Xianhang Wang

Resistance evaluation of Chinese wild Vitis genotypes against Botrytis cinerea and different responses of resistant and susceptible hosts to the infection

The necrotrophic fungus Botrytis cinerea is a major threat to grapevine cultivation worldwide. A screen of 41 Vitis genotypes for leaf resistance to B. cinerea suggested species independent variation and revealed 18 resistant Chinese wild Vitis genotypes, while most investigated V. vinifera, or its hybrids, were susceptible. A particularly resistant Chinese wild Vitis, “Pingli-5” (V. sp. [Qinling grape]) and a very susceptible V. vinifera cultivar, “Red Globe” were selected for further study. Microscopic analysis demonstrated that B. cinerea growth was limited during early infection on “Pingli-5” before 24 h post-inoculation (hpi) but not on Red Globe. It was found that reactive oxygen species (ROS) and antioxidative system were associated with fungal growth. O2- accumulated similarly in B. cinerea 4 hpi on both Vitis genotypes. Lower levels of O2- (not H2O2) were detected 4 hpi and ROS (H2O2 and O2-) accumulation from 8 hpi onwards was also lower in “Pingli-5” leaves than in “Red Globe” leaves. B. cinerea triggered sustained ROS production in “Red Globe” but not in “Pingli-5” with subsequent infection progresses. Red Globe displayed little change in antioxidative activities in response to B. cinerea infection, instead, antioxidative activities were highly and timely elevated in resistant “Pingli-5” which correlated with its minimal ROS increases and its high resistance. These findings not only enhance our understanding of the resistance of Chinese wild Vitis species to B. cinerea, but also lay the foundation for breeding B. cinerea resistant grapes in the future.

Constitutive expression of a grape aspartic protease gene in transgenic Arabidopsis confers osmotic stress tolerance

Aspartic proteases are involved in various processes of plant senescence, programmed cell death, reproduction and stress responses. We previously identified a salt and drought stress induced gene, VlAP17, encoding a Group C aspartic protease. Here, we report its functional analysis through the characterization of transgenic Arabidopsisthaliana plants overexpressing VlAP17 under the control of constitutive promoter. The transgenic plants showed enhance salt and drought stress tolerance during seed germination as well as the seedling and mature plant stage. In additional, various stress responses indicators were analyzed and results suggested that osmotic stress caused less damage to the plasma membrane of transgenic seedlings than to that of wild type plants. VlAP17 overexpression also resulted in increased ABA levels, a reduction in average stomatal aperture size, and elevated expression levels of stress-response genes involved in the ABA-dependent pathway, as well as higher activities of several antioxidases: superoxide dismutase, catalase and peroxidase. Taken together, these findings suggest that VlAP17 plays a role in protecting the integrity of plasma membrane and it may also be involved in, or have a function upstream of, the ABA biosynthetic pathway.

Expression of a grape bZIP transcription factor, VqbZIP39, in transgenic Arabidopsis thaliana confers tolerance of multiple abiotic stresses

The basic region/leucine zipper (bZIP) transcription factors are known to play key roles in response to abiotic stress. In this study, a bZIP gene (VqbZIP39) was isolated from grape (Vitis quinquangularis) and constitutively expressed in Arabidopsis under control of the cauliflower mosaic virus 35S promoter. The transgenic Arabidopsis thaliana plants showed enhance salt and drought stress tolerance during seed germination and in the seedling and mature plant stages. Various physiological parameters related to stress responses were analyzed to gain further insight into the role of VqbZIP39 and it was found that osmotic stress caused less damage to the transgenic seedlings than to the corresponding wild type plants. This correlated with an increase in endogenous ABA content as a consequence of the constitutive over-expression of VqbZIP39, and the up-regulated expression of stress-inducible target genes associated with tolerance of drought, high-salt, and oxidative stresses. Our results suggest that the expression of VqbZIP39 in A. thaliana likely enhances the tolerance to multiple abiotic stresses through the ABA signaling pathway, and may therefore have a similar function in the response to abiotic stresses in grape.

Expression of a grape (Vitis vinifera) bZIP transcription factor, VlbZIP36, in Arabidopsis thaliana confers tolerance of drought stress during seed germination and seedling establishment.

