一种高效的柑橘绿霉菌基因敲除体系的构建

Improvement of a gene targeting system for genetic manipulation in Penicillium digitatum # # 一种高效的柑橘绿霉菌基因敲除体系的构建 first-author corresp ‡Corresponding Author ‡Corresponding Author †E-mail:hyli@zju.edu.cn †E-mail:hyli@zju.edu.cn hyli@zju.edu.cn Institute of Biotechnology, Zhejiang University, Hangzhou 310058, China Institute of Biotechnology, Zhejiang University, Hangzhou 310058, China 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 (Δ PdKu80 ) of P. digitatum . However, using Δ PdKu80 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 Δ PdKu80 strain would be a good candidate for rapid gene function analysis in P. digitatum . 研究目的 2 提高柑橘绿霉菌基因敲除效率。 创新要点 2 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（Δ PdKu80 ）。 研究方法 2 与野生型菌株相比，以 ΔPdKu80 作为出发菌株，提高柑橘绿霉病菌 PdbrlA 和 PdmpkA 的基因敲除效率（见表1）。 重要结论 2 Δ PdKu80 的营养生长、产孢和致病性与野生型菌株基本一致。Δ PdKu80 作为出发菌株，能显著提高柑橘绿霉菌的敲除效率。 Penicillium digitatum Efficiency Gene targeting Non-homologous end-joining (NHEJ) pathway Ku80 指状青霉 基因敲除 非同源末端链接（NHEJ） Ku80 Introduction 1 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 1989 ; Kanetis et al., 2007 2007 ; Macarisin et al., 2007 2007 ). To date, a limited amount of information regarding the molecular mechanisms of pathogenesis is available for this important pathogen (Wang et al., 2012 2012 ; Zhang et al., 2013a 2013a ; 2013b 2013b ; 2013c 2013c ). The availability of genome sequence and genetic transformation has accelerated the progress in molecular biology of P. digitatum (Wang and Li, 2008 2008 ; Marcet-Houben et al., 2012 2012 ; Sun et al., 2013 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 2000 ; Nakaune et al., 2002 2002 ) or conidia transformation mediated by Agrobacterium tumefaciens (Wang and Li, 2008 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 2006 ; Meyer et al., 2007 2007 ; Maruyama and Kitamoto, 2008 2008 ; Villalba et al., 2008 2008 ). The DNA-dependent protein kinase complexes as well as a Ku70-Ku80 heterodimer are the central components of NHEJ (Haber, 2000 2000 ; Jones et al., 2001 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 2006 ; Nayak et al., 2006 2006 ; Meyer et al., 2007 2007 ; Chang, 2008 2008 ; Maruyama and Kitamoto, 2008 2008 ), Botrytis cinerea (Choquer et al., 2008 2008 ), Magnaporthe oryzae (Villalba et al., 2008 2008 ), Penicillium decumbens (Li et al., 2010 2010 ), and Neurospora crassa (Ninomiya et al., 2004 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 2011 ). The objective of this study was to explore the new strategy for increasing the gene-targeting frequency in P. digitatum . Materials and methods 1 Fungal strains and culture conditions 2 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 (1×10 6 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. Cloning and analysis of Ku80 homolog in P. digitatum 2 All oligonucleotide primers used in this study are listed in Table S1 S1 . The P. digitatum Ku80 ortholog, PdKu80 , was found by the BLASTp search of the genomic sequence of P. digitatum (Sun et al., 2013 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. Construction of PdKu80, PdbrlA , and PdmpkA disruption vectors 2 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 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 S1 and Fig. 1a 1a ). After digestion with Kpn I and Sac I restriction enzymes, the PCR fragment was inserted into the Kpn I- Sac I 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 S1 and Fig. 1a 1a ). After digestion with Spe I and Xho I restriction enzymes, the PCR fragment was inserted into the Spe I- Xho I site of the plasmid pTFCM-Ku80-up to generate the PdKu80 disruption plasmid pTFCM-Δ PdKu80 (Fig. 1a 1a ). (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: Δ PdKu80 -14; Lane 3: Δ PdKu80 -22; Lane 4: Δ PdKu80 -25 Before the construction of the Δ PdbrlA and Δ PdmpkA 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 2011 ) using primer pair Trpc-neo- Bam HI/Trpc-neo- Xba I (Table S1 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 Δ PdbrlA and Δ PdmpkA plasmids followed the method described in the construction of a disruption vector of Δ PdKu80 . 