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ABSTRACT: For dispersal and host infection plant pathogens largely depend on asexual spores. Pathogenesis and sporulation are complex processes that are governed by cellular signaling networks including G-protein and phospholipid signaling. Oomycetes possess a family of novel proteins called GPCR-PIPKs (GKs) that are composed of a seven trans-membrane spanning (7-TM) domain fused to a phosphatidylinositol phosphate kinase (PIPK) domain. Based on this domain structure GKs are anticipated to link G-protein and phospholipid signal pathways; however, their functions are currently unknown. Expression analyses of the 12 GK genes in Phytophthora infestans and their orthologs in Phytophthora sojae, revealed differential expression during asexual development. PiGK1 and PiGK4 were fused to monomeric red fluorescent protein (mRFP) and ectopically expressed in P. infestans. In growing hyphae different subcellular distribution patterns were observed indicating that these two GKs act independently during development. We focused on the functional analyses of PiGK4. Its localization suggested involvement in cell differentiation and elongation and its 7-TM domain showed a canonical GPCR membrane topology. Silencing of GK4 and overexpression of full-length and truncated constructs in P. infestans revealed that PiGK4 is not only involved in spore germination and hyphal elongation but also in sporangia cleavage and infection.
Molecular Microbiology 02/2013; · 5.01 Impact Factor
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ABSTRACT: Soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs) are central components of the machinery mediating membrane fusion and key factors for vesicular trafficking in all eukaryotic cells. Taking advantage of the available whole genome sequence of the oomycete plant pathogen Phytophthora sojae, 35 genes encoding putative SNARE proteins were identified in the genome of this organism. PsYKT6, one of the most conserved SNARE proteins, was functionally characterized by homology-dependent gene silencing. The phenotype analysis showed that PsYKT6 is important for proper asexual development, sexual reproduction, and pathogenesis on host soybean cultivars.
Fungal Genetics and Biology 03/2011; 48(3):241-51. · 3.74 Impact Factor
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ABSTRACT: Hexokinases are conserved proteins functioning in glucose sensing and signaling. The rice blast fungus Magnaporthe oryzae contains several hexokinases, including MoHxk1 (hexokinase) and MoGlk1 (glucokinase) encoded respectively by MoHXK1 and MoGLK1 genes. The heterologous expression of MoGlk1 and MoHxk1 in Saccharomyces cerevisiae confirmed their conserved functions. Disruption of MoHXK1 resulted in growth reduction in medium containing fructose as the sole carbon source, whereas disruption of MoGLK1 did not cause the similar defect. However, the ΔMoglk1 mutant displayed decreased proton extrusion and a lower biomass in the presence of ammonium, suggesting a decline in the utilization of ammonium. Additionally, the MoGLK1 allele lacking catalytic activity restored growth to the ΔMoglk1 mutant. Moreover, the expression of MoPMA1 encoding a plasma membrane H(+)-ATPase decreased in the ΔMoglk1 mutant that can be suppressed by glucose and G-6-P. Thus, MoGlk1, but not MoHxk1, regulates ammonium utilization through a mechanism that is independent from its catalytic activity.
PLoS ONE 01/2011; 6(7):e22809. · 4.09 Impact Factor
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ABSTRACT: The C(2)H(2) zinc finger proteins form one of the largest families of transcriptional regulators in eukaryotes. We identified a Phytophthora sojae C(2)H(2) zinc finger (PsCZF1), that is highly conserved in sequenced oomycete pathogens. In transformants of P. sojae containing the PsCZF1 promoter fused to the beta-glucuronidase (GUS) reporter gene, GUS activity was highly induced in the P. sojae oospore stage and upregulated after infection. To elucidate the function of PsCZF1, its expression was silenced by introducing anti-sense constructs into P sojae. PsCZF1-silenced transformants did not exhibit altered cell size or morphology of sporangia and hyphae; however, hyphal growth rate was reduced by around 50% in the mutants. PsCZF1-deficient mutants were also impaired in production of oospores, swimming zoospores and germinating cysts, indicating that the gene is involved in various stages of the life cycle. Furthermore, we found that PsCZF1-deficient mutants lost virulence on host soybean cultivars. Our results suggest that this oomycete-specific C(2)H(2)-type zinc finger protein plays an important role in growth, development, and pathogenesis; therefore, PsCZF1 might be an attractive oomycete-specific target for chemical fungicide screening.
