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Bot. Bull. Acad. Sin. (2004) 45: 23-31

Lu et al. — Coniothyrium minitans xylanase in Arabidopsis

Segregation patterns for integration and expression of Coniothyrium minitans xylanase gene in Arabidopsis thaliana transformants

Z.-X. Lu, A. Laroche*, and H.C. Huang

Agriculture and Agri-Food Canada, Lethbridge Research Centre, P.O. Box 3000, Lethbridge, Alberta T1J 4B1, Canada

(Received October 8, 2002; Accepted August 26, 2003)

Abstract. A xylanase gene cxy1, isolated from the mycoparasitic fungus Coniothyrium minitans, has been transferred into Arabidopsis thaliana by Agrobacterium-mediated transformation. Ten A. thaliana transformants with herbicide resistance were selected, and the transformation efficiency of the modified "Floral Dip" method was about 0.08 %. Most transformants had one or two copies of T-DNA inserts with 3R:1S or 15R:1S segregation ratios while high proportions of susceptible progenies were also observed in some transgenic plants. The pMB4-2 transformant segregated as 1R:2S ratio in its T2 and T3 generations, suggesting that only one copy of T-DNA was integrated into this line's genome and the insertion likely interrupted a gene important or essential to its gametophytic development or alternatively, affected the transmission of such gene. The genetic analysis of the pMB4/cxy1-6 transformant indicated that two copies of T-DNAs were inserted independently into this line's genome, in which the resistant homozygotes on one locus were non-viable. The RBB-Xylan assay for six pMB4/cxy1 transformants indicated that two lines (pMB4/cxy1-3 and pMB4/cxy1-6) expressed the cxy1 gene, and variations for the xylanase activity were observed among T2 progenies of pMB4/cxy1-6 transformant.

Keywords: Agrobacterium-mediated transformation; Arabidopsis thaliana; Coniothyrium minitans; Xylanase gene.

Introduction

Xylan is a complex polysaccharide which consists of a backbone of xylose residues linked by b-1,4-glycosidic bonds. Xylan fibers constitute a significant portion of plant hemi-cellulose and contribute to the strength of plant secondary walls (Thomson, 1993). The hydrolysis of xylan by xylanases (E.C. 3.2.1.8) produced by fibrolytic bacteria and fungi is necessary in the degradation of plant tissue. Xylanolytic enzymes have numerous applications in industry and biotechnology, such as in woody biopulping processes (Bieley, 1985), forage fibre digestions (Gilbert and Hazelwood, 1991), and agricultural waste degradation.

Agrobacterium-mediated gene transfer is the most commonly used technique for plant transformation (Zambryski, 1992; Zupan and Zambryski, 1995). The `Agrobacterium Vacuum Infiltration', a non-tissue culture approach for in planta transformation, was first achieved in Arabidopsis thaliana (L.) Heynh. (Feldmann and Marks, 1987; Bechtold et al., 1993; Chang et al., 1994; Katavic et al., 1994). Later, a relatively simplified protocol called the "Floral Dip" was described by Clough and Bent (1998). However, it still re

quires sterile conditions for seed germination and transformant selection. Recently, we successfully modified this method by germinating A. thaliana seeds and selecting transgenic seedlings in Cornell Peat-Lite Mix in plastic pots under greenhouse conditions.

Coniothyrium minitans Campbell is a mycoparasite which attacks the sclerotia (Huang, 1977; Huang and Kokko, 1987) and hyphae (Huang and Hoes, 1976; Huang and Kokko, 1988) of Sclerotinia sclerotiorum (Lib.) de Bary, an important fungal pathogen of higher plants (Purdy, 1979). A novel xylanase gene cxy1 has been isolated from C. minitans (Laroche et al., 2000) and expressed in Pichia pastoris (Guilliermond) Phaff (Lu et al., 1999). The objective of our study is to explore the impacts of cxy1 gene for resistance to S. sclerotiorum in plants and for improvement of forage utilization in animal production. This report describes the experimental results for cxy1 integration and expression in A. thaliana.

Materials and Methods

Construction of Ti Plasmid

The Agrobacterium tumefaciens (Smith & Townsend) Conn strain EHA105 was provided by Dr. F. Eudes (Lethbridge Research Centre, Agriculture and Agri-Food Canada), and the binary vector pMB4 was obtained from "The Baker Lab" at the Gene Expression Center (Berkeley, CA). Strain EHA105 already contains the helper vector,

Lethbridge Research Centre, Canada, Contribution No. 387-01021.

