A simple and effective method for total RNA isolation of appressoria in Magnaporthe oryzae

Appressorium formation is an important event in establishing a successful interaction between the rice blast fungus, Magnaporthe oryzae, and its host plant, rice. An understanding of molecular events occurring in appressorium differentiation will give new strategies to control rice blast. A quick and reliable method to extract total RNA from appressorium is essential for studying gene expression during appressorium formation and its mechanism. We found that duplicate film is an efficient substratum for appressorium formation, even when inoculated with high density conidia. When inoculated with conidia at 1 × 106 ml^-1, the percentages of conidium germination and appressorium formation were （97.98±0.67）% and （97.88±0.45）%, respectively. We applied Trizol before appressorium collection for total RNA isolation, and as much as 113.6 lag total RNA was isolated from the mature appressoria at 24 h after inoculation. Functional analysis of two genes, MNH6 and MgATG1, isolated from the cDNA subtractive library, revealed that the quantity of RNA was good enough to construct a cDNA （complementary DNA） library or a cDNA subtractive library. This method may be also applicable for the appressorium RNA isolation of other pathogenic fungi in which conidia differentiate into appressoria in the early stages of host infection.     

MoFLP1, encoding a novel fungal fasciclin-like protein, is involved in conidiation and pathogenicity in Magnaporthe oryzae

Fasciclin family proteins have been identified as cell adhesion molecules in various organisms. In this study, a novel Magnaporthe oryzae fasciclin-like protein encoding gene, named MoFLP1, was isolated from a subtractive suppressive cDNA library and functionally analyzed. Sequence analysis showed that the MoFLP1 gene contains an open reading frame (ORF) of 1050 nucleotides encoding 349 amino acids with a calculated molecular weight of 35.85 kDa and a pI of 7.76. The deduced MoFLP1 protein contains a 17-amino acid secretion signal sequence and an 18-amino acid sequence with the characteristics of a glycosylphosphotidylinositol (GPI) anchor additional signal at its N- and C-terminuses, respectively. Potential N-glycosylation sites and domains involving cell adhesion were also identified in MoFLP1. Sequence analysis and subcellular localization by the expression of MoFLP1-GFP fusion construct in M. oryzae indicated that the MoFLP1 protein is probably localized on the vacuole membrane. Two MoFLP1 null mutants generated by targeted gene disruption exhibited marked reduction of conidiation, conidial adhesion, appressorium turgor, and pathogenicity. Our results indicate that fasciclin proteins play important roles in fungal development and pathogenicity in M. oryzae.     

细胞自噬的调节机制与选择性降解

细胞自噬是一个多步骤的降解过程.当细胞自噬发生时,具有双层膜结构的自噬小体先把待降解的细胞内容物包裹起来,然后与溶酶体融合将内容物降解.细胞自噬起初仅被认为是对营养缺乏的适应性机制,是一个非选择性降解过程.近些年来不断深入的研究表明,细胞自噬同时还参与了抵抗炎症、肿瘤、神经退行性变及心脏病等重要生理过程.随着细胞自噬受体蛋白p62的发现,人们进一步认识到细胞自噬还是一个具有高度选择性的降解过程.本文综述了细胞自噬的调节机制及其对一些蛋白聚合体的选择性降解.     

Functional genomics in the rice blast fungus to unravel the fungal pathogenicity

A rapidly growing number of successful genome sequencing projects in plant pathogenic fungi greatly increase the demands for tools and methodologies to study fungal pathogenicity at genomic scale. Magnaporthe oryzae is an economically important plant pathogenic fungus whose genome is fully sequenced. Recently we have reported the development and application of functional genomics platform technologies in M. oryzae. This model approach would have many practical ramifications in design and implementation of upcoming functional genomics studies of filamentous fungi aimed at understanding fungal pathogenicity.     

Resistance to rice blast (Pyricularia oryzae) caused by the expression of trichosanthin gene in transgenic rice plants transferred through agrobacterium method

The gene of trichosanthin has been transferred into rice plants through agrobacterium method. The single copy insertion and the expression of foreign gene have been proved in regenerated plants. In antifungal assay the degrees of rice blast (Pyricularia oryzae) infection of the transgenic plants expressing trichosanthin and expressingGUS gene as control have been evaluated. The differences such as the time of disease symptom observed, the number of infected plants and damaged leaves, the growth of infected plants of the two transgenic plants after being inoculated by rice blast (Pyricularia oryzae) are significant. The transgenic plants with trichosanthin gene grew faster than the plants withGUS gene, even when humidity environment was removed. The results show that the transgenic plants that expressed trichosanthin are able to delay the infection of rice blast compared with the plants as control. In addition, no damage caused by the expression of trichosanthin gene in transgenic plants has been observed.     

