The utilisation of di/tripeptides by Stagonospora nodorum is dispensable for wheat infection

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Abstract

A gene required for di/tripeptide transport in the necrotrophic wheat pathogen Stagonospora nodorum has been cloned, characterised and inactivated by homologous recombination. Recent genome sequencing projects have revealed the presence of fungal homologues though Ptr2 is the first di/tripeptide transporter to be cloned and function characterised from a fungus. Analysis of Ptr2 expression in vitro revealed strong expression in the absence of nitrogen and in the presence of carbon; interestingly there was very low expression in the absence of both nitrogen and carbon. The expression of Ptr2 during infection showed the gene was significantly up-regulated during the initial stages of infection before decreasing to a lower constitutive level suggesting the fungus may be nitrogen starved during the pre-penetration stage of the infection. Ptr2 was inactivated by homologous gene recombination resulting in the strain S. nodorum ptr2. Peptide uptake studies of S. nodorum ptr2 suggest that Ptr2 is solely responsible for the uptake of di/tripeptides. The ability of S. nodorum ptr2 to cause infection was also examined. Pathogenicity assays revealed that the mutant strain was fully pathogenic. As the gene has been shown to be fully responsible for di/tripeptide transport, this implies that the uptake of these small peptides is not required for S. nodorum pathogenicity on wheat leaves.

Introduction

To successfully colonise a host plant, a fungal phytopathogen must overcome a series of physical and chemical barriers. The primary physical barrier the fungus encounters is the surface of the host. Once inside the leaf the host can often respond with various means of chemical defence such as the production of reactive oxygen species, fungal cell wall degrading enzymes and phytoalexins. After successfully overcoming these, the fungus can then go into accessing the nutrients provided by the plant. It had long been assumed that once the fungus had penetrated and was established internally, there was an abundance of nutrients to feed on. However, several key reports around this time though discovered that the expression of various genes required for pathogenicity were only expressed in vitro under nutrient limiting conditions, particularly nitrogen [1], [2], [3], [4]. This led to the counter-assumption that the infecting fungus was instead growing under starvation conditions during infection. Very few reports have since directly measured the nutrient composition of the host during infection. One recent report that measured the nitrogen composition of the tomato leaf apoplast during infection by Cladosporium fulvum revealed the presence of abundant amino acids and other nitrogen sources [5]. However this organism is a unique biotroph and may not be representative of other fungal phytopathogens [6].

Phaeosphaeria nodorum (Müller) Hejaroude (anamorph Stagonospora nodorum (Berk.) Castellani and Germano) [7] is a necrotrophic phytopathogen that is the causal agent of leaf and glume blotch on wheat and is responsible for $60 M (AUD) of crop loss in Australia each year. Surprisingly, though being an economically important pathogen and comparatively simple to work with, very little is known at a molecular level about how the fungus infects wheat.

As with other fungal phytopathogens, S. nodorum must be able to acquire nutrients during colonisation of wheat to complete its lifecycle. A previous report has shown that S. nodorum does produce extracellular proteases during infection [8] and consequently peptides could play a role in nutrition. During the sequencing of a cDNA library of S. nodorum, a clone was sequenced with strong similarity to the peptide transporter, PTR2 from the yeast Saccharomyces cerevisiae. Molecular and biochemical evidence has been gathered showing that PTR2 is required for the uptake of di/tripeptide in yeast [9], [10]. The acquisition of a putative S. nodorum homologue of PTR2 provided the opportunity to determine if di/tripeptides played any role in the pathogenicity of S. nodorum on wheat. Peptide transporters have been previously cloned and characterised from several sources including yeasts, plants and mammals [9], [10], [11], [12], [13], [14], [15], [16], [17], [18]. Interestingly though while orthologues of Ptr2 have been sequenced in fungal genome sequencing projects, none have been functionally characterised from a filamentous fungus. In this study, we have examined the possibility that small peptides act as a nitrogen source for S. nodorum during infection by isolating and characterising a di/tripeptide transporter gene Ptr2.

Section snippets

Fungal strains and media

S. nodorum SN15 was provided by the Department of Agriculture, Western Australia. The fungus was routinely grown on CzV8CS (Czapek Dox agar (Oxoid) 45.4 g l−1, agar 10.0 g l−1, CaCO3 3.0 g l−1, Campbell's V8 juice 200 ml l−1, casamino acids 20.0 g l, peptone 20 g l−1, yeast extract 20 g l−1, adenine 3 g l−1, biotin 0.02 g l−1, nicotinic acid 0.02 g l−1, p-aminobenzoic acid 0.02 g l−1, pyrodoxine 0.02 g l−1, thiamine 0.02 g l−1) containing 1.5% agar. Plates were incubated at 22 °C in 12 h cycles

Nucleotide analysis

A cDNA orthologous to the di/tripeptide transporter PTR2 from S. cerevisiae was identified during sequencing of the S. nodorum wheat cell-wall library. A genomic library was screened with the Ptr2 cDNA clone. Four clones were isolated and one (pPtrg1) was used for subsequent nucleotide analysis. Sequencing revealed a Ptr2 open reading frame of 2026 bases that has been subsequently submitted to Genbank (Accession no. AY187281) (Fig. 2). Comparison of the genomic and cDNA sequences revealed the

Discussion

Since the observation that some genes required for pathogenicity were expressed in vitro only under nitrogen limiting conditions, the nitrogen status of the infecting fungus has received considerable attention. The aim of this study was further to characterize how fungal phytopathogens acquire nutrients in planta.

A comprehensive review of peptide transporters has been previously undertaken [22]. The review described how, peptide transporters could be grouped into two distinct families based

Acknowledgements

The authors would like to thank the Grains Research and Development Corporation for funding and Dr Barbara Howlett for the spore PCR protocol.

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