Hericium erinaceus polysaccharide improves the microstructure, immune function, proliferation and reduces apoptosis of thymus and spleen tissue cells of immunosuppressed mice.

In order to study the effect of Hericium erinaceus polysaccharide (HEP) on the immune and antioxidation functions of immunosuppressed mice. The control group received distilled water orally and the model and experimental groups I, II, and III received 0, 80, 160, and 320 mg/kg HEP respectively for a fortnight after re-molding with cyoclphosphnalide (CTX). Compared with the control group, the secretion of IL-2, IL-4, and IFN-γ, the activity or content of T-AOC, T-SOD, and GSH-PX, and the expression of PCNA mRNA in the thymus and spleen were reduced in immunosuppressed mice (P < .05 or P < .01). Compared with immunosuppressed mice, the levels of IL-2, IFN-γ, and GSH-PX and the PCNA mRNA expression of spleen and thymus were increased (P < .05 or P < .01), and the microstructure were also obviously improved in the experimental group III. Overall, 320 mg/kg of HEP significantly improved the immune and antioxidant functions.

Interleukin-1 receptor-associated kinase 2 promotes inflammatory reactions by activating the nuclear factor kappa-B signaling pathway in diabetic nephropathy.

Diabetic nephropathy (DN) is a major complication of diabetes. Interleukin-1 receptor-associated kinase 2 (IRAK2) has been implicated in various diseases. This study aimed to investigate the role of IRAK2 in DN progression and its association with inflammation and the nuclear factor-kappa B (NF-κB) signaling pathway. DN model mice were generated by intraperitoneal injection of streptozotocin. IRAK2 expression was upregulated in the DN model mice. IRAK2 knockdown increased weight and reduced blood glucose levels in DN model mice. In addition, IRAK2 downregulation improved glomerular morphology in DN mice. IRAK2 knockdown reduced the levels of kidney damage biomarkers (24-h urinary protein, urine albumin-creatinine ratio, and plasma creatinine) and inflammatory cytokines (IL-6, tumor necrosis factor [TNF]-α, TNF-1R, and TNF-2R). Moreover, IRAK2 activated the NF-κB signaling pathway in DN model mice. Overexpression of NF-κB exacerbated DN progression, and IRAK2 knockdown reversed these effects. IRAK2 promoted DN progression and inflammation by activating the NF-κB signaling pathway. These findings suggest that IRAK2 is a potential therapeutic target for DN treatment.

Sgh1, an SR-like Protein, Is Involved in Fungal Development, Plant Infection, and Pre-mRNA Processing in Fusarium graminearum.

Serine/arginine (SR) proteins are essential pre-mRNA splicing factors in eukaryotic organisms. Our previous studies have shownthat the unique SR-specific protein kinase Srk1 is important for RNA splicing and gene transcription in Fusarium graminearum, and interacts with two SR proteins, FgSrp1 and FgSrp2. In this study, we have identified an SR-like protein called Sgh1 in F. graminearum, which is orthologous to budding yeast paralogous Gbp2 and Hrb1. Our data have shownthat the Sgh1 is involved in vegetative growth, conidiation, sexual reproduction, DON synthesis, and plant infection. Moreover, the Sgh1 is mainly localized to the nucleus. RNA-seq analysis has shownthat the expression of over 1100 genes and the splicing efficiency in over 300 introns were affected in the Δsgh1 mutant. Although the RS domain and all three of the RRM domains are important for the Sgh1 functions, only the RS domain is responsible for its nuclear localization. Finally, we verified that the Sgh1 interacts with the unique SR-specific kinase Srk1 in F. graminearum by the yeast-two hybrid (Y2H) and bimolecular fluorescence complementation (BiFC) assays. Taken together, our results have revealed that the Sgh1 regulates the fungal development, plant infection, and the pre-mRNA processing, and the RS domain regulates the function of the Sgh1 by modulating its nucleocytoplasmic shuttling.

