Xiao‐Hong Liu

ABSTRACT We isolated an Mg ATG1 gene encoding a serine/threonine protein kinase from the rice blast fungus Magnaporthe grisea . In the ΔMg atg1 mutant, in which the Mg ATG1 gene had been deleted, autophagy was blocked; the mutant also showed fewer lipid droplets in its conidia, lower turgor pressure of the appressorium, and such defects in morphogenesis as delayed initiation and slower germination of conidia. As a result of lower turgor pressure of the appressorium, the ΔMg atg1 mutant lost its ability to penetrate and infect the two host plants, namely, rice and barley. However, normal values of the parameters and infective abilities were restored on reintroducing an intact copy of the Mg ATG1 gene into the mutant. Autophagy is thus necessary for turnover of organic matter during the formation of conidia and appressoria and for normal development and pathogenicity in M. grisea .

The interaction between pathogens and their host plants is a ubiquitous process. Some plant fungal pathogens can form a specific infection structure, such as an appressorium, which is formed by the accumulation of a large amount of glycerin and thereby the creation of an extremely high intracellular turgor pressure, which allows the penetration peg of the appressorium to puncture the leaf cuticle of the host. Previous studies have shown that autophagy energizes the accumulation of pressure by appressoria, which induces its pathogenesis. Similar to other eukaryotic organisms, autophagy processes are highly conserved pathways that play important roles in filamentous fungal pathogenicity. This review aims to demonstrate how the autophagy process affects the pathogenicity of plant pathogens.

Chromosome 17 is unusual among the human chromosomes in many respects. It is the largest human autosome with orthology to only a single mouse chromosome, mapping entirely to the distal half of mouse chromosome 11. Chromosome 17 is rich in protein-coding genes, having the second highest gene density in the genome. It is also enriched in segmental duplications, ranking third in density among the autosomes. Here we report a finished sequence for human chromosome 17, as well as a structural comparison with the finished sequence for mouse chromosome 11, the first finished mouse chromosome. Comparison of the orthologous regions reveals striking differences. In contrast to the typical pattern seen in mammalian evolution, the human sequence has undergone extensive intrachromosomal rearrangement, whereas the mouse sequence has been remarkably stable. Moreover, although the human sequence has a high density of segmental duplication, the mouse sequence has a very low density. Notably, these segmental duplications correspond closely to the sites of structural rearrangement, demonstrating a link between duplication and rearrangement. Examination of the main classes of duplicated segments provides insight into the dynamics underlying expansion of chromosome-specific, low-copy repeats in the human genome.

There was a large scale outbreak of the highly pathogenic porcine reproductive and respiratory syndrome (PRRS) in China and Vietnam during 2006 and 2007 that resulted in unusually high morbidity and mortality among pigs of all ages. The mechanisms underlying the molecular pathogenesis of the highly virulent PRRS virus (H-PRRSV) remains unknown. Therefore, the relationship between pulmonary gene expression profiles after H-PRRSV infection and infection pathology were analyzed in this study using high-throughput deep sequencing and histopathology.H-PRRSV infection resulted in severe lung pathology. The results indicate that aberrant host innate immune responses to H-PRRSV and induction of an anti-apoptotic state could be responsible for the aggressive replication and dissemination of H-PRRSV. Prolific rapid replication of H-PRRSV could have triggered aberrant sustained expression of pro-inflammatory cytokines and chemokines leading to a markedly robust inflammatory response compounded by significant cell death and increased oxidative damage. The end result was severe tissue damage and high pathogenicity.The systems analysis utilized in this study provides a comprehensive basis for better understanding the pathogenesis of H-PRRSV. Furthermore, it allows the genetic components involved in H-PRRSV resistance/susceptibility in swine populations to be identified.

The development and pathogenicity of the fungus Magnaporthe oryzae, the causal agent of destructive rice blast disease, require it to perceive external environmental signals. Opy2, an overproduction-induced pheromone-resistant protein 2, is a crucial protein for sensing external signals in Saccharomyces cerevisiae. However, the biological functions of the homologue of Opy2 in M. oryzae are unclear. In this study, we identified that MoOPY2 is involved in fungal development, pathogenicity, and autophagy in M. oryzae. Deletion of MoOPY2 resulted in pleiotropic defects in hyphal growth, conidiation, germ tube extension, appressorium formation, appressorium turgor generation, and invasive growth, therefore leading to attenuated pathogenicity. Furthermore, MoOpy2 participates in the Osm1 MAPK pathway and the Mps1 MAPK pathway by interacting with the adaptor protein Mst50. The interaction sites of Mst50 and MoOpy2 colocalized with the autophagic marker protein MoAtg8 in the preautophagosomal structure sites (PAS). Notably, the ΔMoopy2 mutant caused cumulative MoAtg8 lipidation and rapid GFP-MoAtg8 degradation in response to nitrogen starvation, showing that MoOpy2 is involved in the negative regulation of autophagy activity. Taken together, our study revealed that MoOpy2 of M. oryzae plays an essential role in the orchestration of fungal development, appressorium penetration, autophagy and pathogenesis.

Autophagy is a vacuolar/lysosomal cytoplasmic recycling system in eukaryotic cells. ScATG9 is indispensable for autophagy in Saccharomyces cerevisiae. Here, we deleted MgATG9, the orthologue of ScATG9, via targeted gene replacement in the phytopathogenic filamentous fungus Magnaporthe oryzae, and then analyzed the cellular distribution pattern of EGFP-MgAtg9 in the Mgatg9Delta mutant. We detected an expression profile of multiple green dots in the conidial cell inoculated in rich media and in the appressoria differentiated from the conidia in H(2)O. Concurrent with the punctation, we found some fluorescent signals localized on the central vacuole of the submerged hyphae from the conidia cultured in rich media. Next, we introduced DsRed2-MgAtg8 into the Mgatg9Delta mutant expressing EGFP-MgAtg9 and observed partial overlap at multiple sites in the conidial cell, reminiscent of that in the mammalian system. Our findings further led to the postulation that the multiple sites where the two fusions colocalized tend to merge as a central structure in the conidial cell. Finally, we tested the expression of EGFP-MgAtg9 in null mutants of MgATG1, 2, 13 and 18, respectively. We speculate that MgAtg1, 2 and 18, but not MgAtg13, is required for MgAtg9 cycling through the multiple colocalization sites to its storage pools in the conidial cell of M. oryzae, and fusion of these colocalization sites into a central structure could be governed through other unidentified mechanisms.

The ubiquitin-proteasome system and the autophagy system are the two primary mechanisms used by eukaryotes to maintain protein homeostasis, and both are closely related to the pathogenicity of the rice blast fungus. In this research, we identified MoCand2 as an inhibitor of ubiquitination in Magnaporthe oryzae. Through this role, MoCand2 participates in the regulation of autophagy and pathogenicity. Specifically, we found that deletion of MoCand2 increased the ubiquitination level in M. oryzae, whereas overexpression of MoCand2 inhibited the accumulation of ubiquitinated proteins. Interaction analyses showed that MoCand2 is a subunit of Cullin-RING ligases (CRLs). It suppresses ubiquitination by blocking the assembly of CRLs and downregulating the expression of key CRL subunits. Further research indicated that MoCand2 regulates autophagy through ubiquitination. MoCand2 knockout led to over-ubiquitination and over-degradation of MoTor, and we confirmed that MoTor content was negatively correlated with autophagy level. In addition, MoCand2 knockout accelerated the K63 ubiquitination of MoAtg6 and strengthened the assembly and activity of the phosphatidylinositol-3-kinase class 3 complex, thus enhancing autophagy. Abnormal ubiquitination and autophagy in ΔMocand2 resulted in defects in growth, conidiation, stress resistance, and pathogenicity. Finally, sequence alignment and functional analyses in other phytopathogenic fungi confirmed the high conservation of fungal Cand2s. Our research thus reveals a novel mechanism by which ubiquitination regulates autophagy and pathogenicity in phytopathogenic fungi.

Rice blast is a devastating disease worldwide, threatening rice production and food security. The blast fungus Magnaporthe oryzae invades the host via the appressorium, a specialized pressure-generating structure that generates enormous turgor pressure to penetrate the host cuticle. However, owing to ongoing evolution of fungicide resistance, it is vitally important to identify new targets and fungicides. Here, we show that Trs85, a subunit of the transport protein particle III complex, is essential for appressorium-mediated infection in M. oryzae. We explain how Trs85 regulates autophagy through Ypt1 (a small guanosine triphosphatase protein) in M. oryzae. We then identify a key conserved amphipathic α helix within Trs85 that is associated with pathogenicity of M. oryzae. Through computer-aided screening, we identify a lead compound, SP-141, that affects autophagy and the Trs85-Ypt1 interaction. SP-141 demonstrates a substantial capacity to effectively inhibit infection caused by the rice blast fungus while also exhibiting wide-ranging potential as an antifungal agent with broad-spectrum activity. Taken together, our data show that Trs85 is a potential new target and that SP-141 has potential for the control of rice blast. Our findings thus provide a novel strategy that may help in the fight against rice blast.

