Agrobacterium tumefaciens-Mediated Transformation ...

Curr Microbiol (2014) 68:769–776 DOI 10.1007/s00284-014-0541-8

Agrobacterium tumefaciens-Mediated Transformation of the Causative Agent of Valsa canker of Apple Tree Valsa mali var. mali Yang Hu • Qingqing Dai • Yangyang Liu • Zhe Yang Na Song • Xiaoning Gao • Ralf Thomas Voegele • Zhensheng Kang • Lili Huang

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Received: 4 September 2013 / Accepted: 10 December 2013 / Published online: 20 February 2014 Ó Springer Science+Business Media New York 2014

Abstract Valsa mali var. mali (Vmm), which is the causative agent of Valsa canker of apple tree, causes heavy damage to apple production in eastern Asia. In this article, we report Agrobacterium tumefaciens-mediated transformation (ATMT) of Vmm and expression of gfp (green fluorescent protein) in this fungus. The transformation system was optimized to a transformation efficiency of approximately 150 transformants/106 conidia, and a library containing over 4,000 transformants was generated. The tested transformants were mitotically stable. One hundred percent hph (hygromycin B phosphotransferase) integration into Vmm was identified by PCR and five single-copy integration of T-DNA was detected in the eighteen transformants by Southern blot. To our knowledge, this is the first report of ATMT of Vmm. Furthermore, this library has been used to identify genes involved in the virulence of the pathogen, and the transformation system may also be useful to the transformation of other species of the genus Valsa.

Y. Hu Q. Dai Y. Liu N. Song X. Gao Z. Kang L. Huang (&) State Key Laboratory of Crop Stress Biology for Arid Areas, College of Plant Protection, Northwest A&F University, Yangling 712100, People’s Republic of China e-mail: [email protected]; [email protected] Q. Dai e-mail: [email protected] Z. Yang State Key Laboratory of Crop Stress Biology for Arid Areas, College of Life Science, Northwest A&F University, Yangling 712100, People’s Republic of China R. T. Voegele Fachgebiet Phytopathologie, Institut fu¨r Phytomedizin, Universita¨t Hohenheim, 70599 Stuttgart, Germany

Introduction Valsa canker, caused by Valsa spp., affects more than 70 species of woody shrubs and trees [3], including apple and pear trees. For apple trees, Valsa canker is one of the most destructive diseases in eastern Asia including, China [41], Japan [1], and Korea [23], and leads to heavy damage to apple production [1, 7, 23]. Though this disease is economically important, the causative agent was not clear until Wang and co-workers [42] confirmed that Valsa mali var. mali (Vmm) was the causal pathogen of Valsa canker on apple in China. It has been reported that all apple varieties tested are susceptible to Vmm [4, 26]. Because the pathogen penetrates into the host phloem and xylem [19, 39], disease control by chemical treatments is inefficient [1]. A better understanding of pathogenic mechanisms is needed to develop approaches to improve control strategy of this disease. Although several pathogenic factors have been reported in Vmm, such as degradation products of phlorizin [22, 31], isocoumarins [32], and pectinase [19, 24, 45], further characterization by molecular approaches is necessary to confirm their role in plant infection. Recently, polyethylene glycol (PEG)-mediated protoplast transformation was reported for Vmm [15]. However, it has been well documented that transformation of protoplasts often leads to untagged genetic changes [5]. Agrobacterium tumefaciens-mediated transformation (ATMT) has been widely used as a tool to tag fungal genes so that mutants defective in pathogenicity can be identified [5, 29, 34, 37]. Compared with other common tools used for fungal transformation, such as restriction enzymemediated integration (REMI), PEG-mediated protoplast transformation, and biolistic, ATMT has many advantages such as flexibility in choosing the starting materials

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(protoplasts, hyphae, spores, or blocks of mycelial tissue [2, 11, 25, 35, 40], high transformation efficiency [5], high frequency of single-copy T-DNA insertions [35], and easy identification of flanking sequences of T-DNA [9]. Therefore, ATMT is thought to be an effective tool to explore the function of fungal pathogenicity-related genes. Nevertheless, this tool has not yet been applied to the Valsa genus. In this study, an ATMT system was established and optimized for Vmm. The transformation efficiency was about 150 transformants per 106 conidia, and an ATMT library containing more than 4,000 transformants was generated with the A. tumefaciens strain EHA105 carrying pBIG2RHPH2-GFP-GUS. Expression of gfp in Vmm transformants treated with A. tumefaciens strain SK1044 harboring the binary vector pSK1044 was detected. Results described in this article will help us gain more knowledge of this fungus at the molecular level, monitor fungal colonization in apple trees, and establish the transformation of other species in the Valsa genus. To the best of our knowledge, this is the first report of A. tumefaciens-mediated transformation of Vmm.

