Oospore Production Research Articles

The novel roles of RNA m6A modification in regulating the development, infection, and oxidative DNA damage repair of Phytophthora sojae.

N6-methyladenosine (m6A), a vital post-transcriptional regulator, is among the most prevalent RNA modifications in eukaryotes. Nevertheless, the biological functions of m6A in oomycetes remain poorly understood. Here, we showed that the PsMTA1 and PsMTA2 genes are orthologs of human METTL4, while the PsMET16 gene is an ortholog of human METTL16. These genes are implicated in m6A modification and play a critical role in the production of sporangia and oospores, the release of zoospores, and the virulence of Phytophthora sojae. In P. sojae, m6A modifications are predominantly enriched in the coding sequence and the 3' untranslated region. Notably, the PsMTA1 knockout mutant exhibited reduced virulence, attributed to impaired tolerance to host defense-generated ROS stress. Mechanistically, PsMTA1-mediated m6A modification positively regulates the mRNA lifespan of DNA damage response (DDR) genes in reaction to plant ROS stress during infection. Consequently, the mRNA abundance of the DDR gene PsRCC1 was reduced in the single m6A site mutant ΔRCC1/RCC1A2961C, resulting in compromised DNA damage repair and reduced ROS adaptation-associated virulence in P. sojae. Overall, these results indicate that m6A-mediated RNA metabolism is associated with the development and pathogenicity of P. sojae, underscoring the roles of epigenetic markers in the adaptive flexibility of Phytophthora during infection.

Identification of Growing Media Favorable for the Growth of Pythium aphanidermatum, a Telluric Pathogen of Papaya (Carica papaya L.) in Côte d’Ivoire

Pawpaw (Carica papaya L.) is an economically important plant in Côte d'Ivoire. However, its cultivation is subject to numerous biotic constraints, including root and crown rot, caused by several species of Pythium, Phytophthora, and Rhizoctonia. Of all these fungi, Pythium aphanidermatum is the most dreaded in Ivorian plantations, causing root, crown, trunk, and fruit rot. This causes enormous production losses, reducing farmers’ incomes. The present study was initiated with a view to finding a suitable culture medium for the development of P. aphanidermatum, which is responsible for root and crown rot of papaya trees, through the study of its morphology and biology. To this end, the mycelial growth of P. aphanidermatum was evaluated on different culture media (PDA, V8, CMA and Papaya). The results showed that all these culture media were favorable for the mycelial growth of P. aphanidermatum. However, in terms of propagule production, PDA and V8 culture media were the most favorable. The CMA medium produced fewer propagules than the PDA and V8 media. Papaya culture medium inhibited oospore production. Sporocysts and chlamydospores were observed in all media. The culture medium did not influence the mycelial growth of P. aphanidermatum but rather oospore production.

Autophagy-related protein PlATG2 regulates the vegetative growth, sporangial cleavage, autophagosome formation, and pathogenicity of peronophythora litchii

ABSTRACT Autophagy is an intracellular degradation process that is important for the development and pathogenicity of phytopathogenic fungi and for the defence response of plants. However, the molecular mechanisms underlying autophagy in the pathogenicity of the plant pathogenic oomycete Peronophythora litchii, the causal agent of litchi downy blight, have not been well characterized. In this study, the autophagy-related protein ATG2 homolog, PlATG2, was identified and characterized using a CRISPR/Cas9-mediated gene replacement strategy in P. litchii. A monodansylcadaverine (MDC) staining assay indicated that deletion of PlATG2 abolished autophagosome formation. Infection assays demonstrated that ΔPlatg2 mutants showed significantly impaired pathogenicity in litchi leaves and fruits. Further studies have revealed that PlATG2 participates in radial growth and asexual/sexual development of P. litchii. Moreover, zoospore release and cytoplasmic cleavage of sporangia were considerably lower in the ΔPlatg2 mutants than in the wild-type strain by FM4–64 staining. Taken together, our results revealed that PlATG2 plays a pivotal role in vegetative growth, sporangia and oospore production, zoospore release, sporangial cleavage, and plant infection of P. litchii. This study advances our understanding of the pathogenicity mechanisms of the phytopathogenic oomycete P. litchii and is conducive to the development of effective control strategies.

