Cloning and Partial Characterization of Endopolygalacturonase Genes from Botrytis cinerea

Botrytis cinerea is a plant-pathogenic fungus infecting over 200 different plant species. We use a molecular genetic approach to study the process of pectin degradation by the fungus. Recently, we described the cloning and characterization of an endopolygalacturonase (endoPG) gene from B. cinerea (Bcpg1) which is required for full virulence. Here we describe the cloning and characterization of five additional endoPG-encoding genes from B. cinerea SAS56. The identity at the amino acid level between the six endoPGs of B. cinerea varied from 34 to 73%. Phylogenetic analysis, by using a group of 35 related fungal endoPGs and as an outgroup one plant PG, resulted in the identification of five monophyletic groups of closely related proteins. The endoPG proteins from B. cinerea SAS56 could be assigned to three different monophyletic groups. DNA blot analysis revealed the presence of the complete endoPG gene family in other strains of B. cinerea, as well as in other Botrytis species. Differential gene expression of the gene family members was found in mycelium grown in liquid culture with either glucose or polygalacturonic acid as the carbon source.     

Genomic analysis of the necrotrophic fungal pathogens Sclerotinia sclerotiorum and Botrytis cinerea

Sclerotinia sclerotiorum and Botrytis cinerea are closely related necrotrophic plant pathogenic fungi notable for their wide host ranges and environmental persistence. These attributes have made these species models for understanding the complexity of necrotrophic, broad host-range pathogenicity. Despite their similarities, the two species differ in mating behaviour and the ability to produce asexual spores. We have sequenced the genomes of one strain of S. sclerotiorum and two strains of B. cinerea. The comparative analysis of these genomes relative to one another and to other sequenced fungal genomes is provided here. Their 38-39 Mb genomes include 11,860-14,270 predicted genes, which share 83% amino acid identity on average between the two species. We have mapped the S. sclerotiorum assembly to 16 chromosomes and found large-scale co-linearity with the B. cinerea genomes. Seven percent of the S. sclerotiorum genome comprises transposable elements compared to <1% of B. cinerea. The arsenal of genes associated with necrotrophic processes is similar between the species, including genes involved in plant cell wall degradation and oxalic acid production. Analysis of secondary metabolism gene clusters revealed an expansion in number and diversity of B. cinerea-specific secondary metabolites relative to S. sclerotiorum. The potential diversity in secondary metabolism might be involved in adaptation to specific ecological niches. Comparative genome analysis revealed the basis of differing sexual mating compatibility systems between S. sclerotiorum and B. cinerea. The organization of the mating-type loci differs, and their structures provide evidence for the evolution of heterothallism from homothallism. These data shed light on the evolutionary and mechanistic bases of the genetically complex traits of necrotrophic pathogenicity and sexual mating. This resource should facilitate the functional studies designed to better understand what makes these fungi such successful and persistent pathogens of agronomic crops.     

Functional analysis of an extracellular catalase of Botrytis cinerea

There is evidence that the necrotrophic fungal pathogen Botrytis cinerea is exposed to oxidative processes within plant tissues. The pathogen itself also generates active oxygen species and H₂O₂ as pathogenicity factors. Our aim was to study how the pathogen may defend itself against cellular damage caused by the accumulation of H₂O₂ and the role of an extracellular catalase in its detoxification during the infection of tomato and bean plants by B. cinerea. Chloronaphthol staining followed by light microscopy showed that H₂O₂ accumulates in the infection zone in tomato and bean leaves. An extracellular catalase gene (denominated Bccat2) was cloned from B. cinerea. Exposure of mycelium to H₂O₂ in liquid culture resulted in increased Bccat2 mRNA levels in a concentration-dependent manner. Bccat2 mRNA was detected at early stages of tomato leaf infection, suggesting that B. cinerea experiences oxidative stress. Bccat2-deficient mutants were generated by transformation-mediated gene disruption. Mutants were more sensitive then the wild-type strain to H₂O₂in vitro, but they partly compensated for the absence of BcCAT2 by activating other protective mechanisms in the presence of H₂O₂. Bccat2-deficient mutants did not display a consistent reduction of virulence on bean and tomato leaves. Cerium chloride staining of infected leaf tissue for ultrastructural studies showed that Bccat2-deficient mutants were exposed to H₂O₂ comparably to the wild-type. The results suggest that B. cinerea is a robust pathogen adapted to growing in hostile oxidizing environments in host tissues.     

