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Text S1. Detailed description of A. nidulans strain constructions.
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We constructed the pkcA-ts mutants as follows. A 4.4-kb fragment containing the
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coding region of an N-terminus of PkcA was amplified from the total DNA of the A26
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strain using primers, 5pkcAF and pkcA-plR. A 1.0-kb fragment containing the coding
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region of a C-terminus of PkcA and a downstream of pkcA was amplified from the total
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DNA of the A26 strain using primers pkcA-plF and 3pkcA518R-friboB. A 2.1-kb
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fragment containing riboB was amplified from the total DNA of the A26 strain using
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primers, riboBF and riboBR. A 0.9-kb fragment containing a downstream of pkcA was
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amplified from the total DNA of the A26 strain using primers, 3pkcA518F-friboB and
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3pkcA1405R. These amplified fragments were fused by the fusion PCR using primers,
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5npkcAF and 3pkcA1405R-n. The A1145 strain was transformed with the fused
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fragment. The total DNA of the transformants was extracted as described previously [1]
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and Southern blot analysis was performed as described previously [2]. The three
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transformants in which a single copy of the transformed fragment was integrated into
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the pkcA locus confirmed by Southern blot analysis were designated pkcA-ts-2,
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pkcA-ts-3, and pkcA-ts-5 (Fig. S6A and B).
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We constructed strains in which pkcA was expressed under the control of alcA(p) as
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follows: A 1.1-kb fragment containing the upstream of pkcA was amplified from the
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total DNA of the A26 strain using primers 5pkcAF and 5pkcAR-friboB. A 2.1-kb riboB
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fragment was amplified from the total DNA of the A26 strain using primers riboBF and
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riboBR. A 1.4-kb fragment, in which a coding region of the 5’ terminus of pkcA was
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fused to the alcA(p), was amplified from pPAALP (Ichinomiya et al., 2007) using
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primers, ALpkcAF-friboB and pkcA1020R. These amplified fragments were fused by
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the fusion PCR using primers 5npkcAF and pkcA1020R-n. The A1145 strain was
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transformed with the fused fragment. The two transformants in which a single copy of
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the transformed fragment was integrated into the pkcA locus confirmed by Southern blot
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analysis were designated alcA(p)-pkcA-3, and alcA(p)-pkcA-4 (Fig. S6C and D).
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We constructed the bckA deletion mutants as follows: The A1149 strain was
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transformed with a 6.2-kb HindIII-SpeI fragment of pbckA::pyroA. The two
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transformants in which wild-type bckA was replaced with a single copy of the
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bckA::pyroA fragment at the bckA locus confirmed by Southern blot analysis were
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designated ΔbckA-1 and ΔbckA-2 (Fig. S6E and F).
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We constructed the mpkA deletion mutants as follows: A 1.2-kb fragment containing
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the upstream of mpkA was amplified from the total DNA of the A26 strain using primers
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5mpkAF and 5mpkAR-fpyrG. A 2.0-kb fragment containing pyrG was amplified from
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the total DNA of the A26 strain using primers pyrG-481 and pyrG-r-new. A 1.1-kb
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fragment containing the downstream of mpkA was amplified from the total DNA of the
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A26 strain using primers 3mpkAF-fpyrG and 3mpkAR. These amplified fragments
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were fused by the fusion PCR using primers 5mpkAF-n and 3mpkAR-n. The A1149
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strain was transformed with the fused fragment. The three transformants in which a
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single copy of the fragment was integrated into the mpkA locus confirmed by Southern
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blot analysis were designated ΔmpkA-1, ΔmpkA-2 and ΔmpkA-8 (Fig. S6G and H).
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We constructed strains in which Lifeact-EGFP was expressed under the control of the
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alcA(p) as follows: The A1149 strain and the pkcA-ts-2 mutant were transformed with a
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3.0-kb fragment that was amplified from ppyrGLA using primers pyrG5 and pyrGRn.
