The ssgB gene is required for the early stages of sporangium formation in Actinoplanes missouriensis.

In Streptomyces, multiple paralogs of SsgA-like proteins (SALPs) are involved in spore formation from aerial hyphae. However, the functions of SALPs have not yet been elucidated in other actinobacterial genera. Here, we report the primary function of an SsgB ortholog (AmSsgB) in Actinoplanes missouriensis, which develops terminal sporangia on the substrate mycelia via short sporangiophores. Importantly, AmSsgB is the sole SALP in A. missouriensis. The transcription of AmssgB was upregulated during sporangium formation, consistent with our previous findings that AmssgB is a member of the AmBldD regulon. The AmssgB null mutant (ΔAmssgB) strain formed non-globose irregular structures on the substrate mycelium. Transmission electron microscopy revealed that the irregular structures contained abnormally septate hypha-like cells, without an intrasporangial matrix. These phenotypic changes were restored by complementation with AmssgB. Additionally, analysis of the heterologous expression of seven SALP-encoding genes from Streptomyces coelicolor A3(2) (ssgA-G) in the ΔAmssgB strain revealed that only ssgB could compensate for AmSsgB deficiency. This indicated that SsgB of S. coelicolor A3(2) and AmSsgB have comparable functions in A. missouriensis. In contrast to the ΔAmssgB strain, the ftsZ-disrupted strain showed a severe growth defect and produced small sporangium-like structures that swelled to some extent. These findings indicate that AmSsgB is crucial for the early stages of sporangium formation, not for spore septum formation in the late stages. We propose that AmSsgB is involved in sporangium formation by promoting the expansion of the "presporangium" structures formed on the tips of the substrate hyphae. IMPORTANCE: SsgB has been proposed as an archetypical SsgA-like protein with an evolutionarily conserved function in the morphological development of spore-forming actinomycetes. SsgB in Streptomyces coelicolor A3(2) is involved in spore septum formation. However, it is unclear whether this is the primary function of SsgBs in actinobacteria. This study demonstrated that the SsgB ortholog (AmSsgB) in Actinoplanes missouriensis is essential for sporangium expansion, which does not seem to be related to spore septum formation. However, the heterologous expression of ssgB from S. coelicolor A3(2) restored morphological abnormalities in the ΔAmssgB mutant. We propose that the primary function of SsgB is to initiate sporulation in differentiating cells (e.g., aerial hyphae in Streptomyces and "presporangium" cells in A. missouriensis) although its molecular mechanism remains unknown.

Architecture of Actinoplanes missouriensis sporangia and zoospores visualized using quick-freeze deep-etch electron microscopy.

The architecture of sporangia and zoospores of Actinoplanes missouriensis was analyzed at a high resolution using quick-freeze deep-etch replica electron microscopy. This analysis revealed that (i) sporangia were surrounded by at least 2 membranous layers with smooth surfaces, (ii) zoospores were enclosed by a fibrillar layer, and (iii) flagella were generated in a restricted area on the zoospore surface.

Modeling the dynamic changes in Plasmopara viticola sporangia concentration based on LSTM and understanding the impact of relative factor variability.

Reliable disease management can guarantee healthy plant production and relies on the knowledge of pathogen prevalence. Modeling the dynamic changes in spore concentration is available for realizing this purpose. We present a novel model based on a time-series modeling machine learning method, i.e., a long short-term memory (LSTM) network, to analyze oomycete Plasmopara viticola sporangia concentration dynamics using data from a 4-year field experiment trial in North China. Principal component analysis (PCA)-based high-quality input screening and simulation result calibration were performed to ensure model performance, obtaining a high determination coefficient (0.99), a low root mean square error (0.87), and a low mean bias error (0.55), high sensitivity (91.5%), and high specificity (96.5%). The impact of the variability of relative factors on daily P. viticola sporangia concentrations was analyzed, confirming that a low daily mean air temperature restricts pathogen development even during a long period of high humidity in the field.

Involvement of BldC in the Formation of Physiologically Mature Sporangium in Actinoplanes missouriensis.