Seronegative hepatitis--non-A, non-B, non-C, non-D, non-E hepatitis--is poorly characterized but strongly associated with serious complications. We collected 92 sera specimens from patients with non-A-E hepatitis in Chongqing, China between 1999 and 2007. Ten sera pools were screened by Solexa deep sequencing. We discovered a 3,780-bp contig present in all 10 pools that yielded BLASTx E scores of 7e-05-0.008 against parvoviruses. The complete sequence of the in silico-assembled 3,780-bp contig was confirmed by gene amplification of overlapping regions over almost the entire genome, and the virus was provisionally designated NIH-CQV. Further analysis revealed that the contig was composed of two major ORFs. By protein BLAST, ORF1 and ORF2 were most homologous to the replication-associated protein of bat circovirus and the capsid protein of porcine parvovirus, respectively. Phylogenetic analysis indicated that NIH-CQV is located at the interface of Parvoviridae and Circoviridae. Prevalence of NIH-CQV in patients was determined by quantitative PCR. Sixty-three of 90 patient samples (70%) were positive, but all those from 45 healthy controls were negative. Average virus titer in the patient specimens was 1.05 e4 copies/µL. Specific antibodies against NIH-CQV were sought by immunoblotting. Eighty-four percent of patients were positive for IgG, and 31% were positive for IgM; in contrast, 78% of healthy controls were positive for IgG, but all were negative for IgM. Although more work is needed to determine the etiologic role of NIH-CQV in human disease, our data indicate that a parvovirus-like virus is highly prevalent in a cohort of patients with non-A-E hepatitis.

Abstract Autophagy is the major intracellular degradation system by which cytoplasmic materials are delivered to and degraded in the vacuole/lysosome in eukaryotic cells. MoAtg14 in M. oryzae , a hitherto uncharacterized protein, is the highly divergent homolog of the yeast Atg14 and the mammal BARKOR. The MoATG14 deletion mutant exhibited collapse in the center of the colonies, poor conidiation and a complete loss of virulence. Significantly, the Δ Moatg14 mutant showed delayed breakdown of glycogen, less lipid bodies, reduced turgor pressure in the appressorium and impaired conidial autophagic cell death. The autophagic process was blocked in the Δ Moatg14 mutant, and the autophagic degradation of the marker protein GFP-MoAtg8 was interrupted. GFP-MoAtg14 co-localized with mCherry-MoAtg8 in the aerial hypha. In addition, a conserved coiled-coil domain was predicted in the N-terminal region of the MoAtg14 protein, a domain which could mediate the interaction between MoAtg14 and MoAtg6. The coiled-coil domain of the MoAtg14 protein is essential for its function in autophagy and pathogenicity.

Summary Pyricularia oryzae is the causal pathogen of rice blast disease. Autophagy has been shown to play important roles in P . oryzae development and plant infection. The P. oryzae endosomal system is highly dynamic and has been shown to be associated with conidiogenesis and pathogenicity as well. To date, the crosstalk between autophagy and endocytosis has not been explored in P. oryzae . Here, we identified three P. oryzae VPS9 domain‐containing proteins, PoVps9, PoMuk1 and PoVrl1. We found that PoVps9 and PoMuk1 are localized to vesicles and are each co‐localized with PoVps21, a recognized marker of early endosomes. Deletion of PoVPS9 resulted in severe defects in endocytosis and autophagosome degradation and impaired the localization of PoVps21 to endosomes. Additionally, deletion of the PoMUK1 gene in the Δ Povps9 mutant background exhibited more severe defects in development, autophagy and endocytosis compared with the Δ Povps9 mutant. Pull‐down assay showed that PoVps9 interacts with PoVps21, PoRab11 and PoRab1, which have been verified to participate in endocytosis. Furthermore, yeast two‐hybrid and co‐immunoprecipitation assays confirmed that PoVps9 directly interacts with the GDP form of PoVps21. Thus, PoVps9 is a key protein involved in autophagy and in endocytosis.

The SNF1/AMPK pathway has a central role in response to nutrient stress in yeast and mammals. Previous studies on SNF1 function in phytopathogenic fungi mostly focused on the catalytic subunit Snf1 and its contribution to the derepression of cell wall degrading enzymes (CWDEs). However, the MoSnf1 in Magnaporthe oryzae was reported not to be involved in CWDEs regulation. The mechanism how MoSnf1 functions as a virulence determinant remains unclear. In this report, we demonstrate that MoSnf1 retains the ability to respond to nutrient-free environment via its participation in peroxisomal maintenance and lipid metabolism. Observation of GFP-tagged peroxisomal targeting signal-1 (PTS1) revealed that the peroxisomes of ΔMosnf1 were enlarged in mycelia and tended to be degraded before conidial germination, leading to the sharp decline of peroxisomal amount during appressorial development, which might impart the mutant great retard in lipid droplets mobilization and degradation. Consequently, ΔMosnf1 exhibited inability to maintain normal appressorial cell wall porosity and turgor pressure, which are key players in epidermal infection process. Exogenous glucose could partially restore the appressorial function and virulence of ΔMosnf1. Toward a further understanding of SNF1 pathway, the β-subunit MoSip2, γ-subunit MoSnf4, and two putative Snf1-activating kinases, MoSak1 and MoTos3, were additionally identified and characterized. Here we show the null mutants ΔMosip2 and ΔMosnf4 performed multiple disorders as ΔMosnf1 did, suggesting the complex integrity is essential for M. oryzae SNF1 kinase function. And the upstream kinases, MoSak1 and MoTos3, play unequal roles in SNF1 activation with a clear preference to MoSak1 over MoTos3. Meanwhile, the mutant lacking both of them exhibited a severe phenotype comparable to ΔMosnf1, uncovering a cooperative relationship between MoSak1 and MoTos3. Taken together, our data indicate that the SNF1 pathway is required for fungal development and facilitates pathogenicity by its contribution to peroxisomal maintenance and lipid metabolism in M. oryzae.

The Skp1-Cul1-F-box-protein (SCF) ubiquitin ligases are important parts of the ubiquitin system controlling many cellular biological processes in eukaryotes. However, the roles of SCF ubiquitin ligases remain unclear in phytopathogenic Magnaporthe oryzae. Here, we cloned 24 F-box proteins and confirmed that 17 proteins could interact with MoSkp1, showing their potential to participate in SCF complexes. To determine their functions, null mutants of 21 F-box-containing genes were created. Among them, the F-box proteins MoFwd1, MoCdc4 and MoFbx15 were found to be required for growth, development and full virulence. Fluorescent-microscopy observations demonstrated that both MoFbx15 and MoCdc4 were localized to the nucleus, compared with MoFwd1, which was distributed in the cytosol. MoCdc4 and MoFwd1 bound to MoSkp1 via the F-box domain, the deletion of which abrogated their function. Race tube and qRT-PCR assays confirmed that MoFwd1 was involved in circadian rhythm by regulating transcription and protein stability of the core circadian clock regulator MoFRQ. Moreover, MoFWD1 also orchestrates conidial germination by influencing conidial amino acids pools and oxidative stress release. Overall, our results indicate that SCF ubiquitin ligases play indispensable roles in development and pathogenicity in M. oryzae.

The process of protein translocation into the endoplasmic reticulum (ER) is the initial and decisive step in the biosynthesis of all secretory proteins and many soluble organelle proteins. In this process, the Sec61 complex is the protein-conducting channel for transport. In this study, we identified and characterized the β subunit of the Sec61 complex in Magnaporthe oryzae (MoSec61β). Compared with the wild-type strain Guy11, the ΔMosec61β mutant exhibited highly branched mycelial morphology, reduced conidiation, high sensitivity to cell wall integrity stress, severely reduced virulence to rice and barley, and restricted biotrophic invasion. The turgor pressure of ΔMosec61β was notably reduced, which affected the function of appressoria. Moreover, ΔMosec61β was also sensitive to oxidative stress and exhibited a reduced ability to overcome plant immunity. Further examination demonstrated that MoSec61β affected the normal secretion of the apoplastic effectors Bas4 and Slp1. In addition, ΔMosec61β upregulated the level of ER-phagy. In conclusion, our results demonstrate the importance of the roles played by MoSec61β in the fungal development and pathogenesis of M. oryzae.

In fungi, the methylcitrate cycle converts cytotoxic propionyl-coenzyme A (CoA) to pyruvate, which enters gluconeogenesis. The glyoxylate cycle converts acetyl-CoA to succinate, which enters gluconeogenesis. The tricarboxylic acid cycle is a central carbon metabolic pathway that connects the methylcitrate cycle, the glyoxylate cycle, and other metabolisms for lipids, carbohydrates, and amino acids. Fungal citrate synthase and 2-methylcitrate synthase as well as isocitrate lyase and 2-methylisocitrate lyase, each evolved from a common ancestral protein. Impairment of the methylcitrate cycle leads to the accumulation of toxic intermediates such as propionyl-CoA, 2-methylcitrate, and 2-methylisocitrate in fungal cells, which in turn inhibits the activity of many enzymes such as dehydrogenases and remodels cellular carbon metabolic processes. The methylcitrate cycle and the glyoxylate cycle synergistically regulate carbon source utilization as well as fungal growth, development, and pathogenic process in pathogenic fungi.

The present study aimed to demonstrate the role of mepiquat chloride (MC) on the alleviation of the damaging effects of salinity on the germination of four cotton cultivars. Based on the ecophysiological data, CCRI44, CCRI49, and Z571 were less sensitive to salt stress and relied on a salt tolerance strategy to combat salinity at germination. Contrary to those salt-tolerance cultivars, seed germination of CZ91 was significantly inhibited by salt stress but most un-germinated seeds recovered completely when transferred to distilled water, indicating CZ91 follow a salt-avoiding strategy. Priming seeds with MC resulted in nullifying the negative effects of salt stress in salt-tolerance cultivars (especially in the cultivar Z571), but no significant response was observed in the salt-avoiding cultivar CZ91. At the mechanistic level, three complementary physiological traits are modulated by MC-priming upon exposure to salt stress: (i) better OA via increasing the water permeability and solute (i.e., Na+ or K+) mobility probably by regulating PIPs-regulated water channels; (ii) higher tissue tolerance ability via positively maintaining Na+ and K+ homeostasis by co-ordinated regulation of multiple ion transporters (e.g. SOS, HKT1, NHX1, AKT1 and HAK5); and (iii) further enhance the function of ion transport systems via inducing the gene expression of H+-ATPase. The strength of the three physiological traits varied among the MC pretreated cultivars, conferring differential ameliorating roles. Taken together, our observations confirmed that the effect of MC-priming on the alleviation of salinity stress is quite complex and dependent on the cultivar. It also shed light on transcriptional regulation of water and key ion transport systems involved in the maintenance of water absorption and K+/Na+ homeostasis as a key attribute for inducing salt tolerance by MC-priming in cotton cultivars.