Materials and Methods Fungal Isolate and Cultivation 03-8, a virulent strain of Vmm, isolated from apple tree [42, 43, 45, 46], was used as a recipient strain for fungal transformation. For conidiation, potato dextrose agar (PDA) cultures were incubated for one month at 25 °C under continuous fluorescent light. Conidia were harvested from pycnidia into ddH2O using a sterilized inoculation needle. Conidial suspensions were adjusted to a concentration of 106 conidia/mL and used for transformation within 24 h. To determine the minimum inhibitory concentration of hygromycin B for the wild-type strain of Vmm, 106 conidia or culture blocks were inoculated onto PDA plates supplemented with hygromycin B at different concentrations (0, 30, 60, and 90 lg/mL).

Y. Hu et al.: ATMT of Vmm

The A. tumefaciens strain SK1044 harboring the binary vector pSK1044 (a gift from Dr. Jinrong Xu, Purdue University, USA) was cultured at 28 °C in LB supplemented with 20 lg/mL rifampicin and 50 lg/mL kanamycin. pSK1044 carries both the hygromycin B phosphotransferase (hph) and gfp genes in its T-DNA region. A. tumefaciens-Mediated Transformation of Vmm A. tumefaciens-mediated transformation of filamentous fungi has been widely reported since 1998. To date, more than 125 fungal species have been transformed by ATMT [14]. The transformation procedure is based on the protocol described by Michielse et al. [28] and Park and Kim [33], with some modifications. A. tumefaciens carrying a binary vector was cultivated in 10 mL of LB medium containing appropriate concentrations of kanamycin and rifampicin overnight. Cells were collected by centrifugation (12,0009g, 5 min, 25 °C) and resuspended in Induction Medium (IM: 10 mM K2HPO4, 10 mM KH2PO4, 11.1 mM glucose, 5 mM MgSO4, 5.1 mM NaCl, 0.068 mM CaCl2, 6.25 mM NH4NO3, 0.36 lM FeSO4, 10.25 mM 2-(Nmorpholino) ethanesulfonic acid (MES), 0.5 % (v/v) glycerol, pH 5.5, adapted from Bundock et al. [6]) with or without 200 lM acetosyringone (AS), Agrobacterium cells were collected and washed twice with IM. Thereafter, the bacterial suspension was adjusted to an OD660 of 0.5–0.9 and incubated for 6 h at 28 °C under agitation (220 rpm) to preinduce virulence of A. tumefaciens. Bacteria were diluted to an OD660 of 0.3 and 100 lL of this suspension was mixed with a suspension of fungal conidia (106 conidia/mL) in a 1:1 ratio. The Agrobacterium-conidia mixture was spread onto co-cultivation medium (CM; same as IM except that it contains glucose at 5.55 mM and 1.5 % (w/v) agar) containing 200 lM AS. Co-cultivation plates were incubated for 48 h at 25 °C, and then overlaid with 13 mL PDA amended with 60 lg/mL hygromycin B and 500 lg/mL cefotaxime. Fungal colonies grown on the selective medium were transferred to PDA amended with the same antibiotics for secondary screening. Positive transformants were then stored in 20 % (v/v) glycerol at -80 °C.

A. tumefaciens Strains and Binary Vectors Mitotic Stability Test The A. tumefaciens strain EHA105 carrying pBIG2RHPH2GFP-GUS [12] was cultured at 28 °C in Luria–Bertani (LB) medium supplemented with 50 lg/mL rifampicin and 50 lg/mL kanamycin. The binary vector pBIG2RHPH2GFP-GUS was provided by Dr. Fengming Song at Zhejing University, China. This vector confers kanamycin resistance as bacterial selection marker and carries the hygromycin B resistance cassette and a gus (beta-glucuronidase):green fluorescent protein (gfp) fusion cassette between the right and left borders.