Mining oomycete proteomes for phosphatome leads to the identification of specific expanded phosphatases in oomycetes.

Phosphatases are important regulators of protein phosphorylation and various cellular processes, and they serve as counterparts to kinases. In this study, our comprehensive analysis of oomycete complete proteomes unveiled the presence of approximately 3833 phosphatases, with most species estimated to have between 100 and 300 putative phosphatases. Further investigation of these phosphatases revealed a significant increase in protein serine/threonine phosphatases (PSP) within oomycetes. In particular, we extensively studied the metallo-dependent protein phosphatase (PPM) within the PSP family in the model oomycete Phytophthora sojae. Our results showed notable differences in the expression patterns of PPMs throughout 10 life stages of P. sojae, indicating their vital roles in various stages of oomycete pathogens. Moreover, we identified 29 PPMs in P. sojae, and eight of them possessed accessory domains in addition to phosphate domains. We investigated the biological function of one PPM protein with an extra PH domain (PPM1); this protein exhibited high expression levels in both asexual developmental and infectious stages. Our analysis confirmed that PPM1 is indeed an active protein phosphatase, and its accessory domain does not affect its phosphatase activity. To delve further into its function, we generated knockout mutants of PPM1 and validated its essential roles in mycelial growth, sporangia and oospore production, as well as infectious stages. To the best of our knowledge, this study provides the first comprehensive inventory of phosphatases in oomycetes and identifies an important phosphatase within the expanded serine/threonine phosphatase group in oomycetes.

ToGC: a pipeline to correct gene model for functional excavation of dark GPCRs in Phytophthora sojae1

The accuracy of genomic annotation is crucial for subsequent functional investigations; however, computational protocols used in high-throughput annotation of open reading frames (ORFs) can introduce inconsistencies. These inconsistencies, which lead to non-uniform extension or truncation of sequence ends, pose challenges for downstream analyses. Existing strategies to rectify these inconsistencies are time-consuming and labor-intensive, lacking specific approaches. To address this gap, we developed toGC, a tool that integrates genomic annotation with RNA-seq datasets to rectify annotation inconsistencies. Using toGC, we achieved an accuracy of nearly 100% accuracy in correcting inconsistencies in published P. sojae ORFs. We applied this innovative pipeline to the GPCR-bigrams gene family, which was predicted to have 42 members in the P. sojae genome but lacked experimental validation. By employing toGC, we identified 32 GPCR-bigram ORFs with inconsistencies between previous annotations and toGC-corrected sequences. Notably, among these were 5 genes (GPCR-TKL9, GPCR-TKL15, GPCR-PDE3, GPCR-AC3, and GPCR-AC4) showed substantial inconsistencies. Experimental gene annotation confirmed the effectiveness of toGC, as sequences obtained through cloning matched those annotated by toGC. Importantly, we discovered two novel GPCRs (GPCR-AC3 and GPCR-AC4), which were previously mispredicted as a single gene. CRISPR/Cas9-mediated knockout experiments revealed the involvement of GPCR-AC4 but not GPCR-AC3 in oospore production, further confirming their status as two separate genes. In addition to P. sojae, the reliability of the toGC pipeline in Phytophthora capsici and Pythium ultimum further emphasizes the robustness of this pipeline. Our findings highlight the utility of toGC for reliable gene model correction, facilitating investigations into biological functions and offering potential applications in diverse species analyses.

Phytophthora infestans Culture Media: A Comparative Study of Cost-Effective V8 Agar and Rye Agar

Phytophthora infestans is a pathogen that causes late blight in crops, such as potatoes and tomatoes, posing a serious threat to the Solanaceae family. Researchers have attempted to create an affordable and effective medium for isolating and cultivating this pathogen. The aim of this study was to evaluate the efficacy of low-cost V-8 agar for mycelial growth, sporangia production, and oospore production of P. infestans and compare it with Rye A medium. To achieve this, V-8 medium was modified by replacing V-8 juice with tomato. Mycelial growth and sporulation were measured on both media after 14 days of inoculation. It was observed as a white, fluffy growth in both media. The mean radial growth was larger on the low-cost V-8 medium (76 mm) than on Rye A medium (4.4 mm). The lemon-shaped sporangia with a semipapilated structure were recorded with an average length-to-width ratio of 28.6 μm to 30.6 μm, respectively. Oospore production was greater in the low-cost V-8 medium (5x104) than in Rye A medium (1x104). The present study demonstrated that low-cost, locally available materials such as tomato can be used as an alternative nutrient medium for the growth of P. infestans.

