Turkish Journal of Biochemistry – Türk Biyokimya Dergisi 2015; 40(4): 290–297

Biochemistry Research Article – 92604

Ying-Qing Yang, Bo Lan, Yan-Li Jian, Shui-Xiu Hu, Dong-Dong Chang, Shun-Liang Zhang, Xiang-Min Li* Establishment of Agrobacterium tumefaciens- mediated transformation system for asparagi, the pathogen of stem blight

Asparagus (Kuşkonmaz) pas hastalığına neden olan Phomopsis asparagi için Agrobacterium tumefaciens’e dayalı transformasyon sistemi geliştirilmesi

Abstract: Objective: The transformation system for the tive controls, whereas no corresponding DNA band could asparagus stem blight pathogen Phomopsis asparagi be amplified from the 10 transformants. The results of (Sacc.) Bubak has not yet been reported. In the present the two PCR amplifications clearly showed that T-DNA study, we intend to achieve and optimize the genetic was indeed inserted into the genome of target isolate FJ2. transformation of P. asparagi. This study aims at establi- The stability result revealed that transformants still dis- shing the foundation for understanding the pathogenic played high resistance to hygromycin B and could grow mechanism of P. asparagi, which will be of great theoreti- normally after subculture for five generations. cal and practical significance. Conclusion: A stable and efficient ATMT transformation Methods: The Agrobacterium tumefaciens-mediated trans- system for P. asparagus was constructed systematically, formation (ATMT) system for P. asparagi was constructed in which a high transformation rate was achieved. For at three aspects, i.e. condition optimization, insertion improving this system, the trasformation conditions verification and transformant stability. were optimized through gradient experiments, T-DNA insertion was verified through dual PCR and the inser- Results: The optimal conditions for this ATMT system tion segment containing hph gene in the transformant were as follows: 8 h of pre-induction, 48 h of co-culture, was proved hereditary stable through subculture. This 200 μmol/L AS in ISM, co-culture at 25–28oC and pH system layed a foundation for the research on patho- 5.6–5.8 of ISM at the co-culture phase. The PCR result of genic mechanism and pathogenicity-related genes of P. the hph gene revealed that an expected band of about asparagi. 500 bp was amplified from all the 10 transformants selected at random, and the PCR result of the Vir gene Keywords: Asparagus stem blight, Phomopsis asparagi, revealed that an expected band of about 730 bp was Agrobacterium tumefaciens-mediated transformation, amplified from the four strains of A. tumefacien as posi- transformation efficiency, dual PCR verification

*Corresponding author: Xiang-Min Li: Institute of Plant Protection, Shui-Xiu Hu: Institute of Plant Protection, Jiangxi Academy of Jiangxi Academy of, Agricultural Sciences, Nanchang, China, Agricultural Sciences, Nanchang, China; Jiangxi Agricultural e-mail: [email protected] University, College of Biological Science and Engineering, Ying-Qing Yang: Institute of Plant Protection, Jiangxi Academy of Nanchang, China, e-mail: [email protected] Agricultural Sciences, Nanchang, China, e-mail:[email protected] Dong-Dong Chang: Institute of Plant Protection, Jiangxi Academy Bo Lan: Institute of Plant Protection, Jiangxi Academy of Agricultural of Agricultural Sciences, Nanchang, China; Jiangxi Agricultural Sciences, Nanchang, China, e-mail: [email protected] University, College of Biological Science and Engineering, Yan-Li Jian: Institute of Plant Protection, Jiangxi Academy of Nanchang, China, e-mail: [email protected] Agricultural Sciences, Nanchang, China; Jiangxi University of Shun-Liang Zhang: Institute of Plant Protection, Jiangxi Finance and Economics, College of Accounting, Nanchang, China, Academy of Agricultural Sciences, Nanchang, China, e-mail: e-mail:[email protected] [email protected] Ying-Qing Yan et al.: Establishment of ATMT system for P. asparagi 291

