Ubiquitin-dependent Argonauteprotein MEL1 degradation is essential for rice sporogenesis and phasiRNA target regulation.

MEIOSIS ARRESTED AT LEPTOTENE1 (MEL1), a rice (Oryza sativa) Argonaute (AGO) protein, has been reported to function specifically at premeiotic and meiotic stages of germ cell development and is associated with a novel class of germ cell-specific small noncoding RNAs called phased small RNAs (phasiRNAs). MEL1 accumulation is temporally and spatially regulated and is eliminated after meiosis. However, the metabolism and turnover (i.e. the homeostasis) of MEL1 during germ cell development remains unknown. Here, we show that MEL1 is ubiquitinated and subsequently degraded via the proteasome pathway in vivo during late sporogenesis. Abnormal accumulation of MEL1 after meiosis leads to a semi-sterile phenotype. We identified a monocot-specific E3 ligase, XBOS36, a CULLIN RING-box protein, that is responsible for the degradation of MEL1. Ubiquitination at four K residues at the N terminus of MEL1 by XBOS36 induces its degradation. Importantly, inhibition of MEL1 degradation either by XBOS36 knockdown or by MEL1 overexpression prevents the formation of pollen at the microspore stage. Further mechanistic analysis showed that disrupting MEL1 homeostasis in germ cells leads to off-target cleavage of phasiRNA target genes. Our findings thus provide insight into the communication between a monocot-specific E3 ligase and an AGO protein during plant reproductive development.

Plastid Transformation of Sporelings from the Liverwort Marchantia polymorpha L.

We describe a simple and efficient plastid transformation method for the liverwort, Marchantia polymorpha L. Use of rapidly proliferating cells such as sporelings, which are immature thalli developing from spores, as targets made plastid transformation by particle bombardment efficient. Selection on a sucrose-free medium and linearization of the transformation vector significantly improved the recovery rate of plastid transformants. With the method described here, a few plastid transformants are obtained from a single bombardment of sporelings. Homoplasmic transformants of thalli are obtained immediately after primary selection.

The zoospores of the thraustochytrid Aurantiochytrium limacinum: Transcriptional reprogramming and lipid metabolism associated to their specific functions.

Aurantiochytrium limacinum (Thraustochytriaceae, class Labyrinthulomycetes) is a marine Stramenopile and a pioneering mangrove decomposer. Its life cycle involves a non-motile stage and zoospore production. We observed that the composition of the medium, the presence of amino acids in particular, affects the release of zoospores. Two opposite conditions were defined, one with a cell population mainly composed of zoospores and another one with almost only non-motile cells. In silico allelic frequency analysis and flow cytometry suggest that zoospores and non-motile cells share the same ploidy level and are diploid. Through an RNA-seq approach, the transcriptional reprogramming accompanying the formation of zoospores was investigated, with a particular focus on their lipid metabolism. Based on a differential expression analysis, zoospores are characterized by high motility, very active signal transduction, an arrest of the cell division, a low amino acid metabolism and low glycolysis. Focusing on lipid metabolism, genes involved in lipase activities and peroxisomal ß-oxidation are upregulated. qRT-PCR of selected lipid genes and lipid analyses during the life span of zoospores confirmed these observations. These results highlight the importance of the lipid dynamics in zoospores and show the metabolic processes required to use these energy-dense molecules as fuel for zoospore survival during their quest of new territories.

Cross-kingdom signalling regulates spore germination in the moss Physcomitrella patens.

Plants live in close association with microorganisms that can have beneficial or detrimental effects. The activity of bacteria in association with flowering plants has been extensively analysed. Bacteria use quorum-sensing as a way of monitoring their population density and interacting with their environment. A key group of quorum sensing molecules in Gram-negative bacteria are the N-acylhomoserine lactones (AHLs), which are known to affect the growth and development of both flowering plants, including crops, and marine algae. Thus, AHLs have potentially important roles in agriculture and aquaculture. Nothing is known about the effects of AHLs on the earliest-diverging land plants, thus the evolution of AHL-mediated bacterial-plant/algal interactions is unknown. In this paper, we show that AHLs can affect spore germination in a representative of the earliest plants on land, the Bryophyte moss Physcomitrella patens. Furthermore, we demonstrate that sporophytes of some wild isolates of Physcomitrella patens are associated with AHL-producing bacteria.

