Search

Pierce’s disease is a deadly disease of grapevines caused by the bacterial pathogen Xylella fastidiosa (Xf). A Pierce’s disease resistance locus from Vitis arizonica/candicans b43-17 segregated as a single dominant gene and mapped as PdR1a and PdR1b in two F1 sibling selections. The physical mapping of the PdR1b allele allowed the identification of five ORFs of the Leucine-Rich Repeat Receptor Kinase gene family. Two ORFs: V.ari-RGA14 and V.ari-RGA18 were used to transform embryogenic callus of V. vinifera Chardonnay (CH) and Thompson Seedless (TS) and V. rupestris St George (SG) via Agrobacterium tumefaciens. Regenerated plants were inoculated with Xf under greenhouse conditions. Genetic transformation with RGA14 and 18 did not generate resistance in CH and TS, although some lines of CH showed significantly lower stem bacterial concentration and/or exhibited reduced symptoms. In transgenic SG14, improved regrowth was accompanied with lower bacterial titers and decreased pectin lyase and ß-1,3-glucanase 3 gene expression. The limited effects of the transgenes on PD resistance could be explained by the lack of suitable partners or the presence of susceptibility factors that could not be overcome under these experimental conditions. The involvement of RGA17 in b43-17 resistance to Xf should not be discarded.

Vigor control in grapevine may become especially important under climate change. A better understanding of gene-phenotype relationships is required in order to exploit plant genomics for breeding purposes. This research aims to use quantitative trait loci (QTLs) for vigor identified in the progeny from a cross of Ramsey (Vitis champinii) × Riparia Gloire (V. riparia). Genes located 700 kb up and downstream from each QTL position were interrogated for functional enrichment through ShinyGO online tool, based on the gene ontology annotation of Vitis vinifera PN40024. Key biological processes like phloem and xylem development, cell cycle, response to hormones, amino acid transport, tissue development, sugar metabolism, nitrogen transport, and stress/immune responses, showed functional enrichment. Integral response to light and auxin might be required for fine molecular tuning of vegetative growth in Vitis. Fifty out of 1318 candidate genes were prioritized, reducing their amount to a manageable number of candidates for further directed breeding strategies. Highlights Plant vigor control may become especially important under climate change. Genes from various vigor-related QTLs were interrogated for functional enrichment. The analysis reduced candidate gene number based on marker proximity and functional enrichment, constituting a suitable shortcut for target-directed genome-guided breeding strategies. Three TFs are strong candidates for targeted breeding: TIF - HY5, TIF - SUS1, TIF - VOZ1 potentially enhance growth by relating light response to hormone activation, and then to photosynthesis and morphogenesis.

Preference host is an important factor affecting population dynamics in plant-insect interactions. In this research, the two main phylloxera genotypes found in Argentina (gB and gD) were tested on Vitis vinifera cvs. Cabernet Sauvignon, Malbec, Pedro Gimenez, Chardonnay, Cereza, Criolla Grande, and rootstocks 1103 Paulsen, 101-14 Mgt, and SO4 using an excised root assay. Statistical analysis showed that survivorship was affected by the interaction of Cultivar and Time, while hatching was influenced by the interaction of Cultivar and Phylloxera genotype. Cultivar also influenced maximum number of adults, developmental time, as well as gross and net reproductive rates. Phylloxera genotype gB developed faster than gD, however, no other significant differences were found between both genotypes. Principal component analysis showed that Pedro Gimenez and Criolla Grande grouped closely with high values of reproduction-related variables, suggesting they are the most preferable hosts, followed by Cabernet Sauvignon; while Cereza, Malbec, and Chardonnay were less suitable. None of the rootstocks allowed the establishment of phylloxera colonies. This study will help improve the biological understanding of phylloxera in Argentina and help develop tools for its management.