Özet: Amaç: Asparagus (kuşkonmaz) pas hastalığına Introduction sebep olan Phomopsis asparagi (Sacc.) Bubak patojeni için şimdiye kadar bir transformasyon sistemi rapor Asparagus officinalis Linn belongs to the family Liliaceae edilmemiştir. Bu çalışmada, P. asparagi’nin genetik and the genus Asparagus; it is one of the ten most world- transformasyonu gerçekleştirilmiştir ve optimizasyo- wide famous dishes known as the “king of vegetables” in nuh yapılmıştır. Bu çalışma P. asparagi’nin patojenite the international market. It is very popular and apprecia- mekanimasının anlaşılması için zemin oluşturacak nite- ted for high nutritional value and many properties such as likte olup teorik ve pratik alanda oldukça büyük öneme lung moistening, cough relief, phlegm elimination, tumor sahiptir. inhibition, among others [1,2]. Plant disease occurrence is steadily increasing with Metod: P. asparagi için Agrobacterium tumefaciens’e the expansion of asparagus cultivation areas. Especially, dayalı transformasyon (ATMT) sisteminin geliştirilmesi üç stem blight disease has seriously affected the yield and açıdan ele alınmıştır: transformasyon koşullarının opti- quality of asparagus in recent years [2–5]. Asparagus mizasyonu, genin yerleşmesinin doğrulanması ve trans- stem blight is caused by P. asparagi (Sacc.) Bubak [5–7]. formantların stabilitesi. It is a worldwide devastating disease [8–10] and more serious in China, Japan, Thailand, Indonesia and other Bulgular: ATMT sitemi için optimum koşullar 8 saat ön Asian countries. In China, this disease occurs widely, indüksiyon ile birlikte, 25–28oC’de, 200 μmol/L AS içeren and is more pronounced in South compared with North. ve pHsı 5.6–5.8 olan ISM besiyerinde 48 lik ko-kültür It has been observed that diseased plants rapidly die in a süresi olarak tespit edilmiştir. Rastgele seçilen 10 trans- severely damaged field, which results in the destruction formanttan yapılan PCR sonucunda hph geni için bekle- of the whole field [4]. nen yaklaşık 500 bp’lik DNA fragmenti elde edilmiştir. In recent years, with the development of mole- Vir geni için yapılan PCR sonucunda ise pozitif kontrol cular biology and its application in plant pathology, olarak kullanılan dört A. tumefaciens suşundan beklenen fungal transformation has been paid more attention yaklaşık 730 bp’lik DNA fragmenti elde edilmiştir, ancak due to the relative simplicity of this method to obtain aynı fragment 10 transformanttan elde edilememiştir. target genes. Currently, the transformation of more Bu iki PCR sonucu T-DNA’nın FJ2 izolatının genomuna than 100 filamentous fungi has been carried out suc- yerleştiğini göstermektedir. Yapılan stabilite deneyleri cessfully [11–13]. Many approaches have been used in sonucunda transformantların higromisim B’ye karşı fungal transformation, such as PEG mediated trans- yüksek direnç gösterdikleri ve beş generasyon sonra bile formation, Restriction Enzyme-Mediated Integration bu antibiyotik varlığında normal şekilde üreyebildikleri (REMI), A. tumefaciens-mediated transformation görülmüştür. (ATMT) and so on. Of these approaches, ATMT has been becoming more and more popular for its advan- Sonuç: P. asparagus için yüksek transformasyon oranına tages such as simple manipulation, high transforma- sahip kalıcı ve yeterli bir ATMT transformasyon sistemi tion efficiency and high single-copy rate. Therefore it geliştirilmiştir. Bu sistemin geliştirilmesi için transfor- has become the most powerful tool for fungal trans- masyon koşulları optimize edilmiş, T-DNA’nın genoma formation [12,14]. yerleştiği PCR ile doğrulanmış ve genoma yerleştirilen The transformation system of many plant pathogens hph genini içeren segmentin alt kültürlerde de kalıcı has been counstructed successfully and their transforma- olduğu gösterilmiştir. Geliştirilen bu transformasyon tion conditions have been optimized [13,15–20]. In Phom- sistemi P. asparagi’nin patojenite mekanizmasının araştı- opsis, P. viticola was transformed with successfully [21,22], rılmasını amaçlayan çalışmalar için temel oluşturacaktır. and A. tumefaciens-mediated transformation system for the soybean pathogen P. longicolla was constructed [23]. Anahtar Kelimeler: Asparagus stem blight (Kuşkonmaz However, the transformation system of P. asparagi has pas hastalığı); Phomopsis asparagi; Agrobacterium tume- not yet been reported. In the present study, we intend faciens’e dayalı transformasyon, transformasyon verimi, to achieve and optimize the genetic transformation of P. çift PCR doğrulama asparagi. This study aims at establishing the foundation for understanding the pathogenic mechanism of P. aspa- doi 10.1515/tjb-2015-0018 ragi, which will be of great theoretical and practical sig- Received September 12, 2014; accepted April 13, 2015 nificance. 292 Ying-Qing Yan et al.: Establishment of ATMT system for P. asparagi