2-Phenylethyl Isothiocyanate Exerts Antifungal Activity against Alternaria alternata by Affecting Membrane Integrity and Mycotoxin Production.

Black spot caused by Alternaria alternata is one of the important diseases of pear fruit during storage. Isothiocyanates are known as being strong antifungal compounds in vitro against different fungi. The aim of this study was to assess the antifungal effects of the volatile compound 2-phenylethyl isothiocyanate (2-PEITC) against A. alternata in vitro and in pear fruit, and to explore the underlying inhibitory mechanisms. The in vitro results showed that 2-PEITC significantly inhibited spore germination and mycelial growth of A. alternata-the inhibitory effects showed a dose-dependent pattern and the minimum inhibitory concentration (MIC) was 1.22 mM. The development of black spot rot on the pear fruit inoculated with A. alternata was also significantly decreased by 2-PEITC fumigation. At 1.22 mM concentration, the lesion diameter was only 39% of that in the control fruit at 7 days after inoculation. Further results of the leakage of electrolyte, increase of intracellular OD260, and propidium iodide (PI) staining proved that 2-PEITC broke cell membrane permeability of A. alternata. Moreover, 2-PEITC treatment significantly decreased alternariol (AOH), alternariolmonomethyl ether (AME), altenuene (ALT), and tentoxin (TEN) contents of A. alternata. Taken together, these data suggest that the mechanisms underlying the antifungal effect of 2-PEITC against A. alternata might be via reduction in toxin content and breakdown of cell membrane integrity.

Effects of sophorolipids on fungal and oomycete pathogens in relation to pH solubility.

AIMS: The objective of this study was to determine the effects of sophorolipids on several fungal and oomycete plant pathogens and the relationship between sophorolipids at different pH and antimicrobial activities. METHODS AND RESULTS: Sophorolipids had different solubility at different pH with a dramatic increase in solubility when pH was 6 or higher. Inhibition of mycelial growth of Phytophthora infestans by sophorolipids was affected by pH values, showing that when the pH value was higher, the inhibition rate was lower. Sophorolipids inhibited spore germination and mycelial growth of several fungal and oomycete pathogens in vitro including Fusarium sp., F. oxysporum, F. concentricum, Pythium ultimum, Pyricularia oryzae, Rhizoctorzia solani, Alternaria kikuchiana, Gaeumannomyces graminis var. tritici and P. infestans and caused morphological changes in hyphae by microscope observation. Sophorolipids reduced ß-1,3-glucanase activity in mycelia of P. infestans. In greenhouse studies, foliar application of sophorolipids at 3 mg ml-1 reduced severity of late blight of potato caused by P. infestans significantly. CONCLUSION: Sophorolipids influenced spore germination and hyphal tip growth of several plant pathogens and pH solubility of sophorolipids had an effect on their efficacy. Application of sophorolipids reduced late blight disease on potato under greenhouse conditions. SIGNIFICANCE AND IMPACT OF THE STUDY: The findings indicated that sophorolipids have the potential to be developed as a convenient and easy-to-use formulation for managing plant diseases.

G protein α subunit suppresses sporangium formation through a serine/threonine protein kinase in Phytophthora sojae.

