Journal of Experimental Botany, Vol. 54, No. 384, pp. 1101-1111,
March 1, 2003
© 2003 Oxford University Press
Greenhouse and field testing of transgenic wheat plants stably expressing genes for thaumatin-like protein, chitinase and glucanase against Fusarium graminearum
Received 2 September 2002; Accepted 2 December 2002
1 Department of Biochemistry, Kansas State University, 104 Willard Hall, Manhattan, KS 66506, USA
2 Department of Plant Pathology, Kansas State University, Manhattan, KS 66506, USA
3 To whom correspondence should be addressed. Fax: +1 785 532 7278. E-mail: smk{at}ksu.edu
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Abstract |
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Genes encoding pathogenesis-related (PR-) proteins isolated from a cDNA library of Fusarium graminearum-infected wheat spikes of scab-resistant cultivar ‘Sumai-3’ were transformed into susceptible spring wheat, ‘Bobwhite’ using a biolistic transformation protocol, with the goal of enhancing levels of resistance against scab. Twenty-four putative transgenic lines expressing either a single PR-protein gene or combinations thereof were regenerated. Transgene expression in a majority of these lines (20) was completely silenced in the T1 or T2 generations. Four transgenic wheat lines showed stable inheritance and expression of either a single transgene or transgene combinations up to four generations. One line co-expressing a chitinase and ß-1,3-glucanase gene combination, when bioassayed against scab showed a delay in the spread of the infection (type II resistance) under greenhouse conditions. This line and a second transgenic line expressing a rice thaumatin-like protein gene (tlp) which had moderate resistance to scab in previous greenhouse trials, along with susceptible and resistance checks were evaluated for resistance to scab under field conditions. None of the transgenic lines had resistance to scab in the field under conditions of strong pathogen, suggesting these plants lacked effective resistance to initial infection (type I resistance) under these conditions. As far as is known, this is the first report of field evaluation of transgenic wheat expressing genes for PR-proteins against disease resistance.
Key words: Chitinase, field evaluation, genetic transformation, ß-1,3-glucanase, transgene silencing, wheat scab.
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Introduction |
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Fusarium head blight (FHB) or scab, caused by Fusarium graminearum Schwabe (teleomorph Gibberella zeae) is a major disease of wheat in many areas of wheat production in USA and throughout the world (Bai and Shaner, 1994; Parry et al., 1995; McMullen et al., 1997). Under favourable conditions this disease can cause losses of up to $1 billion per year (McMullen et al., 1997). Host resistance and cultural practices over a long time have been used to manage the disease with only moderate success. Effective control of FHB will require better knowledge of the epidemiology of the disease and the use of the modern tools of genetic engineering (Parry et al., 1995; Shah, 1997; Shaner and Buechley, 1999; Patnaik and Khurana, 2001). One strategy is to enhance the natural defences of wheat for disease control. The transcripts levels for many pathogenesis-related (PR-) protein genes were shown to be increased upon scab infection of wheat suggesting their importance in plant defence (Li et al., 2001; Pritsch et al., 2000, 2001). Different classes of PR-proteins including PR-1, PR-2 (ß-1,3-glucanases), PR-3 (chitinases), PR-5 (thaumatin-like protein), and PR-9 (peroxidases) were induced within 6–12 h of inoculation and reached peak levels within 36–48 h after inoculation. Similarly, a 3-fold increase in the polyphenol oxidase (PPO) activity was detected in the resistant wheat cultivars upon inoculation with F. graminearum conidia when compared with the non-inoculated controls (Mohammadi and Kazemi, 2002).
The availability of genes encoding PR-proteins and the demonstration that PR-proteins exhibit strong in vitro anti-fungal activity (Mauch et al., 1988) has led to their deployment for enhancing disease resistance in crop plants. Several groups have reported that the introduction of a single transgene encoding different antimicrobial proteins including PR-proteins in wheat resulted in enhanced resistance (as measured in greenhouse trials) to a wide range of disease resistance including powdery mildew (Bliffeld et al., 1999; Schweizer et al., 1999; Bieri et al., 2000; Oldach et al., 2001), Tilletia tritici (Clausen et al., 2000), barley stripe mosaic virus (Zhang et al., 2001), scab (Chen et al., 1999), and other fungal pathogens (Leckband and Lorz, 1998). The increase in resistance to the pathogen varied widely and in most cases resistance was only partial.
