SOLiD-SAGE of Endophyte-Infected Red Fescue Reveals Numerous Effects on Host Transcriptome and an Abundance of Highly Expressed Fungal Secreted Proteins

<div><p>One of the most important plant-fungal symbiotic relationships is that of cool season grasses with endophytic fungi of the genera <em>Epichloë</em> and <em>Neotyphodium.</em> These associations often confer benefits, such as resistance to herbivores and improved drought tolerance, to the hosts. One benefit that appears to be unique to fine fescue grasses is disease resistance. As a first step towards understanding the basis of the endophyte-mediated disease resistance in <em>Festuca rubra</em> we carried out a SOLiD-SAGE quantitative transcriptome comparison of endophyte-free and <em>Epichloë festucae</em>-infected <em>F. rubra</em>. Over 200 plant genes involved in a wide variety of physiological processes were statistically significantly differentially expressed between the two samples. Many of the endophyte expressed genes were surprisingly abundant, with the most abundant fungal tag representing over 10% of the fungal mapped tags. Many of the abundant fungal tags were for secreted proteins. The second most abundantly expressed fungal gene was for a secreted antifungal protein and is of particular interest regarding the endophyte-mediated disease resistance. Similar genes in <em>Penicillium</em> and <em>Aspergillus</em> spp. have been demonstrated to have antifungal activity. Of the 10 epichloae whole genome sequences available, only one isolate of <em>E. festucae</em> and <em>Neotyphodium gansuense</em> var <em>inebrians</em> have an antifungal protein gene. The uniqueness of this gene in <em>E. festucae</em> from <em>F. rubra</em>, its transcript abundance, and the secreted nature of the protein, all suggest it may be involved in the disease resistance conferred to the host, which is a unique feature of the fine fescue–endophyte symbiosis.</p> </div

Detection of an alternatively spliced variant of <i>Ef-AFP</i>.

<p><b>A.</b> Structural features of the <i>Ef-AFP</i> gene and the alternatively spliced variant. Exons are depicted as dark boxes, introns as lines, and sizes are given in bp. Arrows indicate positions of primers used for PCR amplification. <b>B.</b> PCR products of (1) <i>Ef-AFP</i> using <i>E. festucae</i> genomic DNA, and (2) <i>E. festucae</i>-infected plant cDNA generated from oligo(dT) as templates; (3) partial <i>Ef-</i>AFP alternatively spliced variant using <i>E. festucae</i> genomic DNA, (4) <i>E. festucae</i>-infected plant cDNA generated from a gene-specific primer (Alt <i>Ef-AFP</i> Reverse), and (5) <i>E. festucae</i>-infected plant cDNA generated from oligo(dT) as templates.</p

Sequences of oligonucleotide primers used in this study.

<p>Sequences of oligonucleotide primers used in this study.</p

Accession numbers of the MCM7 and antifungal protein sequences used in the phylogenetic analyses presented in Fig. 2.

<p>NA indicates not applicable.</p>1<p>Species for which whole genome sequences are available.</p

Gene ontology (GO) categorization of the 209 differentially expressed plant genes found by SOLiD-SAGE.

<p>The SAGE tags for each differentially expressed gene can be found in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0053214#pone.0053214.s001" target="_blank">Table S1</a>.</p

The 20 most abundant <i>E. festucae</i> transcripts found in the endophyte-infected plant S1139RC.

<p>The 20 most abundant <i>E. festucae</i> transcripts found in the endophyte-infected plant S1139RC.</p

Characteristics of the 454 sequences.

<p>Characteristics of the 454 sequences.</p

The phylogenetic relationships of the MCM7 and antifungal protein coding sequences.

<p>A. Rooted 50% majority rule maximum parsimony phylogenetic tree of the MCM7 coding sequences. The <i>Tu. melanosporum</i> sequence was designated as the outgroup for rooting the tree. The numbers at the nodes are the bootstrap percentages based on 1,000 replications. The presence (+) or absence (−) of an antifungal protein gene is indicated for each species in the tree. B. The single most parsimonious phylogenetic tree recovered from an exhaustive search of the antifungal protein coding sequences. The tree is midpoint rooted. Accession numbers of the sequences used for both trees are given in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0053214#pone-0053214-t005" target="_blank">Table 5</a>.</p

Gel analysis of <i>F. rubra</i> and <i>E. festucae</i> antisense transcripts.

<p>The diagram illustrates primer design for detection of sense and antisense transcripts. The “A” primers were used for strand specific synthesis of cDNA from the RNA sample. The “A” and “B” primers were used for cDNA amplification. cDNAs generated from gene-specific primers for the <i>F. rubra</i> metallothionein (MT) and the <i>E. festucae</i> NC12, antifungal protein (AFP), and subtilisin-like protease were used as templates for PCR amplification.</p

Physiological differences between two backcross genotypes contrasting in heat tolerance.

<p>Physiological differences between the heat tolerant backcross genotype 169 (black diamonds) and heat sensitive backcross genotype 190 (gray boxes) for 2 weeks of heat stress for (a) TQ, (b) CHL, and (c) EL. Bars represent standard deviations, and asterisks indicate significant differences between the two genotypes (n = 4, P < 0.05).</p