Simultaneous editing of two DMR6 genes in grapevine results in reduced susceptibility to downy mildew.

The reduction of pesticide treatments is of paramount importance for the sustainability of viticulture, and it can be achieved through a combination of strategies, including the cultivation of vines (Vitis vinifera) that are resistant or tolerant to diseases such as downy mildew (DM). In many crops, the knock-out of Downy Mildew Resistant 6 (DMR6) proved successful in controlling DM-resistance, but the effect of mutations in DMR6 genes is not yet known in grapevine. Today, gene editing serves crop improvement with small and specific mutations while maintaining the genetic background of commercially important clones. Moreover, recent technological advances allowed to produce non-transgenic grapevine clones by regeneration of protoplasts edited with the CRISPR/Cas9 ribonucleoprotein. This approach may revolutionize the production of new grapevine varieties and clones, but it requires knowledge about the targets and the impact of editing on plant phenotype and fitness in different cultivars. In this work we generated single and double knock-out mutants by editing DMR6 susceptibility (S) genes using CRISPR/Cas9, and showed that only the combined mutations in VviDMR6-1 and VviDMR6-2 are effective in reducing susceptibility to DM in two table-grape cultivars by increasing the levels of endogenous salicylic acid. Therefore, editing both genes may be necessary for effective DM control in real-world agricultural settings, which could potentially lead to unwanted phenotypes. Additional research, including trials conducted in experimental vineyards, is required to gain a deeper understanding of DMR6-based resistance.

Regeneration of non-chimeric plants from DNA-free edited grapevine protoplasts.

The application of New Breeding Techniques (NBTs) in Vitis vinifera is highly desirable to introduce valuable traits while preserving the genotype of the elite cultivars. However, a broad application of NBTs through standard DNA-based transformation is poorly accepted by public opinion and law regulations in Europe and other countries due to the stable integration of exogenous DNA, which leads to transgenic plants possibly affected by chimerism. A single-cell based approach, coupled with a DNA-free transfection of the CRISPR/Cas editing machinery, constitutes a powerful tool to overcome these problems and maintain the original genetic make-up in the whole organism. We here describe a successful single-cell based, DNA-free methodology to obtain edited grapevine plants, regenerated from protoplasts isolated from embryogenic callus of two table grapevine varieties (V. vinifera cv. Crimson seedless and Sugraone). The regenerated, non-chimeric plants were edited on the downy- and powdery-mildew susceptibility genes, VviDMR6 and VviMlo6 respectively, either as single or double mutants.

Grapevine DMR6-1 Is a Candidate Gene for Susceptibility to Downy Mildew.

Grapevine (Vitis vinifera) is a valuable crop in Europe for both economical and cultural reasons, but highly susceptible to Downy mildew (DM). The generation of resistant vines is of critical importance for a sustainable viticulture and can be achieved either by introgression of resistance genes in susceptible varieties or by mutation of Susceptibility (S) genes, e.g., by gene editing. This second approach offers several advantages: it maintains the genetic identity of cultivars otherwise disrupted by crossing and generally results in a broad-spectrum and durable resistance, but it is hindered by the poor knowledge about S genes in grapevines. Candidate S genes are Downy mildew Resistance 6 (DMR6) and DMR6-Like Oxygenases (DLOs), whose mutations confer resistance to DM in Arabidopsis. In this work, we show that grapevine VviDMR6-1 complements the Arabidopsis dmr6-1 resistant mutant. We studied the expression of grapevine VviDMR6 and VviDLO genes in different organs and in response to the DM causative agent Plasmopara viticola. Through an automated evaluation of causal relationships among genes, we show that VviDMR6-1, VviDMR6-2, and VviDLO1 group into different co-regulatory networks, suggesting distinct functions, and that mostly VviDMR6-1 is connected with pathogenesis-responsive genes. Therefore, VviDMR6-1 represents a good candidate to produce resistant cultivars with a gene-editing approach.

Mining Grapevine Downy Mildew Susceptibility Genes: A Resource for Genomics-Based Breeding and Tailored Gene Editing.

