The grape powdery mildew resistance loci Ren2, Ren3, Ren4D, Ren4U, Run1, Run1.2b, Run2.1, and Run2.2 activate different transcriptional responses to Erysiphe necator.

Multiple grape powdery mildew (PM) genetic resistance (R) loci have been found in wild grape species. Little is known about the defense responses associated with each R locus. In this study, we compare the defense mechanisms associated with PM resistance in interspecific crosses segregating for a single R locus from Muscadinia rotundifolia (Run1, Run1.2b, Run2.1, Run2.2), Vitis cinerea (Ren2), V. romanetii (Ren4D and Ren4U), and the interspecific hybrid Villard blanc (Ren3). By combining optical microscopy, visual scoring, and biomass estimation, we show that the eight R loci confer resistance by limiting infection at different stages. We assessed the defense mechanisms triggered in response to PM at 1 and 5 days post-inoculation (dpi) via RNA sequencing. To account for the genetic differences between species, we developed for each accession a diploid synthetic reference transcriptome by incorporating into the PN40024 reference homozygous and heterozygous sequence variants and de novo assembled transcripts. Most of the R loci exhibited a higher number of differentially expressed genes (DEGs) associated with PM resistance at 1 dpi compared to 5 dpi, suggesting that PM resistance is mostly associated with an early transcriptional reprogramming. Comparison of the PM resistance-associated DEGs showed a limited overlap between pairs of R loci, and nearly half of the DEGs were specific to a single R locus. The largest overlap of PM resistance-associated DEGs was found between Ren3 +, Ren4D +, and Ren4U + genotypes at 1 dpi, and between Ren4U + and Run1 + accessions at 5 dpi. The Ren3 +, Ren4D +, and Ren4U + were also found to have the highest number of R locus-specific DEGs in response to PM. Both shared and R locus-specific DEGs included genes from different defense-related categories, indicating that the presence of E. necator triggered distinct transcriptional responses in the eight R loci.

Vitis Species from the Southwestern United States Vary in Their Susceptibility to Powdery Mildew.

The European grapevine (Vitis vinifera L.) has been cultivated in North America for about 500 years. One of the major limitations to its culture is the powdery mildew (PM) fungus, Erysiphe necator Schw. This study reports on the most extensive screening of Vitis species from the southwestern United States and northern Mexico for resistance to PM, testing 147 accessions of 13 Vitis species. In addition, Vitis vinifera cv. Carignane, a highly susceptible wine grape cultivar, was used as a reference to evaluate the effect of the inoculum 14 days postinoculation. Inoculation was done with a vacuum-operated settling tower using a broadly virulent isolate of E. necator, the C-strain. Resistant accessions (nine), moderately susceptible accessions (39), and highly susceptible accessions (99) were detected. The resistant accessions were then inoculated with an additional fungal isolate, e1-101, and they retained their resistance. Vitis species susceptibility was not associated with a North-South gradation, but Western species were more susceptible than Midwestern and Eastern species. All five of the V. monticola accessions were susceptible, as were the accessions of V. treleasei. The species V. acerifolia, V. candicans, V. cinerea, and V. × doaniana had significantly more resistant to moderately susceptible accessions compared with V. arizonica, V. berlandieri, V. californica, V. × champinii, V. girdiana, V. riparia, and V. rupestris, which had relatively more susceptible accessions than the other species. This research identified new sources of PM resistance in Vitis from the southwestern United States that could be incorporated into PM resistance breeding programs throughout the world.

Field-Collected Glassy-Winged Sharpshooters (Hemiptera: Cicadellidae) Perform More Xylella fastidiosa-Inoculating Behaviors on Susceptible Vitis vinifera cv. 'Chardonnay' Than on Resistant Vitis champinii Grapevines.

