Large-scale gene discovery in the Septoria tritici blotch fungus Mycosphaerella graminicola with a focus on in planta expression
Kema, G.H.J. ; Lee, T.A.J. van der; Mendes, O. ; Verstappen, E.C.P. ; Klein Lankhorst, R.M. ; Sandbrink, H. ; Burgt, A. van der; Zwiers, L.H. ; Csukai, M. ; Waalwijk, C. - \ 2008
Molecular Plant-Microbe Interactions 21 (2008)9. - ISSN 0894-0282 - p. 1249 - 1260.
major facilitator superfamily - rice blast fungus - genome sequence - fusarium-graminearum - magnaporthe-grisea - stagonospora-nodorum - phytophthora-sojae - transporter genes - botrytis-cinerea - toxic compounds
The foliar disease septoria tritici blotch, caused by the fungus Mycosphaerella graminicola, is currently the most important wheat disease in Europe. Gene expression was examined under highly different conditions, using 10 expressed sequence tag libraries generated from M. graminicola isolate IPO323 using seven in vitro and three in planta growth conditions. To identify fungal clones in the interaction libraries, we developed a selection method based on hybridization with the entire genomic DNA of M. graminicola, to selectively enrich these libraries for fungal genes. Assembly of the 27,007 expressed sequence tags resulted in 9,190 unigenes, representing 5.2 Mb of the estimated 39-Mb genome size of M. graminicola. All libraries contributed significantly to the number of unigenes, especially the in planta libraries representing different stages of pathogenesis, which covered 15% of the library-specific unigenes. Even under presymptomatic conditions (5 days postinoculation), when fungal biomass is less than 5%, this method enabled us to efficiently capture fungal genes expressed during pathogenesis. Many of these genes were uniquely expressed in planta, indicating that in planta gene expression significantly differed from in vitro expression. Examples of gene discovery included a number of cell wall-degrading enzymes, a broad set of genes involved in signal transduction (n = 11) and a range of ATP-binding cassette (n = 20) and major facilitator superfamily transporter genes (n = 12) potentially involved in protection against antifungal compounds or the secretion of pathogenicity factors. In addition, evidence is provided for a mycovirus in M. graminicola that is highly expressed under various stress conditions, in particular, under nitrogen starvation. Our analyses provide a unique window on in vitro and in planta gene expression of M. graminicola.
MgAtr7, a new type of ABC transporter from Mycosphaerella graminicola involved in iron homeostasis
Zwiers, L.H. ; Roohparvar, R. ; Waard, M.A. de - \ 2007
Fungal Genetics and Biology 44 (2007)9. - ISSN 1087-1845 - p. 853 - 863.
major facilitator superfamily - atp-binding cassette - multidrug-resistance - siderophore biosynthesis - filamentous fungi - toxic compounds - gene-cluster - spore wall - wheat - virulence
The ABC transporter-encoding gene MgAtr7 from the wheat pathogen Mycosphaerella graminicola was cloned based upon its high homology to ABC transporters involved in azole-fungicide sensitivity. Genomic and cDNA sequences indicated that the N-terminus of this ABC transporter contains a motif characteristic for a dityrosine/pyoverdine biosynthesis protein. This makes MgAtr7 the first member of a new class of fungal ABC transporters harboring both a transporter and a biosynthetic moiety. A homologue of MgAtr7 containing the same biosynthetic moiety was only found in the Fusarium graminearum genome and not in any other fungal genome examined so far. The gene structure of both orthologous transporters is highly conserved and the genomic area surrounding the ABC transporter exhibits micro-synteny between M. graminicola and F. graminearum. Functional analyses revealed that MgAtr7 is neither required for virulence nor involved in fungicide sensitivity but indicated a role in maintenance of iron homeostasis.
Control of Mycosphaerella graminicola on wheat seedlings by medical drugs known to modulate the activity of ATP-binding cassette transporters
Roohparvar, R. ; Huser, A. ; Zwiers, L.H. ; Waard, M.A. de - \ 2007
Applied and Environmental Microbiology 73 (2007)15. - ISSN 0099-2240 - p. 5011 - 5019.
major facilitator superfamily - natural toxic compounds - multidrug-resistance - abc transporters - botrytis-cinerea - efflux pump - fungicide sensitivity - virulence - plant - reversal
Medical drugs known to modulate the activity of human ATP-binding cassette (ABC) transporter proteins (modulators) were tested for the ability to potentiate the activity of the azole fungicide cyproconazole against in vitro growth of Mycosphaerella graminicola and to control disease development due to this pathogen on wheat seedlings. In vitro modulation of cyproconazole activity could be demonstrated in paper disk bioassays. Some of the active modulators (amitriptyline, flavanone, and phenothiazines) increased the accumulation of cyproconazole in M. graminicola, suggesting that they reversed cyproconazole efflux. However, synergism between cyproconazole and modulators against M. graminicola on wheat seedlings could not be shown. Despite their low in vitro toxicity to M. graminicola, some modulators (amitriptyline, loperamide, and promazine) did show significant intrinsic disease control activity in preventive and curative foliar spray tests with wheat seedlings. The results suggest that these compounds have indirect disease control activity based on modulation of fungal ABC transporters essential for virulence and constitute a new class of disease control agents.