Genome-Wide Analysis of AGC Kinases Reveals that MoFpk1 Is Required for Development, Lipid Metabolism, and Autophagy in Hyperosmotic Stress of the Rice Blast Fungus Magnaporthe oryzae.

During eukaryotic evolution, the TOR-AGC kinase signaling module is involved in the coordinated regulation of cell growth and survival. However, the AGC kinases in plant-pathogenic fungi remain poorly understood. In this study, we have identified 20 members of the AGC family of protein kinases. Evolutionary and biological studies have revealed that AGC kinases are highly conserved and involved in the growth (8 genes), conidiation (13 genes), conidial germination (9 genes), appressorium formation (9 genes), and pathogenicity (5 genes) of Magnaporthe oryzae, in which a subfamily protein of the AGC kinases, MoFpk1, the activator of flippase, specifically exhibited diverse roles. Two kinase sites were screened and found to be critical for MoFpk1: 230K and 326D. Moreover, MoFpk1 is involved in cell wall integrity through the negative regulation of MoMps1 phosphorylation. The deletion of MoFpk1 resulted in defective phosphatidylacetamide (PE) and phosphatidylserine (PS) turnover and a series of lipid metabolism disorders. Under hyperosmotic stress, since the ΔMofpk1 mutant is unable to maintain membrane asymmetry, MoYpk1 phosphorylation and MoTor activity were downregulated, thus enhancing autophagy. Our results provide insights into the evolutionary and biological relationships of AGC kinases and new insight into plasma membrane (PM) homeostasis, i.e., responses to membrane stress and autophagy through lipid asymmetry maintenance. IMPORTANCE Our identification and analysis of evolutionary and biological relationships provide us with an unprecedented high-resolution view of the flexible and conserved roles of the AGC family in the topmost fungal pathogens that infect rice, wheat, barley, and millet. Guided by these insights, an AGC member, MoFpk1, was found to be indispensable for M. oryzae development. Our study defined a novel mechanism of plasma membrane homeostasis, i.e., adaptation to stress through the asymmetric distribution of phospholipids. Furthermore, defects in the asymmetric distribution of phospholipids in the membrane enhanced autophagy under hyperosmotic stress. This study provides a new mechanism for the internal linkage between lipid metabolism and autophagy, which may help new fungicide target development for controlling this devastating disease.

De Novo Purine Nucleotide Biosynthesis Pathway Is Required for Development and Pathogenicity in Magnaporthe oryzae.

Purine nucleotides are indispensable compounds for many organisms and participate in basic vital activities such as heredity, development, and growth. Blocking of purine nucleotide biosynthesis may inhibit proliferation and development and is commonly used in cancer therapy. However, the function of the purine nucleotide biosynthesis pathway in the pathogenic fungus Magnaporthe oryzae is not clear. In this study, we focused on the de novo purine biosynthesis (DNPB) pathway and characterized MoAde8, a phosphoribosylglycinamide formyltransferase, catalyzing the third step of the DNPB pathway in M. oryzae. MoAde8 was knocked out, and the mutant (∆Moade8) exhibited purine auxotroph, defects in aerial hyphal growth, conidiation, and pathogenicity, and was more sensitive to hyperosmotic stress and oxidative stress. Moreover, ∆Moade8 caused decreased activity of MoTor kinase due to blocked purine nucleotide synthesis. The autophagy level was also impaired in ∆Moade8. Additionally, MoAde5, 7, 6, and 12, which are involved in de novo purine nucleotide biosynthesis, were also analyzed, and the mutants showed defects similar to the defects of ∆Moade8. In summary, de novo purine nucleotide biosynthesis is essential for conidiation, development, and pathogenicity in M. oryzae.

A Putative D-Arabinono-1,4-lactone Oxidase, MoAlo1, Is Required for Fungal Growth, Conidiogenesis, and Pathogenicity in Magnaporthe oryzae.

Magnaporthe oryzae is the causal agent of rice blast outbreaks. L-ascorbic acid (ASC) is a famous antioxidant found in nature. However, while ASC is rare or absent in fungi, a five-carbon analog, D-erythroascorbic acid (EASC), seems to appear to be a substitute for ASC. Although the antioxidant function of ASC has been widely described, the specific properties and physiological functions of EASC remain poorly understood. In this study, we identified a D-arabinono-1,4-lactone oxidase (ALO) domain-containing protein, MoAlo1, and found that MoAlo1 was localized to mitochondria. Disruption of MoALO1 (ΔMoalo1) exhibited defects in vegetative growth as well as conidiogenesis. The ΔMoalo1 mutant was found to be more sensitive to exogenous H2O2. Additionally, the pathogenicity of conidia in the ΔMoalo1 null mutant was reduced deeply in rice, and defective penetration of appressorium-like structures (ALS) formed by the hyphal tips was also observed in the ΔMoalo1 null mutant. When exogenous EASC was added to the conidial suspension, the defective pathogenicity of the ΔMoalo1 mutant was restored. Collectively, MoAlo1 is essential for growth, conidiogenesis, and pathogenicity in M. oryzae.

Endosomal sorting complexes required for transport-0 (ESCRT-0) are essential for fungal development, pathogenicity, autophagy and ER-phagy in Magnaporthe oryzae.