The glassy-winged sharpshooter, Homalodisca vitripennis (Germar) (Hemiptera: Cicadellidae: Cicadellinae), is an introduced vector of the xylem-dwelling bacterium Xylella fastidiosa Wells et al. (Xanthomonadales: Xanthomonadaceae) in California. Once acquired, X. fastidiosa colonizes the functional foregut of the vector. Bacteria can be inoculated directly into grapevine xylem during the xylem cell acceptance process in sharpshooter stylet probing, represented by the X wave using electropenetrography (EPG). Since 2001, an effort has been underway to develop PD-resistant grapevines, Vitis vinifera L., through classical breeding of various species of resistant wild grapevines with more susceptible V. vinifera. The present study used EPG to compare H. vitripennis stylet probing behaviors in a factorial experiment between V. champinii (a V. candicans/V. rupestris natural hybrid with moderate trichomes) and V. vinifera cv. 'Chardonnay' (which lacks trichomes) that had been gently scraped to remove trichomes or was not scraped. Results showed that sharpshooters performed significantly more X waves/X. fastidiosa inoculation behaviors of overall longer duration on Chardonnay than on V. champinii, regardless of shaving or not-shaving to remove trichomes. In addition, trichomes caused more frequent standing/walking/test-probing behaviors on V. champinii, whose xylem was rapidly accepted for sharpshooter ingestion once probing began. Thus, EPG can detect a novel type of grapevine resistance to X. fastidiosa-to the vector's probing process and inoculation of bacteria-in addition to the bacterial infection and symptom development processes that are the basis for most resistance breeding today. Future research could use EPG to screen grapevines for this novel type of resistance.

Inherent and Stress-Induced Responses of Fine Root Morphology and Anatomy in Commercial Grapevine Rootstocks with Contrasting Drought Resistance.

Some grapevine rootstocks perform better than others during and after drought events, yet it is not clear how inherent and stress-induced differences in root morphology and anatomy along the length of fine roots are involved in these responses. Using a variety of growing conditions and plant materials, we observed significant differences in root diameter, specific root length (SRL) and root diameter distribution between two commonly used commercial grapevine rootstocks: Richter 110 (110R; drought resistant) and Millardet et de Grasset 101-14 (101-14Mgt; drought sensitive). The 110R consistently showed greater root diameters with smaller SRL and proportion of root length comprised of fine lateral roots. The 110R also exhibited significantly greater distance from tip to nearest lateral, longer white root length, and larger proportion of root length that is white under drought stress. Mapping of fine root cortical lacunae showed similar patterns between the rootstocks; mechanical failure of cortical cells was common in the maturation zone, limited near the root tip, and increased with drought stress for both genotypes; however, lacuna formed under wetter soil conditions in 110R. Results suggest that drought resistance in grapevine rootstocks is associated with thick, limitedly branched roots with a larger proportion of white-functional roots that tend to form lacuna under more mild water deficit, all of which likely favor continued resource acquisition at depth.

Differences in grapevine rootstock sensitivity and recovery from drought are linked to fine root cortical lacunae and root tip function.

Structural changes during severe drought stress greatly modify the hydraulic properties of fine roots. Yet, the physiological basis behind the restoration of fine root water uptake capacity during water recovery remains unknown. Using neutron radiography (NR), X-ray micro-computed tomography (micro-CT), fluorescence microscopy, and fine root hydraulic conductivity measurements (Lpr ), we examined how drought-induced changes in anatomy and hydraulic properties of contrasting grapevine rootstocks are coupled with fine root growth dynamics during drought and return of soil moisture. Lacunae formation in drought-stressed fine roots was associated with a significant decrease in fine root Lpr for both rootstocks. However, lacunae formation occurred under milder stress in the drought-resistant rootstock, 110R. Suberin was deposited at an earlier developmental stage in fine roots of 101-14Mgt (i.e. drought susceptible), probably limiting cortical lacunae formation during mild stress. During recovery, we found that only 110R fine roots showed rapid re-establishment of elongation and water uptake capacity and we found that soil water status surrounding root tips differed between rootstocks as imaged with NR. These data suggest that drought resistance in grapevine rootstocks is associated with rapid re-establishment of growth and Lpr near the root tip upon re-watering by limiting competing sites along the root cylinder.

Genetic analysis reveals an east-west divide within North American Vitis species that mirrors their resistance to Pierce's disease.