FgCsn12 Is Involved in the Regulation of Ascosporogenesis in the Wheat Scab Fungus Fusarium graminearum.

Fusarium head blight (FHB), caused by the fungal pathogen Fusarium graminearum, is a destructive disease worldwide. Ascospores are the primary inoculum of F. graminearum, and sexual reproduction is a critical step in its infection cycle. In this study, we characterized the functions of FgCsn12. Although the ortholog of FgCsn12 in budding yeast was reported to have a direct interaction with Csn5, which served as the core subunit of the COP9 signalosome, the interaction between FgCsn12 and FgCsn5 was not detected through the yeast two-hybrid assay. The deletion of FgCSN12 resulted in slight defects in the growth rate, conidial morphology, and pathogenicity. Instead of forming four-celled, uninucleate ascospores, the Fgcsn12 deletion mutant produced oval ascospores with only one or two cells and was significantly defective in ascospore discharge. The 3'UTR of FgCsn12 was dispensable for vegetative growth but essential for sexual reproductive functions. Compared with those of the wild type, 1204 genes and 2240 genes were up- and downregulated over twofold, respectively, in the Fgcsn12 mutant. Taken together, FgCsn12 demonstrated an important function in the regulation of ascosporogenesis in F. graminearum.

Transcriptome Profiling of Duodenum Reveals the Importance of Boron Supplementation in Modulating Immune Activities in Rats.

As an essential trace element, appropriate boron supplementation can promote immune function of animals. To illustrate the effects of boron in a rat model, RNA-Seq was conducted for the RNA from duodenum after treatment with different concentration of boron in which boron was given in the form of boric acid. More than 47 million reads were obtained in 0, 10, and 320 mg/L boron (0, 57.21, and 1830.66 mg/L boric acid) treatment groups that produced 58 965 402, 48 607 328, and 46 760 660 clean reads, respectively. More than 95% of the clean reads were successfully matched to the rat reference genome and assembled to generate 32 662 transcripts. A total of 624 and 391 differentially expressed candidate genes (DEGs) were found between 0 vs.10 and 0 vs. 320 mg/L boron comparison groups. We also identified transcription start site, transcription terminal site, and skipped exons as the main alternative splicing events. GO annotations revealed most of DEGs were involved in the regulation of immune activity. The DEGs were enriched in influenza A, herpes simplex infection, cytosolic DNA-sensing pathway, and antigen processing and presentation signaling pathways. The expression levels of genes enriched in these signaling pathways indicate that lower doses of boron could achieve better effects on promoting immune response in the duodenum. These effects on the immune system appear to be mediated via altering the expression patterns of genes involved in the related signaling pathways in a dose-dependent pattern. These data provide more insights into the molecular mechanisms of immune regulation in rats in response to dietary boron treatment.

FgSfl1 and Its Conserved PKA Phosphorylation Sites Are Important for Conidiation, Sexual Reproduction, and Pathogenesis in Fusarium graminearum.

The fungal plant pathogen, Fusarium graminearum, contains two genes, FgCPK1 and FgCPK2, encoding the catalytic subunits of cAMP-dependent protein kinase A. FgCPK1 and FgCPK2 are responsible for most of the PKA activities and have overlapping functions in various cellular processes in F. graminearum. The cpk1 cpk2 double mutant was significantly reduced in growth, rarely produced conidia, and was non-pathogenic. In this study, we found that the cpk1 cpk2 double mutant was unstable and produced fast-growing spontaneous sectors that were defective in plant infection. All spontaneous suppressor strains had mutations in FgSFL1, a transcription factor gene orthologous to SFL1 in yeast. Thirteen suppressor strains had non-sense mutations at Q501, three suppressor strains had frameshift mutations at W198, and five suppressor strains had mutations in the HSF binding domain of FgSfl1. Only one suppressor strain had both a non-synonymous mutation at H225 and a non-sense mutation at R490. We generated the SFL1 deletion mutant and found that it produced less than 2% of conidia than that of the wild-type strain PH-1. The sfl1 mutant was significantly reduced in the number of perithecia on carrot agar plates at 7 days post-fertilization (dpf). When incubated for more than 12 days, ascospore cirrhi were observed on the sfl1 mutant perithecia. The infection ability of the sfl1 deletion mutant was also obviously defective. Furthermore, we found that in addition to the S223 and S559 phosphorylation sites, FgSFL1 had another predicted phosphorylation site: T452. Interestingly, the S223 phosphorylation site was responsible for sexual reproduction, and the T452 phosphorylation site was responsible for growth and sexual reproduction. Only the S559 phosphorylation site was found to play an important role in conidiation, sexual reproduction, and infection. Overall, our results indicate that FgSFL1 and its conserved PKA phosphorylation sites are important for vegetative growth, conidiation, sexual reproduction, and pathogenesis in F. graminearum.