Following the initial reports of significant improvement in disease resistance in transgenic plants expressing PR-proteins (Broglie et al., 1991; Zhu et al., 1994; Jach et al., 1995; Jongedijk et al., 1995; Lin et al., 1995) there have been numerous studies that have exploited a similar strategy (reviewed in Datta et al., 1999). The results have been mixed with some notable failures (Neuhaus et al., 1991). The current consensus is that combinations of PR-proteins are required to achieve effective disease control.
This paper reports the production of four fertile transgenic wheat lines which stably and constitutively express one or both of two PR-protein genes (for a chitinase and a ß-1,3-glucanase). As will be described, among the different combinations of genes for PR-proteins tested, only a line expressing a rice thaumatin-like protein and a specific combination of a wheat chitinase and a wheat glucanase had partial resistance to scab in greenhouse evaluations. However, field trials of the same lines failed to show any improved resistance under heavy inoculum loads. This is the first report of the field evaluation of transgenic wheat plants expressing PR-proteins.
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Plasmids
The plasmid constructs pAHCUbi194 and pAHCUbi383 (containing wheat chitinase genes) and pAHCUbi289 and pAHCUbi638 (containing wheat ß-1,3-glucanase genes) were used to transform wheat calli. The clones encoding the chitinases and ß-1,3-glucanases used in this study were isolated from a cDNA library constructed from scab-infected Sumai-3 wheat (Li et al., 2001). These cDNAs, a 2.0 kb fragment containing the maize ubiquitin promoter-intron and another 0.25 kb fragment containing the cauliflower mosaic virus transcription terminator (CaMV polyA) fragment were ligated to pBluescript vector. The HindIII fragments carrying the promoter–cDNA–CaMV polyA cassette were recovered and inserted into the unique HindIII site of the plasmid pAHC20 (Christensen et al., 1992). The plasmid pAHC20 contains the bar gene under the control of the maize ubiquitin promoter-intron. The bar gene confers resistance to the herbicide glufosinate (Liberty®, Aventis, Research Triangle Park, NC). The plasmids, pAHCUbi194 and pAHCUbi383, contain the opening reading frames (ORFs) from the two wheat cDNA clones 194 and 383 encoding a class VII and a class IV chitinase, respectively (Fig. 1A, B). Plasmids pAHCUbi289 and pAHCUbi638 (Fig. 1C, D) contain the ORFs from the two wheat glucanase genes 289 and 638, respectively (Li et al., 2001). These plasmids were used singly or in different combinations for wheat transformation.
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Plant materials and transformation
Wheat plants (Triticum aestivum L. cv. ‘Bobwhite’) were grown in growth chambers with a 16 h light period, at a light intensity of 600 µE m–2 s–1 and a temperature regime of 20:18 °C (light:dark). Spikes were collected approximately 14 d post-anthesis and immature caryopses were sterilized in a solution containing 20% commercial bleach and 0.1% Tween 20 for 20 min and then rinsed three times with sterile, distilled water. Immature embryos were excised and placed on CM4 medium (Zhou et al., 1995), with the embryo-side in contact with the medium, for 5–7 d in the dark at 25 °C to initiate callus formation. Embryogenic calli were transferred to CM4 + osmoticum (0.2 M mannitol, 0.2 M sorbitol) 4–16 h prior to genetic transformation. Genes were introduced into embryogenic calli with the particle inflow gun (Finer et al., 1992).
The methods for selection and recovery of transgenic plants were similar to those of Altpeter et al. (1996) with slight modifications. Sixteen hours after particle bombardment, wheat calli were placed on CM4 medium containing 5 mg l–1 glufosinate and cultured in the dark at 25 °C for 8–10 d. Cultures were then transferred to shoot production medium (MSP), containing MS (Murashige and Skoog, 1962) basal salt mixture, B5 vitamins (Gamborg et al., 1968), 0.2 mg l–1 2,4-D, 40 g l–1 maltose, 100 mg l–1 ascorbic acid, 10 mM MES, 5 mg l–1 glufosinate, and 2.0 g l–1 Gelrite (pH 5.7) and cultured in a 16 h light period at 25 °C for 2 weeks. Upon induction of green shoots, the cultures were transferred to elongation and rooting medium (MSE) containing 5 mg l–1 glufosinate and cultured for 2–3 weeks in the light (MSE medium is the same as MSP except for the omission of 2,4-D). Healthy looking plantlets were transferred to soil and grown in environmentally controlled growth rooms (16 h light periods, at a light intensity of 600 µE m–2 s–1).
