NiaA, the structural nitrate reductase gene of Phytophthora infestans: isolation, characterization and expression analysis in Aspergillus nidulans.

The nitrate reductase (NR) gene niaA of the oomycete Phytophthora infestans was selected from a gene library by heterologous hybridization. NiaA occurs as a single-copy gene ant its expression is regulated by the nitrogen source. The nucleotide sequence of niaA was determined and comparison of the deduced amino-acid sequence of 902 residues with NRs of higher fungi and plants revealed a significant homology, particularly within the three cofactor-binding domains for molybdenum, heme and FAD. The P. infestans niaA gene was used as a model gene to test whether oomycete genes are functional in the ascomycete Aspergillus nidulans, a fungus which is highly accessible for molecular genetic studies. The complete niaA gene was stably integrated into the genome of a nia- deletion mutant of A. nidulans. However, transformants containing one or more copies of the niaA gene were not able to complement the nia- mutant. This suggests that there is no functional expression of the introduced niaA gene in A. nidulans. In addition, the activity of two other oomycete gene promoters was analyzed in a transient expression assay. Plasmids containing chimaeric genes with the promoter of the P. infestans ubiquitin gene ubi3R, or the Bremia lactucae ham34 gene, fused to the coding sequence of the Escherichia coli beta-glucuronidase (GUS) reporter gene, were transferred to A. nidulans protoplasts. No significant GUS activity was detectable indicating that the ubi3R and ham34 promoters are not active in A. nidulans. Apparently, the regulatory sequences which are sufficient for gene activation in oomycetes are not functional in the ascomycete A. nidulans.

Structure and genomic organization of the ipiB and ipiO gene clusters of Phytophthora infestans.

Two in planta-induced (ipi) genes, designated ipiB and ipiO, of the potato late blight fungus, Phytophthora infestans (Mont.) de Bary, were isolated from a genomic library by a differential hybridization procedure [Pieterse et al., Physiol. Mol. Plant Pathol. (1993a) in press]. Both genes are expressed at high levels in the early phases of the pathogenic interaction of P. infestans with its host plant potato, suggesting that their gene products have a function in the early stages of the infection process. Here, we describe the nucleotide (nt) sequence and genomic organization of ipiB and ipiO. The ipiB gene belongs to a small gene family consisting of at least three genes, designated ipiB1, ipiB2 and ipiB3, which are clustered in a head-to-tail arrangement. The three ipiB genes are highly homologous throughout the coding regions and 5' and 3' flanking regions. The P. infestans genome contains two very similar ipiO genes, ipiO1 and ipiO2, which are closely linked and arranged in an inverted orientation. The ipiB genes encode three novel, highly similar Gly-rich proteins of 301, 343 and 347 amino acids (aa), respectively. The Gly-rich domains of the IPI-B proteins are predominantly composed of two repeats with the core sequences, A/V-G-A-G-L-Y-G-R and G-A-G-Y/V-G-G. The ipiO genes code for two almost identical 152-aa proteins which do not have any homology with sequences present in data libraries. IPI-B, as well as IPI-O, contains putative signal peptides of 20 and 21 aa, respectively, suggesting that they are transported out of the cytoplasm. In the promoter regions of ipiB and ipiO, a 16-nt sequence motif, matching the core sequence, GCTCATTYYNCAWTTT (where N = A or C or G or T; W = A or T; Y = C or T), was found. This sequence motif appears to be present around the transcription start point (tsp) of seven out of eight oomycetous genes for which the tsp have been determined, suggesting that oomycetes have a sequence preference for transcription initiation.

Differential accumulation of mRNAs encoding extracellular and intracellular PR proteins in tomato induced by virulent and avirulent races of Cladosporium fulvum.

Tomato leaves infected by the fungal pathogen Cladosporium fulvum contain several types of intracellular and extracellular pathogenesis-related (PR) proteins. Previously, we reported the purification and serological characterization of five extracellular PR proteins: P2, P4, P6, a chitinase and a beta-1,3-glucanase [22, 23]. Here we describe the purification of a basic intracellular 33 kDa beta-1,3-glucanase and the isolation and characterization of cDNA clones encoding the two extracellular P14 isomers P4 and P6, the extracellular acidic beta-1,3-glucanase and a basic 35 kDa beta-1,3-glucanase, different from the purified 33 kDa protein. Southern blot analysis demonstrated that tomato PR proteins are not encoded by large gene families, as is the case in tobacco. The number of genes corresponding to each protein was estimated to vary between one and three. A northern blot analysis indicated that the mRNAs for the extracellular PR proteins (P4, P6 and acidic beta-1,3-glucanase) accumulate to similar levels in compatible and incompatible tomato-C. fulvum interactions, although the maximum level of expression is reached much faster in the incompatible interaction. On the other hand, the mRNA for the basic 35 kDa beta-1,3-glucanase is induced rapidly to high levels in both interactions, but declines in time to background levels only in the incompatible interaction. The relevance of this difference in relation to plant defence is discussed.