Immunomagnetic purification of Colletotrichum lindemuthianum appressoria

We developed a method to purify appressoria of the bean anthracnose fungus Colletotrichum lindemuthianum for biochemical analysis of the cell surface and to compare appressoria with other fungal structures. We used immunomagnetic separation after incubation of infected bean leaf homogenates with a monoclonal antibody that binds strongly to the appressoria. Preparations with a purity of >90% could be obtained. Examination of the purified appressoria by transmission electron microscopy showed that most had lost their cytoplasm. However, the plasma membrane was retained, suggesting that there is some form of attachment of this membrane to the cell wall. The purified appressoria can be used for studies of their cell surface, and we have shown that there are clear differences in the glycoprotein constituents of cell walls of appressoria compared with mycelium.     

西瓜炭疽病菌圆刺盘孢分生孢子产生条件的研究

为研究圆刺盘孢Colletotrichum orbiculare的分生孢子产生条件,通过改变培养基的成分、理化性状及培养条件等方法,进行了诱导产孢试验,并对所产生的分生孢子量进行测定。结果表明,C.orbiculare在西瓜茎叶煎汁培养基上25℃恒温黑暗培养30d可产生大量分生孢子,产孢量高达7.15×109个/m L。在显微镜下观察发现,病菌在西瓜茎叶煎汁培养基上和在西瓜茎秆病斑上产生的分生孢子在形态上存在差异,前者产生的分生孢子稍短和略宽,两端钝圆;而后者产生的分生孢子相对长而细,两端较尖。试验证明寄主西瓜茎叶成分对病菌分生孢子的产生具有明显促进作用。     

Interleukin-4 regulates lipid metabolism by inhibiting adipogenesis and promoting lipolysis

Long-term cytokine-mediated inflammation is a risk factor for obesity and type 2 diabetes mellitus (T2DM). Our previous studies reveal significant associations between promoter single nucleotide polymorphisms (SNPs) of interleukin (IL)-4 and T2DM, as well as between SNPs in genes encoding IL-4/IL-4 receptor and high density lipoproteins. Our animal study reveals that IL-4 regulates glucose/lipid metabolism by promoting glucose tolerance and inhibiting lipid deposits. The above results strongly suggest the involvement of IL-4 in energy homeostasis. In the present study, we focus on examining the regulatory mechanism of IL-4 to lipid metabolism. Our results show that IL-4 inhibits adipogenesis by downregulating the expression of peroxisome proliferator-activated receptor-γ and CCAAT/enhancer-binding protein-α. Additionally, IL-4 promotes lipolysis by enhancing the activity and translocation of hormone sensitive lipase (HSL) in mature adipocytes, which suggests that IL-4 plays a pro-lipolytic role in lipid metabolism by boosting HSL activity. Our results demonstrate that IL-4 harbors pro-lipolysis capacity by inhibiting adipocyte differentiation and lipid accumulation as well as by promoting lipolysis in mature adipocytes to decrease lipid deposits. The above findings uncover the novel roles of IL-4 in lipid metabolism and provide new insights into the interactions among cytokine/immune responses, insulin sensitivity, and metabolism.     

Quantification of substratum contact required for initiation of Colletotrichum graminicola appressoria

Colletotrichum graminicola, like many plant pathogenic fungi develop appressoria on germling apices, to facilitate penetration of their host. Induction of these structures occurs after contact with the host surface has been established by the germling. Surface contact and subsequent development of appressoria by germlings of C. graminicola was assessed using interference-reflection microscopy (IRM) and microfabricated pillared silicon substrata. Observations with IRM revealed that under low nutrient conditions, 90% of the germlings developed appressoria once they established 4.5 microm of continuous contact with the substratum. Substrata bearing pillars < or =5 microm in width supported < or =10% appressoria; however, as pillar width was increased the percentage of appressoria formed increased in a sigmoid fashion to a maximum of 80%. The percentage of appressoria produced experimentally on these surfaces was compared to data sets generated from a model designed to calculate the probability of appressorium development on similar pillar arrays at various germ tube contact lengths. These results indicate that germ tubes of C. graminicola require more than 4microm of continuous contact with a hydrophobic substratum for induction of appressoria.     

Temporal and fluoride control of secondary metabolism regulates cellular organofluorine biosynthesis

Elucidating mechanisms of natural organofluorine biosynthesis is essential for a basic understanding of fluorine biochemistry in living systems as well as for expanding biological methods for fluorine incorporation into small molecules of interest. To meet this goal we have combined massively parallel sequencing technologies, genetic knockout, and in vitro biochemical approaches to investigate the fluoride response of the only known genetic host of an organofluorine-producing pathway, Streptomyces cattleya. Interestingly, we have discovered that the major mode of S. cattleya's resistance to the fluorinated toxin it produces, fluoroacetate, may be due to temporal control of production rather than the ability of the host's metabolic machinery to discriminate between fluorinated and non-fluorinated molecules. Indeed, neither the acetate kinase/phosphotransacetylase acetate assimilation pathway nor the TCA cycle enzymes (citrate synthase and aconitase) exclude fluorinated substrates based on in vitro biochemical characterization. Furthermore, disruption of the fluoroacetate resistance gene encoding a fluoroacetyl-CoA thioesterase (FlK) does not appear to lead to an observable growth defect related to organofluorine production. By showing that a switch in central metabolism can mediate and control molecular fluorine incorporation, our findings reveal a new potential strategy toward diversifying simple fluorinated building blocks into more complex products.     