Peroxisomes are ubiquitous organelles in eukaryotic cells that fulfill multiple important metabolisms. Pex13 and Pex14 are key components of the peroxisomal docking complex in yeasts and mammals. In the present work, we functionally characterized the homologues of Pex13 and Pex14 (Mopex13 and Mopex14) in the rice blast fungus Magnaporthe oryzae. Mopex13 and Mopex14 were peroxisomal membrane distributed and were both essential for the maintenance of Mopex14/17 on the peroxisomal membrane. Mopex13 and Mopex14 interacted with each other, and with Mopex14/17 and peroxisomal matrix protein receptors. Disruption of Mopex13 and Mopex14 resulted in a cytoplasmic distribution of peroxisomal matrix proteins and the Woronin body protein Hex1. In the ultrastructure of Δmopex13 and Δmopex14 cells, peroxisomes were detected on fewer occasions, and the Woronin bodies and related structures were dramatically affected. The Δmopex13 and Δmopex14 mutants were reduced in vegetative growth, conidial generation and mycelial melanization, in addition, Δmopex13 showed reduced conidial germination and appressorial formation and abnomal appressorial morphology. Both Δmopex13 and Δmopex14 were deficient in appressorial turgor and nonpathogenic to their hosts. The infection failures in Δmopex13 and Δmopex14 were also due to their reduced ability to degrade fatty acids and to endure reactive oxygen species and cell wall-disrupting compounds. Additionally, Mopex13 and Mopex14 were required for the sexual reproduction of the fungus. These data indicate that Mopex13 and Mopex14, as key components of the peroxisomal docking complex, are indispensable for peroxisomal biogenesis, fungal development and pathogenicity in the rice blast fungus.

Abstract Magnaporthe oryzae is a pathogenic fungus that seriously harms rice production. Phosphatases and carbon metabolism play crucial roles in the growth and development of eukaryotes. However, it remains unclear how serine/threonine phosphatases regulate the catabolism of triglycerides, a major form of stored lipids. In this study, we identified a serine/threonine protein phosphatase regulatory subunit, Smek1, which is required for the growth, conidiation, and virulence of M. oryzae . Deletion of SMEK1 led to defects in the utilization of lipids, arabinose, glycerol, and ethanol. In glucose medium, the expression of genes involved in lipolysis, long‐chain fatty acid degradation, β‐oxidation, and the glyoxylate cycle increased in the Δ smek1 mutant, which is consistent with Δ creA in which a carbon catabolite repressor CREA was deleted. In lipid medium, the expression of genes involved in long‐chain fatty acid degradation, β‐oxidation, the glyoxylate cycle, and utilization of arabinose, ethanol, or glycerol decreased in the Δ smek1 mutant, which is consistent with Δ crf1 in which a transcription activator CRF1 required for carbon metabolism was deleted. Lipase activity, however, increased in the Δ smek1 mutant in both glucose and lipid media. Moreover, Smek1 directly interacted with CreA and Crf1, and dephosphorylated CreA and Crf1 in vivo. The phosphatase Smek1 is therefore a dual‐function regulator of the lipid and carbohydrate metabolism, and controls fungal development and virulence by coordinating the functions of CreA and Crf1 in carbon catabolite repression (CCR) and derepression (CCDR).

Autophagy is a ubiquitous and evolutionarily conserved process found in all eukaryotic cells that allows for the degradation and recycling of old proteins and organelles. Starvation can induce autophagy, and autophagic pathway is an essential process for cellular function under starvation. In Magnaporthe grisea, starvation is one of the key induced factors for the germ tube tip to differentiate into an appressorium. Considering the importance of the rice blast fungus as a primary model for host-pathogen interaction, the role of autophagy in fungal development, appressorium turgor generation and pathogenicity of M. grisea via its role in organelle and protein turnover is a very significant subject.

Mnh6, a nonhistone protein containing an HMG1 box, was isolated from the rice blast fungus, Magnaporthe grisea. In the current study, we utilized an MNH6-deletion mutant to investigate the role of Mnh6 in the disease cycle of M. grisea. The Δmnh6 mutant exhibited pleiotropic effects on fungal morphogenesis, including reduction in mycelial growth, conidiation, appressorium development, plant penetration, and infectious growth in host cells. Furthermore, Δmnh6 mutant had greatly reduced pathogenicity on barley and rice compared to the wild-type. The reintroduction of an intact copy of MNH6 into the Δmnh6 mutant restored morphological features and pathogenicity, suggesting that Mnh6 is required for fungal development, effective pathogenicity, and completion of the disease cycle of M. grisea.

Summary Pyricularia oryzae is a plant pathogen causing rice blast, a serious disease spreading in cultivated rice globally. Transcription factors play important regulatory roles in fungal development and pathogenicity. Here, we characterized the biological functions of Crf1, a basic helix–loop–helix (bHLH) transcription factor, in the development and pathogenicity of P. oryzae with functional genetics, molecular and biochemical approaches. We found that CRF1 is necessary for virulence and plays an indispensable role in the regulation of carbohydrate and lipid metabolism in P. oryzae . Deletion of CRF1 led to defects in utilization of lipids, ethanol, glycerol and L‐arabinose, and down‐regulation of many important genes in lipolysis, β ‐oxidation, gluconeogenesis, as well as glycerol and arabinose metabolism. CRF1 is also essential for peroxisome and vacuole function, and conidial cell death during appressorium formation. The appressorium turgor, penetration ability and virulence in Δ crf1 were restored by supplementation of exogenous glucose. The virulence of Crf1 mutant was also recovered by adding exogenous D‐xylose, but not by addition of ethanol, pyruvate, leucine or L‐arabinose. These data showed that Crf1 plays an important role in the complex regulatory network of carbohydrate and lipid metabolism that governs fungal development and pathogenicity.

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.

Ca2+ is a second messenger in pathways that transduce external signals and activate cellular processes in plants and animals. Ca2+-mediated signal transduction is involved in key pathways that contribute to a variety of fundamental physiological processes in eukaryotic cells. However, little is known about the molecular mechanisms of Ca2+-mediated signal transduction in filamentous fungi. In this study, the MoCMK1 gene, encoding a putative Ca2+/calmodulin-dependent kinase, was identified in the rice blast fungus Magnaporthe oryzae. Three MoCMK1 deletion mutants were obtained by a targeted gene replacement. Colonies of the MoCMK1 mutants had sparse aerial hyphae and fewer conidia than the wild-type strain on complete medium. Conidial germination and appressorial formation were delayed in the ΔMocmk1 mutants. In spray inoculation tests, ΔMocmk1 mutants exhibited a weakened ability to infect the susceptible rice cultivar CO-39, compared to the wild-type strain Guy11. These results showed that MoCMK1 plays key roles in the pathogenicity of the rice blast fungus.

Autophagy is a highly conserved process in lower to higher eukaryotic organisms, and occurs in many types of cells as tissues are remodeled during development. In this study, we investigated the functional role of the Trichoderma reesei TrATG5 gene, which encodes an essential protein required for autophagy. TrATG5 is conserved in structure and function in the filamentous fungi and might clearly rescue the pathogenicity function of MgATG5 in Magnaporthe oryzae. Target gene disruption was used to study the functions of TrATG5. It was found that the autophagic process was blocked in the TrATG5 deletion mutant. The mutant was sensitive to nutrient starvation, with abnormal conidiophores and reduced production of conidia. This new evidence might help to elucidate the molecular machinery of autophagy in filamentous fungi.

Summary 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 .

Pine wood nematode (PWN), Bursaphelenchus xylophilus, is a major pathogen of pine wilt disease (PWD), which is a devastating disease affecting pine trees. Eco-friendly plant-derived nematicides against PWN have been considered as promising alternatives to control PWD. In this study, the ethyl acetate extracts of Cnidium monnieri fruits and Angelica dahurica roots were confirmed to have significant nematicidal activity against PWN. Through bioassay-guided fractionations, eight nematicidal coumarins against PWN were separately isolated from the ethyl acetate extracts of C. monnieri fruits and A. dahurica roots, and they were identified to be osthol (Compound 1), xanthotoxin (Compound 2), cindimine (Compound 3), isopimpinellin (Compound 4), marmesin (Compound 5), isoimperatorin (Compound 6), imperatorin (Compound 7), and bergapten (Compound 8) by mass and nuclear magnetic resonance (NMR) spectral data analysis. Coumarins 1–8 were all determined to have inhibitory effects on the egg hatching, feeding ability, and reproduction of PWN. Moreover, all eight nematicidal coumarins could inhibit the acetylcholinesterase (AChE) and Ca2+ ATPase of PWN. Cindimine 3 from C. monnieri fruits showed the strongest nematicidal activity against PWN, with an LC50 value of 64 μM at 72 h, and the highest inhibitory effect on PWN vitality. In addition, bioassays on PWN pathogenicity demonstrated that the eight nematicidal coumarins could effectively relieve the wilt symptoms of black pine seedlings infected by PWN. The research identified several potent botanical nematicidal coumarins for use against PWN, which could contribute to the development of greener nematicides for PWD control.

