Functional Analysis Of Mitogen-Activated Protein Kinases, PSSAK1and PSMPK1, In Phytophthora Sojae

Phytophthora sojae is the soybean pathogen responsible for the soybean root rot disease that causes around$1-2billion in losses per year and is a threat to soybean production all over the world. In the field, zoospores, formed from sporangia on the tips of flooded hyphae, are the main means of dispersal and inoculum. Elucidating signal transduction pathway that govern this pathogen’s decelopment and pathogenicity will eventually be useful in designing novel control strategies. Mitogen-activated protein kinases (MAPKs) are universal and highly conserved in eukaryotic cells. They play important roles in regulating proliferation, development and are key elements that control adaptation to environment stress. In this thesis, bioinformatics approaches, real time RT-PCR technologies and gene silencing method are used to explore MAPKs in the genome of Phytophthora sojae. Finally, two of MAPKs, PsSAKl and PsMPKl, are functionally characterized.Bioinformatics analysis MAPK genes in the genome of Phytophthora sojae: Soybean root rot disease caused by oomycete pathogen Phytophthora sojae is one of the most destructive diseases in agricultural production, however, the mechanism of pathogenisis at the molecular level is less understood. In this study, we characterize the mechanism of the development and pathogenicity of P. sojae exploiting the key molecule of. signal transduction, MAPK (mitogen-activated protein kinase). MAP kinase signal transduction pathways play a critical role in eukaryotic cells to properly adapt to changing environments. Employing proper bioinformatics methods, we have identified14gene encoding MAP kinase, and the gene number is similar to that of plant sequenced genomes, but many more than that of fungal genomes. Like most MAP kinases, the phosphorylation site of10MAPK of P. sojae is TEY, however, other peculiar phosphorylation site is SEY, TEH (2) and TKH, respectively. Based on phylogenetic dendrograme and domain analysis, we found a novel family of MPAKs in which contain PH (pleckstrin homology) domain in the N-terminus, which was not found in known MAPKs so far. All of these14MAPK genes were randomly distributed in the genome, and the gene structures were various. The bioinformatics of MAPK in P. sojae provided a good platform for further characterization.Transcriptional analysis of MAPKs in Phytophthora sojae:Based on previous bioinformatics of MAPKs in the genome of Phytophthora sojae, in this study, we made use of SYBR green real-time RT-PCR to analyze their transcriptional pattern during the differential development and infections. The results showed, in all14MAPKs in P. sojae, only one gene (PsMPK9) was not detected in all tested samples, the others exhibited different transcriptional patterns. In the zoospore stage,9genes showed differentially transcriptional patterns, for example,6genes (PsMPK2,3,7,10,11,13) upregulated,3genes (PsMPK1,4,12) down-regulated. In the germinated cysts,6genes (PsMPK2,3,7,10,12,13) showed differentially transcriptional patterns. At6h compatible interaction,3genes (PsMPK1,2,3) were significantly upregulated, and at24h,3genes (PsMPK1,2,3) were strikingly upregulated. Theses results indicated that MAPKs would participate in the development and infection of P. sojae in complicated ways; at the same time, we can choose the genes of interest in a systematic way to characterize the function based on these transcriptional patterns.PsSAKl, a stress-activated MAP kinase, controls zoospore viability and pathogenicity in Phytophthora sojae:In this study, PsSAKl, encoding a stress-activated mitogen-activated protein kinase of Phytophthora sojae, is identified. PsSAK1is highly conserved in oomycetes, and it represents a novel MAPK due to its pleckstrin homology (PH) domain. RT-PCR analysis showed that PsSAKl expression was up-regulated in zoospores, cysts and during the early infection. In addition, its expression was induced by osmotic and oxidative stress mediated by NaCl and H2O2. To elucidate the function, the expression of PsSAKl was silenced by stable transformation of P. sojae. The silencing of PsSAKl did not impair hyphal growth, sporulation or oospore production but severely affected zoospore development, in that the silenced strains showed quicker encystment and less efficient germination than the wild type. PsSAK1-silenced mutants were unable to colonize wounded or intact soybean leaves, in contrast to wild types cultures. Further more, although germinated cysts of PsSAK1-silenced mutants produced much longer germ tubes than the wild type strain and the control strain, the mutants could not penetrate epidermis cells. Our results indicate that PsSAK1is an important regulator of zoospore development and pathogenicity in P. sojae. A MAP kinase encoding gene PsMPKl regulates morphogenesis and pathogenicity in Phytophthora sojae:Based on the bioinformatics analysis and transcriptional analysis of whole genomic MAP kinase encoding genes, one gene PsMPKl, which encoded a typical MAP kinase and was upregulated in sporulating hyphae and early stage (6h) of infection, was isolated and functionally characterized. PsMPKl is highly conserved in oomycetes. Three mutants (T21, T40and T64) of P. sojae stably silenced for PsMPK1were obtained using a homology-dependent gene silencing method. Phenotypes of the silenced transformants in comparison to non-silenced strains were evaluated throughtout the life cycle. The silencing of PsMPKl was not associated with differences in the swimming of zoospores, chemotaxis and cyst germination. There also appeared to be no effects on sexual development, as PsMPK1-silenced and non-silenced lines produced equivalent numbers of oospores. However, the hyphal growth rate, zoosporegenesis and the hyphal morphology were markedly affected by silencing. Swelling hyphae, abnormal hyphal structures, were much more common to see in PsMPK1-silenced mutants than in non-silenced lines when they were cultured in10%V8juice media for4to5days. PsMPK1-silenced mutants were also more sensitive to cell wall degradation enzymes, including glucanase and cellulose. Pathogenicity test showed that PsMPK1-silenced mutants could not infect intact soybean leaves of Williams but could infect wounded soybean leaves.