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Received: 2013-08-12

Revision Accepted: 2013-12-06

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Article info.
1.  Introduction
2.  Materials and methods
3.  Results and discussion
4. Reference List
Open peer comments

Journal of Zhejiang University SCIENCE B 2014 Vol.15 No.2 P.116-124

http://doi.org/10.1631/jzus.B1300213


Improvement of a gene targeting system for genetic manipulation in Penicillium digitatum * #


Author(s):  Qian Xu, Cong-yi Zhu, Ming-shang Wang, Xue-peng Sun, Hong-ye Li

Affiliation(s):  . Institute of Biotechnology, Zhejiang University, Hangzhou 310058, China

Corresponding email(s):   hyli@zju.edu.cn

Key Words:  Penicillium digitatum , Efficiency, Gene targeting, Non-homologous end-joining (NHEJ) pathway, Ku80


Qian Xu, Cong-yi Zhu, Ming-shang Wang, Xue-peng Sun, Hong-ye Li. Improvement of a gene targeting system for genetic manipulation in Penicillium digitatum[J]. Journal of Zhejiang University Science B, 2014, 15(2): 116-124.

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publisher="Zhejiang University Press & Springer",
doi="10.1631/jzus.B1300213"
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A1 - Qian Xu
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PB - Zhejiang University Press & Springer
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DOI - 10.1631/jzus.B1300213


Abstract: 
Penicillium digitatum is the most important pathogen of postharvest citrus. gene targeting can be done in P. digitatum using homologous recombination via Agrobacterium tumefaciens mediated transformation (ATMT), but the frequencies are often very low. In the present study, we replaced the Ku80 homolog (a gene of the non-homologous end-joining (NHEJ) pathway) with the hygromycin resistance cassette (hph) by ATMT. No significant change in vegetative growth, conidiation, or pathogenicity was observed in Ku80-deficient strain (ΔPdKu80) of P. digitatum. However, using ΔPdKu80 as a targeting strain, the gene-targeting frequencies for both genes PdbrlA and PdmpkA were significantly increased. These results suggest that Ku80 plays an important role in homologous integration and the created ΔPdKu80 strain would be a good candidate for rapid gene function analysis in P. digitatum.

一种高效的柑橘绿霉菌基因敲除体系的构建

研究目的:提高柑橘绿霉菌基因敲除效率。
创新要点:低效的基因敲除与丝状真菌非同源末端链接(NHEJ)的DNA双链断裂修复途径有关。为提高柑橘绿霉病菌基因敲除效率,本研究利用农杆菌介导的转化体系,获得NHEJ途径中关键因子Ku80的缺失突变体(ΔPdKu80)。
研究方法:与野生型菌株相比,以ΔPdKu80作为出发菌株,提高柑橘绿霉病菌PdbrlAPdmpkA的基因敲除效率(见表1)。
重要结论:ΔPdKu80的营养生长、产孢和致病性与野生型菌株基本一致。ΔPdKu80作为出发菌株,能显著提高柑橘绿霉菌的敲除效率。

关键词:指状青霉;基因敲除;非同源末端链接(NHEJ);Ku80

Darkslateblue:Affiliate; Royal Blue:Author; Turquoise:Article

Article Content

1.  Introduction

  Penicillium digitatum (Pers.:Fr.) Sacc., causing a green mold in citrus, is the most destructive pathogen in the citrus industry and is responsible for the majority of decay losses of postharvest citrus (Eckert and Eaks, 1989; Kanetis et al., 2007; Macarisin et al., 2007). To date, a limited amount of information regarding the molecular mechanisms of pathogenesis is available for this important pathogen (Wang et al., 2012; Zhang et al., 2013a; 2013b; 2013c). The availability of genome sequence and genetic transformation has accelerated the progress in molecular biology of P. digitatum (Wang and Li, 2008; Marcet-Houben et al., 2012; Sun et al., 2013).

 Targeted gene knockout/replacement is an efficient and essential strategy for the functional characterization of interested genes in filamentous fungi. P. digitatum can be genetically transformed with a plasmid containing a selectable marker conferring resistance to antibiotics or herbicides via protoplast transformation of DNA (Hamamoto et al., 2000; Nakaune et al., 2002) or conidia transformation mediated by Agrobacterium tumefaciens (Wang and Li, 2008). However, the transformation via protoplast is usually very difficult for unknown reasons (our unpublished data) and the gene disruption frequency was very low (<4%) or even negligible amongst transformants recovered through A. tumefaciens mediated transformation (ATMT) in many cases. This limitation greatly hampered the functional characterization of targeted genes in P. digitatum.