Microbes employ effectors to disrupt immune responses and promote host colonization. Conserved motifs including RXLR, LFLAK-HVLVxxP (CRN), Y/F/WxC, CFEM, LysM, Chitin-bind, DPBB_1 (PNPi), and Cutinase have been discovered to play crucial roles in the functioning of effectors in filamentous fungi. Nevertheless, little is known about effectors with conserved motifs in endophytes. This research aims to discover the effector genes with conserved motifs in the genome of rice endophyte Falciphora oryzae. SignalP identified a total of 622 secreted proteins, out of which 227 were predicted as effector candidates by EffectorP. By utilizing HMM features, we discovered a total of 169 effector candidates with conserved motifs and three novel motifs. Effector candidates containing LysM, CFEM, DPBB_1, Cutinase, and Chitin_bind domains were conserved across species. In the transient expression assay, it was observed that one CFEM and one LysM activated cell death in tobacco leaves. Moreover, two CFEM and one Chitin_bind inhibited cell death induced by Bax protein. At various points during the infection, the genes’ expression levels were increased. These results will help to identify functional effector proteins involving omics methods using new bioinformatics tools, thus providing a basis for the study of symbiosis mechanisms.

The cell cycle is pivotal to cellular differentiation in plant pathogenic fungi. Cell wall integrity (CWI) signaling plays an essential role in coping with cell wall stress. Autophagy is a degradation process in which cells decompose their components to recover macromolecules and provide energy under stress conditions. However, the specific association between cell cycle, autophagy and CWI pathway remains unclear in model pathogenic fungi Magnaporthe oryzae. Here, we have identified MoSwe1 as the conserved component of the cell cycle in the rice blast fungus. We have found that MoSwe1 targets MoMps1, a conserved critical MAP kinase of the CWI pathway, through protein phosphorylation that positively regulates CWI signaling. The CWI pathway is abnormal in the ΔMoswe1 mutant with cell cycle arrest. In addition, we provided evidence that MoSwe1 positively regulates autophagy by interacting with MoAtg17 and MoAtg18, the core autophagy proteins. Moreover, the S phase initiation was earlier, the morphology of conidia and appressoria was abnormal, and septum formation and glycogen degradation were impaired in the ΔMoswe1 mutant. Our research defines that MoSWE1 regulation of G1/S transition, CWI pathway, and autophagy supports its specific requirement for appressorium development and virulence in plant pathogenic fungi. Video Abstract.

Abstract Csn5 is subunit 5 of the COP9 signalosome (CSN), but the mechanism by which it strictly controls the pathogenicity of pathogenic fungi through autophagy remains unclear. Here, we found that Csn5 deficiency attenuated pathogenicity and enhanced autophagy in Magnaporthe oryzae. MoCSN5 knockout led to overubiquitination and overdegradation of MoTor (the core protein of the TORC1 complex [target of rapamycin]) thereby promoted autophagy. In addition, we identified MoCsn5 as a new interactor of MoAtg6. Atg6 was found to be ubiquitinated through linkage with lysine 48 (K48) in cells, which is necessary for infection-associated autophagy in pathogenic fungi. K48-ubiquitination of Atg6 enhanced its degradation and thereby inhibited autophagic activity. Our experimental results indicated that MoCsn5 promoted K48-ubiquitination of MoAtg6, which reduced the MoAtg6 protein content and thus inhibited autophagy. Aberrant ubiquitination and autophagy in Δ Mocsn5 led to pleiotropic defects in the growth, development, stress resistance, and pathogenicity of M. oryzae . In summary, our study revealed a novel mechanism by which Csn5 regulates autophagy and pathogenicity in rice blast fungus through ubiquitination.

TOR, a widely conserved eukaryotic protein kinase, forms TORC1 and TORC2 to regulate diverse cell signaling. TORC1 controls protein synthesis, cell cycle, and autophagy, whereas TORC2 manages cell polarity, cytoskeleton, and membrane structure. Our previous research found that MoVast2, along with MoVast1, regulates TOR in rice blast fungus Magnaporthe oryzae, maintaining lipid and autophagy balance. Lst8, a key TOR complex component in yeast and mammalian cells. However, the precise role of MoLst8 in M. oryzae is still unclear. In this study, we obtained the DeltaMolst8 mutant through high-through gene knockout strategies. The results showed that loss of MoLST8 leading to a series of defects, such as growth and sporulation reduction, abnormal conidia, and loss of virulence. In addition, this mutant is highly sensitive to rapamycin, leading to growth arrest and autophagy impairment, indicated that MoLst8 positively regulates TORC1 for cellular growth, metabolism, and autophagy. Lipidomics analysis in the mutant revealed lipid metabolism dysregulation, sphingolipid reduction, disrupting membrane tension and homeostasis, suggested that TORC2 mediated lipid regulation is disordered in DeltaMolst8 mutant. Additionally, the study explored TOR-MAPK crosstalk, finding that the mutant shows heightened cell wall stress sensitivity but fails to restore integrity despite MAPK activation. These findings offer insights into MoLst8's role in fungal pathogenesis, contributing to an understanding of fungal biology and disease control strategies.

Calpains are ubiquitous and well-conserved proteins that belong to the calcium-dependent, non-lysosomal cysteine protease family. In this study, 8 putative calpains were identified using Pfam domain analysis and BlastP searches in M. oryzae. Three single gene deletion mutants (ΔMocapn7, ΔMocapn9 and ΔMocapn14) and two double gene deletion mutants (ΔMocapn4ΔMocapn7 and ΔMocapn9ΔMocapn7) were obtained using the high-throughput gene knockout system. The calpain disruption mutants showed defects in colony characteristics, conidiation, sexual reproduction and cell wall integrity. The mycelia of the ΔMocapn7, ΔMocapn4ΔMocapn7 and ΔMocapn9ΔMocapn7 mutants showed reduced pathogenicity on rice and barley.

Casein kinases are serine/threonine protein kinases that are evolutionarily conserved in yeast and humans and are involved in a range of important cellular processes. However, the biological functions of casein kinases in the fungus Magnaporthe oryzae, the causal agent of destructive rice blast disease, are not characterized. Here, two casein kinases, MoYCK1 and MoHRR25, were identified and targeted for replacement, but only MoYCK1 was further characterized due to the possible nonviability of the MoHRR25 deletion mutant. Disruption of MoYCK1 caused pleiotropic defects in growth, conidiation, conidial germination, and appressorium formation and penetration, therefore resulting in reduced virulence in rice seedlings and barley leaves. Notably, the MoYCK1 deletion triggered quick lipidation of MoAtg8 and degradation of the autophagic marker protein GFP-MoAtg8 under nitrogen starvation conditions, in contrast to the wild type, indicating that autophagy activity was negatively regulated by MoYck1. Furthermore, we found that HOPS (homotypic fusion and vacuolar protein sorting) subunit MoVps41, a putative substrate of MoYck1, was co-located with MoAtg8 and positively required for the degradation of MoAtg8-PE and GFP-MoAtg8. In addition, MoYCK1 is also involved in the response to ionic hyperosmotic and heavy metal cation stresses. Taken together, our results revealed crucial roles of the casein kinase MoYck1 in regulating development, autophagy and virulence in M. oryzae.

Magnaporthe oryzae is an important plant pathogenic fungus that greatly threatens the world's food security. Both genome-wide and individual gene studies have shown that the pathogenicity of the fungus is severely dependent on the intracellular autophagy process during appressoria development. This protocol discusses a systematic methodology to discover and monitor autophagy-related (ATG) genes in M. oryzae.

Magnaporthe oryzae has been used as a primary model organism for investigating fungus-plant interaction. Many researches focused on molecular mechanisms of appressorium formation to restrain this fungal pathogen. Autophagy is a very high conserved process in eukaryotic cells. Recently, autophagy has been considered as a key process in development and differentiation in M. oryzae. In this report, we present and discuss the current state of our knowledge on gene expression in appressorium formation and the progress in autophagy of rice blast fungi.

Calpains are intracellular, cysteine proteases found in plants, animals and fungi functioning as signal transduction components in different cellular pathways including sporulation and alkaline adaptation in fungi. Calpains-related MoCAPN1 (MGG_14872), MoCAPN3 (MGG_15810) and MoCAPN4 (MGG_04818) genes from Magnaporthe oryzae genome which are 2604, 3513 and 771-bp in length and encoding identical proteins of 867, 1170 and 256 amino acids were functionally characterized for different phenotypes through gene disruption method. All the mutants except those for MoCAPN1 showed normal phenotypes. In pathogenicity test, the mutants did not lead to any visible changes in phenotypes causing similar blast lesions on blast susceptible rice and barley leaves as those of the Guy-11 strain suggesting no major role in pathogenicity. Germ tubes formation, appressorium formation, mycelium radial growth and mating with 2539 strain were indistinguishable among the mutants and Guy-11 strains. Cell wall integrity (congo red) test, stress response under chemical pressure (ZnSO4, CuSO4 and CdCl2), osmotic and oxidative (NaCl and H2O2) stress response, growth response on glucose and nitrogen deficient media resulted in similar results in the mutants and Guy-11 strains. However, mutants for ΔMoCAPN1 gene produced reduced (0.57±0.15B and 0.54±0.05B) conidia compared to that (1.69±0.13A) of the Guy-11 strain showing its involvement in conidiation.