Magnaporthe oryzae MTP1 gene encodes a type III transmembrane protein involved in conidiation and conidial germination

In this study the MTP1 gene, encoding a type III integral transmembrane protein, was isolated from the rice blast fungus Magnaporthe oryzae. The Mtp 1 protein is 520 amino acids long and is comparable to the Ytp 1 protein of Saccharomyces cerevisiae with 46% sequence similarity. Prediction programs and MTP1-GFP （green fluorescent protein） fusion expression results indicate that Mtp 1 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 Amtpl 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.     

A Gγ subunit promoter T-DNA insertion mutant――A1-412 of Magnaporthe grisea is defective in appressorium formation, penetration and pathogenicity

Heterotrimeric G-proteins consisting of α, β and γ-subunits are essential for the transduction of ex- tracellular signals to various downstream intracellular effectors in eukaryotes. Previous studies showed that Gα and Gβ were involved in regulating     

Influence factor of diesel soot formation in diesel engine combustion predicted by multistep soot model with highlight in soot surface activity

As more strict regulations on soot emission with increasing emphasis on the emitted soot particle size have been imposed, diesel engine partially premixed combustion had been proven to achieve ultra-low NOx and soot emissions with high thermal efficiency simulta- neously, by synergy control of mixing and chemical parameters. It calls for further study in the effect of com- bustion boundaries on soot formation. In this study, soot formation characteristic was investigated by CFD code coupled with reduced n-heptane model and improved multistep soot model. History of acetylene, which is taken as the main species of PAH formation and soot surface growth, plays more important roles on soot prediction. The revised parameter of fraction of active sites αCH was introduced as the indicator of soot surface activity in diesel soot formation. The effects of combustion boundaries on soot surface activity and soot surface growth were explored in this study. When the mixture was quite homogeneous, lowered combustion temperature was the main factor for reduced soot formation due to the lowered specific surface growth rate RCH, in spite of ec/4 increasing slightly due to the slowed decay rate of surface activity. As the inhomo- geneity of the mixture was increased, more unburned hydrocarbons were produced, promoting the formation of acetylene and soot surface activity. It was the dominated reason for higher soot surface growth rate, resulted wors- ened engine-out soot. In addition, residual of CO in later combustion phase impeded the re-oxidation of soot.     

Ambra1 regulates autophagy and development of the nervous system

Autophagy is a self-degradative process involved both in basal turnover of cellular components and in response to nutrient starvation or organelle damage in a wide range of eukaryotes. During autophagy, portions of the cytoplasm are sequestered by double-membraned vesicles called autophagosomes, and are degraded after fusion with lysosomes for subsequent recycling. In vertebrates, this process acts as a pro-survival or pro-death mechanism in different physiological and pathological conditions, such as neurodegeneration and cancer; however, the roles of autophagy during embryonic development are still largely uncharacterized. Beclin1 (Becn1; coiled-coil, myosin-like BCL2-interacting protein) is a principal regulator in autophagosome formation, and its deficiency results in early embryonic lethality. Here we show that Ambra1 (activating molecule in Beclin1-regulated autophagy), a large, previously unknown protein bearing a WD40 domain at its amino terminus, regulates autophagy and has a crucial role in embryogenesis. We found that Ambra1 is a positive regulator of the Becn1-dependent programme of autophagy, as revealed by its overexpression and by RNA interference experiments in vitro. Notably, Ambra1 functional deficiency in mouse embryos leads to severe neural tube defects associated with autophagy impairment, accumulation of ubiquitinated proteins, unbalanced cell proliferation and excessive apoptotic cell death. In addition to identifying a new and essential element regulating the autophagy programme, our results provide in vivo evidence supporting the existence of a complex interplay between autophagy, cell growth and cell death required for neural development in mammals.     

Bioactivity and constituents of several common seaweeds

Bioactivity and constituents of 8 common seaweeds from Dalian intertidal zone of northern Yellow Sea were investigated. In the anti-methicillin-resistant Staphylococcus aureus (MRSA) test, Symphyocladia latiuscula and Enteromorpha intestinalis showed obvious activities with MICs much lower than 1.0 mg·mL-1 . In the DNA damage repair test (DDRT), Chondrus ocellatu showed selective inhibitory activity against the DNA repair-defective E. coli strain vs. the wild-type E. coli strain; while Sym. latiuscula, Enteromorpha intestinalis and Sar. kjellmanianum showed significant anti-E. coli activity with MICs of 64-128 g·mL-1 . In the anti-Pyricularia oryzae test, Sym. latiuscula and Rh. confervoides strongly inhibited the germination of the spores of P. oryzae on agar plate. In the brine shrimp larvicidal test, Sym. latiuscula, Rh. confervoides and Sar. kjellmanianum exhibited potent toxicity against brine shrimp larvae, with LC50 much lower than 1 mg·mL-1 . The HPTLC analysis revealed their diversified secondary metabolites. The HPLC-DAD-MS analysis of the strongest species Sym. latiuscula and database searching showed that it can produce quite diversified metabolites, including halogenated ones, some of which may be new natural products. The results demonstrated the potentials of these seaweeds in the development of new antibiotics, antitumor drugs, agricultural fungicides and pesticides.     