*Corresponding author. Tel: + 1 (403) 317-2224; Fax: + 1 (403) 382-3156; E-mail: laroche@agr.gc.ca

Botanical Bulletin of Academia Sinica, Vol. 45, 2004

which encodes the gene conferring rifampicin resistance (Hood et al., 1993). The pMB4 vector, a large Ti plasmid (12,547 bp) with a 4,000 bp T-DNA, was originally maintained in Escherichia coli Migula strain DH5a on Luria and Broth (LB) medium (Difco Laboratories) with kanamycin (50 µg/ml). The bar gene conferring herbicide resistance (De Block et al., 1987; Thompson et al., 1987) was inserted after the first 35S promoter, and the second 35S promoter was used for the expression of the xylanase gene cxy1.

Both pMB4 vector and the cxy1 cDNA in pBluescript (lZAP cDNA Cloning Kit, Stratagene) were digested with two enzymes (Xho I and Xma I), and ligated to form Ti plasmid (Figure 1). The constructed pMB4 Ti plasmid was transformed into E. coli strain DH5a, and positive clones were selected on LB medium for kanamycin (50 µg/ml) resistance. The plasmids were extracted from the positive DH5a clones, and transformed into A. tumefaciens strain EHA105 by electroporation. The positive EHA105 clones were selected on LB medium for resistance to both kanamycin and rifampicin (50 µg/ml each). An EHA105 culture (250 ml) was prepared overnight (16 h) in LB medium, collected and resuspended (D600 = 1.0) in 5% sucrose solution with 0.2% surfactant Citowet-plus (BASF Canada, Inc.) for the floral dip procedure.

Floral Dip Transformation

The transformation protocol performed in this study was adopted from the method of Clough and Bent (1998), with the modification of using surfactant Citowet-plus instead of Silwet L-77 and growing A. thaliana seedlings in Cornell Peat-Like Mix (Boodley and Sheldrake, 1977) in 12-cm-diameter plastic pots instead of growing the seedlings in Petri dishes with 1/2 Murashige and Skoog (MS) medium (Sigma M-5519).

Seedlings of A. thaliana (ecotype Columbia) were grown in Cornell Peat-Like Mix in plastic pots and kept in a growth cabinet (16 h light with 120 mE/m2/s at 24ºC and 8 h dark at 20ºC) until they reached the flowering stage (about 5 weeks). No fertilizer was supplied during the experiment, and plants were watered when needed.

Two A. tumefaciens treatments, (1) EHA105 strain transformed with pMB4 T-DNA only and (2) EHA105 strain transformed with pMB4 T-DNA plus cxy1 gene, were applied to A. thaliana seedlings independently. The above-ground tissues of eight seedlings were dipped into EHA105 suspension for 3 to 5 s, and the dipped seedlings were covered with clear plastic film (Saran Wrap, Dow Chemical, Inc., Canada) to maintain high humidity for 24 h. The floral dip was repeated again 5 days later. The

Figure 1. pMB4 T-DNA construction of A. tumefaciens with C. minitans xylanase gene (cxy1) for transformation of A. thaliana and locations of the degenerated primers. RB, right border; LB, left border; P35S, 35S promoter of the Cauliflower Mosaic Virus; NOS, terminator of the nopaline systhase gene; UTR, untranslated region; ORF, open reading frame. PCR primer positions are indicated in parentheses and primer sequences are listed in Table 1.

Lu et al. — Coniothyrium minitans xylanase in Arabidopsis

EHA105-treated seedlings were kept in the growth cabinet until maturity.

Evaluation of Transgenic Seedlings

All T1 seeds, collected from the EHA105-treated A. thaliana plants, were sown in Cornell Peat-Like Mix in 52 × 26 cm plastic trays and stratified at 4ºC for two days in darkness to improve their germination. The T1 seedlings were grown under the same conditions as mentioned above.