Eukaryotic heterotrimeric guanine nucleotide-binding proteins consist of α, ß, and γ subunits, which act as molecular switches to regulate a number of fundamental cellular processes. In the oomycete pathogen Phytophthora sojae, the sole G protein α subunit (Gα; encoded by PsGPA1) has been found to be involved in zoospore mobility and virulence, but how it functions remains unclear. In this study, we show that the Gα subunit PsGPA1 directly interacts with PsYPK1, a serine/threonine protein kinase that consists of an N-terminal region with unknown function and a C-terminal region with a conserved catalytic kinase domain. We generated knockout and knockout-complemented strains of PsYPK1 and found that deletion of PsYPK1 resulted in a pronounced reduction in the production of sporangia and oospores, in mycelial growth on nutrient poor medium, and in virulence. PsYPK1 exhibits a cytoplasmic-nuclear localization pattern that is essential for sporangium formation and virulence of P. sojae. Interestingly, PsGPA1 overexpression was found to prevent nuclear localization of PsYPK1 by exclusively binding to the N-terminal region of PsYPK1, therefore accounting for its negative role in sporangium formation. Our data demonstrate that PsGPA1 negatively regulates sporangium formation by repressing the nuclear localization of its downstream kinase PsYPK1.

Topical Application of Adult Cecal Contents to Eggs Transplants Spore-Forming Microbiota but Not Other Members of the Microbiota to Chicks.

The intestinal microbiota plays an essential role in the metabolism and immune competence of chickens from the first day after hatching. In modern production systems, chicks are isolated from adult chickens, instead hatching in a clean environment. As a result, chicks are colonized by environmental bacteria, including potential pathogens. There is a need to investigate methods by which chicks can be exposed to a more appropriate microbial community at hatching. Such methods must be easy to apply in a hatchery and produce consistent results. The development of the intestinal microbiota of chicks hatched from eggs sprayed with dilute adult cecal content during incubation was observed at 0, 3, 7, and 14 days posthatching (dph) across two experiments. High-throughput Illumina sequencing was performed for the V4 hypervariable region of the 16S rRNA gene. A topical treatment of dilute adult cecal content was sufficient to transplant spore-forming bacteria such as Lachnospiraceae and Ruminococcaceae However, this treatment was not able to transplant other taxa that are considered to be core elements of the chicken cecal microbiota, such as Bacteroidaceae, Lactobacillaceae, Bifidobacteriaceae, and Burkholderiaceae The topical treatment significantly altered the microbiota of chicks immediately posthatching and accelerated the normal development of the microbiota with earlier colonization by Ruminococcaceae in the cecum and "Candidatus Arthromitus" in the ileum. The effect of the treatment on the cecal microbiota was maximal at 3 dph but diminished over time.IMPORTANCE Over the last 60 years poultry production has intensified in response to increased demand for meat. In modern systems, chicks hatch without contacting chickens and their gut bacteria. Consequently, they are colonized by environmental bacteria that may cause disease. The normal bacteria that live in the gut, or intestinal microbiota, play an important role in the development of the immune system. Therefore, it is essential to find easy ways to expose chicks to the more appropriate bacteria at hatching. This experiment investigated whether spraying eggs with adult cecal contents was sufficient to transfer an adult microbiota to chicks. Our findings show that spore-forming bacteria were transplanted, but other members of the microbiota were not. In this respect, the spray application was partially successful, but the timing of the spray needs to be modified to ensure that more bacteria are transferred.

Multimetal bioremediation and biomining by a combination of new aquatic strains of Mucor hiemalis.

Here we describe a unique microbial biotechnology for simultaneous bioremediation and biomining of twelve ionic metals overcoming the obstacles of multimetal toxicity to microbes. After a thorough search of key microorganisms in microbiomes of many sulfidic springs in Bavaria (Germany) over an area of 200 km2, we found three new strains EH8, EH10 and EH11 of Mucor hiemalis physiologically compatible and capable of multimetal-remediation and enrichment. We combined the multimetal-resistance, hyper-accumulation and elicitation power of EH8, EH10 and EH11 to develop a novel biotechnology for simultaneous removal, fractionation and enrichment of metal ions. As a first step we showed the intracellular fixing and deposition of mercury as nanospheres in EH8's sporangiospores. Scanning Electron Microscopy-Energy-Dispersive X-Ray analysis revealed binding and precipitation of other applied metal ions as spherical nano-particles (~50-100 nm) at the outer electro-negative cellwall-surface of EH8, EH10 and EH11 sporangiospores. Microbiomes, germinated spores and dead insoluble cellwalls of these strains removed >81-99% of applied Al, Cd, Co, Cr, Cu, Hg, Ni, Pb, U, and Zn simultaneously and furthermore enriched precious Ag, Au and Ti from water all within 48 h, demonstrating the potential of new biotechnologies for safe-guarding our environment from metal pollution and concentrating precious diluted, ionic metals.

