Autophagy provides nutrients for nonassimilating fungal structures and is necessary for plant colonization but not for infection in the necrotrophic plant pathogen Fusarium graminearum

The role of autophagy in necrotrophic fungal physiology and infection biology is poorly understood. We have studied autophagy in the necrotrophic plant pathogen Fusarium graminearum in relation to development of nonassimilating structures and infection. We identified an ATG8 homolog F. graminearum ATG8 whose first 116 amino acids before the predicted ATG4 cleavage site are 100% identical to Podospora anserina ATG8. We generated a ΔFgatg8 mutant by gene replacement and showed that this cannot form autophagic compartments. The strain forms no perithecia, has reduced conidia production and the aerial mycelium collapses after a few days in culture. The collapsing aerial mycelium contains lipid droplets indicative of nitrogen starvation and/or an inability to use storage lipids. The capacity to use carbon/energy stored in lipid droplets after a shift from carbon rich conditions to carbon starvation is severely inhibited in the ΔFgatg8 strain demonstrating autophagy-dependent lipid utilization, lipophagy, in fungi. Radial growth rate of the ΔFgatg8 strain is reduced compared with the wild type and the mutant does not grow over inert plastic surfaces in contrast to the wild type. The ability to infect barley and wheat is normal but the mutant is unable to spread from spikelet to spikelet in wheat. Complementation by inserting the F. graminearum atg8 gene into a region adjacent to the actin gene in ΔFgatg8 fully restores the WT phenotype. The results showed that autophagy plays a pivotal role for supplying nutrients to nonassimilating structures necessary for growth and is important for plant colonization. This also indicates that autophagy is a central mechanism for fungal adaptation to nonoptimal C/N ratios.     

Metabolomic analysis reveals a common pattern of metabolic re-programming during invasion of three host plant species by Magnaporthe grisea

The mechanisms by which biotrophic and hemi-biotrophic fungal pathogens simultaneously subdue plant defences and sequester host nutrients are poorly understood. Using metabolite fingerprinting, we show that Magnaporthe grisea , the causal agent of rice blast disease, dynamically re-programmes host metabolism during plant colonization. Identical patterns of metabolic change occurred during M. grisea infections in barley, rice and Brachypodium distachyon . Targeted metabolite profiling by GC-MS confirmed the modulation of a conserved set of metabolites. In pre-symptomatic tissues, malate and polyamines accumulated, rather than being utilized to generate defensive reactive oxygen species, and the levels of metabolites associated with amelioration of redox stress in various cellular compartments increased dramatically. The activity of NADP-malic enzyme and generation of reactive oxygen species were localized to pathogen penetration sites, and both appeared to be suppressed in compatible interactions. Early diversion of the shikimate pathway to produce quinate was observed, as well as accumulation of non-polymerized lignin precursors. These data are consistent with modulation of defensive phenylpropanoid metabolism by M. grisea and the inability of susceptible hosts to mount a hypersensitive reaction or produce lignified papillae (both involving reactive oxygen species) to restrict pathogen invasion. Rapid proliferation of M. grisea hyphae in plant tissue after 3 days was associated with accelerated nutrient acquisition and utilization by the pathogen. Conversion of photoassimilate into mannitol and glycerol for carbon sequestration and osmolyte production appear to drive hyphal growth. Taken together, our results suggest that fungal pathogens deploy a common metabolic re-programming strategy in diverse host species to suppress plant defence and colonize plant tissue.     

