Study On The Function Of Effectors In Phytophthora Sojae

Phytophthora sojae is one of pathogens that can cause destructive disease of soybean. Despite similar growth habits with filamentus fungus, P. sojae is evolutionarily very distant from’true’fungi, which is closed to algae and is classified in kingdom Stramenopila, Phylum, Oomycota. At present, it can cause around＄1-2 billion in losses per year worldwide. P. sojae can secret a large number of effector moleculars that facilitate the infection of the hosts. Some effectors can be recogniazed by host plant thus were called avirulence (Avr) proteins. These effectors shared a similar molecular pattern with conserved motif (RXLR) at the N terminus. More than 350 candidate genes encode RxLR effector were bioinformatically identified in the genomes of P. sojae. The function of these effectors during the interaction between the host and P. sojae remain unknown. In this study, the the suppression of plant immunity responses, transcriptional profiles, sequence polymorphism and the protein domains of representative effectors were analyzed in details.Functional screening of P. sojae effectors on the suppression of hypersensitive response (HR) on Nicotiana benthamiana 50 effectors were screened to suppress HR triggered by Bax, INF1, Avh238 and Avh241 by using dual-virus Potato virus X expression vector in N. benthamiana. The results showed that 50 candidate effectors can be divided into eight categories based on the different suppression spectrum. Among these effectors, 20 effectors can suppress plant defense responses triggered by INF1 and Avh238 and Avh241; 7 effectors can suppress plant defense responses triggered by Avh238 and Avh241; two suppress HR triggered by INF1, Avh238 and Avh241,7 effectors suppress HR triggered by Avh238 and Avh241, one effector suppresses HR triggered by INF1 and Avh241,4 effectors to suppress HR triggered by Avh238,2 effectors to suppress HR triggered by Avh241; with the rest seven effectors can not suppress any kinds of HR. The results showed that different effectors may target on diversified signaling pathways to manipulate different kinds of HR.Analysis on the transcriptome and sequence polymorphism in P. sojae To further understand the role of effectors in the process of interaction with host and pressures facing the choice of the host, the transcriptome, sequence polymorphisms were analyzed. Firstly, RNA-seq data of the 50 selected effectors was collected and analyzed; it showed that the majority of effectors are highly expressed during the growth and infection stages. However, the transcription of eleven effectors including Avh6, Avh7bl and Avh141 were not detected. The sequence polymorphisms of effectors with different function were analyzed, and the selection pressures are also detected. The result did not demonstrate there is any kind of relationship between genes function and sequence polymorphism. However, the W motif in the RxLR effectors might contribute to the suppression plants defense response according to our current data.Analysis on the functional domain of RxLR effector Avh241 can induce HR in Nicotiana benthamiana Previous data showed that Avh241 can induce HR in N. benthamiana, suggesting it may be recognized by the host during the interaction with pathogen. To explore the key residue sites of Avh241, the bioinformatics analysis of Avh241 was made. The result showed that Avh241 have typical signal peptide region, conserved RxLR-dEER domain and two W motif and two Y motif, as well as the amino acid sequence contains 10αhelices and a B folding in the secondary structure. Based on the above result, the deletion version of Avh241 was screened. The result showed that Avh241 mutant with deletion W, Y motif andβ-sheet in secondary structure can not induce HR in N. benthamiana. In the addition, more than 1,300 mutants were derived from Avh24159-188, a functional fragment lacking of signal peptide and RxLR-dEER motif were screened and sequenced. Nine mutants failed to induce HR with some point mutation were obtained while solvent accessibility of the mutant Avh24159-188 was predicted by Jnet software. The results showed that the site in the mutant with loss function (W94、L110 etc.) was less than 25% of the solubility, suggesting that these amino acid sites play the role in the formation of protein secondary structure and the site W94 and D153 play some important function.The fuctional analysis of effector Avh38 and Avh283 in P. sojae Based on previous screening results of effector suppression HR in N. benthamiana and the analysis on the transcriptional profiling and sequence polymorphism of effectors, the effectors Avh38 and Avh283 shared the same suppression spectrum and similar transcriptional patterns and both effectors are under positive selection, so these two effectors were selected for a further study. By using double-stranded RNA-mediated transient gene silencing (dsRNA) technology, Avh38 and Avh283 transient silencing transformants were obtained, respectively. The phenotype data demonstrated that there is no significant difference between silencing lines and control lines in pathogenicity, asexual development and the chemotaxis patterns of zoospore, suggesting that Avh38 and Avh283 might not contribute to pathogenicity. Therefore, the biological function of these effectors remains to be studied.