Evolutionary Analysis Of Effector Protein Family In Phytophthora Sojae And Genomic Characterizations Of Pythium Guiyangense

Oomycetes, falling within the kingdom Stramenopiles, include more than 800 pathogens that infect plants and animals. Although they share similar growth habits and nutritional strategies with fungi, they are evolutionarily distant from’true’fungi, and close to Heterokontophyta. Due to the biological and physiological differences between oomycetes and fungi, oomycete pathogens are unaffected by the majority of fungicides. With the development of high-throughput sequencing and genomic research, more and more oomycete species are sequenced. By using the bioinformatics tools, the sequence feature and evolution could be investigated through the genomic level, which provide new methods for oomycete research, and also provide better candidates for further functional analysis to improve the efficiency. Phytophthora sojae and other oomycetes secrete large number of effectors to help infection, but the pathogenic mechanisms are still unclear. In this paper, we detailedly investigated CRN effector family and the intron characterizations in P. sojae and the genome of Pythium guiyangense (Py. guiyangense), and the main results are showed as follows:1. Gene duplication and fragment recombination drive the expansion and functional diversification of CRN effector family in P. sojae. CRN gene is a kind of effector which located in plant cytoplasm, and could induce cell death in plants. However, little is known regarding the function and molecular mechanism of the CRN effectors. CRN family is widely distributed in oomycetes, especially in Phytophthora species, and lots of pseudogenes were found, leading to the question how was such a large family evolved? By comparing the CRN effectors in Phytophthora species, they were unequally distributed in the three species, in which each underwent large number of gene gains or losses, indicating that the CRN genes expanded after species evolution in Phytophthora and evolved through pathoadaptation. Compared to RxLR effectors, the CRN effectors were relatively conserved. The CRN family in P. sojae was classified into two subgroups, Class I and Class II. Class I is mainly composed of functional genes with high transcript levels, suggesting important roles during the infection. Furthermore, we demonstrated that most expanded genes underwent gene duplication or/and fragment recombination, and these expanded genes exhibited diverse functions under the selective pressure. Only few CRN effectors could induce plant cell death, while most other effectors could suppress the cell death induced by Bax, Avh241, PsojNIP, PsCRN63 and Avr3a-R3a protein interaction. Overall, these results suggest that gene duplication and fragment recombination may be two mechanisms that drive the expansion and neofunctionalization of the CRN family in P. sojae, which aids in understanding the roles of CRN effectors within each oomycete pathogen.2. The introns in P. sojae exhibit distinct features. Intron is widely distributed in Eukaryotes, however, little is known regarding the introns in oomycetes. We identified 4,013 introns in P. sojae by a comparative analysis of genomic sequences and expressed sequence tags, and 96.6% of the introns contained canonical’GT-AG’splice sites. The P. sojae genome possessed features distinct from other organisms at 5’splice sites, polypyrimidine tracts, branch sites and 3’splice sites. Diverse repeating sequences, ranging from 2 to 10 nucleotides in length, were found at more than half of the intron-exon boundaries, which was not reported in other organisms previously. Furthermore,122 genes which underwent alternative splicing were divided into four splicing patterns. These data indicate that P. sojae has unique splicing mechanisms, which could help improve the annotations of P. sojae and other oomycete genomes, leading to more accurate genomic structures. These results are also helpful for feature and evolution research of pathogenic genes including effectors in oomycetes.3. Py. guiyangense genome exhibits features adapted to its insect pathogenesis. During the infection of oomycete pathogens, plant pathogens and animal pathogens share some common pathways, while also have distinct features. Currently, we mainly focus on the pathogenic genes including PAMP and effectors in plant pathogenic oomycetes. By the comparison of plant pathogens and animal pathogens, we can investigate the similarities and differences, which help understand more about oomycetes. In this study, we performed do novo genome sequencing of Py. guiyangense. Based on the phylogenetic analysis of conserved single copy housekeeping genes, Py. guiyangense clustered with know Pythium species, and is close to Py. irregular and Py. iwayamai. Its genome size is 87 Mb, and encodes 26,056 genes. Due to its insect host, a large number of gene families are changed. The number of plant cell wall degrading enzymes is largely reduced in Py. guiyangense. The predicted proteome includes a large repertoire of protein kinases and proteases involved in animal pathogen interactions, such as subtilisin and trypsin proteases. Py. guiyangense genome does not encode any classical RxLR effectors, although, surprisingly, contains some CRN effectors which are more diverse compared to that from other Pythium species, suggesting evolutionary adaptation to the animal host. Furthermore, Py. guiyangense lacks the ability to synthesize sterols, but secretes large number of Elicitin proteins to uptake cholesterol from mosquitos during the infection process in order to assure proper development of survival propagules. These results lay a foundation for research of pathogenic mechanism of Py. guiyangense, and provide theoretical support for biological control of mosquitoes.