Molecular Dissection Of The Events Occurred During Phytophthora Sojae Early Infection Of Soybean

Phytophthora sojae causes destructive disease of soybean in the world. Oomycetes, including Phytophthora, are evolutionarily very distant from fungi and fall within the kingdom Stramenopiles. Common fungicide applied in fungi can not be used for controlling the diseases caused by oomycetes. Learning the mechanism underlying the development stages and pathogenicity of P. sojae will help researches to find new targets of chemical control and provide good ideas for designing new control strategies.Protoplast preparation of P. sojae and biological characterization of regenerated cells. Protoplasts of the well-studied strain PS2 were prepared and biological characters of regenerated strains were investigated. Protoplasts of 4×10⁶ per mL of osmotica were produced by digesting the cell wall for 3h at 30℃with a 15 mg·mL^-1 solution of Driselase and Lysing enzyme, which provided better effect than Driselase used singly. Nuclei of the generated protoplasts were observed by fluorescence microscopy. Nuclei were found in the fully developed and uniform protoplasts. Regeneration was made at a rate of 1.0% in the medium applied with 1 M mannitol. Regenerated strains that formed gray, orbicular, and felty colonies grew on the medium and showed pathogenicity to the host plant similarly as did their parent.Green fluorescent protein (GFP) as vital marker for studying microbe-plant interaction in the soybean pathogen P. sojae. Transgenic P. sojae strains that produce green fluorescent protein (GFP) were obtained after stable DNA integration using the PEG-mediated transformation method. The expression of GFP during different stages of P. sojae was observed using fluorescent microscopy. Based on this reporter system, the pathogen histopathologic events occurred in soybean leaves, hypocotyls and roots were determined. Meanwhile, the difference of resistance between different soybean cultivars against P. sojae was analyzed microscopically in roots. The results show GFP can be stably expressed in zoosporangium, zoospores, cysts, hyphae and oospores of P.sojae. Using GFP marker, the infecting pathogens in leaves, hypocotyls and roots of host can be distinctly observed. The germ length of cysts germinating on the roots of resistant cultivar Nannong8848 (48.5μm) was longer than that on the roots of susceptible cultivar Hefeng35 (31.3μm), with the similar results from the interactions between Williams82 (resistant)/Williams (susceptible) and the isolate containing GFP.Characters of early interactions with host and nonhosts of P. sojae. To develop improved methods for the control of Phytophthora infection, it is essential to understand the mechanisms by which P. sojae breaks down the plant's defenses. Many of the crucial interactions between the pathogen and the host occur at the earliest time points, including pre-infection. Prior to soybean infection, P. sojae recognizes host signals and forms infection-related structures; it must also confront preformed or induced toxic secondary metabolites in the host plants. In addition, the fungus must adapt to nutrient-deficient conditions in the early stage of host infection. The pathogen regulates the expression of its own genes to deal with the host and environmental stress, and to avoid recognition. Here, firstly exploring the mechanisms underlying the early compatible and incompatible interactions of P.sojae with host (soybean) and nonhost (tomato and non-heading Chinese cabbage) plants, leaf-inoculation method was used to analyze the early characters of interacting organisms. During the early compatible and incompatible interactions of P. sojae with host and nonhost plants, the tips of interacting hyphae became smaller than controls, and reactive oxygen species (ROS) bursted , cell death of all tested plants occurred. To learn the effect of ROS from host or nonhost on infection of P. sojae, zoospores were treated with hydrogen peroxide (final concentration with 1mM), which leads to rapid premature encystment and non-germination of cysts lasting for 10 h. However, the germination rate of cysts treated with 0.3 mM of hydrogen peroxide was higher than that of untreated cysts, and meanwhile, the germ length of former was longer than latter. The result indicates hydrogen peroxide may play an important role in the recognition of the host by the zoospores, germination of cysts and infection stages of P.sojae. Obvious appressorium or appressorium-like structure formation was observed during inoculating the leaf, hypocotyls and root of soybean with zoospores.Induction of the oxidative stress response associated with compatible and incompatible interactions between soybean (Glycine max ) and P. sojae. To ascertain if activated oxidative species play a role in soybean Phytophthora root rot caused by P. sojae, H₂O₂ burst, host cell death, the degree of lipid peroxidation (malondialdehyde [MDA] formation) and two antioxidant enzymes, namely catalase and glutathione reductase, nonenzymatic antioxidants (ascorbate and glutathione) were determined spectrophotometrically in leaves of Hefeng35 (susceptible) and Nannong 493-1 (resistant) soybean cultivars inoculated with the highly virulent pathogen PS2 of the pathogen. Moreover, the host oxidative burst and cell death was also analyzed by stainings and the transcriptional patterns of pathogenesis-related protein genes (PRs) in leaves, the effect of exogenous reductants on the interactions between the soybeans and pathogen were also determined. Under pathogen infection conditions, H₂O₂ accumulation was higher in Nannong 493-1 than in Hefeng 35 and cell death was more severe for Nannong 493-1 during early infection period; however, MDA levels were higher in Hefeng 35 than in Nannong 493-1 at late experiment period. Pathogen infection induced pathogenesis-related genes expression, varying depending on crop, duration of pathogen infection. Exogenous treatment of soybean with reductants (DTT, ascorbate, glutathione) could facilitate the infection of the pathogen, which indicated that activated oxygen species (AOS) might play an important role in the interactions of soybean and P. sojae.Identification of P. sojae genes upregulated during the earlystage of soybean infection. To explore the molecular mechanisms that are involved in the pathogenicity of P. sojae, a suppression subtractive hybridization (SSH) method was developed to screen for P. sojae genes that are differentially expressed in the early stage of Glycine max (soybean) infection. A cDNA library enriched for upregulated parasite genes was generated; of the 73 genes that were found to be upregulated, 66 are significantly similar to sequences in the P. sojae genome, and seven have no significant similarities in the databases examined. These sequences are predicted to encode proteins involved in protein biosynthesis, energy production, cell signaling, cell-wall biogenesis, and transcription regulation. These results provide an important insight into the genes expressed during P. sojae infection of soybean, which may be involved in oomycete pathogenesis.Cloning, expression profiles and functional analysis of a C₂H₂-type zinc finger protein from P. sojae. Two genes Pszf1 and Psbadh1 from SSH library were cloned, and alignment with orthologs, Southern blots, transcriptional levels and functions were determined for each genes. Pszf1 is one of C₂H₂-type zinc finger protein genes of P. sojae. To elucidate the function of, PsZF1-deficient mutants were obtained by transcriptional gene silencing. The PsZF1-silenced mutants showed increased sensitivity to hydrogen peroxide compared with wild type. The results prove that Pszf1 is crucial for early infection of P. sojae with soybean and for oxidative stress modulation in the important oomycete plant pathogen.Cloning and functional analysis of an osmotic stress-related gene from P. sojae.Psbadh1, an osmotic stress-related gene, encodes a putative betaine aldehyde dehydrogenase. The predicted amino acid sequence of Psbadh1 in P. sojae is 55% identical to a protein from Arabidopsis thialiana and has conserved domain sequences 'xxLELGGKSP' among different plants and microbes. The PsBADH1 showed BADH activity based on enzyme activity assay. The gene is unique in the P. sojae genome. Psbadh1 was silenced in P. sojae through stable transformation based on internuclear gene-silencing theory. Two PsBADH1-silenced transformants, which reduced abundance of Psbadh1 mRNA, showed sensitivity to salt stress, which indicated P. sojae required Psbadh1 for osmotic modulation.