Drought is one of the most serious factors that limit agricultural productivity and there is considerable interest in understanding the molecular bases of drought responses and their regulation. While numbers of basic leucine zipper (bZIP) transcription factors (TFs) are known to play key roles in response of plants to various abiotic stresses, only a few group K bZIP TFs have been functionally characterized in the context of stress signaling. In this study, we characterized the expression of the grape (Vitis vinifera) group K bZIP gene, VlbZIP36, and found evidence for its involvement in response to drought and the stress-associated phytohormone abscisic acid (ABA). Transgenic Arabidopsis thaliana lines over-expressing VlbZIP36 under the control of a constitutive promoter showed enhanced dehydration tolerance during the seed germination stage, as well as in the seedling and mature plant stages. The results indicated that VlbZIP36 plays a role in drought tolerance by improving the water status, through limiting water loss, and mitigating cellular damage. The latter was evidenced by reduced cell death, lower electrolyte leakage in the transgenic plants, as well as by increased activities of antioxidant enzymes. We concluded that VlbZIP36 enhances drought tolerance through the transcriptional regulation of ABA-/stress-related genes.

CRISPR/Cas9-mediated efficient targeted mutagenesis in grape in the first generation

Summary The clustered regularly interspaced short palindromic repeats‐associated protein 9 (CRISPR/Cas9) system is a powerful tool for editing plant genomes. Efficient genome editing of grape (Vitis vinifera) suspension cells using the type II CRISPR/Cas9 system has been demonstrated; however, it has not been established whether this system can be applied to get biallelic mutations in the first generation of grape. In this current study, we designed four guide RNAs for the VvWRKY52 transcription factor gene for using with the CRISPR/Cas9 system, and obtained transgenic plants via Agrobacterium‐mediated transformation, using somatic embryos of the Thompson Seedless cultivar. Analysis of the first‐generation transgenic plants verified 22 mutant plants of the 72 T‐DNA‐inserted plants. Of these, 15 lines carried biallelic mutations and seven were heterozygous. A range of RNA‐guided editing events, including large deletions, were found in the mutant plants, while smaller deletions comprised the majority of the detected mutations. Sequencing of potential off‐target sites for all four targets revealed no off‐target events. In addition, knockout of VvWRKY52 in grape increased the resistance to Botrytis cinerea. We conclude that the CRISPR/Cas9 system allows precise genome editing in the first generation of grape and represents a useful tool for gene functional analysis and grape molecular breeding.

Ectopic Expression of the Wild Grape WRKY Transcription Factor VqWRKY52 in Arabidopsis thaliana Enhances Resistance to the Biotrophic Pathogen Powdery Mildew But Not to the Necrotrophic Pathogen Botrytis cinerea

WRKY transcription factors are known to play important roles in plant responses to biotic stresses. We previously showed that the expression of the WRKY gene, VqWRKY52, from Chinese wild Vitis quinquangularis was strongly induced 24 h post inoculation with powdery mildew. In this study, we analyzed the expression levels of VqWRKY52 following treatment with the defense related hormones salicylic acid (SA) and methyl jasmonate, revealing that VqWRKY52 was strongly induced by SA but not JA. We characterized the VqWRKY52 gene, which encodes a WRKY III gene family member, and found that ectopic expression in Arabidopsis thaliana enhanced resistance to powdery mildew and Pseudomonas syringae pv. tomato DC3000, but increased susceptibility to Botrytis cinerea, compared with wild type (WT) plants. The transgenic A. thaliana lines displayed strong cell death induced by the biotrophic powdery mildew pathogen, the hemibiotrophic P. syringe pathogen and the necrotrophic pathogen B. cinerea. In addition, the relative expression levels of various defense-related genes were compared between the transgenic A. thaliana lines and WT plants following the infection by different pathogens. Collectively, the results indicated that VqWRKY52 plays essential roles in the SA dependent signal transduction pathway and that it can enhance the hypersensitive response cell death triggered by microbial pathogens.