The investigation of media components for optimal metabolite production of Aspergillus terreus ATCC 20542.

PURPOSE: This study aimed to assess the effect of nitrogen, salt and pre-culture conditions on the production of lovastatin in A. terreus ATCC 20542. METHODS: Different combinations of nitrogen sources, salts and pre-culture combinations were applied in the fermentation media and lovastatin yield was analysed chromatographically. RESULT: The exclusion of MnSO4 ·5H2O, CuSO4·5H2O and FeCl3·6H2O were shown to significantly improve lovastatin production (282%), while KH2PO4, MgSO4·7H2O, and NaCl and ZnSO4·7H2O were indispensable for good lovastatin production. Simple nitrogen source (ammonia) was unfavourable for morphology, growth and lovastatin production. In contrast, yeast extract (complex nitrogen source) produced the highest lovastatin yield (25.52 mg/L), while powdered soybean favoured the production of co-metabolites ((+)-geodin and sulochrin). Intermediate lactose: yeast extract (5:4) ratio produced the optimal lovastatin yield (12.33 mg/L) during pre-culture, while high (5:2) or low (5:6) lactose to yeast extract ratio produced significantly lower lovastatin yield (7.98 mg/L and 9.12 mg/L, respectively). High spore concentration, up to 107 spores/L was shown to be beneficial for lovastatin, but not for co-metabolite production, while higher spore age was shown to be beneficial for all of its metabolites. CONCLUSION: The findings from these investigations could be used for future cultivation of A. terreus in the production of desired metabolites.

The oospore stage of Plasmopara obducens, impatiens downy mildew.

Impatiens downy mildew is caused by Plasmopara obducens, a pathogen known in the United States for over a hundred years, but newly attacking ornamental Impatiens walleriana in production and in the landscape. Little is known about the life cycle of P. obducens; thus, in this study an attempt was made to determine whether the pathogen is homothallic or heterothallic. Fourteen single-sporangium isolates and three single-zoospore isolates were used in single and dual inoculations of stem tissue to see whether the pathogen was homothallic or heterothallic; all isolates tested were able to produce oospores when inoculated singly, suggesting homothally. Cold treatment at 0 C for at least 1 mo induced oospores to germinate and produce primary sporangia. Inoculation of plant tissue with germinating oospores resulted in infection. Other incubation temperatures (-10, 10, and 20 C) did not induce germination, but fluctuating temperatures (between -10 and 0 C, or 0 and 10 C) induced some germination. Spores incubated at -10 C had significantly thicker walls than spores incubated at other temperatures. Evidence suggests that oospores can serve as an overwintering stage.

Key innovations in transition from homospory to heterospory.

Heterospory (i.e. dimorphic spores) is a long-lasting topic discussed in plant biology. It is observed in many of ferns, fern allies, and seed plants. The rise of heterospory and the mechanisms underlying its success in plant evolution are not clearly elucidated. In this short communication, an attempt is made to shed some light on these two questions.

The Effect of pH on Spore Germination, Growth, and Infection of Strawberry Roots by Fusarium oxysporum f. sp. fragariae, Cause of Fusarium wilt of Strawberry.

Previous work has shown that raising the pH of acidic soil to near neutrality can reduce the incidence of Fusarium wilt. The basis for this effect has not been established. The present study assessed effects of pH on spore germination, growth, and infection of strawberry roots by Fusarium oxysporum f. sp. fragariae, the cause of Fusarium wilt of strawberry. There was not a significant effect of pH (5 versus 7) on the rate of spore germination at either 20 or 25°C for any of the three tested isolates (one representative of each clonal lineage of F. oxysporum f. sp. fragariae found in California). Likewise, pH did not have a significant effect on fungal growth at 20°C. At 25°C, two isolates grew faster at pH 7 than at pH 5. Growth of the third isolate was unaffected by the difference in pH. For the strawberry cultivar Albion, the frequency of root infection was significantly higher for plants grown in acidified soil (near pH 5) than for plants grown in soil near neutrality. The higher frequency of root infection in acidified soil was associated with a lower level of microbial activity, as measured by hydrolysis of fluorescein diacetate.

