Pulsed field gel electrophoresis reveals chromosome length differences between strains of Cladosporium fulvum (syn. Fulvia fulva)

Summary Methods are described for the electrophoretic separation of chromosome-sized DNA molecules from the fungal tomato pathogen Cladosporium fulvum (syn. Fulvia fulva). Using a hexagonal electrode array and switching times of 75 min at 45 V for 14 days, nine bands could be resolved. By comparison with co-electrophoresed Aspergillus nidulans chromosomal DNA (which was resolved into seven bands), the sizes of the C. fulvum bands are estimated to be between 1.9 Mb and 5.4 Mb. The two largest bands are believed to be doublets, giving a minimum genome size of 44 Mb. Cloned probes for the ribosomal DNA repeat, an anonymous single copy fragment and a newly discovered retrotransposon were hybridized to blots of the pulsed field gels, demonstrating the use of this technique for genomic mapping. Most strains of C. fulvum had an identical pattern of bands. Two strains exhibited two polymorphisms which could be due to a translocation.     

Nitrogen limitation induces expression of the avirulence gene avr9 in the tomato pathogen Cladosporium fulvum

The avirulence gene avr9 of the fungal tomato pathogen Cladosporium fulvum encodes a race-specific peptide elicitor that induces the hypersensitive response in tomato plants carrying the complementary resistance gene Cf9. The avr9 gene is not expressed under optimal growth conditions in vitro, but is highly expressed when the fungus grows inside the tomato leaf. In this paper we present evidence for the induction of avr9 gene expression in C. fulvum grown in vitro under conditions of nitrogen limitation. Only growth medium with very low amounts of nitrogen (nitrate, ammonium, glutamate or glutamine) induced the expression of avr9. Limitation of other macronutrients or the addition of plant factors did not induce the expression of avr9. The induced expression of avr9 is possibly mediated by a positive-acting nitrogen regulatory protein, homologous to the Neurospora crassa NIT2 protein, which induces the expression of many genes under conditions of nitrogen limitation. The avr9 promoter contains several putative NIT2 binding sites. The expression of avr9 during the infection process was explored cytologically using transformants of C. fulvum carrying an avr9 promoter-β-glucuronidase reporter gene fusion. The possibility that expression of avr9 in C. fulvum growing in planta is caused by nitrogen limitation in the apoplast of the tomato leaf is discussed.     

The Hydrophobin HCf-1 of Cladosporium fulvum Is Required for Efficient Water-Mediated Dispersal of Conidia

Six hydrophobin genes (HCf-1 to -6) have thus far been identified in the tomato pathogen Cladosporium fulvum. HCf-1 to -4 are Class I hydrophobins and HCf-5 and -6 are Class II hydrophobins. In this paper we describe the isolation of deletion mutants that lack HCf-1, HCf-2, or both these genes. Global down-regulation of the expression of Class I hydrophobins is achieved by homology-dependent gene silencing. Analysis of the mutant strains shows that HCf-1 confers hydrophilic character to the conidia and this facilitates the dissemination of conidia on the surface of water droplets. Other Class I hydrophobins, such as HCf-3 or HCf-4, may be involved in the development and germination of conidia.     

Resistance in tomato to Xanthomonas campestris pv vesicatoria is determined by alleles of the pepper-specific avirulence gene avrBs3

Bacterial spot disease of tomato and pepper caused by Xanthomonas campestris pv vesicatoria is prevented by resistance genes in the plant that match genes for avirulence in the bacterium. Based on DNA homology to the avirulence gene avrBs3, which induces the resistance response on pepper, we have isolated another avirulence gene from X. c. vesicatoria, designated avrBs3-2. This gene differs in specificity from avrBs3 in inducing the hypersensitive response on tomato but not on pepper. Sequence analysis of the avrBs3-2 gene revealed a high degree of conservation: the 3480 by open reading frame contains an internal region of 17.5 nearly identical 102 bp repeat units that differ in their order from those present in the avrBs3 gene. The coding region is 97% identical to avrBs3 and expresses constitutively a 122 kDa protein, thus representing a natural allele of this gene. The previously isolated 1.7 kb avrBsP gene from X. c. vesicatoria is 100% identical to the corresponding avrBs3-2 sequence, indicating that these genes might be identical. Interestingly, derivatives of avrBs3-2 lacking the C-terminal region and part of the repetitive region are still able to confer incompatibility in tomato. The avrBs3-2 gene is compared with the sequence of avrBs3 derivatives generated by deletion of repeat units that also have avirulence activity on tomato. Both genes, avrBs3 and avrBs3-2, are flanked by a 62 by long inverted repeat, which prompts speculations about the origin of the members of the avrBs3 gene family.     