Pierce's disease (PD) caused by the bacterium Xylella fastidiosa is a deadly disease of grapevines. This study used 20 SSR markers to genotype 326 accessions of grape species collected from the southeastern and southwestern United States, Mexico and Costa Rica. Two hundred sixty-six of these accessions, and an additional 12 PD resistant hybrid cultivars developed from southeastern US grape species, were evaluated for PD resistance. Disease resistance was evaluated by quantifying the level of bacteria in stems and measuring PD symptoms on the canes and leaves. Both Bayesian clustering and principal coordinate analyses identified two groups with an east-west divide: group 1 consisted of grape species from the southeastern US and Mexico, and group 2 consisted of accessions collected from the southwestern US and Mexico. The Sierra Madre Oriental mountain range appeared to be a phylogeographic barrier. The state of Texas was identified as a potential hybridization zone. The hierarchal STRUCTURE analysis on each group showed clustering of unique grape species. An east-west divide was also observed for PD resistance. With the exception of Vitis candicans and V. cinerea accessions collected from Mexico, all other grape species as well as the resistant southeastern hybrid cultivars were susceptible to the disease. Southwestern US grape accessions from drier desert regions showed stronger resistance to the disease. Strong PD resistance was observed within three distinct genetic clusters of V. arizonica which is adapted to drier environments and hybridizes freely with other species across its wide range.

Impact of phenolic compounds on progression of Xylella fastidiosa infections in susceptible and PdR1-locus containing resistant grapevines.

Pierce's disease is of major concern for grapevine (Vitis vinifera) production wherever the bacterial pathogen Xylella fastidiosa and its vectors are present. Long-term management includes the deployment of resistant grapevines such as those containing the PdR1 locus from the wild grapevine species Vitis arizonica, which do not develop Pierce's disease symptoms upon infection. However, little is understood about how the PdR1 locus functions to prevent disease symptom development. Therefore, we assessed the concentrations of plant defense-associated compounds called phenolics in healthy and X. fastidiosa-infected PdR1-resistant and susceptible grapevine siblings over time. Soluble foliar phenolic levels, especially flavonoids, in X. fastidiosa-infected PdR1-resistant grapevines were discovered to be significantly lower than those in infected susceptible grapevines. Therefore, it was hypothesized that PdR1-resistant grapevines, by possessing lowered flavonoid levels, affects biofilm formation and causes reduced X. fastidiosa intra-plant colonization, thus limiting the ability to increase pathogen populations and cause Pierce's disease. These results therefore reveal that differences in plant metabolite levels might be a component of the mechanisms that PdR1 utilizes to prevent Pierce's disease.

Genetic mapping and survey of powdery mildew resistance in the wild Central Asian ancestor of cultivated grapevines in Central Asia.

Cultivated grapevines (Vitis vinifera) lack resistance to powdery mildew (PM) with few exceptions. Resistance to this pathogen within V. vinifera has been reported in earlier studies and identified as the Ren1 locus in two Central Asian table grape accessions. Other PM-resistant cultivated varieties and accessions of the wild ancestor V. vinifera subsp. sylvestris were soon identified raising questions regarding the origin of the resistance. In this study, F1 breeding populations were developed with a PM susceptible V. vinifera subsp. vinifera breeding line and a PM-resistant subsp. sylvestris accession. Genotyping was carried out with five Ren1 locus linked SSR markers. A PM resistance locus explaining up to 96% of the phenotypic variation was identified in the same genomic position, where the Ren1 locus was previously reported. New SSR marker alleles linked with the resistance locus were identified. We report results of PM resistance in multiple accessions of subsp. sylvestris collected as seed lots or cuttings from five countries in the Caucasus and Central Asia. A total of 20 females from 11 seed lots and 19 males from nine seed lots collected from Georgia, Armenia, and Azerbaijan were resistant to PM. Three male and one female plant collected as cuttings from Afghanistan and Iran were also resistant to PM. Allelic analysis of markers linked with the Ren1 locus in conjunction with disease evaluation data found a high diversity of allelic haplotypes, which are only possible via recombination events occurring over a long time period. Sequence analysis of two alleles of the SSR marker that cosegregates with the resistance found SNPs that were present in the wild progenitor and in cultivated forms. Variable levels of PM resistance among the tested accessions were also observed. These lines of evidence suggest that the powdery mildew fungus may have been present in Asia for a longer time than currently thought, giving the wild progenitor V. vinifera subsp. sylvestris time to coevolve with and develop resistance to this pathogen.

