| Aldehyde dehydrogenases (ALDHs) catalyze the irreversible oxidation of a broad spectrum of reactive aldehydes to their corresponding carboxylic acids. In plants, several studies indicate that plant ALDHs participate in diverse and important pathways and play crucial roles in development, growth and stress responses. Although many plant ALDHs have been characterized, only one gene ALDH7B4 from Arabidopsis was predicted to be involved in pathogen and stress tolerance. In a previous study, an ALDH gene, VpALDH2a (accession no. DQ150256), was identified in our lab by studying the gene differential expression of Chinese wild Vitis pseudoreticulata accession Baihe-35-1leaves infected by U. necator in different periods using mRNA differential display method. To further utilize VpALDH2a, we tested this gene function in transgenic Arabidopsis and grapevine. In addition, we also studied the post-invasion resistance mechanisms in Arabidopsis. The main results are as follows:1. Results of qRT-PCR showed that expression levels of VpALDH2a gene are significantly enhanced in Chinese wild V. pseudoreticulata accession Baihe-35-1 leaves after inoculation by powdery mildew U. necator and downy mildew P. viticola respectively. Induction of VpALDH2a by U. necator was peaked at 48 hpi, whereas induction of VpALDH2a by P. viticola was peaked at 72 hpi, and the later is twice than the former at the same time course.2. Phylogenetic analysis showed that VpALDH2a belong to ALDH2 mitochondria group. Transient expression of VpALDH2a showed that VpALDH2a -YFP fusion protein is localized in the mitochondria of Arabidopsis epidermal cells by using particle bombardment. E. coli expressed GST-VpALDH2a exhibited ALDH activity in vitro and was capable of utilizing malondialdehyde (MDA), acetaldehyde and glyceraldehydes as its substrate.3. Ectopic over-expression of VpALDH2a in Arabidopsis resulted in hypersensitive response-like cell death, and showed enhanced resistance to downy mildew isolate Hyaloperonospora arabidopsidis noco2 and powdery mildew isolate Golovinomyces cichoracearum (Gc) UCSC1 presumably via SA-signaling pathway.4. Ectopic over-expression of VpALDH2a in Arabidopsis showed enhanced salt tolerance, which is accompanied by less MDA accumulation and upregulation of the stress-responsive superoxide dismutase (SOD) activity.5. The optimum media of leaves regeneration of'Thompson Seedless'is MS+BAP 5.0 mg·l-1+NAA 0.5 mg·l-1 and the frequency of adventitious bud induction was 22.4%; the optimum media of leaves somatic embryogenesis regeneration of'Thompson Seedless'is MS+TDZ 1.75 mg·l-1+NAA 0.1 mg·l-1 and the percentage of embryogenesis was 4.6%.6. A study of two parameters (induction medium and hormone) was carried out to determine the best combination for the embryogenesis induction of'Thompson seedless'anthers (Vitis vinifera L.). As average, there is no significiant difference between the percentage of embryogenesis on NN69-derived medium and MS-derived medium. The best combination of hormones for embryogenic callus induction from anthers of'Thompson seedless'is MS+BAP 1.0 mg·l-1+ 2,4-D 1.0 mg·l-1,and the induction rate was 11.5%. Different development stages of anthers from four grapevine (Vitis vinifera L.) varieties, Thompson seedless, Emerald Seedless, Chardonnay and Merlot, were used to optimize parameters influencing somatic embryogenesis in Vitis. Among the different stages, Stage III explants produced the maximum embryogenic response regardless of genotype, but embryogenic competence was dependent on variety. Of varieties evaluated, a greater number produced embryogenic cultures from anthers of Merlot (13.2%). Embryogenic cultures initiated from anthers of Thompson seedless were used to optimize the transformation system with Agrobacterium. The results indicated that pre-cultivation 2 d, co-cultivation 48 h and Hygromycin 16 mg·l-1 were benefit to transformation of grapevine.7. Transgenic grape were obtained from somatic embryogenesis by GUS, PCR and Southern blot analysis of VpALDH2a gene. No significance was observed between non-transformed and transgenic lines after inoculation of powdery mildew U. necator, but over-expression of VpALDH2a in grapevine maybe enhance resistance to downy mildew P. viticola.8. A sow thistle powdery mildew isolate was identified and designated Golovinomyces cichoracearum UMSG1(Gc UMSG1) that has largely overcome penetration resistance but is invariably stopped by post-invasion resistance of Arabidopsis thaliana. The post-invasion nonhost resistance is mainly manifested as the formation of a callosic encasement of the haustorial complex (EHC) and hypersensitive response (HR), which appears to be controlled by both salicylic acid (SA)-dependent and SA-independent defense pathways. While the broad-spectrum resistance protein RPW8.2 is dispensable for post-penetration resistance against G. cichoracearum UMSG1, and its specific targeting to the extrahaustorial membrane is physically blocked by the EHC. Our work suggests that EHC formation is a conserved subcellular defense evolved in plants against haustorial invasion.
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