Identification of genes involved in fungal responses to strigolactones using mutants from fungal pathogens.

Strigolactones (SLs) as components of root exudates induce hyphal branching of arbuscular mycorrhizal (AM) fungi which is thought to favor the establishment of the beneficial symbiosis. Little is known on how AM fungi respond to SLs. Since AM fungi are poor model systems due to their obligate biotrophism and the lack of genetic transformation protocols, we took advantage of the sensitivity of several phytopathogenic fungi to GR24, a synthetic SLs analog. With the aim to identify the molecular determinants involved in SLs response in AM fungi and assuming conserved mechanisms in the fungal kingdom, we exploited the fungal pathogens Botrytis cinerea and Cryphonectria parasitica, for which mutant collections are available. Exposure of B. cinerea and C. parasitica to GR24 embedded in solid medium led to reduction of fungal radial growth. We set up the screening of a set of well-characterized gene deletion mutants to isolate genotypes with altered responses to SLs. Two B. cinerea mutants (defective of BcTrr1, a thioredoxin reductase and BcLTF1, a GATA transcription factor) turned out to be less responsive to GR24. One feature shared by the two mutants is the overproduction of reactive oxygen species (ROS). Indeed, an oxidizing effect was observed in a B. cinerea strain expressing a redox-sensitive GFP2 in the mitochondrial intermembrane space upon exposure to GR24. ROS and mitochondria are, therefore, emerging as mediators of SLs actions.

The transcriptome of the arbuscular mycorrhizal fungus Glomus intraradices (DAOM 197198) reveals functional tradeoffs in an obligate symbiont.

• The arbuscular mycorrhizal symbiosis is arguably the most ecologically important eukaryotic symbiosis, yet it is poorly understood at the molecular level. To provide novel insights into the molecular basis of symbiosis-associated traits, we report the first genome-wide analysis of the transcriptome from Glomus intraradices DAOM 197198. • We generated a set of 25,906 nonredundant virtual transcripts (NRVTs) transcribed in germinated spores, extraradical mycelium and symbiotic roots using Sanger and 454 sequencing. NRVTs were used to construct an oligoarray for investigating gene expression. • We identified transcripts coding for the meiotic recombination machinery, as well as meiosis-specific proteins, suggesting that the lack of a known sexual cycle in G. intraradices is not a result of major deletions of genes essential for sexual reproduction and meiosis. Induced expression of genes encoding membrane transporters and small secreted proteins in intraradical mycelium, together with the lack of expression of hydrolytic enzymes acting on plant cell wall polysaccharides, are all features of G. intraradices that are shared with ectomycorrhizal symbionts and obligate biotrophic pathogens. • Our results illuminate the genetic basis of symbiosis-related traits of the most ancient lineage of plant biotrophs, advancing future research on these agriculturally and ecologically important symbionts.

Evaluación de la virulencia de hongos entomopatógenos del pulgón del ciprés, de dos regiones ecológicas de Chile. II. / Evaluation of the virulence of entomopathogenic fungi of cypress aphid, from two ecological regions of Chile. II

El pulgón del ciprés Cinara cupressi, se considera uno de los insectos invasores más dañinos del mundo, ha provocado una gran mortalidad en especies Cupresáceas nativas y exóticas en varios países. En Chile, fue detectado el año 2003 y para el año 2008 esta plaga ya se encontraba distribuida en todo el país, afectando especies cupresáceas forestales exóticas como Cupressus macrocarpa y especies nativas, tales como, Austrocedrus chilensis (Ciprés de la Cordillera) y Fitzroya cupressoides (Alerce), que están categorizadas en el estado de conservación vulnerable y en peligro respectivamente. El área forestal de Chile ha potenciado y privilegiado el uso de controladores biológicos como parte del manejo integrado de plagas. Es por esto que se ha utilizado el parasitoide Pauesia juniperorum. Sin embargo, se han detectado bajos niveles de parasitismo. Se propone el uso de hongos entomopatógenos como alternativa y complemento al control biológico. Se prospectaron hongos entomopatógenos en la Región ecológica mediterránea per-húmeda y en la Región oceánica con influencia mediterránea. Como resultado se han identificado seis géneros de hongos descritos como patógenos de insectos. Sin embargo, bajo condiciones de laboratorio sólo cepas del género Verticillium fueron virulentas, causando sobre el 80 por ciento de mortalidad acumulada a los 7 días. No hubo diferencias significativas entre las cepas de Verticillium y un producto químico (pirimicarb) aplicado como control, aunque el hongo fue más lento. Las cepas más virulentas fueron identificadas como Verticillium lecanii, Ve 1 y Ve 2, con un TL50 de: 3.2 y 3.1 días y un DL50 : 1.24 y 1.37 conidias mL-1 respectivamente, sugiriendo el uso de estos hongos para controlar C. cupressi en Chile.