VlbZIP30 of grapevine functions in dehydration tolerance via the abscisic acid core signaling pathway

Drought stress limits the growth and development of grapevines, thereby reducing productivity, but the mechanisms by which grapevines respond to drought stress remain largely uncharacterized. Here, we characterized a group A bZIP gene from “Kyoho” grapevine, VlbZIP30, which was shown to be induced by abscisic acid (ABA) and dehydration stress. Overexpression of VlbZIP30 in transgenic Arabidopsis thaliana enhanced dehydration tolerance. Transcriptome analysis revealed that a major proportion of ABA-responsive and/or drought-responsive genes are transcriptionally regulated by VlbZIP30 during ABA or mannitol treatment at the cotyledon greening stage. We identified an A. thaliana G-box motif (CACGTG) and a potential grapevine G-box motif (MCACGTGK) in the promoters of the 39 selected A. thaliana genes upregulated in the transgenic plants and in the 35 grapevine homologs, respectively. Subsequently, using two grapevine-related databases, we found that 74% (23/31) and 84% (21/25) of the detected grapevine genes were significantly upregulated by ABA and drought stress, respectively, suggesting that these genes are involved in ABA or dehydration stress and may be regulated by VlbZIP30 in grapevine. We propose that VlbZIP30 functions as a positive regulator of dehydration-responsive signaling in the ABA core signaling pathway.Crop genetics: drought tolerance gene found in grapeResearchers in China have identified a key gene involved in drought resistance in grapes. After exposing grape plants to drought, Xiping Wang’s team at Northwest A&F University observed increased expression of the VlbZIP30 gene. VlbZIP30 expression also increased in response to the plant hormone ABA, a known regulator of stress response. Further investigation demonstrated that VlbZIP30 probably regulates ABA signaling, though its precise activity remains unclear. The researchers discovered a genetic sequence consistently present in the promoters of genes activated by VlbZIP30. They also identified over 30 genes regulated by VlbZIP30. While many of these genes are probably involved in drought tolerance, more work will be needed to clarify their functions. These findings may serve to guide efforts to breed or engineer grape varieties with improved drought tolerance.

The Grape VlWRKY3 Gene Promotes Abiotic and Biotic Stress Tolerance in Transgenic Arabidopsis thaliana

WRKY transcription factors are known to play important roles in plant responses to various abiotic and biotic stresses. The grape WRKY gene, WRKY3 was previously reported to respond to salt and drought stress, as well as methyl jasmonate and ethylene treatments in Vitis labrusca × V. vinifera cv. ‘Kyoho.’ In the current study, WRKY3 from the ‘Kyoho’ grape cultivar was constitutively expressed in Arabidopsis thaliana under control of the cauliflower mosaic virus 35S promoter. The 35S::VlWRKY3 transgenic A. thaliana plants showed improved salt and drought stress tolerance during the germination, seedling and the mature plant stages. Various physiological traits related to abiotic stress responses were evaluated to gain further insight into the role of VlWRKY3, and it was found that abiotic stress caused less damage to the transgenic seedlings than to the wild-type (WT) plants. VlWRKY3 over-expression also resulted in altered expression levels of abiotic stress-responsive genes. Moreover, the 35S::VlWRKY3 transgenic A. thaliana lines showed improved resistance to Golovinomyces cichoracearum, but increased susceptibility to Botrytis cinerea, compared with the WT plants. Collectively, these results indicate that VlWRKY3 plays important roles in responses to both abiotic and biotic stress, and modification of its expression may represent a strategy to enhance stress tolerance in crops.

Current Progress and Future Prospects for the Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) Genome Editing Technology in Fruit Tree Breeding

Abstract Difficulties in generating woody perennial fruit trees and a lack of molecular markers have impeded functional analysis of agronomically important genes and genetic engineering for improved fruit cultivars. The clustered regularly interspaced short palindromic repeats (CRISPR) technology has been applied in editing fruit tree genomes, providing timely opportunities for studying the functions of the genes in fruit development and stress tolerance. It also leads to the new era of genetic engineering of fruit trees. In this review, we intend to present recent progress in the application of CRISPR to genome editing of fruit trees. We reviewed the most widely used CRISPR systems developed in other plant species that may be adopted for fruit trees and describe three genetic transformation methods used for fruit trees that have potential application value for different CRISPR systems. We also discuss the potential of the CRISPR system to study basic fruit tree biology and for precision fruit tree breeding.