Identification Of AVR5and AVR1C And Polymorphism Of AVR3C In Phytophthora Sojae

Soybean(Glycine max)is an important world-wide crop involved in the food, industry and energy. Phytophthora sojae is the casual agent of soybean stem rot and root rot disease. It is one of the most destructive diseases that affect soybean production around the world. In the soybean-P. sojae pathosystem, P. sojae exhibits a gene for gene interaction with soybean, in which Avr gene products from the pathogen are recognized by the host plant expressing the cognate resistant gene. Developing resistant cultivars of soybean was used as a main strategy to control the disease. Research on the interaction between soybean and P. sojae will greatly benefit our understanding of pathogenesis mechanisms and will provide important tools for breeding and disease management in soybeans. In this thesis, genetic mapping data, genome sequence assemblies, haplotype analysis and the predicted RXLR secretome of P. sojae hypothesized that the PsAvr3a and PsAvr5genes are alleles. The co-bombardment assays and P. sojae genetic transformation assays provided functional evidence for the hypothesis that PsAvr3a and PsAvr5are alleles; Genetic analysis of Fi and F2progeny showing that PsAvr1c segregates as a dominant allele at a single locus, the analysis of PsAvr1c candidates laid a foundation for identifying this avirulence gene; research about the polymorphism of PsAvr3c in China could benefit us to understand the mechanism of overcoming Rps3c at the molecular level.Phytophthora sojae is an oomycete and a plant pathogen that could cause root and stem rot disease of soybean. The perception of P. sojae avirulence (Avr) gene products by corresponding soybean resistance (Rps) gene products causes effector triggered immunity. Past studies had shown that the PsAvr3a and PsAvr5genes of P. sojae are genetically linked.Genetic mapping data, genome sequence assemblies, haplotype analysis and the predicted RXLR secretome of P. sojae hypothesized that the PsAvr3a and PsAvr5genes are alleles. The co-bombardment assays provided functional evidences for the hypothesis that PsAvr3a and PsAvr5are alleles, PsAvr3ap6497could interact with Rps5, but PsAvr3aP7064and PsAvr3aACR12not. The approach of P. sojae stable genetic transformation obtained additional evidences that PsAvr3aP6497gene product could interact with Rps5. Over-expression of heterologous PsAvr3aP6497in a P. sojae strain P7074that is normally virulent on Rps3a. and Rps5causes gain of avirulence in the presence of Rps3a and Rps5; whereas silencing of PsAvr3aP6497in a P. sojae strain P6497that is normally avirulent on Rps3a. and Rps5causes loss of avirulence in the presence of Rps3a. and Rps5. Sequence analysis showed that two crucial residues in the effector domain distinguish recognition by Rps3a and Rps5.Phytophthora sojae isolates ACR10avirulent on Rpslc and P7076virulent on Rpslc were used to make a cross to identify an avirulence gene PsAvrlc. Among a total of25F1progeny,12F1progeny were avirulent on Rpslc, one F1was virulent on Rpslc and others showed inconsistent virulence phenotype or the loss of general virulence. An F1(F1-62) individual showing avirulence on Rpslc was used to create40F2progeny. A total of22F2progeny were avirulent on Rpslc,6F2progeny were virulent on Rpslc and others showed inconsistent virulence phenotype or the loss of general virulence. Based on the summary of28F2progeny virulence phenotype, we conclude that the F2segregation ratio fits3:1significantly (P>0.05). So we suggest that PsAvr1c segregates as a dominant allele at a single locus. A total of75Avh genes were selected as the candidates of PsAvr1c, showing sequence polymorphisms among P. sojae isolates R2(P6497), R7(P7064), R17(P7074) and R19(P7076) that were potentially consistent with the virulence phenotype to Rpslc.CAPs analysis showed that20of75Avh genes were not PsAvr1c and20CAPs markers were unlinked with the PsAvr1c locus. A total of46Avh genes from P. sojae were analyzed by RT-PCR in the mycelia cultures of isolate ACR10and P7076to test for transcriptional polymorphisms between avirulent and virulent strains. The predicted gene PsAvhlll displayed transcriptional polymorphism, as the mRNA was detectable in ACR10but not in P7076. However, this transcriptional polymorphism for PsAvhlll was not evident among randomly chosen avirulent and virulent F2progeny and thus PsAvh111was also eliminated as a candidate for PsAvr1c. Due to the lack of success in finding any cosegregation or genetic linkage between Avh candidate genes and virulence phenotypes on Rpslc, we decided to perform a bulked segregant analysis in order to discover DNA markers associated with the PsAvr1c gene. An avirulent pool and a virulent pool had been made and three SNPs were found to be associated with PsAvr1c locus.We suggested that PsAvr1c was located in an approximately94kb region of the genome to which the three SNPs pointed. Thus, all the data laid a foundation for identifying this avirulence gene PsAvr1c.22of P. sojae isolates were collected from various geographic regions in China and their genomic DNA was isolated as templates of PCR. A pair of specific primers was used to amplify the full-length and a few flanking regions of P. sojae avirulence gene Avr3c.There were three types of nucleotide sequence polymorphism about Avr3c based on sequencing data, including Avr3c-1, Avr3c-2and Avr3c-3. The isolate showing avirulence on Rps3c has the type of Avr3c-1. Among the isolates showing virulence on Rps3c,15of them have the type of Avr3c-1,5of them have the type of Avr3c-2which shares similarity of97%with the type of Avr3c-1and1of them has the type of Avr3c-3which shares similarity of96%with the type of Avr3c-1.Compared with the type of Avr3c-1from the avirulent isolate, the type of Avr3c-1from the virulent isolates was the same as it, the type of Avr3c-2from the virulent isolates had amino acid substitution at14sites, and the type of Avr3c-3from the virulent isolate has amino acid substitution at21sites and the deletion at one site. The transcript of Avr3c-1allele in two virulent isolates wasn’t detectable. dN/dS analysis based on the full-length sequence showed that Avr3c was under the positive selection from the host. With the evolutionary models,21positively selected amino-acid residues were identified in Avr3c (P>99%).