Identification of Proteins Involved in the Symbiotic Interaction Between the Fungal Endophyte Neotyphodium Lollii with Perennial Ryegrass (Lolium Perenne)

Abstract

Secreted proteins have important roles in a large number of plant-fungal interactions ranging from pathogenesis to symbiosis and biocontrol. These proteins serve a myriad of functions such as fungal morphogenesis, adhesion to and/or degradation of the plant cell wall, eliciting or countering plant defence-related responses, and nutrient acquisition. It is likely that secreted proteins, and those involved in cell wall biogenesis, play an important role in communication in the symbiotic interaction between the fungal endophyte Neotyphodium lolii with perennial ryegrass (Lolium perenne) particularly given the close association between fungal hyphae and the plant cell wall. In addition, these proteins are likely to be responsible for establishing and maintaining a normal symbiotic relationship. In this study, a proteomics approach was used to identify secreted proteins involved in symbiosis. Initially, total and secreted proteins from in vitro fungal cultures were examined using two-dimensional electrophoresis in order to develop a set of proteomic tools specific to the N. lolii/L. perenne system before examining infected vs. uninfected plant protein fractions. As the Neotyphodium genome sequence was unavailable and there were very few Neotyphodium or Epichloë proteins in publicly available databases, two culture-derived cDNA libraries were constructed and used to create a fungal EST database containing over 7300 sequences. This database was successfully used to identify fungal proteins analysed using MALDI-TOF mass fingerprinting and MS/MS mass spectrometry. Proteomic analysis of enriched fractions from infected vs. uninfected plants using MALDI-TOF mass fingerprinting by comparison to public databases resulted in the identification of four plant proteins with altered expression during symbiosis. In addition, a novel fungal trypsin-like serine protease was identified following de novo MS/MS peptide sequencing g and interrogation of a proprietary AgResearch/Agriculture Victoria Plant Genomics Programme database. Subsequent expression analysis and sequencing of genomic DNA showed that the tspA gene was highly upregulated in planta compared to in culture. The function of tspA in symbiosis was examined by over-expressing the gene under the control of the Aureobasidium pullulans translation elongation factor 1-alpha gene promoter. No major differences were observed between plants infected with the parent strain or endophyte strains transformed with the tspA over-expression construct. Expression of the full-length peptide in E. coli was also undertaken and polyclonal antisera to tspA generated. In addition to the proteomics studies, a reverse genetics approach was also used to identify secreted proteins and proteins predicted to be involved in cell wall biogenesis. The fungal EST database was mined using key word searches for genes known to be involved in other plant-microbe interactions and nineteen genes chosen for expression analysis. Nine genes (celA, chiA, chiB, abpA, spsA, nlhA, rhgA, ptfA and nlhB) with relatively high expression in planta compared to their expression in culture were chosen for further analysis. These were subsequently sequenced and putative function assigned to each gene using bioinformatic analysis. Eight of the genes were previously uncharacterised in Neotyphodium and Epichloë spp. The rhgA gene was chosen for further characterisation using gene disruption. In planta analysis of rhgA- mutants showed that disruption of the rhgA gene resulted in unregulated hyphal growth. This phenotype was fully complemented by insertion of the rhgA gene. This suggests that the rhgA gene is important for maintaining normal hyphal growth and regulation during symbiosis.