Function Analysis Of GPCR-PIPKs Family And PsYKT6 In Phytophthora Sojae

Phytophthora sojae is the casual agent of damping off of the seedlings and root rot disease of soybean, which is an economically important crop around the world. It is one of the most notorious pathogens which caused tremendous economic loss in soybean production annually. P. sojae switch the life stages by sensing hormone, temperature, humidity and other environmental conditions.During infection of their hosts, P. sojae secreted a variety of extracellular proteins to promote the pathogenicity. Therefore, to study the mechanism that P. sojae sense the environmental factors and the signal transduction pathway, and the process that P. sojae secretie extracellular proteins will provide theoretical bases for disease control. Methods for reverse genetics involving gene disruption are not currently feasible in oomycetes due to low rates of homologous recombination during transformation and diploidy. The molecular mechanisms underlying development and infection are largely unknown. As the increasing mount of the genome sequence and transcription profile data for P. sojae available, a feasible and efficient genetics protocols is critical for studying the biology, pathogenicity, and evolution of these organisms.Double-stranded RNA mediated gene silencing in Phytophthora sojae. Double-stranded (ds)RNA mediated gene silencing, a post-transcriptional and highly conserved process in eukaryotes, results in specific gene silencing through degradation of the target mRNA. Recently, we set up a dsRNA mediated transient gene silencing protocol combined with in vitro dsRNA synthesis and a polyethylene glycol (PEG) mediated transformation system. Single-gene silencing is accomplished by introducing unique dsRNA fragments into protoplasts, and multiple-gene silencing is performed with chimeric overlapping dsRNA fragments. The cell cycle regulated gene PsCdcl4 (a protein phosphatase that regulates the mitosis and the cell cycle) and the avirulence gene PsAvr3a (an RxLR effector that is recognized by the soybean Rps3a resistance proteins) are transiently silenced by introducing in vitro synthesized dsRNA into P. sojae protoplasts. The results showed that there is no reduction in PsCdc14 or PsAvr3a mRNA in the transformants until 8 days after the specific gene dsRNA was transferred into protoplasts. From 9 to 15 days, the transformants exhibited significant gene silencing, with mRNA levels reduced by 50% to 95%. All the silencing transformants showed recovery of gene expression by 17 days. Thus, the gene silencing protocols could significantly reduce the mRNA level of PsCdcl4 and PsAvr3a, yielding detectable phenotypes. The successful setup of transient gene silencing could make P. sojae high-throughput gene function elucidation work feasible, facilitating our understanding of the development and pathogenicity mechanisms of this organism.Bioinformatics prediction and transcription analysis of GPCR candidate coding gene in phytophthora sojae.G protein-coupled receptors (GPCRs) represent the largest family of transmembrane receptors in eukaryote and are responsible for transducing extracellular signals into intracellular responses that involve complex intracellular-signaling networks, affecting the development and metabolic pathway. Phytophthora sojae changes the life stages by sensed the hormone, temperature, humidity and other environmental condition. Consequently, to study the mechanism of P. sojae sense the environmental information and the signal transduction pathway will provide theoretical bases for disease control. In this study, we predicted many putative GPCRs coding genes in the genome of P. sojae, P. ramorum and P. infestans according to the sequences and domains from reported GPCR in other organisms. The ortholog alleles and the gene genomic distribution patterns of the putative GPCR genes of P. sojae, P. ramorum and P. infestans were analyzed. In P. sojae, the transcription profile of putative GPCR was also performed by RNA-sequencing and qRT-PCR. These results provide a new study way for analysis function gene, which is crucial to disease control and drug development.Two novel protens, PsPIPK4 and PsPIPK10 are respectively involved in asexual development and sexual reproduction in Phytophthora sojae. P. sojae, zoospores have the characteristic of chemotaxis, which can swim chemotactically toward compounds released by roots of their host plants, and then encyst on the root surface from where the hyphae penetrate the root directly from the cyst. Sexual oospores are often thick walled and thus more durable than vegetative structures. Also, sexual oospores are the main agents of dispersal for P. sojae. The genome sequencing of P. sojae provide a great opportunity to identify a novel group of GPCRs, termed as GPCR-PIPKs, which fused a typical GPCR 7-transmembrane domain at the N-terminusl combined with a phosphatidylinositol phosphate kinase (PIPK) domain at the C-terminus. PsPIPK4, one of GPCR-PIPks, was upregualted in cyst and cyst germination. The function analysis indicated zoospore encystment and cyst germination were badly affected, resulting in the inability of chemotaxis and infecting soybean in PsPIPK4-silenced mutants. PsPIPK10, another GPCR-PIPks, was constitutive expression in asexual development and infection stages. PsPIPK10-silenced mutants exhibit remarkable reduction of oospore number, just around 1-5% production compared to the wild type. This is the first reported that putative GPCRs are involved in asexual development and sexual reproduction according to our current knomledge.Genome-wide identification and transcriptional profiling of SNARE family genes and functional characterization of PsYKT6 in Phytophthora sojae. In eukaryotic cells, the intracellular vesicle trafficking is essential for many aspects of cellular processes including polarized growth and secretion of extracellular proteins. SNAREs (soluble N-ethylmaleimide-sensitive factor attachment protein receptors) are central components of the machinery mediating membrane fusion in all eukaryotic cells. Here, we identified 35 genes, which might be a total set, encoding putative SNARE proteins that are key factors for vesicular trafficking, taking advantage of available whole genome sequence in the oomycete plant pathogen Phytophthora sojae. The subsequent systematic analysis to determine its transcription in different development and infection stages revealed that most putative SNAREs show important roles in P. sojae. One of the most conserved SNARE proteins (PsYKT6) was functionally characterized by homology-dependent gene silencing method. The phenotype analysis showed that PsYKT6 plays key roles in both asexual development and sexual reproduction, and in pathogenesis on host soybean cultivars.