Fap7, an important ribosome assembly factor, plays a vital role in pre-40S small ribosomal subunit synthesis in Saccharomyces cerevisiae via its ATPase activity. Currently, the biological functions of its homologs in filamentous fungi remain elusive. Here, MoFap7, a homologous protein of ScFap7, was identified in the rice blast fungus Magnaporthe oryzae, which is a devastating fungal pathogen in rice and threatens food security worldwide. ΔMofap7 mutants exhibited defects in growth and development, conidial morphology, appressorium formation and infection, and were sensitive to oxidative stress. In addition, site-directed mutagenesis analysis confirmed that the conserved Walker A motif and Walker B motif in MoFap7 are essential for the biological functions of M. oryzae. We further analyzed the regulation mechanism of MoFap7 in pathogenicity. MoFap7 was found to interact with MoMst50, a regulator functioning in the MAPK Pmk1 signaling pathway, that participates in modulating plant penetration and cell-to-cell invasion by regulating the phosphorylation of MoPmk1. Moreover, MoFap7 interacted with the GTPases MoCdc42 and MoRac1 to control growth and conidiogenesis. Taken together, the results of this study provide novel insights into MoFap7-mediated orchestration of the development and pathogenesis of filamentous fungi.

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.

The COP9 signalosome (CSN) is a highly conserved protein complex in eukaryotes, affecting various development and signaling processes. To date, the biological functions of the COP9 signalosome and its subunits have not been determined in Magnaporthe oryzae. In this study, we characterized the CSN in M. oryzae (which we named MoCsn6) and analyzed its biological functions. MoCsn6 is involved in fungal development, autophagy, and plant pathogenicity. Compared with the wild-type strain 70-15, ΔMocsn6 mutants showed a significantly reduced growth rate, sporulation rate, and germ tube germination rate. Pathogenicity assays showed that the ΔMocsn6 mutants did not cause or significantly reduced the number of disease spots on isolated barley leaves. After the MoCSN6 gene was complemented into the ΔMocsn6 mutant, vegetative growth, sporulation, and pathogenicity were restored. The Osm1 and Pmk1 phosphorylation pathways were also disrupted in the ΔMocsn6 mutants. Furthermore, we found that MoCsn6 participates in the autophagy pathway by interacting with the autophagy core protein MoAtg6 and regulating its ubiquitination level. Deletion of MoCSN6 resulted in rapid lipidation of MoAtg8 and degradation of the autophagic marker protein green fluorescent protein-tagged MoAtg8 under nutrient and starvation conditions, suggesting that MoCsn6 negatively regulates autophagic activity. Taken together, our results demonstrate that MoCsn6 plays a crucial role in regulating fungal development, pathogenicity, and autophagy in M. oryzae. IMPORTANCE Magnaporthe oryzae, a filamentous fungus, is the cause of many cereal diseases. Autophagy is involved in fungal development and pathogenicity. The COP9 signalosome (CSN) has been extensively studied in ubiquitin pathways, but its regulation of autophagy has rarely been reported in plant-pathogenic fungi. Investigations on the relationship between CSN and autophagy will deepen our understanding of the pathogenic mechanism of M. oryzae and provide new insights into the development of new drug targets to control fungal diseases. In this study, the important function of Csn6 in the autophagy regulation pathway and its impact on the pathogenicity of M. oryzae were determined. We showed that Csn6 manages autophagy by interacting with the autophagy core protein Atg6 and regulating its ubiquitination level. Furthermore, future investigations that explore the function of CSN will deepen our understanding of autophagy mechanisms in rice blast fungus.

Calcineurin, a key regulator of the calcium signaling pathway, is involved in calcium signal transduction and calcium ion homeostasis. Magnaporthe oryzae is a devastating filamentous phytopathogenic fungus in rice, yet little is known about the function of the calcium signaling system. Here, we identified a novel calcineurin regulatory-subunit-binding protein, MoCbp7, which is highly conserved in filamentous fungi and was found to localize in the cytoplasm. Phenotypic analysis of the MoCBP7 gene deletion mutant (ΔMocbp7) showed that MoCbp7 influenced the growth, conidiation, appressorium formation, invasive growth, and virulence of M. oryzae. Some calcium-signaling-related genes, such as YVC1, VCX1, and RCN1, are expressed in a calcineurin/MoCbp7-dependent manner. Furthermore, MoCbp7 synergizes with calcineurin to regulate endoplasmic reticulum homeostasis. Our research indicated that M. oryzae may have evolved a new calcium signaling regulatory network to adapt to its environment compared to the fungal model organism Saccharomyces cerevisiae.

The peroxisomal matrix proteins involved in many important biological metabolism pathways in eukaryotic cells are encoded by nucleal genes, synthesized in the cytoplasm and then transported into the organelles. Targeting and import of these proteins depend on their two peroxisomal targeting signals (PTS1 and PTS2) in sequence as we have known so far. The vectors of the fluorescent fusions with PTS, i.e., green fluorescence protein (GFP)-PTS1, GFP-PTS2 and red fluorescence protein (RFP)-PTS1, were constructed and introduced into Magnaporthe oryzae Guy11 cells. Transformants containing these fusions emitted fluorescence in a punctate pattern, and the locations of the red and green fluorescence overlapped exactly in RFP-PTS1 and GFP-PTS2 co-transformed strains. These data indicated that both PTS1 and PTS2 fusions were imported into peroxisomes. A probable higher efficiency of PTS1 machinery was revealed by comparing the fluorescence backgrounds in GFP-PTS1 and GFP-PTS2 transformants. By introducing both RFP-PTS1 and GFP-PTS2 into Δmgpex6 mutants, the involvement of MGPEX6 gene in both PTS1 and PTS2 pathways was proved. In addition, using these transformants, the inducement of peroxisomes and the dynamic of peroxisomal number during the pre-penetration processes were investigated as well. In summary, by the localization and co-localization of PTS1 and PTS2, we provided a useful tool to evaluate the biological roles of the peroxisomes and the related genes.

To investigate the clinical phenotype and ACAT1 gene mutation in a family affected with beta-ketothiolase deficiency (BKTD).Clinical features and laboratory test data were collected. The probands were monozygotic twin brothers. Genomic DNA was isolated from peripheral blood leukocytes obtained from the probands and their family members. Molecular genetic testing of the ACAT1 gene was carried out.The probands have presented with fever, vomiting and severe ketoacidosis. By arterial blood gas testing, pH was determined to be 7.164, bicarbonate was 4.0 mmol/L, and urine ketone was ++++. Urinary organic acid gas chromatography-mass spectrometry analysis showed excessive excretion of 3-hydroxybutyric acid, 2-methyl-3-hydroxybutyric acid and tiglylglycine. Increased 3-hydroxybutyrylcarnitine (C4-OH), tiglylcarnitine(C5:1) and 3-hydroxyisovalerylcarnitine (C5-OH) levels. The clinical phenotype of proband's parents were both normal, but an elder sister turned out to be an affected patient. Genetic analysis has identified two heterozygous mutations [c.622C>T(p.R208X) and c.653C>T (p.S218F)] in the proband, which were respectively detected in the mother and father. The c.653C>T (p.S218F) mutation was not found among the 100 healthy controls and has not been included in the Human Gene Mutation Database(HGMD).The primary clinical manifestations of BKTD is ketoacidosis. Urine organic acid and blood acylcarnitine analyses play an important role in the diagnosis of the disease. The compound heterozygous of ACAT1 gene mutations probably underlie the BKTD in our patient.

Septins, a conserved family of GTP-binding proteins, are widely recognized as an essential cytoskeletal component, playing important roles in a variety of biological processes, including division, polarity, and membrane remodeling, in different eukaryotes. Although the roles played by septins were identified in the model organism Saccharomyces cerevisiae, their importance in other fungi, especially pathogenic fungi, have recently been determined. In this review, we summarize the functions of septins in pathogenic fungi in the cell cycle, autophagy, endocytosis and invasion host-microbe interactions that were reported in the last two years in the field of septin cell biology. These new discoveries may be expanded to investigate the functions of septin proteins in fungal pathogenesis and may be of wide interest to the readers of Microbiology and Molecular Pathology.

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.

The appressorium is a specialized structure that is differentiated from Magnaporthe oryzae spores that can infect host cells. In the process of cellular transformation from spore to appressorium, the contents inside the spores are transferred into appressoria, accompanied by major differences in the gene expression model. In this study, we reported a transcription factor (TF), Pcf1, which was depressed at the transcription level and degraded at the protein level in nuclei of incipient appressoria at four hpi (hours post inoculation). To investigate its degradation mechanism, the interacting proteins of Pcf1 were identified using an immunoprecipitation-mass spectrometry (IP-MS) assay. Yeast two-hybrid (Y2H) and co-IP (co-immunoprecipitation) assays confirmed that Pcf1 interacted with the casein kinase 2 (CK2) holoenzyme through direct combination with the CKb2 subunit. Moreover, Pcf1 was ubiquitinated in the hyphae. These changes in Pcf1 protein levels in nuclei provide a new clue of how TFs are degraded during appressorium formation: temporarily unnecessary TFs in spores are phosphorylated through interacting with CK2 enzyme and are then ubiquitinated and digested by the ubiquitin proteasome system (UPS).

Magnaporthe oryzae is an important pathogen that causes a devastating disease in rice. It has been reported that the dual-specificity LAMMER kinase is conserved from yeast to animal species and has a variety of functions. However, the functions of the LAMMER kinase have not been reported in M. oryzae. In this study, we identified the unique LAMMER kinase MoKns1 and analyzed its function in M. oryzae. We found that in a MoKNS1 deletion mutant, growth and conidiation were primarily decreased, and pathogenicity was almost completely lost. Furthermore, our results found that MoKns1 is involved in autophagy. The ΔMokns1 mutant was sensitive to rapamycin, and MoKns1 interacted with the autophagy-related protein MoAtg18. Compared with the wild-type strain 70−15, autophagy was significantly enhanced in the ΔMokns1 mutant. In addition, we also found that MoKns1 regulated DNA damage stress pathways, and the ΔMokns1 mutant was more sensitive to hydroxyurea (HU) and methyl methanesulfonate (MMS) compared to the wild-type strain 70−15. The expression of genes related to DNA damage stress pathways in the ΔMokns1 mutant was significantly different from that in the wild-type strain. Our results demonstrate that MoKns1 is an important pathogenic factor in M. oryzae involved in regulating autophagy and DNA damage response pathways, thus affecting virulence. This research on M. oryzae pathogenesis lays a foundation for the prevention and control of M. oryzae.