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Transformants were randomly selected for subculturing for five generations on PDA plates without hygromycin B. Subcultures were then assayed for growth on PDA plates containing 60 lg/mL hygromycin B to verify mitotic stability. Microscopy For observation of GFP fluorescence, transformants were grown for three days at room temperature. Mycelium was


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viewed under the fluorescence microscope (Olympus Corporation, Japan) equipped with filter blocks with spectral properties matching those of GFP (excitation filter 485 nm, dichromic mirror 510 nm, and barrier filter 520 nm). The wild type was used as control. Genomic DNA Extraction and Molecular Analysis of Transformants To extract total genomic DNA, transformants were grown on cellophane membranes placed on PDA for 3 days at 25 °C. Hyphae were harvested with sterilized toothpicks and ground to a fine powder in liquid nitrogen with a mortar and pestle. Fungal genomic DNA was isolated following the cetyltrimethylammonium bromide (CTAB) method [17]. For Southern blot analysis, genomic DNA was digested with EcoRI, which cuts once in the T-DNA. Electrophoresis and blotting followed standard protocols [36]. The hph gene fragment amplified with primers, H852 (50 -AACTCACCGC GACGTCTGTC-30 ) and H850 (50 -TTGTCCGTCAGGACA TTGTT-30 ) from pBIG2RHPH2-GFP-GUS, was labeled with digoxigenin (DIG)-dUTP using the DIG DNA Labeling and Detection Kit II (Roche, Mannheim, Germany). Hybridization and detection were carried out according to the manufacturer’s instructions. To confirm chromosomal insertion of transforming T-DNA, PCR amplifications were carried out by amplifying about 1000-bp region of the hph gene located on the T-DNA with primers Hyg-F (50 -CCTGA ACTCACCGCGACGTC-30 ) and Hyg-R (50 -CTATCCTTTGCCCTC GGACGAGTG-30 ). The PCR reaction condition was designed as follows: 1 cycle at 94 °C for 5 min; 30 cycles of 95 °C for 30 s; 60 °C for 1 min and 72 °C for 2 min; and 1 final cycle at 72 °C for 10 min. The presence of the full-length gfp was confirmed by PCR with primers EGFPX (50 -GCC CTCTAGACAGA CACAATGGTGAGCAAGGGCGAG-30 ) and GFP-STOP (50 -GGCGGATCCTTACTTGTACAGCTCGTCCAT-30 ) [21]. The PCR reaction condition were as follows: 1 cycle at 94 °C for 5 min; 35 cycles of 94 °C for 1 min; 55 °C for 1 min and 72 °C for 1 min; and 1 final cycle at 72 °C for 10 min.

Results and Discussion Hygromycin B Sensitivity Test In PDA cultures inoculated with either hyphal blocks and conidia, Vmm growth was completely inhibited by 30 lg/ mL of hygromycin B after incubation for 10 days at 25 °C. However, the false positive rate was very high (about 50 %) in preliminary ATMT experiments. Therefore, the

Fig. 1 Effect of co-cultivation time on transformation efficiency. Each experiment was repeated three times to calculate the average number of transformants per plate (90 mm). Error bars indicate standard error

selection of transformants was increased to 60 lg/mL concentration of hygromycin B, and false positive rate fell below 5 %. Optimization of the Transformation System for Vmm To establish a system of ATMT for Vmm, mycelia were first used as the starting material for ATMT in previous studies in our lab, but no transformants were generated [16]. Therefore, conidia were chosen, and eventually transformants were obtained. Our preliminary ATMT experiments followed the protocol of Mullins and associates [29] with the following modifications: (1) A. tumefaciens strain was EHA105-pBIG2RHPH2-GFP-GUS; and (2) the filter was cellophane. However, several independent experiments indicated that the transformation efficiency using this procedure was very low (about 6 transformants/106 conidia) and bacteria often showed excessive growth on the cellophane placed on PDA amended with 60 lg/mL hygromycin B and 500 lg/mL cefotaxime. Therefore, we suspect that the excessive growth of bacteria might be due to the cellophane. In previous studies, filters used for co-incubation (such as nitrocellulose, cellophane, and Hybond N?) were considered as one of the most important factors affecting transformation efficiency [27, 37, 44]. However, in the protocol of Park and Kim [33], no filters were used. Similarly, Khang et al. [20] suggested the use of nitrocellulose membrane not to be essential for successful transformation of Magnaporthe grisea and Fusarium oxysporum. Therefore, we performed experiments according to the protocol of Park and Kim [33], and no filters were used. Using this procedure, the transformation efficiency was approximately 120 transformants/106 conidia. The Agrobacterium-