 Homologous recombination (HR) of exogenous DNA into the genome occurs easily in the yeast species, but it is not a ready mechanism for filamentous fungi since the non-homologous end-joining (NHEJ) pathway seems to be dominant over the HR pathway for repairing of DNA double-strand breaks (DSBs) in filamentous fungi (Ishibashi et al., 2006; Meyer et al., 2007; Maruyama and Kitamoto, 2008; Villalba et al., 2008). The DNA-dependent protein kinase complexes as well as a Ku70-Ku80 heterodimer are the central components of NHEJ (Haber, 2000; Jones et al., 2001). Previous studies indicated that destroying the NHEJ machinery by disrupting the Ku70- or Ku80-coding gene could increase the gene-targeting frequency in a number of filamentous fungi, such as Aspergillus spp. (da Silva Ferreira et al., 2006; Nayak et al., 2006; Meyer et al., 2007; Chang, 2008; Maruyama and Kitamoto, 2008), Botrytis cinerea (Choquer et al., 2008), Magnaporthe oryzae (Villalba et al., 2008), Penicillium decumbens (Li et al., 2010), and Neurospora crassa (Ninomiya et al., 2004). However, disruption of Ku70 in Alternaria alternata, the pathogenic fungus causing brown spots on citrus, did not apparently improve the efficiency of gene targeting (Wang et al., 2011). The objective of this study was to explore the new strategy for increasing the gene-targeting frequency in P. digitatum.


2.  Materials and methods

2.1.  Fungal strains and culture conditions

 The wild strain PdKH8 of P. digitatum was obtained from infected citrus of Ponkan (Citrus reticulata Blanco) in Quzhou, Zhejiang Province, China. The strain was stored as conidial suspensions in 15% glycerol at −80 °C, and recovered on regular potato dextrose agar (PDA). Mycelium was obtained by inoculating 5 μl (1×106 conidia/ml) in 150 ml liquid potato dextrose broth (PDB), which was incubated on a rotary shaker at 160×g and 25 °C. Conidia were harvested in sterile water from a 7-d-old culture by scrapping the culture with a sterile glass rod, and the desired conidial concentration was adjusted by counting cells using a haematocytometer.

2.2.  Cloning and analysis of Ku80 homolog in P. digitatum

 All oligonucleotide primers used in this study are listed in Table S1. The P. digitatum Ku80 ortholog, PdKu80, was found by the BLASTp search of the genomic sequence of P. digitatum (Sun et al., 2013) using Ku80 homolog from Aspergillus oryzae (AoKu80, BAE78503.1). The amplified fragments in this study were cloned into the vector pMD18-T (TaKaRa Biotech. Co., Dalian, China) and the absence of polymerase-derived errors was checked by DNA sequencing. The PdKu80 sequence was submitted into the GenBank/EMBL/DDBJ database under accession No. KF019685.

2.3.  Construction of PdKu80, PdbrlA, and PdmpkA disruption vectors

 The PdKu80 disruption vector was constructed by inserting the two flanking sequences of PdKu80 into the up- or downstream side of the hygromycin resistance cassette (hph) in the vector pTFCM (Wang and Li, 2008). The primers used to amplify the homologous arms were synthesized according to the genomic sequence of P. digitatum (unpublished data). Briefly, a 1.2-kb DNA fragment containing the upstream flanking sequence of PdKu80 was amplified from the P. digitatum genomic DNA by polymerase chain reaction (PCR) using primers Ku80-up-F/Ku80-up-R (Table S1 and Fig. 1a). After digestion with KpnI and SacI restriction enzymes, the PCR fragment was inserted into the KpnI-SacI site of pTFCM to generate the plasmid pTFCM-Ku80-up. Subsequently, a 1-kb fragment representing the downstream flanking sequence of the PdKu80 gene was obtained using primers Ku80-down-F/Ku80-down-R (Table S1 and Fig. 1a). After digestion with SpeI and XhoI restriction enzymes, the PCR fragment was inserted into the SpeI-XhoI site of the plasmid pTFCM-Ku80-up to generate the PdKu80 disruption plasmid pTFCM-ΔPdKu80 (Fig. 1a).