Can fern spores develop hydration memory in response to priming?

Fern spores and seeds initiate germination with fast water uptake, followed by a stationary phase with no appreciable water uptake and biochemical and metabolic processes that precede germination. After that, seed, germination is avoided by dehydration, as part of the priming treatments. After dehydration, seeds maintain their metabolic advances (hydration memory). As a result, rehydrated seeds germinate rapidly. We hypothesized that, as seeds, fern spores may be capable of developing hydration memory. To assess priming, spores of six fern species were exposed to: four or eight days of hydration in water (hydro-priming) or in a soil matrix (matrix-priming); or 1 month of hydration in the soil of the collection site (natural-priming). At the end of the treatments, the spores were dehydrated in the dark and germinated under laboratory conditions. Germination was evaluated using lag-time, germination rate and germination percentage. Priming treatments shortened lag time and/or increased germination rate or germination percentage in relation to the controls. Matrix-priming (8 days) reduced the spore germination percentage in three species. Our results provide evidence that fern spores possess a hydration memory that probably evolved in the soil bank and suggests that hydration-dehydration cycles within the natural soil might provide advantages for successful germination.

A quantitative method to assess the role of indoor air decontamination to simultaneously reduce contamination of environmental surfaces: testing with vegetative and spore-forming bacteria.

Indoor air can spread pathogens, which can be removed/inactivated by a variety of means in healthcare and other settings. We quantitatively assessed if air decontamination could also simultaneously reduce environmental surface contamination in the same setting. Two types of vegetative bacteria (Staphylococcus aureus and Acinetobacter baumannii), and a bacterial spore-former (Geobacillus stearothermophilus) were tested as representative airborne bacteria. They were separately aerosolized with a Collison nebulizer into a 24-m3 aerobiology chamber and air samples collected with a programmable slit-to-agar sampler. Settling airborne particles were collected on culture plates placed at, and collected from, five different locations on the floor of the chamber with a custom-built remote plate-placement and -retriever system. Experimentally contaminated air in the chamber was decontaminated for 45 min with a device based on HEPA filtration and UV light. The plates were incubated and CFU counted. The device reduced the viability levels of all tested bacteria in the air by >3 log10 (>99·9%) in 45 min. Based on two separate tests, the average reductions in surface contamination for S. aureus, A. baumannii and G. stearothermophilus were respectively, 97, 87 and 97%. We thus showed that air decontamination could substantially and simultaneously reduce the levels of surface contamination in the same setting irrespective of the type of pathogen present. SIGNIFICANCE AND IMPACT OF THE STUDY: The innovative and generic test protocol described can quantitatively assess the reduction in environmental surface contamination from microbial decontamination of indoor air in the same setting. This added advantage from air decontamination has implications for infection prevention and control in healthcare and other settings without the need for additional expense or effort. Continuous operation of an air decontamination device, such as the one tested here, can lead to ongoing reductions in pathogens in air and on environmental surfaces.

New host records of three Kudoa spp. (K. yasunagai, K. thalassomi, and K. igami) with notable variation in the number of shell valves and polar capsules in spores.