Several pathogens continuously threaten viticulture worldwide. Until now, the investigation on resistance loci has been the main trend to understand the interaction between grapevine and the mildew causal agents. Dominantly inherited gene-based resistance has shown to be race-specific in some cases, to confer partial immunity, and to be potentially overcome within a few years since its introgression. Recently, on the footprint of research conducted in Arabidopsis, putative genes associated with downy mildew susceptibility have been discovered also in the grapevine genome. In this work, we deep-sequenced four putative susceptibility genes-namely VvDMR6.1, VvDMR6.2, VvDLO1, VvDLO2-in 190 genetically diverse grapevine genotypes to discover new sources of broad-spectrum and recessively inherited resistance. Identified Single Nucleotide Polymorphisms were screened in a bottleneck analysis from the genetic sequence to their impact on protein structure. Fifty-five genotypes showed at least one impacting mutation in one or more of the scouted genes. Haplotypes were inferred for each gene and two of them at the VvDMR6.2 gene were found significantly more represented in downy mildew resistant genotypes. The current results provide a resource for grapevine and plant genetics and could corroborate genomic-assisted breeding programs as well as tailored gene editing approaches for resistance to biotic stresses.

Abnormal Endogenous Repression of GA Signaling in a Seedless Table Grape Cultivar with High Berry Growth Response to GA Application.

Gibberellin (GA) application is routinely used in the table grape industry to increase berry size and cluster length. Although grapevine cultivars show a wide range of growth responsiveness to GA3 application, the reasons for these differences is unclear. To shed light on this issue, two commercial grapevine cultivars with contrasting berry response to GA were selected for comparative analysis, in which we tested if the differences in response: (1) is organ-specific or cultivar-related; (2) will be reflected in qualitative/quantitative differences in transcripts/proteins of central components of GA metabolism and signaling and levels of GA metabolites. Our results showed that in addition to the high response of its berries to GA, internodes and rachis of cv. Black finger (BF) presented a greater growth response compared to that of cv. Spring blush (SB). In agreement, the results exposed significant quantitative differences in GA signaling components in several organs of both cultivars. Exceptionally higher level of all three functional VvDELLA proteins was recorded in young BF organs, accompanied by elevated VvGID1 expression and lower VvSLY1b transcripts. Absence of seed traces, low endogenous GA quantities and lower expression of VvGA20ox4 and VvGA3ox3 were also recorded in berries of BF. Our results raise the hypothesis that, in young organs of BF, low expression of VvSLY1b may be responsible for the massive accumulation of VvDELLA proteins, which then leads to elevated VvGID1 levels. This integrated analysis suggests causal relationship between endogenous mechanisms leading to anomalous GA signaling repression in BF, manifested by high quantities of VvDELLA proteins, and greater growth response to GA application.

Gibberellin metabolism in Vitis vinifera L. during bloom and fruit-set: functional characterization and evolution of grapevine gibberellin oxidases.

Gibberellins (GAs) are involved in the regulation of flowering and fruit-set in grapes (Vitis vinifera L.), but the molecular mechanisms behind this process are mostly unknown. In this work, the family of grapevine GA oxidases involved in the biosynthesis and deactivation of GAs was characterized. Six putative GA 20-oxidase (GA20ox), three GA 3-oxidase (GA3ox), and eight GA 2-oxidase (GA2ox) proteins, the latter further divided into five C19-GA 2ox and three C20-GA2ox proteins, were identified. Phylogenetic analyses suggest a common origin of the GA3ox and C19-GA2ox groups and challenge previous evolutionary models. In vitro analysis revealed that all GA3ox and GA20ox enzymes prefer substrates of the non-13-hydroxylation pathway. In addition, ectopic expression of GA2ox genes in Arabidopsis thaliana confirmed the activity of their encoded proteins in vivo. The results show that bioactive GA1 accumulates in opening grapevine flowers, whereas at later developmental stages only GA4 is detected in the setting fruit. By studying the expression pattern of the grapevine GA oxidase genes in different organs, and at different stages of flowering and fruit-set, it is proposed that the pool of bioactive GAs is controlled by a fine regulation of the abundance and localization of GA oxidase transcripts.

Loss of plastoglobule kinases ABC1K1 and ABC1K3 causes conditional degreening, modified prenyl-lipids, and recruitment of the jasmonic acid pathway.

