Molecular Mechanism Of The Response Of WRKYs To Phylloxera Damage In Grapevine Roots

Phylloxera(Daktulosphaira vitifoliae Fitch) is a monophagous aphidthat feeds on grape(Vitis L.) roots and leaves and caused devastating damage on European grapevines in the 19 th century. Application of resistant rootstocks is a common-used method to control phylloxera. However, little information is available on the molecular mechanism involved in the resistance of rootstocks to phylloxera. The WRKY family is one of the largest transcription factor families in plant. Here, we investigated the expression profile of grape WRKY genes in different genotype grapes attacked by phylloxera, sensentive cultivar Crimson Seedless and resistant rootstock 1103 Paulsen, the main results prestnted in this thesis were as follows.1. A total of 63 grape WRKY genes were identified and were classified into three groups and seven subgroups according to the classification in Arabidopsis except VvWRKY32-2. These WRKYs were unevenly located on 18 chromosomes and an unknown chromosome except chromosome 3. Software prediction indicated that the majority of grape WRKY TFs were located in nuclear. The changes in exon numbers and protein length implied that sequence deletion existed in family evolution and the whole genome duplication event contributed to the expansion of this gene family. Grape WRKY genes showed differences in expression abundance in different parts of the developmental fruit berry, and the expression levels of some genes presented spatial and temporal expression patterns.2. Grape WRKY genes responded to the interaction between grapevine and phylloxera. More than 30 WRKY genes were altered at expression levels in Crimson Seedless infected with phylloxera, and expression of most induced WRKY genes was up-regulated while expressions of VvWRKY20, 24, 42, 49 and 51-2 were highly down-regulated. The expression changes of WRKY genes in rootstock 1103 Paulsen were consistent with that in Crimson Seedless. However, VvWRKY2-2 and VvWRKY42 exhibited totally different expression patterns in 1103 Paulsen compared with Crimson Seedless, indicating their potential key regulators to determine the resistance of different grape genotypes to phylloxera. Some WRKY genes showed different expression dynamics during the formation of phylloxera-induced galls. In comparison to non-inoculated root, the phylloxera-induced galls exhibited increased expressions of VvWRKY2 and Vv WRKY46 and decreased expressions of VvWRKY51-1,-53 and-55, suggesting that WRKY genes contributed negatively or positively to grape defense response to phylloxera. Additionally, similar expression patterns were detected in the non-infected root adjacent to gall, which indicated that WRKY genes were systemically induced by phylloxera infection.3. In order to better understand how grape resistance to phylloxera can affect the constitutive expression patterns of WRKY genes, the expression abundances of some WRKY genes in leaves and roots were evaluated among sensitive, middle-resistant and immune grapes. The expression abundances of WRKY genes in different phylloxera-resistant grape leaves and roots exhibited no correlation with their natural resistances. Therefore, it was inferred that phylloxera attack promotered the induced defense response mediated by grape WRKY genes.4. The SA-related genes were involved in the interaction between grapevine and phylloxera. Phylloxera transient infection repressed the expression of the genes related to SA accumulation, like VvPAD4 and VvMATE, and the total SA concentrations in root and leaf down 24% and 58%, respectively. Meanwhile, some Pathogen-related(PR) genes like VvG1, VvCHI1 B, VvCHI1B1 and VvPR3 were up-regulated in 1103 Paulsen infected with phylloxera. In addition, exogenous SA and Me-JA application lowered the survival rate of phylloxera larvae by 50% and 24%, respectively. In summary, up-regulation of PR genes in transcription level was regulated by some other factors in addition to SA. Besides, the expression of VvCHI1 B was strongly up-regulated by 50-fold in phylloxera-induced gall of 1103 Paulsen while it was down-regulated in Crimson Seedless root gall.5. It is predicted that VvWRKY40-1 and VvWRKY46 might participate in SA signal pathway based on function analysis of Arabidopsis orthologous genes and the facts that both of them were continuously induced by phylloxera attack in 1103 Paulsen. The results of rapid amplification of complementary DNA ends indicated that VvWRKY40-1 and Vv WRKY46 respectively contains an ORF of 954 bp and 1050 bp, and both of them were located in nuclear. Consistent findings of transient expression and in vitro experiment verified that VvWRKY40-1 and VvWRKY46 proteins combined with the cis-element Wbox(T)(T)TGAC(C/T) and initiated expression of downstream gene. Both VvWRKY40-1 and VvWRKY46 proteins combined and activated the promoter sequence of VvCHI1 B in one-hybrid yeast system and co-transformation experiments, which confirmed the fact that grape WRKY TF directly regulated PR genes at transcription level against phylloxera attack.