Leaf painting assay
To examine the expression of the bar gene in the transgenic plants, a freshly prepared aqueous solution of the herbicide, Liberty® (0.2% v/v) was applied on the mid-lamina portion (about 2.5 cm long) of the second/third youngest leaf using a cotton plug. The painted area was marked using a marker pen and visual observations were recorded 3–4 d after painting.
Molecular characterization of the transgenic plants
Plant materials and methods of protein extraction, SDS-polyacrylamide gel electrophoresis and western and Southern blot analyses were carried out as described in Chen et al. (1998). For western blot analysis, polyclonal antibodies raised in rabbits against a barley chitinase (Swegle et al., 1992) and a barley ß-1,3-glucanase (a gift from Dr Murray Ballance, University of Manitoba, Winnipeg) respectively, were used. The antibodies were used at 1:1000 (v/v) dilutions. For RNA blot hybridization, total RNA was extracted from 150 mg leaves or spikes with TRIZOL® reagent (Gibco BRL, Grand Island, NY) according to the manufacturer’s instructions. Aliquots of RNA (10 µg) were analysed by formaldehyde-agarose gel electrophoresis and blotted on to Hybond-N+ nylon membranes (Amersham, Arlington Heights, IL). Membranes were probed with 32P-labelled coding region fragments of the wheat chitinase or glucanase genes. The other steps for RNA hybridization were similar to those for DNA hybridization (Chen et al., 1998). Reverse-transcriptase (RT-) PCR reactions were performed with 0.5 µg of total RNA using the Advantage One-Step, RT-PCR kit (Clonetech, Palo Alto, CA) as per the manufacturer’s instructions. For distinguishing the transgene-specific transcripts, a forward primer in the first exon of the ubiquitin promoter, 5'-CGTGTTGTTCGCAGCGCACAC-3', and a reverse primer in the CaMV polyA fragment 5'-GCTCAACACATGAGCGAAACCC-3' (Fig. 1), were used in the RT-PCR reaction. The reverse transcriptase reactions were performed in a thermal cycler at 50 °C for 1 h, followed by PCR amplification of 15–25 cycles at 94 °C for 0.5 min, 64.7 °C for 0.5 min and 1 min at 72 °C. A complete final extension for the PCR products was performed at 72 °C for 10 min. A semi-quantitative competitor RT-PCR assay (Igaz et al., 1998) using concentrations ranging from 10 pg to 80 pg of plasmid DNA used for transformation as the competitor were performed to quantify the transgene-specific mRNAs. After the reverse transcriptase reaction the competitor DNA were added into the reaction mixture and PCR amplified for 15 cycles. The transgene-specific transcripts ranged in sizes from 1.1 kb to 1.4 kb for the different cDNA clones, which were resolved on 1.6% w/v agarose gels.
The detection of the bar gene and genes of interest by PCR amplification was carried out using 200 ng of total genomic DNA as template and BIOLASETM DNA polymerase (Bioline, Springfield, NJ) as per the manufacturer’s instruction using the following primer combinations; Bar F, 5'-CCTGCCTTCATACGCTATTTATTTGCC-3' (forward primer); Bar R, 5'-CTTCAGCAGGTGGGTGTAGAGCGTG-3' (reverse primer) and Ubi A, 5'-GCCCTGCCT TCATACGCTAT-3' (forward primer in the intron of ubiquitin promoter) and PolyA-R, 5'-GGAATTCAAGCTTCATCGAGCTCGGTA-3' (reverse primer in the CaMV polyA fragment). DNA amplifications were performed in a thermal cycler using initial denaturation at 94 °C for 5 min, followed by 30 cycles of 1.0 min at 94 °C, 1.5 min at 62.1 °C (for bar gene)/62.4 °C (genes of interest) and 2.0 min at 72 °C. One additional complete extension cycle was performed for 10 min at 72 °C. The transgene products ranged in sizes from 0.6 kb, 1.1–1.4 kb for the bar gene and different cDNA clones, which were resolved on a 0.8% or 1.4% w/v agarose gel and visualized by ethidium bromide staining.