The transgenic seedlings were screened and selected by spraying 0.1% Liberty (Registered mix: Venture 25DG, 15% glufosinate ammonium, AgrEvo) directly onto above-ground tissues of 5-day old T1 seedlings. The spray was repeated twice at 5-day intervals. The T1 transformants were transplanted into Cornell Peat-Like Mix in 12-cm-diameter plastic pots (one plant per pot) to produce T2 progenies. The same procedure was followed to obtain T3 populations from the T2 generation.

Genetic analysis for herbicide resistance was conducted on T2 segregating populations of each pMB4 or pMB4/cxy1 transformant independently. Further genetic studies on T3 progenies selected from four T2 families were conducted. The segregation ratios for resistance to the herbicide Liberty were determined for each transformant and statistically analyzed by c2 test for the hypotheses of Mendelian segregation.

Genomic DNA was isolated from fresh leaf tissues of T1, T2, and T3 transgenic seedlings using the FastDNA Kit (BIO 101, Inc., Cat. 6540-400). The transformants were further confirmed by PCR amplifications with specific primers (Figure 1; Table 1). Primers pMB4-F1, pMB4-F2, and pMB4-R1 were generated from the pMB4 sequence to detect the cxy1 insert after the second 35S promoter region. Primers Bar-F1 and Bar-R1 were generated from the bar gene sequence to determine the T-DNA fragment after the first 35S promoter. Primers Ffam-F1, Ffam-R1, Ffam-R2, and Ffam-R3 were generated from the cxy1 sequence to verify the cxy1 inserts in A. thaliana pMB4/cxy1 transformants.

Xylanase Expression in Transformants

Enzymatic solutions of A. thaliana T2 and T3 seedlings were extracted from 0.5 mg leaf tissues with 1 ml of 100 mM NaPO4 buffer (pH 6.5), in the presence of protease inhibitor at the recommended concentration of 10 ml/tablet (Roche Molecular Biochemicals, Complete, Mini, EDTA-free, Cat. No. 1836170).

Xylanase activity was analyzed by the RBB-Xylan assay (Lu et al., 1999). The substrate solution was prepared as 10 mg/ml Remazol Brillian Blue R-D-Xylan (RBB-Xylan, Sigma M-5019) in 100 mM NaPO4 buffer (pH 6.5). The mixture of sample supernatant and substrate solution (100 µl each) was incubated at 37ºC for 60 min, and the enzymatic reaction was terminated with 800 µl 95% ethanol. The supernatant for each reaction was collected to measure the xylanase activity at 570 nm. Xylanase (E.C. 3.2.1.8) (Sigma X-4001) was used as the positive and standard control. One unit of xylanase activity was defined as liberating 1 µmole of the reducing sugar measured as xylose equivalents from xylan per minute at pH 6.5 at 37°C.

Results

Efficiency of Floral Dip Transformation

Transformations of the xylanase gene cxy1 into A. thaliana genome using A. tumefaciens strain EHA105 were successful in this study, and the efficiency of the modified Floral Dip method was consistent in two independent experiments (Table 2). When the results of two experiments were combined, herbicide resistance was detected in 4 out of 5269 T1 seedlings in the pMB4 T-DNA treatment while 6 out of 7477 T1 seedlings displayed this resistance in the pMB4 T-DNA plus cxy1 insert treatment. The overall transformation efficiency was approximately 0.08%, higher than the efficiency of in planta transformation reported by Feldmann and Marks (1987) and similar to the results of the Floral Dip transformation reported by Clough and Bent (1998).

Botanical Bulletin of Academia Sinica, Vol. 45, 2004

Molecular Analysis of Transformants

PCR amplification of the pMB4 T1 and T2 DNAs indicated that the bar gene was detected in A. thaliana transformant genome. The predicted DNA fragments of 276 bp and 669 bp were verified in T1 pMB4 transformants with the (1) Bar-F1 and Bar-R1 primers and (2) pMB4-F1 and pMB4-R1 primers, respectively. When further tested on T2 populations of the pMB4-2 transformant, these two fragments were also detected in T2 herbicide resistant plants while no PCR amplification was observed in T2 susceptible plants (Figure 2).

PCR amplification of the pMB4/cxy1 T1 and T2 DNAs indicated that the cxy1 gene was integrated into A. thaliana transformants selected with herbicide resistance. With the pMB4-F2 and pMB4-R1 primers, the 1,559 bp fragment was detected from pMB4/cxy1 T1 transformants while the 275 bp fragment was amplified from transformants with pMB4 (data not shown). In addition, the predicted fragment sizes of 920 bp, 949 bp, 1343 bp, and 526 bp were verified in the four primer combinations: (I) pMB4-F1 and Ffam-R1, (II) pMB4-F2 and Ffam-R2, (III) pMB4-F1 and Ffam-R2, and (IV) pMB4-F2 and Ffam-R1, respectively (Figure 3).