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Fig. 2 Effect of cell density of A. tumefaciens (OD660) on transformation efficiency. Each experiment was repeated three times to calculate the average number of transformants per plate (90 mm). Error bars indicate standard error

conidiophore mixture can be directly spread onto CM, and then overlaid with PDA amended with 60 lg/mL hygromycin B and 500 lg/mL cefotaxime. This procedure is simple and highly efficient. We determined the influences of the following three factors on the transformation efficiency without any fliter: (1) the effect of co-cultivation time (Fig. 1); (2) the effect of the number of Agrobacterium cells (OD660) (Fig. 2); and (3) the effect of preinduction of Agrobacterium cells (IM ? AS or IM - AS). Our results demonstrate that higher transformation efficiencies are obtained after 36 h of co-cultivation (Fig. 1). While optimizing the parameter ‘‘number of Agrobacterium cells (OD660)’’, we found the transformation efficiency was relatively low (Fig. 2). This may have been caused by prolonged concentration adjusting operation on the bacterial suspension. According to our results, co-cultivation for two days with a cell density of OD660 = 0.3 results in a relative high transformation efficiency (about 150 transformants/106 conidia) in a short period of time (6 days). Compared with the previously published PEG-mediated protoplast transformation of Vmm [15], the ATMT transformation system developed in this study has a much higher transformation efficiency as well as a simpler procedure. To determine whether AS is essential for A. tumefaciens-mediated Vmm transformation, the inducer was omitted from the IM in which the bacterial cells were grown before co-cultivation. We found the transformation efficiency of the group with AS was 120 transformants/106 conidia, and the other was 150 transformants/106 conidia. According to the results, addition of AS in the IM is not essential. A similar phenomenon was reported in previous studies [27–30, 47]. More importantly, two reports on ATMT transformation of Colletotrichum graminicola

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Fig. 3 The colonies of 8 putative transformants grown on PDA plate containing 60 lg/mL hygromycin for 3 days after subcultured five times. The center of the plate is the wild type 03-8

showed that the omission of AS in IM (IM - AS) may avoid tandem integrations and improve single-copy integration of T-DNA [13, 30]. Meanwhile, the transformation efficiency of Vmm with or without AS in IM was similar in this article (P \ 0.01, t test). This is consistent with the results of Combier et al. [10] and Takahara et al. [38]. Mitotic Stability of Transformants With this optimized ATMT system, a library containing more than 4,000 transformants was generated using EHA105-pBIG2RHPH2-GFP-GUS. 249 randomly selected transformants were repeatedly subcultured every 3 days for five times on PDA plates without hygromycin B, and these subcultured transformants were again transferred to PDA selective plates containing 60 lg/mL hygromycin B. The results show that 243 putative transformants could grow on selective medium (partial set of transformants illustrated in Fig. 3), thus showing that transformants by the ATMT method are mitotically stable. Verification of hph Insertion by PCR A total of 20 randomly selected transformants were analyzed for the insertion of hph. A single expected band of about 1 kb was amplified from all of them using primers Hyg-F and Hyg-R. Plasmid pBIG2RHPH2-GFP-GUS was used as the positive control (not shown), and the wild-type strain 03-8 was used as the negative control (partial PCR


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Fig. 4 PCR analysis of the hph gene in hygromycin B-resistant colonies. The 1,000-bp hph fragment was amplified in all transformants but not the wild type. WT wild-type strain 03-8; M marker