Effect of water flow on the release flux of dense non-aqueous phase liquids from the riverbed-take dichloromethane as an example.

This research is intended to study the effect of water flow on the release flux of DNAPLs, which have been deposited on the riverbed surface after sudden water pollution accidents. Those contaminants will slowly diffuse from the riverbed into the overlying water body through hydrodynamic action, causing ongoing and serious water pollution. By taking dichloromethane as a typical contaminant, the release form under different hydrodynamic conditions was observed through flume experiments, and the response relationship between the release flux and hydrodynamic factors was analyzed, with an emphasis on parameterizing the release rate. The results suggested that stronger water disturbance significantly enhanced the release of contaminants. And the relationship between the release flux and hydrodynamic factors generally followed an exponential distribution (R2 > 0.94). Besides, the mathematical relationships between the release flux and the average flow velocity, shear force and turbulent intensity were established as follows: F=183.63×e0.332u-, F=617.78×e22.292τ and F=119.03×e2.127TKE. Thus, this study has offered a solution to solve the source term quantification problem of the differential equation of convective diffusion, which can provide the basis for further developing the mathematical models of this kind of pollutants.

Effect of Boron on Microstructure, Immune Function, Expression of Tight Junction Protein, Cell Proliferation and Apoptosis of Duodenum in Rats.

Boron is an essential trace element for animals. Appropriate boron supplementation can produce beneficial effects on the animal body, while a high dose of boron has adverse and even toxic effects. Our aim was to investigate the impact of different doses of boron on the microstructure of duodenum in rats, expression of secretory immunoglobulin A (SIgA) and tight junction protein, cell proliferation and apoptosis. Eighty newly weaned clean Sprague-Dawley (SD) rats were given distilled water supplemented with 0, 10, 20, 40, 80, 160, 320, and 640 mg/L of boron for 60 days. We found that supplementation of 40 and 80 mg/L boron could increase the height of duodenal villi and the crypt depth, the number of intraepithelial lymphocytes (IELs) and goblet cells, the expression of SIgA, Zonula occludens-1 (ZO-1) and occludin, and proliferating cell nuclear antigen (PCNA) in duodenum of rats; decrease expression of Caspase-3 mRNA and the number of Caspase-3-positive cells, but supplementation of 320 and 640 mg/L boron could have the opposite effect in these indexes. The results showed that supplemented with 40 and 80 mg/L of boron could improve the structure and function of duodenum, while supplemented with 320-640 mg/L had a significant inhibitory effect.

Genetically increased circulating 25(OH)D level reduces the risk of type 2 diabetes in subjects with deficiency of vitamin D: A large-scale Mendelian randomization study.