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The two transformants of the wild-type strain, in which a single copy of the amplified
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fragment was integrated into the pyrG locus confirmed by Southern blot analysis, were
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designated A1149LA-1 and A1149LA-2 (Fig. S5I and J). The two transformants of the
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pkcA-ts mutant, in which a single copy of the amplified fragment was integrated into the
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pyrG locus confirmed by Southern blot analysis, were designated pkcA-tsLA-1 and
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pkcA-tsLA-2.
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We constructed the bckA deletion mutants in which Lifeact-EGFP was expressed
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under the control of the alcA(p) as follows: The A1149LA-1 strain was transformed
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with the 6.2-kb HindIII-SpeI fragment of pbckA::pyroA. The two transformants in
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which wild-type bckA was replaced with a single copy of the bckA::pyroA fragment at
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the bckA locus confirmed by Southern blot analysis were designated ΔbckALA-1 and
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ΔbckALA-2.
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We constructed strains in which MpkA-FLAG was expressed from its own promoter
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as follows: A 1.5-kb fragment containing mpkA was amplified from pMPKA-pyroA
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using primers mpkA5 and 3FLAG-mpkA. A 3xFLAG fragment was amplified from
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p3xFLAG-myc-CMV-26 (Sigma) using primers 3xFLAGF and 3xFLAGR. A 4.5-kb
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fragment containing pyroA and the downstream region of mpkA was amplified from
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pMPKA-pyroA using primers mpkA3 and 5mpkA-FLAG. These amplified fragments
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were fused by the fusion PCR using primers mpkA5 and mpkA3. The A1149 strain and
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the pkcA-ts-2 mutant were transformed with the fused fragment. The two transformants
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of the wild-type strain, in which a single copy of the fused fragment was integrated into
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the mpkA locus confirmed by Southern blot analysis, were designated A1149/MF-1 and
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A1149/MF-2. The two transformants of the pkcA-ts mutant, in which a single copy of
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the fused fragment was integrated into the mpkA locus confirmed by Southern blot
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analysis, were designated pkcA-ts/MF-1 and pkcA-ts/MF-2.
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We constructed wild-type strains, which were auxotrophic for pyrimidine as follows:
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The A1149 strain was transformed with the 2.0-kb EcoRV-PstI fragment of ppyrG+750
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containing pyrG (Yamazaki H., Ohta A., Horiuchi H., unpublished). The three
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transformants in which a single copy of the fragment was integrated into the pyrG locus
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confirmed by Southern blot analysis were designated A1149/pyrG-1, A1149/pyrG-2 and
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A1149/pyrG-8.
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We constructed the heterokaryons containing both wild-type and pkcA-deleted nuclei
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as follows: A 2.1-kb fragment containing riboB was amplified from the total DNA of
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the A26 strain using primers, riboBF and riboBR. The 1.1-kb upstream and 1.1-kb
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downstream fragment of the pkcA coding region was amplified from the total DNA of
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the A26 strain using primers, 5pkcAF and 5pkcAR-friboB, and 3pkcAF-friboB and
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3pkcAR respectively. These amplified fragments were fused by the fusion PCR using
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primers, 5npkcAF and 3pkcAR. The A1145 strain was transformed with the fused
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fragment.
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untransformed-pkcA alleles confirmed by Southern blot analysis, were designated
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ΔpkcA-h1, ΔpkcA-h2, ΔpkcA-h3.
The
three
transformants,
which
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4
contain
both
transformed-
and
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References
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1. Oakley CE, Weil CF, Kretz PL, Oakley BR (1987) Cloning of the riboB locus of
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Aspergillus nidulans. Gene 53: 293-298.
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2. Horiuchi H, Fujiwara M, Yamashita S, Ohta S, Takagi M (1999) Proliferation of
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intrahyphal hyphae caused by disruption of csmA, which encodes a class V chitin
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synthase with a myosin motor-like domain in Aspergillus nidulans. J Bacteriol 181:
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3721-3729.
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