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emi12169-sup-0001-si.tiff592K

Fig. S1. Phylogenetic analysis of Beauveria bassiana BbGPCR3. (a) Neighbour joining analysis of BbGPCR3 with other GPCRs. Bootstrap values > 50% from 1000 replicates are shown as numbers at each supported branch. Abbreviations for organism species followed by GenBank accession numbers of their respective gene are shown as follows; fungi (Ac: Ajellomyces capsulatus, Ad: A. dermatitidis, Af: Aspergillus fumigatus, Ao: Arthroderma otae, Bb: Beauveria bassiana, Cm: Cordyceps militaris, Gg: Gaeumannomyces graminis, Ma: Metarhizium anisopliae, Mg: Magnaporthe grisea, Nc: Neurospora crassa, Tat: Trichoderma atroviride, Tr: T. reesei, Tv: T. virens, Te: Trichophyton equinum); slime molds (Da: Dictyostelium aureostipes, Dd: D. discoideum, Df: D. fasciculatum); plants (At: Arabidopsis thaliana, Os: Oryza sativa, Ps: Pisum sativum Sb, Sorghum bicolor, Zm: Zea mays); metazoa (Tad: Trichoplax adhaerens). (b) Phylogenetic analysis of BbGPCR3 with fungal GPCRs that have been at least partially genetically characterized. GenBank accession numbers follow the organism and gene abbreviations as in (a).

emi12169-sup-0002-si.tif227K

Fig. S2. Molecular manipulations of B. bassiana BbGPCR3 gene. (a) Schematic diagram of disruption vector, BbGPCR3 genomic locus, and homologous recombination event. (b) PCR confirmation of correct recombination events. Lane 1: WT strain; lane 2: ΔBbGPCR3; lane 3: complemented (ΔBbGPCR3::BbGPCR3) strain, and lane M: DNA molecular size standards. (c) Southern blotting analysis of XhoI-digested genomic DNA from WT strain (lane1), ΔBbGPCR3 (lane2) and ΔBbGPCR3::BbGPCR3 strains (lane3). The electrophoretic positions and sizes of the DNA bands are indicated.

emi12169-sup-0003-si.tiff2221K

Fig. S3. Representative images for growth phenotypes of WT strain (WT), ΔBbGPCR3 mutant (KO) and ΔBbGPCR3::BbGPCR3 strain (CP). (a) 7 day growth on inidated carbon and nitrogen sources, (b) 7 day old growth under indicated stress conditions, (c) 10 day growth on menadione (top panel) and H2O2 (bottom panel). Culture media were prepared as described in the Methods section.

emi12169-sup-0004-si.tif363K

Fig. S4. Effect of cAMP on stress response. Conidia (1 μl, 107 conidia ml−1) of the WT (hashed bar), ΔBbGPCR3 (open bars) and ΔBbGPCR3::BbGPCR3 (dotted bars) were spotted onto CZA plates containing the indicated stress reagent (NaCl, sorbitol, H2O2, menadione, Congo Red) and various concentrations of cAMP. The diameters of the colonies after 7 d of growth are presented. Error bars: SD of the mean from three replicate assays.

emi12169-sup-0005-si.tif644K

Fig. S5. Effect of growth carbon substrate (top panels) and cAMP concentration (bottom panels) on the time-course of blastospore production in B. bassiana. The carbon source in SDB (glucose) was replaced with different carbons including, trehalose, sucrose, fructose, glycerol and lactate. cAMP was included in SDB media at the indicated final concentrations (0 to 7.5 mM). Cultures were inoculated using conidia derived from the WT (○), ΔBbGPCR3 (□) and ΔBbGPCR3::BbGPCR3 strains (●) and were grown at 25°C with aeration. Blastospore concentration was quantified over the indicated time course (24, 30, 36, 48, 60 and 72h) via direct counting as described in the Methods section.

emi12169-sup-0006-si.tif9997K

Fig. S6. Effect of amino acids on blastospore production. Media (CZB) containing glucose (4%) supplemented with the indicated amino acids (GAAx), arginine, tryptophan, glycine, and proline at various final concentrations (w/v: 0.25, 0.50, 0.75 and 1.00%) were inoculated with conidia derived from WT (striped lines), ΔBbGPCR3 mutant (open bar) and ΔBbGPCR3::BbGPCR3 (dotted bar) strain and blastospore production was quantified after 72 h of growth. Different letters on column bars indicate significant difference (Tukey's honestly significant difference: P < 0.05). Error bars: SD of the mean from three replicate assays.

emi12169-sup-0007-si.tif625K

Fig. S7. Time course of blastospore production in the presence of various amino acids (final time point shown in Fig. S5. Amino acid concentrations are given on top of each graph. Blastospore concentrations at indicated time points for WT (○), ΔBbGPCR3 mutant (□) and ΔBbGPCR3::BbGPCR3 (●) strains.

emi12169-sup-0008-si.docx33K

Table S1. PCR primers used for the gene cloning, generation and identification of gene replacement mutants.

Table S2. Primers for qRT-PCR analysis.

Table S3. Enriched pathway analysis. KEGG annotation showed that there were 5631 genes with pathway annotation in B. bassiana genome, 1450 DEGs with pathway annotation in paired library of WTSDBBbGPCR3SDB and 2943 DEGs with pathway annotation in paired library of ΔBbGPCR3SDBBbGPCR3TRE.

emi12169-sup-0009-si.xlsx13K

Excel File S1. Pfam analysis.

emi12169-sup-0010-si.xlsx206K

Excel File S2. Summary of overlapping genes.

emi12169-sup-0011-si.xlsx21K

Excel File S3. Summary of gene ontology (GO) enrichment terms.

emi12169-sup-0012-si.xlsx42K

Excel File S4. PHI database analysis.

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