Characterization of pathogenesis-related protein 1 genes expressed in Korean wild grapevine genotypes inoculated with Phakopsora euvitis

We described the cloning and characterization of pathogenesis-related protein 5 gene in maize, named ZmPR5 (GenBank Accession Number: HM230665). Molecular and bioinformatic analyses of ZmPR5 revealed an open reading frame (ORF) of 525 bp, encoding a protein of 175 amino acids (aa) and a deduced molecular mass of 17.5 KDa. Homology analysis of the Zea mays L. deduced amino acid sequence, indicated homology between 40 and 74% with Oryza sativa, Hordeum vulgare , and Triticum aestivum , among others. Real-time reverse transcription-polymerase chain reaction (RT-PCR) analysis showed that the expression of ZmPR5 is constantly higher in the maize resistant inbred line R15 compared with that in the susceptible inbred line 478. Moreover, the ZmPR5 gene was up-regulated after it was challenged by Rhizoctonia solani. We subsequently purified the recombinant protein and analyzed its antimicrobial activities in vitro. The results obtained show that the recombinant protein inhibited hyphal growth of R. solani. This study suggests that the expression of ZmPR5 is closely related to maize sheath blight resistance caused by R. solani . Further, the antifungal activity of ZmPR5 showed that ZmPR5 plays an important role in the disease resistance response. Key words: Maize, ZmPR5, banded leaf and sheath blight, pathogenesis-related protein, Rhizoctonia solani.

Genome-wide identification and characterization of pathogenesis related protein 1 gene family in Brassica juncea.

The family of pathogenesis related protein 4 (PR4) is a group of proteins with a Barwin domain in C terminus and generally thought to be involved in plant defense responses. In the present study, PR4 (des ignated as PgPR4) cDNA was isolated from the leaf of Panax ginseng C.A. Meyer. and characterized. The ORF is 513 bp with a deduced amino acid sequence of 170 residues. A GenBank BlastX search revealed that the deduced amino acid of PgPR4 shares the highest sequence similarity to PR4 of Sambucus nigra (72% identity). Sequence and structural analysis indicated that PgPR4 belongs to class II of PR4 proteins. This is the first report on the isolation of PR4 gene from the P. ginseng genome. The high level expression of PgPR4 was observed in the root as revealed by quantitative real time PCR. The temporal expression analysis dem onstrated that PgPR4 expression could be up regulated by pathogen infection, salt, wounding, and hormone stresses. These results suggest that PgPR4 could play a role in the molecular defense response of ginseng to abiotic stress and pathogen attack. Keywrds: Panax ginseng, abiotic stress, biotic stress, pathogenesis related protein 4, gene expression DOI: 10.1134/S1021443714050100 Abbreviations: JA—jasmonic acid; PR—pathogenesis related; SA—salicylic acid. RESEARCH PAPERS

A pathogenesis-related protein (PgPR5) gene that isolated from the leaf of Panax ginseng was characterized. The ORF is 756 bp with a deduced amino acid sequence of 251 residues. The calculated molecular mass of the matured protein is approximately 27.5 kDa with a predicated isoelectric point of 7.80. A GenBank BlastX search revealed that the deduced amino acid of PgPR5 shares highest sequence similarity to PR5 of Actinidia deliciosa (80% identity, 87% similarity). PgPR5 has a C-terminal and N-terminal signal peptide, suggesting that it is a vacuolar secreted protein. The expression of PgPR5 under various environmental stresses was analyzed at different time points using real-time PCR. Our results reveal that PgPR5 is induced by salt stress, chilling stress, heavy metal, UV, and pathogen infection. These results suggest that the PgPR5 could play a role in the molecular defence response of ginseng to abiotic and pathogen attack. This is the first report of the isolation of PR5 gene from the P. ginseng.

The family of pathogenesis-related protein 4 (PR4) is a group of proteins with a Barwin domain in C-terminus and generally thought to be involved in plant defense responses. In the present study, PR4 (designated as PgPR4) cDNA was isolated from the leaf of Panax ginseng C.A. Meyer. and characterized. The ORF is 513 bp with a deduced amino acid sequence of 170 residues. A GenBank BlastX search revealed that the deduced amino acid of PgPR4 shares the highest sequence similarity to PR4 of Sambucus nigra (72% identity). Sequence and structural analysis indicated that PgPR4 belongs to class II of PR4 proteins. This is the first report on the isolation of PR4 gene from the P. ginseng genome. The high-level expression of PgPR4 was observed in the root as revealed by quantitative real-time PCR. The temporal expression analysis demonstrated that PgPR4 expression could be up-regulated by pathogen infection, salt, wounding, and hormone stresses. These results suggest that PgPR4 could play a role in the molecular defense response of ginseng to abiotic stress and pathogen attack.