Effectiveness of Volatiles Emitted by Streptomyces abikoensis TJGA-19 for Managing Litchi Downy Blight Disease.

Litchi is a fruit of significant commercial value; however, its quality and yield are hindered by downy blight disease caused by Peronophythora litchii. In this study, volatile organic compounds (VOCs) from Streptomyces abikoensis TJGA-19 were investigated for their antifungal effects and studied in vitro and in planta for the suppression of litchi downy blight disease in litchi leaves and fruits. The growth of P. litchii was inhibited by VOCs produced by TJGA-19 cultivated on autoclaved wheat seeds for durations of 10, 20, or 30 days. Volatiles from 20-day-old cultures were more active in inhibition effect against P. litchii than those from 10- or 30-day-old cultures. These volatiles inhibit the growth of mycelia, sporulation, and oospore production, without any significant effect on sporangia germination. Additionally, the VOCs were effective in suppressing disease severity in detached litchi leaf and fruit infection assays. With the increase in the weight of the wheat seed culture of S.abikoensis TJGA-19, the diameters of disease spots on leaves, as well as the incidence rate and disease indices on fruits, decreased significantly. Microscopic results from SEM and TEM investigations showed abnormal morphology of sporangia, mycelia, and sporangiophores, as well as organelle damage in P. litchii caused by VOCs of TJGA-19. Spectroscopic analysis revealed the identification of 22 VOCs produced by TJGA-19, among which the most dominant compound was 2-Methyliborneol. These findings indicated the significant role of TJGA-19 compounds in the control of litchi downy blight disease and in improving fruit quality.

Evaluating the contribution of historical and contemporary temperature to the oospore production of self-fertile Phytophthora infestans.

Reproductive systems play an important role in the ecological function of species, but little is known about how climate change, such as global warming, may affect the reproductive systems of microbes. In this study, 116 Phytophthora infestans isolates sampled from five different altitudes along a mountain were evaluated under five temperature regimes to determine the effects of historical and experimental temperature on the reproductive system of the pathogen. Both altitude, a proxy for historical pathogen adaptation to temperature, and temperature used in the experiment affected the sexual reproduction of the pathogen, with experimental temperature, that is, contemporary temperature, playing a role several times more important than historical temperature. Furthermore, the potential of sexual reproduction, measured by the number of oospores quantified, increased with the temperature breadth (i.e., difference between the highest and lowest temperature at which sexual reproduction takes place) of the pathogen and reached the maximum at the experimental temperature of 21°C, which is higher than the annual average temperature in many potato-producing areas. The results suggest that rising air temperature associated with global warming may increase the potential of sexual reproduction in P. infestans. Given the importance of sexuality in pathogenicity and ecological adaptation of pathogens, these results suggest that global warming may increase the threat of P. infestans to agricultural production and other ecological services and highlight that new epidemiological strategies may need to be implemented for future food security and ecological resilience.

A major facilitator superfamily transporter PlMFS1 contributes to growth, oosporogenesis, and pathogenesis of Peronophythora litchii

Major facilitator superfamily (MFS) transporters are secondary active membrane transporters that play an important role in solute interchange and energy metabolism. Peronophythora litchii causes the most destructive disease on lichi, litchi downy blight. PlM90 was reported as a key oosporogenesis regulator. Here, we identified an MFS transporter gene PlMFS1, which is up-regulated during oospore formation at the late infection stage, while down-regulated in the PlM90 mutant. To investigate PlMFS1 function, we generated PlMFS1 knockout mutants using CRISPR/Cas9-mediated genome editing technology. Compared with the wild-type strain SHS3, PlMFS1 deletion impaired mycelium growth, zoospore release, oospore production and pathogenicity. Furthermore, PlMFS1 deletion significantly affected P. litchii utilization of fructose, lactose and maltose, and may be the PlMFS1 mechanism involved in mycelial growth. PlMFS1 gene deletion also led to deceased laccase activity, laccase-encoding gene downregulation and impaired P. litchii pathogenicity. To our knowledge, this is the first report of an MFS transporter involved in sugar utilization, sexual reproduction, asexual reproduction and pathogenesis in oomycetes.