To date, 26 Kudoa spp. (Myxozoa: Myxosporea: Multivalvulida) have been recorded in edible marine fishes in Japan. In the future, it is likely that even more marine fish multivalvulid myxosporeans will be characterized morphologically and genetically, which will aid the precise understanding of their biodiversity and biology. We examined 60 individuals of six fish species collected from the Philippine Sea off Kochi or from the border between the Philippine Sea and East China Sea around Miyako Island, Okinawa, i.e., the southern part of Japan. Newly collected parasite species included Kudoa yasunagai from the brain of Japanese meagre (Argyrosomus japonicus) and Japanese parrotfish (Calotomus japonicus), Kudoa miyakoensis n. sp. and Kudoa thalassomi from the brain and trunk muscle, respectively, of bluespine unicornfish (Naso unicornis), and Kudoa igami from the trunk muscle of Carolines parrotfish (Calotomus carolinus), African coris (Coris gaimard), and Pastel ringwrasse (Hologymnosus doliatus). With the exception of Japanese parrotfish for K. yasunagai, all these fish are new host records for each kudoid species. Notable variation in the number of shell valves (SV) and polar capsules (PC) was observed for all four kudoid species. In particular, spores with seven or eight SV/PC were prominent in K. igami isolates, despite the original Japanese parrotfish-derived description characterizing it as having spores with six, or less commonly five, SV/PC. However, molecular genetic characterization based on the ribosomal RNA gene (rDNA) and mitochondrial DNA (cytochrome c oxidase subunit 1 and ribosomal RNA small and large subunits) found no significant differences in the nucleotide sequences of isolates with different phenotypical features as far as examined in the present study. A newly erected species, K. miyakoensis n. sp., was determined to be phylogenetically closest to brain-parasitizing species, such as K. chaetodoni, K. lemniscati, and K. yasunagai based on rDNA nucleotide sequences, but differed from them morphologically.

Effectiveness of an ultraviolet-C disinfection system for reduction of healthcare-associated pathogens.

BACKGROUND: Healthcare-associated infections caused by multidrug-resistant (MDR) pathogens are significantly associated with increased mortality and morbidity. Environmental cleaning can reduce transmission of these pathogens but is often inadequate. Adjunctive methods are warranted to enhance the effectiveness of disinfection particularly in hospital settings where healthcare-associated infections are of major concern. METHODS: We conducted a study to examine the effectiveness of a mobile, automatic device, Hyper Light Disinfection Robot (model: Hyper Light P3), which utilized ultraviolet-C (UV-C) to kill MDR-Pseudomonas aeruginosa, MDR- Acinetobacter baumannii, methicillin-resistant Staphylococcus aureus (MRSA), vancomycin-resistant Enterococcus faecium (VRE), Mycobacterium abscessus and Aspergillus fumigatus. The performance of this device in disinfecting hospital rooms previously admitted by patients harboring MRSA and VRE was also assessed. RESULTS: Except for VRE and M. abscessus, more than 3 log10 reduction of vegetative bacteria colonies was observed after UV-C irradiation of 5 min at a distance of 3 m from the device. At the distance of 1 m, substantial and comparable reduction of colonies was observed across all tested microorganisms regardless of exposure time. The killing effect was less pronounced for A. fumigatus particularly at the distance of 2-3 m. In uncleaned hospital rooms, there was significant reduction in the number of bacteria colonies sampled from different surfaces after UV-C irradiation for 15 min. CONCLUSIONS: UV-C disinfection system was effective in killing MDR pathogens. Further study is warranted to confirm its effectiveness as an adjunctive method in disinfecting hospital environment.

Ultrasensitive Response of Developing Myxococcus xanthus to the Addition of Nutrient Medium Correlates with the Level of MrpC.

