1Cooperative Research Centre for Tropical Plant Pathology, The University of Queensland, Brisbane, QLD. 4072, Australia
2CSIRO Tropical Agriculture, 306 Carmody Rd, Brisbane, QLD. 4067, Australia
Expression of genes encoding antimicrobial peptides offers an alternative strategy for genetic engineering of canola against fungal pathogens. This approach is especially attractive for the control of canola pathogens for which limited natural resistance source is available. To identify plant proteins with antifungal activities against canola pathogens such as Sclerotinia sclerotiorum and Leptosphaeria maculans we have screened crude extracts of approximately 250 accessions of Australian native plants. As a result of this screening, several peptides with significant activity against canola pathogens were identified. Among these, peptides purified from the nuts of an Australian native tree species, Macadamia integrifolia were found especially active against a range of pathogens. One of the peptides characterised in detail was MiAMP1 (macadamia antimicrobial peptide 1). This peptide has 76 aa residues and expressed as a precursor protein with an additional 26 aa signal peptide. Purified MiAMP1 inhibited the growth of L. maculans and S. sclerotiorum, suggesting that it can be used as a novel gene to provide tolerance against these pathogens. MiAMP1 gene has been introduced into double haploid canola lines and expression was confirmed using Northern and Western blot analyses. High MiAMP1 expressing transgenic lines of canola were selected. These lines are currently being characterised in inoculation experiments to determine whether the level of resistance to Leptosphaeria maculans and Sclerotinia sclerotiorum has changed. In addition to MiAMP1, other antifungal peptides from macadamia were isolated. These new peptides are currently being characterised.
KEYWORDS: Leptosphaeria maculans, Sclerotinia sclerotiorum, Brassica napus, macadamia, transgenic plants.
Canola (Brassica napus L.) is a major crop grown for its oil in Canada, India, China Europe and other parts of the world. One of the major factors restricting the cultivation of this crop is its susceptibility to Leptosphaeria maculans (Phoma lingam)-causative agent of the black leg disease and Sclerotinia sclerotiorum- causative agent of the white rot disease. It has been estimated that crop losses due to these two diseases may reach up to 95% (Grison et al. 1996). Application of fungicides is not very effective for eliminating the infections caused by these diseases. Additionally, fungicide application increases the cost of production and may have adverse effects on environment. Breeding for resistance to blackleg has been partially successful (Stringham et al. 1995) while breeding for resistance to S. sclerotiorum was ineffective due to very limited source of resistance available in the germplasm. Therefore, identification and introduction of genes for novel resistance to canola through genetic engineering methods provide an attractive alternative for improving resistance against these two diseases.
GENETIC ENGINEERING OF CANOLA FOR ENHANCED DISEASE RESISTANCE
Genes that can be potentially employed in any transgenic plant for enhanced disease resistance may include a) genes encoding antifungal proteins, b) genes encoding inhibitors of pathogenicity factors and c) genes for initiation and enhancing plant’s natural defence responses (Bushnell et al. 1998). Because of the unavailability of the latter two classes of genes for L. maculans and S. sclerotiorum, expression of the genes encoding antifungal proteins in transgenic canola appears to be the only option for increased resistance against these two pathogens. Antifungal proteins with direct antimicrobial activity on pathogens are produced in the plants after infection. Transgenic expression of an antifungal gene encoding chitinase was found effective in enhancing the tolerance of transgenic canola to L. maculans, S. sclerotiorum (Grison et al. 1996) and Rhizoctonia solani (Brogglie et al. 1991). However, large-scale exposure of transgenic plants expressing defensive proteins to pathogenic fungi may render these proteins less effective. Therefore, novel forms of resistance should be continuously identified for potential deployment in transgenic plants against L. maculans and S. sclerotiorum. Once new proteins with antimicrobial properties are identified, genes encoding such proteins can be cloned and expressed in transgenic canola plants. In this paper we discussed application of antimicrobial peptide technology to the genetic engineering of canola for enhanced disease resistance.