Gene editing with an oxathiapiprolin resistance selection marker reveals that PuLLP, a loricrin-like protein, is required for oospore development in Pythium ultimum

Oomycetes, such as Pythium species, contain numerous devastating plant pathogens that inflict substantial economic losses worldwide. Although CRISPR/Cas9-based genome editing is available, the selection markers available for genetic transformation in these species are limited. In this study, a mutated version of the Phytophthora capsici oxysterol-binding protein-related protein 1 (PcMuORP1), known to confer oxathiapiprolin resistance, was introduced into the CRISPR/Cas9 system for in situ complementation in Pythium ultimum. We targeted PuLLP, which encodes a loricrin-like protein, and showed significant downregulation when the Puf RNA-binding protein-encoding gene PuM90 was knocked out. The PuLLP knockout mutants could not produce oospores, indicating a similar biological function as PuM90. The reintroduction of PuLLP into the knockout mutant using PcMuORP1 as a selection marker restored oospore production. Further comparisons with the conventional selection marker NPTII indicated that PcMuORP1 could be applied at a lower concentration and cost, resulting in a higher screening efficiency. Successive subculturing in the absence of selective pressure showed that PcMuORP1 had little long-term effect on the fitness of transformants. Hence, it could be reused as an alternative selection marker. This study demonstrates the successful implementation of the PcMuORP1 gene as a selection marker in the genetic transformation of Py. ultimum and reveals the loricrin-like protein PuLLP as a sexual reproduction-related factor downstream of the Puf RNA-binding protein PuM90. Overall, these results will help accelerate the functional genomic investigation of oomycetes.

A Novel FYVE Domain-Containing Protein Kinase, PsZFPK1, Plays a Critical Role in Vegetative Growth, Sporangium Formation, Oospore Production, and Virulence in Phytophthora sojae.

Proteins containing both FYVE and serine/threonine kinase catalytic (STKc) domains are exclusive to protists. However, the biological function of these proteins in oomycetes has rarely been reported. In the Phytophthora sojae genome database, we identified five proteins containing FYVE and STKc domains, which we named PsZFPK1, PsZFPK2, PsZFPK3, PsZFPK4, and PsZFPK5. In this study, we characterized the biological function of PsZFPK1 using a CRISPR/Cas9-mediated gene replacement system. Compared with the wild-type strain, P6497, the PsZFPK1-knockout mutants exhibited significantly reduced growth on a nutrient-rich V8 medium, while a more pronounced defect was observed on a nutrient-poor Plich medium. The PsZFPK1-knockout mutants also showed a significant increase in sporangium production. Furthermore, PsZFPK1 was found to be essential for oospore production and complete virulence but dispensable for the stress response in P. sojae. The N-terminal region, FYVE and STKc domains, and T602 phosphorylation site were found to be vital for the function of PsZFPK1. Conversely, these domains were not required for the localization of PsZFPK1 protein in the cytoplasm. Our results demonstrate that PsZFPK1 plays a critical role in vegetative growth, sporangium formation, oospore production, and virulence in P. sojae.