Upon depletion of nutrients, Myxococcus xanthus forms mounds on a solid surface. The differentiation of rod-shaped cells into stress-resistant spores within mounds creates mature fruiting bodies. The developmental process can be perturbed by the addition of nutrient medium before the critical period of commitment to spore formation. The response was investigated by adding a 2-fold dilution series of nutrient medium to starving cells. An ultrasensitive response was observed, as indicated by a steep increase in the spore number after the addition of 12.5% versus 25% nutrient medium. The level of MrpC, which is a key transcription factor in the gene regulatory network, correlated with the spore number after nutrient medium addition. The MrpC level decreased markedly by 3 h after adding nutrient medium but recovered more after the addition of 12.5% than after 25% nutrient medium addition. The difference in MrpC levels was greatest midway during the period of commitment to sporulation, and mound formation was restored after 12.5% nutrient medium addition but not after adding 25% nutrient medium. Although the number of spores formed after 12.5% nutrient medium addition was almost normal, the transcript levels of "late" genes in the regulatory network failed to rise normally during the commitment period. However, at later times, expression from a reporter gene fused to a late promoter was higher after adding 12.5% than after adding 25% nutrient medium, consistent with the spore numbers. The results suggest that a threshold level of MrpC must be achieved in order for mounds to persist and for cells within to differentiate into spores.IMPORTANCE Many signaling and gene regulatory networks convert graded stimuli into all-or-none switch-like responses. Such ultrasensitivity can produce bistability in cell populations, leading to different cell fates and enhancing survival. We discovered an ultrasensitive response of M. xanthus to nutrient medium addition during development. A small change in nutrient medium concentration caused a profound change in the developmental process. The level of the transcription factor MrpC correlated with multicellular mound formation and differentiation into spores. A threshold level of MrpC is proposed to be necessary to initiate mound formation and create a positive feedback loop that may explain the ultrasensitive response. Understanding how this biological switch operates will provide a paradigm for the broadly important topic of cellular behavior in microbial communities.

Ecophysiology and lipid dynamics of a eukaryotic mangrove decomposer.

Aurantiochytrium limacinum is an osmo-heterotrophic Stramenopile and a pioneering mangrove decomposer which is taxonomically assigned to the family of Thraustochytriaceae (class: Labyrinthulomycetes). The life cycle of A. limacinum involves different cell types including mono- and multi-nucleated cells as well as flagellated zoospores which colonize new fallen leaves. The ecological relevance of thraustochytrids is underestimated and eclipsed by their biotechnological importance, due to their ability to accumulate large amount of lipids, mainly triacylglycerols (TAGs). In this study, we aimed to understand the ecophysiological parameters that trigger zoospore production and the interplay between the life cycle of A. limacinum and its lipid metabolism. When grown in a rich medium, cells accumulated large amounts of TAGs at the end of their growth period, but no zoospores were produced. In poor media such as artificial sea water, zoospores were produced in massive quantities. In the absence of organic carbon, the zoospores remained swimming for at least 6 days, consuming their TAGs in the process. Addition of glucose rapidly triggered the maturation of the zoospores. On the basis of these data, we propose a life cycle for A. limacinum integrating the potential perturbations/changes in the environment surrounding a mangrove leaf that could lead to the production of zoospores and colonization of new areas.

Transcriptomic and proteomic analysis reveals wall-associated and glucan-degrading proteins with potential roles in Phytophthora infestans sexual spore development.

Sexual reproduction remains an understudied feature of oomycete biology. To expand our knowledge of this process, we used RNA-seq and quantitative proteomics to examine matings in Phytophthora infestans. Exhibiting significant changes in mRNA abundance in three matings between different A1 and A2 strains compared to nonmating controls were 1170 genes, most being mating-induced. Rising by >10-fold in at least one cross were 455 genes, and 182 in all three crosses. Most genes had elevated expression in a self-fertile strain. Many mating-induced genes were associated with cell wall biosynthesis, which may relate to forming the thick-walled sexual spore (oospore). Several gene families were induced during mating including one encoding histidine, serine, and tyrosine-rich putative wall proteins, and another encoding prolyl hydroxylases which may strengthen the extracellular matrix. The sizes of these families vary >10-fold between Phytophthora species and one exhibits concerted evolution, highlighting two features of genome dynamics within the genus. Proteomic analyses of mature oospores and nonmating hyphae using isobaric tags for quantification identified 835 shared proteins, with 5% showing >2-fold changes in abundance between the tissues. Enriched in oospores were ß-glucanases potentially involved in digesting the oospore wall during germination. Despite being dormant, oospores contained a mostly normal complement of proteins required for core cellular functions. The RNA-seq data generated here and in prior studies were used to identify new housekeeping controls for gene expression studies that are more stable than existing normalization standards. We also observed >2-fold variation in the fraction of polyA+ RNA between life stages, which should be considered when quantifying transcripts and may also be relevant to understanding translational control during development.