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| PpMID1-silenced transformants produce less aerial mycelia and mycelia in liquid culture are highly branched. (A) Colony morphology. Photographs were taken 7 days after subculture. (B) PpMID1-silenced transformants produced less aerial mycelia on solid medium. Mycelia disks (5 mm id.) of silenced transformants (PpMID1_7 and PpMID1_46), EV, and wild-type Phytophthora parasitica (94069) were transferred to 20% V8 juice agar and grown in the dark at 25 @BULLET C. Photographs for the side view of each colony were taken 3 days after subculture. Arrowheads, colony margins of silenced transformants. Bar = 5 mm. (C) Mycelia produced by PpMID1-silenced transformants in liquid medium are highly branched. Mycelia disks (5 mm id.) of PpMID1_7, PpMID1_46, EV, and the wild type (94069) were transferred to 20% V8 juice medium and grown at 25 @BULLET C in the dark. Photographs were taken under a microscope 1 day later. Arrowheads, branched hyphae. Bar = 100 µm.
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Phytophthora parasitica is a notorious oomycete pathogen that causes severe disease in a wide variety of crop species. Infection of plants involves mainly its asexual life stage, including papillate sporangia and biflagellated zoospores, which are the primary dispersal and infection agents of this pathogen. Calcium signaling has been thought as the...
Citations
... RNA-seq of P. infestans life stages showed that > 22% (> 4,000) genes were upregulated in sporangia compared to hypha, more than one-third of these genes were upregulated > 100-fold [29]. Many genes, including those encoding cell cycle regulators, PiCdc14 [30] and PcSDA1 [31], G protein β and γ subunits [32], Myb and MADSbox transcription factors [33], the loricrin-like protein PiLLP [34], the mating pheromoneinduced death 1 protein PpMID1 [35], and the catalase PiCAT2 [36], are involved in sporangia development in Phytophthora. However, despite its importance in the Phytophthora disease cycle, the primary regulator of the dramatically transcriptional changes during sporangia formation remains poorly understand, the clarification of the regulatory mechanism could strongly benefit the design of oomycetes-specific microbicide to combat with the diseases through inhibiting the development of Phytophthora. ...
Proper transcription orchestrated by RNA polymerase II (RNPII) is crucial for cellular development, which is rely on the phosphorylation state of RNPII’s carboxyl-terminal domain (CTD). Sporangia, developed from mycelia, are essential for the destructive oomycetes Phytophthora , remarkable transcriptional changes are observed during the morphological transition. However, how these changes are rapidly triggered and their relationship with the versatile RNPII-CTD phosphorylation remain enigmatic. Herein, we found that Phytophthora capsici undergone an elevation of Ser5-phosphorylation in its uncanonical heptapeptide repeats of RNPII-CTD during sporangia development, which subsequently changed the chromosomal occupation of RNPII and primarily activated transcription of certain genes. A cyclin-dependent kinase, PcCDK7, was highly induced and phosphorylated RNPII-CTD during this morphological transition. Mechanistically, a novel DCL1-dependent microRNA, pcamiR1, was found to be a feedback modulator for the precise phosphorylation of RNPII-CTD by complexing with PcAGO1 and regulating the accumulation of PcCDK7. Moreover, this study revealed that the pcamiR1-CDK7-RNPII regulatory module is evolutionarily conserved and the impairment of the balance between pcamiR1 and PcCDK7 could efficiently reduce growth and virulence of P . capsici . Collectively, this study uncovers a novel and evolutionary conserved mechanism of transcription regulation which could facilitates correct development and identifies pcamiR1 as a promising target for disease control.
... Golgi reassembly stacking protein PsGRASP is associated with zoosporogenesis (Si et al., 2021a). Soluble N-ethylmaleimide-sensitive factor attachment protein receptor PsYKT6, Mating pheromone-induced death 1 PpMID1, catalase PiCAT2 and two chitin synthase PcCHS/PsCHS1 regulate both processes (Zhao et al., 2011b;Hwu et al., 2017;Cheng et al., 2019;Wang et al., 2020). The above studies identify the pathways from membrane receptor, signal transduction to the downstream transcriptional regulation involved in sporangium formation and cleavage. ...
Oomycetes cause hundreds of destructive plant diseases, threatening agricultural production and food security. These fungus-like eukaryotes show multiple sporulation pattern including the production of sporangium, zoospore, chlamydospore and oospore, which are critical for their survival, dispersal and infection on hosts. Recently, genomic and genetic technologies have greatly promoted the study of molecular mechanism of sporulation in the genus Phytophthora and Peronophythora . In this paper, we characterize the types of asexual and sexual spores and review latest progress of these two genera. We summarize the genes encoding G protein, mitogen-activated protein kinase (MAPK) cascade, transcription factors, RNA-binding protein, autophagy-related proteins and so on, which function in the processes of sporangium production and cleavage, zoospore behaviors and oospore formation. Meanwhile, various molecular, chemical and electrical stimuli in zoospore behaviors are also discussed. Finally, with the molecular mechanism of sporulation in Phytophthora and Peronophythora is gradually being revealed, we propose some thoughts for the further research and provide the alternative strategy for plant protection against phytopathogenic oomycetes.