Rice is an important food crop all over the world. It can be infected by the rice blast fungus Magnaporthe oryzae, which results in a significant reduction in rice yield. The infection mechanism of M. oryzae has been an academic focus for a long time. It has been found that G protein, AMPK, cAMP-PKA, and MPS1-MAPK pathways play different roles in the infection process. Recently, the function of TOR signaling in regulating cell growth and autophagy by receiving nutritional signals generated by plant pathogenic fungi has been demonstrated, but its regulatory mechanism in response to the nutritional signals remains unclear. In this study, a yeast amino acid permease homologue MoGap1 was identified and a knockout mutant of MoGap1 was successfully obtained. Through a phenotypic analysis, a stress analysis, autophagy flux detection, and a TOR activity analysis, we found that the deletion of MoGap1 led to a sporulation reduction as well as increased sensitivity to cell wall stress and carbon source stress in M. oryzae. The ΔMogap1 mutant showed high sensitivity to the TOR inhibitor rapamycin. A Western blot analysis further confirmed that the TOR activity significantly decreased, which improved the level of autophagy. The results suggested that MoGap1, as an upstream regulator of TOR signaling, regulated autophagy and responded to adversities such as cell wall stress by regulating the TOR activity.

One new capsaicinoid, N-vanillyl-4E,6E-dien-8-methylnonanamide (4), along with nine known capsaicinoids, capsaicin (1), dihydrocapsaicin (2), N-vanillyloctanamide (3), nordihydrocapsaicin (5), N-vanillyldecanamide (6), homocapsaicin (7), N-vanillyl-4,8-dimethylnonanamide (8), homodihydrocapsaicin II (9), and homodihydrocapsaicin I (10) were isolated from the fruits of Capsicum annuum using semi-preparative high-performance liquid chromatography. The structural characterizations of the isolated compounds were elucidated by spectroscopic data and comparison with the literatures. Bioassays showed that the isolated capsaicinoids significantly reduced the radical length of Lactuca sativa seedling, this inhibition being dose-dependent.

Magnaporthe oryzae has been used as a model fungal pathogen to study the molecular basis of plant-fungus interactions due to its economic and genetic importance. In this study, we identified a novel gene, Moplaa, which is the homologue of Homo sapiens PLAA encoding a phospholipase A(2)-activating protein. Moplaa is conserved in some eukaryotic organisms by multiple alignment analysis. The function of the Moplaa gene was studied using the gene target replacement method. The Moplaa deletion mutant exhibited retarded growth and conidial germination, reduced conidiation, appressorial turgor pressure and pathogenicity to rice CO-39. Reintroduction of the gene restored defects of the Moplaa deletion mutant.

100-kernel weight (KW) is one of the most important agronomic traits in maize ( Zea mays L.), related to yield. To realize its genetic basis, in this study, a recombinant inbred line (RIL) population derived from the cross between Mo17 and Huangzao4 was used for quantitative trait locus (QTL) mapping for KW under high and low nitrogen (N) regimes. As a result, five QTLs were identified on chromosomes 3, 4, 7 and 9, of which three were detected under both N environments, while the other two QTLs were respectively detected under high and low N regimes. These QTLs could explain phenotypic variance rom 4.47 to 14.47%. Due to additive effects, the three QTLs from Mo17, including two on chromosome 3 and one on chromosome 4, could increase KW from 0.64 to 1.01 g, while the other two from Huangzao4 on chromosomes 7 and 9 could decrease KW from 0.62 to 1.07 g. These results are beneficial for understanding the genetic basis of KW and developing the markers linked with KW for marker-assisted selection breeding in maize. Key words : Maize ( Zea mays L.), 100-kernel weight, quantitative trait locus (QTL), recombinant inbred line (RIL), nitrogen regime.

Amino acids are vital components in cell metabolism. Leucine is a regulatory factor that generates significant impact on protein synthesis/turnover, modulates diverse cellular signalling pathways and participates in oxidative processes and immune responses. Here, we identified and characterized the functions of a leucine-associated Zn2 Cys6 -type transcription factor, MoLeu3. Disruption of MoLEU3 resulted in significantly reduced pathogenicity in barley and rice. Quantitative RT-PCR showed that the expression levels of the putative leucine biosynthesis-related genes, MoLEU1, MoLEU2 and MoLEU4 were downregulated in the ΔMoleu3 mutant. We used high-throughput gene knockout method to generate the null mutants of MoLEU1, MoLEU2 and MoLEU4 respectively. The ΔMoleu1, ΔMoleu2 and ΔMoleu4 mutants are leucine auxotroph and showed similar phenotypic characterizations, including reduced conidiation, delayed mobilization and degradation of glycogen and lipid droplets, limited appressorium-mediated penetration, and restricted invasive hyphae growth within host cells. Collectively, MoLEU1, MoLEU2, and MoLEU4 regulated by MoLEU3 play crucial roles in fungal development and infectious processes through modulation of leucine biosynthesis in Magnaporthe oryzae.

In this study the MTP1 gene, encoding a type III integral transmembrane protein, was isolated from the rice blast fungus Magnaporthe oryzae. The Mtp1 protein is 520 amino acids long and is comparable to the Ytp1 protein of Saccharomyces cerevisiae with 46% sequence similarity. Prediction programs and MTP1-GFP (green fluorescent protein) fusion expression results indicate that Mtp1 is a protein located at several membranes in the cytoplasm. The functions of the MTP1 gene in the growth and development of the fungus were studied using an MTP1 gene knockout mutant. The MTP1 gene was primarily expressed at the hyphal and conidial stages and is necessary for conidiation and conidial germination, but is not required for pathogenicity. The Deltamtp1 mutant grew more efficiently than the wild type strain on non-fermentable carbon sources, implying that the MTP1 gene has a unique role in respiratory growth and carbon source use.

Magnaporthe oryzae is the causal agent of rice blast, leading to significant reductions in rice and wheat productivity. Nap1 is a conserved protein in eukaryotes involved in diverse physiological processes, such as nucleosome assembly, histone shuttling between the nucleus and cytoplasm, transcriptional regulation, and the cell cycle. Here, we identified Nap1 and characterized its roles in fungal development and virulence in M. oryzae. MoNap1 is involved in aerial hyphal and conidiophore differentiation, sporulation, appressorium formation, plant penetration, and virulence. ΔMonap1 generated a small, elongated, and malformed appressorium with an abnormally organized septin ring on hydrophobic surfaces. ΔMonap1 was more sensitive to cell wall integrity stresses but more resistant to microtubule stresses. MoNap1 interacted with histones H2A and H2B and the B-type cyclin (Cyc1). Moreover, a nuclear export signal (NES) domain is necessary for Nap1's roles in the regulation of the growth and pathogenicity of M. oryzae. In summary, NAP1 is essential for the growth, appressorium formation, and pathogenicity of M. oryzae.

To explore pathogenic mutation in a family affected with 2-hydroxyglutaric aciduria.Exons of 3 candidate genes, including L2HGDH, D2HGDH and SLC25A1, were amplified with polymerase chain reaction and subjected to direct sequencing.DNA sequencing has found that the proband and his affected younger brother have both carried a heterozygous mutation c.845G>A (p.R282Q) in the exon 7 of the L2HGDH gene. The same mutation was not detected in the his sister who was healthy. Pedigree analysis has confirmed that the above mutation was inherited from the mother. No mutation was detected in exons and flanking sequences of the D2HGDH and SLC25A1 genes.Mutation of the L2HGDH gene probably underlies the 2-hydroxyglutaric aciduria in this family.

Benign familial infantile convulsions (BFIC) is a form of idiopathic epileptic syndrome characterized by onset of afebrile seizures between 3 and 12 months of life, Spontaneous remission after several weeks or months, and autosomal dominant mode of inheritance. Previous linkage analysis in western countries defined three susceptible loci on chromosomes 19q12.0-13.1, 16p12-q12, and 2q23-31, but studies performed in several Chinese families with BFIC got negative results of these previously reported loci. The authors investigated the relation of voltage-gated potassium channel gene KCNQ2 to BFIC in a Chinese family and thus to understand the molecular pathogenesis of BFIC.A four-generation Chinese BFIC family was investigated. All the affected 17 members had similar pattern of seizures starting from 2 to 6 months of age. In 15 of them, the seizures disappeared spontaneously within the first year of life. The phenotype extended beyond infancy only in two patients. Blood sample was collected from the 41 family members and 75 unassociated normal individuals. Polymerase chain reaction (PCR)-DNA direct sequencing was performed to screen all exons and their flanking introns of KCNQ2 gene for mutation analysis. Polymerase chain reaction-single strand conformation polymorphism (PCR-SSCP) was used to ascertain the co-segregation of genotype and phenotype and to exclude polymorphism.PCR amplification and subsequent direct sequencing of KCNQ2 from the DNA of proband revealed a heterozygous guanine to thymine nucleotide exchange (G812T) in exon 5, leading to the substitution of glycine by valine at amino acid position 271 (G271V) of the predicted protein. The same mutation with a comparable localization has been previously described for KCNQ3 in benign familial neonatal convulsions (BFNC). The glycine at this position (G271) is located in pore region of KCNQ2 protein and is evolutionarily highly conserved. The same SSCP variant as that of the proband was shown in the rest of the affected members of this family but not in the unaffected members enrolled in the study of this family and all the 75 unrelated normal individuals.Previously reported mutations of KCNQ2 were mainly identified in BFNC family in which at least one individual had an onset of seizures during the first week of life, a hallmark of the BFNC disorder. The results of the present study suggest the possibility that KCNQ2 mutation exist in patients with BFIC diagnosis. G812T of KCNQ2 gene is a novel mutation found in BFIC and functional expression of KCNQ2 G812T is required for understanding the mechanism of BFIC and other idiopathic epilepsy.