Fig.1
Schematic diagram showing the construction of PdKu80 disruption plasmid and molecular identification of ΔPdKu80 by Southern blot analysis
(a) Construction of PdKu80 disruption plasmid. Black arrow represented the PdKu80, while the white arrow represented a hygromycin resistance cassette (hph). The positions of the PCR primers were indicated with small arrows. Probes were indicated with black bars. (b) Identification by Southern blot using a probe specific to hph. (c) Identification by Southern blot using a probe specific to the 3' part of the PdKu80 gene. Lane 1: wild-type strain PdKH8; Lane 2: ΔPdKu80-14; Lane 3: ΔPdKu80-22; Lane 4: ΔPdKu80-25

 Before the construction of the ΔPdbrlA and ΔPdmpkA vectors, a plasmid pNEO1300 was constructed by removing hph and inserting the neomycin resistant gene (neo) into the multiple cloning sites (MCSs) in pCAMBIA1300 (CAMBIA, Canberra, Australia). The neo was amplified from plasmid pCA-neo (Jiang et al., 2011) using primer pair Trpc-neo-BamHI/Trpc-neo-XbaI (Table S1). The sequences of brlA (PdbrlA, JX298844) and mpkA (PdmpkA, KF019686) homologs in P. digitatum were obtained from a genomic database (unpublished data). The construction of ΔPdbrlA and ΔPdmpkA plasmids followed the method described in the construction of a disruption vector of ΔPdKu80. Briefly, the up- and downstream flanking sequences (0.7 and 0.9 kb, respectively) of PdbrlA were amplified using primers brlA-up-F/brlA-up-R and brlA-down-F/brlA-down-R (Table S1 and Fig. S1a), while the up- and downstream flanking sequences (1.0 and 0.9 kb, respectively) of PdmpkA were amplified using primers mpkA-up-F/mpkA-up-R and mpkA-down-F/mpkA-down-R (Table S1 and Fig. S2a). The amplified sequences were then ligated to the KpnI-BamHI and XbaI-HindIII sites of pNEO1300 (Figs. S1a and S2a), respectively.

2.4.  Disruptions of PdKu80, PdbrlA, and PdmpkA in P. digitatum

 The constructed disruption plasmids were transformed into an A. tumefaciens strain AGL-1 by the electroporation method described by Wang and Li (2008). To disrupt PdKu80 in P. digitatum, the procedure of ATMT was conducted as the method described previously (Wang and Li, 2008). To disrupt PdbrlA and PdmpkA in PdKu80-disrupted strain (ΔPdKu80) of P. digitatum, a similar approach was also used, but instead of hygromycin B, 67 μg/ml of neomycin was added into the PDA for the selection of the putative PdbrlA- or PdmpkA-disrupted transformants.

 The disruptions of PdKu80, PdbrlA, and PdmpkA in P. digitatum were identified by PCR and followed by a Southern blot. For identification of ΔPdKu80, primer pairs Ku80-up-F/Ku80-i-R, Ku80-up-F/Ku80-i2-R, and Ku80-V-F/hph-R1 (Table S1 and Fig. S3) were used; for ΔPdbrlA, primers brlA-i-F/brlA-i-R and brlA-V-F/brlA-V-R (Table S1 and Fig. S1b) were used; for ΔPdmpkA, primers mpkA-i-F/mpkA-i-R and mpkA-V-F/mpkA-V-R (Table S1, Figs. S2b and S2c) were used. For Southern blot analysis, genomic DNA (100 μg) was digested with XhoI for 24 h, the fragments were separated on a 0.8% (w/v) agarose gel, and transferred to a Nylon membrane (Millipore, Billerica, MA, USA). The probes (Fig. 1a) were labeled by PCR using the primers of hph-F/hph-R2 and Ku80-down-F/Ku80-down-R (Table S1), respectively, with the PCR DIG probe synthesis kit I (Roche, Mannheim, Germany), according to the manufacturer’s instruction. Aqueous hybridization was performed at 42 °C overnight and detected using the DIG luminescent detection kit (Roche) according to the manufacturer’s instruction.