Plastoglobules (PGs) are plastid lipid-protein particles. This study examines the function of PG-localized kinases ABC1K1 and ABC1K3 in Arabidopsis thaliana. Several lines of evidence suggested that ABC1K1 and ABC1K3 form a protein complex. Null mutants for both genes (abc1k1 and abc1k3) and the double mutant (k1 k3) displayed rapid chlorosis upon high light stress. Also, k1 k3 showed a slower, but irreversible, senescence-like phenotype during moderate light stress that was phenocopied by drought and nitrogen limitation, but not cold stress. This senescence-like phenotype involved degradation of the photosystem II core and upregulation of chlorophyll degradation. The senescence-like phenotype was independent of the EXECUTER pathway that mediates genetically controlled cell death from the chloroplast and correlated with increased levels of the singlet oxygen-derived carotenoid ß-cyclocitral, a retrograde plastid signal. Total PG volume increased during light stress in wild type and k1 k3 plants, but with different size distributions. Isolated PGs from k1 k3 showed a modified prenyl-lipid composition, suggesting reduced activity of PG-localized tocopherol cyclase (VTE1), and was consistent with loss of carotenoid cleavage dioxygenase 4. Plastid jasmonate biosynthesis enzymes were recruited to the k1 k3 PGs but not wild-type PGs, while pheophytinase, which is involved in chlorophyll degradation, was induced in k1 k3 and not wild-type plants and was localized to PGs. Thus, the ABC1K1/3 complex contributes to PG function in prenyl-lipid metabolism, stress response, and thylakoid remodeling.

Identification and characterization of the defensin-like gene family of grapevine.

Defensins are a class of small and diverse cysteine-rich proteins found in plants, insects, and vertebrates, which share a common tertiary structure and usually exert broad-spectrum antimicrobial activities. We used a bioinformatic approach to scan the Vitis vinifera genome and identified 79 defensin-like sequences (DEFL) corresponding to 46 genes and allelic variants, plus 33 pseudogenes and gene fragments. Expansion and diversification of grapevine DEFL has occurred after the split from the last common ancestor with the genera Medicago and Arabidopsis. Grapevine DEFL localization on the 'Pinot Noir' genome revealed the presence of several clusters likely evolved through local duplications. By sequencing reverse-transcription polymerase chain reaction products, we could demonstrate the expression of grapevine DEFL with no previously reported record of expression. Many of these genes are predominantly or exclusively expressed in tissues linked to plant reproduction, consistent with findings in other plant species, and some of them accumulated at fruit ripening. The transcripts of five DEFL were also significantly upregulated in tissues infected with Botrytis cinerea, a necrotrophic mold, suggesting a role of these genes in defense against this pathogen. Finally, three novel defensins were discovered among the identified DEFL. They inhibit B. cinerea conidia germination when expressed as recombinant proteins.

Arabidopsis thaliana deficient in two chloroplast ascorbate peroxidases shows accelerated light-induced necrosis when levels of cellular ascorbate are low.

Arabidopsis chloroplasts have a multi-layered defense against hydrogen peroxide (H(2)O(2)) that includes a stromal and thylakoid ascorbate peroxidase (sAPX and tAPX). Single and double null mutants in SAPX and TAPX (sapx and tapx) were each crossed with ascorbate deficient vtc2. The single, double and triple mutants did not show visual light stress phenotypes when grown at control or high light intensities (CL and HL; 120 and 1,000 micromol photons m(-2) s(-1)). Upon shift from CL to HL, mesophyll of expanded leaves of the triple mutant bleached within hours, with exclusion of the major vein areas; this contrasts to reported patterns of cell death under ozone treatment and calatase deficiency. tapx-vtc2 and sapx-vtc2, but not tapx-sapx or single mutants, showed limited bleaching. Bleaching and necrosis were accompanied by accumulation of H(2)O(2). Cellular concentrations of alpha-tocopherol, ascorbate and glutathione showed dramatic increase in response to HL in all eight genotypes and the four vtc2 genotypes accumulated more glutathione under CL than the others. Transcript analysis of other ROS responsive genes in vtc2 and the triple mutant showed up to 20-fold induction after transition to HL, generally irrespective of genotype. We conclude that chloroplast APX proteins in Arabidopsis can be effectively compensated by other endogenous H(2)O(2) detoxification systems, but that low cellular ascorbate levels in absence of chloroplast APX activity are detrimental to the cell during excess light.

Isolation of chloroplast proteins from Arabidopsis thaliana for proteome analysis.

This chapter describes a simple protocol for large-scale chloroplast purification for proteome analysis. The protocol has not been tested for protein import activity but is optimized for chloroplast proteome yield and purity and minimal protein degradation.