Fig. 5 Southern blot analysis of 18 PCR positive transformants transformants generated with EHA105. One band (lanes marked with an asterisk) represents a single T-DNA integration event. WT wild-type strain 03-8

analytic results illustrated in Fig. 4). These results indicate that the hph gene can be integrated into Vmm by ATMT. Analysis of T-DNA Insertion Events by Southern Blot DNA from eighteen PCR positive transformants and a wild-type strain digested with EcoRI was subjected to Southern blot analysis. The number of hybridization signals showed that five transformants had single-copy insertion, and another 13 had two or more insertions, confirming T-DNA transfer into the Vmm genome. No hybridizing band was detected in the lane loaded with DNA from the untransformed wild-type strain. The demonstration of integration patterns of eighteen selected transformants is shown in Fig. 5. Two reports on ATMT transformation of C. graminicola showed that the omission of AS in IM (IM - AS) may avoid tandem integrations and improve single-copy integration of T-DNA [13, 29].

However, Southern blot analysis in this study indicated that addition of AS in IM did not generate obvious tandem integrations. Moreover, five transformants with single-copy T-DNA insertion were identified, which further implicated the transformation system established in this article was suitable to study the pathogenicity-related genes in Vmm. Confirmation the Presence of gfp by PCR In all of the randomly selected transformants transformed with pBIG2RHPH2-GFP-GUS (more than 20), gfp expression could not be detected. Chen and associates [8] as well as Hanif and associates [18] reported similar results. Therefore, PCR analysis was performed to detect the gfp gene in transformants with primers EGFPX and GFP-STOP [21]. The full-length gfp fragment was amplified from 18 out of 19 randomly selected transformants. The wild-type strain was used as a negative control and

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Fig. 6 PCR analysis of the presence of gfp gene in hygromycin B-resistant colonies transformed with EHA105. M maker; WT wild-type strain 03-8

Fig. 7 Expression of gfp in Vmm. a hyphae of the transformant observed using a fluorescence microscopy; b hyphae of the transformant observed in bright field. Scale bar corresponds to 100 lm

showed no corresponding products (Fig. 6). The binary vector pBIG2RHPH2-GFP-GUS used in this study carries a gus::gfp fusion cassette between the right and left border, and expression of gfp could be detected in transformants when used to transform F. graminearum and C. lagenarium [12]. However, it remains to be investigated why gfp expression could not be detected in the selected transformants obtained with EHA105-pBIG2RHPH2-GFP-GUS. In order to further confirm gfp expression, Vmm was transformed with SK1044-pSK104. Approximately 200 transformants were obtained in two independent experiments. gfp expression could be detected in all of the 20 randomly selected transformants using a fluorescence microscopy (excitation filter 485 nm, dichromic mirror 510 nm, barrier filter 520 nm) (One of the transformants is depicted in Fig. 7). This indicates that Vmm was successfully tagged by gfp. Expression of the gfp reporter gene can not only allow rapid identification of transformants, but also help us monitor fungal colonization. In conclusion, we report for the first time the establishment of genetic transformation of Vmm by ATMT and gfp tagging of Vmm. This transformation strategy will help to gain more knowledge of the Valsa apple canker pathogen at the molecular levels, such as the investigation of the pathogenicity-

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related genes and monitoring fungal colonization in apple trees. Moreover, it may be useful to transform other Valsa species. In the long run, this transformation system may contribute to a more effective management of Valsa canker. Acknowledgments The authors thank Fengming Song (Zhejiang University, China) for providing the pBIG2RHPH2-GFP-GUS plasmid; Jinrong Xu (Purdue-NWAFU Joint Research Center, Northwest A&F University, China) for providing A. tumefaciens SK1044 harboring the binary vector pSK1044; undergranduates Ms. Dianru Wang, Mr. Jie Zhou, and Mr. Decheng Ren for their help in the collection of transformants; Ms. Na Song, a doctoral student, for microscopic observation; and Ms. Jing Gao for testing the hygromycin B sensitivity of Vmm. The authors also thank Dr. Jinrong Xu and Dr. Xiaoyu Qiang for correcting the grammatical and spelling errors as well as critical reading of the manuscript. This study was funded by the National Natural Science Foundation of China (No. 31171796), Specialized Research Fund for the Doctoral Program of Higher Education (20120204110002), and the Special Fund for Agro-scientific Research in the Public Interest of China (nyhyzx201203034-03).

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