Scab bioassay
Resistance to scab in greenhouse-grown plants was evaluated by a ‘single floret injection inoculation method’ (Xu and Chen, 1993) with the indicated number of plants. Two plants of each transgenic line and of the controls were transplanted per one gallon (8-inch) pot. A 4' highx4' widex10' long, plastic-covered, mist chamber was constructed on a greenhouse bench. A single central floret of the spikelets of wheat entries were inoculated at anthesis with 10 µl of a conidial suspension of isolate Z-3639 of Fusarium graminearum. The pots were randomly picked for each entry at anthesis and inoculated with the conidial suspension. In the first experiment, the conidial suspension contained 1.6x104 conidia ml–1 (a minimal concentration was used in the initial experiment for standardizing the inoculum procedure and detection of the disease), while in the second and third experiments, the conidial suspension contained 5x105 conidia ml–1. New sets of plants at similar physiological stage of anthesis were picked randomly on three different days and injected with the same inoculum. A minimum of six heads per entry was inoculated on each day. Plants were then placed overnight in the mist chamber with 15 s of mist every 12 min. The chamber was opened during the day and misted each night for a total of three nights. The plants were then placed in a greenhouse. The infected spikelets per inoculated head were counted 7, 10 and 14 d after inoculation. The percentage of infected spikelets per head was also calculated during the same period. Data collected from different dates were analysed separately by analysis of variance (ANOVA) with mean separation by LSD (P=0.05) using Statistical Analysis System (SAS Institute, 1998).
Field testing of transgenic and control lines were carried out in spring 2002 at the Plant Pathology Experimental Farm located near Manhattan, KS, USA. A randomized complete block design was used with 20 replicates for each treatment. Each plot consisted of a ‘hill’ separated in all directions by 45 cm. One to three plants of each entry were grown in the greenhouse in 15 cm pots for about 3 weeks and then transplanted to the field on March 15 (one pot per ‘hill’). Corn kernels (93 g m–2) colonized by F. graminearum were applied to the soil on April 1. To provide head wetness, impact sprinklers irrigated the area during anthesis for 3 min h–1 from 21.00–06.00 h. When scab began to develop, the percentage of spikelets affected was rated for each hill at 3 d intervals for about 2 weeks. Data collected from different dates were analysed separately by analysis of variance (ANOVA) with mean separation by LSD (P=0.05) using Statistical Analysis System (SAS Institute, 1998).
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Results |
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Transformation and characterization of T0 plants
Twenty-four independent, fertile, primary transgenic lines with the selectable marker bar gene and single PR-protein gene or gene combinations were identified. PCR analysis using gene-specific primer combinations designed for detection of the bar gene (data not shown) and PR-protein genes was used for characterization of the 24 transgenic lines (results from a few T1 plants transformed with single PR-protein gene or their combinations are shown in Fig. 2). Four transgenic lines contained only the 383 chitinase and three had only the 638 glucanase gene, while the remaining 17 lines came from the co-bombardment of three different PR-protein pairs (194:383, 383:638 and 289:383) of transgene constructs (Table 1). PCR analysis for the genes of interest in 17 lines from the multiple plasmid bombardment experiments detected the presence of all the expected transgenes in all lines except two that had one plasmid (194 chitinase or 638 glucanase). High co-transformation efficiency (15 out of 17) in the transgenic wheat plants could thus be demonstrated.
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Western blot analyses were subsequently carried out with extracts from leaves of these T0 transgenic plants for detecting the transgene-encoded proteins using appropriate controls including the escapes (bar-minus plants) and untransformed parental lines. All 24 lines (T0 plants) were positive for the bar gene by PCR and had expression (to varying extents) of either one or two transgenes as expected from the PCR results (data not shown).
Progeny analyses of transgenic plants
Ten to 20 seeds from each T0 generation plant were germinated for the 24 transgenic lines containing the bar gene. The leaf painting assay and PCR analyses of the genes of interest were performed to detect the segregation of Liberty resistant/sensitive and genes of interest (plus/minus) plants. T1 progeny of 18 lines were completely susceptible to Liberty even though a majority (about 75%) had the bar gene and transgene as revealed by PCR. The presence of expected transgenes was confirmed (in most, but not in all cases) by carrying out PCR analysis of genomic DNA as templates using Ubi A and PolyA-R primers which yielded PCR-fragments of distinct sizes for each of the four transgenes (Fig. 2). Progenies from only six lines survived painting with Liberty. The segregation patterns for Liberty resistance/susceptibility of T1 plants from these six lines were consistent with one or two transgene loci (segregation ratio of 3:1 or 15:1, Table 2). Results of PCR analyses for the bar gene and genes of interest, as well as the data from RT-PCR, Southern blotting and western blot analysis for transgene-encoded proteins were also consistent with the presence of one or two transgene loci (data not presented). The 18 Liberty-sensitive lines had no detectable transgene-encoded proteins as revealed by western blot analyses, presumably resulting from silencing of bar and transgenes. These results indicated that a vast majority of the transgenic lines had undergone transgene inactivation in the T1 generation.