The cypress aphid, Cinara cupressi, is considered one of the most important invasive species causing high mortality in exotic and native species of Cupressaceae in several countries in the world. In Chile it was detected in 2003 and in 2008 was distributed throughout the country affecting the exotic forest species Cupressus macrocarpa and the native forest species Austrocedrus chilensis (Ciprés de la Cordillera) and Fitzroya cupressoides (Alerce), both classified as vulnerable and endangered species respectively. Efforts to their management have focused on biological control by using the parasitoid Pauesia juniperorum but until now it has not reached satisfactory control. We propose using entomopathogenic fungi, as alternative and complementary biocontrol. Entomopathogenic fungi were prospected in the ecological region Mediterranean per-humid and in the ecological region Oceanic with mediterranean influence in both colonies of C. cupressi. There were identified six genera of fungi described as insect pathogens. However, in laboratory assays only Verticillium strains were virulent, causing about 80 percent cumulative mortality at seven days. There were not significant differences among strains of Verticillium and chemical (pirimicarb) applied as control, although fungi were slower. The most virulent strains were two Verticillium lecanii, Ve 1 and Ve 2, with an LT50 of: 3.2 and 3.1 days and LD50 : 1.24 and 1.37 conidia mL-1 respectively, suggesting the use of these fungi to control C. cupressi in Chile.

Prospección de hongos entomopatógenos del pulgón del ciprés en dos regiones ecológicas de Chile: I / Prospection of entomopathogenic fungi of cypress aphid in two ecological regions of Chile: I

El pulgón del ciprés (Cinara cupressi), es una plaga exótica distribuida en todo el territorio continental chileno que ha provocado daños importantes tanto enespecies forestales introducidas como nativas. Se han estudiado algunos aspectos de la biología de estepulgón, mediante control biológico y productos químicos, los cuales no presentan actualmente resultadossatisfactorios. Durante el tiempo que esta plaga se ha establecido en Chile, no se ha realizado un estudio sistemático de hongos entomopatógenos asociados aeste pulgón, aunque se cuenta con antecedentes de un control promisorio en pulgones con estos agentes enotros países. Con el objetivo inicial de aislar en una primera fase del presente estudio hongos patogénicospotenciales para el control biológico de C. cupressi, iniciamos su búsqueda en dos regiones ecológicas del sur de Chile (Región ecológica Mediterránea Per-Húmeda y Región ecológica Oceánica con influencia Mediterránea), seleccionándose en cada una de ellas 6sitios de muestreo donde en cada uno se recolectaron 10 ramas con colonias de pulgones que fueron mantenidas en una cámara bioclimática (20 +/- 2 °C, 16:8 h) por 7 días, para estimular el desarrollo fúngico. Se aislaron integrantes de lo géneros Verticillium, Paecilomyces(ambos con mayor frecuencia de presencia), Fusarium y un entomophthoral en estudio.

The cypress aphid (Cinara cupressi) is an exotic plague distributed throughout the Chilean continental territory which has caused significant damage both inintroduced forest species as well as in native ones. Some characteristics as to the biology of this aphid have beenstudied by means of biological control and chemicals yet they have not revealed any satisfactory results up tonow. During the time of occurrence of this plague in Chile systematic study on entomopathogenic fungi associated to this aphid has failed to be accomplishedalthough information about a promissory control in aphids with these agents has been reported in other countries. In order to carry out an isolation of potential pathogenic fungi for the biological control of C.cupressiin the first phase of this present research, we began to look for them in two ecological regions from southernChile (Ecological Mediterranean Per-humid Region and Ecological Oceanic Region having Mediterraneaninfluence). Six sampling sites were selected in each of them while 10 branches infected with aphid colonies were kept in a bioclimatic chamber (20+2ºC, 16:8 h) for 7 days to stimulate fungal growth. Fungi of generaVerticillium, Paecilomyces (both showing the highest occurrence frequency), Fusarium and an entomophtoral under study were isolated.

Expression profiles of a phosphate transporter gene (GmosPT) from the endomycorrhizal fungus Glomus mosseae.