茄科雷尔氏菌脂酰-CoA合成酶的功能鉴定

【目的】茄科雷尔氏菌是一种常见的农作物致病菌,引起植物青枯病。研究其脂肪酸代谢途径将有助于寻找新的抗菌药物靶点,为防治青枯病害提供新的思路。【方法】利用大肠杆菌FadD序列,进行同源比对发现茄科雷尔氏菌GMI1000中RSc2857(RsFadD)具有较高的相似性,推测其具有脂酰-CoA合成酶活性。采用PCR扩增方法获得RsfadD基因,连入表达载体pBAD24M后互补大肠杆菌fadD突变株,并检测转化子的生长情况。RsfadD与pET-28b连接后,在大肠杆菌BL(DE3)中表达,并利用Ni-NTA纯化获得带有组氨酸标签的RsFadD,体外测定RsFadD的活性。利用同源重组方法,获得RsfadD敲除突变株,分析突变株的生长性状。【结果】RsfadD异体互补大肠杆菌fadD突变株,恢复突变株在以脂肪酸为碳源的基础培养基上生长。体外活性测定RsFadD具有脂酰-CoA合成酶活性,对不同链长的脂肪酸都具有活性,但活性低于大肠杆菌FadD。RsfadD突变株在添加不同链长脂肪酸的基础培养上仅能微弱生长,而在丰富培养基上生长无差异。【结论】茄科雷尔氏菌中RsfadD编码脂酰-CoA合成酶,在脂肪酸利用过程中发挥重要作用。但RsfadD突变株在基础培养基上微弱生长,说明茄科雷尔氏菌基因组中还有其他的脂酰-CoA合成酶基因。以上研究结果为进一步研究茄科雷尔氏菌中脂酰-CoA合成酶以及脂肪酸利用机制奠定了基础。     

Acceleration effect of amino acid supplementation on glycerol assimilation by Escherichia coli in minimal medium

Growth of Escherichia coli BL21 in a glycerol minimal medium was accelerated following supplementation with trace amounts of amino acid (0.35 mM). Of 12 amino acids tested, Arg and Ser gave the highest response, increasing cell growth by 63 and 53 %, respectively, compared to control cells. The ability of amino acids to accelerate cell growth was “switch-like” and was achieved by promoting glycerol utilization, which may be applied to shorten the long lag-phase when glycerol is used as carbon source. Acceleration of cell growth following amino acid supplementation was also observed using lactose minimal medium.     

Autophagy provides nutrients for nonassimilating fungal structures and is necessary for plant colonization but not for infection in the necrotrophic plant pathogen Fusarium graminearum

The role of autophagy in necrotrophic fungal physiology and infection biology is poorly understood. We have studied autophagy in the necrotrophic plant pathogen Fusarium graminearum in relation to development of nonassimilating structures and infection. We identified an ATG8 homolog F. graminearum ATG8 whose first 116 amino acids before the predicted ATG4 cleavage site are 100% identical to Podospora anserina ATG8. We generated a ΔFgatg8 mutant by gene replacement and showed that this cannot form autophagic compartments. The strain forms no perithecia, has reduced conidia production and the aerial mycelium collapses after a few days in culture. The collapsing aerial mycelium contains lipid droplets indicative of nitrogen starvation and/or an inability to use storage lipids. The capacity to use carbon/energy stored in lipid droplets after a shift from carbon rich conditions to carbon starvation is severely inhibited in the ΔFgatg8 strain demonstrating autophagy-dependent lipid utilization, lipophagy, in fungi. Radial growth rate of the ΔFgatg8 strain is reduced compared with the wild type and the mutant does not grow over inert plastic surfaces in contrast to the wild type. The ability to infect barley and wheat is normal but the mutant is unable to spread from spikelet to spikelet in wheat. Complementation by inserting the F. graminearum atg8 gene into a region adjacent to the actin gene in ΔFgatg8 fully restores the WT phenotype. The results showed that autophagy plays a pivotal role for supplying nutrients to nonassimilating structures necessary for growth and is important for plant colonization. This also indicates that autophagy is a central mechanism for fungal adaptation to nonoptimal C/N ratios.     

Pseudomonas fluorescens CHA0 maintains carbon delivery to Fusarium graminearum-infected roots and prevents reduction in biomass of barley shoots through systemic interactions