AmBldD is a global transcriptional regulator that represses the transcription of several genes required for sporangium formation in Actinoplanes missouriensis. Here, we characterized one of the AmBldD regulons: AMIS_1980, encoding an ortholog of BldC, which is a transcriptional regulator involved in the morphological development of Streptomyces. We determined the transcriptional start point of the bldC ortholog by high-resolution S1 nuclease mapping and found an AmBldD box in its 5'-untranslated region. Reverse transcription-quantitative PCR analysis revealed that the transcription of bldC is activated during sporangium formation. A bldC null mutant (ΔbldC) strain formed normally shaped sporangia, but they exhibited defective sporangium dehiscence; under a dehiscence-inducing condition, the number of spores released from the sporangia of the ΔbldC strain was 2 orders of magnitude lower than that from the sporangia of the wild-type strain. RNA sequencing analysis indicated that BldC functions as a transcriptional activator of several developmental genes, including tcrA, which encodes a key transcriptional activator that regulates sporangium formation, sporangium dehiscence, and spore dormancy. Using electrophoretic mobility shift assay (EMSA), we showed that a recombinant BldC protein directly binds to upstream regions of at least 18 genes, the transcription of which is downregulated in the ΔbldC strain. Furthermore, using DNase I footprinting and EMSA, we demonstrated that BldC binds to the direct repeat sequences containing an AT-rich motif. Thus, BldC is a global regulator that activates the transcription of several genes, some of which are likely to be required for sporangium dehiscence. IMPORTANCE BldC is a global transcriptional regulator that acts as a "brake" in the morphological differentiation of Streptomyces. BldC-like proteins are widely distributed throughout eubacteria, but their orthologs have not been studied outside streptomycetes. Here, we revealed that the BldC ortholog in Actinoplanes missouriensis is essential for sporangium dehiscence and that its regulon is different from the BldC regulon in Streptomyces venezuelae, suggesting that BldC has evolved to play different roles in morphological differentiation between the two genera of filamentous actinomycetes.

The Evolution of euAPETALA2 Genes in Vascular Plants: From Plesiomorphic Roles in Sporangia to Acquired Functions in Ovules and Fruits.

The field of evolutionary developmental biology can help address how morphological novelties evolve, a key question in evolutionary biology. In Arabidopsis thaliana, APETALA2 (AP2) plays a role in the development of key plant innovations including seeds, flowers, and fruits. AP2 belongs to the AP2/ETHYLENE RESPONSIVE ELEMENT BINDING FACTOR family which has members in all viridiplantae, making it one of the oldest and most diverse gene lineages. One key subclade, present across vascular plants is the euAPETALA2 (euAP2) clade, whose founding member is AP2. We reconstructed the evolution of the euAP2 gene lineage in vascular plants to better understand its impact on the morphological evolution of plants, identifying seven major duplication events. We also performed spatiotemporal expression analyses of euAP2/TOE3 genes focusing on less explored vascular plant lineages, including ferns, gymnosperms, early diverging angiosperms and early diverging eudicots. Altogether, our data suggest that euAP2 genes originally contributed to spore and sporangium development, and were subsequently recruited to ovule, fruit and floral organ development. Finally, euAP2 protein sequences are highly conserved; therefore, changes in the role of euAP2 homologs during development are most likely due to changes in regulatory regions.

Dependence on clade II bHLH transcription factors for nursing of haploid products by tapetal-like cells is conserved between moss sporangia and angiosperm anthers.

Clade II basic helix-loop-helix transcription factors (bHLH TFs) are essential for pollen production and tapetal nursing functions in angiosperm anthers. As pollen has been suggested to be related to bryophyte spores by descent, we characterized two Physcomitrium (Physcomitrella) patens clade II bHLH TFs (PpbHLH092 and PpbHLH098), to test if regulation of sporogenous cells and the nursing cells surrounding them is conserved between angiosperm anthers and bryophyte sporangia. We made CRISPR-Cas9 reporter and loss-of-function lines to address the function of PpbHLH092/098. We sectioned and analyzed WT and mutant sporophytes for a comprehensive stage-by-stage comparison of sporangium development. Spore precursors in the P. patens sporangium are surrounded by nursing cells showing striking similarities to tapetal cells in angiosperms. Moss clade II bHLH TFs are essential for the differentiation of these tapetal-like cells and for the production of functional spores. Clade II bHLH TFs provide a conserved role in controlling the sporophytic somatic cells surrounding and nursing the sporogenous cells in both moss sporangia and angiosperm anthers. This supports the hypothesis that such nursing functions in mosses and angiosperms, lineages separated by c. 450 million years, are related by descent.