... The production and release of zoospores in vitro represents a specific component of the disease cycle. Although several structures are infective, zoospores are believed to be responsible for rapid increases in infection, due to their production in greater number, and more efficient germination [26,27]. Zoospore production per colony area (cm 2 ) was assessed to identify phenotypic variation within isolates from Hawaii, and among isolates collected worldwide. ...
Commercial production of cacao in Hawaii has doubled in the past 10 years, and farmers are receiving premium prices for their beans from the expanding local confectionery industry. Black pod, caused by Phytophthora spp., is the only major cacao disease that has been reported in Hawaii but distribution and molecular identification are lacking. To determine the species of Phytophthora affecting Theobroma cacao, a sampling trip was conducted on Hawaii Island and Oahu. Ten isolates of Phytophthora palmivora were obtained from diseased cacao on Hawaii Island, but none from Oahu, despite the presence of symptomatic pods. No other Phytophthora species were found. Laboratory studies showed that all isolates produced lesions on unwounded cacao pods, but they differed in terms of their temperature–growth responses. Fungicide sensitives for a subset of isolates (n = 4) were determined using media amended with a range of fungicide concentrations. The Hawaiian isolates of P. palmivora were more sensitive to mefenoxam, chlorothalonil, and fosetyl-Al, than isolates from Ghana (n = 2) and Mexico (n = 1). This study identifies P. palmivora as a causal agent of black pod in Hawaii based on molecular data and provides valuable preliminary information on fungicide resistance and temperature response that can be used to improve disease management.
... Gene silencing of a homologue of this channel subunit in P. parasitica prevented cold shock-induced zoosporogenesis. This effect could be partially rescued by increasing the concentration of extracellular Ca 2+ [17]. Analysis of changes in mRNA expression involved in P. infestans zoosporogenesis revealed significant upregulation of 15 genes. ...
In eukaryotes, two sources of Ca2+ are accessed to allow rapid changes in the cytosolic levels of this second messenger: the extracellular medium and intracellular Ca2+ stores, such as the endoplasmic reticulum. One class of channel that permits Ca2+ entry is the transient receptor potential (TRP) superfamily, including the polycystic kidney disease (PKD) proteins, or polycystins. Channels that release Ca2+ from intracellular stores include the inositol 1,4,5-trisphosphate/ryanodine receptor (ITPR/RyR) superfamily. Here, we characterise a family of proteins that are only encoded by oomycete genomes, that we have named PKDRR, since they share domains with both PKD and RyR channels. We provide evidence that these proteins belong to the TRP superfamily and are distinct from the ITPR/RyR superfamily in terms of their evolutionary relationships, protein domain architectures and predicted ion channel structures. We also demonstrate that a hypothetical PKDRR protein from Phytophthora infestans is produced by this organism, is located in the cell-surface membrane and forms multimeric protein complexes. Efforts to functionally characterise this protein in a heterologous expression system were unsuccessful but support a cell-surface localisation. These PKDRR proteins represent potential targets for the development of new “fungicides”, since they are of a distinctive structure that is only found in oomycetes and not in any other cellular organisms.
... sojae P7076) was cultivated at 25 • C on 10% (v/v) V8 juice agar in a polystyrene dish (Förster et al., 1994). P. sojae P7076 infection assay was carried out as previously described with some modifications (Jia et al., 2013;Fang et al., 2017). Briefly, 20 µL 5 Tween 20 was dripped on middle of 10-day-old detached leaves, and then transferred 5-day-old fresh mycelial disks approximately 5.5 mm diameter with a 200 µL Eppendorf pipette tip on the middle of detached leaves. ...
Lesion mimic mutants provide ideal genetic materials for elucidating the molecular mechanism of cell death and disease resistance. Here, we isolated a Glycine max lesion mimic mutant 2-1 (Gmlmm2-1), which displayed a light-dependent cell death phenotype. Map-based cloning revealed that GmLMM2 encods a coproporphyrinogen III oxidase and participates in tetrapyrrole biosynthesis. Knockout of GmLMM2 led to necrotic spots on developing leaves of CRISPR/Cas9 induced mutants. The GmLMM2 defect decreased the chlorophyll content by disrupting tetrapyrrole biosynthesis and enhanced resistance to Phytophthora sojae. These results suggested that GmLMM2 gene played an important role in the biosynthesis of tetrapyrrole and light-dependent defense in soybeans.