Materials and Methods NH4NO3 0.5 g; CaCl2 0.01 g; MgSO4·7H2O 0.6 g; NaCl 0.3 g;

(NH4)2SO4 0.5 g; Glucose 1 g; MES 7.808 g; total volume of 1000 mL adjusted with ddH2O; final pH value 5.6 adjus- Fungal isolate and plasmid ted with 4 mol/L NaOH and 4 mol/L HCl], followed by o pre-induction at 28 C for 8 h; when the OD600 value was The virulent isolate FJ2 of P. asparagi used in this study was about 0.3, it was suitable for co-culture. The P. asparagi conserved by our laboratory [4,5]. The plasmid pTHPR1, isolates was cultured on PDA plates for 16 days, when pyc- with hph gene that confered resistance to hygromycin B, nidia grew up. The pycnidia were scraped with a slide and was kindly provided by Professor Lian-Hui Zhang at the bundled with a piece of gauze, followed by adding 20 mL Institute of Molecular and Cell Biology (IMCB), Singa- sterile water and pounding with a pounder, making the pore. A. tumefaciens strains of AGL-1, EHA105, MP90 and spores released. After filtrated though 3 layers of gauzes at LBA4404 were conserved by our laboratory. the aseptic condition, followed by addition of sterile water for dilution until the concentration of the conidium solu- tion reached to 1×106 per mL, then 100 μL of the conidium Susceptibility of P. asparagi to hygromycin B solution and 100 μL of the pre-induced A. tumefaciens solution were mixed and spread onto the nitrocellulose The appropriate amount of hygromycin B (Roche, membrane placed on the surface of an solid induction

Germany) was added into the potato dextrose agar medium (SIM) [K2HPO4 2.05 g; KH2PO4 1.45 g; NH4NO3 0.5 medium (PDA) [24] for adjusting the hygromycin B con- g; CaCl2 0.01 g; MgSO4·7H2O 0.6 g; NaCl 0.3 g; (NH4)2SO4 0.5 centrations to 0, 10, 20, 30, 40, 50, 60, 70, 80, 90 and 100 g; Glucose 1 g; MES 7.808 g; agar 18 g; total volume of 1000

μg/mL respectively. The wild type isolate FJ2 of P. asparagi mL adjusted with ddH2O; final pH value 5.6 adjusted with was incubated on PDA plates for 5 d and mycelial plugs 4 mol/L NaOH and 4 mol/L HCl] plate, amended with ace- were cut from the colony edge with a 5-mm-diameter tosyringone (AS) at 200 μmol/L. After co-culture at 25oC borer, and then transferred onto the centre of PDA plates for 48 h, the nitrocellulose membrane was transferred to with hygromycin B at different concentrations and incu- a PDA plate amended with 100 μg/mL hygromycin B and bated at 25oC for 8 d. For further determining the screening 300 μg/mL cephamycin, and was incubated at 25oC for 6 concentration of hygromycin B, 100 μL of the conidium d, followed by re-screening on PDA plates amended with suspension at 106 per mL was spread evenly onto the PDA 100 μg/mL hygromycin B and 300 μg/mL cephamycin. The plate with hygromycin B at 0, 10, 20, 30, 40, 50, 60, 70, 80, screened transformants were subcultured for five genera- 90 and 100 μg/mL respectively, and incubated at 25oC for tions and conserved at –70oC. 8 d. Three replications of each concentration were taken and all the experiments were repeated twice. Effects of different factors on transformation efficiency Transformation method Effects of different factors on the transformation efficiency The transformation method described by Mullins et al. were investigated. These factors included pre-induction [25] was followed with minor modifications. The A. tume- time (4–12 h), co-culture time (24–72 h), concentrations faciens strain AGL-1 with the plasmid pTHPR1 containing of Acetosyringone (AS) (Sigma, USA) during co-culture the hph gene that confered resistance to hygromycin B (0–400μg/mL), temperatures during co-culture (22–34oC), was incubated in 3 mL minimal medium (MM) solution and pH values during co-culture (5.2–6.4). The transfor-