Clade-Specific Monitoring of Airborne Pseudoperonospora spp. Sporangia Using Mitochondrial DNA Markers for Disease Management of Cucurbit Downy Mildew.

Management of cucurbit downy mildew (CDM) caused by Pseudoperonospora cubensis, relies on an intensive fungicide program. In Michigan, CDM occurs annually due to an influx of airborne sporangia and timely alerts of airborne inoculum can assist growers in assessing the need to initiate fungicide sprays. This research aimed to improve the specific detection of airborne P. cubensis sporangia by adapting quantitative real-time polymerase chain reaction (qPCR) assays to distinguish among P. cubensis clades I and II and P. humuli in spore trap samples from commercial production sites and research plots. We also evaluated the suitability of impaction spore traps compared with Burkard traps for detection of airborne sporangia. A multiplex qPCR assay improved the specificity of P. cubensis clade II detection accelerating the assessment of field spore trap samples. After 2 years of monitoring, P. cubensis clade II DNA was detected in spore trap samples before CDM symptoms were first observed in cucumber fields (July and August), while P. cubensis clade I DNA was not detected in air samples before or after the disease onset. In some commercial cucumber fields, P. humuli DNA was detected throughout the growing season. The Burkard spore trap appeared to be better suited for recovery of sporangia at low concentrations than the impaction spore trap. This improved methodology for the monitoring of airborne Pseudoperonospora spp. sporangia could be used as part of a CDM risk advisory system to time fungicide applications that protect cucurbit crops in Michigan.

Further evidence for the hybrid status of the Brazilian native fern Hypolepis ×paulistana (Dennstaedtiaceae).

Hypolepis ×paulistana was described in 2016 as a putative hybrid, known from a single gathering. The hybrid status of these plants was based solely on the intermediate morphology of the sporophyte, when compared to its presumed parent species. These were thought to be H. stolonifera and H. rugosula, but, H. rigescens (Kunze) T. Moore could not be explicitly ruled out either. In the present work, we tested the hybrid status of Hypolepis ×paulistana adding palynological evidence and by using chloroplast sequences to unambiguously identify the maternal progenitor of the species. We find that sporangia of Hypolepis ×paulistana contain both well-formed spores, as well as spores with morphological and developmental anomalies. The size of the regular spores and the abnormal spores suggest that H. ×paulistana is likely a diploid, and probably infertile hybrid. The ornamentation of the regular spores of H. ×paulistana is similar to that of H. stolonifera. The chloroplast sequences of H. ×paulistana are identical to those of H. stolonifera, as well as their sister position within the global phylogeny of the genus. Thus, we provide new evidence for the hybrid status of H. ×paulistana, and we corroborate the earlier finding that H. stolonifera is the maternal parent.

Involvement of three FliA-family sigma factors in the sporangium formation, spore dormancy and sporangium dehiscence in Actinoplanes missouriensis.