Mapping strategy for resistance genes in tomato based on RFLPs between cultivars: Cf9 (resistance to Cladosporium fulvum) on chromosome 1

Summary The contribution of introgressed regions derived from wild species to the genetic variation within the species of Lycopersicon esculentum was investigated by comparing the RFLP patterns of 2 introgression-free, obsolete cultivars (Moneymaker and Premier) and a modern cultivar (Sonatine) that carries at least 5 introgressed resistance genes. In this analysis 195 mapped nuclear markers were used in combination with 6 restriction enzymes. Among the 1170 probe-enzyme combinations tested, only 3 showed a polymorphism between the 2 introgression-free cultivars. On the other hand 24 probe-enzyme combinations were found to exhibit polymorphisms between Moneymaker and Sonatine. These represented ten polymorphic loci distributed among 5 linkage groups on chromosomes 1, 3, 4, 6, and 9.On the assumption that most of the polymorphic loci corresponded to introgressed chromosome segments of wild species carrying resistance genes, linkages between these loci and the component resistance genes were examined by RFLP analysis of pairs of near-isogenic lines differing only for one particular resistance gene, and a variety of commercial cultivars having different resistance gene compositions. Two of the polymorphic linkage groups could thus be ascribed to resistance genes whose map positions were already known: Cf2 on chromosome 6 and Tm2a on chromosome 9, whereas another marker, TG301 on chromosome 1, could be assigned to the Cladosporium fulvum resistance gene Cf9 with a hitherto disputable map position. By linkage analysis of a segregating F₂ population the genetic distance between the Cf9 gene and the marker TG301 was estimated at 5.5 ± 2.3 cM.     

Nutrient limitation of Microcystis aeruginosa in northern California Klamath River reservoirs

Nutrient limitations were investigated in Copco and Iron Gate Reservoirs, on the Klamath River in California, where blooms of the toxin-producing cyanobacterium Microcystis aeruginosa were first reported in 2005. Nutrient enrichment experiments conducted in situ in June and August, 2007 and 2008, determined responses in phytoplankton biomass, Microcystis abundance and microcystin concentration to additions of phosphorus and different forms of nitrogen (NH₄⁺, NO₃, and urea). Microcystis abundance was determined using quantitative PCR targeting the phycocyanin intergenic spacer cpcBA.Total phytoplankton biomass increased with additions of N both before and during Microcystis blooms, with no primary effects from P, suggesting overall N limitation for phytoplankton growth during the summer season. NH₄⁺ generally produced the greatest response in phytoplankton growth, while Microcystis abundance increased in response to all forms of N. Microcystis doubling time in the in situ experiments was 1.24–1.39 days when N was not limiting growth. The results from this study suggest availability of N during the summer is a key growth-limiting factor for the initiation and maintenance of toxic Microcystis blooms in Copco and Iron Gate Reservoirs in the Klamath River.     

Induction of tomato stress protein mRNAs by ethephon, 2,6-dichloroisonicotinic acid and salicylate

To study the possible involvement of plant hormones in the synthesis of stress proteins in tomato upon inoculation with Cladosporium fulvum, we investigated the induction of mRNAs encoding PR proteins and ethylene biosynthesis enzymes by ethephon, 2,6-dichloroisonicotinic acid (INA) and salicylic acid (SA) by northern blot analysis. Ethephon slightly induced some but not all mRNAs encoding intra- and extracellular PR proteins. INA induced all PR protein mRNAs analysed, except for intracellular chitinase and extracellular PR-4. SA induced all PR protein mRNAs analyzed, except for intracellular chitinase and osmotin. None of the inducers affected the expression of ACC synthase mRNA, whereas all three induced ethylene-forming enzyme (EFE) mRNA.Abbreviations ACC 1-aminocyclopropane-1-carboxylic acid - EFE ethylene-forming enzyme - HR hypersensitive response - INA 2,6-dichloroisonicotinic acid - PR pathogenesis-related - SA salicylic acid - SAR systemic acquired resistance     