Soil bacteria such as pseudomonads may reduce pathogen pressure for plants, both by activating plant defence mechanisms and by inhibiting pathogens directly due to the production of antibiotics. These effects are hard to distinguish under field conditions, impairing estimations of their relative contributions to plant health. A split-root system was set up with barley to quantify systemic and local effects of pre-inoculation with Pseudomonas fluorescens on the subsequent infection process by the fungal pathogen Fusarium graminearum. One root half was inoculated with F. graminearum in combination with P. fluorescens strain CHA0 or its isogenic antibiotic-deficient mutant CHA19. Bacteria were inoculated either together with the fungal pathogen or in separate halves of the root system to separate local and systemic effects. The short-term plant response to fungal infection was followed by using the short-lived isotopic tracer (11)CO(2) to track the delivery of recent photoassimilates to each root half. In the absence of bacteria, fungal infection diverted carbon from the shoot to healthy roots, rather than to infected roots, although the overall partitioning from the shoot to the entire root system was not modified. Both local and systemic pre-inoculation with P. fluorescens CHA0 prevented the diversion of carbon as well as preventing a reduction in plant biomass in response to F. graminearum infection, whereas the non-antibiotic-producing mutant CHA19 lacked this ability. The results suggest that the activation of plant defences is a central feature of biocontrol bacteria which may even surpass the effects of direct pathogen inhibition.     

Nutrition of Acetobacter rancens S3 and F11 Isolated from Tanks for Vinegar Production

The strains S3 and F11 which were isolated respectively from static and submerged tanks for vinegar production were identified as Acetobacter rancens. Neither strain grew in an ammonium defined medium containing ethanol, glucose, glycerol or organic acids as the sole carbon source. When casamino acids were added, they grew luxuriantly with lactate, ethanol or glycerol as the carbon source and less well with acetate or glucose. They grew, forming much acetic acid, in defined ethanol medium when alanine was supplied in place of casamino acids, but strain S3 showed a longer lag time than strain Fl1. This lag time could be shortened by addition of aspartate and glutamate. These amino acids could be replaced by succinate, fumarate, malate, lactate, pyruvate or propionate but not by glucose. Both strains required lactate or pyruvate in defined glucose medium but many other organic acids, which were effective in defined ethanol medium, were ineffective or slightly effective in glucose medium.     

Requirement of siderophore biosynthesis for plant colonization by Salmonella enterica

Contaminated fresh produce has become the number one vector of nontyphoidal salmonellosis to humans. However, Salmonella enterica genes essential for the life cycle of the organism outside the mammalian host are for the most part unknown. Screening deletion mutants led to the discovery that an aroA mutant had a significant root colonization defect due to a failure to replicate. AroA is part of the chorismic acid biosynthesis pathway, a central metabolic node involved in aromatic amino acid and siderophore production. Addition of tryptophan or phenylalanine to alfalfa root exudates did not restore aroA mutant replication. However, addition of ferrous sulfate restored replication of the aroA mutant, as well as alfalfa colonization. Tryptophan and phenylalanine auxotrophs had minor plant colonization defects, suggesting that suboptimal concentrations of these amino acids in root exudates were not major limiting factors for Salmonella replication. An entB mutant defective in siderophore biosynthesis had colonization and growth defects similar to those of the aroA mutant, and the defective phenotype was complemented by the addition of ferrous sulfate. Biosynthetic genes of each Salmonella siderophore, enterobactin and salmochelin, were upregulated in alfalfa root exudates, yet only enterobactin was sufficient for plant survival and persistence. Similar results in lettuce leaves indicate that siderophore biosynthesis is a widespread or perhaps universal plant colonization fitness factor for Salmonella, unlike phytobacterial pathogens, such as Pseudomonas and Xanthomonas.     

Peroxisomal fatty acid beta-oxidation is not essential for virulence of Candida albicans