Application of promoter trapping based on transformation in Magnaporthe grisea is reported in this paper. Two promoter-trapping vectors, designated as pCBGFP and pEGFPHPH, were constructed and transformed into protoplasts of M. Grisea. A library of 1077 transformants resistant to hygromycin B was generated. Of which, 448 transformants were found to express eGFP gene in different structures of M. grisea. Three transformants grew slowly, 5 transformants decreased in conidiation and 7 transformants reduced in pathogenicity greatly among these 448 transformants. Eleven transformants were checked by genomic southern blot randomly, and 9 of which were single-copy insertions. The promoter trapping technique has been applied successfully in M. Grisea and can be used as a tool for functional genomic analysis.

Abstract Rice blast, known as rice “cancer”, is caused by Magnaporthe oryzae and is particularly serious in Asian and African rice regions. China is also a frequently occurring region of rice blast. Rice blast not only seriously threatens the yield and quality of rice but also affects food security in China. In M. oryzae , the Mst11-Mst7-Pmk1 MAPK signaling pathway mediates pathogenicity by regulating the formation of appressorium and the development of infection hyphae. Stomatal cytokinesis defective 2 (Scd2, also called Ral3 or Bem1) is a component of the Scd complex, which has been proven to be closely related to the MAPK signaling pathway. However, its biological roles in M. oryzae remain elusive. Here, we identified MoScd2, a homologous protein of Schizosaccharomyces pombe Scd2, and preliminarily revealed its role in the development of rice blast fungus. We found that MoScd2 was involved in colony growth, sporulation, spore morphology, spore germination, appressorium formation, turgor in appressoria, mobilization of glycogen from spores to appressoria and pathogenicity. The deletion of MoScd2 resulted in a reduction in Pmk1 and Mps1 phosphorylation levels. In addition, MoScd2 was confirmed to interact with MoMst50, which is a key component of the MAPK signaling pathway in M. oryzae . In summary, MoScd2 was involved in the MAPK signaling pathway of M. oryzae via interaction with MoMst50 to participate in the influence of pathogenicity. In addition, MoScd2 also influences M. oryzae pathogenicity by participating in autophagy.

The complete mitochondrial genome of Pyricularia oryzae Cavara 1892 strain Guy11 is 34,865 bp in length (GenBank accession number OP095391), containing 29 tRNA genes, 2 rRNA genes, and 15 protein-coding genes (PCGs). The gene order and orientation are novel compared to other Sordariomycetes species with sequenced mitogenomes in the GenBank database. Phylogenetic analysis suggests that P. oryzae Guy11 and 19 other Sordariomycetes species form a monophyletic group. The complete mitochondrial sequence of P. oryzae Guy11 will be a valuable resource for species identification, population genetics, phylogenetics, and comparative genomics studies in Sordariomycetes and Magnaporthales.

Recalcitrant rice blast disease is caused by Magnaporthe oryzae, which has a significant negative economic reverberation on crop productivity. In order to induce the disease onto the host, M. oryzae positively generates many types of small secreted proteins, here named as effectors, to manipulate the host cell for the purpose of stimulating pathogenic infection. In M. oryzae, by engaging with specific receptors on the cell surface, effectors activate signaling channels which control an array of cellular activities, such as proliferation, differentiation and apoptosis. The most recent research on effector identification, classification, function, secretion, and control mechanism has been compiled in this review. In addition, the article also discusses directions and challenges for future research into an effector in M. oryzae.

The complete mitochondrial genome of <i>Pyricularia oryzae</i> Cavara 1892 strain Guy11 is 34,865 bp in length (GenBank accession number OP095391), containing 29 tRNA genes, 2 rRNA genes, and 15 protein-coding genes (PCGs). The gene order and orientation are novel compared to other Sordariomycetes species with sequenced mitogenomes in the GenBank database. Phylogenetic analysis suggests that <i>P. oryzae</i> Guy11 and 19 other Sordariomycetes species form a monophyletic group. The complete mitochondrial sequence of <i>P. oryzae</i> Guy11 will be a valuable resource for species identification, population genetics, phylogenetics, and comparative genomics studies in Sordariomycetes and Magnaporthales.

MGTA1, a putative fungal Zn(II) 2 Cys 6 transcriptional activator-encoding gene, was isolated from rice blast pathogen Magnaporthe grisea, which is homologous to CLTA1 from Colletotrichum lindemuthianum with 51% identity at protein level.MGTA1 cassette contains a 2370 bp open reading frame, consisting of 6 exons, coding a 790 amino acid peptide.MGTA1 gene exists as a single copy in genomes of 7 strains of M. grisea, and is expressed in tip hyphae, conidia, and mature appressoria of strain Guy11.

To identify the types of OTC gene mutations in three male patients with late onset ornithine transcarbamylase deficiency (OTCD, MIM #311250).Genomic DNA was extracted from peripheral blood leukocytes. The 10 exons and their flanking sequences of the OTC gene were amplified with polymerase chain reaction and subjected to direct DNA sequencing.Based on DNA sequence analysis, all of the three patients have carried OTC gene mutations. Patients 1 and 2 were both hemizygous for mutation c.586G> A(p.D196N). A novel mutation c.800G> C(p.S267T) were confirmed in patient 3.p.S267T mutation has affected the conserved amino acid motif of the OTC protein, and is therefore a pathogenic mutation.

The MDMV (Maize Dwarf Mosaic Virus, MDMV) CP (Coat Protein, CP) gene was cloned by RT-PCR method and introduced into the embryonic calli derived from immature embryos of elite inbred 18-599hong and 18-599bai via particle bombardment. Bombarded calli were selected on selection medium containing 5-10 mg/L (PPT) Bialaphos. From resistant calli, 79 plantlets were regenerated. 18 of 79 were grown and harvested. The results of Southern blotting and PCR analysis demonstrated that MDMV CP have been integrated into the genome of the transgenic plants. PCR-positive progeny plants were artificially inoculated with MDMV strain B, and the average chlorosis of the functional leaves of each plant was investigated. The typical symptoms were observed from the leaves of the control inbreds. while, the presence of the MDMV CP gene provided resistance to inoculation with MDMV strain B.

In this study, we analyzed the physical interactions of the dominant negative isoform of MoYpt7. Our results show that MoYpt7 interacts with MoGdi1. The dominant negative isoform of MoYpt7 (dominant negative isoform, N125I) is essential for colony morphology, conidiation, and pathogenicity in the rice blast fungus. These results further demonstrate the biological functions of MoYpt7 in Magnaporthe oryzae.

Abstract Dominant white phenotype in pigs is considered to be caused by two structural mutations in KIT gene, including a 450-kb duplication encompassing the entire KIT gene, and a splice mutation (G &gt; A) at the first base in intron 17, which leads to the deletion of exon 17 in mature KIT mRNA, and the production of KIT protein lacking a critical catalytic domain of kinase. However, this speculation has not yet been validated by functional studies. Here, by using CRISPR/Cas9 technology, we created two mouse models mimicing the structural mutations of KIT gene in dominant white pigs, including the splice mutation mouse model KIT D17/+ with exon 17 of one allele of KIT gene deleted, and duplication mutation mouse model KIT Dup/+ with one allele of KIT gene coding sequence (CDS) duplicated. We found that each mutation individually can not cause dominant white phenotype. Splice mutation homozygote is lethal and heterozygous mice present piebald coat. Inconsistent with previous speculation, we found KIT gene duplication mutation did not confer the patched phenotype, and had no obvious impact on coat color. Interestingly, combination of these two mutations lead to dominant white phenotype. Further molecular analysis revealed that combination of these two structural mutations could inhibit the kinase activity of the KIT protein, thus reduce the phosphorylation level of PI3K and MAPK pathway associated proteins, which may be related to the observed impaired migration of melanoblasts during embryonic development, and eventually lead to dominant white phenotype. Our study provides a further insight into the underlying genetic mechanisms of porcine dominant white coat colour. Author summary KIT plays a critical role in control of coat colour in mammals. Two mutation coexistence in KIT are considered to be the cause of the Dominant white phenotype in pigs. One mutation is a 450-kb large duplication encompassing the entire KIT gene, another mutation is a splice mutation causing the skipping of KIT exon 17. The mechanism of these two mutations of KIT on coat color formation has not yet been validated. In this study, by using genome edited mouse models, we found each structural mutation individual does not lead dominant white phenotype, but combination of these two mutations could lead to a nearly complete white coat colour similar to pig dominant white phenotype, possibly due to the inhibition of the kinase activity of the KIT protein, thus its signalling function on PI3K and MAPK pathways, leading to impaired migration of melanoblasts during embryonic development, and eventually lead to dominant white phenotype. Our study provides a further insight into the underlying genetic mechanisms of porcine dominant white coat colour.