The Cladosporium fulvum resistance gene Cf-ECP3 is part of the Orion cluster on the short arm of tomato Chromosome 1

The resistance of tomato to the pathogenic fungus Cladosporiumfulvum complies with the gene-for-gene relationship. Race specificresistance is based on Cf-gene mediated recognition ofsecreted avirulence products, resulting in a hypersensitive response (HR).Besides the avirulence gene products, C. fulvum secretes anumber of extra cellular proteins (ECPs) into the apoplast. Two L.esculentum accessions have previously been identified that reactedwith a HR upon injection with purified ECP3. The corresponding resistance genedesignated Cf-ECP3 was mapped by using an F₂population composed of 192 plants from the cross of susceptible MoneyMaker toresistant L. esculentum G1.1153.Cf-ECP3 inherited monogenically, cosegragated with theChromosome 1 Cleaved Amplified Polymorphic Sequence (CAPS) marker CT116 and wasmapped accurately at Orion, a locus harbouring Cf-ECP2 inother genotypes. RFLP anaysis with a Cf-9 probe furtherdemonstrated cosegregation of Cf-ECP3 with anHcr9 (Homologue of Cladosporiumfulvumresistance gene Cf-9) indicating that this gene is likelyan Hcr9. Thus in addition to the Milky Way locusharbouringthe Cf-4, Cf-4A andCf-9 resistance genes targeted against AVR4, AVR4A andAVR9, Orion is another complex locus on the short arm of Chromosome 1 thatharbours at least two functional Cf-genes,Cf-ECP2 and Cf-ECP3, targeted againstthe fungal excreted proteins ECP2 and ECP3.     

柠条锦鸡儿F3H基因克隆及功能分析

柠条锦鸡儿为豆科灌木,对各种环境胁迫具有较强的适应能力,类黄酮是天然的抗氧化剂,花青素属类黄酮化合物,逆境胁迫会影响植物体内花青素的合成,而黄烷酮3-羟化酶(F3H)是花青素生物合成所必需的一种关键酶。该研究成功分离克隆了柠条锦鸡儿的F3H基因,命名为CkF3H。CkF3H基因的开放阅读框(ORF)为1095 bp,编码364个氨基酸,推测的蛋白质分子量为41.3 kDa,理论等电点为5.9。生物信息学分析发现,CkF3H基因序列与其它植物F3H有较高的一致性,推测CkF3H蛋白与其它植物F3H蛋白具有相似的功能。利用染色体步移法克隆得到CkF3H起始密码子ATG上游468 bp的启动子序列,PlantCARE软件分析表明,该序列具有启动子的基本元件CAAT-box和TATA-box以及多种与逆境胁迫相关的顺式调控元件。实时荧光定量PCR分析表明,CkF3H在柠条的根、茎和叶中均有表达,没有组织特异性;CkF3H的表达受低温、高盐、干旱和高温胁迫的诱导,并且在低温胁迫下,CkF3H的表达还受到光周期的影响。综上所述,研究结果表明CkF3H基因在柠条锦鸡儿适应低温、高盐、干旱和高温胁迫的过程中发挥作用。     

ThehapC gene ofAspergillus nidulans is involved in the expression of CCAAT-containing promoters