ABSTRACT: Observational studies have reported that Vitamin D deficiency and the risk type 2 diabetes are associated, but the causation is unclear. Mendelian randomization (MR) involving genetic variants as instrument variables (IVs) overcomes the reverse-casualty and unmeasured confounding. However, with limited sample size and IVs, previous MR studies showed inconsistent results. Leveraging by a largely increased sample size for both stages, we aim to provide an updated and precise estimate for the causality between Vitamin D and type 2 diabetes.A 2-sample multi-IVs MR was performed. IVs for circulating 25-hydroxyvitamin D (25(OH)D) were obtained from a genome-wide association study from UK biobank involving 329,247 subjects of European ancestry. The causal effect of 25(OH)D and type 2 diabetes was estimated using traditional inverse variance weighting and MR pleiotropy residual sum and outlier (MR-PRESSO) framework which provides a robust estimate by systematically filtering out IVs identified with potential pleiotropy effects.A higher genetically instrumented 25(OH)D was causally linked to reduced risk of type 2 diabetes risk by MR-PRESSO [odds ratio (OR) per standard deviation (SD) = 0.950, 95% confidence interval (CI) = 0.913-0.988, P = .010] after removing 13 (13/193) invalid IVs. In addition, we confirmed the causal role Vitamin D using 2 synthesis-related single-nucleotide polymorphisms (SNPs) which are consistent with previous MR studies [OR per SD = 0.894, 95% CI = 0.816-0.979, P = .016].With a largely improved sample size, our results confirmed that genetically increased 25(OH)D concentration reduced the risk of type 2 diabetes and provided a more precise estimate for the effect size. The updated result empowers the role of Vitamin D and provides nontrivial evidence for interventional studies.

GPR30 mediated effects of boron on rat spleen lymphocyte proliferation, apoptosis, and immune function.

Supplementing different quantities of boron can significantly affect immune function in rat spleen, but the mechanism of action behind this effect remains unclear. Our purpose was to study the involvement of the estrogen membrane receptor GPR30 in the effect of boron on the proliferation, apoptosis, and immune function of rat spleen lymphocytes. Results showed that the addition of 0.4 mmol/L boron had a beneficial effect on the immune function and proliferation of spleen lymphocytes, but the addition of 40 mmol/L boron had opposite effect. After using G-15 to selectively inhibit GPR30, the proportions of CD4+ and CD8+ T cells, the content of IL-2 and IFN-γ, and the expression of PCNA protein were significantly decreased, while lymphocyte apoptosis rate increased significantly (p < 0.05 or p < 0.01). After G-15 treatment, the addition of 0.4 mmol/L boron had no effects on T cell subsets, lymphocyte proliferation, PCNA protein expression, and IgG and cytokine content (P > 0.05), while the addition of 40 mmol/L boron did not change the effects on lymphocyte subsets, proliferation and apoptosis. The results suggested that GPR30 mediates the effects of 0.4 mmol/L boron boron on the proliferation, apoptosis and immune function of spleen lymphocytes.

AVP(4-8) Improves Cognitive Behaviors and Hippocampal Synaptic Plasticity in the APP/PS1 Mouse Model of Alzheimer's Disease.

Memory deficits with aging are related to the neurodegeneration in the brain, including a reduction in arginine vasopressin (AVP) in the brain of patients with Alzheimer's disease (AD). AVP(4-8), different from its precursor AVP, plays memory enhancement roles in the CNS without peripheral side-effects. However, it is not clear whether AVP(4-8) can improve cognitive behaviors and synaptic plasticity in the APP/PS1 mouse model of AD. Here, we investigated for the first time the neuroprotective effects of AVP(4-8) on memory behaviors and in vivo long-term potentiation (LTP) in APP/PS1-AD mice. The results showed that: (1) APP/PS1-AD mice had lower spontaneous alternation in the Y-maze than wild-type (WT) mice, and this was significantly reversed by AVP(4-8); (2) the prolonged escape latency of APP/PS1-AD mice in the Morris water maze was significantly decreased by AVP(4-8), and the decreased swimming time in target quadrant recovered significantly after AVP(4-8) treatment; (3) in vivo hippocampal LTP induced by high-frequency stimulation had a significant deficit in the AD mice, and this was partly rescued by AVP(4-8); (4) AVP(4-8) significantly up-regulated the expression levels of postsynaptic density 95 (PSD95) and nerve growth factor (NGF) in the hippocampus of AD mice. These results reveal the beneficial effects of AVP(4-8) in APP/PS1-AD mice, showing that the intranasal administration of AVP(4-8) effectively improved the working memory and long-term spatial memory of APP/PS1-AD mice, which may be associated with the elevation of PSD95 and NGF levels in the brain and the maintenance of hippocampal synaptic plasticity.

