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X. X. H. B. P. B. H. P. 5’ UTR. Cut2. 3’ UTR. S. S. HygB. A. Guy11/pPS20. pCB1636. 1 kb. B. Guy11. T3. T40. T41. 6 kb. 4.47 kb. 3 kb. C. Absolute transcript abundance. Guy11. cut2. cut2/ CUT2.

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  1. X X H B P B H P 5’ UTR Cut2 3’ UTR S S HygB A Guy11/pPS20 pCB1636 1 kb B Guy11 T3 T40 T41 6 kb 4.47 kb 3kb C Absolute transcript abundance Guy11 cut2 cut2/CUT2

  2. Supplementary figure 1. Targeted gene disruption and complementation of M. grisea CUT2. (A) Organization of the CUT2 locus: CUT2 gene is cross-hatched. The restriction enzymes are BbuI (B), HindIII (H), PstI (P), SalI (S) and XhoI (X). The SalI sites in pCB1636 and the XhoI sites in pPS20 were used for the construction of the CUT2-disruption plasmid. Scale bar is 1 kb. (B) Southern blot analysis of DNA from wild-type strain Guy11 and selected transformants digested with HindIII and hybridized to the 1.4 kb Hygromycin B gene released from pCB1636. Guy11 shows no hybridization, whereas transformants T3 and T40 show multiple integration of the CUT2-disruption plasmid. One band (4470 bp) is detected in the T41 transformant, resulting from the single introduction of the Hygromycin B selectable marker into the CUT2 locus. T41 is designated the cut2 mutant strain. (C) QrtRT-PCR monitored expression of the CUT2and βTUB (control) genes in WT Guy11, cut2 and cut2/CUT2strains collected from barley epidermal peels at 8 hpi. Gene disruption in T41 has resulted in the complete loss of CUT2transcript.

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