稻瘟菌致病性变异突变体的筛选及共分离分析

通过筛选稻瘟菌(Magnaporthe grisea)P131小种的REMI(Restriction Enzyme MediatedIntegration)转化体库获得对水稻品种梅雨明致病性变异的突变体,命名为PX1.与野生型菌株P131相比,该突变体对水稻品种梅雨明致病性丧失,在洋葱表皮上侵染钉形成率显著降低,而孢子萌发率和附着胞形成率差异不显著.遗传分析表明,该突变体的突变表型和潮霉素抗性标记共分离,说明突变是由于外源质粒插入引起的,因此,可以此为标记克隆控制该表型的基因.     

空心阴极放电自脉冲现象的实验研究

研究了氩气中圆柱形空心阴极放电的自脉冲现象.测试了空心阴极放电不同阶段的伏安特性及自脉冲频率和电流脉冲上升沿随放电平均电流、气压和电路电容的变化;讨论了自脉冲的形成机理. 结果表明,自脉冲现象出现在空心阴极放电的负阻特性阶段,并具有稳定的频率.自脉冲频率随平均电流的增加而线性增大,气压和孔径的乘积pD值对频率的影响与其大小有关,但电极间的并联电容不影响脉冲频率;电流脉冲上升沿几乎与平均电流、气压或并联电容无关. 实验结果表明,自脉冲现象是空心阴极本身的一种特征放电过程.     

蜡蚧轮枝菌侵染黑刺粉虱体表过程的显微观察

采用扫描电镜研究了蜡蚧轮枝菌侵染黑刺粉虱成虫的过程。结果表明：接种蜡蚧轮枝菌孢子12h后,附着在虫体上的分生孢子萌发形成芽管,24h后,在复眼、腹部气孔、足的基节和翅的基部等部位菌丝侵入黑刺粉虱体内,接种72h后虫体被厚厚的菌丝层覆盖。分生孢子可以直接以芽管侵入表皮,也可以产生附着胞再侵入。分生孢子在黑刺粉虱体表萌发形成芽管,芽管顶端可形成附着胞,附着胞再形成侵入钉穿透寄主体壁。寄主体表结构影响形成附着胞的形状及穿透体壁时芽管长度。穿刺点处的小孔及附着胞周围发现粘状分泌物,为蜡蚧轮枝菌在入侵黑刺粉虱过程中穿透体壁时对寄主表皮产生降解作用的酶。     

Toll-like receptor signalling in macrophages links the autophagy pathway to phagocytosis

Phagocytosis and autophagy are two ancient, highly conserved processes involved, respectively, in the removal of extracellular organisms and the destruction of organisms in the cytosol. Autophagy, for either metabolic regulation or defence, involves the formation of a double membrane called the autophagosome, which then fuses with lysosomes to degrade the contents, a process that has similarities with phagosome maturation. Toll-like-receptor (TLR) engagement activates a variety of defence mechanisms within phagocytes, including facilitation of phagosome maturation, and also engages autophagy. Therefore we speculated that TLR signalling might link these processes to enhance the function of conventional phagosomes. Here we show that a particle that engages TLRs on a murine macrophage while it is phagocytosed triggers the autophagosome marker LC3 to be rapidly recruited to the phagosome in a manner that depends on the autophagy pathway proteins ATG5 and ATG7; this process is preceded by recruitment of beclin 1 and phosphoinositide-3-OH kinase activity. Translocation of beclin 1 and LC3 to the phagosome was not associated with observable double-membrane structures characteristic of conventional autophagosomes, but was associated with phagosome fusion with lysosomes, leading to rapid acidification and enhanced killing of the ingested organism.     

Fluorescent co-localization of PTS1 and PTS2 and its application in analysis of the gene function and the peroxisomal dynamic in Magnaporthe oryzae

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 （PTS 1 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 Guy ll cells. Transformants containing these fusions emitted fluorescence in a punctate pattern, and the locations of the red and green fluorescence overlapped exactly in RFP-PTS 1 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 backgrotmds in GFP-PTS1 and GFP-PTS2 transformants. By introducing both RFP-PTS1 and GFP-PTS2 into Amgpex6 mutants, the involvement of MGPEX6 gene in both PTS1 and PTS2 pathways was proved. In addition, using these transformants, the inducement ofperoxisomes 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.     