Xestospongin C, a Reversible IP3 Receptor Antagonist, Alleviates the Cognitive and Pathological Impairments in APP/PS1 Mice of Alzheimer's Disease.

Exaggerated Ca2+ signaling might be one of primary causes of neural dysfunction in Alzheimer's disease (AD). And the intracellular Ca2+ overload has been closely associated with amyloid-ß (Aß)-induced endoplasmic reticulum (ER) stress and memory impairments in AD. Here we showed for the first time the neuroprotective effects of Xestospongin C (XeC), a reversible IP3 receptor antagonist, on the cognitive behaviors and pathology of APP/PS1 AD mice. Male APP/PS1-AD mice (n = 20) were injected intracerebroventricularly with XeC (3µmol) via Alzet osmotic pumps for four weeks, followed by cognition tests, Aß plaque examination, and ER stress-related protein measurement. The results showed that XeC pretreatment significantly improved the cognitive behavior of APP/PS1-AD mice, raising the spontaneous alteration accuracy in Y maze, decreasing the escape latency and increasing the target quadrant swimming time in Morris water maze; XeC pretreatment also reduced the number of Aß plaques and the overexpression of ER stress proteins 78 kDa glucose-regulated protein (GRP-78), caspase-12, and CAAT/enhancer-binding protein (C/EBP) homologous protein (CHOP) in the hippocampus of APP/PS1 mice. In addition, in vitro experiments showed that XeC effectively ameliorated Aß1 - 42-induced early neuronal apoptosis and intracellular Ca2+ overload in the primary hippocampal neurons. Taken together, IP3R-mediated Ca2+ disorder plays a key role in the cognitive deficits and pathological damages in AD mice. By targeting the IP3 R, XeC might be considered as a novel therapeutic strategy in AD.

Stage-specific functional relationships between Tub1 and Tub2 beta-tubulins in the wheat scab fungus Fusarium graminearum.

The filamentous ascomycete Fusarium graminearum contains two ß-tubulin genes TUB1 and TUB2 that differ in functions during vegetative growth and sexual reproduction. To further characterize their functional relationship, in this study we determined the co-localization of Tub1 and Tub2 and assayed their expression levels in different mutants and roles in DON production. Tub1 co-localized with Tub2 to the same regions of microtubules in conidia, hyphae, and ascospores. Whereas deletion of TUB1 had no obvious effect on the transcription of TUB2 and two α-tubulin genes (TUB4 and TUB5), the tub2 mutant was up-regulated in TUB1 transcription. To assay their protein expression levels, polyclonal antibodies that could specifically detect four α- and ß-tubulin proteins were generated. Western blot analyses showed that the abundance of Tub1 proteins was increased in tub2 but reduced in tub4 and tub5 mutants. Interestingly, protein expression of Tub4 and Tub5 was decreased in the tub1 mutant in comparison with the wild type, despite a lack of obvious changes in their transcription. In contrast, deletion of TUB2 had no effect on translation of TUB4 and TUB5. Ectopic expression of Tub2-mCherry partially recovered the growth defect of the tub1 mutant but did not rescue its defect in sexual reproduction. Expression of Tub1-GFP in the tub2 mutant also partially rescued its defects in vegetative growth, suggesting that disturbance in the balance of α- and ß-tubulins contributes to mutant defects. The tub2 but not tub1 mutant was almost blocked in DON biosynthesis. Expression of TRI genes, toxisome formation, and DON-related cellular differentiation were significantly reduced in the tub2 mutant. Overall, our results showed that Tub1 and Tub2 share similar subcellular localization and have overlapping functions during vegetative growth but they differ in functions in DON production and ascosporogenesis in F. graminearum.