The present study aimed to assess 25 grapevine genotypes, representing different Vitis species, for resistance to grapevine leaf rust (GLR), caused by Phakopsora euvitis, using leaf disc assay. Disinfected leaf discs of 12 mm in diameter were placed in agar-water medium. On the abaxial side, a 30-µl drop at a concentration of 30,000 urediniospores/ml was deposited and incubated in a growth chamber under controlled conditions. The genotypes were assessed by the components of resistance latent period, number of pustule per cm2, diameter of pustules (DP; mm), number of urediniospores per disc, severity (%), and area under the disease severity progress curve. The ANOVA revealed a significant difference (p < 0.05) among genotypes for all components of resistance tested. Significant correlation was observed for all components of resistance evaluated. Based on disease severity, the genotypes were classified into four resistance categories: (1) resistant, (2) partially resistant, (3) susceptible, and (4) highly susceptible. None of the genotypes were asymptomatic and 32% were considered resistant or partially resistant. ‘IAC766’ and ‘Seibel 405’ were the most resistant, showing the lowest severity of 0.03 and 1.48%, respectively. ‘Seibel 128’, V. del rioi Sd1, V. slavinii and V. candicans were partially resistant. From the resistant and partially resistant genotypes, only V. candicans has trichomes on the abaxial leaf surface. Particularly, the resistant genotypes are resistance sources to GLR to be explored in future breeding programs and for genetic analysis to localize resistant genes to P. euvitis.

The examination of germplasm within grapevine accessions derived from wild genotypes holds significant importance within the grapevine breeding program, particularly in the improving of cultivars and rootstocks. Due to the greater genetic variation present in wild genotypes, there is an increased possibility of possessing the desired features. The objective of this study was to determine the genetic diversity and population genetic structure of 64 grapevine genotypes using inter-primer binding site (iPBS) retrotransposon markers and simple sequence repeat (SSR) markers. A total of 236 bands were generated using iPBS markers, of which 162 bands exhibited polymorphism. A comprehensive assessment was conducted on a total of 126 SSR alleles using the SSR markers, revealing that 91 of these exhibited polymorphisms. Despite the similarity in mean values between polymorphic bands generated by iPBS (6.48) and SSR markers (6.5), the iPBS markers exhibited a greater polymorphism information content (PIC: 0.39) in comparison to SSR markers (0.29). The UPGMA analysis classified the genotypes into two primary groups at a similarity index of 0.62 based on combined data. The rootstocks utilized as points of reference are consolidated inside a singular cluster (A), distinct from both the Mediterranean wild population and cultivars. The comparison of genetic variation, represented by FST values, revealed that the maximum differentiation was observed between subpopulations SP3 and SP5. The wild grapevine population grown in the Southeast Mediterranean Region of Turkey exhibited significant differentiation. Both marker systems employed in this study were highly polymorphic and useful for genetic characterization and mapping of grapevine populations.

&lt;p style="text-align: justify;"&gt;&lt;strong&gt;Aim&lt;/strong&gt;: To evaluate the suitability of wild grapevine genotypes to saline conditions by measuring leaf ion content and salt injury symptoms.&lt;/p&gt;&lt;p style="text-align: justify;"&gt;&lt;strong&gt;Methods and results&lt;/strong&gt;: Vines of nine wild (&lt;em&gt;Vitis vinifera&lt;/em&gt; L. ssp. sylvestris) genotypes were planted in pots and after the good establishment, salinity treatments (0, 50, 100 and 150 mM NaCl) started. At the end of the experimental period, the K&lt;sup&gt;+&lt;/sup&gt;, Na&lt;sup&gt;+&lt;/sup&gt;, NO&lt;sub&gt;3&lt;/sub&gt;&lt;sup&gt;-&lt;/sup&gt; and Cl&lt;sup&gt;-&lt;/sup&gt; content of leaves and visible symptoms of salt injury were recorded. Leaf Na&lt;sup&gt;+&lt;/sup&gt; and Cl&lt;sup&gt;-&lt;/sup&gt; content increased with increasing salinity but levels and accumulation rates were different among genotypes.&lt;/p&gt;&lt;p style="text-align: justify;"&gt;&lt;strong&gt;Conclusion&lt;/strong&gt;: Based on Na&lt;sup&gt;+&lt;/sup&gt; and Cl&lt;sup&gt;-&lt;/sup&gt; content and salt symptoms, genotypes 4 and 7 showed less symptoms than the other genotypes at moderate (50-100 mM) NaCl concentration and none could tolerate high salt (150 mM) concentration.&lt;/p&gt;&lt;p style="text-align: justify;"&gt;&lt;strong&gt;Significance and impact of the study&lt;/strong&gt;: Under saline conditions, ion accumulation in the leaves significantly varied among wild genotypes and some of them could be recommended as saline tolerant genotypes.&lt;/p&gt;