[K2HPO4 2.05 g; KH2PO4 1.45 g; NH4NO3 0.5 g; CaCl2 0.01 g; mation method described by Mullins et al. (2001) was

Glucose 2 g; (NH4)2SO4 0.3 g; FeSO4 0.01 g; 5 mL Z-buffer taken as the original method and the optimization of the

(ZnSO4·7H2O, CuSO4, H3BO3 and MnSO4·H2O at the concen- posterior factors was based on the frontier results. For the tration of 0.01% respectively); total volume of 1000 mL effect of pre-induction time on the transformation effici- adjusted with ddH2O; final pH value 6.7 adjusted with 4 ency, the IM solution was used for pre-induction, and mol/L NaOH and 4 mol/L HCl] amended with spectino- culured for 4, 6, 8, 10 and 12 h separately. mycin at 100 μg/mL and rifampicin at 30 μg/mL at 28oC The transformants in each dish were counted and ten for 48 h, when the OD600 value was above 0.8, the bacteria replicates were maintained for the statistics of transfor- suspension was diluted to OD600 value of 0.15 with induc- mants’ number, while three replicates were maintained 2 tion medium (IM) solution [K HPO4 2.05 g; KH2PO4 1.45 g; for the statistics of the OD600 values in the A. tumefaciens Ying-Qing Yan et al.: Establishment of ATMT system for P. asparagi 293 solution. All the experiments were repeated twice. B and incubated at 25oC for 6 d to detect the resistance on The transformation efficiency was expressed as the hygromycin B. The colony diameter in each dish from each numbers of transformants per 105 conidia [Transformation generation was measured and ten replicates were main- efficiency=(Numbers of transformants/Numbers of conida tained for the statistics of colony diameters from transfor- per dish)×105]. mants and the wild type FJ2. This experiment was repea- ted twice.

Statistical methods

The data were analyzed for significant difference at 5% Results level by Duncan’s multiple range test (DMRT) using SAS 9.0. Sensibility of P. asparagi to hygromycin B

Dual PCR verification on transformants After incubation for 8 d, the mycelia could not grow on the PDA plates containing 90 μg/mL hygromycin B, but could Ten transformants were randomly selected, subcultured grow on the PDA plates with hygromycin B lower than 90 for five generations, and their genomic DNAs were extrac- μg/mL at different levels. Similarly, after incubation for 8 ted using the Universal DNA Extraction Kit from TaKaRa d, the conidia could not grow on the PDA plates contai- (Japan). The genomic DNAs of A. tumefaciens strains of ning 90 μg/mL hygromycin B, but could grow on the PDA AGL-1, EHA105, MP90 and LBA4404 were extracted with plates with hygromycin B lower than 90 μg/mL at different Bacterium Genomic DNA Extraction Kit from TIANGEN levels. To guarantee the resistant quality, 100 μg/mL was (Beijing, China). selected as the hygromycin B concentration for later scree- Specific primers of hph-F (5’-GCAAGACCTGCCTG- ning of transformants. AAACCG-3’) and hph-R (5’-GGTCAAGACCAATGCGGAGC-3’) were designed according to the hph gene sequence. PCR was carried out in a volume of 20 μL containing 10 mmol/L Effects of several factors on the transforma-