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Transgene silencing persists even in the advanced generations in some lines
All the six Liberty-resistant lines expressing the transgenes in the T1 generation showed co-segregation for both the bar gene and the gene(s) of interest (Table 2). The T1 plants expressing the highest levels of transgene-encoded protein(s) were selected by western blotting from each of the six lines and selfed to obtain T2 progenies. Liberty painting, PCR for transgenes, RT-PCR, and western blotting were used for the analyses of progeny of subsequent generations to determine whether these lines stably expressed the transgene(s). When compared to their T1 parents, T2 progenies from two of these six lines (Nos 26 and 62) had very low levels of transgene-encoded protein and transcripts as revealed by RT-PCR and western blotting. Presumably, additional transgene silencing has occurred in the T2 generation plants. In the T3 generation, no evidence for a functional transgene was found even though the transgenes were detected by PCR in these two silenced transgenic lines.
Generation of lines homozygous for transgene loci
For the identification of homozygous lines, seeds were collected from all the T2 plants of the four lines that showed high levels of transgene expression and from line 26 that had low activity of transgenes. Analyses of seedlings of the T3 generation of these transgenic lines (Nos 26, 32A, 76, 78, and 82), revealed that the progeny from the plant 32A-2–4 were homozygous. T3 progeny from T2 parents of the remaining four lines showed segregation in leaf painting assays and western blot analyses for protein expression for the transgenes. Homozygous T3 parents were identified by analyses of T4 generation plants for the four lines (Nos 26, 76, 78, and 82). As discussed in the last section, only low levels of transgene-encoded protein were detected in the T3 progenies of line 26, and this line was completely silenced for the transgenes in homozygous T4 progeny (data not shown).
Detection of transgenes in homozygous lines
Genomic DNA from representative T3 (or T4) generation plants was digested with HindIII (which releases the fragment containing the intact ubiquitin-promoter linked to PR-protein transgene from the 5.7 kb vector-bar gene fragment; Fig. 1) and subjected to Southern blotting using a bar probe or PR-protein cDNA probes. The six lines had distinct patterns of transgene bands (except 32A and 32C which had identical banding patterns for four different enzyme digests; data not presented). The estimation of the transgene copy number using known amounts of transforming plasmids as standards ranged from 3 (line 32A) to over 10 (line 76) for the intact bar gene, in addition to rearranged copies (Fig. 3A, B). Even though it was difficult to estimate the copy number of the PR-protein genes accurately, because of the presence of endogenous homologous genes in wheat (Fig. 3C), comparison of the intensities of the 3.2 kb band (containing the intact ubiquitin promoter-PR protein gene-polyadenylation signal sequences) and upon digestion with different enzyme combinations indicated that copy numbers also ranged 3–10 (data not presented).
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Transgene expression in the homozygous lines
Detection of transgene-encoded chitinase and glucanase was carried out using appropriate polyclonal antibodies. A low level of 26 kDa (expected size) of 383 chitinase protein was detected in the non-transgenic control and line No. 82. In the heterozygous T2 plants of line No. 26, and in the homozygous plants of lines 32A, 76 and 78 the intensity of this 26 kDa chitinase band was higher. Line No. 32A had the highest levels of 383 protein followed by line No. 78 (Fig. 4A). With the ß-1,3-glucanases antibody, multiple bands within the size range of approximately 30–32 kDa (expected size range for 638 and 289 glucanases) was detected in the transgenic lines 32A, 76, 78, and 82 (basal level expression was detected in line 26 and non-transgenic controls). The highest levels of 638 protein were seen in line 32A followed by line 82 (which had no chitinase transgene expression). Even though the predicted size difference between the two transgenic glucanase proteins is 2 kDa, the presence of multiple bands precluded unambiguous identification of 638 and 289 glucanases in these blots (Fig. 4B).
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The highest levels of both transgene-encoded proteins were found in line 32A which was transformed with the 383 chitinase and 638 glucanase genes. Transgene-specific mRNA levels in this line were estimated using a semi-quantitative competitor RT-PCR assay (Igaz et al., 1998) using concentrations ranging from 10–80 pg of the plasmid DNA used for the transformation as the competitor. The intensities of the PCR bands derived from the transgenes were compared to those derived from the plasmid (which includes the intron) using the BioRad gel documentation unit. Based upon these observations it is predicted that about 0.8% and 0.25% of the total mRNA (assuming mRNA is 1% of the total RNA and the amplified transcripts are close to the average sizes of mRNA) came from the contributions of 638 glucanase and 383 chitinase transgenes (Fig. 5). This line contained approximately three copies of each of the transgenes (Fig. 3C; and data not shown). RT-PCR using 0.5 µg of total RNA from line No. 32A yielded strong PCR bands for the transgene specific transcripts within 15 cycles. However, in all the other lines using similar amounts of total RNA, 20–25 cycles of PCR following the reverse transcription were required to amplify and detect the transgene transcripts. Northern blot analyses also confirmed the expression of both the chitinase and glucanase genes in all the four expressing lines (data not shown).
