Whole Genome Sequencing Of Plasmopara Viticola And Functional Analysis Of RXLR Effector Protein R7

The Grape Downy Mildew(GDM), which is caused by Plasmopara viticola, is economically important as one of the most devastating grapevine diseases world-wide. Successful control of the disease is one of the key factors for the development of grape industry. Infection of young inflorescences, berries and leaves cause 20-80% losses or even crop failure if outbreak of epidemic. In the study, purified sporangia were subjected to two sequencing methods, and the bioinformatics analysis had been conducted to those attained data which aimed to predict and obtain the RXLR effectors of P. viticola. The functional identification of candidate protein R7(named ourselves) was carried out. The study served as basic data for the study of pathogenic mechanism of P. viticola and other oomycetes. Results indicated that:1. Whole genome sequencing and data analysis of P.viticolaWe sequenced P. viticola genomic DNA using 454 pyrosequencing and Illumina Genome Analyzer. In total, 4 libraries were built and 10.37 G data were generated. The present results confirm that the final genome size is about 240 M, which is far more than estimated by Feulgen Absorbance Cytophotometry. Homologous annotations according Nt genome library found that P. viticola genome has high homology with some bacteria, such as Microbacterum tesaceum and Variovorax paradoxus. While homology with Phytophthora infestans genome is 3% and with the existing P. viticola DNA sequences is only 1.28%, which also shows that the P. viticola gene sequence contained in Nt library is very rare.2. The prediction of putative RXLR effector proteins of P.viticolaUsing bioinformatics approaches, 1822 putative RXLR/QXLR effector proteins were identified, including 97 RXLR/QXLR-d EER effector proteins, which is more than the number of most sequenced oomycetes.3. Functional validation of 7 RXLR effector protein candidatesFunctional validation of signal peptides, sub-cellular localization and tobacco transient expression assay were conducted to verify the functionality of 7 RXLR effector proteins. Results show that signal peptides from R1 and R2 were non-functional. R3 effector is distributed in the nucleus and cell membrane, and the remaining candidates are localized in the nucleus. R5 and R7 can induce chlorosis and necrosis on N.benthamiana, suggesting that these two effectors can interact with grapevine. R3, R4 and R6, however, show no necrotic lesions and can not inhibit hypersensitive response induced by Bax.4. The Functional verification of R7 effector proteinThe function of R7 candidate effector was verified in detail. Data suggested that RFLR-DEER mutant does not affect the internalization of R7 effector after being secreted into the apoplast. We predicted nuclear localization signal of R7 in silico and it is found that nuclear localization signal is essential to R7 induced tobacco cell necrosis. Mutants produced by deleting a few amino acid proved amino acid in the position between 101 and 125 and the position between 509 and 583 are likely to be key domains that induce tobacco necrosis.5. Cell death inhibition assays induced by R7 effector proteinTo determine cell defense mechanism in the process of infection by R7, a certain concentration of catalase, lanthanum chloride and DPI were added to inoculation treatment. Results showed that adding CAT and DPI respectively, R7 can still induce tobacco necrosis, while leaf does not produce necrosis after adding La Cl3, which shows R7 induced tobacco cell necrosis is associated with calcium channel pathway.