Background and Aims Grafted grapevines are a key component of modern viticulture. Grafting provides not only resistance to soil‐borne pests, but can also improve adaptability to the environment and can improve fruit yield and quality. Graft incompatibility induced by grapevine viruses causes damage to vineyards worldwide. The aim of the present study was to create a rapid and reliable means of evaluating graft incompatibility resulting from grafting virus‐infected scions onto healthy grape rootstocks. Methods and Results Scions of Control (healthy Cabernet Franc), LR131 (Cabernet Franc infected with GLRaV‐1) and LR132 (Cabernet Franc infected with GLRaV‐1 and GVA) were grafted onto different rootstocks by micrografting, green grafting and bench grafting methods. All the micrografts infected with LR132 failed, while LR131 and Control micrografts had similar survival rates. The impact of LR132 was less severe on green grafted plants, followed by bench grafts. Analysis of variance showed an interaction of grafting method × virus variety × rootstock, affecting grafting survival. When grafted onto Freedom and 101‐14, LR131 inhibited the growth of both scion and rootstock, but no significant impact was seen when LR131 was grafted onto St. George and AXR1. Well‐established callus was visible between the surfaces of LR131 and the rootstocks. The virus GLRaV‐1 was detected moving from the LR131 scion source to rootstocks 7 days after micrografting. Callus formation was delayed by LR132 between scion and rootstock surfaces, and a scion‐rootstock vascular connection did not establish, resulting in the death of all micrografts. Conclusions Micrografting is a reliable and sensitive method to screen for virus tolerant graft combinations compared with green and bench grafting. Significance of the Study The results of the present study validate micrografting as a means of producing credible information for selection of virus tolerant rootstocks, and ultimately vineyards with higher rates of compatible grafts.

Background and Aims The North American insect pest grape phylloxera was introduced into Europe in the 19th century and devastated the Vitis vinifera‐based vineyards. It has since become widely distributed among the world’s vineyards. Although it is present in Argentina, it has not caused any major damage. This work aims: to characterise the genetic diversity of Argentinean phylloxera; to determine population structure and reproductive mode; to compare Argentinean genotypes with samples from seven other countries; and to examine relationships between the infestation level and genetic profile with ecological factors. Methods and Results One hundred and twenty‐nine samples of phylloxera from Argentinean provinces were analysed with 21 microsatellite markers. Seventeen multilocus genotypes were identified. Unweighted pair group method with arithmetic mean analysis identified two major groups. Principal coordinates analysis grouped together Peruvian, California biotypes A and B and Argentinean phylloxera, showing dissimilarity with other foreign samples. Associations between infestation level, berry colour and climatic region, and associations between genetic clusters and climatic region were established. Conclusions Endemic Argentinean phylloxera genotypes showed genetic diversity and were different from other samples assessed. Parthenogenesis is proposed as the main reproductive mode, but rare sexual reproduction is not ruled out. No particular structure was observed in the phylloxera populations. Significance of the Study This is the first detailed report on the genetic characterisation of phylloxera populations in Argentina using microsatellites markers. It provides the foundation for further studies.

Vigour control in grapevine can be achieved by grafting with particular rootstocks, and may become especially important under climate change. This research studied the F1 progeny from the cross of 'Ramsey' × 'Riparia Gloire de Montpellier' rootstocks, known to confer high and low vigour, respectively. We hypothesized that vigour correlates with growth rate, leaf area, biomass partitioning, plant hydraulics and gas exchange; and that these variables could be associated with genetic markers. We evaluated 138 seedlings from this cross, three replicates each, for 60 days in a greenhouse at UC Davis, California, during summer 2014 and 2015. Each plant was pruned to a single shoot and watered daily. Shoot growth rate, leaf area and dry biomass were measured for the complete population, both years. In 2014, after day 45, 40 genotypes were subjected to a 50% water deficit, based from initial weight at full pot capacity, and plant and root hydraulic conductance's, stomatal conductance and water potential were measured. The progeny showed transgressive segregation and significant differences in vigour. A PCA analysis showed a strong role for shoot growth rate, specific leaf area, plant hydraulics and partitioning indices for vigour determination. Under water stress, larger plants showed less specific hydraulic and stomatal conductance's, indicating higher sensibility upon drought. Significant QTLs for leaf area, specific leaf area and partitioning indices were found on chromosomes 1, 4, 16, and 5; accounting for 20% of explained variability for leaf area, and from 10 to 14% of explained variability for specific leaf area and partitioning indices in 2014. Mapping data from 2015 is being analysed.

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.

A protocol was standardized to regenerate six grape cultivars through meristematic bulk (MB) induction, which was used for genetic transformation. Meristematic bulk induction worked best with Vitis vinifera ‘Thompson Seedless’ (98.4 %), followed by ‘Chardonnay’ (97.6 %), ‘Redglobe’ (90.2 %) and ‘Cabernet Sauvignon’ (86.2 %), and was less successful with Vitis rupestris ‘St. George’ (85.4 %) and ‘101-14 Millardet et de Grasset (Vitis riparia × V. rupestris)’ (79.6 %). Benzylaminopurine and naphthaleneacetic acid was the most effective combination of cytokinin and auxin for MB formation. 100 µg/ml kanamycin was a better antibiotic selection agent than 2.0 µg/ml hygromycin during transformation. The expression of green fluorescent protein was evaluated with in vitro leaves and roots. Transformation efficiency using meristematic slices was a function of the genotype. Transformation efficiency was greatest in Chardonnay (51.7 %), followed by Thompson Seedless (42.3 %), St. George (41.6 %), Redglobe (40 %), Cabernet Sauvignon (35.6 %) and 101-14 Mgt (29.9 %). This study found that MB induction was a fast and simple alternative for genetic transformation of grape cultivars.