Modeling vegetative vigour in grapevine: unraveling underlying mechanisms.

Mechanistic modeling constitutes a powerful tool to unravel complex biological phenomena. This study describes the construction of a mechanistic, dynamic model for grapevine plant growth and canopy biomass (vigor). To parametrize and validate the model, the progeny from a cross of Ramsey (Vitis champinii) × Riparia Gloire (V. riparia) was evaluated. Plants with different vigor were grown in a greenhouse during the summer of 2014 and 2015. One set of plants was grafted with Cabernet Sauvignon. Shoot growth rate (b), leaf area (LA), dry biomass, whole plant and root specific hydraulic conductance (kH and Lpr), stomatal conductance (gs), and water potential (Ψ) were measured. Partitioning indices and specific leaf area (SLA) were calculated. The model includes an empirical fit of a purported seasonal pattern of bioactive GAs based on published seasonal evolutionary levels and reference values. The model provided a good fit of the experimental data, with R = 0.85. Simulation of single trait variations defined the individual effect of each variable on vigor determination. The model predicts, with acceptable accuracy, the vigor of a young plant through the measurement of Lpr and SLA. The model also permits further understanding of the functional traits that govern vigor, and, ultimately, could be considered useful for growers, breeders and those studying climate change.

The genomic diversification of grapevine clones.

BACKGROUND: Vegetatively propagated clones accumulate somatic mutations. The purpose of this study was to better appreciate clone diversity and involved defining the nature of somatic mutations throughout the genome. Fifteen Zinfandel winegrape clone genomes were sequenced and compared to one another using a highly contiguous genome reference produced from one of the clones, Zinfandel 03. RESULTS: Though most heterozygous variants were shared, somatic mutations accumulated in individual and subsets of clones. Overall, heterozygous mutations were most frequent in intergenic space and more frequent in introns than exons. A significantly larger percentage of CpG, CHG, and CHH sites in repetitive intergenic space experienced transition mutations than in genic and non-repetitive intergenic spaces, likely because of higher levels of methylation in the region and because methylated cytosines often spontaneously deaminate. Of the minority of mutations that occurred in exons, larger proportions of these were putatively deleterious when they occurred in relatively few clones. CONCLUSIONS: These data support three major conclusions. First, repetitive intergenic space is a major driver of clone genome diversification. Second, clones accumulate putatively deleterious mutations. Third, the data suggest selection against deleterious variants in coding regions or some mechanism by which mutations are less frequent in coding than noncoding regions of the genome.

Differences in hydraulic traits of grapevine rootstocks are not conferred to a common Vitis vinifera scion.

Cultivars of grapevine are commonly grafted onto rootstocks to improve resistance against biotic and abiotic stress, however, it is not clear whether known differences in hydraulic traits are conferred from rootstocks to a common scion. We recently found that Vitis riparia and Vitis champinii differed in drought-induced embolism susceptibility and repair, which was related to differences in root pressure generation after rewatering (Knipfer et al. 2015). In the present study, we tested whether these and other physiological responses to drought are conferred to a common V. vinifera scion (Cabernet Sauvignon) grafted on V. riparia and V. champinii rootstocks. We measured xylem embolism formation/repair using in vivo microCT imaging, which was accompanied with analysis of leaf gas exchange, osmotic adjustment and root pressure. Our data indicate that differences in scion physiological behaviour for both rootstock combinations were negligible, suggesting that the sensitivity of Cabernet Sauvignon scion to xylem embolism formation/repair, leaf gas exchange and osmotic adjustment is unaffected by either V. riparia or V. champinii rootstock in response to drought stress.

Population Structure, Diversity and Reproductive Mode of the Grape Phylloxera (Daktulosphaira vitifoliae) across Its Native Range.

