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Supporting Information Figs S1-S5

Fig. S1 Correction for PTIO quenching effects on DAR-4M-T fluorescence.(a) Spores loaded with DAR-4M-AM were allowed to germinate in the presence of increasing concentrations of the NO-scavenger PTIO. At low PTIO concentrations there was a stimulation in fluorescence above the control with no PTIO added, whilst at PTIO concentrations above 5-10 µM, there was both an instantaneous drop in signal, and a decrease in the rate of fluorescence increase. (b) Addition of increasing concentrations of PTIO at the end of the experiment caused an instantaneous drop in DAR-4M-T fluorescence due to absorption and/or quenching effects. (c) Following baseline subtraction, the change in fluorescence was used to construct a quench calibration curve. The data were fitted with a mono-exponential curve, which, when inverted, was used to generate a concentration dependent correction coefficient.

Fig. S2 Cross-Kingdom phylogenetic tree of organisms carrying proteins with nitric oxide synthase domain architecture.Phylogenetic tree of sequences revealed by iterative Hidden-Markov search of metazoans, plants, fungi, eubacteria and archaebacteria using PFAM seed alignment of the nitric oxide synthase domain (PF02898). The sequences were analysed for the presence of domains necessary for nitric oxide production and reveal 6 clades, including nitric oxide synthases and a fungal-specific clade of putative bifunctional p450 reductases, named fungal NOS-like (NOL; wherein chytridiomycetes are shown in blue; zygomycetes in turquoise; ascomycetes in red and basidiomycetes in green).

Samalova et al..Fig. S3

Fig. S3 Nitric oxide synthase clade. Detailed phylogenetic tree of the nitric oxide synthase clade shown in Fig. S2, revealing that three ascomycete fungi contain a rudimentary nitric oxide synthase domain. PFAM domains are indicated.

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Samalova et al. Fig. S4

Fig. S4 Phylogenetic trees of nitrate and nitrite reductase in fungi.Phylogenetic trees of nitrate and nitrite reductases as revealed by Hidden-Markov search of fungi residing in Table S1, using (a) M. oryzae acc MGG_06062 (NIA1) and (b) MGG_00634 (NII1). Values at the nodes represent percentage bootstrap support, from 100 bootstrap replicates. PFAM domains are indicated. Chytridiomycetes are shown in blue; zygomycetes in turquoise; ascomycetes in red and basidiomycetes in green

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Fig. S5 Southern blot analysis to confirm single targeted replacement event.Wildtype Guy11 or Δku70 and mutant gDNA was digested with appropriate restriction enzymes and hybridised to α32P [dCTP] labelled hygromycin (for Δnol3 and Δnii1) or bialophos (for Δnol2 and Δnia1) probe. The fragment sizes of the integrating selectable markers are indicated with an arrow; the star indicates the knock-outs used in this study, where more than one mutant was generated for a given gene(s).