2.5.  Assays of vegetative growth and sporulation

 To determine the effect of the deletion of PdKu80 on vegetative growth and sporulation, mycelial plugs (0.5 cm in diameter) of both wild-type PdKH8 and ΔPdKu80 strains of P. digitatum were prepared and transferred on PDA, Czapek yeast extract agar (CYA), and oat agar (OA) plates as described previously (Zhang et al., 2009). The plates were incubated at 25 °C for 5 d on PDA, and for 8 d on CYA and OA. Sporulation was assayed at the same day as above, following the method described previously (Zhang et al., 2013b). The experiment was conducted twice with at least three replicates.

2.6.  Assay of the tolerance to salt stress

 To determine whether PdKu80 is involved in the tolerance to salt stress, mycelial plugs (0.5 cm in diameter) of both wild-type PdKH8 and ΔPdKu80 strains of P. digitatum were prepared and transferred on PDA plates supplemented with 0.5 mol/L NaCl and KCl. The growth on PDA with a non-supplement of either NaCl or KCl was used as control. The plates were incubated at 25 °C for 5 d, and the colony diameter in each plate was measured. Three replicates were used for each strain and the experiments were repeated twice.

2.7.  Virulence assay

 Mature and un-wounded Ponkan fruits (C. reticulata) were wounded with a bunch of five needles (2–3 mm in depth). Conidial suspensions (1×106 conidia/ml) of the wild-type PdKH8 and ΔPdKu80 of P. digitatum were collected from 7-d-old PDA plates. Three microliters of the conidial suspensions were added onto each wounded site. The inoculated citruses were incubated at room temperature and disease symptoms were observed daily, and the lesion diameters were recorded at Day 5 after inoculation. Thirty fruits were used for each strain and the experiment was repeated twice.


3.  Results and discussion

3.1.  Identification and analysis of the putative PdKu80 gene in P. digitatum

 An exhaustive search via the BLASTp program was made to identify the Ku80 homolog in the P. digitatum genome (http://www.ncbi.nlm.nih.gov/) using AoKu80 gene (BAE78503.1) as a reference sequence. The gene was a bidirectional best hit with AoKu80, shared the highest similarity with AoKu80 (E-value 0, identity 68%, similarity 82%, 718 amino acids), and was assumed as the putative PdKu80 (AGT79985.1) through conserved domain analysis using a Pfam database (Bateman et al., 2004). Results showed that the putative PdKu80 gene was 2673 bp in length, which contained 10 introns and encoded 712 amino acids.

 The possible conserved functional domains, Ku70/Ku80 N-teminal α/β domain (positions 6 to 190), Ku70/Ku80 β-barrel domain (positions 274 to 440), and Ku C terminal domain (positions 590 to 710) were presented in this gene, suggesting that it was definitely a Ku80 gene in P. digitatum. Sequence alignment revealed that PdKu80 had 64%, 46%, 45%, and 41% identity to its homologs identified in A. oryzae (BAE78503.1), Verticillium albo-atrum (XP_003001068.1), Fusarium oxysporum (EGU85850.1), and Neurospora crassa (BAD16623.1), respectively.

3.2.  Creation of PdKu80-disrupted strain (ΔPdKu80)

  PdKu80 targeting plasmid was constructed by substituting the sequence encoding PdKu80 with hph (Fig. 1a). The constructed targeting plasmid pTFCM-ΔPdKu80 was transformed into the wild-type strain PdKH8 by the ATMT method. Eighty-three transformants selected from a hygromycin-contained medium were primarily identified by PCR with primer pairs, Ku80-up-F/Ku80-i-R and Ku80-up-F/Ku80-i2-R (Fig. 1a and Table S1). With these primer pairs, the fragments of 1.5 and 1.9 kb were amplified from the PdKH8 and ectopic transformants, while none fragment could be amplified from the PdKu80-disrupted mutants (ΔPdKu80; Fig. S3a). To get a positive result, primer pair Ku80-V-F/hph-R1 (Fig. 1a and Table S1) was used to amplify a fragment of 2.2 kb from the PdKu80-disrupted mutants, while none fragment could be amplified from the PdKH8 or ectopic transformants (Fig. S3b). Based on these PCR results, the transformants ΔPdKu80-14, ΔPdKu80-22, and ΔPdKu80-25 were identified as ΔPdKu80s. Southern blot analysis using a probe specific to hph verified the insertion of hph (Fig. 1b), and the Southern blot analysis using a probe located at the 3′ region of PdKu80 (Fig. 1a) indicated that additional ectopic integration of the replacement cassette sequence did not occur in these transformants (Fig. 1c). The growth rates of three ΔPdKu80s were identical, and the data collected from ΔPdKu80-25 were presented hereafter.