Grape Phylloxera, Daktulosphaira vitifoliae, is a gall-forming insect that feeds on the leaves and roots of many Vitis species. The roots of the cultivated V. vinifera cultivars and hybrids are highly susceptible to grape phylloxera feeding damage. The native range of this insect covers most of North America, and it is particularly abundant in the eastern and central United States. Phylloxera was introduced from North America to almost all grape-growing regions across five of the temperate zone continents. It devastated vineyards in each of these regions causing large-scale disruptions to grape growers, wine makers and national economies. In order to understand the population diversity of grape phylloxera in its native range, more than 500 samples from 19 States and 34 samples from the introduced range (northern California, Europe and South America) were genotyped with 32 simple sequence repeat markers. STRUCTURE, a model based clustering method identified five populations within these samples. The five populations were confirmed by a neighbor-joining tree and principal coordinate analysis (PCoA). These populations were distinguished by their Vitis species hosts and their geographic locations. Samples collected from California, Europe and South America traced back to phylloxera sampled in the northeastern United States on V. riparia, with some influence from phylloxera collected along the Atlantic Coast and Central Plains on V. vulpina. Reproductive statistics conclusively confirmed that sexual reproduction is common in the native range and is combined with cyclical parthenogenesis. Native grape phylloxera populations were identified to be under Hardy-Weinberg equilibrium. The identification of admixed samples between many of these populations indicates that shared environments facilitate sexual reproduction between different host associated populations to create new genotypes of phylloxera. This study also found that assortative mating might occur across the sympatric range of the V. vulpina west and V. cinerea populations.

Drought Stress Enhances Up-Regulation of Anthocyanin Biosynthesis in Grapevine leafroll-associated virus 3-Infected in vitro Grapevine (Vitis vinifera) Leaves.

Reddish-purple coloration on the leaf blades and downward rolling of leaf margins are typical symptoms of grapevine leafroll disease (GLD) in red-fruited grapevine cultivars. These typical symptoms are attributed to the expression of genes encoding enzymes for anthocyanins synthesis, and the accumulation of flavonoids in diseased leaves. Drought has been proven to accelerate development of GLD symptoms in virus-infected leaves of grapevine. However, it is not known how drought affects GLD expression nor how anthocyanin biosynthesis in virus-infected leaves is altered. The present study used HPLC to determine the types and levels of anthocyanins, and applied reverse transcription quantitative polymerase chain reaction (RT-qPCR) to analyze the expression of genes encoding enzymes for anthocyanin synthesis. Plantlets of Grapevine leafroll-associated virus 3 (GLRaV-3)-infected Vitis vinifera 'Cabernet Sauvignon' were grown in vitro under PEG-induced drought stress. HPLC found no anthocyanin-related peaks in the healthy plantlets with or without PEG-induced stress, while 11 peaks were detected in the infected plantlets with or without PEG-induced drought stress, but the peaks were significantly higher in infected drought-stressed plantlets. Increased accumulation of total anthocyanin compounds was related to the development of GLD symptoms in the infected plantlets under PEG stress. The highest level of up-regulated gene expression was found in GLRaV-3-infected leaves with PEG-induced drought stress. Analyses of variance and correlation of anthocyanin accumulation with related gene expression levels found that GLRaV-3-infection was the key factor in increased anthocyanin accumulation. This accumulation involved the up-regulation of two key genes, MYBA1 and UFGT, and their expression levels were further enhanced by drought stress.

Overexpression of a thaumatin-like protein gene from Vitis amurensis improves downy mildew resistance in Vitis vinifera grapevine.

Downy mildew is a highly destructive disease in grapevine production. A gene encoding pathogenesis-related (PR) thaumatin-like protein was isolated from the downy mildew-resistant grapevine "Zuoshan-1," a clonal selection from wild Vitis amurensis Rupr. The predicted thaumatin-like protein (VaTLP) has 225 amino acids and it is acidic, with a calculated isoelectric point of 4.8. The full length of the VaTLP gene was transformed into somatic embryogenic calli of V. vinifera 'Thompson Seedless' via Agrobacterium tumefaciens. Real-time RT-PCR confirmed that the VaTLP gene was expressed at a high level in the transgenic grapevines. Improved resistance of the transgenic lines against downy mildew was evaluated using leaf disks and whole plants inoculated with Plasmopara viticola, the pathogen causing grapevine downy mildew disease. Bioassay of the pathogen showed that both hyphae growth and asexual reproduction were inhibited significantly among the transgenic plants. Histological analysis also confirmed this disease resistance by demonstrating the inhibition and malformation of hyphae development in leaf tissue of the transgenic plants. These results indicated that the accumulation of VaTLP could enhance resistance to P. viticola in transgenic 'Thompson Seedless' grapevines.

