNA-sequencing, we sequenced the transcriptome of two larval stages: L1 (probing) and L2-3 (feeding) larvae of two root-feeding grape phylloxera lineages feeding on the rootstock Teleki 5C (biotype C) and V. vinifera Riesling (biotype A). In total, 7501 differentially expressed genes (DEGs) were commonly modulated by the two biotypes. For the probing larvae, we found an increased number of DEGs functionally associated with insect chemoreception traits, such as odorant-binding proteins, chemosensory proteins, ionotropic, odorant, and gustatory receptors. The transcriptomic profile of feeding larvae was enriched with DEGs associated with the primary metabolism. Larvae feeding on the tolerant rootstock Teleki 5C exhibited higher numbers of plant defense suppression-associated DEGs than larvae feeding on the susceptible host. Based on the identified DEGs, we discuss their potential role for the compatible grape phylloxera-Vitis interaction belowground. This study was the first to compare the transcriptomes of two grape phylloxera lineages feeding on a tolerant and susceptible host, respectively, and to identify DEGs involved in the molecular interaction with these hosts. Our data provide a source for future studies on host adaptation mechanisms of grape phylloxera and help to elucidate grape phylloxera resistance further.

The salivary gland proteome of root-galling grape phylloxera (Daktulosphaira vitifoliae Fitch) feeding on Vitis spp.

The successful parasitisation of a plant by a phytophagous insect is dependent on the delivery of effector molecules into the host. Sedentary gall forming insects, such as grape phylloxera (Daktulosphaira vitifoliae Fitch, Phylloxeridae), secrete multiple effectors into host plant tissues that alter or modulate the cellular and molecular environment to the benefit of the insect. The identification and characterisation of effector proteins will provide insight into the host-phylloxera interaction specifically the gall-induction processes and potential mechanisms of plant resistance. Using proteomic mass spectrometry and in-silico secretory prediction, 420 putative effectors were determined from the salivary glands or the root-feeding D. vitifoliae larvae reared on Teleki 5C (V. berlandieri x V. riparia). Among them, 170 conserved effectors were shared between D. vitifoliae and fourteen phytophagous insect species. Quantitative RT-PCR analysis of five conserved effector candidates (protein disulfide-isomerase, peroxidoredoxin, peroxidase and a carboxypeptidase) revealed that their gene expression decreased, when larvae were starved for 24 h, supporting their assignment as effector molecules. The D. vitifoliae effectors identified here represent a functionally diverse group, comprising both conserved and unique proteins that provide new insight into the D. vitifoliae-Vitis spp. interaction and the potential mechanisms by which D. vitifoliae establishes the feeding site, suppresses plant defences and modulates nutrient uptake.

Grape phylloxera (D. vitifoliae) manipulates SA/JA concentrations and signalling pathways in root galls of Vitis spp.

Overcoming host defensive traits is a prerequisite to establish compatible plant-parasite interactions. Following parasite perception, jasmonic (JA) and salicylic acid (SA) signalling pathways mediate biotic stress signals resulting in the activation of host defence responses. Piercing-sucking grape phylloxera (Daktulosphaira vitifoliae) infests Vitis spp. by the formation of organoid root galls. This study aims to investigate whether host defensive SA/JA signalling pathways are affected during D. vitifoliae infestation. We hypothesize that the JA signalling pathway is induced during larval probing (14 hai). Compatible root gall formation (24 hai - 14 dai) involves the reduction of the JA, but the induction of the SA signaling pathway. T5C (V.berlandieri x V.riparia) cuttings are infested with a D. vitifoliae single founder lineage (biotype C). Phytohormone quantification (HPLC-MS) and transcriptional alterations of JA/SA marker genes (qRT-PCR) are determined in root tissues from larval probing (14 hai) until gall formation (>14 dai). Non-infested root tips are considered controls. Our results show a significant induction of all analysed JA marker genes during insect probing (14 hai), but their significant reduction during early gall formation (24 hai). Following gall formation (5-14 dai) SA and JA-Ile increase. However, only the analysed SA marker genes are induced, whereas JA marker gene expression levels are significantly reduced. Based on this data we conclude that the observed suppression of the JA signalling pathway might represent an important step for the compatible D. vitifoliae - Vitis spp. root interaction. We discuss whether the induced SA defences protect nutritive root galls against soil microbes.

Polyphenolic profiling of roots (Vitis spp.) under grape phylloxera (D. vitifoliae Fitch) attack.

Many plants respond to herbivore attacks by the formation of secondary metabolites, such as polyphenols. Grape phylloxera (Daktulosphaira vitifoliaeFitch) induces organoid root galls on fibrous root tips of tolerant Vitis spp. rootstocks. We aim to understand if and how secondary metabolites are involved in the compatible interaction of D. vitifoliae and tolerant Vitis ssp. rootstocks belowground. We hypothise that D. vitifoliae infestation triggers the accumulation of phenolic key compounds in root gall tissue without preventing the compatible host-parasite interaction on two tolerant rootstocks with different genetic background: Teleki 5C (V. berlandieri x V. riparia) and Fercal (B.C. n°1B x 31 Richter). Plants and insects are grown in isolated climate chambers to sample root tips of non-infested plants (control) as well as root tips and galls of D. vitifoliae infested plants (5-14 dai). HPLC-MS-based analyses of phenolic key compounds are compared with gene expression levels of the biosynthetic phenylpropanoid pathway analysing temporal sequences of D. vitifoliae infested root tissue. The results show that the induction of the phenylpropanoid pathway by D. vitifoliae infestation plays an important role in the plant response. Concentrations of phenolic key compounds vary significantly among the rootstocks tested. Both rootstocks display an accumulation of flavan-3-ols and stilbenes in infested root gall tissue. Comparing the host responses of the two rootstocks Fercal shows a stronger accumulation of stilbenes locally in infested root galls, whereas Teleki 5C indicates elevated amounts of stilbenes in non-infested root tip tissue.