Molecular And Cellular Biology Of Interactions Between Phytophthora Sojae And Host Soybean

Phytophthora sojae Kaufmann&Gerdemann is an important oomycete pathogen in soybean production, which causes "damping off" in seedlings and root rot in plants, with an annual loss of more than1billion dollar. P. sojae is a soilbone pathogen and almost obligate to soybean (Glycine max). As being infection source in field, sexual oospores develop asexual zoosporangia in which cytoplasm subdivides into several zoospores. Equipped with flagella, zoospores target host and initiate infection on host. As focusing on cellular biology of interactions between P. sojae and host, not only it can promote researches on molecular mechanism of the interaction, but also it is beneficial to understand and master the discipline of the occurrence and development of this disease, with which we can formulate effective control strategies. In this thesis, we observed the cellular biology of interactions between P. sojae and host with conventional cytological staining methods and molecular fluorescent probes; based on P. sojae GFP-tagging isolates, we elucidate the compatible and incompatible interaction by live-cell imaging.Cellular biology of compatible interaction between Phytophthora sojae and host. As advances of technologies of microscopy and molecular fluorescence, it is entirely possible to assist the research on interaction between P. sojae and host. Based on molecular fluorescent probes and conventional staining methods in cytology, this study successfully researched on cellular morphology and subcellular structure during infection, and cell biology of compatible interaction between P. sojae and susceptible host. According to characteristics of morphology of P. sojae during interaction, the infection process is mainly composed of3stages, pre-infection, penetration, and extension. In stage of pre-infection, as the same as asexual life of P. sojae, the zoosporangium is proliferated from the expansion of hypha apex which cytoplasm in hypha flows into; the zoosporogenesis is achieved by subdivision of cytoplasm in sporangia which destines to develop as primary inoculum. In infection, with assistance of two flagella, zoospores swim freely towards the right host, and attach the groove of host epidermis, encysting and germinating towards a suitable penetration sites. By3h post-inoculation, most of germinated cysts preferentially penetrate anticlinal walls between epidermal cells of host by germ tube, without apparent appressoria. By6h post-inoculation, cysts have penetrated epidermis successfully, and primary invasive hypha grow and extend intercellularly in the space between cortical cells, initiating development of haustoria. By20h post-inoculation, invasive hypha extend intercellularly and intracellularly in the host, even outwards the epidermis. Meanwhile, responses of host at the penetration sites, such as callose deposition, ROS burst, accumulation of peroxidase, can be observed by Aniline Blue and DAB labeling respectively.Genetic stable transformation of Phytophthora sojae with green fluorescent protein. To trace the process of infection with GFP, as a reporter gene, it is one of the most effective ways to study interaction between plant pathogen and host. As redesigned vector pTOR and pHAM34with promoter of ham34gene in plant pathogen Bremia lactucae, we construct vector pTOR::GFP and pHAM34::Flag-GFP both of which could express GFP stably in P. sojae; as been introduced with several restriction sites, vector pHAM34::Flag-GFP could provide an effective way to research on gene function and localization, and subcellular localization of effectors et al.. Through the system of genetic stable transformation in oomycetes by CaCl2-PEG, P.sojae was introduced with vector pTOR::GFP and GFP transformants were obtained. Biological assay, including morphology and diameter of colony, proliferation of zoosporangia, germinated cysts, proliferation of oospores, pathogenic ability, it indicates GFP has no toxic influence on P. sojae. Therefore, GFP isolates could be used to image the interaction between P. sojae and host.Cellular researches on compatible and incompatible interaction between phytophthora sojae GFP-tagging isolate and host soybean. With GFP imaging, we observed that once having penetrated host Phytophthora sojae invasive hypha develop mastoid structures known as haustoria that keep intimate touch with living cells. As in compatible interaction (Williams), invasive hypha can extend to xylem of hypocotyl almost at12h postinoculation without triggering apparent defense response, and appear to develop kinds of intumescent structures in intercellular and intracellular space of host, such as knot-like, rhizomorph-like, broom-like and lobe-like. In incompatible interaction, invasive hypha are mainly restricted in the cortex and fails to extend beyond the endodermis of hypocotyl with triggering strong autofluorescence which is closely relative to resistance of host; though those structures observed in the compatible interaction also can be formed, most of them are covered by unknown materials with autofluorescence.