1.1.33
EXPRESSION OF THE AVR9 GENE OF CLADOSPORIUM FULVUM IS REGULATED BY A GATA-TYPE TRANSCRIPTION FACTOR

A PEREZ-GARCIA1,2, SS SNOEIJERS1,2, T GOOSEN1, HWJ VAN DEN BROEK1 and PJGM DE WIT2

1Laboratory of Genetics, Department of Biomolecular Sciences, Wageningen Agricultural University, Dreijenlaan 2, 6703 HA Wageningen, The Netherlands; 2Department of Phytopathology, Wageningen Agricultural University, Binnenhaven 9, 6709 PD Wageningen, The Netherlands

Background and objectives
From the tomato fungal pathogen Cladosporium fulvum the avirulence genes Avr9 and Avr4 have been isolated [1]. Since the Avr9 gene has been studied the most extensively, initially our research will concentrate on gaining insight into the regulation of this gene. For the Avr9 gene it is known that its expression is not limited to in planta growth. Expression can also be induced in vitro under conditions of nitrogen limitation. Regulation of nitrogen metabolism in filamentous fungi such as Aspergillus nidulans and Neurospora crassa has been studied in great detail. Single positively acting regulatory genes, designated areA in A. nidulans and nit-2 in N. crassa, encoding AREA and NIT2 regulatory proteins, respectively, induce the expression of genes involved in nitrogen metabolism when primary nitrogen sources become limited. Both AREA and NIT2 bind to promoter sequences containing at least two copies of the sequence (TA)GATA [2]. Previous studies have shown that regulated expression of the Avr9 gene in planta is obtained with a 600-bp DNA promoter fragment upstream of the main transcription start. This promoter fragment contains six copies of the TAGATA sequence and an additional six copies of the core sequence GATA in the distal part of the promoter. This study will address the question whether Avr9 expression results from binding to the promoter of a transcription factor homologous to AREA and NIT2. If the nitrogen circuit is conserved in filamentous fungi, regulation of the Avr9 gene can be studied in A. nidulans, for which both an efficient gene-targeting system and a variety of areA mutants are available.

To study whether nitrogen limitation is also the condition inducing in planta Avr9 expression, nitrate reductase-deficient tomato mutants (A29) which show high levels of nitrate accumulation in the intercellular fluid (35 mM) can be used. Crosses were made to combine this mutation with the Cf-9 resistance gene.

Results and conclusions
A 600-bp Avr9 promoter fragment-uidA fusion construct has been introduced into different areA mutants and into the areA wild type of A. nidulans by targeted integration. Quantitative determinations of GUS activity following nitrogen starvation of single-copy transformants showed that induction of the Avr9 promoter was similarly regulated in A. nidulans and in C. fulvum, and that functional sites for binding of a regulatory protein, homologous to AREA, are present in the Avr9 promoter.

Studies with specific point-mutated Avr9 promoter-uidA fusion constructs in A. nidulans indicated that some of the TAGATA elements in the Avr9 promoter are indeed essential for expression, and probably constitute true AREA-binding elements.

To isolate the C. fulvum nit-2 are A homologous gene, a PCR-based strategy has been used to clone the DNA-binding zinc-finger domain of the gene. The deduced amino-acid sequence was found to be identical to that of the zinc-finger region of the AREA protein. Currently, isolation of the entire gene is under way using a complementation strategy in A. nidulans.

References
1. De Wit PJGM, 1995. Advances in Botanical Research 21, 147-185.
2. Marzluf GA, 1997. Microbiology and Molecular Biology Reviews 61, 17-32.