Joan M. Henson

Stress tolerance in plants via habitat-adapted symbiosis

We demonstrate that native grass species from coastal and geothermal habitats require symbiotic fungal endophytes for salt and heat tolerance, respectively. Symbiotically conferred stress tolerance is a habitat-specific phenomenon with geothermal endophytes conferring heat but not salt tolerance, and coastal endophytes conferring salt but not heat tolerance. The same fungal species isolated from plants in habitats devoid of salt or heat stress did not confer these stress tolerances. Moreover, fungal endophytes from agricultural crops conferred disease resistance and not salt or heat tolerance. We define habitat-specific, symbiotically-conferred stress tolerance as habitat-adapted symbiosis and hypothesize that it is responsible for the establishment of plants in high-stress habitats. The agricultural, coastal and geothermal plant endophytes also colonized tomato (a model eudicot) and conferred disease, salt and heat tolerance, respectively. In addition, the coastal plant endophyte colonized rice (a model monocot) and conferred salt tolerance. These endophytes have a broad host range encompassing both monocots and eudicots. Interestingly, the endophytes also conferred drought tolerance to plants regardless of the habitat of origin. Abiotic stress tolerance correlated either with a decrease in water consumption or reactive oxygen sensitivity/generation but not to increased osmolyte production. The ability of fungal endophytes to confer stress tolerance to plants may provide a novel strategy for mitigating the impacts of global climate change on agricultural and native plant communities.

THE ROLE OF FUNGAL SYMBIOSES IN THE ADAPTATION OF PLANTS TO HIGH STRESS ENVIRONMENTS

All plants studied in natural ecosystemsare symbiotic with fungi that either resideentirely (endophytes) or partially(mycorrhizae) within plants. Thesesymbioses appear to adapt to biotic andabiotic stresses and may be responsible forthe survival of both plant hosts and fungalsymbionts in high stress habitats. Here wedescribe the role of symbiotic fungi inplant stress tolerance and present astrategy based on adaptive symbiosis topotentially mitigate the impacts of globalchange on plant communities.

Cloning, Characterization, and Transcription of Three Laccase Genes from Gaeumannomyces graminis var. tritici, the Take-All Fungus

ABSTRACT Gaeumannomyces graminis var. tritici, a filamentous ascomycete, is an important root pathogen of cereals that causes take-all disease and results in severe crop losses worldwide. Previously we identified a polyphenol oxidase (laccase) secreted by the fungus when induced with copper. Here we report cloning and partial characterization of three laccase genes (LAC1, LAC2, and LAC3) from G. graminis var. tritici. Predicted polypeptides encoded by these genes had 38 to 42% amino acid sequence identity and had conserved copper-binding sites characteristic of laccases. The sequence of the LAC2 predicted polypeptide matched the N-terminal sequence of the secreted laccase that we purified in earlier studies. We also characterized expression patterns of these genes by reverse transcription-PCR. LAC1 was transcribed constitutively, and transcription of LAC2 was Cu inducible. All three genes were transcribed in planta; however, transcription of LAC3 was observed only in planta or in the presence of host (wheat) plant homogenate.

Isolation and serological analyses of fungal melanins.

Melanins are notoriously difficult to work with because of their unique physical and chemical properties. The study of melanins is hampered by the scarcity of melanin-specific reagents and serological techniques. In this study we describe modifications to the standard method for the isolation of melanins from in vitro-melanized fungal cells and detail the optimization of serological techniques for the study of melanin compounds. The isolation procedure involves the digestion of melanized cells with a combination of proteolytic and glycolytic enzymes, denaturant, organic extractions, and boiling in 6.0 M HCl. Elemental quantitative analyses suggest that this procedure does not significantly affect the relative elemental composition of melanins. For the serological assays, our goal was to achieve a homogenous distribution of melanin particles on a solid support to maximize their recognition by melanin-binding antibodies. The results from enzyme-linked immunosorbent assays (ELISAs) demonstrate that melanins, in general, disperse more efficiently on, and adhere better to, medium-binding polystyrene surfaces, especially in the presence of trace amounts of salt. Blocking the melanin-coated ELISA plates with the commercially available SuperBlock((R)) Blocking Buffer for 4 h was more efficient at reducing non-specific binding of a negative control monoclonal antibody (mAb) compared to blocking with 2% bovine serum albumin (BSA) and 5% milk. Increasing the ionic strength of the antibody solutions reduced binding to the melanins, indicating that binding is in part mediated by electrostatic interactions. These conditions were also applied to immunofluorescence (IF) analyses of melanins, and the results were consistent with those obtained by ELISA.

PCR detection and analysis of the free-living amoeba Naegleria in hot springs in Yellowstone and Grand Teton National Parks.

ABSTRACT Free-living thermotolerant amoebae pose a significant health risk to people who soak and swim in habitats suitable for their growth, such as hot springs. In this survey of 23 different hot springs in Yellowstone and Grand Teton National Parks, we used PCR with primer sets specific for Naegleria to detect three sequence types that represent species not previously described, as well as a fourth sequence type identified as the pathogen Naegleria fowleri.