A yeast STE11 homologue CoMEKK1 is essential for pathogenesis-related morphogenesis in Colletotrichum orbiculare

Several signal transduction pathways, including mitogen-activated protein kinase (MAPK) pathways, are involved in appressorium development in Colletotrichum orbiculare, the causal agent of cucumber anthracnose disease. In this study, CoMEKK1, a yeast MAPK kinases (MAPKK) kinase STE11 homolog, was identified as a disrupted gene in an Agrobacterium tumefaciens-mediated transformation mutant. The phenotype of comekk1 disruptant was similar to that of cmk1, a Saccharomyces cerevisiae Fus3/Kss1 MAPK homolog mutant. Moreover, comekk1 and cmk1 mutants were sensitive to high osmotic and salinity stresses, indicating that Comekk1p/Cmk1p signal transduction is involved in stress tolerance. The transformants of the wild type and the comekk1 mutant expressing a constitutively active form of the CoMEKK1 showed slower hyphal growth and abnormal appressorium formation, whereas those of the cmk1 disruptant did not. A Cmk1p-green fluorescent protein (GFP) intracellular localization experiment indicated that nuclear localization of the Cmk1p-GFP fusion protein induced by salt stress was diminished in comekk1 mutants. These results indicate that Comekk1p functions upstream of Cmk1p.     

Fungal effector SIB1 of Colletotrichum orbiculare has unique structural features and can suppress plant immunity in Nicotiana benthamiana

Fungal plant pathogens secrete virulence-related proteins, called effectors, to establish host infection; however, the details are not fully understood yet. Functional screening of effector candidates using Agrobacterium-mediated transient expression assay in Nicotiana benthamiana identified two virulence-related effectors, named SIB1 and SIB2 (Suppression of Immunity in N. benthamiana), of an anthracnose fungus Colletotrichum orbiculare, which infects both cucurbits and N. benthamiana. The Agrobacterium-mediated transient expression of SIB1 or SIB2 increased the susceptibility of N. benthamiana to C. orbiculare, which suggested these effectors can suppress immune responses in N. benthamiana. The presence of SIB1 and SIB2 homologs was found to be limited to the genus Colletotrichum. SIB1 suppressed both (i) the generation of reactive oxygen species triggered by two different pathogen-associated molecular patterns, chitin and flg22, and (ii) the cell death response triggered by the Phytophthora infestans INF1 elicitin in N. benthamiana. We determined the NMR-based structure of SIB1 to obtain its structural insights. The three-dimensional structure of SIB1 comprises five β-strands, each containing three disulfide bonds. The overall conformation was found to be a cylindrical shape, such as the well-known antiparallel β-barrel structure. However, the β-strands were found to display a unique topology, one pair of these β-strands formed a parallel β-sheet. These results suggest that the effector SIB1 present in Colletotrichum fungi has unique structural features and can suppress pathogen-associated molecular pattern–triggered immunity in N. benthamiana.     

Establishment of a selection marker recycling system for sequential transformation of the plant-pathogenic fungus Colletotrichum orbiculare

Genome sequencing of pathogenic fungi has revealed the presence of various effectors that aid pathogen invasion by the manipulation of plant immunity. Effectors are often individually dispensable because of duplication and functional redundancy as a result of the arms race between host plants and pathogens. To study effectors that have functional redundancy, multiple gene disruption is often required. However, the number of selection markers that can be used for gene targeting is limited. Here, we established a marker recycling system that allows the use of the same selection marker in successive transformations in the model fungal pathogen Colletotrichum orbiculare, a causal agent of anthracnose disease in plants belonging to the Cucurbitaceae. We identified two C. orbiculare homologues of yeast URA3/pyrG, designated as URA3A and URA3B, which can be used as selection markers on medium with no uridine. The gene can then be removed from the genome via homologous recombination when the fungus is grown in the presence of 5-fluoroorotic acid (5-FOA), a chemical that is converted into a toxin by URA3 activity. The ura3a/b double mutants showed auxotrophy for uridine and insensitivity to 5-FOA. Using the ura3a/b mutants, transformation with the URA3B marker and its removal were successfully applied to disrupt the virulence-related gene, PKS1. The pks1 mutants showed a reduction in virulence, demonstrating that the method can be used to study virulence-related genes in C. orbiculare. The establishment of a URA3-based marker recycling system in plant-pathogenic fungi enables the genetic analysis of multiple genes that have redundant functions, including effector genes.     

