Identification Of Avirulence Gene Of Phytophthora Sojae And The Study On Resistance Mechanism Of Potato Cultivar Zihuabai To Phytophthora Infestans

Oomycetes which are separated from mastigomycotina recently, can infect manyplants. It causes serious problems on natural ecosystems by infect crops, forestry andornamental plant. Soybean blight and potato late blight are two main diseases in soybeansand potatoes caused by Oomycetes fungi Phytophthora sojae and Phytophthora infestansseparately. Currently, the most effective, economic and environmentally friendly way ofprevention and treatment of these two diseases rely on cultivating resistant varieties.Therefore, cloning and identification of avirulence gene in Phytophthora using molecularbiology techniques and unravel the mechanism of potato resistance to late blight diseasecould lay the foundation for soybean and potato disease resistance breeding.In this thesis, two P.sojae avirulence genes Avr1d was cloned by using candidate Avhgenes screening method. Genetic mapping of the candidate Avh genes in F2mappingpopulation suggested that Avh6co-segregates with Avr1d. Transient expression assaysconfirm that Avr1d triggers cell death specifically in Rps1d soybean plants. Alignmentbetween Avr1d and the whole genome shotgun sequencing from5P.sojae strains wereperformed. The results showed that Avr1d gene is present in P. sojae strains that areavirulent to host which contain Rps1d resistance gene, whereas the gene is deleted from thegenome of virulent strains. Two allelee of Avh6were identified in the avirulent strains andthey encoded slightly different protein products. Transient expression of full-length wildtype Avr1d and full-length Avr1d with RFLR (Arg-Phe-Leu-Arg) mutant to AAAA (Ala-Ala-Ala-Ala) showed that RFLR region could not lead the Avr1d protein into the host cells.The liposome binding experiment data indicated that Avr1d could bind to PtdIns (4) P andthe binding is achieved through positive amino acids of effector domain of Avr1d, insteadof by the RFLR motif.Three Avr1c linked single nucleotide polymorphisms (SNPs) were found by BulkSegregate Analysis combined with next-generation sequencing technology. Genetic andphysical mapping results suggested Avh275c co-segregate with Avr1c. Transientexpression experiments also prove that Avh275c is Avr1c. Haplotype sequencing analysisshowed that three alleles of Avr1c exist in the strain collection. Previous work proved thatAvh275c located very close to Avr1a on P.sojae genome. Comparison of the sequences from these two gene suggested Avr1c and Avr1a encoded slightly different protein. Themost variable region of the protein was found within N terminal effector domain.The infection of P.infestans on potato will trigger the host a series of resistanceresponds. To know the resistance mechanism of potato resistant cultivar Zihuabai,accumulation of reactive oxygen species (ROS), activities of oxygen scavenging enzymes,and transcription level of some resistance genes were determined in both resistant andsusceptible potato cultivars inoculated with P. infestans. Our results showed that ROSaccumulation is an important signal pathway that involved in the procedure of Zihuabairesistant to P.infestans. The accumulation of ROS in the resistant cultivar depends on themembrane NADPH oxidase. The results of transcript expression of important protein alsoindicated that signaling molecules also involved in regulating the resistance of potatocultivar Zihuabai to P. infestans.