The rare actinomycete Actinoplanes missouriensis forms sporangia, which open up and release zoospores in response to water. Here, we report a genetic and functional analysis of four FliA-family sigma factors, FliA1, FliA2, FliA3 and FliA4. Transcription of fliA1, fliA2 and fliA3 was directly activated by the global transcriptional activator TcrA during sporangium formation and dehiscence, while fliA4 was almost always transcribed at low levels. Gene disruption analysis showed that (a) deletion of fliA2 reduced the zoospore swimming speed by half, (b) the fliA1-fliA2 double-deletion mutant formed abnormal sporangia in which mutant spores ectopically germinated and (c) deletion of fliA3 induced no phenotypic changes in the wild-type and mutant strains of fliA1 and/or fliA2. Comparative RNA-Seq analyses among the wild-type and gene deletion mutant strains showed probable targets of each FliA-family sigma factor, indicating that FliA1- and FliA2-dependent promoters are quite similar to each other, while the FliA3-dependent promoter is somewhat different. Gene complementation experiments also indicated that the FliA1 regulon overlaps with the FliA2 regulon. These results demonstrate that A. missouriensis has developed a complex transcriptional regulatory network involving multiple FliA-family sigma factors for the accomplishment of its characteristic reproduction process, including sporangium formation, spore dormancy and sporangium dehiscence.

A new phylogeny of the cladoxylopsid plexus: contribution of an early cladoxylopsid from the Lower Devonian (Emsian) of Quebec.

PREMISE: Cladoxylopsids formed Earth's earliest forests and gave rise to the ancestors of sphenopsids and ferns. Lower Devonian (Emsian) strata of the Battery Point Formation (Quebec, Canada) contain new anatomically preserved cladoxylopsids, one of which is described in this article. To assess the phylogenetic position of this fossil and address questions of cladoxylopsid phylogeny, we conducted a comprehensive phylogenetic study. METHODS: Permineralized axes were studied in serial sections using the cellulose acetate peel technique. We evaluated phylogenetic relationships among cladoxylopsids using a data set of 36 new morphological characters and 31 species, in parsimony-constrained analyses. RESULTS: We describe Adelocladoxis praecox gen. et sp. nov., a cladoxylopsid with small actinostelic axes bearing dichotomously branched, helically arranged ultimate appendages and fusiform sporangia. Adelocladoxis provides the oldest evidence of cladoxylopsid anatomy, including ultimate appendages and sporangia. In agreement with non-phylogenetic classification schemes, our phylogenetic analysis resolves a basal grade of iridopterids and a clade of cladoxylopsids s.s., which includes a pseudosporochnalean cladoxylopsid clade, a cladoxylalean cladoxylopsid clade, and Adelocladoxis. CONCLUSIONS: Our phylogenetic analysis illuminates aspects of tempo and mode of evolution in the cladoxylopsid plexus. Originating prior to the Emsian, cladoxylopsids reached global distribution by the Frasnian. Iridopterids and cladoxylopsids s.s. radiated in the Emsian-Eifelian. The sequence of character change recovered by our phylogeny supports a transition from actinostelic protosteles to dissected steles, associated with an increase in xylem rib number and medullation generating a central parenchymatous area.

Spray-Induced Gene Silencing as a Potential Tool to Control Potato Late Blight Disease.

Phytophthora infestans causes late blight disease on potato and tomato and is currently controlled by resistant cultivars or intensive fungicide spraying. Here, we investigated an alternative means for late blight control by spraying potato leaves with double-stranded RNAs (dsRNA) that target the P. infestans genes essential for infection. First, we showed that the sporangia of P. infestans expressing green fluorescent protein (GFP) can take up in vitro synthesized dsRNAs homologous to GFP directly from their surroundings, including leaves, which led to the reduced relative expression of GFP. We further demonstrate the potential of spray-induced gene silencing (SIGS) in controlling potato late blight disease by targeting developmentally important genes in P. infestans such as guanine-nucleotide binding protein ß-subunit (PiGPB1), haustorial membrane protein (PiHmp1), cutinase (PiCut3), and endo-1,3(4)-ß-glucanase (PiEndo3). Our results demonstrate that SIGS can potentially be used to mitigate potato late blight; however, the degree of disease control is dependent on the selection of the target genes.[Formula: see text] Copyright © 2021 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

A Multiplex TaqMan qPCR Assay for Detection and Quantification of Clade 1 and Clade 2 Isolates of Pseudoperonospora cubensis and Pseudoperonospora humuli.