A field study showed that transgenic grapevine rootstocks can provide trans-graft-mediated protection to a wild type scion against Pierce’s disease (PD) development. We individually field-tested two distinct strategies. The first expressed a chimeric antimicrobial protein (CAP) that targeted the functionality of the lipopolysaccharide (LPS) surface of Xylella fastidiosa (Xf), the causative agent of PD. The second expressed a plant polygalacturonase inhibitory protein (PGIP) that prevents PD by inhibiting breakdown of pectin present in primary cell walls. Both proteins are secreted to the apoplast and then into the xylem, where they migrate past the graft union, transiting into the xylem of the grafted scion. Transgenic Vitis vinifera cv. Thompson Seedless (TS) expressing ether CAP or PGIP were tested in the greenhouse and those lines that showed resistance to PD were grafted with wild type TS scions. Grafted grapevines were introduced into the field and tested over 7 years. Here we present data on the field evaluation of trans-graft protection using four CAP and four PGIP independent rootstock lines, compared to an untransformed rootstock. There was 30 to 95% reduction in vine mortality among CAP- and PGIP-expressing lines after three successive yearly infections with virulent Xf. Shoot tissues grafted to either CAP or PGIP transgenic rootstocks supported lower pathogen titers and showed fewer disease symptoms. Grafted plants on transgenic rootstocks also had more spring bud break following infection, more shoots, and more vigorous growth compared to those grafted to wild type rootstocks. No yield penalty was observed in the transgenic lines and some PGIP-expressing vines had enhanced yield potential. Trans-graft protection is an efficient way to protect grape scions against PD while preserving their valuable varietal genotypes and clonal properties.

9-cis-epoxycarotenoid dioxygenase (NCED) is a key enzyme involved in the biosynthesis of abscisic acid (ABA), which is associated with drought tolerance in plants. An osmotic-inducible VaNCED1 gene was isolated from a drought-resistant cultivar of Vitis amurensis and constitutively overexpressed in a drought-sensitive cultivar of Vitis vinifera. Transgenic plants showed significantly improved drought tolerance, including a higher growth rate and better drought resistant under drought conditions, compared to those of wild-type (WT) plants. After water was withheld for 50 days, the upper leaves of transgenic plants remained green, whereas most leaves of WT plants turned yellow and fell. Besides the increase in ABA content, overexpression of VaNCED1 induced the production of jasmonic acid (JA) and accumulation of JA biosynthesis-related genes, including allene oxide cyclase (AOC) and 12-oxophytodienoate reductase (OPR3). Moreover, transgenic plants possessed advantageous physiological indices, including lower leaf stomatal density, lower photosynthesis rate, and lower accumulation of proline and superoxide dismutase (SOD), compared to those of WT plants, indicating increased resistance to drought stress. Quantitative real time polymerase chain reaction (RT-qPCR) analysis revealed that overexpression of VaNCED1 enhanced the expression of drought-responsive genes, such as ABA-responsive element1 (ABRE1), ABRE binding factors 2 (ABF2), plasma membrane intrinsic proteins 2 (PIP2), C-repeat/DRE-Binding Factor 4 (VvCBF4) and ABA-insensitive 5 (ABI5). Although the development of transgenic plants was delayed by 4 months than WT plants, because of seed dormancy and abnormal seedlings, the surviving transgenic plants provided a solid method for protection of woody plants from drought stress.

