Functional Analysis Of G Protein And Calcium Signal Transduction Pathways In Phytophthora Sojae

Soybean root rot disease caused by oomycete pathogen Phytophthora sojae is one of main diseases which caused annual loss in agricultural production. In the field, sporangia formed on the tips of flooded mycelia, and cleavage to release mobile zoospores, which swim toward new host, through chemotaxis to the isoflavones secreted from soybean root. Consequently, to study the mechanism of sporangia formation and zoospore chemotaxis is crucial to disease control.Bioinformatics analysis of G protein and calcium signal transduction pathways in Phytophthora sojae. G protein and calcium are the most ubiquitous cellular components in eukaryotic cells that execute many important roles in signaling. Genes encoding G protein binding effectors and calcium binding proteins required for signal transduction and metabolism have been characterized in many organisms, but only a few have been described for oomycetes. In this study, the genome sequences of Phytophthora sojae were explored to construct a genome wide inventory of genes involved in G protein and calcium signaling pathways, including those encoding adenylyl cyclase (AC), regulator of G protein signal (RGS), phospholipase D (PLD), calmodulin (CAM) and calcium dependent protein kinase (CMK). The most obvious missing link is a gene encoding G protein y subunit. In all eukaryotic genomes sequenced to date, Gy genes are annotated based on certain conserved features; however, these genes seem to be absent in Phytophthora spp. Analysis of the structural and regulatory domains and domain organization of the predicted isoforms of G protein, AC, RGS, PLD, CAM and CMK revealed many novel features compared with characterized representatives in other eukaryotes. Examples are transmembrane AC proteins with a N-terminal signal peptide. Compared with other sequenced eukaryotes, the genus Phytophthora clearly has several exceptional features in its ATP catalyze enzyme.Transcriptional analysis of G protein and calcium signal pathways in Phytophthora sojae. Characterization of genes involved in development, stress response and pathogenesis is an important step in identifying methods to study Phytophthora sojae. In this study, the relative expression levels of 20 genes involved in G protein and calcium signals were studied using semi-quantitive RT-PCR. From the results of transcriptional analysis,10 genes including G protein a and B subunits were expressed upregulated in sporulating hyphae than that in nutrient hyphae.13 genes including 10 involved in calcium signal were expressed downregulated in zoospore than that in nutrient hyphae or sporulating hyphae. A Ga, a Gβ, a phospholipase, a calcium binding protein, a calcium binding protein kinase and a putative transcriptional factor were expressed upregulated in early infection stage of P. sojae. A Gβ,3 calcium binding protein kinase and 2 phospholipase D were differentially upregulated in early infection stage of P. sojae. Our results indicates that G protein and calcium signals are not only involved in P. sojae sporulating, but also important for early infection. G protein may temporarily suppress calcium signal activation in P. sojae zoospore.Characterization of G protein alpha subunit. For the soybean pathogen Phytophthora sojae chemotaxis of zoospores to isoflavones is believed to be critical for recognition of the host and for initiating infection. However, the molecular mechanisms underlying this chemotaxis are unknown. To investigate the molecular mechanism of chemotaxis to isoflavone, expression of PsGPAl was silenced by introducing antisense constructs into P. sojae. PsGPAl silencing did not disturb hyphal growth or sporulation but severely affected zoospore behavior including chemotaxis to the soybean isoflavone daidzein. Zoospore encystment and cyst germinating were also alternated, causing that the PsGPAl silenced mutants were disabled in infecting soybean. Upregulation of a calmodulin gene in zoospores of PsGPAl-silenced mutants pointed to suppressor activity of Ga targeted at steps in the calcium signaling pathway, indicating that Ga subunit may be a negative regulator of calcium signal pathways.Screening for Ga binding proteins and analysis of Ga regulated signal transduction pathways. G protein a subunit of Phytophthora sojae influences not only sporangia cleavage and zoospore release, but also zoospore motility and cyst germination. The mechanisms that how Ga binds with effectors and regulates downstream signal pathways are unknown. In this study, we constructed a P. sojae cDNA library and used yeast two hybrid method to screen for Ga binding proteins, and got 7 putative Ga binding proteins including ribulose-phosphate 3-epimerase and putative reverse transcriptase. Meanwhile, we compared gene expression profiles of P. sojae zoospores between wild type P6497 and Ga mutants A2 and A27 by cDNA Macroarray. From the results of comparison,6 genes including 3 regulator of G protein (RGS),1 Ca2+/calmodulin protein kinase and 1 Ca2+ ATPase were expressed down-regulated by Ga silencing, however,1 calmodulin,2 Ca2+/calmodulin protein kinase and 1 homolog of transcriptional factor were expressed up-regulated by Ga silencing. These results indicate that RGS activity may be dependent on the activity of Ga in P. sojae. And the relationship between G protein and calcium signal is very complex.