Identification of two novel powdery mildew resistance loci, Ren6 and Ren7, from the wild Chinese grape species Vitis piasezkii.

BACKGROUND: Grapevine powdery mildew Erysiphe necator is a major fungal disease in all grape growing countries worldwide. Breeding for resistance to this disease is crucial to avoid extensive fungicide applications that are costly, labor intensive and may have detrimental effects on the environment. In the past decade, Chinese Vitis species have attracted attention from grape breeders because of their strong resistance to powdery mildew and their lack of negative fruit quality attributes that are often present in resistant North American species. In this study, we investigated powdery mildew resistance in multiple accessions of the Chinese species Vitis piasezkii that were collected during the 1980 Sino-American botanical expedition to the western Hubei province of China. RESULTS: A framework genetic map was developed using simple sequence repeat markers in 277 seedlings of an F1 mapping population arising from a cross of the powdery mildew susceptible Vitis vinifera selection F2-35 and a resistant accession of V. piasezkii DVIT2027. Quantitative trait locus analyses identified two major powdery mildew resistance loci on chromosome 9 (Ren6) and chromosome 19 (Ren7) explaining 74.8 % of the cumulative phenotypic variation. The quantitative trait locus analysis for each locus, in the absence of the other, explained 95.4 % phenotypic variation for Ren6, while Ren7 accounted for 71.9 % of the phenotypic variation. Screening of an additional 259 seedlings of the F1 population and 910 seedlings from four pseudo-backcross populations with SSR markers defined regions of 22 kb and 330 kb for Ren6 and Ren7 in the V. vinifera PN40024 (12X) genome sequence, respectively. Both R loci operate post-penetration through the induction of programmed cell death, but vary significantly in the speed of response and degree of resistance; Ren6 confers complete resistance whereas Ren7 confers partial resistance to the disease with reduced colony size. A comparison of the kinetics of induction of powdery mildew resistance mediated by Ren6, Ren7 and the Run1 locus from Muscadinia rotundifolia, indicated that the speed and strength of resistance conferred by Ren6 is greater than that of Run1 which, in turn, is superior to that conferred by Ren7. CONCLUSIONS: This is the first report of mapping powdery mildew resistance in the Chinese species V. piasezkii. Two distinct powdery mildew R loci designated Ren6 and Ren7 were found in multiple accessions of this Chinese grape species. Their location on different chromosomes to previously reported powdery mildew resistance R loci offers the potential for grape breeders to combine these R genes with existing powdery mildew R loci to produce grape germplasm with more durable resistance against this rapidly evolving fungal pathogen.

Responses of In vitro-Grown Plantlets (Vitis vinifera) to Grapevine leafroll-Associated Virus-3 and PEG-Induced Drought Stress.

Stresses caused by viral diseases and drought have long threatened sustainable production of grapevine. These two stresses frequently occur simultaneously in many of grapevine growing regions of the world. We studied responses of in vitro-grown plantlets (Vitis vinifera) to Grapevine leafroll associated virus-3 (GLRaV-3) and PEG-induced drought stress. Results showed that stress induced by either virus infection or drought had negative effects on vegetative growth, caused significant decreases and increases in total soluble protein and free proline, respectively, induced obvious cell membrane damage and cell death, and markedly increased accumulations of [Formula: see text] and H2O2. Co-stress by virus and drought had much severer effects than single stress on the said parameters. Virus infection alone did not cause significant alternations in activities of POD, ROS, and SOD, and contents of MDA, which, however, markedly increased in the plantlets when grown under single drought stress and co-stress by the virus and drought. Levels of ABA increased, while those of IAA decreased in the plantlets stressed by virus infection or drought. Simultaneous stresses by the virus and drought had co-effects on the levels of ABA and IAA. Up-regulation of expressions of ABA biosynthesis genes and down-regulation of expressions of IAA biosynthesis genes were responsible for the alternations of ABA and IAA levels induced by either the virus infection or drought stress and co-stress by them. Experimental strategies established in the present study using in vitro system facilitate investigations on 'pure' biotic and abiotic stress on plants. The results obtained here provide new insights into adverse effects of stress induced by virus and drought, in single and particularly their combination, on plants, and allow us to re-orientate agricultural managements toward sustainable development of the agriculture.