High light response of the thylakoid proteome in arabidopsis wild type and the ascorbate-deficient mutant vtc2-2. A comparative proteomics study.

The thylakoid proteome of chloroplasts contains multiple proteins involved in antioxidative defense, protein folding, and repair. To understand this functional protein network, we analyzed the quantitative response of the thylakoid-associated proteome of Arabidopsis (Arabidopsis thaliana) wild type and the ascorbate-deficient mutant vtc2-2 after transition to high light (HL; 1,000 micromol photons m(-2) s(-1)). The soluble thylakoid proteomes of wild type and vtc2-2 were compared after 0, 1, 3, and 5 d of HL using two-dimensional gels with three independent experiments, followed by a multivariant statistical analysis and tandem mass spectrometry. After 5 d of HL, both wild-type and vtc2-2 plants accumulated anthocyanins, increased their total ascorbate content, and lost 10% of photosystem II efficiency, but showed no bleaching. Anthocyanin and total ascorbate concentrations in vtc2-2 were respectively 34% and 20% of wild type, potentially leading to enhanced oxidative stress in vtc2-2. Forty-five protein spots significantly changed as a consequence of genotype, light treatment, or both. Independent confirmation was obtained from western blots. The most significant response was the up-regulation of thylakoid YCF37 likely involved in photosystem I assembly, and specific fibrillins, a flavin reductase-like protein, and an aldolase, each located in thylakoid-associated plastoglobules. Fe-superoxide dismutase was down-regulated in vtc2-2, while Cu,Zn-superoxide dismutase was up-regulated. vtc2-2 also showed a systematic up-regulation of a steroid dehydrogenase-like protein. A number of other stress-related proteins, several thylakoid proteases, and lumenal isomerases did not change, while PsbS increased in wild type upon light stress. These findings are discussed in terms of plastid metabolism and oxidative stress defense, and emphasize that understanding of the chloroplast stress-response network must include the enzymatic role of plastoglobules.

The oligomeric stromal proteome of Arabidopsis thaliana chloroplasts.

This study presents an analysis of the stromal proteome in its oligomeric state extracted from highly purified chloroplasts of Arabidopsis thaliana. 241 proteins (88% with predicted cTP), mostly assembled in oligomeric complexes, were identified by mass spectrometry with emphasis on distinguishing between paralogues. This is critical because different paralogues in a gene family often have different subcellular localizations and/or different expression patterns and functions. The native protein masses were determined for all identified proteins. Comparison with the few well characterized stromal complexes from A. thaliana confirmed the accuracy of the native mass determination, and by extension, the usefulness of the native mass data for future in-depth protein interaction studies. Resolved protein interactions are discussed and compared with an extensive collection of native mass data of orthologues in other plants and bacteria. Relative protein expression levels were estimated from spot intensities and also provided estimates of relative concentrations of individual proteins. No such quantification has been reported so far. Surprisingly proteins dedicated to chloroplast protein synthesis, biogenesis, and fate represented nearly 10% of the total stroma protein mass. Oxidative pentose phosphate pathway, glycolysis, and Calvin cycle represented together about 75%, nitrogen assimilation represented 5-7%, and all other pathways such as biosynthesis of e.g. fatty acids, amino acids, nucleotides, tetrapyrroles, and vitamins B(1) and B(2) each represented less than 1% of total protein mass. Several proteins with diverse functions outside primary carbon metabolism, such as the isomerase ROC4, lipoxygenase 2 involved in jasmonic acid biosynthesis, and a carbonic anhydrase (CA1), were surprisingly abundant in the range of 0.75-1.5% of the total stromal mass. Native images with associated information are available via the Plastid Proteome Database.

In-depth analysis of the thylakoid membrane proteome of Arabidopsis thaliana chloroplasts: new proteins, new functions, and a plastid proteome database.