3.3.  Disruption of PdKu80 did not affect vegetative growth or sporulation

 Plate assay showed that the colony morphology of the ΔPdKu80-25 was identical to that of the wild-type strain PdKH8 on PDA, CYA, and OA media (Fig. 2a). The average colony diameter of ΔPdKu80-25 was slightly smaller than that of the wild-type strain PdKH8 on PDA; however, no significant difference was observed on CYA or OA (P>0.05; Figs. 2a and 2b). In addition, the sporulation of the ΔPdKu80-25 was similar to that of the wild-type strain PdKH8 on PDA, CYA, and OA media (P>0.05; Fig. 2c). These results indicated that disruption of PdKu80 did not affect vegetative growth or sporulation in P. digitatum.



Fig.2
Effect of disruption of PdKu80 on the vegetative growth and sporulation
The results showed that disruption of PdKu80 did not affect vegetative growth or sporulation. (a) The colonies of PdKH8 and ΔPdKu80-25 on PDA, CYA, and OA media. (b, c) Columns representing the average colony diameters and number of conidia and bars represented standard errors calculated from three independent colonies. Data were collected 5 d after incubation on PDA and 8 d on CYA and OA at 26 °C

3.4.  Disruption of PdKu80 did not affect the tolerance to salt stress

 The radial growths of ΔPdKu80-25 and the wild-type strain PdKH8 were compared on NaCl- and KCl-contained PDA (0.5 mol/L) to test the effect of PdKu80 disruption on the tolerance of P. digitatum to salt stress. Compared with non-NaCl- and non-KCl-contained PDA, the mycelial growth for both ΔPdKu80-25 and the wild-type strain PdKH8 was severely inhibited (about 60%); however, the inhibition rates were similar for both strains, as indicated that the colony diameters for ΔPdKu80-25 and the wild-type strain PdKH8 were not significantly different (P>0.05; Fig. 3). This result indicated that the disruption of PdKu80 did not affect the tolerance of P. digitatum to NaCl or KCl stress.



Fig.3
Effect of disruption of PdKu80 on the tolerance to salt stress
The results showed that disruption of PdKu80 did not affect the tolerance to salt stress. (a) The colonies of PdKH8 and ΔPdKu80-25 on PDA with or without of 0.5 mol/L NaCl or 0.5 mol/L KCl at 5 d of incubation. (b) Columns representing the average colony diameters and bars representing the standard errors calculated from three independent colonies

3.5.  Disruption of PdKu80 did not affect the virulence

 Pathogenicity assays on mature Ponkan fruits by inoculation of conidial suspensions of the ΔPdKu80-25 and the wild-type strain PdKH8 revealed that the water-soaked lesion occurred on both strain-inoculated spots. At 5 d post-inoculation, the average lesion diameters caused by ΔPdKu80-25 and the wild-type strain PdKH8 were not significantly different (P>0.05) (Fig. 4), indicating that disruption of PdKu80 did not interfere with the virulence of P. digitatum to citrus.



Fig.4
Effect of disruption of PdKu80 on the virulence
The results showed that disruption of PdKu80 did not affect the virulence. (a) Symptoms produced at 5 d after inoculation. (b) Columns represented the average lesion diameters and bars represented the standard errors calculated from three independent colonies, respectively

3.6.  Disruption of PdKu80 improved gene targeting efficiency

 The effect of the disruption of PdKu80 on homologous gene targeting was evaluated by disrupting PdbrlA (JX298844) and PdmpkA (KF019686) genes in both ΔPdKu80-25 and the wild-type strain PdKH8 strains, respectively. The strategies for creating the ΔPdbrlA and ΔPdmpkA targeting cassettes were following the methods described in Section 2, and illustrated in Figs. S1a and S2a. The targeting cassettes of ΔPdbrlA and ΔPdmpkA were integrated into the strains of ΔPdKu80-25 and wild-type PdKH8 by ATMT, respectively. The putative transformants were selected on neomycin-contained media, and analyzed by diagnostic PCR. For ΔPdbrlA, a 0.6-kb fragment was able to be amplified from ectopic transformants but was not able to be amplified from ΔPdbrlA by primers brlA-i-F/brlA-i-R (Table S1 and Fig. S1b), which were located in the replaced region of PdbrlA (Fig. S1a); when using primers brlA-V-F/brlA-V-R (Table S1 and Fig. S1a), two fragments with sizes of 1.0 and 1.2 kb were amplified from ectopic mutants, while only the fragment of 1.2 kb was amplified from ΔPdbrlA, and only 1.0 kb fragment was amplified from the wild-type strain of P. digitatum (Fig. S1c).

