Supplementary Fig. 1. Targeted disruption of Cc.arp9. (A) A diagram of the genomic locus of
Cc.arp9. Black arrows indicate primers used for the PCR experiments in (B). (B) Genomic PCR
experiments confirming the Cc.arp9 disruption. Lane 1, the parental strain ku3-24 as a control;
Lane 2, Cc.arp9d#3; Lane 3, Cc.arp9d#4; and Lanes M on the left and right sides represent size
markers: HindIII/EcoRI on the left and a 100 bp ladder marker (1001000 bp) on the right.
Supplementary Fig. 2. Isolation of a nucleus in which marker excision occurred
through 5-FC counterselection and PCR confirmation. Protoplasts prepared from mycelial cells
of strain Cc.arp9d#3 were subjected to 5-FC counterselection to generate strains in which the
Cc.arp9 disruption construct was removed. After the counterselection, 11 5-FC-resistant strains
were isolated. Nine of the 11 strains exhibited sensitivity to hygromycin B, suggesting that the
construct used for Cc.arp9 was removed through intramolecular homologous recombination.
Genomic PCR using two, Cc.arp9d_rev#1 and Cc.arp9d_rev#2, of the nine strains, as well as
Cc.arp9d#3 and ku3-24 as controls, were then performed. (A) Diagram of the genomic locus of
the Cc.arp9 gene in strain ku3-24, Cc.arp9 disruptants from ku3-24 (before 5-FC
counterselection) and strains in which the Cc.arp9 disruption construct was removed (after
5-FC counterselection). Thin arrows indicate the primers used for PCR. Thick arrows indicate
direct repeat sequences that mediate intramolecular homologous recombination to remove
expression cassettes for Hph and PoFcy1. (B) Genomic PCR experiments confirming that
marker excision occurred. Lane M, size markers (HindIII on the left side, and HindIII/EcoRI
on the right side); Lane 1, Cc.arp9d_rev#1; Lane 2, Cc.arp9d_rev#2; Lane 3, Cc.arp9d#3; and
Lane 4, ku3-24. The reason for the amplification of small amounts of three fragments, indicated
by white arrows and were expected to be amplified from the genomes of strains in which the
construct was removed (after 5-FC counterselection), from genomic DNAs from strain
Cc.arp9d#3 (before 5-FC counterselection) is described in Nakazawa and Honda (2015).
Supplementary Fig. 3. Generation of Cc.arp9d/Cc.snf5d double-gene disruptants by
introducing the Cc.snf5-disrupting construct into Cc.arp9d_rev#2. (A) Diagram of the genomic
locus of the Cc.snf5 gene. Arrows indicate the primers used for PCR experiments in (B). (B)
Genomic PCR experiments confirming that the Cc.arp9d/Cc.snf5d double-gene disruptants had
been generated. We designated this mutation Cc.snf5-2. Lane1, Cc.arp9d/Cc.snf5d#4; Lane 2,
Cc.arp9d/Cc.snf5d#5; Lane 3, Cc.arp9d_rev#2; Lane 4, ku3-24; and Lane M, HindIII size
marker.
Supplementary Fig. 4. The effects of the Cc.arp9-1 mutation on oidiaphore development.
Selected micrographs showing oidiaphores (white arrows) from indicated strains, which shows
that the number of mature oidiaphores produced in Cc.arp9d#3 is less than that produced in its
parental strain ku3-24. For more details, please see Table 4. Scale bars represent 25 µm.
Supplementary Table 1. Primers used in this study.
Primer
Sequence (5’ - 3’)
TN40
ACCCTTTCCCCCAAAATTTGGAAGC
TN41
ACCTTCTGGCATGACCTTTTGATGATCGC
TN95
TACAACGAGATCGGTGCCAGC
TN96
AAGCCGGTCATGAAGAAGTGGAGACG
TN116
AAATACGTGCTCTTCAACCAACTTCAACG
TN117
ACGTCGTGACTGGGAAGAAGACACAACGCCATCAG
TN118
CCTGTGTGAAATTGTTCTTCAACGACAACAGCCAG
TN119
ACGATGGAACAACAGGTTGGTGG
TN128
ACCGTCGTACGTGTATTCCCAGC
TN129
TGGTGGCGAGGGTGGTCAACTAG
TN134
TCTGACGCGAAGCTCTTCCACC
TN135
AACCGCATCCTTGTCATCACGG
TN164
TATGGGCATGGGAGGTGGAAAC
TN165
ACGTCGTGACTGGGAAGTTCTTCCAGGCCGAGGTG
RN166 CCTGTGTGAAATTGTAGGGTATCAAGAGGGAGGGG
TN167 TGGTCATCAAGTTTCATGGAACCGAC
TN168 TGCGAGATTTCGGTTGGGACTC
TN169 TCTGTGAGATGACCAACGAGGAAGAG
TN170 TTACGATCCCGAAGCGTGCC
TN171 AAGAGGAACGAATGGGCTTTGAAG
TN197 AAGCCAGCCATCCCTTCAGC
TN198 TGCGAGATTTCGGTTGGGACTC
TN250 TCTGGGTTCACAGCCATGAGAGC
TN251 ATGCCAGTTCTGAGACAGCACGTTC
M13F
TCCCAGTCACGACGTTGTAAAACGACGG
M13R
ACAATTTCACACAGGAAACAGCTATGACC
arp9_5RACE-1
ACCAGCTGCTTCGCCAGTCG
arp9_5RACE-2
TTGCATCACGCTCGAATTGCT
arp9_3RACE-1
TGCTCTTCAACCAACTTCAACGACG
arp9_3RACE-2
AACGGTTCAATGTACCCGCATTCG
Supplementary Table 2. Effects of the Cc.arp9-1 mutation on clamp cell formation.
Strain
28ºC
37ºC
ku3-24
99.2% (126/127) 1
100% (100/100) 1
Cc.arp9d#3
100% (105/105) 1
99.4% (174/175) 1
Cc.arp9d#4
99.1% (114/115) 1
100% (155/155) 1
Values in parentheses are “no. of septa with clamp cells / no. of septa observed”. For the effects
of Cc.snf5 disruption 1 (Cc.snf5-2) on clamp cell formation, please see Ando et al. (2013).