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The T3 and T4 generation plants of this line (32A) that were homozygous for the transgene locus had a lesion-mimic phenotype. These symptoms developed at the booting stage and appeared to be dependent on the accumulation of high levels of the transgene expression, since heterozygous plants from this line did not have these lesions. The plants derived from the backcross with the parental line, ‘Bobwhite’, which are heterozygous for the transgene loci did not show this phenotype. The stable inheritance and expression of the transgenes was confirmed by the leaf painting assay, PCR and western blotting in heterozygous plants.
Scab bioassay and disease resistance
Three trials were conducted to evaluate the resistance of transgenic lines to scab under greenhouse conditions. In the first set of experiments, two heterozygous T2 lines, line 26 (low expression for 383 chitinase), line 32A (high expression for both 383 chitinase and 638 glucanase) and susceptible check ‘Bobwhite’ plants were tested. In the second set of experiments, homozygous T3 plants of lines 32A, 32C (genetically identical to line 32A, but with no expression for 383 chitinase and 638 glucanase, used as an epigenetic control), a resistant check line, MN99112 (an experimental line received from Dr James Anderson, University of Minnesota, with resistance from Wuhan 3 wheat) and ‘Bobwhite’ were bioassayed in the autumn of 2001. In the third experiment, homozygous T4 plants of lines 76, 78 (moderate expression for both 383 chitinase and 289 glucanase) and line 82 (moderate expression 638 glucanase) with the resistant check (MN99112) and ‘Bobwhite’ were bioassayed against scab in the spring of 2002. Symptoms characteristic of scab appeared on the spikes of transgenic and control plants in the greenhouse on the third day after inoculation with a conidial suspension. In the first greenhouse experiment, the segregating population of T2 progenies of lines 32A and 26 were compared with the control non-transgenic parental line (Table 3). The conidial density used in this experiment (1.6x104 conidia ml–1) resulted in moderate infection. Hyphal growth in the inoculated spikes could be detected within 3–4 d after inoculation (DAI). The mean number of infected spikelets in line 32A was one-third to half the number in line 26 and the susceptible ‘Bobwhite’ control on 10 DAI. The mean number of infected spikelets/head were also found to be still significantly lower (3.6) in line 32A by 14 DAI, when compared to the susceptible check (9.8) or line 26 (11.5). Since no significant differences in disease severity were observed between line 26 and the non-transgenic controls and because the plants homozygous for the transgene loci in this line were completely silenced, line 26 was excluded from further bioassay analyses.
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In the second greenhouse experiment (Table 4A), the resistant check MN99112 had the lowest levels of disease as expected. The number of infected spikelets/head on 10 and 14 DAI in line 32A (3.7 and 7.2) and the resistant check (2.0 and 4.2) were significantly lower than the values for the silenced line 32C and the non-transgenic controls (Table 4A). A delay in both the onset and progression of the infection was seen in line 32A when compared with the susceptible control. The greater disease incidence and faster spread in line 32C, which is genetically identical to line 32A indicates that the observed increase in disease resistance is dependent on transgene expression and is not due to the tissue culture-related effects.
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In the third greenhouse experiment, homozygous T4 plants from three other lines; lines 76 and 78 which express both the chitinase and ß-1,3-glucanase transgenes at moderately high levels, and line 82 which expresses only a ß-1,3-glucanase at a moderately high level, did not show any improved resistance when compared to the susceptible control (Table 4B). The resistant check MN99112, showed minimal infection.
Field evaluation of two transgenic lines
The following treatments were included in this experiment: homozygous T4 plants of line BT-14-18 [(transgenic line expressing the rice tlp transgene) (Chen et al., 1999)]; line 32A; line 32C; non-transgenic ‘Bobwhite’ control plants; MN99112 (resistant check); Scab-7 (resistant check); and Wheaton (susceptible check). Field evaluation of lines 32A and BT-14-18 did not show any improved resistance. The disease ratings for these lines were similar to, or higher than, the non-transgenic ‘Bobwhite’ and susceptible controls (Table 5). A significant reduction in disease incidence was recorded on the third day rating in line 32A. However, by the sixth day of disease rating, the disease severity in the transgenic lines was either similar to or higher than the susceptible controls. Disease developed early and was severe in the transgenic lines and the ‘Bobwhite’ control (parental line) when compared with the susceptible check (Wheaton). The resistant check MN99112 had the lowest disease incidence. Another experimental line, Scab-7 also had low scores until day 14 when disease severity was similar to the susceptible check Wheaton (Table 5).