Genetic Analysis of T2 Populations

Four T2 A. thaliana populations, derived independently from four pMB4 transformants (pMB4-1, pMB4-2, pMB4-

3, and pMB4-4), were analyzed for segregation of herbicide resistance (Table 3). For each transformant, we used the c2 test to analyse the segregation fitness with different segregation ratios (such as 1R:1S, 1R:2S, 1R:3S, 2R:1S, 3R:1S, 11R:1S, 15R:1S) then selected the ratio with the highest P value and listed it in our tables. Two transformants exhibited the inheritance of herbicide resistance as a single T-DNA insert with a 3R:1S ratio, and one transformant (pMB4-1) behaved as one copy of a T-DNA insert with non-viable resistant homozygotes (2R:1S). The pMB4-2 transformant was observed to have 162 resistant and 303 susceptible individuals in its T2 population fitting a 1R:2S segregation ratio.

Six T2 A. thaliana populations, developed independently from six pMB4/cxy1 transformants, were analyzed for segregation of herbicide resistance (Table 3). Four transformants (pMB4/cxy1-2, pMB4/cxy1-3, pMB4/cxy1-4, pMB4/cxy-1-5) inherited as a single T-DNA insert with a 3R:1S ratio. One transformant (pMB4/cxy1-1) behaved as two copies of T-DNA inserts with a 15R:1S ratio. The pMB4/cxy1-6 transformant exhibited 424 resistant and 41 susceptible individuals in its T2 population fitting a 11R:1S segregation ratio.

Genetic analysis of T3 Populations

In order to explore the nature of segregation distortion in the pMB4-2 transformant, 16 T3 populations were derived independently from 16 resistant T2 plants, and their

Figure 2. PCR detection of T-DNA inserts in T2 progenies of A. thaliana pMB4-2 transformant. A refers to PCR with Bar-F1/R1 primers; B refers to PCR with pMB4-F1/R1 primers. M, DNA size marker (100 bp ladder, Life Technology); At, wild A. thaliana (negative control). Fourteen T2 individuals were analyzed, only four (No. 5, 6, 8, 13) exhibited herbicide resistance.

Figure 3. PCR detection of C. minitans xylanase gene (cxy1) in A. thaliana T1 transformants. (I) pMB4-F1 and Ffam-R1 primers; (II) pMB4-F2 and Ffam-R2 primers; (III) pMB4-F1 and Ffam-R2 primers; (IV) pMB4-F2 and Ffam-R1 primers. M, DNA size marker (100 bp ladder, Life Technology); P, A. thaliana pMB4-3 transformant; and F, A. thaliana pMB4/cxy1-3 transformant.

Lu et al. — Coniothyrium minitans xylanase in Arabidopsis

segregation for herbicide resistance was analyzed (Table 4). All 16 populations were observed to segregate for the herbicide resistance, 15 populations with the 1R:2S ratio and 1 population with the1R:3S ratio.

Nineteen T3 pMB4/cxy1-1 populations, derived independently from 19 T2 resistant plants of pMB4/cxy1-1, were analyzed for segregation of herbicide resistance (data not shown). Six out of 19 T3 populations were observed to have all resistant progenies. The other 13 populations exhibited both resistant and susceptible progenies, 7 populations with the 15R:1S ratio, 4 with the 3R:1S ratio, and 2 with the 2R:1S ratio.

Eight T3 pMB4/cxy1-3 populations were derived independently from 8 T2 resistant plants of pMB4/cxy1-3, and their segregations for herbicide resistance were analyzed (data not shown). In these 8 T3 populations, 4 populations were observed to have all resistant progenies, 3 populations were segregated with the 3R: 1S ratio and 1 population with the 2R:1S ratio.

Twenty T3 pMB4/cxy1-6 populations, derived independently from 20 resistant T2 plants of pMB4/cxy1-6, were analyzed for segregation of herbicide resistance (Table 5). Seven out of 20 T3 populations were observed to have all resistant progenies. The other populations exhibited both resistant and susceptible progenies, 5 populations with the 11R:1S ratio, 5 with the 3R:1S ratio, and 3 with the 2R:1S ratio.