Arbuscular mycorrhizal (AM) fungi have long been shown to successfully contribute to phosphate uptake by plant roots. The first step of the fungus-mediated uptake is carried out by fungal membrane Pi transporters (PT) that transfer Pi from the soil into the extraradical hyphae. In the present work we report the identification and characterisation of a PT gene from Glomus mosseae, an AM fungus important for natural and agricultural ecosystems. Degenerate primers and rapid amplification of cDNA ends-polymerase chain reaction (PCR) allowed us to obtain a sequence (GmosPT) showing a highly significant similarity with GiPT and GvPT, the only two other PT genes already isolated from AM fungi. Reverse transcriptase-PCR experiments were carried out to study GmosPT expression profiles in structures corresponding to different fungal life stages (quiescent and germinated sporocarps, intraradical and extraradical hyphae) and in extra- and intraradical hyphae exposed to high and low Pi concentrations. GmosPT showed an expression pattern similar to GiPT, the Glomus intraradices PT gene, since its transcript was more abundant in the extraradical mycelium treated with micromolar Pi levels. In addition, the intraradical mycelium also showed a significant GmosPT expression level that was independent from external Pi concentrations. This finding opens new questions about the role and functioning of high-affinity PT in AM fungi.

Zinc ions alter morphology and chitin deposition in an ericoid fungus.

A sterile mycelium PS IV, an ascomycete capable of establishing ericoid mycorrhizas, was used to investigate how zinc ions affect the cellular mechanisms of fungal growth. A significant reduction of the fungal biomass was observed in the presence of millimolar zinc concentrations; this mirrored conspicuous changes in hyphal morphology which led to apical swellings and increased branching in the subapical parts. Specific probes for fluorescence and electron microscopy localised chitin, the main cell wall polysaccharide, on the inner part of the fungal wall and on septa in control specimens. In Zn-treated mycelium, hyphal walls were thicker and a more intense chitin labelling was detected on the transverse walls. A quantitative assay showed a significant increase in the amount of chitin in metal-treated hyphae.

A combined morphological and molecular approach to characterize isolates of arbuscular mycorrhizal fungi in Gigaspora (Glomales).

• Morphological features of resting spores and information from nucleotide sequences of ribosomal RNA were used to characterize seven mycorrhizal fungal isolates in Gigaspora from different geographical areas. • Detailed observations were made under the light microscope on single spores mounted in Melzer's reagent and polyvinyl alcohol-lactic acid-glycerol medium to resolve size, colour and cell wall structures. Neighbour-joining analyses were carried out on a portion of the 18S gene and on the internal transcribed spacer (ITS) region amplified by PCR from multisporal DNA preparations. • Combined data allowed us to design oligonucleotides that unambiguously distinguished Gi. rosea from Gi. margarita and Gi. gigantea and also identified two isolates as Gi. rosea that had been previously diagnosed as Gi. margarita. ITS sequences revealed substantial genetic variability within clones of a single isolate of Gi. rosea as well as among geographically disjunct Gi. rosea isolates. • The results show how complementary morphological and molecular data can clarify relationships among species of low morphological divergence. Sequence information allowed the extent of genetic divergence within these species to be investigated and provided useful PCR primers for detection and identification.

Detection and identification of bacterial endosymbionts in arbuscular mycorrhizal fungi belonging to the family Gigasporaceae.

Intracellular bacteria have been found previously in one isolate of the arbuscular mycorrhizal (AM) fungus Gigaspora margarita BEG 34. In this study, we extended our investigation to 11 fungal isolates obtained from different geographic areas and belonging to six different species of the family Gigasporaceae. With the exception of Gigaspora rosea, isolates of all of the AM species harbored bacteria, and their DNA could be PCR amplified with universal bacterial primers. Primers specific for the endosymbiotic bacteria of BEG 34 could also amplify spore DNA from four species. These specific primers were successfully used as probes for in situ hybridization of endobacteria in G. margarita spores. Neighbor-joining analysis of the 16S ribosomal DNA sequences obtained from isolates of Scutellospora persica, Scutellospora castanea, and G. margarita revealed a single, strongly supported branch nested in the genus Burkholderia.

Chitin synthase genes in the arbuscular mycorrhizal fungus Glomus versiforme: full sequence of a gene encoding a class IV chitin synthase.

Chitin synthase genes of the arbuscular mycorrhizal fungus Glomus versiforme were sought in an investigation of the molecular basis of fungal growth. Three DNA fragments (Gvchs1, Gvchs2 and Gvchs3) corresponding to the conserved regions of distinct chitin synthase (chs) genes were amplified by means of the polymerase chain reaction (PCR) with two sets of degenerate primers. Gvchs1 and Gvchs2 encode two class I chitin synthases, whereas Gvchs3 encodes a class IV chitin synthase. A genomic library was used to obtain the Gvchs3 complete gene (1194 amino acids), which shows a very close similarity to the class IV chitin synthase from Neurospora crassa.

Intrasporal variability of ribosomal sequences in the endomycorrhizal fungus Gigaspora margarita.