The ability to detect and quantify aerially dispersed plant pathogens is essential for developing effective disease control measures and epidemiological models that optimize the timing for control. There is an acute need for managing the downy mildew pathogens infecting cucurbits and hop incited by members of the genus Pseudoperonospora (Pseudoperonospora cubensis clade 1 and 2 isolates and Pseudoperonospora humuli, respectively). A highly specific multiplex TaqMan quantitative polymerase chain reaction (PCR) assay targeting unique sequences in the pathogens' mitochondrial genomes was developed that enables detection of all three taxa in a single multiplexed amplification. An internal control included in the reaction evaluated whether results were influenced by PCR inhibitors that can make it through the DNA extraction process. Reliable quantification of inoculum as low as three sporangia in a sample was observed. The multiplexed assay was tested with DNA extracted from purified sporangia, infected plant tissue, and environmental samples collected on impaction spore traps samplers. The ability to accurately detect and simultaneously quantify all three pathogens in a single multiplexed amplification should improve management options for controlling the diseases they cause.

Detection of Airborne Sporangia of Pseudoperonospora cubensis and P. humuli in Michigan Using Burkard Spore Traps Coupled to Quantitative PCR.

Cucurbit downy mildew (CDM), caused by the oomycete pathogen Pseudoperonospora cubensis, is a devastating foliar disease on cucumber resulting in reduced yields. In 2004, the pathogen re-emerged in the United States, infecting historically resistant cucumber cultivars and requiring the adoption of an intensive fungicide program. The pathogen cannot overwinter in Michigan fields but because of an influx of airborne sporangia CDM occurs annually. In Michigan, spore traps are used to monitor the presence of airborne P. cubensis sporangia in cucumber growing regions to guide the initiation of a fungicide program. However, Pseudoperonospora humuli sporangia, the causal agent of downy mildew on hop, are morphologically indistinguishable from P. cubensis sporangia. This morphological similarity reduces the ability to accurately detect P. cubensis from spore trap samples when examined with the aid of light microscopy. To improve P. cubensis detection, we adapted a qPCR-based assay to allow the differentiation between P. cubensis and P. humuli on Burkard spore trap samples collected in the field. Specifically, we evaluated the specificity and sensitivity of P. cubensis detection on Burkard spore trap tapes using a morphological-based and quantitative-PCR (qPCR)-based identification assay and determined whether sporangia of P. cubensis and P. humuli on Burkard samples could be distinguished using qPCR. We found that the qPCR assay was able to detect a single sporangium of each species on spore trap samples collected in the field with Cq values <35.5. The qPCR assay also allowed the detection of P. cubensis and P. humuli in samples containing sporangia from both species. However, the number of sporangia quantified using light microscopy explained only 54 and 10% of the variation in the Cq values of P. cubensis and P. humuli, respectively, suggesting a limited capacity of the qPCR assay for the absolute quantification of sporangia in field samples. After 2 years of monitoring using Burkard spore traps coupled with the qPCR in cucumber fields, P. humuli sporangia were detected more frequently than P. cubensis early in the growing season (May and June). P. cubensis sporangia were detected ∼5 to 10 days before CDM symptoms were first observed in cucumber fields during both years. This research describes an improved sporangial detection system that is key for the monitoring and management of P. cubensis in Michigan.

Susceptibility of frogs to chytridiomycosis correlates with increased levels of immunomodulatory serotonin in the skin.

Chytridiomycosis, caused by the fungus Batrachochytrium dendrobatidis (Bd), is a skin disease responsible for the global decline of amphibians. Frog species and populations can vary in susceptibility, but this phenomenon remains poorly understood. Here, we investigated serotonin in the skin of infected and uninfected frogs. In more susceptible frog populations, skin serotonin rose with increasing infection intensity, but decreased in later stages of the disease. The more resistant population maintained a basal level of skin serotonin. Serotonin inhibited both Bd sporangial growth and Jurkat lymphocyte proliferation in vitro. However, serotonin accumulates in skin granular glands, and this compartmentalisation may prevent inhibition of Bd growth in vivo. We suggest that skin serotonin increases in susceptible frogs due to pathogen excretion of precursor tryptophan, but that resistant frogs are able to control the levels of serotonin. Overall, the immunosuppressive effects of serotonin may contribute to the susceptibility of frogs to chytridiomycosis.