The 5 regulatory region of theamdS gene ofAspergillus nidulans, which encodes an acetamidase required for growth on acetamide as a carbon and nitrogen source, contains a CCAAT sequence which is required for setting the basal level ofamdS expression. Mobility shift studies have identified a factor inA. nidulans nuclear extracts which binds to this CCAAT sequence. InSaccharomyces cerevisiae theHAP3 gene encodes one component of a multisubunit complex that binds CCAAT sequences. A search of the EMBL and SwissProt databases has revealed anA. nidulans sequence with significant homology to theHAP3 gene adjacent to the previously cloned regulatory geneamdR. Sequencing of the remainder of this region has confirmed the presence of a gene, designatedhapC, with extensive homology toHAP3. The predicted amino acid sequence of HapC shows extensive identity to HAP3 in the central conserved domain, but shows little conservation in the flanking sequences. A haploid carrying ahapC deletion has been created and is viable, but grows poorly on all media tested. This null mutant grows especially slowly on acetamide as a sole carbon and nitrogen source, indicating thathapC plays a role inamdS expression. In agreement with this notion, it has been shown that thehapC deletion results in reduced levels of expression of anamdS::lacZ reporter gene and this effect is particularly evident under conditions of carbon limitation. Nuclear extracts prepared from thehapC deletion mutant show no CCAAT binding activity to theamdS orgatA promoters, indicating thathapC may encode a component of the complex binding at this sequence.     

The tomato Orion locus comprises a unique class of <Emphasis Type="Italic">Hcr9</Emphasis> genes

Resistance against the tomato fungal pathogen Cladosporium fulvum is often conferred by Hcr9 genes (Homologues of the C. fulvum resistance gene Cf-9) that are located in the Milky Way cluster on the short arm of chromosome 1. These Hcr9 genes mediate recognition of fungal avirulence gene products. In contrast, the resistance gene Cf-Ecp2 mediates recognition of the virulence factor Ecp2 and is located in the Orion (OR) cluster on the short arm of chromosome 1. Here, we report the map- and homology-based cloning of the OR Hcr9 cluster. A method was optimised to generate clone-specific fingerprint data that were subsequently used in the efficient calculation of genomic DNA contigs. Three Hcr9s were identified as candidate Cf-Ecp2 genes. By PCR-based cloning using specific OR sequences, orthologous Hcr9 genes were identified from different Lycopersicon species and haplotypes. The OR Hcr9s are very homologous. However, based on the relative low sequence homology to other Hcr9s, the OR Hcr9s are classified as a new subgroup.Data deposition: The sequence of the Cf-Ecp2 Hcr9 gene cluster and the orthologous Hcr9 sequences have been deposited in the GenBank database (accession No. AY639600..AY639604)     

Comparative studies on metal biosorption by two strains of Cladosporium cladosporioides

Two strains of a fungus, Cladosporium cladosporioides 1 and C. cladosporioides 2 showed different metal biosorption properties. Strain 1 showed preferential sorption of gold and silver, while strain 2 could bind metals such as copper and cadmium in addition to gold and silver. Strain 1 had a cell-wall hexosamine content of 0.1%. X-ray photoelectron spectroscopy (XPS) and Fourier transform infra-red spectroscopy (FTIR) analyses indicated that nitrogen was not involved in metal biosorption by the strain. In strain 2 the cell-wall hexosamine content was 150 times that of strain 1. These results indicated that hexosamine was responsible for non-specific metal binding while cell-wall polymers other than hexosamines had a role in conferring selectivity in precious-metal binding.     

Regulation of nod gene expression in Bradyrhizobium japonicum

Summary The best inducers of nod:: lacZ translational fusions in Bradyrhizobium japonicum are isoflavones, primarily genistein and daidzein. Upstream of the nodABC genes in B. japonicum is a novel gene, nodY, which is coregulated with nodABC. Measurements of the activity of lacZ fusions to the nodD gene of B. japonicum show that this gene is inducible by soybean seed extract and selected flavonoid chemicals. The induction of the nodY ABC and nodD operons appears to require a functional nodD gene, indicating that the nodD gene product controls its own synthesis as well as other nod genes.     

The gltF gene of Klebsiella pneumoniae: cloning and initial characterization

Summary From a gene bank of Klebsiella pneumoniae M5a1, a 1.7 kb gene fragment was isolated which was able to restore the Ntr⁺ phenotype and ammonium (methylammonium) transport, but not glutamate synthase in an Escherichia coli glt mutant (glutamate synthase deficiency). The fragment strongly hybridized with the gltF regulatory gene from E. coli. After subcloning the fragment into an overexpression vector, a protein with a molecular weight of 27000 dalton was identified as the gene product. The results indicate that the fragment cloned contains the gltF gene from K. pneumoniae.