Differential gene expression in sugarcane in response to challenge by the fungal pathogen Ustilago scitaminea (Sydow) (sugarcane smut).

Suppression subtractive hybridisation was used to detect sugarcane differential gene expression in response to challenge by the fungal pathogen, Ustilago scitaminea. Isolated fragments having homology with flavonoid pathway transcription factors were utilised as RFLP probes in a marker discovery effort. A multiple linear regression model included two polymorphisms and explained 25% of the variation in smut resistance ratings within a set of 78 phenotypically well-characterised sugarcane clones. Introduction resistance mechanisms, and the isolation of fullResistance to sugarcane smut, a disease caused by length sequences for use as potential transgenes. the fungus ustilago scitaminea H. & P. Sydow, is thought to be determined by combinations of preformed bud structural characteristics (Waller, 1970), and pre-formed bud phenylpropanoids and glycosylflavonoids (Lloyd and Naidoo, 1983). In addition, Dean (1982) proposed two 'physiological' (inducible) barriers, one of which is partially overcome by wounding the bud prior to inoculation. Smut resistance screening in the plant breeding program results in a continuous resistant to susceptible phenotypic distribution. Excessive inoculation pressure can skew a population towards susceptibility for all but the most resistant genotypes. These observations suggest that resistance is partial and polygenic (rate-limiting, minor gene, non-race specific). However, race specific resistance might be superimposed on a background of partial resistance. Several races of sugarcane smut have been postulated to exist (Gillaspie et al., 1983). As well as physical and chemical pre-formed barriers, partial resistance can include an inducible component thought to involve minor gene-for-minor gene interactions, e.g. specific receptor detection of non-race specific elicitors such as linear P-1,3 glucans from fungal cell walls and oligo-galacturonides from plant cell walls (Klarzynski et al., 2000). Recently discovered receptor kinases (S-receptor kinase-like) possessing domains similar to plant lectins might be capable of detecting such elicitors (Swarup et al., 1996). Given the importance of specific receptors and downstream regulatory elements in race and non-race specific plant-pathogen interactions, differentially expressed sequences homologous to known examples are the focus of this on-going project. These sequences are employed as RFLP probes in the development of markers for resistance and susceptibiIity. Other objectives include gaining insight into Materials and methods Single budded setts of resistant (N521219) and susceptible (Co301) varieties were dipped in 0.01% Tween-20 with or without lo8 teliospores/mL. After seven days, RNA was extracted from breaking buds using TRIzolm (Life Technologies). Since one of the stated goals is to isolate fulllength coding sequences of candidate resistance genes, cDNA synthesis was performed using a Clontech SMARTTM kit in order to generate as high a proportion of full-length products as possible (Ciavatta and Cairney, 2000). SMART cDNA can also serve as the starting material for detection of differential gene expression. The chosen methodology for detecting differential gene expression was Suppression Subtractive Hybridisation (SSH) using a PCR SelectTM kit (Clontech) (Diatchenko et al., 1998). Sequencing of cloned differentially expressed sequences was performed on an ABI 310 Genetic Analyser. Comparison to non-redundant nucleotide, protein and EST databases was by BLASTN and BLASTX at http://www.ncbi. nlm.nih. gov/blast/blast. cgi. Based on resultant homologies, differentially expressed sequences were assigned putative identities and functions. For RFLP analyses, genomic DNA was extracted from a set of 78 phenotypically well-characterised clones which have been or are current parents in the sugarcane breeding programme. Southern blotting and RFLP detection, using identified differentially expressed sequences as probes, was conducted according to standard protocols (Sambrook et al., 1989). Differential expression can be analysed by Northern Blotting (Sambrook et al., 1989). In the absence of sufficient RNA, virtual Northern blots were produced using the same SMART PCR-amplified tester and driver cDNA used for subtraction.