Xylanase Activity in A. thaliana Transformants

The RBB-Xylan assay for six pMB4/cxy1 transformants of A. thaliana indicated that only pMB4/cxy1-3 and pMB4/cxy1-6 expressed xylanase activity (Table 3; Figure 4) although the full length of cxy1 gene was integrated into all pMB4/cxy1 transformants. The pMB4/cxy1-3 was determined to produce xylanase activity of 186 mU, and similar activities were detected in its T2 and T3 progenies (data not shown). Both DNA evidence and xylanase activity verified that the cxy1 gene was integrated in A. thaliana

Botanical Bulletin of Academia Sinica, Vol. 45, 2004

Figure 4. RBB-Xylan assay for A. thaliana transformants. A1, H2O; A2, standard xylanase (Sigma X-4001, 120 mU); A3, A. thaliana only; A4, A. thaliana pMB4-1 transformant; A5-A10, A. thaliana pMB4/cxy1 transformant 1 to 6, in which A7 and A10 exhibited xylanase activity; the same sample order in Row B as in Row A; C1 to F10, 40 T2 progenies of pMB4/cxy1-6, in which C2, C8, F3, and F9 exhibited the xylanase activity. The reactions were 1 h for Row A and 12 h for Row B to Row F.

pMB4/cxy1-3 transformant and could be normally expressed through successive generations.

In contrast, the pMB4/cxy1-6 transformant of A. thaliana had weaker xylanase activity (80 mU), and the segregation was observed in its T2 generation (Figure 4). Of total 40 T2 plants tested, only four were verified to have activity similar to their T1 transformants, and the T3 progenies from these 4 T2 plants were also observed to have activity similar to their parents (data not shown). In addition, nine more were detected to express the minimal xylanase activity after 12 h of incubation, versus 1 h for the standard RBB-Xylan assay.

Discussion

This study demonstrated that the xylanase gene cxy1 can be efficiently transformed into A. thaliana by Agrobacterium infection with our modified Floral Dip method. Most of our transformants had one or two copies of T-DNA inserts with the 3R:1S or 15R:1S segregation ratios, exhibiting Mendelian inheritance in their T2 and T3 populations. For example, pMB4/cxy1-2 transformant was shown to have one copy of the T-DNA insert in the A. thaliana genome. However, the 2R:1S and 11R:1S segregation ratios were also observed in the T2 and T3 progenies of some pMB4 or pMB4/cxy1 transformants (Table

Lu et al. — Coniothyrium minitans xylanase in Arabidopsis

3). Such an exceptional proportion of herbicide susceptible progenies in transgenic plants has been reported by other investigators (Feldmann and Marks, 1987; Valvekens et al., 1988; Errampalli et al., 1991; Katavic et al., 1994; Feldmann et al., 1997; Bonhomme et al., 1998; Howden et al., 1998; Harbord et al., 2000), and may be due to the instability of the inserts, the loss of expression of the resistance gene, or the induction of a recessive mutation for gamete or embryo lethality. In our study, the insertions of T-DNA likely interrupted a gene that is important or essential in plant development; therefore, the homozygous resistant genotypes couldn't survive, resulting in the 2R:1S or 11R:1S distortion ratios.

The 1R:1S ratio is more common for gametophytic lethal in either male or female gametes of A. thaliana transformants (Howden et al., 1998); however, other distorted ratios were also reported in previous studies (Feldmann and Marks, 1987; Katavic et al., 1994; Bonhomme et al., 1998). In this study, segregation of the 1R:2S ratio was unexpected in the T2 and T3 progenies of pMB4-2 transformant (Table 3, 4). Since the herbicide resistance can be inherited in its T2 and T3 generations, the pMB4-2 transformant seems to behave as a stable insertion. In addition, as resistant individuals had the T-DNA insert and susceptible ones didn't in their T3 progenies (Figure 2), loss of bar gene expression is an unlikely cause. Based on genetic and molecular studies, we propose that only one copy of T-DNA was integrated into A. thaliana pMB4-2 transformant, and the insertion likely interrupted a gene important or essential to its gametophytic development. This insertion event could have resulted in a reduction of transmission (Bonhomme et al., 1998; Howden et al., 1998). It is possible that the homozygous resistant genotypes in its T2 and T3 progenies couldn't survive, and the gametophytic development in this transformant may favor the gametes without the T-DNA insert, resulting in the 1R:2S segregation distortion. Further study through reciprocal backcrosses with wild A. thaliana plants can confirm this unequal gamete transmission through both micro- or mega-gametophytes.