Talaromyces omanensis and Aspergillus fumigatus endophytic fungi suppress Pythium aphanidermatum and its induced damping-off diseases of cucumber and radish

Damping-off diseases caused huge losses in crop production in numerous countries. Pythium aphanidermatum represents a major cause of these diseases. In the present investigation, two endophytic fungi Talaromyces omanensis and Aspergillus fumigatus isolated from a desert plant, Rhazya stricta, were investigated for their efficiency in suppressing P. aphanidermatum and its stimulated damping-off diseases in cucumber and radish. The results proved the ability of both endophytes to inhibit P. aphanidermatum growth in both solid and liquid forms, inhibit its oospore production, induce abnormal patterns in its hyphae and suppress its induced damping-off diseases. Moreover, T. omanensis produced an inhibition zone area against P. aphanidermatum and enhanced the fresh weight of cucumber and length, fresh weight and dry weight of radish. Further, A. fumigatus caused cellular leakage in P. aphanidermatum mycelium. The previous results were interpreted through the secretion of β-1,3-glucanase, cellulase and siderophore by both endophytic fungi.

Use of oxathiapiprolin for controlling soybean root rot caused by Phytophthora sojae: efficacy and mechanism of action.

Oxathiapiprolin is a new isoxazoline fungicide developed by DuPont to control oomycete diseases. Although oxathiapiprolin has shown strong inhibitory activity against oomycete pathogens, little is known about its ability to control Phytophthora sojae. Oxathiapiprolin showed high inhibitory activity against Phytophthora sojae, with 50% effective concentration (EC50 ) values ranging from 1.15 × 10-4 to 4.43 × 10-3 μg mL-1 . Oxathiapiprolin inhibited various stages of Phytophthora sojae development, including mycelial growth, sporangium formation, oospore production, and zoospore release. Electron microscopy studies revealed that oxathiapiprolin caused severe morphological and ultrastructural damage to Phytophthora sojae. Oxathiapiprolin affected the cell membrane and wall of Phytophthora sojae, making it more sensitive to osmotic and cell wall stress. Oxathiapiprolin exhibited translocation activity; it was absorbed by soybean roots and then translocated to the leaves. It was effective at reducing soybean Phytophthora root rot under glasshouse and field conditions. Both fungicide seed treatment and foliar spray significantly reduced disease incidence and yield losses compared with untreated controls in the field. Oxathiapiprolin exhibits high inhibitory activity against Phytophthora sojae, and has multiple mechanisms of action including severe mycelial damage and modulation of osmotic and cell wall stress. These results indicate that oxathiapiprolin can be used at low concentrations for highly effective management of soybean Phytophthora root rot caused by Phytophthora sojae. © 2022 Society of Chemical Industry.

Pathogen Adaptation to American (Rpv3-1) and Eurasian (Rpv29) Grapevine Loci Conferring Resistance to Downy Mildew.

Durable resistance is a key objective in genetic improvement for disease resistance in grapevines, which must survive for years in the field in the presence of adaptable pathogen populations. In this study, the adaptation of 72 Northern Italian isolates of Plasmopara viticola, the downy mildew agent, has been investigated into Bianca, possessing Rpv3-1, the most frequently exploited resistance locus for genetic improvement, and Mgaloblishvili, a Vitis vinifera variety possessing the newly discovered Rpv29 locus. Infection parameters (latency period, infection frequency, and disease severity) and oospore production and viability were evaluated and compared to those of Pinot noir, the susceptible reference. The expected levels of disease control were achieved by both resistant cultivars (>90% on Bianca; >25% on Mgaloblishvili), despite the high frequency of isolates able to grow on one (28%) or both (46%) accessions. The disease incidence and severity were limited by both resistant cultivars and the strains able to grow on resistant accessions showed signatures of fitness penalties (reduced virulence, infection frequency, and oospore density). Together, these results indicate an adequate pathogen control but suitable practices must be adopted in the field to prevent the diffusion of the partially adapted P. viticola strains to protect resistance genes from erosion.

High Temporal Variability in Late Blight Pathogen Diversity, Virulence, and Fungicide Resistance in Potato Breeding Fields: Results from a Long-Term Monitoring Study.