Adoption and Use of Mobile Learning in Continuing Professional Development by Health and Human Services Professionals.

INTRODUCTION: Health and human services professionals are increasingly using mobile devices to support clinical decision-making and evidence-based practice. However, research on self-directed learning in an era of growing digital technology utilization is underdeveloped. This study explored the adoption and use of mobile learning as a continuing professional development (CPD) activity. METHODS: A mixed-methods case study using semistructured interviews and a web-based questionnaire was conducted with health and human services professionals in Newfoundland and Labrador, Canada. RESULTS: Respondents reported using a smartphone (53.8%), tablets (50.4%), YouTube (43.0%), and mobile apps (35.8%) for CPD. The highest-rated benefits of mobile learning included improved access to information (M = 3.51); potential for enhanced knowledge acquisition (M = 3.45); staying up to date (M = 3.44); and verifying information (M = 3.40). The greatest barriers included cost of some apps and resources (M = 3.07); websites/programs not functional on mobile devices (M = 2.84); workplace barriers preventing access to digital resources (M = 2.82); and social media use linked to negative perceptions of professionalism (M = 2.65). Interview respondents described the flexibility and convenience of mobile learning, the level of autonomy it offered, and the advantages of learning on their own time. Technical issues, particularly for rural and remote practitioners, and digital professionalism also emerged as potential barriers. DISCUSSION: A systems model organizes the factors influencing the adoption and use of mobile devices and resources to support "just-in-time" learning. Addressing policies, practices, and regulations that enable or inhibit adoption of mobile learning for CPD may foster enhanced use to support better clinical decision-making, improved accuracy, and greater patient safety.

Water Environmental Capacity Calculated Based on Point and Non-Point Source Pollution Emission Intensity under Water Quality Assurance Rates in a Tidal River Network Area.

A mathematical model for simulating hydrodynamics and pollutants migration in a tidal river network was constructed, which takes the temporal and spatial distribution of rainfall runoff and non-point pollutants into consideration. Under the design hydrologic conditions of a typical hydrological year, the daily concentration change process for the control section is obtained. Aiming at the uncertainty of hydrology and water quality parameters such as flow direction, flow rate and concentration change in tidal river network area, a statistical analysis method is used to obtain the maximum allowable concentration of pollutants in the control section under the condition of the water quality standard assurance rate of. Then, a formula for calculating the pollutions emission intensity of point and non-point sources is derived. The method was applied to the pollution source control in a typical region like Taihu in China.

Determination of the absolute configurations of the stereogenic centers of ustilaginoidins by studying the biosynthetic monomers from a gene knockout mutant of Villosiclava virens.

Ustilaginoidins are a kind of mycotoxins with 9,9'-linked bis-naphtho-γ-pyrones structures produced by the rice false smut pathogen Villosiclava virens. These metabolites displayed a wide range of bioactivities, such as teratogenic, cytotoxic, phytotoxic, and antibacterial activities. So far 26 ustilaginoidins have been isolated from V. virens, among which 18 compounds contained stereogenic center(s), however, most of them were unknown for the absolute configurations, except that of ustilaginoidin D. In this study, the absolute structures of these ustilaginoidins were constructed for the first time by analysis of the biosynthetic monomers obtained from a gene knockout mutant (ΔUV_2091) of V. virens. The gene UV_2091 was predicted to encode an enzyme that dimerized the monomeric naphtho-γ-pyrones in V. virens. Knockout of this gene led to the accumulation of three monomers, namely hemiustilaginoidin F (1), epihemiustilaginoidin D (2), and hemiustilaginoidin D (3), but the production of ustilaginoidins was completely blocked. The structures of the monomers were deduced by spectroscopic analysis, in combination with TDDFT ECD calculations for determining the absolute configurations. These compounds were tested for their phytotoxic, cytotoxic, antibacterial, and antifungal activities. Compounds 1 and 3 showed inhibition against the radicle and plumule elongation of rice and lettuce seeds at the tested concentrations. Compound 1 was active against the tested five human cancer cells, with half maximal inhibitory concentrations (IC50s) of 13.2~37.3 µM. Compounds 1~3 inhibited the growth of the tested pathogenic bacteria with minimum inhibitory concentrations of 8~32 µg/mL, while compound 3 exhibited antifungal activity against Magnaporthe oryzae (IC50, 5.21 µg/mL). A comparison of these data with those of the ustilaginoidins provided insights into the structure-bioactivity relationships.

