Epichloë hybrida, sp. nov., an emerging model system for investigating fungal allopolyploidy.

Endophytes of the genus Epichloë (Clavicipitaceae, Ascomycota) frequently occur within cool-season grasses and form interactions with their hosts that range from mutualistic to antagonistic. Many Epichloë species have arisen via interspecific hybridization, resulting in species with two or three subgenomes that retain all or nearly all of their original parental genomes, a process termed allopolyploidization. Here, we characterize Epichloë hybrida, sp. nov., a mutualistic species that has increasingly become a model system for investigating allopolyploidy in fungi. The Epichloë species so far identified as the closest known relatives of the two progenitors of E. hybrida are E. festucae var. lolii and E. typhina. We confirm that the nuclear genome of E. hybrida contains two homeologs of most protein-coding genes from E. festucae and E. typhina, with genome-wide gene expression analysis indicating a slight bias in overall gene expression from the E. typhina subgenome. Mitochondrial DNA is detectable only from E. festucae, whereas ribosomal DNA is detectable only from E. typhina. Inheriting ribosomal DNA from just one parent might be expected to preferentially favor interactions with ribosomal proteins from the same parent, but we find that ribosomal protein genes from both parental subgenomes are nearly all expressed equally in E. hybrida. Finally, we provide a comprehensive set of resources for this model system that are intended to facilitate further study of fungal hybridization by other researchers.

An interspecific fungal hybrid reveals cross-kingdom rules for allopolyploid gene expression patterns.

Polyploidy, a state in which the chromosome complement has undergone an increase, is a major force in evolution. Understanding the consequences of polyploidy has received much attention, and allopolyploids, which result from the union of two different parental genomes, are of particular interest because they must overcome a suite of biological responses to this merger, known as "genome shock." A key question is what happens to gene expression of the two gene copies following allopolyploidization, but until recently the tools to answer this question on a genome-wide basis were lacking. Here we utilize high throughput transcriptome sequencing to produce the first genome-wide picture of gene expression response to allopolyploidy in fungi. A novel pipeline for assigning sequence reads to the gene copies was used to quantify their expression in a fungal allopolyploid. We find that the transcriptional response to allopolyploidy is predominantly conservative: both copies of most genes are retained; over half the genes inherit parental gene expression patterns; and parental differential expression is often lost in the allopolyploid. Strikingly, the patterns of gene expression change are highly concordant with the genome-wide expression results of a cotton allopolyploid. The very different nature of these two allopolyploids implies a conserved, eukaryote-wide transcriptional response to genome merger. We provide evidence that the transcriptional responses we observe are mostly driven by intrinsic differences between the regulatory systems in the parent species, and from this propose a mechanistic model in which the cross-kingdom conservation in transcriptional response reflects conservation of the mutational processes underlying eukaryotic gene regulatory evolution. This work provides a platform to develop a universal understanding of gene expression response to allopolyploidy and suggests that allopolyploids are an exceptional system to investigate gene regulatory changes that have evolved in the parental species prior to allopolyploidization.

Exploring molecular signaling in plant-fungal symbioses using high throughput RNA sequencing.

Plant-fungal symbioses are a common feature in nature. They vary from pathogenic interactions, where fungi subvert plant resources for their own use, to mutualistic associations, where both fungus and host benefit from the interaction. Although the ecological importance of plant-fungal symbioses has long been recognized and the biology of several key associations are now well studied, new technologies have the potential to allow fresh insight into the molecular basis of plant-fungal interactions. One such technique - high throughput RNA sequencing - has recently been used to explore the molecular basis of cross-species communications. Here, we give a brief overview of this emerging technology, and present a general guide for employing the methodology to dissect plant-fungal symbiosis.

Transformation of the ryegrass endophyte Neotyphodium lolii can alter its in planta mycelial morphology.

The fungus Neotyphodium lolii grows in the intercellular spaces of perennial ryegrass as a mutualistic endosymbiont. One of the benefits it conveys to the plant is the production of alkaloids toxic to herbivores. We wanted to determine in planta expression patterns of the N. lolii 3-hydroxy-3-methylglutaryl-CoA reductase (HMG CoA reductase) gene, believed to be involved in the synthesis of two of these alkaloid toxins, lolitrem B and ergovaline. We transformed the N. lolii strain Lp19 with plasmids, in which DNA fragments upstream of the open reading frame of the N. lolii HMG CoA reductase gene controlled expression of the GUS (gusA; Escherichia coli beta-glucuronidase) reporter gene. In exponentially growing cultures, the GUS gene was not expressed if the length of upstream sequence was less than 400 bp, and >1100 bp were required for maximum expression. When reintroduced into ryegrass plants, transformants often showed highly increased hyphal branching compared to the wild-type parent strain, although in culture their growth kinetics and morphology were indistinguishable from that of the wild-type. Deterioration of hyphae and the hypha-plant interface occurred and in one transformant reduced tillering (formation of new plants, referred to in agronomy as tillers) and death of infected plants. We found no evidence that these abnormalities were caused by interference of the construct with the function of the native gene, as judged by analysis of the site of integration of the promoter-GUS cassette, expression of the native gene and lolitrem B and ergovaline levels in infected plants. However, there was some correlation between GUS expression and the degree of hyphal branching, suggesting that high levels of beta-glucuronidase may disturb the symbiotic interaction. Levels of another alkaloid, peramine, were also not significantly affected by transformation. In previous studies increased in planta branching of the endophyte has been shown to be associated with a severe reduction of alkaloid production. Our results show that a plant-endophyte association in which increased branching occurs is still able to produce alkaloids.

Quantitative assessment of in planta distribution of metabolic activity and gene expression of an endophytic fungus.

Using perennial ryegrass infected with an Acremonium transformant carrying the Escherichia coli beta-D-glucuronidase gene (gusA) (GUS system) under control of a constitutive promoter, we have developed methods for the quantitative extraction of endophyte-associated GUS activity from plant material. Fluorometric assays of these extracts allow quantitative assessment of the distribution of endophyte-associated GUS activity within single plants (tillers) with high resolution. Fluorescence microscopy with the dye Imagene Green can in addition visualize individual GUS-expressing hyphae. Since the transformant expresses the GUS gene constitutively, GUS activity can be used as an indicator of in planta endophyte metabolic activity. Using this approach we found that (i) the concentration of endophyte metabolic activity in plant tissue decreases with increasing plant size, (ii) approximately 70% of endophyte metabolic activity present in a plant is located in the leaf sheaths, (iii) basal-apical gradients and lateral (younger to older tissue) gradients of endophyte metabolic activity exist and (iv) basal-apical gradients are established early in leaf development. Our data suggest that the concentration of endophyte in each part of the plant is regulated so that a predetermined threshold of total endophyte activity per plant is not exceeded and a consistent distribution pattern is maintained.