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Discussion |
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Resistance to scab is usually inherited as a quantitative trait. Several chromosomes and QTLs are known to influence this resistance reaction (Bai and Shaner, 1994; Anderson et al., 2001), indicating that multiple genes affect the resistance. This finding is further supported by the induction of different PR-proteins including PR-2, PR-3, PR-4, PR-5, and other PR-genes within 12–24 h after initiation of infection (Li et al., 2001; Pritsch et al., 2001). However, there is no direct evidence to support the functional role of these genes in defence against scab. The aim of the present study was to introduce single PR-protein genes or gene combinations identified from a scab-infected Sumai-3 library (Li et al., 2001) into a susceptible wheat background and characterize their efficiency in defence against scab.
Previous studies involving genes-encoding PR-proteins for enhancing resistance to scab yielded transgenic lines over-expressing a rice tlp gene (PR-5) that showed a delayed resistance to spread in the head (type II resistance, Chen et al., 1999). This promising result encouraged the identification of candidate genes from a scab-resistant genotype (Sumai-3) and to transform wheat with these genes in order to enhance the levels of resistance to scab further in wheat. The major obstacle to this approach was encountered in generating enough lines with stable inheritance and expression of the transgenes. A majority of the lines underwent transgene-silencing at different generations. Of the 24 independent transgenic lines identified, only four lines (about 20%) stably expressed the transgenes in the T3 generation. This report contradicts an earlier conclusion that ubiquitin promoter-driven genes are stably expressed over several generations, whereas CaMV promoter-driven genes were silenced in T1 or subsequent generations (Chen et al., 1998, 1999). The studies reported here indicate that the maize ubiquitin promoter-intron is also prone to random gene silencing in transgenic wheat plants, even though the frequency of gene silencing is somewhat less compared to genes under the control of the CaMV promoter.
Similarly high frequency of co-integration (90%) was detected in 15 of the 17 independent transgenic lines when co-bombarded with multiple plasmids. These results are in accordance with earlier reports on co-transformation of wheat with multiple genes (Barro et al., 1997; Bliffeld et al., 1999). There are previous reports on the co-expression of chitinase and ß-1,3-glucanase genes in tobacco (Jach et al., 1995; Zhu et al., 1994) and tomato (Jongedijk et al., 1995) which are dicotyledonous plants that are more amenable to genetic transformation than is wheat. This is the first report of stable co-expression of two different PR-protein genes (a chitinase and a ß-1,3-glucanase gene) in transgenic wheat plants. Earlier attempts to co-express multiple PR-proteins for disease resistance in wheat resulted in the expression of only a single gene (Bliffeld et al., 1999; Chen et al., 1999; Schweizer et al., 1999). The accumulation of the two PR-protein genes in combination had no deleterious phenotypic effect on the plants, except for line No. 32A. This line, which had the highest levels of both chitinase and ß-1,3-glucanase, was normal up to the T2 generation when the transgene locus was still segregating. However, when this line was rendered homozygous for the transgene loci, expression of the transgene-encoded proteins reached the highest levels and the plants developed a lesion mimic phenotype. This line grew normally in the vegetative phase, but at the booting stage developed necrotic spots that appeared on the second and third leaves from the flag leaf and later spread throughout the plant. The homozygous plants from this line were backcrossed with the parental line ‘Bobwhite’ to determine whether the lesion mimic phenotype was dependent on ‘gene-dosage’. The heterozygous plants derived from the backcross, did not develop the lesions and had a normal seed set.
Earlier attempts with expression of ß-1,3-glucanase gene in wheat resulted in the failure to recover transgenic plants (Bliffeld et al., 1999). It was proposed that constitutive expression of ß-1,3-glucanase interfered with plant regeneration and normal development of transgenic wheat plants. All the lines, including line 32A, set normal spikes (no lesions) with viable seeds. Lines 76 and 78 co-expressing the 383 chitinase and 289 glucanase at moderate levels, line 82 expressing the 638 glucanase only, and the heterozygous plants from line 26 expressing the 383 chitinase had normal phenotypes.
