Identification of maize cDNA clones corresponding to genes encoding chitinase and b-1,3-glucanase

--Shenchuan Wu, Jack M. Widholm and Alan L. Kriz

The degradative enzymes chitinase and b-1,3-glucanase have been found to accumulate in higher plants following pathogen attack and environmental stress, which indicates their involvement in plant defense responses. These enzymes are particularly important for resistance of plants to fungal invasion, as chitin and b-1,3-glucans are major cell wall constituents of certain fungi. As an approach to identifying means for possible enhancement of fungal resistance in maize through the use of genetic engineering technology, we have isolated cDNA clones corresponding to maize genes encoding chitinases and b-1,3-glucanases. These full-length clones were obtained from a cDNA library generated from mRNA isolated from maize seedlings (inbred line Va26) that had been treated with mercuric chloride as an inducing agent. The library was screened with partial clones, corresponding to each gene, that had been generated by PCR amplification of cDNA obtained from the mercuric chloride-induced mRNA sample. Degenerate oligonucleotide primers for the PCRs were designed from published sequences of barley chitinase and b-1,3-glucanase genes. Nucleotide sequence analysis of these clones indicates that the maize and barley chitinase genes exhibit 73% amino acid identity, and that the b-1,3-glucanase sequences from the two species exhibit 63% identity at the amino acid level.

To investigate gene expression patterns, the chitinase and b-1,3-glucanase clones have been used as probes in northern blot analysis of maize RNAs from a variety of tissues and treatments. Various chemical treatments had previously been shown to result in induction of chitinase b-1,3-glucanase expression (e.g., Plant Science 76:211, 1991). While no transcripts corresponding to either gene were present in control 7-day old seedlings, treatment with mercuric chloride resulted in rapid, high-level induction of expression of both genes. Similar responses in b-1,3-glucanase expression patterns were observed with ethephon and salicylic acid treatments, but these latter treatments had little effect on expression of chitinase genes. Neither gene was expressed in maize kernel tissues during normal seed development, but both wounding and infection with the fungal pathogen Aspergillus flavus resulted in accumulation of chitinase and b-1,3-glucanase transcripts. This induction was limited to aleurone tissues, with little or no expression detectable in the embryo or the starchy endosperm of kernels from wounded or inoculated ears.

These preliminary studies on expression patterns of genes involved in fungal defense mechanisms provide information with respect to the control of economically important fungal infections of maize kernels. A. flavus infection, for example, can result in production of the highly carcinogenic compound aflatoxin. Since aflatoxin-contaminated grain is of little value, effective control of A. flavus infection would have a significant impact on the grain industry. Such control may be realized through the use of genetic engineering approaches to enhance and redirect expression of chitinase and b-1,3-glucanase genes to seed tissues through the use of appropriate promoters.

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