ABSTRACT

Forty-five cDNA clones representing 22 different genes were isolated and characterised from a subtracted library of CuCl₂-treated versus H₂O-treated Brassica carinata plants. Database comparison of these sequences by BLAST search identified 13 clones with high similarity to defence genes from other plant species. Similarity to Arabidopsis thaliana genomic DNA sequence of unknown function was found in yet four more clones. Finally, five cDNA clones showed no sequence similarity to database entries. More recently, we used a cDNA-AFLP approach to examine mRNA differential expression in the elicitor-treated plants. Sequence comparison of some of these cDNA fragments has identified 15 additional CuCl₂-treatment induced mRNAs. Further characterisation of one of these clones revealed a high similarity to an A. thaliana resistance gene.

INTRODUCTION

The term elicitor was first applied to compounds that induced the production of phytoalexins in plants. Later, it was discovered that these compounds also induced other defence reactions such as lignin and PR protein production (Kombrink and Hahlbrock, 1986). Elicitors are classified as either biotic or abiotic. Biotic elicitors include such compounds as complex carbohydrates, lipids and proteins (Darvil and Albersheim, 1984). Abiotic elicitors are structurally diverse compounds including heavy metals, such as copper and mercury, and detergents (Yoshikawa, 1978).

Cultivars of Brassica carinata (Ethiopian mustard) are generally highly resistant to attack by the blackleg causal organism Leptosphaeria maculans (Sjödin and Glimelius, 1988). This resistance is attributed to genes on three chromosomes of the b genome (Zhu et al., 1993). Our laboratory is interested in using biotechnology to increase resistance to blackleg and other fungal diseases in canola (B. napus and B. rapa) varieties grown in western Canada. As a first step towards this goal we are isolating and characterising defence genes from B. carinata. The very efficient transformation system developed for B. carinata (Babic et al., 1998) is facilitating our efforts to characterise the genes with unknown function.

METHODS

Three-week-old B. carinata plants were sprayed with 5 mM CuCl₂ to elicit the plants’ defence response. Control plants were sprayed with water. Induction of phytoalexin production was checked at 24 h by HPLC analysis of leaf extracts (Pedras and Séguin-Swartz, 1992). Next a time course for defence gene mRNA induction was performed by RT-PCR. The RNA was extracted from plants beginning 6 h post-treatment and proceeding up to 24 h. The primers for PCR amplification were derived from B. napus genes for chitinase and PR1. The maximal amount of mRNA from both genes was found 12 h post-treatment. This time point was, therefore, used in the preparation of RNA for library construction. A cDNA library from mRNA induced by the CuCl₂-treatment was prepared by subtractive hybridisation with cDNA from H₂O-treated plants. A portion of the library was screened by hybridisation with ³²P-labelled cDNA from mRNA of CuCl₂ and H₂O-treated plants. Clones that hybridised more strongly to the cDNA from CuCl₂-treated than to that of H₂O-treated plants were sequenced. The sequences were compared to each other and then compared to database entries using a BLAST search (Altschul et al., 1997).

cDNA-AFLP analysis of mRNA differential expression was performed essentially as described by Bachem and colleagues (1996). The mRNA was extracted from 4-week-old B. carinata plants, treated as described above, at 12 h post-treatment. The double-stranded cDNA from CuCl₂ and H₂O-treated plants was digested with Taq I and Ase I then ligated to compatible adaptors of known sequence for PCR amplification. Amplification of these fragments employed primers containing a portion of the adaptors’ sequence and a two-base selective extension. cDNA fragments derived from genes that appeared to be more highly expressed in CuCl₂-treated plants were excised from polyacrylamide gels. The same region of the gel was also excised from the lane containing cDNA from the H₂O-treated plants. The DNA from the gel slices was eluted and re-amplified. These PCR products were then analysed for single-strand conformation polymorphism (Mathieu-Daudé et al., 1996). The unique single-strand cDNA fragments in lanes representing mRNA from CuCl₂-treated versus H₂O-treated plants were excised from the gel, amplified and sequenced. These sequences were also compared to each other and then compared to database entries using a BLAST search. A primer from the cDNA fragments of interest and primers from the vector were used to amplify the genes out of the subtracted cDNA library.

RESULTS

Forty-five cDNA clones from a portion of the subtracted library hybridised more strongly to the cDNA from CuCl₂-treated plants than to that of H₂O-treated plants. DNA sequence analysis of the clones showed that they represented 22 different genes. Database comparison of these sequences by BLAST search identified 13 clones with high similarity to defence genes from other plant species. Similarity to Arabidopsis thaliana genomic DNA sequence of unknown function was found in yet four more clones. Finally, five cDNA clones showed no sequence similarity to database entries. The clones with sequence similarity to known defence genes are described in Table 1 and their “Expect” values are given.

