A Ch B Col-0 Cg pen2-1 Supplemental Figure 1. Trypan Blue Staining of Leaves Inoculated with Adapted and Nonadapted Colletotrichum Species.(A) Conidial suspensions of C. higginsianum MAFF305635 (Ch) were drop-inoculated on A. thaliana Col-0. (B) Conidial suspensions of C. gloeosporioides S9275 (Cg) were drop-inoculated on A. thaliana pen2-1 mutant. Inoculated plants were incubated for 60 h and stained with lactophenol trypane blue. Bar = 50 μm. 1
A B C 0.1% Glucose 0.1% Sorbitol 0.1% Sucrose 0.1% Sucrose 0.1% Sorbitol Supplemental Figure 2. The Effects of Sorbitol and Sucrose on Pathogenicity of Cg to the pen2 Mutant Plants. (A) Sorbitol did not enhance lesion development by Cg on the pen2 mutant. Conidial suspensions of Cg were drop-inoculated on the pen2-1 mutant with 0.1 % glucose, 0.1 % sorbitol, or 0.1 % sucrose. Inoculated plants were incubated for 7 days. (B) Sorbitol did not affect infection-related morphogenesis of Cg. Conidial suspensions of Cg expressing RFP were inoculated on A. thaliana Col-0 with 0.1 % sucrose or 0.1 % sorbitol. Inoculated plants were incubated for 18 h, and infection-related morphogenesis of Cg was investigated by a confocal microscopy. Arrows indicate appressoria. Bars = 50 μm. 2
Control 0.1% Glucose Supplemental Figure 3. The Adapted C. higginsianum Develops Melanized Appressoria in the Presence of Glucose. Conidial suspensions of C. higginsianum with 0.1% glucose were inoculated on A. thaliana Col-0, and inoculated plants were incubated for 16 h. As a control, conidial suspensions of C. higginsianum were inoculated without glucose. Bars = 10 μm. 3
Carpropamid - + Supplemental Figure 4. Standard Cg Infection on the pen2 Mutant in the Presence of Carpropamid. Conidial suspensions of Cg without the addition of glucose were inoculated on the pen2 mutant with carpropamid. Inoculated plants were incubated for 4 days. Cg developed slight lesions on the pen2 muatnt in the presence of carpropamid the same as Cg without carpropamid. 4
Plant surface C Intracellular Ih Intracellular Supplemental Figure 5. Plasmolysis Assay on Arabidopsis Cells Invaded by Cg via HTE. Conidia of Cg expressing RFP were inoculated on the pen2 mutant expressing PEN1-GFP. At 13 hpi, inoculated leaves were treated with 0.85 M NaCl for 10 min. Arrowheads indicate plasma membrane of an epidermal cell invaded by Cg. C, Conidium; Ih, Intracellular hypha. Bars = 10 μm. 5
A B Plant surface Intracellular a c ih a c ih Supplemental Figure 6. Cg Develops Intracellular Hyphae underneath Melanized Appressoria on the Host Plant Mulberry. (A) Formation of melanized appressorium by Cg on the host plant mulberry. Conidial suspensions of Cg were inoculated on leaves of mulberry (Morus alba) and inoculated leaves were incubated for 24 hrs. Arrows indicate melanized appressoria of Cg. Bar = 50 μm. (B) Melanized appressoria of Cg invaded the mulberry leaves. Conidial suspensions of Cg were inoculated on mulberry leaves, and inoculated leaves were incubated for 60 hrs. Left photos focused on fungal structures formed on plant surface (Plant surface), whereas right photos focused on intracellular hypha (Intracellular). Bars = 10 μm. a, appressorium; c, conidium; ih, intracellular hypha. 6
Supplemental Figure 7. Amino Acid Sequence of C. orbiculare Mtk1. The deduced amino acid sequence of C. orbiculare (Co) Mtk1 was aligned with a putative fungal MAPKKK Mck1 (MGG_00883) of Magnaporthe oryzae (Mo) by using the CLUSTAL W program. Similar residues are shown on gray backgrounds. Gaps introduced for alignment are indicated by dashes. 7
A 104-T (WT) SCD1REP1-1 (scd1) DMA5 (maf1) B DMT1 (mtk1) WT maf1 WT mtk1 WT scd1 Supplemental Figure 8. The mtk1 Mutant Exhibits Reduced Pathogenicity. (A) Colony phenotype of the mtk1 mutant. The wild-type strain 104-T of C. orbiculare, the mtk1 mutant DMT1, the maf1 mutant DMA5, the scd1 mutant SCD1REP1-1 were grown on potato dextrose agar medium for 1 week. (B) Inoculation assay of the mtk1 mutant on the host plants. Conidial suspensions of tested fungal strains were drop-inoculated on the right side of cucumber cotyledons. As a control, conidial suspensions of the wild-type strain 104-T were inoculated on the left side. Inoculated plants were incubated for 7 days. The mtk1 mutant shows significant reduction in pathogenicity. 8
A 104-T (WT) DMA5 (maf1) DMT1 (mtk1) SCD1REP1-1 (scd1) B 104-T (WT) DMA5 (maf1) DMT1 (mtk1) SCD1REP1-1 (scd1) Supplemental Figure 9. Inoculation Assay of C. orbiculare Pathogenicty Mutants on the Arabidopsis pen2 Mutant. (A) Infection-related morphogenesis of C. orbiculare pathogenicity mutants. Conidial suspension of each strain was incubated on glass for 12 h. The mtk1 mutant DMT1 failed to form appressoria, which is similar to the phenotype of the maf1 mutant DMA5. The scd1 mutant SCD1REP1-1 forms colorless (non-melanized) appressoria. Bars = 20 μm. (B) Inoculation assay on the pen2 mutant. Conidial suspensions of each strain were drop-inoculated on A. thaliana pen2-1 mutant. Inoculated plants were incubated for 4 days. 9
Supplemental Table 1. Primers Used for Quantitative RT-PCR Gene Primer name Sequence Actin2 Actin2f ACCTTGCTGGACGTGACCTTACTGAT Actin2 Actin2r GTTGTCTCGTGGATTCCAGCAGCTT PEN2 PEN2f TAACATGCTTCTAGCGCACGCAG PEN2 PEN2r CATCTGGATCACTCGGATCATATG PEN3 PEN3f GGTGTTAAGAACAGTCTCGTCAC PEN3 PEN3r TCTTCTGACCTCCAGATATACC CYP79B2 CYP79B2f AGGCAACCCATTGCTTACCGCC CYP79B2 CYP79B2r CCATTGCTTTACGGAGAATCTC CYP81F2 CYP81F2f ATTGTCCGCATGGTCACAGGGAG CYP81F2 CYP81F2r GTAGCCGTGTCCGAACACTTTAAG 10