... To construct silencing vectors, partial sequences of ppxyn1 (nt 709-902) and ppxyn2 (nt 719-913) were cloned into the pENTR/D-TOPO vector (Thermo Fisher Scientific), followed by double replacement with pHPdest (Hwu et al. 2017), a Gateway destination cassette, using LR clonase II, to generate pHPdest::ppxyn1 and pHPdest::ppxyn2. ...
... Protoplast preparation was performed as described (Hwu et al. 2017). Three days after growth of P. parasitica in 20% V8 medium, the mycelium was harvested, washed in 0.4 M mannitol for 10 min, and digested at 25 °C for 45 min in 14 volumes of a digestion solution (Hwu et al. 2017). ...
... Protoplast preparation was performed as described (Hwu et al. 2017). Three days after growth of P. parasitica in 20% V8 medium, the mycelium was harvested, washed in 0.4 M mannitol for 10 min, and digested at 25 °C for 45 min in 14 volumes of a digestion solution (Hwu et al. 2017). Protoplasts were collected by filtration through four layers of Miracloth (EMD Millipore, Billerica, MA, USA) and centrifugation at 1000× g for 3 min at 25 °C. ...
Plant cell walls are pivotal battlegrounds between microbial pathogens and their hosts. To penetrate the cell wall and thereby to facilitate infection, microbial pathogens are equipped with a wide array of cell wall-degrading enzymes to depolymerize the polysaccharides in the cell wall. However, many of these enzymes and their role in the pathogenesis of microbial pathogens are not characterized, especially those from Oomycetes. In this study, we analyzed the function of four putative endo-beta-1,4-xylanase-encoding genes (ppxyn1–ppxyn4) from Phytophthora parasitica, an oomycete plant pathogen known to cause severe disease in a wide variety of plant species. All four genes belong to the glycoside hydrolase family 10 (GH10). Recombinant proteins of ppxyn1, ppxyn2, and ppxyn4 obtained from the yeast Pichia pastoris showed degrading activities toward birch wood xylan, but they behaved differently in terms of the conditions for optimal activity, thermostability, and durability. Quantitative RT-PCR revealed upregulated expression of all four genes, especially ppxyn1 and ppxyn2, during plant infection. In contrast, ppxyn3 was highly expressed in cysts and its close homolog, ppxyn4, in germinating cysts. To uncover the role of ppxyn1 and ppxyn2 in the pathogenesis of P. parasitica, we generated silencing transformants for these two genes by double-stranded RNA-mediated gene silencing. Silencing ppxyn1 and ppxyn2 reduced the virulence of P. parasitica toward tobacco (Nicotiana benthamiana) and tomato plants. These results demonstrate the crucial role of xylanase-encoding ppxyn1 and ppxyn2 in the infection process of P. parasitica.
One of the main obstacles often encountered in efforts to increase productivity and quality of tobacco ( Nicotiana tabacum ) yields is the attack of black shank disease by the oomycetes Phytophthora nicotianae . So far, what has been done mainly by tobacco cultivators is the observation of resistance through calculating the rate of death or disease of plants due to pathogen attack, so research is needed to determine the resistance profile of local varieties of tobacco to black shank disease in molecular stage to speed up the screening process of tobacco varieties. This study aim to determine the gene expression profile related to resistance in tobacco varieties Beinhart 1000, Hick Broadleaf, and two local varieties, namely Dark 302 and Dark 314, in response to black shank disease. The research consisted of preparing tobacco and P. nicotianae inoculum, artificial inoculation, followed by semi-quantitative analysis of gene expression related to tobacco resistance, PR1, PR4, PLP2, and PUB24. Tobacco resistant variety, Beinhart 1000, expressed a relatively 285% higher PR1 significantly than the control. The length of time of infection showed that PR1, PR4, PLP2, and PUB24 genes were expressed temporally, and the dynamics of expression of each test gene affected the development of pathological conditions.
The key players of calcium (Ca 2+) homeostasis and Ca 2+ signal generation, which are Ca 2+ channels, Ca 2+ /H + antiporters, and Ca 2+-ATPases, are present in all fungi. Their coordinated action maintains a low Ca 2+ baseline, allows a fast increase in free Ca 2+ concentration upon a stimulus, and terminates this Ca 2+ elevation by an exponential decrease-hence forming a Ca 2+ signal. In this respect, the Ca 2+ signaling machinery is conserved in different fungi. However, does the similarity of the genetic inventory that shapes the Ca 2+ peak imply that if "you've seen one, you've seen them all" in terms of physiological relevance? Individual studies have focused mostly on a single species, and mechanisms elucidated in few model organisms are usually extrapolated to other species. This mini-review focuses on the physiological relevance of the machinery that maintains Ca 2+ homeostasis for growth, virulence, and stress responses. It reveals common and divergent functions of homologous proteins in different fungal species. In conclusion, for the physiological role of these Ca 2+ transport proteins, "seen one," in many cases, does not mean: "seen them all."