Sixteen cDNA fragments from cDNA-AFLP analysis representing CuCl₂-induced gene expression have been characterised so far. The sequence of one fragment matched a cDNA clone isolated in the previous screening. One fragment had similarity to a phenylalanine ammonia lyase gene from rice. The sequence of another fragment was similar to the O-methyltransferase I gene of A. thaliana that is involved in secondary metabolism. A third fragment had high similarity to the A. thaliana amt1 gene for ammonium transport. Amplification out of the cDNA library using a primer derived from another of the fragments and a vector primer yielded a clone with very high similarity to the RPS5 resistance gene of A. thaliana.

DISCUSSION

We have isolated 13 cDNA clones of genes from B. carinata with high similarity to known defence genes in other plant species. These results indicate that CuCl₂, as it is in other plants, is an effective elicitor of the defence response in B. carinata. Subsequent to the elicitor treatment we used two different techniques to isolate cDNAs representing elicitor-induced gene expression. Both the subtractive hybridisation library and the mRNA differential display techniques proved successful in identifying defence genes. Furthermore, there appeared to be little overlap in the genes identified. We compared mRNA expression patterns of genes isolated by the two methods and found that the mRNA differential display technique could reveal subtler quantitative differences in elicitor-induced versus control plant material.

In an effort to identify the functions of the genes with no database similarity we have transformed antisense constructs of them into B. carinata. We are presently evaluating the phenotypes of the self-fertilised progeny of the transformants. Additionally, we are studying the expression patterns of the genes in wild-type plants after treatment with various elicitors and infection by aggressive and non-aggressive fungal pathogens. Finally, we are determining if the nucleotide similarity of our homolog to the resistance gene RPS5 is reflective of a functional similarity.

TABLE 1. Brassica carinata cDNA clones with similarity to defence genes in other plant species.

* The Expect value (E) is a parameter that describes the number of hits one can "expect" to see just by chance when searching a database of a particular size.

ACKNOWLEDGEMENTS

We would like to thank Dr. M. S. C. Pedras for the phytoalexin analysis, Mr. B. Panchuk for DNA sequence analysis and Mr. D. Schwab for oligonucleotide synthesis. This work is supported by a NRC/NSERC Research Partnership Program grant to M. S. C. Pedras and J. L. Taylor.

REFERENCES

1. Altschul, S. F., T. L. Madden, A. A. Schaffer, J. Zhang, Z. Zhang, W. Miller and D. J. Lipman. 1997. Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic Acids Research 25:3389-402.

2. Babic, V., R. S. Datla, G. J. Scoles and W. A. Keller. 1998. Development of an efficient Agrobacterium-mediated transformation system for Brassica carinata. Plant Cell Reports 17:183-189.

3. Bachem, C. W. B., R. S. van der Hoeven, S. M. de Bruijn, D. Vreugdenhil, M. Zabeau and R. G. F. Visser. 1996. Visualisation of differential gene expression using a novel method of RNA fingerprinting based on AFLP: Analysis of gene expression during potato tuber development. The Plant Journal 9:745-753.

4. Darvil, A. G. and P. Albersheim. 1984. Phytoalexins and their elicitors- A defense against microbial infection in plants. Annual Review of Plant Physiology 35:243-275.

5. Kombrink, E. and K. Hahlbrock. 1986. Responses of cultured parsley cells to elicitors from phytopathogenic fungi. Plant Physiology 81:216-221.

6. Mathieu-Daudé, F., R. Cheng, J. Welsh and M. McClelland. 1996. Screening of differentially amplified cDNA products from RNA arbitrarily primed PCR fingerprints using single strand conformation polymorphism (SSCP) gels. Nucleic Acids Research 24:1504-1507.

7. Pedras, M. S. C. and G. Séguin-Swartz. 1992. The blackleg fungus: phytotoxins and phytoalexins. Canadian Journal of Plant Pathology 14:67-75.

8. Sjödin, C. and K. Glimelius. 1988. Screening for resistance to blackleg Phoma lingam (Tode ex Fr.) Desm. within Brassicaceae. Journal of Phytopathology 123:322-332.

9. Yoshikawa, M. 1978. Diverse modes of action of biotic and abiotic phytoalexin elicitors. Nature 275: 546-547.

10. Zhu, J. S., D. Struss and G. Röbbelen. 1993. Studies on resistance to Phoma lingam in Brassica napus-Brassica nigra addition lines. Plant Breeding 111:192-197.