PURIFICATION AND TRANSGENIC EXPRESSION OF ANTIMICROBIAL PEPTIDES IN CANOLA
Antimicrobial peptides (AMPs) are cysteine rich short proteins commonly found in the seeds of many plant species (Broekaeart et al. 1997). These peptides usually have broad antimicrobial activity on a number of pathogenic fungi and thus are promising candidates for use in transgenic plants for managing plant diseases (Terras et al. 1994). To identify new antifungal peptides effective against fungal pathogens of canola, we have screened the crude extracts of approximately 250 accessions of Australian native plants, which represent relatively unexplored source of antimicrobial proteins (Marcus et al. 1997; Harrison et al. 1997). This survey resulted in identification of several peptides showing promising activity against L. maculans and S. sclerotiorum. Amino acid sequences of purified peptides were determined and compared with the sequences of other proteins in the databases. One of the peptides (MiAMP1=macadamia antimicrobial peptide 1) purified from the seeds of macadamia (Macadamia integrifolia) did not show any similarity to any of the previously identified peptides (Marcus et al. 1997). Purified MiAMP1 was active in vitro against L. maculans and S. sclerotiorum when fungal spores or hyphal fragments were incubated in microtitre plates in the presence of varying concentrations of MiAMP1 peptide. Presence of 50 μg/ml of MiAMP1 in the growth medium caused marked distortion of the hyphal growth of both L. maculans and S. sclerotiorum. However, the level of inhibition against S. sclerotiorum diminished rapidly after 24 hrs. Mammalian and tobacco cells treated in vitro with high concentrations of MiAMP1 (100 μM) did not show any loss of viability. These properties of MiAMP1 suggested that it could be used in transgenic plants as a novel resistance gene against L. maculans and S. sclerotiorum.
The amino acid sequence of MiAMP1 contains a 26-amino acid signal peptide and a 76-amino acid mature peptide with six cysteine residues. To express MiAMP1 peptides in transgenic plants, a binary vector carrying MiAMP1 cDNA (including the 26 aa signal peptide) has been constructed under the control of enhanced double CaMV 35S RNA promoter. This construct (35S-MiAMP1) was transferred into canola plants using an Agrobacterium tumefaciens-mediated transformation technique. In these transformation experiments double haploid lines derived from cv. Westar and open pollinated plants of cv. Oscar were used. Consequently, ten Westar and eight Oscar plants were generated using hygromycin selection and transgenic status of the plants were confirmed by molecular analyses. These primary transgenic plants were self-pollinated and their progeny were germinated on a medium containing hygromycin. Hygromycin resistant progeny from these 18 lines were then further characterised by Northern and Western blot analyses. In these analyses seven out of ten Westar and six out of eight Oscar lines showed a detectable level of MiAMP1 expression. Based on these analyses several high MiAMP1 expressing transgenic lines were selected. These plants are currently being evaluated for resistance against L. maculans and S. sclerotiorum. Initial inoculation experiments with L. maculans are being conducted on 8-9 days old seedlings grown in controlled environment rooms. In these experiments, cotyledons of 10-day old seedlings will be wounded and inoculated with L. maculans pycnidiospore suspension. After 9-10 days of inoculation, symptom development will be assessed using a scale divided into nine classes, with one representing complete resistance and nine representing highly susceptible interactions. Sclerotinia sclerotiorum inoculations of the stems of transgenic canola expressing MiAMP1 will also be undertaken with adult plants. Inoculated plants will then be scored for lesion extension. Transgenic lines showing promising levels of resistance will then be tested under field conditions.
CONCLUSIONS and FUTURE PROSPECTS
The use of antimicrobial peptides as a novel source of resistance in transgenic canola represents a potentially promising strategy for the management of canola diseases that are difficult to combat otherwise. However, for successful application of antimicrobial genes into engineering of disease resistance in transgenic canola, AMPs should be evaluated for certain characteristics. Most importantly the peptide should be toxic to the pathogen(s) of interest without having any toxicity to plant and mammalian cells. Correct processing and expression of the peptide at high levels in canola plants are also important for in planta antimicrobial activity. MiAMP1 and other peptides we have recently identified certainly meet these criteria.
The future strategies involving deployment of AMP genes in transgenic canola for enhanced resistance to fungal diseases will be directed towards improvement of antifungal efficacy and increasing the spectrum and the durability of resistance. Site directed mutagenesis of MiAMP1 based on 3D structure has been recently shown to increase the activity of native MiAMP1 (unpublished). Research on the mode of action of MiAMP would be useful in determining the potential target molecules in the pathogen. This knowledge could then be used to design new and more effective AMPs. Durability and spectrum of the AMP-mediated resistance could also be enhanced by combinatorial expression of AMP genes. It may be also important to control and regulate spatial and temporal accumulation of AMPs in transgenic plants for optimal disease control.
This work is partially funded by the Grains Research and Development Corporation (GRDC). We thank Ms. N. Willemsen for her assistance in molecular characterisation of transgenic canola plants and R.D. Beversdorf for supplying double haploid canola lines used in transformation experiments.
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