Targeted Deletion of the USTA and UvSLT2 Genes Efficiently in Ustilaginoidea virens With the CRISPR-Cas9 System.

Ustilaginoidea virens is the causal agent of rice false smut, one of the major fungal diseases of rice. However, there are only limited molecular studies with this important pathogen due to the lack of efficient approaches for generating targeted gene disruption mutants. In this study, we used the CRISPR-Cas9 system to efficiently generate mutants deleted of the USTA ustiloxin and UvSLT2 MAP kinase genes. Three gRNA spacers of USTA, UA01, UA13, and UA21, were expressed with the RNAP III promoter of Gln-tRNA. For all of them, the homologous gene replacement frequency was higher when the Cas9 and gRNA constructs were transformed into U. virens on the same vector than sequentially. UA01, the spacer with the highest on-target score, had the highest knockout frequency of 90%, which was over 200 times higher than that of Agrobacterium tumefaciens-mediated transformation (ATMT) for generating ustA mutants. None of these USTA spacers had predicted off-targets with 1 or 2-nt variations. For predicted off-targets with 3 or 4-nt variations, mutations were not detected in 10 ustA mutants generated with spacer UA13 or UA21, indicating a relatively low frequency of off-target mutations in U. virens. For UvSLT2, the homologous gene replacement frequency was 50% with CRISPR-Cas9, which also was significantly higher than that of ATMT. Whereas ustA mutants had no detectable phenotypes, Uvslt2 mutants were slightly reduced in growth rate and reduced over 70% in conidiation. Deletion of UvSLT2 also increased sensitivity to cell wall stresses but tolerance to hyperosmotic or oxidative stresses. Taken together, our results showed that the CRISPR-Cas9 system can be used as an efficient gene replacement or editing approach in U. virens and the UvSlt2 MAP kinase pathway has a conserved role in cell wall integrity.

The PKR regulatory subunit of protein kinase A (PKA) is involved in the regulation of growth, sexual and asexual development, and pathogenesis in Fusarium graminearum.

Fusarium graminearum is a causal agent of wheat scab disease and a producer of deoxynivalenol (DON) mycotoxins. Treatment with exogenous cyclic adenosine monophosphate (cAMP) increases its DON production. In this study, to better understand the role of the cAMP-protein kinase A (PKA) pathway in F. graminearum, we functionally characterized the PKR gene encoding the regulatory subunit of PKA. Mutants deleted of PKR were viable, but showed severe defects in growth, conidiation and plant infection. The pkr mutant produced compact colonies with shorter aerial hyphae with an increased number of nuclei in hyphal compartments. Mutant conidia were morphologically abnormal and appeared to undergo rapid autophagy-related cell death. The pkr mutant showed blocked perithecium development, but increased DON production. It had a disease index of less than unity and failed to spread to neighbouring spikelets. The mutant was unstable and spontaneous suppressors with a faster growth rate were often produced on older cultures. A total of 67 suppressor strains that grew faster than the original mutant were isolated. Three showed a similar growth rate and colony morphology to the wild-type, but were still defective in conidiation. Sequencing analysis with 18 candidate PKA-related genes in three representative suppressor strains identified mutations only in the CPK1 catalytic subunit gene. Further characterization showed that 10 of the other 64 suppressor strains also had mutations in CPK1. Overall, these results showed that PKR is important for the regulation of hyphal growth, reproduction, pathogenesis and DON production, and mutations in CPK1 are partially suppressive to the deletion of PKR in F. graminearum.

