Analysis Of The Fuction Of A Transcription Factor Gene VpRFP1 From Vitis Pseudoreticulata W.T. Wang

The object of study is the accession Baihe-35-1from Chinese wild Vitis pseudoreticulata in resistance to the pathogens of grape powdery mildew, we analysis the function of the transcription factor gene VpRFP1 from Chinese wild Vitis pseudoreticulata. The bioinformatics analysis revealed that the structure and the conserve motif of the VpRFP1, and the related information of the location in the grape chromosome. GST-VpRFP1 fusion protein expressed in E.coli BL21, purification the fusion protein by electrodialysis assay, and immunized the rabbit with the purification product obtain the polyclonal antibodies, western blotting showed that the antiserum had specific antigen-antibody recognition with the fusion protein. And test the expression variation of VpRFP1 inoculation by U. necator either in resistant grapevines and susceptible grapevines was investigated by Real-Time PCR and Western Blot. o determine whether the VpRFP1 protein is capable of acting as a nuclear factor, the subcellular immunogold labeling of the VpRFP1 protein was examined in V. pseudoreticulata leaves. To investigate the functional role of the VpRFP1 protein as a transcriptional activator, GAL4 fusion proteins were also analyzed in yeast. The overexpression of the VpRFP1 in transgenic Arabidopsis plants showed enhanced resistance to Erysiphe cichoracearum. Additionally, the transgenic lines are more resistant to P. syringae pv. tomato DC3000 than the wild type, and present the cell death resistant to the bacteria. Silence of VpRFP1 homolog in tomato showed that the transgenic plant susceptible pathogen and associated with SA-dependent PR gene declined, whereas the JA-dependent PR gene increased. This research obtained such results as follows.1. The VpRFP1 cDNA of 1,415 nt contains a 170 nt untranslated region at 5'-end and a 192 nt untranslated region at 3'-end. The genomic sequence of the VpRFP1 gene was also cloned directly from the V. pseudoreticulata genomic DNA(GeneBank accession no. GU446678). VpRFP1 is located on the chromosome 17 of the published Pinot Noir whole-genome Alignment of the genomic DNA sequence with the cDNA of VpRFP1 indicated that the VpRFP1 gene contained 2 introns and 3 exons. The predicted open reading frame (ORF) encodes a RING-finger protein of 350 amino acids, which has theoretical pI value of 5.83 and a deduced molecular mass of 38,005 Da. The VpRFP1 amino acid sequence contained a nuclear localization signal (NLS) at N-terminal and the RING-finger motif at C-terminal. Compared to other species, the RING-finger motifs belongs to a novel RING finger variant of a C4C4-type, with the consensus sequence of Cys-X2-Cys-X13-Cys-X1-Cys-X4-Cys-X2-Cys-X10-Cys-X2-Cys. Phylogenetic tree analysis showed that they were classified into three subfamilies. V. pseudoreticulata, V. vinifera and L. esculentum was part of the same subfamily.2. 1053bp of the VpRFP1 open reading frame (ORF) amplified by RT-PCR; clone this sequence into pMD19-T vector, sequenced and make sure it correct, ligation with pGEX-4T-1 vector and transformation into E.coli BL21; expressed a 64kDa fusion protein GST-VpRFP1 induced by IPTG, and its was expressed in inclusion body, purification the fusion protein by electrodialysis assay, and immunized the rabbit with the purification product obtain the polyclonal antibodies, western blotting showed that the antiserum had specific antigen-antibody recognition with the fusion protein.3. For resistant grapevine accession Baihe35-1 of V. pseudoreticulata, VpRFP1 up-regulated expression was detected at 12 hpi, peaked at 24 hpi, and reduced at 48 hpi, then maintained at a relatively low level during 48–144 hpi after U. necator inoculation. In contrast, the susceptible grapevine cultivars Carignane and Thompson Seedless of V. vinifera displayed high transcription level at 0h, with the down-regulation of VpRFP1 expression upon inoculation to reach the lowest levels at 24 hpi and 48 hpi, respectively. The expression of VpRFP1 in V. vinifera cv. Carignane presented a gradually increasing trend after inoculation, and reached the maximum expression at 144 hpi. By contrast, the expression of VpRFP1 in the resistance accession Baihe-35-1 of the V. pseudoreticulata was transiently changeable, and high peak were occurred at 48 hpi and 96 hpi, and low peak were presented at 72 hpi and 120 hpi, respectively.4. Immunogold labeling with VpRFP1 antiserum showed that gold particles were mainly localized in the nuclear. No substantial signal was detected in controls without antiserum or using the pre-immune serum, confirming the specificity of the immunolocalization. The none-infected leaves treated with the specific antiserum against VpRFP1 showed few gold particles in the nucleus and fewer in cytoplasm. At 24 hours after artificial inoculation (hip), pathogen-infected samples showed high density of gold particles in the nucleus; while similar signal was found in the cytoplasm of none infected samples. These results suggested that VpRFP1 was located in the nucleus upon activity.5. The full-length VpRFP1 and most of deletion mutants showed no transcriptional activation activity, whereas the RING finger motif connected with the C-terminal region of the VpRFP1 (pGBKT7-D, and pGBKT7-J) presented transactivation activity in yeast. 6. Overexpression of the VpRFP1 in transgenic Arabidopsis plants conferred the enhanced disease resistance to E. cichoracearum. The transcript of AtPR1, AtPR2 associated with the VpRFP1 presented an increasing trend after infection, which is different from the expression of AtPR3 and AtPDF1.2. At the macroscopic level, cell death in mature infected leaves by trypan blue staining is significantly increased on the transgenic lines compared with wild-type plants. Microscopic analysis indicates that the extent of visible lesions is also significantly increased in the transgenic lines compared with the wild type.7. To test whether the TRV-vector can directly infect the tomato leaves, cDNA was used as a template for RT-PCR with TRV-R2 specific primers. The TRV-R2 specific PCR product (427 bp) was detected only in the TRV-infiltrated, and was absent in the control (no infiltration and treated with suspension solution). To examine whether TRV-vector could be used efficiently to silence genes in L. esculentum, we detected the LeRFL1 by RT-PCR. Plant infiltrated with TRV carrying a fragment of LeRFL1 displayed a weak expression and control's expression normally.Taken together, our results suggested that VpRFP1 may be a transcriptional activator of defense-related genes that are involved in grapevine defense response.