Comparative transcriptomics of Central Asian Vitis vinifera accessions reveals distinct defense strategies against powdery mildew.

Grape powdery mildew (PM), caused by the biotrophic ascomycete Erysiphe necator, is a devastating fungal disease that affects most Vitis vinifera cultivars. We have previously identified a panel of V. vinifera accessions from Central Asia with partial resistance to PM that possess a Ren1-like local haplotype. In this study, we show that in addition to the typical Ren1-associated late post-penetration resistance, these accessions display a range of different levels of disease development suggesting that alternative alleles or additional genes contribute to determining the outcome of the interaction with the pathogen. To identify potential Ren1-dependent transcriptional responses and functions associated with the different levels of resistance, we sequenced and analyzed the transcriptomes of these Central Asian accessions at two time points of PM infection. Transcriptomes were compared to identify constitutive differences and PM-inducible responses that may underlie their disease resistant phenotype. Responses to E. necator in all resistant accessions were characterized by an early up-regulation of 13 genes, most encoding putative defense functions, and a late down-regulation of 32 genes, enriched in transcriptional regulators and protein kinases. Potential Ren1-dependent responses included a hotspot of co-regulated genes on chromosome 18. We also identified 81 genes whose expression levels and dynamics correlated with the phenotypic differences between the most resistant accessions 'Karadzhandahal', DVIT3351.27, and O34-16 and the other genotypes. This study provides a first exploration of the functions associated with varying levels of partial resistance to PM in V. vinifera accessions that can be exploited as sources of genetic resistance in grape breeding programs.

Strategies for RUN1 Deployment Using RUN2 and REN2 to Manage Grapevine Powdery Mildew Informed by Studies of Race Specificity.

The Toll/interleukin-1 receptor nucleotide-binding site leucine-rich repeat gene, "resistance to Uncinula necator 1" (RUN1), from Vitis rotundifolia was recently identified and confirmed to confer resistance to the grapevine powdery mildew fungus Erysiphe necator (syn. U. necator) in transgenic V. vinifera cultivars. However, sporulating powdery mildew colonies and cleistothecia of the heterothallic pathogen have been found on introgression lines containing the RUN1 locus growing in New York (NY). Two E. necator isolates collected from RUN1 vines were designated NY1-131 and NY1-137 and were used in this study to inform a strategy for durable RUN1 deployment. In order to achieve this, fitness parameters of NY1-131 and NY1-137 were quantified relative to powdery mildew isolates collected from V. rotundifolia and V. vinifera on vines containing alleles of the powdery mildew resistance genes RUN1, RUN2, or REN2. The results clearly demonstrate the race specificity of RUN1, RUN2, and REN2 resistance alleles, all of which exhibit programmed cell death (PCD)-mediated resistance. The NY1 isolates investigated were found to have an intermediate virulence on RUN1 vines, although this may be allele specific, while the Musc4 isolate collected from V. rotundifolia was virulent on all RUN1 vines. Another powdery mildew resistance locus, RUN2, was previously mapped in different V. rotundifolia genotypes, and two alleles (RUN2.1 and RUN2.2) were identified. The RUN2.1 allele was found to provide PCD-mediated resistance to both an NY1 isolate and Musc4. Importantly, REN2 vines were resistant to the NY1 isolates and RUN1REN2 vines combining both genes displayed additional resistance. Based on these results, RUN1-mediated resistance in grapevine may be enhanced by pyramiding with RUN2.1 or REN2; however, naturally occurring isolates in North America display some virulence on vines with these resistance genes. The characterization of additional resistance sources is needed to identify resistance gene combinations that will further enhance durability. For the resistance gene combinations currently available, we recommend using complementary management strategies, including fungicide application, to reduce populations of virulent isolates.