Tris-HCl (pH 8.4), 1.5 mmol/L MgCl2, 50 mmol/L KCl, 0.25 tion efficiency mmol/L dNTP, 0.2 mmol/L each primer, 20 ng of template DNA and 1 U of Taq DNA polymerase using the following The A. tumefaciens AGL-1 in the MM solution was cultured program: 95oC for 2 min, then 30 cycles of 94oC for 40 s, continuously for about 48 h until the pH value was over 0.8, 56oC for 40 s and 72oC for 1 min, with a final extension of followed by a dilution with the IM solution for a pH value 72oC for 10 min. of 0.15, and was pre-induced at 28oC. The transformation To exclude the probability of false positive bands efficiency reached a high value when A. tumefaciens was caused by the contamination of A. tumefaciens, speci- pre-induced for 8 h with the OD600 value of 0.329. When fic primers of VCF (5’-ATCATTTGTAGCGACT-3’) and VCR the pre-induced time went up to10 h and 12 h, the OD600 (5’-AGCTCAAACCTGCTTC-3’) were designed based on the value was 0.391 and 0.431 separately (Figure 1a). Though Vir gene published by Sawada et al. [26], and were utilized the transformation efficiency at 10 or 12 h as the pre-induc- for PCR amplification using the four A. tumefaciens strains tion time was a little higher than that of 8 h, the difference with the Vir gene as positive controls, i.e. AGL-1, EHA105, among them was not significant (Figure 1a). Therefore 8 h MP90 and LBA4404. The amplification program was: 95oC was chosen as the optimal pre-induction time. for 2.5 min, then 40 cycles of 95oC for 40 s, 55oC for 1 min The co-culture times were set as 24, 36, 48, 60 and 72 h and 72oC for 2 min, with a final extension of 72oC for 7 min. in the present study. The results indicated that as the incre- asing of the co-culture time, the transformation efficiency improved gradually, and co-culture for 48 h could arrive at Stability test of transformants a higher value compared to 24 and 36 h. Though co-culture for 60 and 72 h could produce a little more transformants, Ten transformants were selected randomly and incuba- 48 h was chosen as the best co-culture time considering ted on PDA plates at 25oC for 6 d, then stored at 4oC for time-saving, higher single copy rates and convenience for 10 d. Another four cycles of such test were repeated. The transformant picking (Figure 1b). transformants after subcultured for five generation were AS concentrations at 0, 50, 100, 200 and 400 μmol/L transferred onto PDA plates with 100 μg/mL hygromycin were set in the present study. The results showed that the 294 Ying-Qing Yan et al.: Establishment of ATMT system for P. asparagi

(a) 25 0.5 (d) 25 Transformants’ number a a a a a 20 OD 0.45 600 20 b a 0.4 conidia conidia b 5 5 15 0.35 b 600 15 10 c c c 0.3 O D c 5 0.25 10 c Transformants’ Transformants’ d 0 0.2 numbers per 10 numbers numbers per 10 numbers 2 4 6 8 10 12 14 5 Pre-inducement time (h) 22 25 28 31 34 Cocultivation temperature oC

(b) 30 a b 25 b (e) 25

conidia 20 5 20 a a conidia

15 c 5 15 10 b

Transformants’ 5 d 10 b b Transformants’

numbers per 10 numbers 0 c 24 36 48 60 72 5 d Cocultivation time (h) per 10 numbers 0 5.2 5.4 5.6 5.8 6 6.2 6.4 Cocultivation pH values (c) 30 25 a a

conidia 20 5 b 15 10 c

Transformants’ 5 d 0 numbers per 10 numbers 0 50 100 200 400 Concentrations of AS μmol/L