Legionella species diversity in an acidic biofilm community in Yellowstone National Park

ABSTRACT Legionella species are frequently detected in aquatic environments, but their occurrence in extreme, acidic, geothermal habitats has not been explored with cultivation-independent methods. We investigated a predominately eukaryotic algal mat community in a pH 2.7 geothermal stream in Yellowstone National Park for the presence of Legionella and potential host amoebae. Our analyses, using PCR amplification with Legionella-specific primers targeting 16S rRNA genes, detected four known Legionella species, as well as Legionella sequences from species that are not represented in sequence databases, in mat samples and cultivated isolates. The nonrandom occurrence of sequences detected at lower (30°C) and higher (35 to 38°C) temperatures suggests that natural thermal gradients in the stream influence Legionella species distributions in this mat community. We detected only one sequence, Legionella micdadei, from cultivated isolates. We cultured and sequenced partial 18S rRNA gene regions from two potential hosts, Acanthamoeba and Euglena species.

Defoliation effects on enzyme activities of the ectomycorrhizal fungus Suillus granulatus in a Pinus contorta (lodgepole pine) stand in Yellowstone National Park

Ectomycorrhizal (EM) basidiomycete fungi are obligate mutualists of pines and hardwoods that receive fixed C from the host tree. Though they often share most recent common ancestors with wood-rotting fungi, it is unclear to what extent EM fungi retain the ability to express enzymes that break down woody substrates. In this study, we tested the hypothesis that the dominant EM fungus in a pure pine system retains the ability to produce enzymes that break down woody substrates in a natural setting, and that this ability is inducible by reduction of host photosynthetic potential via partial defoliation. To achieve this, pines in replicate blocks were defoliated 50% by needle removal, and enzyme activities were measured in individual EM root tips that had been treated with antibiotics to prevent possible bacterial activity. Results indicate that the dominant EM fungal species (Suillus granulatus) expressed all enzymes tested (endocellulase D-glucosidase, laccase, manganese peroxidase, lignin peroxidase, phosphatase and protease), and that activities of these enzymes increased significantly (P < 0.001) in response to defoliation. Thus, this EM fungus (one of the more specialized mutualists of pine) has the potential to play a significant role in C, N and P cycling in this forested ecosystem. Therefore, many above-ground factors that reduce photosynthetic potential or divert fixed C from roots may have wide-reaching ecosystem effects.

DNA hybridization and polymerase chain reaction (PCR) tests for identification of Gaeumannomyces, Phialophora and Magnaporthe isolates

DNA hybridization and PCR tests for Gaeumannomyces graminis and related fungi were developed to aid in their identification and classification. Hybridizations with a Gaeumannomyces graminis var. tritici mitochondrial DNA probe to restriction fragments from other Gaeumannomyces, Phialophora or Magnaporthe isolates demonstrated that almost all of the tested Gaeumannomyces and Phialophora isolates, including Gaeumannomyces cylindrosporus and Gaeumannomyces incrustans , had sequences homologous to the probe. In addition, M. poae had strong homology with the probe. Most isolates of Gaeumannomyces graminis var. tritici , the causative agent of take-all disease of cereals, had identical Eco R I and Msp I hybridization patterns, whereas sizes of hybridizing restriction fragments of Phialophora and Magnaporthe isolates showed more variation. Polymerase chain reaction (PCR) tests with primers developed for G. graminis var. tritici were positive for all G. graminis isolates. PCR tests for G. incrustans, G. cylindrosporus, Phialophora and Magnaporthe were positive, but test conditions in which positive results were obtained were more limited than PCR conditions which amplified G. graminis DNA.

Transformation ofGaeumannomyces graminis to benomyl resistance

SummaryGaeumannomyces graminis var.graminis andtritici were transformed to benomyl resistance using pBT3, a plasmid encoding fungicide-resistant β-tubulin. Either circular or linear plasmid DNA producedG. graminis var.graminis transformants in which plasmid DNA was integrated into the fungal genome. There was no evidence for autonomous plasmid replication in any of the transformants examined. 4/11 linear DNA transformants had a single plasmid copy, whereas 8/9 circular DNA transformants had multiple copies of the plasmid. Integration of transforming DNA occurred by nonhomologous recombination in all (20/20) of these transformants.

Algal Species and Light Microenvironment in a Low-pH, Geothermal Microbial Mat Community

ABSTRACT Unicellular algae are the predominant microbial mat-forming phototrophs in the extreme environments of acidic geothermal springs. The ecology of these algae is not well known because concepts of species composition are inferred from cultivated isolates and microscopic observations, methods known to provide incomplete and inaccurate assessments of species in situ. We used sequence analysis of 18S rRNA genes PCR amplified from mat samples from different seasons and different temperatures along a thermal gradient to identify algae in an often-studied acidic (pH 2.7) geothermal creek in Yellowstone National Park. Fiber-optic microprobes were used to show that light for algal photosynthesis is attenuated to <1% over the 1-mm surface interval of the mat. Three algal sequences were detected, and each was present year-round. A Cyanidioschyzon merolae sequence was predominant at temperatures of ≥49°C. A Chlorella protothecoides var. acidicola sequence and a Paradoxia multisita-like sequence were predominant at temperatures of ≤39°C.