Marteilia cochillia is released into seawater via cockle Cerastoderma edule faeces.

Outbreaks of Marteilia cochillia have caused massive mortalities of common cockle, Cerastoderma edule, in some natural beds in Galicia (NW Spain) since 2012. The life cycle of Marteilia spp. is still unresolved and the most accepted hypothesis suggests that an additional host is involved. Researchers have assumed that sporangia are shed into the environment in the faeces, but details about this process have not been reported previously. Here, we report the massive liberation of Marteilia cochillia sporangia through the exhalant siphon into the environment, packaged as faeces. Using light microscopy observations on fresh samples, imprints and histology, we also describe a thick (ca. 5 µm) transparent envelope covering the sporangia that has not been reported previously. The massive release of encapsulated sporangia reported here ensures that millions of infective stages of M. cochillia cycle through the environment and become available for infection. The elucidation of the role played by the sporangia envelope would be of utmost importance for the understanding M. cochillia life cycle.

Phytophthora infestans Sporangia Produced in Artificial Media and Plant Lesions Have Subtly Divergent Transcription Profiles but Equivalent Infection Potential and Aggressiveness.

Sporangia of the potato late blight agent Phytophthora infestans are often used in studies of pathogen biology and plant responses to infection. Investigations of spore biology can be challenging in oomycetes because their sporangia are physiologically active and change in response to environmental factors and aging. Whether sporangia from artificial media and plant lesions are functionally equivalent has been a topic of debate. To address these issues, we compared the transcriptomes and infection ability of sporangia from rye-sucrose media, potato and tomato leaflets, and potato tubers. Small differences were observed between the mRNA profiles of sporangia from all sources, including variation in genes encoding metabolic enzymes, cell-wall-degrading enzymes, and ABC transporters. Small differences in sporangia age also resulted in variation in the transcriptome. Taking care to use sporangia of similar maturity, we observed that those sourced from media or plant lesions had similar rates of zoospore release and cyst germination. There were also no differences in infection rates or aggressiveness on leaflets, based on single-spore inoculation assays. Such results are discordant with those of a recent publication in this journal. Nevertheless, we conclude that sporangia from plant and media cultures are functionally similar and emphasize the importance of using "best practices" in experiments with sporangia to obtain reliable results.

Phytophthora infestans Sporangia Produced in Culture and on Tomato Leaflet Lesions Show Marked Differences in Indirect Germination Rates, Aggressiveness, and Global Transcription Profiles.

Sporangia of Phytophthora infestans from pure cultures on agar plates are typically used in lab studies, whereas sporangia from leaflet lesions drive natural infections and epidemics. Multiple assays were performed to determine if sporangia from these two sources are equivalent. Sporangia from plate cultures showed much lower rates of indirect germination and produced much less disease in field and moist-chamber tests. This difference in aggressiveness was observed whether the sporangia had been previously incubated at 4°C (to induce indirect germination) or at 21°C (to prevent indirect germination). Furthermore, lesions caused by sporangia from plates produced much less sporulation. RNA-Seq analysis revealed that thousands of the >17,000 P. infestans genes with a RPKM (reads per kilobase of exon model per million mapped reads) >1 were differentially expressed in sporangia obtained from plate cultures of two independent field isolates compared with sporangia of those isolates from leaflet lesions. Among the significant differentially expressed genes (DEGs), putative RxLR effectors were overrepresented, with almost half of the 355 effectors with RPKM >1 being up- or downregulated. DEGs of both isolates include nine flagellar-associated genes, and all were down-regulated in plate sporangia. Ten elicitin genes were also detected as DEGs in both isolates, and nine (including INF1) were up-regulated in plate sporangia. These results corroborate previous observations that sporangia produced from plates and leaflets sometimes yield different experimental results and suggest hypotheses for potential mechanisms. We caution that use of plate sporangia in assays may not always produce results reflective of natural infections and epidemics.