Figure 1: Effects of several factors on the transformation efficiency: (a) Effects of pre-induction time on transformation efficiency. primary axis, 5 the transformants’ numbers per 10 spores at different pre-induction times, secondary axis, the OD600 values at different pre-induction times. (b) Effects of co-culture time on transformation efficiency. X-axis, cocultivation times, Y-axis, the transformants’ numbers per 105 spores. (c) Effects of AS concentration on transformation efficiency during co-culture. X-axis, concentrations of AS, Y-axis, the transformants’ numbers per 105 spores. (d) Effects of cocultivation temperatures on transformation efficiency during co-culture. X-axis, cocultivation temperatures, Y-axis, the transformants’ numbers per 105 spores. (e) Effects of cocultivation pH values on transformation efficiency during co-culture. X-axis, cocultivation pH values, Y-axis, the transformants’ numbers per 105 spores. Ten replicates were maintained for the statistics of transformants’ number, while three replicates were maintained for the statistics of the OD600 values in the A. tumefaciens solution. All the experiments were repeated twice. Data in this figure, representing the mean±SD of ten replications, were analyzed for significant difference by using Duncan’s multiple range test (DMRT). Different capital letters and lowercase letters above the bars indicate significant difference at the 5% level. transformation efficiency reached a relatively high level 25–28 oC was chosen as the optimal temperature during with the AS concentration at 200 μmol/L. Though the co-culture (Figure 1d). transformants at 400 μmol/L AS were slightly higher than The SIM media at pH 5.2, 5.4, 5.6, 5.8, 6.0, 6.2 and 6.4 those at 200 μmol/L, the difference between them was not were prepared and used for co-culture. The results showed significant (Figure 1c). Therefore 200 μmol/L of AS was that as the increasing of the pH value, the transformation selected as the optimal concentration. efficiency improved gradually, and the transformation effici- Temperatures of 22, 25, 28, 31 and 34oC were set ency maximized when the pH value was between 5.6 and during co-culture in the present study. The transforma- 5.8. The transformation efficiency decreased rapidly when tion efficiency reached the peak value at 25oC during the pH value was over 5.8. Therefore 5.6–5.8 was chosen as co-culture, and still kept a high value at 28oC, for which the optimal pH value during co-culture (Figure 1e). Ying-Qing Yan et al.: Establishment of ATMT system for P. asparagi 295

(a) Table 1: Colony diameters of the transformants from different 1 2 3 4 5 6 7 8 9 10 11 12 M generations. 2000 bp 1000 bp 750 bp Strain types Colony diameters after 6 days 500 bp 250 bp Wild type FJ2 7.76±0.43a a 100 bp First generation 7.57±0.39 Second generation 7.60±0.43a Third generation 7.39±0.32a Fourth generation 7.47±0.35a (b) 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 M Fifth generation 7.42±0.42a 2000 bp Ten replicates were maintained for the statistics of colony diameters, 1000 bp 750 bp and the experiment was repeated twice. Data in this table, repre- 500 bp 250 bp senting the average±SD of ten replicates, were analyzed for signifi- 100 bp cant difference by using Duncan‘s Multiple Range Test (DMRT), the data with the same letter are not significantly different at 5% level (P=0.05).