DEX1, a plasma membrane-localized protein, functions in microspore development by affecting CalS5 expression in Arabidopsis thaliana

Microsporogenesis in flowering plants plays important roles in sexual reproduction. It has been reported that DEFECTIVE IN EXINE FORMATION1 (DEX1) is essential for exine pattern formation in Arabidopsis thaliana. However, the functions of DEX1 in regulating microspore development are largely not understood. In this study, we show that DEX1 is strongly expressed in the tapetum by using RNA in situ hybridization. dex1 microspores were degenerated and aborted after release from the tetrads. The callose wall in tetrads was thinner in the dex1 mutant than in the wild type, suggesting that DEX1 affects callose formation at the tetrad stage during anther development. RT-PCR and real-time PCR analyses showed that CalS5, which plays an important role in callose synthesis during microspore development, was greatly down-regulated in dex1 plants. DEX1 encodes a membrane protein with one transmembrane domain, one intracellular domain and one extracellular domain. Collectively, our results demonstrate that DEX1 is essential for microspore development, possibly by regulating the expression of CalS5.     

Noeggerathiales as coal-forming plants in Cathaysia: conclusions from an Early Permian vegetational Pompeii in Inner Mongolia

Noeggerathiales are an extinct group of sporebearing plants of uncertain systematic position that are known from Carboniferous and Permian age Euramerican and Cathaysian floras that occurred in presentday Europe, North America, and East Asia. The order Noeggerathiales includes over 50 species of more than 20 fossil genera, but their paleoecology is not well understood yet. Previously this group had been found only in extrabasinal floras or those inhabiting clastic wetlands. Noeggerathiales have never been recorded in coal ball floras. Thus, it is up to now uncertain whether this group has contributed to the formation of coal. Recent investigations of an Early Permian peatforming flora of the Taiyuan Formation near Wuda, Inner Mongolia, which was preserved in a volcanic ash fall has provided evidence that noeggerathialean plants not only existed in the peatforming vegetation but could even be the dominant group in some areas of the coal swamp. The Noeggerathiales in this particular peatforming forest include Tingia unita, Paratingia wudensis, and a new species of Paratingia. Exceptionally well-preserved specimens indicate that these noeggerathialean plants are small trees with a canopy of compound leaves and strobili near the top of an unbranched （monocaulous） stem.     

γ-氨丙基倍半硅氧烷的水解聚合机理研究及产物结构表征

以γ-氨丙基三乙氧基硅烷(H₂N·CH₂·CH₂·CH₂·Si(OC₂H₅)₃)为原料，通过控制其水解缩聚的条件制备聚γ-氨丙基倍半硅氧烷。本文详细论述了聚γ-氨丙基倍半硅氧烷的水解缩合过程，推断其水解缩合的机理，并利用FTIR，XRD，NMR等手段对聚合产物进行了结构表征，确定了合成产物的结构，证明产物中存在聚倍半硅氧烷结构。     

Ancient DNA sequences of rice from the low Yangtze reveal significant genotypic divergence

Rice (Oryza sativa) was first domesticated in the lower and middle Yangtze regions of China, and rice remains have been found in many Chinese archaeological sites. Until now, only phenotypic archeobotanical evidence, such as the spikelet bases of ancient grains, has been used to speculate on the domestication process and domestication rate of rice. In this study, we sequenced 4 genomic segments from rice remains in Tianluoshan, a site of the local Hemudu Neolithic culture in the low Yangtze and two other archaeological sites (??2400 and 1200 BC, respectively). We compared our sequences with those of the current domesticated and wild rice (O. rufipogon) populations. At least two genotypes were found in the remains from each site, suggesting a heterozygotic state of the rice seeds. One ancient genotype was not found in the current domesticated population and might have been lost. The rice remains belonged to the japonica group, and most if not all were japonica-type, suggesting that the remains might be at an early stage of indica-japonica divergence or an indica-japonica mixture. We also identified sequences with significant similarity to those from species of Sapindales, Zygophyllales, and Brassicales, which is consistent with the identification of other plant remains in the Tianluoshan site and the common rice field weeds such as mustards in southern China.     

Bombyx mori bidensovirus: The type species of the new genus Bidensovirus in the new family Bidnaviridae

Bombyx mori bidensovirus （BmBDV）, which had been assigned to Densovirinae in Parvoviridae previously, replicates mainly in silkworm midgut columnar cells and causes the fatal flacheric disease. In contrast to parvovirus, this virus possesses two single-stranded DNA genome segments and encodes a putative protein-primed DNA polymerase. The accumulating evidence sug- gests that it has unique characteristics different to parvovirus and adopts its own mechanisms for replication. So far, little is known about the replication mechanisms of BmBDV. In this review, we focus on the pathology associated with this virus and the viral biology such as viral genome structure, viral genes, and viral replication and expression strategies.