Regulation of sporangium formation by the orphan response regulator TcrA in the rare actinomycete Actinoplanes missouriensis.

The rare actinomycete Actinoplanes missouriensis forms terminal sporangia containing a few hundred flagellated spores, which can swim in aquatic environments after release from sporangium. However, gene regulation for its characteristic morphological development is largely unknown. Here, we report the functional analysis of an orphan response regulator, TcrA, which is encoded next to the chemotaxis-flagellar gene cluster. The tcrA null (ΔtcrA) mutant formed sporangium, in which sporulation proceeded. However, many distorted spores were produced and some spores ectopically germinated in the mutant sporangia. In addition, spores were hardly released from the mutant sporangia. A comparative RNA-Seq analysis between the wild-type and ΔtcrA strains showed that TcrA upregulated the transcription of more than 263 genes, which were integrated into 185 transcriptional units. In silico searches identified a 21-bp direct repeat sequence, 5'-nnGCA(A/C)CCG-n4 -GCA(A/C)CCGn-3', as the TcrA box, which was confirmed by electrophoretic mobility shift assays. Finally, we identified 34 transcriptional units as the TcrA regulon. TcrA seems to regulate a few hundred genes through the transcriptional activation of three FliA-family sigma factor genes besides its own regulon. We concluded that TcrA is a global transcriptional activator that controls many aspects of sporangium formation, including flagellar biogenesis, spore dormancy and sporangium dehiscence.

Phosphagen kinase function in flagellated spores of the oomycete Phytophthora infestans integrates transcriptional regulation, metabolic dynamics and protein retargeting.

Flagellated spores play important roles in the infection of plants and animals by many eukaryotic microbes. The oomycete Phytophthora infestans, which causes potato blight, expresses two phosphagen kinases (PKs). These enzymes store energy in taurocyamine, and are hypothesized to resolve spatial and temporal imbalances between rates of ATP creation and use in zoospores. A dimeric PK is found at low levels in vegetative mycelia, but high levels in ungerminated sporangia and zoospores. In contrast, a monomeric PK protein is at similar levels in all tissues, although is transcribed primarily in mycelia. Subcellular localization studies indicate that the monomeric PK is mitochondrial. In contrast, the dimeric PK is cytoplasmic in mycelia and sporangia but is retargeted to flagellar axonemes during zoosporogenesis. This supports a model in which PKs shuttle energy from mitochondria to and through flagella. Metabolite analysis indicates that deployment of the flagellar PK is coordinated with a large increase in taurocyamine, synthesized by sporulation-induced enzymes that were lost during the evolution of zoospore-lacking oomycetes. Thus, PK function is enabled by coordination of the transcriptional, metabolic and protein targeting machinery during the life cycle. Since plants lack PKs, the enzymes may be useful targets for inhibitors of oomycete plant pathogens.

Chitin synthase is involved in vegetative growth, asexual reproduction and pathogenesis of Phytophthora capsici and Phytophthora sojae.

Chitin is a structural and functional component of the fungal cell wall and also serves as a pathogen-associated molecular pattern (PAMP) that triggers the innate immune responses of host plants. However, no or very little chitin is found in the fungus-like oomycetes. In Phytophthora spp., the presence of chitin has not been demonstrated so far, although putative chitin synthase (CHS) genes, which encode the enzymes that synthesize chitin, are present in their genomes. Here, we revealed that chitin is present in the zoospores and released sporangia of Phytophthora, and this is most consistent with the transcriptional pattern of PcCHS in Phytophthora capsici and PsCHS1 in Phytophthora sojae. Disruption of the CHS genes indicated that PcCHS and PsCHS1, but not PsCHS2 (which exhibited very weak transcription), have similar functions involved in mycelial growth, sporangial production, zoospore release and the pathogenesis of P. capsici and P. sojae. We also suggest that chitin in the zoospores of P. capsici can act as a PAMP that is recognized by the chitin receptors AtLYK5 or AtCERK1 of Arabidopsis. These results provide new insights into the biological significance of chitin and CHSs in Phytophthora and help with the identification of potential targets for disease control.