 For ΔPdmpkA, with primers mpkA-i-F/mpkA-i-R located in the replaced region of PdmpkA (Table S1 and Fig. S2a), a 0.4-kb fragment was able to be amplified from the wild-type and ectopic transformants but could not from ΔPdmpkA (Fig. S2b). To get a positive result, primer pair mpkA-V-F/neo-R (Table S1 and Fig. S2a) was used to amplify a fragment of 2.0 kb from the mutants of ΔPdmpkA, while no fragments could be amplified from the wild-type and ectopic transformants (Fig. S2c).

 For disrupting genes PdbrlA and PdmpkA, a total of 46 and 50 transformants, respectively, were picked up in the transformation experiment using wild-type PdKH8 as the targeting strain, and only 1 and 2 mutants were demonstrated to be PdbrlA- and PdmpkA-disrupted mutants, respectively, by diagnostic PCR. The efficiencies of PdbrlA and PdmpkA targeting were 2.2% and 4.0%, respectively (Table 1). However, when using ΔPdKu80-25 as the targeting strain, the efficiencies of PdbrlA and PdmpkA targeting were 33.3% and 13.0%, respectively (Table 1).



Table 1

Effect of Ku80 deletion on the efficiency of gene targeting in P. digitatum
Targeting strain PdbrlA
PdmpkA
No. of transformants No. of knockout mutants Targeting frequency (%) No. of transformants No. of knockout mutants Targeting frequency (%)
PdKH8 46 1 2.2 50 2 4.0
ΔPdKu80-25 24 8 33.3 23 3 13.0

 Targeted gene replacement requires the cellular machinery involved in the repair of DNA DSBs. There are two types of cellular machinery involved in DBS repair, the HR pathway and the NHEJ pathway (Kanaar et al., 1998). HR and NHEJ act independently and function competitively (van Dyck et al., 1999), thus making it possible to inactivate gene(s) of one pathway to favour another pathway. Ku70-Ku80 heterodimer and a catalytic subunit comprise a DNA-dependent protein kinase complex, which is the central component of NHEJ (Haber, 2000; Jones et al., 2001). Inactivation of the NHEJ machinery by disrupting the Ku70- or Ku80-coding gene has been shown to increase HR in a number of filamentous fungi (Ninomiya et al., 2004; da Silva Ferreira et al., 2006; Krappmann et al., 2006; Nayak et al., 2006; Meyer et al., 2007; Chang, 2008; Choquer et al., 2008; Villalba et al., 2008; Li et al., 2010).

 The frequency of gene targeting in P. digitatum via ATMT was always very low (our unpublished data). In the present study, the Ku80 homolog in P. digitatum was identified and its deficient mutant strain was generated. Our experiment showed that the deletion of PdKu80 did not affect the basic biological characteristics, such as vegetative growth, sporulation, tolerance to salt stress, or pathogenicity, but significantly increased the gene targeting frequency in P. digitatum, suggesting that the created ΔPdKu80 strain can be used as a recipient strain for large-scale gene function characterization in P. digitatum. The generation of the ΔPdKu80 strain will expedite the progress in the functional genomic studies of this fungus.



* Project supported by the National Natural Science Foundation of China (Nos. 31371961 and 31071649), the China Agriculture Research System (No. CARS-27), and the Special Fund for Agro-scientific Research in the Public Interest (No. 201203034), China# Electronic supplementary materials: The online version of this article (http://dx.doi.org/10.1631/jzus.B1300213) contains supplementary materials, which are available to authorized usersCompliance with ethics guidelines Qian XU, Cong-yi ZHU, Ming-shang WANG, Xue-peng SUN, and Hong-ye LI declare that they have no conflict of interest.


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Open peer comments: Debate/Discuss/Question/Opinion

<1>

Qing-qing Huang<819075736@qq.com>

2015-06-12 10:12:23

Dear author,
Where is the Table S1,I couldn't find it. I want to make it as a reference.I am looking forward to your help

Please provide your name, email address and a comment





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