Characterisation of proteinaceous toxins isolated from Pyrenophora teres f. teres.

Abstract

Pyrenophora teres f. teres (Ptt) causes net form net blotch disease (NFNB), an important disease of barley in Australia and worldwide. This fungus uses proteinaceous toxins to cause necrosis and different isolates of Ptt differ in their ability to cause symptoms on different cultivars of barley. However, little is known about the roles of pathogen growth and individual toxins in symptom development. This project therefore aimed to determine whether there is a relationship between toxin production, fungal growth and virulence in NFNB. Conidial germination, extent of fungal growth and culture filtrate toxicity were compared for six South Australian Ptt isolates with different virulence on the barley cultivar ‘Sloop’. In addition, Ptt toxin production was optimised before identification and selection of virulence-related candidate proteins (VRCPs) for further characterisation. The biological activity of recombinant VRCPs on susceptible and resistant cultivars and VRCPs gene expression during the interaction of Sloop with each isolate were also compared. In general, the more virulent isolates had higher rates of conidial germination (both in vitro and in planta) and fungal development in planta, represented by longer hyphae and more appressoria, compared with less virulent isolates. Similarly, PttGAPDH and its transcript were more abundant during the interaction of barley with more virulent isolates. A proteomics approach was used to identify proteins unique to the more virulent isolate, proteins from bioactive fractions on either susceptible (Sloop) or resistant cultivars (CI9214 and Beecher) and proteins from the intercellular washing fluids (ICWFs) of infected barley. These analyses revealed that Ptt produced proteins between 37 and 150 kDa that have biological activity. Liquid Chromatography-Electrospray Ionisation Ion-Trap Mass Spectrometry (LC-eSI-IT MS), of individual biologically active proteins was used to identify peptides which matched to 17 proteins that belong to three groups of fungal proteins including virulence-related proteins; fungal growth and development proteins; and those with unknown function (hypothetical proteins). However, Ptt toxins were not detected in the ICWF protein profiles suggesting that Ptt toxins were either in trace amounts or might be internalised into the cell. The four VRCPs selected, were identified as hypothetical proteins with unknown function in the Ptt database. Further bioinformatic analysis characterised these VRCPs as an isochorismatase (PttCHFP1), an endo-1, 4-β- xylanase A (PttXyn11A), a glycophosphatidylinositol (GPI)-anchored common in fungal extracellular membrane (CFEM) domain-containing protein (PttGPICFEM) and an unknown proteinaceous secreted (but conserved) hypothetical protein (PttSP1). These VRCPs were heterologously expressed and characterised using gene expression studies. PttXyn11A had strong homology with the well characterised endoxylanases, TrXyn11A from Trichoderma reesei and BcXyn11A from Botrytis cinerea, known to contribute to virulence. A necrosis-inducing region on the surface of the enzyme was also identified in PttXyn11A, suggesting a potential role in necrosis induction. The culture filtrates for more virulent isolates had significantly greater xylanase activity than those from less virulent isolates. Even though heterologously expressed PttXyn11A was toxic to Escherichia coli, xylanase activity was detectable at very low levels and was not enough to cause symptoms in the bioassay. In addition, semi-quantitative reverse transcriptase polymerase chain reaction (RT-PCR) and RT-quantitative PCR (RT-qPCR) analysis demonstrated that PttXyn11A was expressed more abundantly by the more virulent isolates compared with the other isolates in culture and during the plant-pathogen interaction. Together, these results suggest that PttXyn11A plays a role in virulence, either through its ability to degrade the plant cell wall to assist fungal growth or through its necrosisinducing ability. PttCHFP1 showed homology to an isochorismatase, an enzyme that has been proposed to have a role in plant defence via inhibition of salicylic acid production. PttSP1 showed homology to a membrane lipoprotein proposed to have a role in fungal development. Bioassay of recombinant PttCHFP1 and PttSP1 induced chlorosis symptoms in the susceptible barley cultivar (Sloop). The cysteine-rich CFEM domain identified in PttGPI-CFEM has been suggested to have an important role in hyphal attachment and fungal networking. However, E. coli was not able to express this gene probably due to its attachment to the plasma membrane and/or cell wall. Analysis of the gene expression profiles for PttCHFP1, PttGPI-CFEM and PttSP1 showed no significant differences between isolates in vitro and in planta suggesting that all isolates regulated the expression of these genes to the essential level possibly required for pathogenesis. This is the first study to identify the relationship between fungal growth and proteinaceous toxin production, characterise individual proteinaceous toxins in the mixture of Ptt culture filtrate and investigate the expression profiles of genes encoding VRCPs during the Ptt-barley interaction. This study therefore provides a better understanding of the Ptt-barley interaction by identifying the potential toxins which might lead to identify the toxin targets and ultimately support the breeding of resistant cultivars of barley.