МИКРОБИОЛОГИЯ, 2011, том 80, № 1, с. 86-92
= ЭКСПЕРИМЕНТАЛЬНЫЕ СТАТЬИ
AGROBACTERIUM TUMEFACIENS-MEDIATED TRANSFORMATION OF THE PLANT PATHOGENIC FUNGUS ROSELLINIA NECATRIX
© 2011 Sanae Kano*, Takuma Kurita*, Satoko Kanematsu**, Tsutomu Morinaga*, 1
*Faculty of Life and Environmental Sciences, Prefectural University of Hiroshima, Nanatsuka-cho 562, Shobara-shi, Hiroshima, 727-0023, Japan **National Institute of Fruit Tree Science, National Agricultural Research Organization (NARO), 92-24 Shimokuriyagawa, Morioka-shi, Iwate, 020-0123, Japan
Abstract. Rosellinia necatrix is a soil-borne root pathogen affecting a wide range of commercially important plant species. The mycelium of R. necatrix was transformed to hygromycin B resistance by an Agrobacterium tumefaciens-mediated transformation system using a binary plasmid vector containing the hygromycin B phosphotransferase (hph) gene controlled by the heterologous fungal Aspergillus nidulans P-gpd (glyceraldehyde 3-phosphate dehydrogenase) promoter and the trpC terminator. Co-cultivation of R. necatrix strain W1015 and A. tumefaciens strain AGL-1 at 25°C using the binary vector pAN26-CB1300, which contained the hygromycin B resistance cassette based on pAN26 and pCAMBIA1300, resulted in high frequencies of transformation. The presence of the hph gene in the transformants was detected by PCR, and single-copy integration of the marker gene was demonstrated by Southern blot analysis. This report of an Agrobacterium-mediated transformation method should allow the development of T-DNA tagging as a system for insertional mutagenesis in R. necatrix and provide a simple and reliable method for genetic manipulation.
Keywords: Agrobacterium tumefaciens, hygromycin B, Rosellinia necatrix, T-DNA, transformation.
The filamentous ascomycete Rosellinia necatrix (Har-tig) Berlese is a commercially important, soil-borne, root pathogen affecting a wide range of plant species. It is the causal agent of white root rot disease, and host plants infected by the fungus quickly wither and die. In Japan, this fungal disease, which spreads rapidly and is very difficult to prevent, has done great damage to commercially grown grapevines, apple and pear trees, and other crops. Among the few effective control methods for white root rot are fungicides and biological control. However, long-term prevention of this root disease is difficult.
In filamentous fungi, the presence of double-stranded RNA (dsRNA) elements [1, 2] has been reported. The elements are known to reduce the virulence of phytogenic fungi. Such dsRNA elements in R. necatrix have also been reported [3]. Moreover, a novel bipartite dsRNA has been isolated and characterized from R.. necatrix [4]. Valuable knowledge about the molecular basis of the pathogenicity of R. necatrix could be gained from genetic studies such as gene insertional mutagenesis. However, understanding the genetic basis of its pathogenicity has been limited by the lack of a suitable transformation system.
Agrobacterium tumefaciens-mediated transformation (ATMT), which has long been a workhorse in plant science, has been exploited for fungal transformation. A. tumefaciens has the ability to deliver its T-DNA into chromosomes of the budding yeast, Saccharomyces cere-
1 Corresponding author (e-mail: tmorina@pu-hiroshima.ac.jp). Phone/Fax.: +81-824-74-1777.
visiae [5], and diverse filamentous fungi [6—9]. Besides as-comycetes and basidiomycetes, this technique has been successfully applied to transform zygomycetes also [10]. In comparison with Restriction Enzyme Mediated Integration (REMI), ATMT does not require protoplasts and allows a broad spectrum of starting material to be transformed. Protoplasts, hyphae, spores and even blocks of mushroom mycelial tissues [6] were transformed through ATMT with a higher efficiency than through REMI. DNA transfer from A. tumefaciens has been used for both gene knock-out and gene transformation studies in filamentous fungi [5, 7, 9] and is being developed as a system for insertional mutagenesis in filamentous fungi [8, 9].
In this study, we described a successful procedure for the genetic transformation of mycelium from R. necatrix. Transformation efficiency was optimized based on Agro-bacterium strain and co-cultivation temperature. Single-copy T-DNA insertions into fungal genomes suggest that ATMT is a useful insertional mutagenesis and gene disruption tool in R. necatrix.