Phagocytic cells form the first line of defense against infections by the human fungal pathogen Candida albicans. Recent in vitro gene expression data suggest that upon phagocytosis by macrophages, C. albicans reprograms its metabolism to convert fatty acids into glucose by inducing the enzymes of the glyoxylate cycle and fatty acid beta-oxidation pathway. Here, we asked whether fatty acid beta-oxidation, a metabolic pathway localized to peroxisomes, is essential for fungal virulence by constructing two C. albicans double deletion strains: a pex5Delta/pex5Delta mutant, which is disturbed in the import of most peroxisomal enzymes, and a fox2Delta/fox2Delta mutant, which lacks the second enzyme of the beta-oxidation pathway. Both mutant strains had strongly reduced beta-oxidation activity and, accordingly, were unable to grow on media with fatty acids as a sole carbon source. Surprisingly, only the fox2Delta/fox2Delta mutant, and not the pex5Delta/pex5Delta mutant, displayed strong growth defects on nonfermentable carbon sources other than fatty acids (e.g., acetate, ethanol, or lactate) and showed attenuated virulence in a mouse model for systemic candidiasis. The degree of virulence attenuation of the fox2Delta/fox2Delta mutant was comparable to that of the icl1Delta/icl1Delta mutant, which lacks a functional glyoxylate cycle and also fails to grow on nonfermentable carbon sources. Together, our data suggest that peroxisomal fatty acid beta-oxidation is not essential for virulence of C. albicans, implying that the attenuated virulence of the fox2Delta/fox2Delta mutant is largely due to a dysfunctional glyoxylate cycle.     

Requirement for phosphoglucose isomerase of Xanthomonas campestris in pathogenesis of citrus canker

A mutant (XT906) of Xanthomonas campestris pv. citri, the causal agent of citrus canker, was induced by insertion of the transposon Tn5tac1 and isolated. This mutant did not grow or elicit canker disease in citrus leaves but was still able to induce a hypersensitive response in a nonhost plant (the common bean). The mutant was also unable to grow on minimal medium containing fructose or glycerol as the sole carbon source. A 2.5-kb fragment of wild-type DNA that complemented the mutant phenotype of XT906 was isolated. Sequence analysis revealed that this DNA fragment encoded a protein of 562 amino acids that shows homology to phosphoglucose isomerase (PGI). Enzyme activity assay confirmed that the encoded protein possesses PGI activity. Analysis of the activity of the promoter of the pgi gene revealed that it was inhibited by growth in complex medium but induced by culture in plant extract. These results demonstrate that PGI is required for pathogenicity of X. campestris pv. citri.     

Neurospora Mutant Exhibiting Hyperproduction of Amylase and Invertase

A mutant strain of Neurospora crassa has been isolated which is derepressed for amylase and beta-fructofuranosidase (invertase). Large amounts of the two enzymes were secreted into the culture medium upon depletion of exogenous carbon source. The resulting increases of the two extracellular enzymes were prevented by actinomycin D, cycloheximide, and glycerol. The starving cells of the mutant strain produced amylase and invertase de novo, as evidenced by incorporation of radioactive amino acids into the enzymes. Preliminary genetic studies indicate that these elevated enzyme levels described are due to a single gene mutation.     

New insights into the regulation of plant immunity by amino acid metabolic pathways

Besides defence pathways regulated by classical stress hormones, distinct amino acid metabolic pathways constitute integral parts of the plant immune system. Mutations in several genes involved in Asp‐derived amino acid biosynthetic pathways can have profound impact on plant resistance to specific pathogen types. For instance, amino acid imbalances associated with homoserine or threonine accumulation elevate plant immunity to oomycete pathogens but not to pathogenic fungi or bacteria. The catabolism of Lys produces the immune signal pipecolic acid (Pip), a cyclic, non‐protein amino acid. Pip amplifies plant defence responses and acts as a critical regulator of plant systemic acquired resistance, defence priming and local resistance to bacterial pathogens. Asp‐derived pyridine nucleotides influence both pre‐ and post‐invasion immunity, and the catabolism of branched chain amino acids appears to affect plant resistance to distinct pathogen classes by modulating crosstalk of salicylic acid‐ and jasmonic acid‐regulated defence pathways. It also emerges that, besides polyamine oxidation and NADPH oxidase, Pro metabolism is involved in the oxidative burst and the hypersensitive response associated with avirulent pathogen recognition. Moreover, the acylation of amino acids can control plant resistance to pathogens and pests by the formation of protective plant metabolites or by the modulation of plant hormone activity.     

Growth of Trichophyton mentagrophytes on individual amino acids

Trichophyton mentagrophytes was tested for its ability to utilize individual amino acids as a source of carbon and nitrogen in basal medium containing 0.4 mM magnesium sulphate (0.1 g/l) in 0.05 M potassium phosphate buffer at pH6.5. Growth was quantitated by measurement of both dry weight and fungal protein. Seventeen naturally occurring amino acids supported growth, serving as a source of both carbon and nitrogen. Seven amino acids failed to support growth under these conditions; however three of these could be metabolized for nitrogen, but not for carbon.     