The genetic analysis of the pMB4/cxy1-6 transformant indicated that two copies of T-DNAs were inserted into the A. thaliana genome independently, in which the resistant homozygote on one locus was non-viable. If X refers to one T-DNA insertion and Y refers to another independent insertion, and the X insertion is lethal to A. thaliana growth, the T1, T2, and T3 genotypes of pMB4/cxy1-6 can be predicted as follows:

Theoretically, a total of 11 resistant genotypes in the T2 population can produce 3/11 T3 populations with all resistant progenies, 4/11 T3 populations with an 11R:1S ratio, 2/11 T3 populations with an 3R:1S ratio and 2/11 T3 populations with an 2R:1S ratio. The observed segregation of the pMB4/cxy1-6 T2 progenies was observed to fit the 11R:1S ratio (Table 3). In 20 pMB4/cxy1-6 T3 populations derived independently from 20 T2 progenies, 7 were observed to have all resistant progenies, 5 were segregated with an 11R:1S ratio, 4 with a 3R:1S ratio, and 4 with a 2R:1S ratio (Table 5). Therefore, observed segregation of the pMB4/cxy1-6 T2 and T3 progenies support the hypothesis for the above genotypic model.

It is a common observation that not all T-DNA insertions are expressed in plants, and in most cases only one copy of the multiple inserted T-DNAs seems to be functional (Hobbs et al., 1990; Chang et al., 1994; McCabe et al., 1999). Herbicide resistance was used to screen and select the pMB4 or pMB4/cxy1 transgenic genotypes in this study; therefore, the bar gene should have been functionally expressed in all our transformants. However, xylanase activity was only detected in two out of six pMB4/cxy1 transformants, and cxy1 expression obviously varied among T2 progenies of pMB4/cxy1-6 transformant (Figure 4) though PCR amplification confirmed the presence of cxy1 gene in all pMB4/cxy1 transformants (Figure 3). The genetic analyses in this study indicated that pMB4/cxy1-6 had two copies of T-DNA inserts with one locus non-viable for resistant homozygotes; therefore, the segregation for xylanase activity in the T2 generation suggested that the cxy1 gene may be expressed from only one T-DNA insert and that homozygous resistance on this locus was non-viable. The lack of expression of the cxy1 gene, isolated from the fungus C. minitans, in A. thaliana may be due to a difference in codon usage. The cxy1 silencing in most of our transformants is likely due to the gene hyper-methylation (Matzke and Matzke, 1990) or to T-DNA rearrangement, deletion, or mutation (Errampalli et al., 1991) in the A. thaliana genome. Further studies to identify the insertion patterns of pMB4/cxy1 transformants are warranted in order to establish the inheritance of the cxy1 expression in A. thaliana genome.

Pathogenesis-related (PR) proteins are reported to be effective sources for plant defenses under both biotic and abiotic stresses (Yun et al., 1997). As a PR-like protein, our xylanase gene cxy1 was isolated from a special mycoparasite C. minitans (Laroche et al., 2000) and has now been expressed in a higher plant genome, providing a basis for investigating transgenic Arabidopsis plants for antifungal resistance to S. sclerotiorum and other plant pathogens. In addition, our Arabidopsis transformants will be useful in the analysis of cxy1 function and xylan hydrolysis in higher plants since little xylanase activity was detected in the original Arabidopsis plants (ecotype Columbia). The results reported in this study make it possible to use the transgenic Arabidopsis as a model plant to further study the degradation of xylan in higher plants for improvement of forage utilization in animal production.

Botanical Bulletin of Academia Sinica, Vol. 45, 2004

Acknowledgments. We would like to thank Dr. F. Eudes (Lethbridge Research Centre, Agriculture and Agri-Food, Canada) for supplying the A. tumefaciens strain and vector for this study.

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Lu et al. — Coniothyrium minitans xylanase in Arabidopsis