Fig. S1. Sequence analysis of the MaPls1 gene.

A. Alignment of Pls1 tetraspanins. Conserved motifs in transmembrane domains are underlined in black (TM1 to 4). ECL: extracellular loop; ICL: intracellular loop; LEL: large extracellular loop. Conserved amino acids are in black (identical) or grey (similar). CCG motif and two conserved cysteine are indicated with a solid triangle (▲). Charged amino acids in transmembrane domains are labelled with a solid star (★), and domains conserved among fungal tetraspanins are underlined with dots (●●●). Amino acids in insect fungal pathogens different from plant fungal pathogens are labelled with empty stars (☆). Protein sequences were aligned using ClustalX.

B. Transmembrane structure analysis of MaPls1 and MgPls1 proteins using TMHMM-2.0 software.

C. Phylogenetic analysis of Pls1 tetraspanins in Pezizomycotina with MEGA 4.0. MaPls1: Metarhizium acridum; BbPls1: Beauveria bassiana, MgPls1: Magnaporthe grisea; NcPls1: Neurospora crassa; ClPls1: Colletotrichum lindemuthianum; BcPls1: Botryotinia fuckeliana; SsPls1: Sclerotinia sclerotiorum.


Fig. S2. Construction and identification of ΔMaPls1 and complemented strains.

A. Restriction maps of the MaPls1 locus, replacement vector pK2-PB-Pls1, and complementary cassette. BI: BamHI; XI: XbaI; EV: EcoRV; EI: EcoRI. PMaPls1: MaPls1 promoter.

B. PCR analysis of two ΔMaPls1 transformants. PCR was conducted with primer pairs L1/pR (lane L1 and L2) and R1/BF (lane R1 and R2) to verify the homologous recombination of the left and right regions of MaPls1 respectively.

C. Southern blot. Genomic DNA from WT, ΔMaPls1 and complemented strains were digested with BamHI/XbaI. A 462 bp fragment of the MaPls1 downstream flanking sequence amplified with TF/TR was used as the probe.


Fig. S3. Plate growth and stress tolerance.

A. Radial growth on PDA and PDA supplemented with 1 mol l−1 NaCl, 500 μg ml−1 Congo Red or 6 mmol l−1 H2O2 respectively. Bar = 5 mm.

B. Heat tolerance assay. Conidial suspensions were treated at 45°C for 2 h, 4 h, 6 h or 8 h respectively, and germination rate was determined on PDA according to Liu and colleagues (2010).

C. UV-B irradiation tolerance assay. Conidia were spread on PDA and exposed to UV-B (1350 mWm−2) for 2 h, 4 h, 6 h or 8 h respectively, and grown at 28°C for 20 h. Germination was assayed and compared. Each trial was repeated three times. Error bars are standard deviations of three trials. a, b: significant difference, P < 0.05.


Fig. S4. GO annotation of differentially expressed genes.


Table S1. Major characteristics of WT and ΔMaPls1 DGE tag libraries.

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Table S2. Differentially expressed genes by DGE.


Table S3. Verification of DGE results by qRT-PCR.


Table S4. BLAST analysis of differentially expressed genes by PHI database


Table S5. Primers used for construction and analysis of engineered strains.

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