Microbial Pathogenesis 06/2009; 47(2):78-86. · 1.94 Impact Factor
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ABSTRACT: Sensing chemical signal secreted from host root and find the best site for penetration are crucial for initiating infection of Phytophthora zoospore. G protein alpha subunit of P. sojae participates in not only the chemotaxis to soybean isoflavone, but also finding penetrating site. Furthermore, although calcium signal pathways are influenced by Galpha, other signal pathways also influenced by G protein remain to be discovered. In this addendum, we describe an RGS protein, PsRGS6, is expressed downregulated in zoospores of Galpha silenced mutant. This result indicates that the expression of Galpha and RGS protein may be influenced by each other. Some differences between Galpha mutants of P. infestans and P. sojae may be due to the different developmental procedures.
Communicative & integrative biology 04/2009; 2(2):91-3.
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ABSTRACT: 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 largely unknown. To investigate the role of G-protein and calcium signaling in chemotaxis, we analyzed the expression of several genes known to be involved in these pathways and selected one that was specifically expressed in sporangia and zoospores but not in mycelium. This gene, named PsGPA1, is a single-copy gene in P. sojae and encodes a G-protein alpha subunit that shares 96% identity in amino acid sequence with that of Phytophthora infestans. To elucidate the function, expression of PsGPA1 was silenced by introducing antisense constructs into P. sojae. PsGPA1 silencing did not disturb hyphal growth or sporulation but severely affected zoospore behavior, including chemotaxis to the soybean isoflavone daidzein. Zoospore encystment and cyst germination were also altered, resulting in the inability of the PsGPA1-silenced mutants to infect soybean. In addition, the expressions of a calmodulin gene, PsCAM1, and two calcium- and calmodulin-dependent protein kinase genes, PsCMK3 and PsCMK4, were increased in the mutant zoospores, suggesting that PsGPA1 negatively regulates the calcium signaling pathways that are likely involved in zoospore chemotaxis.
Eukaryotic Cell 11/2008; 7(12):2133-40. · 3.60 Impact Factor
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ABSTRACT: Soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs) are central components of the machinery mediating membrane fusion and key factors for vesicular trafficking in all eukaryotic cells. Taking advantage of the available whole genome sequence of the oomycete plant pathogen Phytophthora sojae, 35 genes encoding putative SNARE proteins were identified in the genome of this organism. PsYKT6, one of the most conserved SNARE proteins, was functionally characterized by homology-dependent gene silencing. The phenotype analysis showed that PsYKT6 is important for proper asexual development, sexual reproduction, and pathogenesis on host soybean cultivars.
Fungal Genetics and Biology.
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ABSTRACT: G protein coupled receptors (GPCRs) present a large family of receptors involved in a broad spectrum of cell signaling, which are responsive for transmitting the extracellular signals to intracellular response by stimuli. Despite exhibiting striking diversity in primary sequence and biology function, all GPCRs possess the conserved fundamental architecture consisting of seven transmembrane domains and share common mechanisms of signal transduction. This class of protein were primarily identified in the gnome of Phytophthora sojae, the destructive cause of stem and root rot in soybean. P. sojae have approximately 41 seven transmembrane proteins, which are classified into five classes based on sequence homology, including metabotropic glutamate receptors, rhodopsin-like superfamily, cyclic AMP receptors, one novel class phosphatidylinositol phosphate kinases with a G-protein coupled receptor, and other unclassified proteins. Subsequently, the expression profiles were evaluated during asexual lifestages, stresses treatments and infection stage. The results showed that some genes were constitutively expressed; in contrast, the other genes are induced during zoosporogenesis or stresses. Finally, genetic transformation of homology-dependent gene silencing of P. sojae were performed and the gene-silenced mutants of five genes were generated respectively. We found that out of five genes, three genes are probably involved in sexual reproduction and pathogenicity, while two genes are not obviously affected the phenotypes including chemotaxis to isofalvone. Further study on these mutants, and other GPCRs genes will be focused on to elucidate the functions of all GPCRs genes in P. sojae growth, development, stress response, chemotaxis and pathogenesis.
