Supplementary material.
1. UV-irradiation
1.1. Method
1,000 fresh conidia obtained from both the V. dahliae wild-type and the ΔVdku70 strains were
plated on PDA. Next, plates were incubated at room temperature for 1 h. In order to determine the
conidial survival of the V. dahliae wild-type and the ΔVdku70 strains in response to UV, they were
then exposed in 50–200 J/m2 UV radiation with a UV Crosslinker (Spectrolinker™ XL-1000A,
Spectronics Corporation, NY, USA). It was followed by incubation for 7 days at room temperature
before colony forming units (cfu) were counted on each plate. The UV-irradiation assay was
repeated three times independently.
1.2. Result
Colony survival of the ΔVdku70 strain after UV exposure was significantly less than that of
the wild type strain (p < 0.01). Specifically, only 1.2% of the UV-treated ΔVdKu70 colonies
survived at 50 J/m2 UV irradiation, compared with 11.5% survival rate for wild-type strain
colonies under the same intensity of UV-irradiation (Supplementary Fig.3A). These results showed
that the growth and development of Δ VdKu70 strain were impaired under UV-irradiation
condition (Supplementary Fig.3A).
2. Temperature-sensitivity culture
2.1. Method
Individually, a total of 2 μL wild-type or ΔVdKu70 conidial suspensions (5×106cfu/ml) were
inoculated at the center of agar plates. Based on the previous study, V. dahliae grows at 10~30°C
and the optimum temperature for the growth of V. dahliae is 20~25°C, and most strains do not
grow at 33°C (Francl et al. 1990; Shi. 1995). In this work, plates were incubated at 25 °C or 30 °C
respectively, and colony phenotypes were observed after 7-days incubation. The assay was
repeated three times independently.
2.2. Result
The growth of the wild type and theΔVdku70 strains was compared at 25 °C and 30 °C
culture conditions. We observed that the ΔVdku70 strain has no obvious growth defects under
normal growth condition, at 25 °C. However, with rising in temperature, at 30 °C, both the wild
type and the ΔVdku70strains grow slowly, notably the growth rate of ΔVdku70 strain was much
lower than that of the wild type strain (Supplementary Fig.3B). Previous studies have showed that
yku70 mutant strain displays temperature-sensitive growth defect in S. cerevisiae (Boulton and
Jackson 1996; Teo and Jackson 1997). Our results also showed that ΔVdKu70 strain exhibits
similar growth defect (Supplementary Fig.3B).
References
Boulton SJ and Jackson SP (1996) Saccharomyces cerevisiae Ku70 potentiates illegitimate DNA
double-strand break repair and serves as a barrier to error-prone DNA repair pathways.
EMBO Journal 15:5093-5103.
Francl LJ, Madden LV, Rowe RC, and Riedel RM (1990) Correlation of growing season
environmental variables and the effect of early dying on potato yield. Phytopathology 80:
425-432.
Shi LY (1995) Research Progress on Cotton Verticillium Wilt in China. Acta Gossypii Sinica
7:243-245.
Teo SH and Jackson SP (1997) Identification of Saccharomyces cerevisiae DNA ligase IV:
involvement in DNA double-strand break repair. EMBO Journal 16:4788–4795.
Supplementary Fig.1 Phylogenetic tree of Verticillium dahliae Ku70 and Ku70
orthologs in other fungi. The tree was created with MEGA5.01 analysis software using
the Neighbor-Joining method. Source organisms and accession numbers for
homologous Ku proteins were as follows: VaKu70, Verticillium alfalfa (EEY22158);
VdKu70, Verticillium dahliae (EGY20618); CgKu70, Colletotrichum gloeosporioides
(XP_007274178); CoKu70, Colletotrichum orbiculare (ENH83415); BbKu70,
Beauveria bassiana (XP_008600211); MaKu70, Metarhizium anisopliae
(XP_007817332); VvKu70, Villosiclava virens (KDB15942); CpKu70, Cryphonectria
parasitica (EU091476); OpKu70, Ophiostoma piceae (EPE10157); TrKu70,
Trichoderma reesei (ETS02014); NtKu70, Neurospora tetrasperma (XP_009849399);
PaKu70, Podospora anserine, (CDP30650); MgKu70, Magnaporthe grisea,
(BAH86596); OpKu70, Ophiostoma piceae (EPE10157); BcKu70, Botrytis cinerea
(XP_001545469); AdKu70, Ajellomyces dermatitidis (EEQ86026); AcKu70,
Ajellomyces capsulatus (EGC44870); AsKu70, Aspergillus sojae (BAE78502);
AsKu70, Cryptococcus neoformans (P0CO51); and KlKu70, Kluyveromyces lactis
(CAH01326).
Supplementary Fig.2 A single ΔVdku70 transformant detected in a total of 206
screened V. dahliae transformants. A. V. dahliae transformants. B. PCR screening and
confirmation of the ΔVdku70 transformant with the primer pair Ku70-J-F/Ku70-J-R.
Supplementary Fig.3 TheΔVdku70 strain is temperature-sensitive and
hypersensitive to UV- irradiation. A. Colony survival rates for V. dahliae wild-type
and ΔVdKu70 strains after UV exposure for 50, 100, 150, 200J/m2. Values represent
the mean ± SD from three independent replicates. ** t test significant at p < 0.01. B.
Colony phenotypes for the V. dahliae wild-type and ΔVdKu70 strains after7-days
incubation fat 25 °C or 30 °C.
Supplementary Fig.4 The primers efficiency of the N. benthamiana actin gene (A)
and V. dahliae ribosomal RNA gene (B) was analyzed using qRT-PCR.