Long-term site-specific studies describing changes in the phenotypic variability of Phytophthora infestans populations allow quantitative predictions of pathogen spread and possible outbreaks of epidemics, and provide key input for regional resistance breeding programs. Late blight samples were collected from potato (Solanum tuberosum) breeding fields in Estonia during a twelve-year study period between 2001 and 2014. In total, 207 isolates were assessed for mating type and 235 isolates for metalaxyl resistance and 251 isolates for virulence factors. The frequency of mating types strongly fluctuated across the years, whereas the later period of 2010–2014 was dominated by the A2 mating. Despite fluctuations, both mating types were recorded in the same fields in most years, indicating sustained sexual reproduction of P. infestans with oospore production. Metalaxyl-resistant and intermediately resistant strains dominated in the first years of study, but with the progression of the study, metalaxyl-sensitive isolates became dominant, reaching up to 88%. Racial diversity, characterized by normalized Shannon diversity index decreased in time, varying from 1.00 in 2003 to 0.43 in 2013. The frequency of several virulence factors changed in a time-dependent manner, with R2 increasing and R6, R8, and R9 decreasing in time. Potato cultivar resistance background did not influence the frequency of P. infestans mating type, response to metalaxyl, and racial diversity. However, the diversity index decreased in time among isolates collected from resistant and susceptible cultivars, and remained at a high level in moderately resistant cultivars. These data demonstrate major time-dependent changes in racial diversity, fungicide resistance, and virulence factors in P. infestans, consistent with alterations in the control strategies and popularity of potato cultivars with different resistance.

Highly Diverse Phytophthora infestans Populations Infecting Potato Crops in Pskov Region, North-West Russia.

There is limited understanding of the genetic variability in Phytophthora infestans in the major potato cultivation region of north-western Russia, where potato is grown primarily by small households with limited chemical treatment of late blight. In this study, the mating type, sensitivity to metalaxyl, and genotype and population genetic diversity (based on 12 simple sequence repeat (SSR) markers) of 238 isolates of P. infestans from the Pskov region during the years 2010–2013 were characterized. The aim was to examine the population structure, phenotypic and genotypic diversity, and the prevalent reproductive mode of P. infestans, as well as the influence of the location, time, and agricultural management practices on the pathogen population. The frequency of the A2 mating was stable over the four seasons and ranged from 33 to 48% of the sampled population. Both mating types occurred simultaneously in 90% of studied fields, suggesting the presence of sexual reproduction and oospore production in P. infestans in the Pskov region. Metalaxyl-sensitive isolates prevailed in all four years (72%), however, significantly fewer sensitive isolates were found in samples from large-scale conventional fields. A total of 50 alleles were detected in the 141 P. infestans isolates analyzed for genetic diversity. Amongst the 83 SSR multilocus genotypes (MLGs) detected, 65% were unique and the number of MLGs varied between locations from 3 to 20. These results, together with the high genotypic diversity observed in all the locations and the lack of significance of linkage disequilibrium, suggest that sexual recombination is likely responsible for the unique MLGs and the high genetic diversity found in the Pskov region population, resembling those of north-eastern European populations.

Phytophthora tropicalis: Causal agent of persimmon fruit rot in Brazil

AbstractPersimmon fruit (Diospyros kaki L.) from commercial orchards located in southern Brazil, in Curitiba, Paraná, showed dark firm rot. The incidence in the field was about 2% in an evaluation in the 2016 growing season, and from these infected fruit, three Phytophthora sp. isolates were obtained. Therefore, this study aimed to identify the causal agent of the disease through morphophysiological and molecular analysis of the isolates. Initially, the pathogenicity test was performed on persimmon fruit to fulfil Koch's postulates. Mycelial growth at eight temperatures ranging from 8°C to 35°C was evaluated for taxonomic purposes. Next, the morphological characteristics of sporangia, chlamydospores and oospores were evaluated. For molecular characterization, sequencing of the ITS‐rDNA region and portions of the COXI and TEF1α genes and phylogenetic analysis were performed. All isolates were pathogenic, causing symptoms of firm and dark rot similar to those observed in the orchards. Mycelial growth was not observed at 35°C. There was an abundant production of ellipsoid, papillate, deciduous sporangia with long pedicels and the formation of globose chlamydospores. The isolates were heterothallic, all belonging to group A1, in which the production of plerotic oospores with amphigenous antheridia was verified. The morphophysiological and molecular characterization allowed the identification of the isolates as Phytophthora tropicalis. To our knowledge, this is the first report of persimmon fruit rot caused by P. tropicalis.