Isolation and characterization of a mutant strain of Streptococcus uberis,which fails to utilize a plasmin derived beta-casein peptide for the acquisition of methionine

AIMS: To isolate and characterize a mutant of Streptococcus uberis strain 0140J which fails to utilize a plasmin derived beta-casein peptide for the acquisition of methionine. METHODS AND RESULTS: Random insertional mutagenesis was used to isolate a mutant strain of Strep. uberis 0140J which was unable to utilize methionine from within a casein-derived peptide. The altered gene in the mutant strain showed homology to an oligopeptide permease gene of Streptococcus pyogenes (oppF). The mutant was unable to obtain specific amino acids from defined peptides of various lengths and its growth yield in skimmed milk was between 1 and 10% that of the wild-type strain, but was restored following the inclusion of these amino acids. CONCLUSIONS: The oligopeptide permease homologue of Strep. uberis 0140J is necessary for the utilization of amino acids from within specific peptides. Efficient acquisition of essential amino acids by Strep. uberis 0140J is required for the bacterium to achieve an optimum yield in milk. SIGNIFICANCE AND IMPACT OF THE STUDY: Streptococcus uberis is a major agent of bovine mastitis with a corresponding high economic loss. By targeting metabolic pathways essential to the growth of Strep. uberis it may be possible to prevent the establishment of growth of the bacterium in milk. This study has identified the acquisition of essential amino acids as playing a role in the growth of Strep. uberis in milk.     

Phenotype microarray profiling of Staphylococcus aureus menD and hemB mutants with the small-colony-variant phenotype

Standard biochemical tests have revealed that hemin and menadione auxotrophic Staphylococcus aureus small-colony variants (SCVs) exhibit multiple phenotypic changes. To provide a more complete analysis of the SCV phenotype, two genetically defined mutants with a stable SCV phenotype were comprehensively tested. These mutants, generated via mutations in menD or hemB that yielded menadione and hemin auxotrophs, were subjected to phenotype microarray (PM) analysis of over 1,500 phenotypes (including utilization of different carbon, nitrogen, phosphate, and sulfur sources; growth stimulation or inhibition by amino acids and other nutrients, osmolytes, and metabolic inhibitors; and susceptibility to antibiotics). Compared to parent strain COL, the hemB mutant was defective in utilization of a variety of carbon sources, including Krebs cycle intermediates and compounds that ultimately generate ATP via electron transport. The phenotype of the menD mutant was similar to that of the hemB mutant, but the defects in carbon metabolism were more pronounced than those seen with the hemB mutant. In both mutant strains, hexose phosphates and other carbohydrates that provide ATP in the absence of electron transport stimulated growth. Other phenotypes of SCV mutants, such as hypersensitivity to sodium selenite, sodium tellurite, and sodium nitrite, were also uncovered by the PM analysis. Key results of the PM analysis were confirmed in independent growth studies and by using Etest strips for susceptibility testing. PM technology is a new and efficient technology for assessing cellular phenotypes in S. aureus.     

CaSRB9基因的克隆及其在酿酒酵母形态发生中的作用

白色念珠菌是一种重要的人体致病真菌 ,致病机制与其形态发生紧密相关。酿酒酵母Flo8因子在其形态发生中起重要作用 ,我们把白色念珠菌基因组DNA导入酿酒酵母flo8基因缺失株中 ,筛选能够互补 flo8侵入生长缺陷的基因 ,分离到了一个与酿酒酵母SRB9同源的新基因 ,命名为CaSRB9。该基因全长 4998bp ,编码一种16 6 5个氨基酸的蛋白质。在双倍体酿酒酵母中CaSRB9可以部分互补MAPK途径基因缺失株以及 flo8缺失株的菌丝生长缺陷 ;在单倍体酿酒酵母中表达能够互补 flo8缺失株的侵入生长缺陷 ,但在MAPK途径基因缺失株中不能形成侵入生长