Flavonols are a group of grape phenolics that play an important role in young red wines, as they are involved in copigmentation of the flavylium form of anthocyanins. A study on the flavonol composition of grape skins in several wild grapevine genotypes from different Iberian natural populations, preserved at El Encin Germoplasm Bank, has been carried out in 2012. Flavonol glycosides contained in grape skins were determined by HPLC-DAD and HPLC-MS, through a previous phase of purification, using ion exchange chromatographic columns to retain anthocyanins and other phenolic compounds, so that flavonols do not suffer co-elution with other components, improving HPLC analysis. Thus, it was possible to separate 12 flavonol glycosides, and eight of them were successfully identified. The major flavonols were quercetin-3-O -glucoside, quercetin-3-O -glucoronide and myricetin-3-O -glucoside. The diversity and number of flavonols differed for each genotype. The total content of flavonols ranged from 25 to 350 mg/kg grapes; the richest genotype was three times richer than Tempranillo grapes, used as a reference. The most significant difference between wild genotypes and reference cultivars was that, in many cases, myricetin-3-O -glucoside or quercetin-3-O -glucuronide predominated in wild genotypes.

A total of 411 grapevine genotypes were screened in vitro for resistance to the rust pathogen Phakopsora euvitis. They included both rootstock and hybrid selections from 14 different Vitis species, principally V. vinifera. The majority tested were susceptible or highly susceptible. Not one was asymptomatic. The interspecific hybrid selections ‘41B’ and ‘Seibel 128’ (both rootstocks) and ‘Aurore’ showed the highest level of resistance to the pathogen.

Understanding the genetic diversity and relationships between genotypes is an effective step in designing effective breeding programs. Insertional polymorphisms of retrotransposons were studied in 75 cultivated and wild grape genotypes using retrotransposon-microsatellite amplified polymorphism (REMAP) technique. In the morphological part of work, seven pomological traits with a high breeding interest were also analyzed in the cultivated genotypes. A total of 328 markers were produced by 42 primer pairs, out of which 313 markers (95.43%) were polymorphic. Number of markers ranged from 4 in loci Tvv1Fa-873, Vine1-811, Gret1Ra-855 and Tvv1Fa-890 to 12 in locus Vine1Ra-841 with an average value of 7.45. Similarity values based on Dice's coefficient among all 75 grapevine genotypes varied from 0.41 to 0.77. Classification of genotypes using unweighted pair-group method using complete-linkage clustering led to six distinct groups. Some wild and cultivated varieties placed in the same groups. It seems there are close relationship between wild and cultivated genotypes and maybe wild genotypes are ancestor of native grapevines. Grouping of grapevine genotypes based on molecular marker data was not in agreement with clustering by agro-morphological data indicating that the most of multiplied sequences are confined to the non-coding regions of transposon elements. Results showed a substantial level of genetic diversity at molecular and pomological level and the potential of this diversity for future grape breeding programs.

Grapevine is one of the major horticultural crops of the world with the cultivated area exceeding 7.5 million ha used for a myriad of products ranging through fresh table grape, preserves, juice, wine, and raisins. The main objective of this study was to introduce twenty-eight grapevine cultivars (ten wild, ten wine, four table, and four raisin grapes) into Gedeo Zone for the first time and ampelographically characterize them in Dilla and Yirgacheffe agroecological conditions in Gedeo Zone, Southern Ethiopia, from August 2018 to July 2021. Ten Vitis abyssinica wild grapevine cultivars were collected from Adama, Addis Ababa, Alamata, Arba Minch, Bahir Dar, Dire Dawa, Gondar, Hawassa, Jimma, and Jinka areas. Additional ten world class wine grapes were gathered from Ziway Castel Winery, and four table and four raisin grapes were also collected from Raya Horti Farm and Koka Vineyard at the same time. The experiment was a 2 × 28 factorial arranged in randomized complete block design (RCBD) with three replications, and data were analyzed using the R-software. The analysis of variance revealed that the interaction of cultivar and location significantly (P < 0.001) affected grapevine plant height, leaf number, number of fruits per plant, and tendril number per vine, while grapevine trunk diameter, flower cluster, root length, and number of suckers per vines were not significantly (P > 0.05) influenced by the interaction of the two factors. Generally, the wine grapevine cultivars had lower canopy such as plant height, leaf number, number of tendrils, and suckering vines while these registered a higher number of fruits per plant, trunk diameter, flower cluster, and root length compared to the wild grapevine cultivars. The results of the present study suggested that Syrah, Chenin Blanc, and Grenache can produce high grapevine berry yield and wine quality in Gedeo Zone agroecology particularly in Dilla location. The wild grapevines collected from Dire Dawa, Arba Minch, Jinka, and Alamata were the potential candidates for the world class wine, raisin, and table grapevines which could open new frontiers in the future for Ethiopian native Vitis abyssinica wild grapevine breeding and genetic engineering that will help to move the national and international viticulture and enology industry forward. As the Ethiopian native grapevines are at the risk of total extinction, adequate conservation strategies are required. Breeding, detailed identification, and introducing the potential grapes in different regions of the country are needed. This finding represents a step forward in efforts to understand hybridization of Vitis abyssinica grapevine with Vitis vinifera and other new world Vitis species.

