1.8.34
THE ROLE OF ABC TRANSPORTERS IN PATHOGENICITY AND SEXUAL REPRODUCTION OF MYCOSPHAERELLA GRAMINICOLA, THE CAUSAL AGENT OF SEPTORIA TRITICI LEAF BLOTCH OF WHEAT

K VENEMA and MA DE WAARD

Department of Phytopathology, Wageningen Agricultural University, Wageningen, The Netherlands

Background and objectives
ABC transporters are members of the ATP-binding superfamily of transporters [1]. ABC transporters occur abunduntly in both pro- and eukaryotic organisms, and are involved in the secretion of both non-toxic substrates (e.g. mating factors) and toxic substrates (e.g. antibiotics, secondary metabolites). ABC transporters also counteract the passive accumulation of exogenous toxins (e.g. antibiotics, drugs, fungicides) in organisms by active secretion, reducing their cytoplasmic concentrations and so rendering the target organisms less sensitive. ABC transporters, therefore, are a natural means of defence against toxic compounds made by plants and microbes. However, ABC transporters became known worldwide for their role in multi-drug resistance (MDR) in mammalian tumour cells against anti-tumour drugs, a phenomenon which is of major concern in cancer therapy. Recently, MDR was described in plant pathogenic fungi [2, 3], indicating that ABC transporters may play an important role in pathogenesis and disease control. ABC transporters in plant pathogenic fungi may have roles in reducing the accumulation of fungitoxic plant defence compounds in mycelium, and the secretion of pathogenicity factors, such as toxins and mating factors, from fungal cells. ABC transporters may, therefore, be important new pathogenicity factors. The aim of this research is to raise our understanding of the biological role of ABC transporters in Mycosphaerella graminicola, by cloning ABC transporter-encoding genes involved in pathogenicity and/or sexual reproduction.

Materials and methods
In PCR, using degenerate primers against ABC domains, fragments encoding these domains were amplified. The fragments were cloned and sequenced. Those fragments encoding novel ABC transporters were used in hybridization of Nothern blots. RNA on these Northern blots was isolated from several M. graminicola-wheat interactions at different time points after infection.

Results and conclusion
In addition to the ABC domains from the ABC transporter-encoding genes Mgatr1 and Mgatr2 (see poster by Zwiers and De Waard), several new ABC domains have been isolated using a PCR strategy, and degenerate primers derived from homologous regions in the ABC domain. The fact that the ABC domains were also recovered from the previously cloned Mgatr1 and Mgatr2 validates the procedure. The novel fragments and several heterologous probes were used to screen a phage library of M. graminicola DNA. Several positive clones were isolated for further characterization.

M. graminicola strain IP0323 was inoculated on wheat cultivar Obelisk (compatible interaction) and cultivar Kavkaz/K4500 (incompatible interaction). In addition, a mixture of M. graminicola strains IP0323 and IP094269, of opposite mating type, was inoculated on cultivar Obelisk. RNA was isolated from these M. graminicola-wheat interactions at 6, 8, 10, 12, 14, 16, 18 and 22 days after inoculation of 10-day-old seedlings and analysed by Northern blotting.

References
1. Gottesman MM, Pastan I, 1993. Annual Review of Biochemistry 62, 385-427.
2. De Waard MA, Van Nistelrooy JGM, Langeveld CR et al. (eds), Modern Fungicides and Antifungal Compounds. Intercept, Andover, UK, pp. 293-299.
3. Del Sorbo G, Andrade A, Van Nistelrooy JGM et al., 1997. Molecular and General Genetics 254, 417-426.