Overexpression of calreticulin modulates protein kinase B/Akt signaling to promote apoptosis during cardiac differentiation of cardiomyoblast H9c2 cells

Calreticulin is a Ca(2+)-binding molecular chaperone of the lumen of the endoplasmic reticulum. Calreticulin has been shown to be essential for cardiac and neural development in mice, but the mechanism by which it functions in cell differentiation is not fully understood. To examine the role of calreticulin in cardiac differentiation, the calreticulin gene was introduced into rat cardiomyoblast H9c2 cells, and the effect of calreticulin overexpression on cardiac differentiation was examined. Upon culture in a differentiation medium containing fetal calf serum (1%) and retinoic acid (10 nm), cells transfected with the calreticulin gene were highly susceptible to apoptosis compared with controls. In the gene-transfected cells, protein kinase B/Akt signaling was significantly suppressed during differentiation. Furthermore, protein phosphatase 2A, a Ser/Thr protein phosphatase, was significantly up-regulated, implying suppression of Akt signaling due to dephosphorylation of Akt by the up-regulated protein phosphatase 2A via regulation of Ca(2+) homeostasis. Thus, overexpression of calreticulin promotes differentiation-dependent apoptosis in H9c2 cells by suppressing the Akt signaling pathway. These findings indicate a novel mechanism by which cytoplasmic Akt signaling is modulated to cause apoptosis by a resident protein of the endoplasmic reticulum, calreticulin.     

广州南沙番石榴枯萎病的病原菌

近年来,以枝条萎蔫、枝干干枯直至整株枯死为典型症状的番石榴枯萎病在广州南沙区严重发生。为明确该病害的病因,我们进行了病害调查、标本采集、病原菌分离和致病性测定;通过病原菌的显微形态比较和基于多基因系统发育分析,鉴定了病原菌种类;通过对寄主不同部位接种,测定了病原菌对寄主根、茎干和果的侵染能力。结果表明,引起广州南沙番石榴枯萎病的病原菌为番石榴纳氏霉Nalanthamala psidii,该菌不仅能从有伤的枝条侵染造成整株枯死,还能从根部侵染,使植株生长明显减缓,最终造成植株枯死,此外还可以侵染果实造成腐烂症状。本研究明确了广州南沙发生的番石榴枯萎病与中国台湾、马来西亚和南非报道的番石榴立枯病是一种病害,均是由番石榴纳氏霉侵染引起,为该病害的侵染特性及防控技术研究提供理论依据。     

一株谦逊迷孔菌Daedalea modesta的分离及其杀线虫的作用

为了研究野生担子菌对线虫的作用效果,从腐木桩上采集担子菌,应用常规组织分离法获得纯培养菌丝,以全齿复活线虫作靶标,初步筛选到1株对线虫有高活性的菌株,应用形态学方法和分子生物学方法进行了种的鉴定,测定了其对全齿复活线虫Panagrellus redivivus、松材线虫Bursaphelenchus xylophilus、爪哇根结线虫Meloidogyne javanica的杀线虫活性,并测定了其在不同条件下的发酵液对全齿复活线虫、松材线虫和爪哇根结线虫二龄幼虫的作用效果。经鉴定该菌株为谦逊迷孔菌Daedalea modesta,其菌丝在平板上能够迅速杀死线虫,平板菌落上接种线虫48h,菌丝对全齿复活线虫、松材线虫和爪哇根结线虫的致死率分别为95.61%、85.75%和100%;其马铃薯葡萄糖液体发酵液25℃处理全齿复活线虫、松材线虫和爪哇根结线虫二龄虫24h线虫死亡率均达100%,发酵液稀释10×时,对爪哇根结线虫仍有致死效果。本研究为该菌株用于线虫防治的进一步研究提供了科学参考。     

Sodium diethyldithiocarbamate protects against the MPTP-induced inhibition of immune responses in mice

The effects of 1-methyl-4-phenyl - 1,2,3,6-tetrahydropyridine (MPTP) on immune parameters, and the restorative influence of sodium diethyldithiocarbamate (DTC) or deprenyl were evaluated in mice. The concentrations of dopamine (DA), 3-methoxytyramine (3-MT), 3-4-dihydroxyphenyl acetic acid (DOPAC), and homovanillic acid (HVA), were concomitantly measured in the striatum. MPTP depressed T-cell responses. DTC restored these responses as well as the concentration of striatal DA. Deprenyl had no effect on the concentrations of DA and its metabolites, yet it modified the immune responses alike MPTP. The findings suggest a dopamine pathway could be involved in the brain-controlled immunostimulation afforded by DTC.