The cyclase-associated protein FgCap1 has both protein kinase A-dependent and -independent functions during deoxynivalenol production and plant infection in Fusarium graminearum.

Fusarium graminearum is a causal agent of wheat scab and a producer of the trichothecene mycotoxin deoxynivalenol (DON). The expression of trichothecene biosynthesis (TRI) genes and DON production are mainly regulated by the cyclic adenosine monophosphate-protein kinase A (cAMP-PKA) pathway and two pathway-specific transcription factors (TRI6 and TRI10). Interestingly, deletion mutants of TRI6 show reduced expression of several components of cAMP signalling, including the FgCAP1 adenylate-binding protein gene that has not been functionally characterized in F. graminearum. In this study, we show that FgCap1 interacts with Fac1 adenylate cyclase and that deletion of FgCAP1 reduces the intracellular cAMP level and PKA activity. The Fgcap1 deletion mutant is defective in vegetative growth, conidiogenesis and plant infection. It also shows significantly reduced DON production and TRI gene expression, which can be suppressed by exogenous cAMP, indicating a PKA-dependent regulation of DON biosynthesis by FgCap1. The wild-type, but not tri6 mutant, shows increased levels of intracellular cAMP and FgCAP1 expression under DON-producing conditions. Furthermore, the promoter of FgCAP1 contains one putative Tri6-binding site that is important for its function during DON biosynthesis, but is dispensable for hyphal growth, conidiogenesis and pathogenesis. In addition, FgCap1 shows an actin-like localization to the cortical patches at the apical region of hyphal tips. Phosphorylation of FgCap1 at S353 was identified by phosphoproteomics analysis. The S353A mutation in FgCAP1 has no effect on its functions during vegetative growth, conidiation and DON production. However, expression of the FgCAP1S353A allele fails to complement the defects of the Fgcap1 mutant in plant infection, indicating the importance of the phosphorylation of FgCap1 at S353 during pathogenesis. Taken together, our results suggest that FgCAP1 is involved in the regulation of DON production via cAMP signalling and subjected to a feedback regulation by TRI6, but the phosphorylation of FgCap1 at S353 is probably unrelated to the cAMP-PKA pathway because the S353A mutation only affects plant infection.

MoCDC14 is important for septation during conidiation and appressorium formation in Magnaporthe oryzae.

As a typical foliar pathogen, appressorium formation and penetration are critical steps in the infection cycle of Magnaporthe oryzae. Because appressorium formation and penetration are closely co-regulated with the cell cycle, and Cdc14 phosphatases have an antagonistic relationship with cyclin-dependent kinases (CDKs) on proteins related to mitotic exit and cytokinesis, in this study, we functionally characterized the MoCDC14 gene in M. oryzae. The Mocdc14 deletion mutant showed significantly reduced growth rate and conidiation. It was also defective in septum formation and nuclear distribution. Septation was irregular in Mocdc14 hyphae and hyphal compartments became multi-nucleate. Mutant conidia often showed incomplete septa or lacked any septum. During appressorium formation, the septum delimiting appressoria from the rest of the germ tubes was often formed far away from the neck of the appressoria or not formed at all. Unlike the wild-type, some mutant appressoria had more than one nucleus at 24 h. In addition to appressoria, melanization occurred on parts of the germ tubes and conidia, depending on the irregular position of the appressorium-delimiting septum. The Mocdc14 mutant was also defective in glycogen degradation during appressorium formation and appressorial penetration of intact plant cells. Similar defects in septum formation, melanization and penetration were observed with appressorium-like structures formed at hyphal tips in the Mocdc14 mutant. Often a long fragment of mutant hyphae was melanized, together with the apical appressorium-like structures. These results indicate that MoCDC14 plays a critical role in septation, nuclear distribution and pathogenesis in M. oryzae, and correct septum formation during conidiogenesis and appressorium formation requires the MoCdc14 phosphatase.