The complete mitogenome sequence of Pseudohynobius jinfo, which is endemic to China, is sequenced in the present study. The genome was 16,393 bp in length, including 13 typical vertebrate protein-coding genes, 2 rRNA genes, 22 tRNA genes, and 1 non-coding control region. Except for ND6 and eight tRNA genes, all other mitochondrial genes were encoded on the heavy strand. The gene order and composition of P. jinfo was similar to that of the most Hynobiidae. Seven genes (ND1, ND2, COII, COIII, ND3, ND4 and Cytb) had an incomplete stop codon. Base composition of the genome was A (33.6%), T (31.8%), C (20.6%) and G (14.1%) with an A + T-rich feature (65.4%) as that of other vertebrate mitochondrial genomes. There was a long non-coding region (126 bp) between tRNA-Thr and tRNA-Pro genes.

Objective To review the clinical features of a Chinese family with glutaric acidemia type Ⅰ and analyze the glutaryl-CoA dehydrogenase(GCDH) gene mutation. Methods The patient's clinical data were collected from glutaric acidemia type Ⅰ family, including data of brain computer tomography (CT), magnetic resonance imaging (MRI) examination, urine organic acid and blood carnitine tandem mass analysis.The family members' genomic DNA was extracted from peripheral blood leukocytes.The 11 exons and their flanking sequences of GCDH gene were amplified with PCR and subjected to direct DNA sequencing. Results The proband's head circumference and visible exte-rior were normal.The Glasgow Coma Scale(GCS) score of the patient was 15.The four limbs activities and muscular tension were normal.The muscle strength was grade V. The Babinski sign, Brinell syndrome and Klinefelter syndrome were negative.CT findings revealed widened bilateral frontotemporal subdural interval, mild hydrocephalus, equidensite arc shadow at the right frontal parietal.MRI findings demonstrated bilateral frontotemporal atrophy, broadened cerebral sulci, fissures, sylvian fissure and subarachnoid space in the front temporal lobe.The T1, T2 and diffusion weighted image showed abnormal signals in the bilateral globus pallidus and the central white matter of the frontal lobe.The density of cerebral hemisphere white matte was attenuated.In the temporal parietal subdural, equal T1, moderate T2 and high intensity fluid-attenuated inversion recovery signals were detected.The inherited metabolic diseases screening showed high urinary glutaric acid excretion.The blood glutarylcarnitine was 0.34 μmol/L (0-0.20 μmol/L as normal reference) which was detected by tandem mass spectrometry.GCDH gene sequencing analysis confirmed that the proband was compound heterozygous mutations with c. 1205G>A and IVS10-2A>C.The pedigree analysis revealed that the proband's monozygotic twin little sister was also an glutaric acidemia type Ⅰ patient.The genotype of the little sister was completely consistent with proband.The clinical symptoms and disease severity were similar between the monozygotic twins. Conclusions The monozygotic twins who shared the same mutation and genetic background can have similar phenotypes and clinical symptoms.IVS10-2A>C is the prevalent GCHD gene mutation type in Chinese glutaric acidemia type I patients. Key words: Glutaric acidemia type Ⅰ; Glutaryl-CoA dehydrogenase; Monozygotic twins; Inborn errors metabolism; Compound heterozygous

OBJECTIVE The congenital long QT syndrome (LQTs) is a hereditary disorder in which most affected family members have delayed ventricular repolarization manifested on the electrocardiogram (ECG) as QT interval prolongation. The disorder is associated with an increased propensity to arrhythmogenic syncope, polymorphous ventricular tachycardia (torsade de pointes), and sudden arrhythmic death. LQTs is due to mutations involving principally the myocyte ion-channels, and this monogenetic disorder has an autosomal inheritance pattern. This study investigated the gene mutation of a Chinese family of LQTs with multiple phenotypes including dilated cardiomyopathy (DCM) and cardiac conduction defects, thus to understand the molecular pathogenesis of the diseases. METHODS A three-generation Chinese LQTs family with multiple phenotypes was investigated. Blood sample was collected from the 8 family members and 100 unassociated normal individuals. Polymerase chain reaction (PCR)-DNA direct sequencing was performed to screen all exons and their flanking introns of SCN5A gene for mutation analysis. Polymerase chain reaction-single strand conformation polymorphism (PCR-SSCP) was used to exclude polymorphism. RESULTS PCR amplification and subsequent direct sequencing of SCN5A from proband revealed a heterozygous deletion of nine base pairs (CAGAAGCCC) in exon 26, corresponding to the three amino acid residues Gln1507-Lys1508-Pro1509 (QKP). This mutation is localized in the linker region between DIII-DIV of SCN5A. The same mutation was found in another patient (her grandmother) and excluded in the remaining living subjects in this family. This mutation was confirmed using SSCP in 100 unassociated healthy individuals. Similar analysis excluded possible mutations that would lead to amino acid changes in KCNQ1, KCNH2 and LAMIN A/C commonly associated with LQTs and DCM with conduction disorders, no new mutations that would lead to amino acid changes was found. CONCLUSION The result of the present study suggests that SCN5A mutation delQKP1507-1509 exists in patients with LQTs. The delQKP1507-1509 of SCN5A is a novel mutation in Chinese people. The same mutation was previously reported in a French family with only a single LQTs phenotype. Further studies on functional expression of SCN5A mutation delQKP1507-1509 will be helpful to understand the mechanism of the multiple phenotypes.

Peroxisomes are ubiquitous organelles in eukaryotic cells that fulfill multiple important metabolisms. Pex13 and Pex14 are key components of the peroxisomal docking complex in yeasts and mammals. In the present work, we functionally characterized the homologues of Pex13 and Pex14 (Mopex13 and Mopex14) in the rice blast fungus <i>Magnaporthe oryzae</i>. Mopex13 and Mopex14 were peroxisomal membrane distributed and were both essential for the maintenance of Mopex14/17 on the peroxisomal membrane. Mopex13 and Mopex14 interacted with each other, and with Mopex14/17 and peroxisomal matrix protein receptors. Disruption of Mopex13 and Mopex14 resulted in a cytoplasmic distribution of peroxisomal matrix proteins and the Woronin body protein Hex1. In the ultrastructure of Δmopex13 and Δmopex14 cells, peroxisomes were detected on fewer occasions, and the Woronin bodies and related structures were dramatically affected. The Δmopex13 and Δmopex14 mutants were reduced in vegetative growth, conidial generation and mycelial melanization, in addition, Δmopex13 showed reduced conidial germination and appressorial formation and abnomal appressorial morphology. Both Δmopex13 and Δmopex14 were deficient in appressorial turgor and nonpathogenic to their hosts. The infection failures in Δmopex13 and Δmopex14 were also due to their reduced ability to degrade fatty acids and to endure reactive oxygen species and cell wall-disrupting compounds. Additionally, Mopex13 and Mopex14 were required for the sexual reproduction of the fungus. These data indicate that Mopex13 and Mopex14, as key components of the peroxisomal docking complex, are indispensable for peroxisomal biogenesis, fungal development and pathogenicity in the rice blast fungus.

We introduced MDMV CP gene into calli derived from immature embryos of elite inbredlines18-599Hong and18-599Bai by microprojectile bombardment.12fertile transformants of18-599Hong and6fertile trans-formants of18-599Bai were obtained from resistant calli which had been subcultured on selecting medium contain-ing8,10and5mg /L Bialaphos for3cycles with3weeks per cycle.The result of PCR amplification implied that the CP gene had been introduced into maize inbred lines18-599Hong and18-599Bai.

The process of protein translocation into the endoplasmic reticulum (ER) is the initial and decisive step in the biosynthesis of all secretory proteins and many soluble organelle proteins. In this process, the Sec61 complex is the protein-conducting channel for transport. In this study, we identified and characterized the β subunit of the Sec61 complex in <i>Magnaporthe oryzae</i> (MoSec61β). Compared with the wild-type strain Guy11, the Δ<i>Mosec61β</i> mutant exhibited highly branched mycelial morphology, reduced conidiation, high sensitivity to cell wall integrity stress, severely reduced virulence to rice and barley, and restricted biotrophic invasion. The turgor pressure of Δ<i>Mosec61β</i> was notably reduced, which affected the function of appressoria. Moreover, Δ<i>Mosec61β</i> was also sensitive to oxidative stress and exhibited a reduced ability to overcome plant immunity. Further examination demonstrated that MoSec61β affected the normal secretion of the apoplastic effectors Bas4 and Slp1. In addition, Δ<i>Mosec61β</i> upregulated the level of ER-phagy. In conclusion, our results demonstrate the importance of the roles played by MoSec61β in the fungal development and pathogenesis of <i>M. oryzae</i>.

The process of protein translocation into the endoplasmic reticulum (ER) is the initial and decisive step in the biosynthesis of all secretory proteins and many soluble organelle proteins. In this process, the Sec61 complex is the protein-conducting channel for transport. In this study, we identified and characterized the β subunit of the Sec61 complex in <i>Magnaporthe oryzae</i> (MoSec61β). Compared with the wild-type strain Guy11, the Δ<i>Mosec61β</i> mutant exhibited highly branched mycelial morphology, reduced conidiation, high sensitivity to cell wall integrity stress, severely reduced virulence to rice and barley, and restricted biotrophic invasion. The turgor pressure of Δ<i>Mosec61β</i> was notably reduced, which affected the function of appressoria. Moreover, Δ<i>Mosec61β</i> was also sensitive to oxidative stress and exhibited a reduced ability to overcome plant immunity. Further examination demonstrated that MoSec61β affected the normal secretion of the apoplastic effectors Bas4 and Slp1. In addition, Δ<i>Mosec61β</i> upregulated the level of ER-phagy. In conclusion, our results demonstrate the importance of the roles played by MoSec61β in the fungal development and pathogenesis of <i>M. oryzae</i>.