The sequence variability of the ribosomal internal transcribed spacer (ITS) region, which comprises the 5.8 gene and the flanking regions ITS1 and ITS2, was investigated in the arbuscular mycorrhizal fungus Gigaspora margarita. DNA analysis of a multispore preparation and three single spores led to the identification of 11 slightly different sequences (three variants within a single spore), indicating substantial intersporal and intrasporal genetic variability (up to 9% sequence divergence). The sequence variations inside a single spore may be higher than that observed between spores. Even so, primers designed on the ITS1 and ITS2 regions identified Gi. margarita isolates and detected the endophyte during colonization.

Chitin synthase homologs in three ectomycorrhizal truffles.

Degenerate PCR primers were used to amplify a conserved gene portion coding chitin synthase from genomic DNA of six species of ectomycorrhizal truffles. DNA was extracted from both hypogeous fruitbodies and in vitro growing mycelium of Tuber borchii. A single fragment of about 600 bp was amplified for each species. The amplification products from Tuber magnatum, T. borchii and T. ferrugineum were cloned and sequenced, revealing a high degree of identity (91.5%) at the nucleotide level. On the basis of the deduced amino acid sequences these clones were assigned to class II chitin synthase. Southern blot experiments performed on genomic DNA showed that the amplification products derive from a single copy gene. Phylogenetic analysis of the nucleotide sequences of class II chitin synthase genes confirmed the current taxonomic position of the genus Tuber, and suggested a close relationship between T. magnatum and T. uncinatum.

Generation of RAPD-PCR primers for the identification of isolates of Glomus mosseae, an arbuscular mycorrhizal fungus.

Mycorrhizal fungi are usually identified on the basis of the morphological characters shown by fruit bodies, spores, vegetative mycelia or symbiotic structures. The development of molecular techniques provides a valuable and alternative approach to identify mycorrhizal fungi, especially when it is difficult to gather a sufficient number of data on morphological features. Short arbitrary oligonucleotides were used as primers for the amplification of genomic DNA extracted from spores of arbuscular fungi. The RAPD fingerprints showed banding patterns which allowed us to distinguish between species and even isolates within Glomales. In order to identify mycorrhizal fungi during their symbiotic phase, a nonpolymorphic RAPD band identified as marker for some isolates of Glomus mosseae was purified from agarose gels and cloned in a bluescript vector. The fragment was sequenced and specific primers (PO-M3) were designed for the mycorrhizal fungus. They specifically and successfully amplified the DNA not only from G. mosseae spores, but also from roots of pea, clover, leek and onion plants when they were colonized by G. mosseae isolates.

DNA probes for identification of the ectomycorrhizal fungus Tuber magnatum Pico.

The random amplified polymorphic DNA (RAPD) technique was used to develop DNA probes for the identification of ectomycorrhizal fungi belonging to the genus Tuber. RAPD fingerprinting revealed a high degree of interspecific variability and a low degree of intraspecific variability. One band (approximately 1.5 kb), consistently appearing when genomic DNA was amplified with an aspecific primer (OPA-18), was found to be a good marker for Tuber magnatum, and was used as a probe in Southern hybridization experiments. The specificity of the results suggests that this probe may be useful in developing specific primers for PCR amplifications.

Multiple DNA-protein interactions at the CpG island of the human pseudoautosomal gene MIC2.

The human MIC2 gene is pseudoautosomal and in females it escapes X inactivation. At the 5' end of the gene a 1.2-kb-long CpG island has been identified that is unmethylated on the active X, the inactive X, and on the Y chromosome. We have demonstrated by 5' RACE experiments that this region contains the transcription start site of the gene. To better characterize this CpG island, we have investigated the interaction between this region and nuclear proteins in vitro by using DNA gel mobility shift and DNase I footprinting techniques. Band shift experiments with HeLa cell nuclear extract have indicated that all the island is involved in multiple interactions with nuclear proteins. Experiments with a eukaryotic purified Sp1 protein have shown that this factor specifically binds to several sites of the island. Three DNase I protected footprints have been identified in the region between nucleotides -122 and +34 with respect to the transcription initiation site. By using a recombinant Sp1 protein, we have shown that all the footprints are due to the binding of Sp1. The sequences of two footprints correspond to the decanucleotide binding site for Sp1, the sequence of the third one does not contain any published Sp1 recognition site.

An erythroid specific enhancer upstream to the gene encoding the cell-type specific transcription factor GATA-1.

The transcription factor GATA-1 is expressed in a subset of hemopoietic cells, where it mediates the cell-type specific expression of several genes. We have cloned the mouse and human GATA-1 genes. A region upstream to the first exon, and highly conserved between mouse and man, acts as an erythroid specific enhancer in transient assays, if linked to the GATA-1 or to the SV40 promoter. The activity of the enhancer is almost completely dependent on the integrity of a dimeric GATA-1 binding site.