SOLiD-SAGE of endophyte-infected red fescue reveals numerous effects on host transcriptome and an abundance of highly expressed fungal secreted proteins

Abstract

One of the most important plant-fungal symbiotic relationships is that of cool season grasses with endophytic fungi of the genera Epichloë and Neotyphodium. 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 Festuca rubra we carried out a SOLiD-SAGE quantitative transcriptome comparison of endophyte-free and Epichloë festucae-infected F. rubra. 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 Penicillium and Aspergillus spp. have been demonstrated to have antifungal activity. Of the 10 epichloae whole genome sequences available, only one isolate of E. festucae and Neotyphodium gansuense var inebrians have an antifungal protein gene. The uniqueness of this gene in E. festucae from F. rubra, 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.

Figure 1. Gel analysis of F. rubra and E. festucae antisense transcripts.

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 F. rubra metallothionein (MT) and the E. festucae NC12, antifungal protein (AFP), and subtilisin-like protease were used as templates for PCR amplification.

Figure 2. The phylogenetic relationships of the MCM7 and antifungal protein coding sequences.

A. Rooted 50% majority rule maximum parsimony phylogenetic tree of the MCM7 coding sequences. The Tu. melanosporum 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 Table 5.

Figure 3. Detection of an alternatively spliced variant of Ef-AFP.

A. Structural features of the Ef-AFP 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. PCR products of (1) Ef-AFP using E. festucae genomic DNA, and (2) E. festucae-infected plant cDNA generated from oligo(dT) as templates; (3) partial Ef-AFP alternatively spliced variant using E. festucae genomic DNA, (4) E. festucae-infected plant cDNA generated from a gene-specific primer (Alt Ef-AFP Reverse), and (5) E. festucae-infected plant cDNA generated from oligo(dT) as templates.