Magnaporthe oryzae is an important plant pathogen that causes rice blast. Hse1 and Vps27 are components of ESCRT-0 involved in the multivesicular body (MVB) sorting pathway and biogenesis. To date, the biological functions of ESCRT-0 in M. oryzae have not been determined. In this study, we identified and characterized Hse1 and Vps27 in M. oryzae. Disruption of MoHse1 and MoVps27 caused pleiotropic defects in growth, conidiation, sexual development and pathogenicity, thereby resulting in loss of virulence in rice and barley leaves. Disruption of MoHse1 and MoVps27 triggered increased lipidation of MoAtg8 and degradation of GFP-MoAtg8, indicating that ESCRT-0 is involved in the regulation of autophagy. ESCRT-0 was determined to interact with coat protein complex II (COPII), a regulator functioning in homeostasis of the endoplasmic reticulum (ER homeostasis), and disruption of MoHse1 and MoVps27 also blocked activation of the unfolded protein response (UPR) and autophagy of the endoplasmic reticulum (ER-phagy). Overall, our results indicate that ESCRT-0 plays critical roles in regulating fungal development, virulence, autophagy and ER-phagy in M. oryzae.

Afterload-related cardiac performance predicts prognosis in critical ill patients with sepsis: A prospective observational pilot study.

ABSTRACT: To investigate the usefulness of afterload-related cardiac performance (ACP) for assessing cardiac impairment and predicting prognosis in septic patients.Adult patients with sepsis in the intensive care unit were included. Cardiac output, cardiac index, cardiac power index, and ACP were calculated at the time of admission (D0) and 48-72 h after admission (D3). They were correlated with Acute Physiology and Chronic Health Evaluation II and sequential organ failure assessment scores, then the prognostic values were analyzed.A total of 41 patients with sepsis were selected. ACP showed a stronger negative correlation with Acute Physiology and Chronic Health Evaluation II and sequential organ failure assessment scores than cardiac output, cardiac index, and cardiac power index. ACP predicted 28-day mortality with an area under the curve of 0.775 and 0.976 on D0 and D3, respectively. In addition, most non-survivors had emergent cardiac impairment (ACP ≤ 80%) on D0, and cardiac function was deteriorated on D3. Survival analysis showed that the patients with a decreased ACP from D0 to D3 had the highest mortality. The decrease of ACP on D3 was an independent risk factor for mortality (hazard ratio, 11.89; P = .0028).ACP can be used to assess the severity of cardiac impairment in sepsis. Continued decline of ACP during the first 3 days strongly suggests a poor prognosis.

Isolation and Functional Analysis of Effector Proteins of Magnaporthe oryzae.

In nature, plants have evolved a myriad of preformed and induced defenses to protect themselves from microbes. Upon microbial infection, the recognition of the microbe-associated molecular patterns (MAMPs) by the pattern recognition receptors (PRRs) triggers the first stage of defense response (Dodds and Rathjen, Nat Rev Genet 11:539-548, 2010). However, in order to develop microbial delivery, effectors target PRRs for deregulating immune responses and facilitating host colonization (Thomma et al., Plant Cell 23:4-15, 2011). Here, we contribute a protocol for the screening system of Magnaporthe oryzae effectors and construct a fluorescent system to trace secretory proteins in the sheath infection samples. Using the tobacco rattle virus (TRV) system, the proteins including LysM, Chitin, Cutinase, and CFEM domains were selected and divided into two kinds according to the results of cell death induced or inhibited test in Nicotiana benthamiana. Then, candidate effectors can be deleted or overexpressed in M. oryzae. The barley or rice infection with M. oryzae, rice leaf sheath inoculation, and subcellular localization during infection can be performed to explore the functions of these effectors.

[Effects of continuous veno-venous hemofiltration on the hemodynamics of sheep with endotoxic shock].

OBJECTIVE: To study the influence of continuous veno-venous hemofiltration (CVVH) on the hemodynamics and the prognosis of sheep with endotoxic shock. METHODS: Endotoxic shock was induced by intravenous infusion of lipopolysaccharide, L-2880, to sheep, and the animals were randomly divided to control group (NHF, endotoxin only) or treatment group (HF, endotoxin+CVVH). In the latter groups a zero-balanced CVVH was instituted with an ultrafiltration rate of 40 ml x kg(-1) x h(-1) and a blood flow rate of 80 ml/min. The hemodynamic parameters were monitored. RESULTS: After the administration of endotoxin, the mean arterial pressure (MAP) was decreased significantly, while mean pulmonary artery pressure (MPAP) and cardiac index (CI) increased significantly in both groups (P<0.05 or P<0.01). With CVVH, MAP and stroke volume (SV) were increased significantly, and were significantly higher than that of NHF group (P<0.05 or P<0.01). CI declined progressively in NHF group and significantly lower than that of HF group, in which there was no significant change(P<0.05). No significant difference in MPAP was found between two groups (P>0.05). Moreover, plasma lactate levels were lower in NHF group than in HF group (P<0.05). After 5 hours, the sheep in HF group all survived, and 2 sheep of NHF group died. CONCLUSION: Intravenous infusion of endotoxin could produce endotoxic shock in sheep. CVVH was shown to have a beneficial effect on the hemodynamics, and it gave a better prognosis in endotoxic, indicating that it might be important method to treat endotoxic shock.