The availability of only four lines with stable expression of transgenes limited the number of combinations of PR-protein genes that could be assayed for their contributions to defence against scab. Among these four lines, only the line with the highest level of expression of 383 chitinase and 638 glucanase had a significant increase in scab resistance as measured in greenhouse trials. The other lines, with lower levels of expression of both transgenes and one line expressing only a ß-1,3-glucanase showed no improvement in disease resistance. Thus, it appears that a combination of chitinase and ß-1,3-glucanase genes is necessary to enhance host resistance to scab. Even then transgene expression must approach the levels seen in the heterozygous plants of line 32A because lines 76 and 78 which had lower levels of both chitinase and glucanase were quite susceptible to scab.
It was expected that plants homozygous for the transgene loci with higher levels of expression would have greater resistance to scab than heterozygous plants. However, disease severity was similar in both heterozygous and homozygous plants of line 32A (Tables 3, 4A) relative to the Bobwhite control. This may be the maximum level of protection that can be achieved, possibly because these enzymes only delay the spread of disease but do not eliminate it.
The greenhouse studies reported in this study are similar to the protective synergistic interactions of chitinase/glucanase as antifungal defences in vitro reported by other groups (Leah et al., 1991; Mauch et al., 1988) and in vivo studies with transgenic tobacco, tomato and carrots (Zhu et al., 1994; Jach et al., 1995; Jongedijk et al., 1995; Melchers and Stuvier, 2000). To improve the protective effects of over-expression of PR-protein genes, there are ongoing studies for combining different antifungal protein genes via crosses of homozygous expressing parents with different combinations of PR-protein and other antifungal genes. For example, crosses have been carried out between the moderately resistant tlp transgenic lines (Chen et al., 1999) with line No. 32A for pyramiding all three PR-protein genes into one line and to evaluate the interaction of these PR-protein genes as antifungal defences.
The two lines that were identified as moderately resistant to scab under greenhouse conditions (No. 32A and BT-14-18) did not show any improved resistance to scab when bioassayed against scab in a scab field nursery. No significant reduction in symptoms was observed except for line No. 32A on day 3. But on day 6 and beyond, the transgenic plants were overwhelmed by the disease. These differences between the results of greenhouse studies versus field evaluations are attributed to the differences in the inoculation techniques employed. In the greenhouse studies, a conidial suspension (104–105 conidia ml–1) was injected into the young spikelets after anthesis as a single application. On the other hand, in the field inoculation, the corn kernels provide a continuous inoculum of ascospores, which land on the spikes, germinate and infect the spikes under conducive environmental conditions. The infection process is continuous and emerging spikes get infected at different stages and ages. Therefore, greenhouse assays screen for type II resistance (resistance to fungal spread from a single inoculation point), while field tests screen for both type II and type I (resistance to inoculation and spread). It is likely that the transgenic wheat line No. 32A displays type II resistance but not type I. Sharp et al. (2002) in an evaluation of transgenic wheat plants expressing viral coat proteins against wheat mosaic virus reported that while these plants were more resistant in greenhouse studies, they offered no protection in the field. As far as is known, this is the first report of field evaluation of a cereal plant expressing genes for PR-proteins against disease resistance.
The poor performance of line 32A in these field evaluations might have been due to the lesion-mimic phenotype. These plants which are presumably undergoing programmed cell death may have compromised ability to withstand continuous pathogen pressure which occurs under field conditions. The lesion-mimic phenotype is only seen in the homozygous and not in heterozygous plants, which are equally resistant to scab in greenhouse bioassays. Next spring it is planned to conduct field evaluation of segregating populations of plants derived from line 32A to determine whether they will withstand the severe conditions of the disease in the scab nursery. In addition, there is a plan to evaluate plants with three different PR-protein genes namely chitinase, ß-1,3-glucanase and tlp, because this combination of PR-protein genes would target not only the fungal cell wall, but also the fungal plasma membrane, the known target of TLP (Vigers et al., 1991; Yun et al., 1998). It is likely that PR-proteins may be effective only in combination with several other PR-proteins.
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The authors thank JS Essig, Dr ML Main, and RD Wamsley for wheat transformation and regeneration. Dr B Friebe, Dr RL Bowden, Mr D Wilson, and Mr MA Davis are sincerely acknowledged for their expertise and assistance with scab testing and evaluation. We also appreciate the help from B Narasimhamoorthy with the statistical analyses. This research was funded in part by the Kansas Wheat Commission and, the US Wheat and Barley Scab Initiative. This is contribution No. 03-106-J of the Kansas Agricultural Experiment Station.
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