Trehalose biosynthesis is involved in sporulation of Stagonospora nodorum

doi:10.1016/j.fgb.2009.02.002

Fungal Genetics and Biology

Volume 46, Issue 5, May 2009, Pages 381-389

https://doi.org/10.1016/j.fgb.2009.02.002 Get rights and content

Abstract

Stagonospora nodorum is a necrotrophic fungal pathogen that is the causal agent of leaf and glume blotch on wheat. S. nodorum is a polycyclic pathogen, whereby rain-splashed pycnidiospores attach to and colonise wheat tissue and subsequently sporulate again within 2–3 weeks. As several cycles of infection are needed for a damaging infection, asexual sporulation is a critical phase of its infection cycle. A non-targeted metabolomics screen for sporulation-associated metabolites identified that trehalose accumulated significantly in concert with asexual sporulation both in vitro and in planta. A reverse-genetics approach was used to investigate the role of trehalose in asexual sporulation. Trehalose biosynthesis was disrupted by deletion of the gene Tps1, encoding a trehalose 6-phosphate synthase, resulting in almost total loss of trehalose during in vitro growth and in planta. In addition, lesion development and pycnidia formation were also significantly reduced in tps1 mutants. Reintroduction of the Tps1 gene restored trehalose biosynthesis, pathogenicity and sporulation to wild-type levels. Microscopic examination of tps1 infected wheat leaves showed that pycnidial formation often halted at an early stage of development. Further examination of the tps1 phenotype revealed that tps1 pycnidiospores exhibited a reduced germination rate while under heat stress, and tps1 mutants had a reduced growth rate while under oxidative stress. This study confirms a link between trehalose biosynthesis and pathogen fitness in S. nodorum.

Introduction

Stagonospora nodorum is a necrotrophic fungal pathogen that is the causative agent of S. nodorum blotch of wheat. This disease costs the Australian wheat industry up to AUD $60 million per year in losses, and also has a significant effect on wheat production in North America (Bhathal et al., 2003, Brennan and Murray, 1998). The disease is typically initiated by wind-borne sexual ascospores that land on the leaf surface, but can also be initiated from infected seed (Solomon et al., 2006a). Under ideal conditions, multiple rounds of asexual reproduction will occur on the plant host during one growing season. This allows the pathogen to keep pace with the growth of the host, promoting colonisation of the flag leaf and glume. The polycyclic nature of S. nodorum infection means that successful asexual reproduction is a key factor in a damaging infection. With the goal of controlling the pathogen in mind, we have used a broad range of molecular techniques to elucidate the mechanisms of S. nodorum pathogenicity. This has revealed many factors with an influence on sporulation. A cohort of signalling proteins, the G-alpha subunit Gna1, the mitogen-activated protein kinase Mak2, and the calcium/calmodulin-dependant kinases CpkA and CpkC have been shown to be involved in asexual sporulation in S. nodorum (Solomon et al., 2006c, Solomon et al., 2004b, Solomon et al., 2005b). Work studying the catabolism/anabolism of mannitol confirmed a requirement for mannitol 1-phosphate dehydrogenase, and mannitol dehydrogenase activities for asexual sporulation (Solomon et al., 2005a, Solomon et al., 2006d). Interestingly, trehalose was more abundant in strains lacking mannitol 1-phosphate dehydrogenase. Recently, the short-chain dehydrogenase Sch1, which is up-regulated by Gna1-dependant signalling, has been shown to be required for asexual sporulation (Tan et al., 2008). Also of interest is the mode of action of malayamycin, a novel antifungal molecule which, in S. nodorum, acts primarily though inhibition of sporulation (Li et al., 2008, Loiseleur et al., 2007).

A non-targeted metabolomics study was completed to further characterise metabolic functions required for sporulation. The disaccharide trehalose was found to markedly increase during sporulation both in vitro and in planta. S. nodorum strains lacking a predicted trehalose 6-phosphate synthase, Tps1, were shown to be affected in their ability to develop lesions and were unable to develop pycnidia and pycnidiospores at the wild-type rate. Trehalose levels in planta and in vitro were greatly reduced in tps1 strains, and the ability to resist oxidative stress was also diminished.

Section snippets

Strains and culture conditions

S. nodorum SN15 was obtained and cultured on CZV8CS medium or minimal medium as described previously (Solomon et al., 2004a).

Infection conditions

Whole-plant spray and detached leaf assays were conducted as described previously (Solomon et al., 2004a, Solomon et al., 2004b). Infections were conducted at least twice.

Gene deletion and complementation constructs

The pTPSKO knockout plasmid was constructed as follows: A 1056 bp region 5′ of the Tps1 start codon was amplified by PCR with SN15 gDNA and primers TPSKO5′F (5′-CTCGAGAGATCTAATAGATGCCATAA-3′) and TPSKO5′R

Trehalose is a sporulation-associated metabolite in S. nodorum

A non-targeted analysis of SN15 metabolism during sporulation both in vitro and in planta was performed in order to identify sporulation-specific metabolites. For in vitro sporulation, SN15 was grown on solid minimal medium and tissue harvested prior to sporulation occurred (4 dpi), during the initiation of sporulation (11 dpi), and at the peak of asexual sporulation (18 dpi). For in planta sporulation, wheat cv. Amery was infected with SN15 in the latent period assay format, and lesions excised

Trehalose in fungi

Trehalose is a characteristic metabolite of fungi and is much more abundant and frequent in this kingdom than elsewhere. However the role(s) of trehalose remain ill defined. It is a key metabolite in the sporulation of several fungi where it can accumulate to high concentrations in hyphae and spores. It also accumulates in response to environmental stress. In Saccharomyces cerevisiae trehalose accumulates to high levels in stationary-phase cultures and spores, and in cells subjected to heat

Acknowledgments

The authors would like to thank the Grains Research and Development Corporation for financial support.

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¹: Present address: Rothamsted Research, Harpenden, Hertfordshire, AL5 2JQ, UK.

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Trehalose biosynthesis is involved in sporulation of Stagonospora nodorum

Abstract

Introduction

Section snippets

Strains and culture conditions

Infection conditions

Gene deletion and complementation constructs

Trehalose is a sporulation-associated metabolite in S. nodorum

Trehalose in fungi

Acknowledgments

J. Biol. Chem.

J. Biol. Chem.

Trends Biotechnol.

Anal. Biochem.

Trend. Biochem. Sci.

Yield reduction in wheat in relation to leaf disease from yellow (tan) spot and Septoria nodorum blotch

Eur. J. Plant Pathol.

Economic Importance of Wheat Diseases in Australia

Trehalose metabolism is important for heat stress tolerance and spore germination of Botrytis cinerea

Microbiology

The ontogeny of Stagonospora nodorum pycnidia in culture

Sydowia

New insights on trehalose: a multifunctional molecule

Glycobiology

Trehalose is required for the acquisition of tolerance to a variety of stresses in the filamentous fungus Aspergillus nidulans

Microbiology

Trehalose synthesis and metabolism are required at different stages of plant infection by Magnaporthe grisea

EMBO J.

Dothideomycete plant interactions illuminated by genome sequencing and EST analysis of the wheat pathogen Stagonospora nodorum

Plant Cell