Figure 2: PCR amplifications of genomic DNAs from transformants: (a) PCR for hph gene. Lane 1, Wild type isolate FJ2 of P. asparagi; Lane 2, Plasmid pTHPR1 (positive control for hph gene); Lanes 3 to Stability of transformants 12, Ten randomly selected transformants; M, DL2000 marker. (b) PCR for Vir gene. Lanes 1 to 4, A. tumefaciens strains of AGL-1, EHA105, Ten transformants selected at random were subcultured MP90 and LBA4404 (positive controls for Vir gene), respectively; on PDA plates without hygromycin B for five generations, Lanes 5, Wild type isolate FJ2 of P. asparagi; Lanes 6 to 15, Ten ran- and then transferred onto PDA plates with 100 μg/mL hyg- domly selected transformants; M, DL2000 marker. romycin B. It was observed that the 10 transformants after subcultured for five generations could grow normally on Dual PCR verification on transformants PDA plates containing 100 μg/mL hygromycin B with an average colony diameter of about 7.42 cm after incubated The specific PCR amplification of hph gene revealed that for 6 d (Table 1), which indicated clearly that the T-DNA an expected band of about 500 bp could be amplified from was indeed inserted into the genome of P. asparagi and all genomic DNAs extracted from 10 transformants of P. the transformants behaved stable mitosis. asparagi and the pTHPR1 plasmid DNA, whereas no cor- responding DNA band was amplified from that of the wild type isolate FJ2 (Figure 2a). Discussion To eliminate the possibility that the genomic DNAs from 10 transformants were contaminated by that of A. Asparagus stem blight, caused by P. asparagi, is a world- tumefaciens (containing plasmid pTHPR1), the wild type wide devastating disease and the genetic breeding based FJ2 and 10 transformants were tested for the presence of on the pathogenic genes is the most economical and Vir gene, which existed in A. tumefaciens, using four A. effective control method [2]. However, there has been no tumefaciens strains with Vir gene as the positive control, report on pathogenic genes of P. asparagi. To explicit the i.e. AGL-1, EHA105, MP90 and LBA4404. The specific PCR pathogenic genes of P. asparagi, a transformation system amplification of Vir gene showed that an expected band of has to be constructed firstly. The transformation system about 730 bp could be amplified from four A. tumefaciens for P. asparagi was constructed successfully in this study, strains, whereas no corresponding DNA band was ampli- which would promote the research progress on patho- fied from the wild type isolate FJ2 and 10 transformants genic mechanism and pathogenicity related genes of P. (Figure 2b), indicating that the genomic DNAs from the asparagi. wild type isolate FJ2 and 10 transformants were not con- In respect to pre-induction time, 6 h was taken as taminated by that of A. tumefaciens. the pre-induction time in some previous reports [15,20], From the results of the two PCR amplifications men- whereas 8 h was confirmed as the best pre-induction time tioned above, we concluded that T-DNA insertion indeed through gradient experiments of pre-induction time in the existed in the genomes of 10 transformants, which proved present study, and the pre-induction time more than 8 h that the constructed transformation system was efficient could not improve transformation efficiency observably. in carrying out the transformation of P. asparagi. In regarding to co-culture time, we observed that with 296 Ying-Qing Yan et al.: Establishment of ATMT system for P. asparagi the increase of co-culture time, the transformation effici- in this study the A. tumefaciens-mediated transformation ency increased, which supported the previous conclusions system for P. asparagi was constructed and the transfor- in the transformation of other fungi [15,16,20]. The results mation rate was high at around 20–30 transformants per on Aspergillus giganteus showed that there was no trans- 105 spores. On transformation effectiveness, this study formant produced when the co-culture time was ess than supported the frontier report, in which a high transforma- 24 h or more than 72 h [17]. Some reporters thought that it tion rate could be achieved with hph gene, whereas a cor- was difficult to screen single colony of transformant due to respondingly low rate could be achieved with Bmlr gene the excessive growth of mycelia when the co-culture time or GFP gene [21,22]. exceeded 48 h, and with the extension of co-culture time, A stable and efficient ATMT transformation system for it was prone to more multi-copy and false positive transfor- P. asparagus was constructed through condition optimi- mants [18,20]. In this study, co-culture time of 48 h could zing, insertion verifying and stability confirming, which produce enough transformants, which suggested that co- established the foundation for understanding the patho- culture time of 48 h was enough for efficient transformation genic mechanism of P. asparagi and would be of great the- of P. asparagi and supported the previous views [17,18,20]. oretical and practical significance. In general, AS and its concentration during co-culture contributed to transformation efficiency greatly and the transformation efficiency improved with the increase of Acknowledgement: The authors thank Professor Lian- AS concentrations [13,15,20]. The results of our research Hui Zhang at the Institute of Molecular and Cell Biology suggested that high transformation efficiency of P. aspa- (IMCB), Singapore for providing A. tumefaciens isolate ragi could be achieved with AS included at the co-culture AGL-1 and plasmid pTHPR1 with hph gene. This research phase. The transformation efficiency improved with the was supported by the National Natural Science Foundation increasing of AS concentrations, which was identical with of China (Grant No. 31460456), the Natural Science Foun- the views of most researchers [12,19,27,28]. dation of Jiangxi Province (Grant No. 20142BAB214022), In respect to pH values during co-culture, pH 5.5 was the National Basic Research Program of China (Grant No. the best co-culture pH value in transformation of Fusa- 2011CB111603) and the Special Fund for Agro-scientific rium oxysporum [20]. In this study, it was concluded that Research in the Public Interest from the Ministry of Agri- pH 5.6–5.8 during co-culture was the optimal pH value for culture of China (Grant No. 201003074). transformation of P. asparagi. The pH values during co- Conflict of Interest: The authors have no conflict of interest. culture could affect the expression of VirA protein, and subsequently affect the transfer of T-DNA [29]. 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