Isolation And Functional Analysis Of Resistance Genes Analog From Grapevine Resistant To Downy Mildew

Downy mildew caused by the oomycete Plasmopara viticola (Berk. and Curt.) Berl. and de Toni is the most economically important fungal disease of grapes(Vitis spp.). This devastating disease causes partial or total crop losses and has a severe secondary environmental impact due to the repeated fungicide applications required as a control measure. To achieve sustainable grapevine production, the isolation and incorporation of genes into grapevine that confer resistance to downy mildew would be of considerable economic and environmental benefit. The study was carried out to resistance genes from the resistant Vitis riparia cv Beta by homology-based method and cDNA amplified fragments length polymorphism (cDNA-AFLP) analysis. The expression patterns of the test fragments were conducted through real-time PCR, for further functional analysis. The main results were discrived as follows:1. Degenerate primers that were designed based on the conserved motifs in the NBS domain of resistance proteins were used to isolate NBS-type sequences. A total of10RGA fragments were identified, and their predicted amino acid sequences compared to each other and to the amino acid sequences of known R-genes revealed significant sequence similarity. Phylogenetic analysis demonstrated that10RGAs dispersed along the phylogram on the two major branches of either TIR or non-TIR type of the NBS-LRR proteins. Quantitative real-time polymerase chain reaction (qRT-PCR) analysis showed that transcripts of RGA1, RGA2, RGA5and RGA23were specifically induced after infection with P. viticola.2. The full length sequence of disease resistance homology gene in the cDNA from Beta was4787bp named as RGA1which was obtained by the rapid amplification cDNA ends (RACE) based on RGA1fragment. It deduced1514amino acids. RGA2cDNA was3883bp, encoding1200amino acids, RGA5cDNA was4282bp, encoding a polypeptide of1335amino acids, while RGA23was1607bp, encoding897amino acids.3. The predicted RGA1, RGA2and RGA5proteins has been divided into three domains:N-terminus Drosophila Toll and mammalian interleukin-1receptor (TIR); nucleotide binding site (NBS); C-terminus leucine-rich repeat (LRR). RGA1, RGA2and RGA5belong to the TIR-NBS-LRR resistance protein. The predicted RGA23protein shows all the characteristic domains of NBS-LRR protein. The conserved NBS motifs are highly similar to those of other NBS-LRR resistance proteins. The LRR domain consists of13repeats. A coiled-coil (CC) region is located between residues8and97, indicating that RGA23belongs to the CC subfamily of NBS-LRR resistance protein.4. The expression of RGA1, RGA2, RGA5and RGA23under P. viticola infection and abiotic stresses was analyzed at different time points using quantitative real-time polymerase chain reaction (qRT-PCR). Results showed that the P. viticola treatment and four tested abiotic stimuli, SA, MeJA, ABA, and H2O2, triggered a significant induction of RGA1, RGA2, RGA5and RGA23within12d of inoculation.5. We carried out cDNA-AFLP analysis on artificially infected leaves of the resistant Vitis riparia cv Beta at the oil spot stage, on water-treated leaves and on a sample of pure sporangia as controls. Selective amplifications with306primer combinations allowed the visualization of about15300transcript-derived fragments (TDFs) in infected leaves,1370of which were differentially expressed.6. We sequenced674fragments,489of which were identified as grapevine transcripts after homology searching. All TDFs were classified, energy and metabolism, disease/defense, transporters, cell structure, transcription, energy gene, signal transduction, protein storage were supposed to correspond to the expression of the disease-resistant genes. Many grapevine genes spanning almost all functional categories were downregulated during infection, especially genes involved in photosynthesis.7. The expression level of28modulated grapevine transcripts was analyzed further by real-time RT-PCR, to validate cDNA-AFLP expression profiles. This study provides global catalogue of grapevine genes expressed during infection, together with their functional annotations. This will help to elucidate the molecular basis of the infection process and identify genes and chemicals that could help to inhibit the pathogen.