Overwintering of an endangered charophyte during milder winters in Central Europe enhances lake water quality

The aims of our research were: 1) to study the changes in the quantitative and qualitative structure of submerged macrophytes relative to the physical and chemical characteristics of water in a model lake dominated by the endangered charophyte Lychnothamnus barbatus, 2) to test the overwintering of total macrophytes and L. barbatus in different winter conditions, and 3) to determine the spatial and seasonal variation in the oospore production by L. barbatus across key environmental gradients.Two gradients proved to be responsible for the macrophyte structure: 1) the depth and light gradient, and 2) the seasonality of water trophy. The depth of occurrence, light conditions and interspecies competition are the key factors determining the production of L. barbatus biomass and oospores. In 2018 mass overwintering of total macrophytes and L. barbatus in mild winter conditions significantly improved lake water quality, indicating that macrophyte communities can protect lakes from the negative consequences of global warming.This research thus demonstrated that macrophytes are a key factor in improving and stabilising water quality of a temperate mesotrophic lake which hosts one of the largest populations of the endangered charophyte.

The consensus Nglyco -X-S/T motif and a previously unknown Nglyco -N-linked glycosylation are necessary for growth and pathogenicity of Phytophthora.

Asparagine (Asn, N)-linked glycosylation within Nglyco -X-S/T; X≠P motif is a ubiquitously distributed post-translational modification that participates in diverse cellular processes. In this work, N-glycosylation inhibitor was shown to prevent Phytophthora sojae growth, suggesting that N-glycosylation is necessary for oomycete development. We conducted a glycoproteomic analysis of P. sojae to identify and map N-glycosylated proteins and to quantify differentially expressed glycoproteins associated with mycelia, asexual cyst, and sexual oospore developmental stages. A total of 355 N-glycosylated proteins was found, containing 496 glycosites, potentially involved in glycan degradation, carbon metabolism, glycolysis, or other metabolic pathways. Through PNGase F deglycosylation assays and site-directed mutagenesis of a GPI transamidase protein (GPI16) upregulated in cysts and a heat shock protein 70 (HSP70) upregulated in oospores, we demonstrated that both proteins were N-glycosylated and that the Nglyco -N motif is a target site for asparagine - oligosaccharide linkage. Glycosite mutations of Asn 94 Nglyco -X-S/T in the GPI16 led to impaired cyst germination and pathogenicity, while mutation of the previously unknown Asn 270 Nglyco -N motif in HSP70 led to decreased oospore production. In addition to providing a map of the oomycete N-glycoproteome, this work confirms that P. sojae has evolved multiple N-glycosylation motifs essential for growth.

The Mitogen-Activated Protein Kinase PlMAPK2 Is Involved in Zoosporogenesis and Pathogenicity of Peronophythoralitchii.

As an evolutionarily conserved pathway, mitogen-activated protein kinase (MAPK) cascades function as the key signal transducers that convey information by protein phosphorylation. Here we identified PlMAPK2 as one of 14 predicted MAPKs encoding genes in the plant pathogenic oomycete Peronophythora litchii. PlMAPK2 is conserved in P. litchii and Phytophthora species. We found that PlMAPK2 was up-regulated in sporangium, zoospore, cyst, cyst germination and early stage of infection. We generated PlMAPK2 knockout mutants using the CRISPR/Cas9 method. Compared with wild-type strain, the PlMAPK2 mutants showed no significant difference in vegetative growth, oospore production and sensitivity to various abiotic stresses. However, the sporangium release was severely impaired. We further found that the cleavage of the cytoplasm into uninucleate zoospores was disrupted in the PlMAPK2 mutants, and this developmental phenotype was accompanied by reduction in the transcription levels of PlMAD1 and PlMYB1 genes. Meanwhile, the PlMAPK2 mutants exhibited lower laccase activity and reduced virulence to lychee leaves. Overall, this study identified a MAPK that is critical for zoosporogenesis by regulating the sporangial cleavage and pathogenicity of P. litchii, likely by regulating laccase activity.