9-cis-epoxycarotenoid dioxygenase (NCED) is a key enzyme involved in the biosynthesis of abscisic acid (ABA), which is associated with drought tolerance in plants. An osmotic-inducible VaNCED1 gene was isolated from a drought-resistant cultivar of Vitis amurensis and constitutively overexpressed in a drought-sensitive cultivar of Vitis vinifera. Transgenic plants showed significantly improved drought tolerance, including a higher growth rate and better drought resistant under drought conditions, compared to those of wild-type (WT) plants. After water was withheld for 50 days, the upper leaves of transgenic plants remained green, whereas most leaves of WT plants turned yellow and fell. Besides the increase in ABA content, overexpression of VaNCED1 induced the production of jasmonic acid (JA) and accumulation of JA biosynthesis-related genes, including allene oxide cyclase (AOC) and 12-oxophytodienoate reductase (OPR3). Moreover, transgenic plants possessed advantageous physiological indices, including lower leaf stomatal density, lower photosynthesis rate, and lower accumulation of proline and superoxide dismutase (SOD), compared to those of WT plants, indicating increased resistance to drought stress. Quantitative real time polymerase chain reaction (RT-qPCR) analysis revealed that overexpression of VaNCED1 enhanced the expression of drought-responsive genes, such as ABA-responsive element 1 (ABRE1), ABRE binding factors 2 (ABF2), plasma membrane intrinsic proteins 2 (PIP2), C-repeat/DRE-Binding Factor 4 (VvCBF4) and ABA-insensitive 5 (ABI5). Although the development of transgenic plants was delayed by 4 months than WT plants, because of seed dormancy and abnormal seedlings, the surviving transgenic plants provided a solid method for protection of woody plants from drought stress.

A protocol for in vitro induction of tetraploids of the grape rootstock 101-14 Millardet et de Grasset (Vitis riparia × V. rupestris) (101-14 Mgt) (2n = 2x = 38) × Muscadinia rotundifolia cv. Trayshed (2n = 2x = 40) hybrids was established. Different explant materials were exposed to the antimitotic chemical agents colchicine and oryzalin, at three concentrations, for 24, 48 and 72 h. Treated in vitro shoot tips, anthers and pre-embryogenic cultures were regenerated, and their ploidy levels were confirmed by chromosome counts and flow cytometry. It is generally considered that anatomical characteristics of leaves, including leaf stomatal size and number of chloroplasts in guard cells, correlate with ploidy levels of grape plants. Chromosome counts and flow cytometry are labor intensive and difficult, while the number of chloroplasts in stomatal guard cells and stomatal size are easily measured and can be stable and reliable markers. Analysis of stomata revealed clear differences between regenerated diploid and tetraploid plants, and these measurements were used as a preliminary screen, followed by chromosome counts and flow cytometry for confirmation. This study presents the results obtained by various combinations of explants, antimitotic agents, exposure time, concentration and ploidy analysis to evaluate in vitro chromosome doubling. These results provide an easy and effective method for production of tetraploid, potentially fertile Vitis × Muscadinia hybrids for utilization in rootstock breeding programs.

Overexpression of vacuolar Na+/H+ antiport AtNHX1 has been reported to improve plant salt tolerance. We produced transgenic plants of Vitis vinifera ?Thompson Seedless? via Agrobacterium tumefaciens carrying the gene encoding for AtNHX1 under the control of the constitutive promoter CaMV35S. Independent transgenic lines with improved tolerance to salt stress were selected in first round evaluations. The objective of this study was to analyze the response of the selected lines to NaCl stress in hydroponics and potted soils. Untransformed and transgenic plants were acclimated to greenhouse conditions and grown hydroponically using Long Ashton nutrient solution. In the salinity treatments, NaCl concentrations were increased stepwise by 25 mM every 10 days up to 150 mM. In addition, untransformed and transgenic plants were grown in a mix of 7-3 grape pomace-sand soil and watered with Long Ashton nutrient solution with and without increasing concentrations of NaCl. Analysis of plant growth variables confirmed the improved tolerance of the transgenic lines, which showed higher shoot length, leaf number, leaf area, and fresh and dry weight of shoots and roots. However, their improved performance was not correlated with higher Na accumulation in root and/or shoot tissue compared to the untransformed controls. Na and Cl tissue contents were higher in plants grown in hydroponics; and roots accumulated 1.5-2.5 times more NaCl than aerial parts