The physiological and molecular response to salt stress was studied in two wild grapevine (Vitis vinifera L. ssp. sylvestris or Vitis sylvestris) accessions “Khedhayria” and “Houamdia”, previously identified as salt-tolerant and salt-sensitive pair wise. Plants from both accessions were subjected to a progressive salt stress by the use of a nutritional solution containing up to 150 mM NaCl for 2 weeks. Salt stress adversely affected growth and water potential since the first day of exposure to 150 mM NaCl. However, chlorophyll fluorescence parameters were unchanged until 14 days of salt exposure. At that time point the predawn water potential (ΨPD), the non-photochemical quenching of fluorescence (NPQ) and the coefficient of photochemical quenching (qp) were significantly less altered in the tolerant accession. At the molecular level semi-quantitative RT-PCR assays revealed a differential expression of (Vs α-gal/SIP and Vs DHN) genes within these contrasting accessions after exposure to 24 h and 14 days of salt. Comparably, the Vs RD22 gene had increased slightly after only 14 days of treatment in both accessions. These results were the first pieces of information reported on the early and late regulation of salt response genes in wild grapevines. Furthermore, genotype-dependent parameters such as NPQ, qp, mRNA levels of Vs α-gal/SIP and Vs DHN could be used to screen salt-tolerant wild grapevine genotypes.

Background and Aims Wild grapevine, considered the ancestor relative of cultivated vines, has a large gene pool that is currently endangered in Europe. These plants can contribute to improving adaptation capacity to stresses due to climate change. The aim of this study was to evaluate the key ampelographic traits to identify true wild individuals supporting the preservation and use of wild populations. Methods and Results Prospections performed since 2002 enabled the inventory of 51 localities with wild grapevines, most of which were located along Spanish riverbank forests. A morphological study of 192 individuals grafted in the grapevine collection of El Encin (Alcala de Henares, Spain) was carried out ex situ, and results were compared with data from 182 Spanish commercial cultivars grown in the same collection. Wild individuals presented morphological differences with cultivars, but only a few significant differences were found within wild individuals when comparing their geographic origin and plant sex. Ten morphological traits were relevant to discriminate wild and cultivated specimens. Conclusions Ampelography, supported by previous molecular screening, is recommended to identify wild grape plants, although it is not advisable to establish relationships among wild genotypes by geographical location or gender. Significance of the Study Results are expected to contribute to improved discovery, preservation and use of this important phytogenetic resource.

Resveratrol and its derivatives are the most important phytoalexins with a crucial role in plant defense mechanisms. These compounds can occur either naturally or in response to abiotic stresses. Among them, salinity is one of the major threats to the sustainability and productivity of agro-economically important species, particularly those involved in the vini-viticulture sector. Understating salinity tolerance mechanisms in plants is required for the development of novel engineering tools. This study aimed to investigate the potential role of resveratrol derivatives in salinity tolerance of wild grapevines. Our data revealed that the tolerant Tunisian wild grapevine genotype "Ouchtata" exhibited an increased accumulation of resveratrol derivatives (glycosylated and non-glycosylated resveratrol and t-ɛ-viniferin and hydroxylated t-piceatannol) in both stems and roots, along with an increased total antioxidant activity (TAA) compared to the sensitive genotype "Djebba" under stress conditions, suggesting an involvement of these stilbenes in redox homeostasis, thereby, protecting cells from salt-induced oxidative damage. Overall, our study revealed, for the first time, an active role for resveratrol derivatives in salt stress tolerance in wild grapevine, highlighting their potential use as metabolic markers in future grapevine breeding programs for a sustainable vini-viticulture in salt-affected regions.