FAT SIGNALS--lipases and lipolysis in lipid metabolism and signaling

Lipolysis is defined as the catabolism of triacylglycerols stored in cellular lipid droplets. Recent discoveries of essential lipolytic enzymes and characterization of numerous regulatory proteins and mechanisms have fundamentally changed our perception of lipolysis and its impact on cellular metabolism. New findings that lipolytic products and intermediates participate in cellular signaling processes and that "lipolytic signaling" is particularly important in many nonadipose tissues unveil a previously underappreciated aspect of lipolysis, which may be relevant for human disease.     

Rosiglitazone regulates IL-6-stimulated lipolysis in porcine adipocytes

Interleukin (IL)-6, a proinflammatory cytokine, stimulates adipocyte lipolysis and induces insulin resistance in obese and diabetic subjects. However, the effects of the anti-diabetic drug rosiglitazone on IL-6-stimulated lipolysis and the underlying molecular mechanism are largely unknown. In this study, we demonstrated that rosiglitazone suppressed IL-6-stimulated lipolysis in differentiated porcine adipocytes by inactivation of extracellular signal-related kinase (ERK). Meanwhile, rosiglitazone enhanced the lipolysis response of adipocytes to isoprenaline. In addition, rosiglitazone significantly reversed IL-6-induced down-regulation of several genes such as perilipin A, peroxisome proliferators activated receptor gamma (PPARγ), and fatty acid synthetase, as well as the up-regulation of IL-6 mRNA. However, mRNA expression of PPARγ coactivator-1 alpha (PCG-1α) was enhanced by rosiglitazone in IL-6-stimulated adipocytes. These results indicate that rosiglitazone suppresses IL-6-stimulated lipolysis in porcine adipocytes through multiple molecular mechanisms.     

DHA regulates lipogenesis and lipolysis genes in mice adipose and liver

Docosahexaenoic acid (DHA) is one kind of ω-3 polyunsaturated fatty acids (PUFAs) and plays an important role in lipid metabolism. In this research, mice were daily intragastric administrated with DHA for 3 weeks. Subcutaneous adipose tissue and liver were separated every week, RNA was extracted. Peroxisome proliferator-activated receptor (PPARγ), Sterol regulatory element binding protein-1c (SREBP-1c), Fatty acid synthetase (FAS), Hormone sensitive lipase (HSL) and triglyceride hydrolase TGH genes expression were detected by quantitative PCR. Data showed that, DHA up-regulated PPARγ, HSL and TGH in adipose tissue, but it had no effect on SREBP-1c and FAS expression. However, in liver there were some differences in regulating these genes. PPARγ, SREBP-1c and FAS were down-regulated, HSL was up-regulated and TGH had no change. These results indicated that DHA played different regulating roles in lipid metabolism in different tissues. In adipose tissue, DHA increased the expression of lipogenesis and lipolysis genes. In liver lipogenesis genes were decreased, but lipolysis genes were increased by DHA. In conclusion, DHA could reduce body fat mass through regulating lipogenesis and lipolysis genes.     

H3K4 trimethylation by CclA regulates pathogenicity and the production of three families of terpenoid secondary metabolites in Colletotrichum higginsianum

The role of histone 3 lysine 4 (H3K4) methylation is poorly understood in plant pathogenic fungi. Here, we analysed the function of CclA, a subunit of the COMPASS complex mediating H3K4 methylation, in the brassica anthracnose pathogen Colletotrichum higginsianum. We show that CclA is required for full genome-wide H3K4 trimethylation. The deletion of cclA strongly reduced mycelial growth, asexual sporulation and spore germination but did not impair the morphogenesis of specialized infection structures (appressoria and biotrophic hyphae). Virulence of the ΔcclA mutant on plants was strongly attenuated, associated with a marked reduction in appressorial penetration ability on both plants and inert cellophane membranes. The secondary metabolite profile of the ΔcclA mutant was greatly enriched compared to that of the wild type, with three different families of terpenoid compounds being overproduced by the mutant, namely the colletochlorins, higginsianins and sclerosporide. These included five novel molecules that were produced exclusively by the ΔcclA mutant: colletorin D, colletorin D acid, higginsianin C, 13-epi-higginsianin C and sclerosporide. Taken together, our findings indicate that H3K4 trimethylation plays a critical role in regulating fungal growth, development, pathogenicity and secondary metabolism in C. higginsianum.     

Histochemical Studies of the Non‐Host Resistance and the Role of Actin Cytoskeleton in Pepper Against Colletotrichum orbiculare

The penetration process and defence reactions (hypersensitive response, oxidative burst and cell wall fortification) of Colletotrichum orbiculare were studied histochemically on pepper cultivar ‘A11’ (non‐host) and susceptible cucumber cultivar ‘Changchun Thorn’ (host). The results indicate that C. orbiculare could hardly penetrate the non‐host pepper leaves. It was papillae rather than hypersensitive response and H₂O₂ that played an important role in resisting the colonization and development of C. orbiculare on the non‐host pepper. The depolymerization of the actin microfilament weakened the papilla deposition of pepper and allowed successful penetration of the non‐adapted C. orbiculare, suggesting that the actin cytoskeleton of pepper is significant in preventing the invasion of the non‐host pathogen C. orbiculare.