An extensive analysis of the Arabidopsis thaliana peripheral and integral thylakoid membrane proteome was performed by sequential extractions with salt, detergent, and organic solvents, followed by multidimensional protein separation steps (reverse-phase HPLC and one- and two-dimensional electrophoresis gels), different enzymatic and nonenzymatic protein cleavage techniques, mass spectrometry, and bioinformatics. Altogether, 154 proteins were identified, of which 76 (49%) were alpha-helical integral membrane proteins. Twenty-seven new proteins without known function but with predicted chloroplast transit peptides were identified, of which 17 (63%) are integral membrane proteins. These new proteins, likely important in thylakoid biogenesis, include two rubredoxins, a potential metallochaperone, and a new DnaJ-like protein. The data were integrated with our analysis of the lumenal-enriched proteome. We identified 83 out of 100 known proteins of the thylakoid localized photosynthetic apparatus, including several new paralogues and some 20 proteins involved in protein insertion, assembly, folding, or proteolysis. An additional 16 proteins are involved in translation, demonstrating that the thylakoid membrane surface is an important site for protein synthesis. The high coverage of the photosynthetic apparatus and the identification of known hydrophobic proteins with low expression levels, such as cpSecE, Ohp1, and Ohp2, indicate an excellent dynamic resolution of the analysis. The sequential extraction process proved very helpful to validate transmembrane prediction. Our data also were cross-correlated to chloroplast subproteome analyses by other laboratories. All data are deposited in a new curated plastid proteome database (PPDB) with multiple search functions (http://cbsusrv01.tc.cornell.edu/users/ppdb/). This PPDB will serve as an expandable resource for the plant community.

Clp protease complexes from photosynthetic and non-photosynthetic plastids and mitochondria of plants, their predicted three-dimensional structures, and functional implications.

Tetradecameric Clp protease core complexes in non-photosynthetic plastids of roots, flower petals, and in chloroplasts of leaves of Arabidopsis thaliana were purified based on native mass and isoelectric point and identified by mass spectrometry. The stoichiometry between the subunits was determined. The protease complex consisted of one to three copies of five different serine-type protease Clp proteins (ClpP1,3-6) and four non-proteolytic ClpR proteins (ClpR1-4). Three-dimensional homology modeling showed that the ClpP/R proteins fit well together in a tetradecameric complex and also indicated unique contributions for each protein. Lateral exit gates for proteolysis products are proposed. In addition, ClpS1,2, unique to land plants, tightly interacted with this core complex, with one copy of each per complex. The three-dimensional modeling show that they do fit well on the axial sites of the ClpPR cores. In contrast to plastids, plant mitochondria contained a single approximately 320-kDa homo-tetradecameric ClpP2 complex, without association of ClpR or ClpS proteins. It is surprising that the Clp core composition appears identical in all three plastid types, despite the remarkable differences in plastid proteome composition. This suggests that regulation of plastid proteolysis by the Clp machinery is not through differential regulation of ClpP/R/S gene expression, but rather through substrate recognition mechanisms and regulated interaction of chaperone-like molecules (ClpS1,2 and others) to the ClpP/R core.

A new approach for plant proteomics: characterization of chloroplast proteins of Arabidopsis thaliana by top-down mass spectrometry.

A recently developed methodology for the characterization of complex proteomes, top-down Fourier transform mass spectrometry (FTMS), is applied for the first time to a plant proteome, that of the model plant Arabidopsis thaliana. Of the 3000 proteins predicted by the genome sequence, 97 were recently identified in two separate "bottom-up" mass spectrometry studies in which the proteins were purified and digested and in which the mass spectrometry-measured mass values of the resulting peptides matched against those expected from the DNA-predicted proteins. In the top-down approach applied here, molecular ions from a protein mixture are purified, weighed exactly (+/-1 Da), and fragmented in the FTMS. Of the 22 molecular weight values found in three isolated mixtures, 7 were chosen, and their primary structures were fully characterized; in only one case was the bottom-up structure in full agreement. The top-down technique is not only efficient for identification of the DNA-predicted precursors, such as that of a protein present as a 5% mixture component, but also for characterization of the primary structure of the final protein. For two proteins the previously predicted cleavage site for loss of the signal peptide was found to be incorrect. Two 27-kDa proteins are fully characterized, although they are found to differ by only 12 residues and 6 Da in mass in a 3:1 ratio; the bottom-up studies did not distinguish these proteins. Direct tandem mass spectrometry dissociation of two 15-kDa molecular ions showed >90% sequence similarity, whereas three-stage mass spectrometry traced their +14-Da molecular mass discrepancies to an unusual N-methylation on the N-terminal amino group; the bottom-up approach identified only one precursor protein. The high potential of the top-down FTMS approach for characterization as well as identification of complex plant proteomes should provide a real incentive for its further automation.