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 S1 and Fig. S1a 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 S1 and Fig. S2a S2a ). The amplified sequences were then ligated to the Kpn I- Bam HI and Xba I- Hin dIII sites of pNEO1300 (Figs. S1a S1a and S2a S2a ), respectively. Disruptions of PdKu80 , PdbrlA , and PdmpkA in P. digitatum 2 The constructed disruption plasmids were transformed into an A . tumefaciens strain AGL-1 by the electroporation method described by Wang and Li ( 2008 2008 ). To disrupt PdKu80 in P. digitatum , the procedure of ATMT was conducted as the method described previously (Wang and Li, 2008 2008 ). To disrupt PdbrlA and PdmpkA in PdKu80 -disrupted strain (Δ PdKu80 ) 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 Δ PdKu80 , primer pairs Ku80-up-F/Ku80-i-R, Ku80-up-F/Ku80-i2-R, and Ku80-V-F/hph-R1 (Table S1 S1 and Fig. S3 S3 ) were used; for Δ PdbrlA , primers brlA-i-F/brlA-i-R and brlA-V-F/brlA-V-R (Table S1 S1 and Fig. S1b S1b ) were used; for Δ PdmpkA , primers mpkA-i-F/mpkA-i-R and mpkA-V-F/mpkA-V-R (Table S1 S1 , Figs. S2b and S2c S2b and S2c ) were used. For Southern blot analysis, genomic DNA (100 μg) was digested with Xho I 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 1a ) were labeled by PCR using the primers of hph-F/hph-R2 and Ku80-down-F/Ku80-down-R (Table S1 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. Assays of vegetative growth and sporulation 2 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 Δ PdKu80 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 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 2013b ). The experiment was conducted twice with at least three replicates. Assay of the tolerance to salt stress 2 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 Δ PdKu80 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. Virulence assay 2 Mature and un-wounded Ponkan fruits ( C. reticulata ) were wounded with a bunch of five needles (2–3 mm in depth). Conidial suspensions (1×10 6 conidia/ml) of the wild-type PdKH8 and Δ PdKu80 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. Results and discussion 1 Identification and analysis of the putative PdKu80 gene in P. digitatum 2 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/ 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 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. Creation of PdKu80 -disrupted strain (Δ PdKu80 ) 2 PdKu80 targeting plasmid was constructed by substituting the sequence encoding PdKu80 with hph (Fig. 1a 1a ). The constructed targeting plasmid pTFCM-Δ PdKu80 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 1a and Table S1 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 (Δ PdKu80 ; Fig. S3a S3a ). To get a positive result, primer pair Ku80-V-F/hph-R1 (Fig. 1a 1a and Table S1 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 S3b ). Based on these PCR results, the transformants Δ PdKu80 -14, Δ PdKu80 -22, and Δ PdKu80 -25 were identified as Δ PdKu80s . Southern blot analysis using a probe specific to hph verified the insertion of hph (Fig. 1b 1b ), and the Southern blot analysis using a probe located at the 3′ region of PdKu80 (Fig. 1a 1a ) indicated that additional ectopic integration of the replacement cassette sequence did not occur in these transformants (Fig. 1c 1c ). The growth rates of three Δ PdKu80s were identical, and the data collected from Δ PdKu80 -25 were presented hereafter. Disruption of PdKu80 did not affect vegetative growth or sporulation 2 Plate assay showed that the colony morphology of the Δ PdKu80 -25 was identical to that of the wild-type strain PdKH8 on PDA, CYA, and OA media (Fig. 2a 2a ). The average colony diameter of Δ PdKu80 -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 2a and 2b ). In addition, the sporulation of the Δ PdKu80 -25 was similar to that of the wild-type strain PdKH8 on PDA, CYA, and OA media ( P > 0.05; Fig. 2c 2c ). These results indicated that disruption of PdKu80 did not affect vegetative growth or sporulation in P. digitatum . The results showed that disruption of PdKu80 did not affect vegetative growth or sporulation. (a) The colonies of PdKH8 and Δ PdKu80 -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 Disruption of PdKu80 did not affect the tolerance to salt stress 2 The radial growths of Δ PdKu80 -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 Δ PdKu80 -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 Δ PdKu80 -25 and the wild-type strain PdKH8 were not significantly different ( P > 0.05; Fig. 3 3 ). This result indicated that the disruption of PdKu80 did not affect the tolerance of P. digitatum to NaCl or KCl stress. The results showed that disruption of PdKu80 did not affect the tolerance to salt stress. (a) The colonies of PdKH8 and Δ PdKu80 -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 Disruption of PdKu80 did not affect the virulence 2 Pathogenicity assays on mature Ponkan fruits by inoculation of conidial suspensions of the Δ PdKu80- 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 Δ PdKu80 -25 and the wild-type strain PdKH8 were not significantly different ( P > 0.05) (Fig. 4 4 ), indicating that disruption of PdKu80 did not interfere with the virulence of P. digitatum to citrus. 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 Disruption of PdKu80 improved gene targeting efficiency 2 The effect of the disruption of PdKu80 on homologous gene targeting was evaluated by disrupting PdbrlA (JX298844) and PdmpkA (KF019686) genes in both Δ PdKu80 -25 and the wild-type strain PdKH8 strains, respectively. The strategies for creating the Δ PdbrlA and Δ PdmpkA targeting cassettes were following the methods described in Section 2, and illustrated in Figs. S1a S1a and S2a S2a . The targeting cassettes of Δ PdbrlA and Δ PdmpkA were integrated into the strains of Δ PdKu80 -25 and wild-type PdKH8 by ATMT, respectively. The putative transformants were selected on neomycin-contained media, and analyzed by diagnostic PCR. For Δ PdbrlA , a 0.6-kb fragment was able to be amplified from ectopic transformants but was not able to be amplified from Δ PdbrlA by primers brlA-i-F/brlA-i-R (Table S1 S1 and Fig. S1b S1b ), which were located in the replaced region of PdbrlA (Fig. S1a S1a ); when using primers brlA-V-F/brlA-V-R (Table S1 S1 and Fig. S1a 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 Δ PdbrlA , and only 1.0 kb fragment was amplified from the wild-type strain of P. digitatum (Fig. S1c S1c ). For Δ PdmpkA , with primers mpkA-i-F/mpkA-i-R located in the replaced region of PdmpkA (Table S1 S1 and Fig. S2a S2a ), a 0.4-kb fragment was able to be amplified from the wild-type and ectopic transformants but could not from Δ PdmpkA (Fig. S2b S2b ). To get a positive result, primer pair mpkA-V-F/neo-R (Table S1 S1 and Fig. S2a S2a ) was used to amplify a fragment of 2.0 kb from the mutants of Δ PdmpkA , while no fragments could be amplified from the wild-type and ectopic transformants (Fig. S2c 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 Pdmpk A targeting were 2.2% and 4.0%, respectively (Table 1 1 ). However, when using Δ PdKu80 -25 as the targeting strain, the efficiencies of PdbrlA and PdmpkA targeting were 33.3% and 13.0%, respectively (Table 1 1 ). 2 Targeting strain 3 PdbrlA 3 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 Δ PdKu80 -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 1998 ). HR and NHEJ act independently and function competitively (van Dyck et al., 1999 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 2000 ; Jones et al., 2001 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 2004 ; da Silva Ferreira et al., 2006 2006 ; Krappmann et al., 2006 2006 ; Nayak et al., 2006 2006 ; Meyer et al., 2007 2007 ; Chang, 2008 2008 ; Choquer et al., 2008 2008 ; Villalba et al., 2008 2008 ; Li et al., 2010 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. 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This article does not contain any studies with human or animal subjects performed by any of the authors. 10.1631/jzus.B1300213 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 2013-08-12 2014-01-17 2013-12-06 2022-01-05 Copyright © Zhejiang University and Springer-Verlag Berlin Heidelberg 2014 2014 Journal of Zhejiang University-SCIENCE B (Biomedicine and Biotechnology) 02 15