SUMMARY Polygalacturonase-inhibiting proteins (PGIPs) are plant cell-wall proteins that specifically inhibit fungal endo-polygalacturonases (PGs) that contribute to the aggressive decomposition of susceptible plant tissues. The inhibition of fungal PGs by PGIPs suggests that PGIPs have a role in plant tolerance to fungal infections and this has been observed in transgenic plants expressing PGIPs. Xylella fastidiosa, the causal agent of Pierce's disease (PD) in grapevines, has genes that encode cell-wall-degrading enzymes, including a putative PG. Therefore, we hypothesized that PGIP expression could confer tolerance against this bacterium as well as against the fungal pathogen Botrytis cinerea. To test this hypothesis, Vitis vinifera cvs. 'Thompson Seedless' and 'Chardonnay' were transformed to express pear fruit PGIP-encoding gene (pPGIP) under the control of the CaMV 35S promoter. Substantial pear PGIP (pPGIP) activity was found in crude extracts from leaves and in xylem exudate of transgenic lines obtained from independent transformation events, but not in untransformed controls. pPGIP activity was detected in xylem exudate of untransformed scions grafted on to transgenic rootstocks expressing pPGIP. Leaves of transgenic plants infected with B. cinerea had reduced rates of lesion expansion. The development of PD was delayed in some transgenic lines with increased pPGIP activity. PD-tolerant transgenic lines had reduced leaf scorching, lower Xylella titres and better re-growth after pruning than the untransformed controls.

PG-inhibiting proteins (PGIPs) are leucine rich repeat plant cell wall proteins that specifically inhibit fungal polygalacturonases (PGs). Their role in plant defense response suggests that they may be useful for genetic engineering to obtain transgenic plants with increased tolerance to fungal infection. In addition, the fact that Xylella fastidiosa, the causal agent of Pierce’s Disease (PD) in grapevines, has genes putatively encoding PG and other cell wall-degrading enzymes led us to the hypothesis that PGIP could confer resistance against this bacterium. In order to test this hypothesis, proembryogenic calli originating from anthers of Vitis vinifera cvs. Thompson Seedless and Chardonnay were co-cultivated with Agrobacterium tumefaciens strain EHA 101 harboring binary plasmid pDU94.0928 that contains the pear pgip gene under the control of the CaMV 35S promoter. Plants from 51 independently isolated lines have been transferred to the greenhouse. Putative transgenic lines growing in the greenhouse were analyzed by PCR, Western blotting and PGIP activity assays. Western blot analysis demonstrated the presence of the protein in roots, leaves and young stems of the transgenic plants but not in untransformed controls. High levels of enzyme activity have been found in crude extracts from leaves of transgenic lines obtained from independent transformation events but not in untransformed controls. Preliminary results have shown that the development of PD in the transgenic lines analyzed so far is delayed. We also present results regarding the disease response of greenhouse-grown plants after inoculation with Botrytis cinerea.

The effects of polyamines on gynogenetic embryogenesis and regeneration of plantlets in onion were studied. Whole flowers from two onion genotypes, ‘Valcatorce INTA’ cultivar and ‘Torrentina’ population, were used as initial explants. Embryo induction was greatest with a combined treatment of 2 mM putrescine and 0.1 mM spermidine. Addition of putrescine alone, with a few exceptions, did not have any significant effect on either embryo induction or haploid plantlet production for both onion genotypes. ‘Torrentina’ showed a higher embryo generation capacity (9.5%) than ‘Valcatorce INTA’ (2.8%). Fast regeneration of embryos was achieved (from 60 to 90 days) as compared to a previously reported time of 46‐152 days. The use of spermidine (0.1 mM) after 15 days of culture promoted further embryo maturation and plantlet formation. ‘Torrentina’ produced more haploid plants (1.90%). This is the first report of successful use of polyamines for induction of gynogenic embryos and regeneration of onion plantlets. © 2000 Elsevier Science Ireland Ltd. All rights reserved.

We postulated that a synergistic combination of two innate immune functions, pathogen surface recognition and lysis, in a protein chimera would lead to a robust class of engineered antimicrobial therapeutics for protection against pathogens. In support of our hypothesis, we have engineered such a chimera to protect against the Gram-negative Xylella fastidiosa (Xf), which causes diseases in multiple plants of economic importance. Here we report the design and delivery of this chimera to target the Xf subspecies fastidiosa (Xff), which causes Pierce disease in grapevines and poses a great threat to the wine-growing regions of California. One domain of this chimera is an elastase that recognizes and cleaves MopB, a conserved outer membrane protein of Xff. The second domain is a lytic peptide, cecropin B, which targets conserved lipid moieties and creates pores in the Xff outer membrane. A flexible linker joins the recognition and lysis domains, thereby ensuring correct folding of the individual domains and synergistic combination of their functions. The chimera transgene is fused with an amino-terminal signal sequence to facilitate delivery of the chimera to the plant xylem, the site of Xff colonization. We demonstrate that the protein chimera expressed in the xylem is able to directly target Xff, suppress its growth, and significantly decrease the leaf scorching and xylem clogging commonly associated with Pierce disease in grapevines. We believe that similar strategies involving protein chimeras can be developed to protect against many diseases caused by human and plant pathogens.