Hybridization of cultivated Vitis vinifera with wild V. californica and V. girdiana in California.

Hybridization of introduced domesticates and closely related natives is well documented in annual crops. The widespread introduction of the domesticated grapevine, Vitis vinifera, into California where it overlaps with two native congenerics, with which it is interfertile, provides opportunity to investigate hybridization between woody perennials. Although geographically widespread, the introduction over the past two centuries has been limited to a few elite clonal cultivars, providing a unique opportunity to study the effects of hybridization on the native species. The amount of hybridization with V. vinifera and the genetic diversity of wild-growing Vitis californica and Vitis girdiana were examined using nineteen microsatellite markers. STRUCTURE analysis was used to define hybrid and introgressed individuals and to analyze genetic structure of the native species. FAMOZ software was used to identify which V. vinifera cultivars served as parents of F 1 hybrids. The three species were clearly distinguished by STRUCTURE analysis. Thirty percent of 119 V. californica vines were hybrids. The domesticated parent was identified for 16 F 1 hybrid vines; the original California cultivar, 'Mission', was the parent of eight. Backcrosses were also found, showing introgression into subsequent generations. Similar results were obtained for a small sample of V. girdiana. Removing hybrids greatly reduced the genetic variation of the presumed pure species, among which there was essentially no genetic structure. Limited genetic variability indicates the California natives may be threatened by genetic erosion. The discovery of F 1 hybrids of 'Mission', a cultivar not grown in the areas for ~100 years, suggests long generation times for wild vines that, often, grow into expansive liana and propagate by layering, all factors that limit recruitment in populations already disjunct by habitat lose. Hermaphroditic flowers and fruit that is more attractive to birds may favor the production of backcross seed and establishment of introgressed individuals.

Adaptive genomic structural variation in the grape powdery mildew pathogen, Erysiphe necator.

BACKGROUND: Powdery mildew, caused by the obligate biotrophic fungus Erysiphe necator, is an economically important disease of grapevines worldwide. Large quantities of fungicides are used for its control, accelerating the incidence of fungicide-resistance. Copy number variations (CNVs) are unbalanced changes in the structure of the genome that have been associated with complex traits. In addition to providing the first description of the large and highly repetitive genome of E. necator, this study describes the impact of genomic structural variation on fungicide resistance in Erysiphe necator. RESULTS: A shotgun approach was applied to sequence and assemble the genome of five E. necator isolates, and RNA-seq and comparative genomics were used to predict and annotate protein-coding genes. Our results show that the E. necator genome is exceptionally large and repetitive and suggest that transposable elements are responsible for genome expansion. Frequent structural variations were found between isolates and included copy number variation in EnCYP51, the target of the commonly used sterol demethylase inhibitor (DMI) fungicides. A panel of 89 additional E. necator isolates collected from diverse vineyard sites was screened for copy number variation in the EnCYP51 gene and for presence/absence of a point mutation (Y136F) known to result in higher fungicide tolerance. We show that an increase in EnCYP51 copy number is significantly more likely to be detected in isolates collected from fungicide-treated vineyards. Increased EnCYP51 copy numbers were detected with the Y136F allele, suggesting that an increase in copy number becomes advantageous only after the fungicide-tolerant allele is acquired. We also show that EnCYP51 copy number influences expression in a gene-dose dependent manner and correlates with fungal growth in the presence of a DMI fungicide. CONCLUSIONS: Taken together our results show that CNV can be adaptive in the development of resistance to fungicides by providing increasing quantitative protection in a gene-dosage dependent manner. The results of this work not only demonstrate the effectiveness of using genomics to dissect complex traits in organisms with very limited molecular information, but also may have broader implications for understanding genomic dynamics in response to strong selective pressure in other pathogens with similar genome architectures.