MATERIALS AND METHODS
Strains and culture conditions. The R. necatrix strains (W97, W1015, W370T1 and W779) used in this study (Table 1) were originally obtained from Dr. Hitoshi Nakamu-ra (National Institute of Agro-Environmental Sciences, Japan) and were routinely grown on potato dextrose agar (PDA; potato extract, 2% glucose, 2% agar) at 25°C. Mycelia for DNA extraction were grown on a cellophane
Table 1. Strains and plasmids used in this study
Strain or plasmid
Characteristics (locality)
Origin or provider
Rosellinia necatrix W97 W1015 W370T1 W779
Field-isolated original strain (Japan) W779 protoplast-regenerated strain W370 single hypha-isolated strain Field-isolated original strain (Japan)
Dr. H. Nakamura Dr. H. Nakamura Dr. H. Nakamura Dr. H. Nakamura
Agrobacterium tumefaciens (Rhizobium radiobacter)
AGL-1 EHA105 LBA4404 AGL0 recA::bla pTiBo542AT-region Mop+ CbR L,L-succinamopine type, derivative of pTiBo542 Ti plasmid pAL4404, derivative of pTiAch5 ATCC[11] Dr. Y Niimi [12] Dr. Y Niimi [12]
Plasmid
pAN26 hph gene FGSC [13]
pBI121 Binary plasmid Dr. Y Niimi [14]
pCAMBIA1300 Binary plasmid CAMBIA [15]
pAN26-BI121 Binary plasmid This study
pAN26-CB1300 Binary plasmid This study
sheet overlaid on PDA. A. tumefaciens strain AGL-1 (ATCC BAA-101) was supplied by the American Type Culture Collection [11]. The A. tumefaciens strains EHA105 and LBA4404 [12] were kindly provided by Dr. Yoshiyuki Niimi (Prefectural University of Hiroshima, Japan), and are listed in Table 1. These Agrobacterium strains were routinely grown on Luria-Bertani (LB) agar (1% sodium chloride, 1% tryptone, and 0.5% yeast extract, pH 7.0) containing 50 ^g/ml kanamycin to maintain the plasmid vector for transformation experiments.
Plasmid construction. The plasmid vectors pAN26 [13], pBI121 [14] and pCAMBIA1300 [15] were supplied by the Fungal Genetics Stock Center (Kansas City, KS, USA), Dr. Yoshiyuki Niimi (Japan) and the Center for Application of Molecular Biology to International Agriculture (Canberra, Australia), respectively (Table 1). The plasmids pAN26-BI121 and pAN26-CB1300 contain a hygromycin В resistance cassette, which is based on pAN26 for the selection ofresistant fungal clones (Fig. 1), and a kanamycin resistance gene for selection in Agrobacterium. The hygromycin В resistance cassette contains the hygromycin phosphotransferase gene (hph) under the control ofthe glyceraldehyde 3-phosphate dehydrogenase promoter (P-gpd) and the trpC terminator (T-trpC) from Aspergillus nidulans (Eidam) Winter. A. tumefaciens strains AGL-1, EHA105 and LBA4404 were transformed by electroporation with constructed binary vectors using a Gene Pulser system (Bio-Rad, Tokyo, Japan) according to the supplier's protocol [16].
A. tumefaciens-mediated fungal transformation. The transformation procedure was based on the protocol described by Chen et al. [6], with some modifications. A single colony of A. tumefaciens (strains AGL-1, EHA105 and LBA4404) carrying the plasmid pAN26-BI121 or pAN26-CB1300 was grown in 30 ml LB broth containing
50 ^g/ml kanamycin overnight at 28°C with shaking at 120 rpm. The cells were collected again and suspended in induction medium [5] to an optical density at 600 nm of 0.5—0.8. The bacterial suspension was incubated for 5— 6 h at 28°C under agitation (120 rpm) to preinduce the virulence of A. tumefaciens.
The R. necatrix strains were grown in PD (potato extract and 2% glucose) medium for 1 week or on the surface of a cellophane sheet on PDA for 4 days at 25°C. The culture broth containing the mycelial mat was homogenized with a Nissei Homogenizer (AM-12) at 8000 rpm for 2 min. The homogenized mycelium and the virulence-preinduced A. tumefaciens were mixed and collected by centrifugation. The resultant pellet containing R. necatrix mycelium and A. tumefaciens was transferred onto a cellophane sheet overlaid on IM agar medium (IM + 2% agar). After 3 days of co-culture on these IM agar media at each temperature, 17°C, 20°C and 25°C, the cellophane sheets were transferred onto selection medium (SM: PDA containing 40 ^g/ml hygromycin B for selection of transformants and 25 ^g/ml meropenem for killing A tumefaciens [17]). Hygromycin-resistant colonies appear after approximately 2 weeks. These colonies were transferred to and maintained on PDA containing 50 ^g/ml hygromycin B.
To calculate the efficiency ofthe transformation, small mycelial cubes cut with a Pasteur pipette from the margins of young R. necatrix mycelium were inoculated onto cellophane sheets on PDA [18]. After overnight incubation at 25°C, the cellophane sheets with the young mycelial fragments were transferred to IM agar medium (Fig. 2B-a, b). Young mycelial fragments were co-cultivated by pouring the virulence-preinduced A tumefaciens onto the IM agar medium containing the young mycelial fragments. The cellophane sheets were transferred onto SM after 3 days at
BamHI
XbaI
pAN26 ■
P-gpd hph T-trpC
BIR1hph3F RB^—-
pAN26-BI121
ca. 3.8 kbp
\7
Hygromycin B-resistant cassette
P-gpd
Probe
hph
BIR1hph4R
LB
T-trpC
pAN26-CB1300
RB
Probe
T-trpC
hph
XbaI
EcoRI
LB
P-gpd
BamHI
XhoI
Fig. 1. Schematic representation of pAN26 and construction of pAN26-BI121 and pAN26-CB1300 used in the transformation of R. necatrix. pAN26-BI121 vector was constructed with the In-Fusion PCR Cloning System using the primers BIR1hph3F and BIR1hph4R which are indicated by arrows. pAN26-CB1300 was constructed using a restriction enzyme-based method. Bars correspond with hybridization
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