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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.

The ipiB and ipiO genes of the potato late blight fungus Phytophthora infestans (Mont.) de Bary were isolated from a genomic library in a screen for genes induced in planta. Expression of these genes was studied during pathogenesis on various host tissues and different host plants, some of which show specific resistance against P. infestans infection. During pathogenesis on leaves and tubers of the fully susceptible potato cultivar (cv.) Ajax and on leaves of the fully susceptible tomato cv. Moneymaker, the P. infestans ipiB and ipiO genes show a transient expression pattern with highest mRNA levels in the early stages of infection. During the interaction with leaves of the partially resistant potato cv. Pimpernel, the expression is also transient but accumulation and disappearance of the mRNAs is delayed. Also in P. infestans inoculated onto a race-specific resistant potato cultivar and onto the nonhost Solanum nigrum, ipiB and ipiO mRNA is detectable during the initial stages of infection. Apparently, the expression of the ipiB and the ipiO genes is activated in compatible, incompatible and nonhost interactions. In encysted zoospores, ipiB and ipiO mRNA accumulation was not detectable, but during cyst germination and appressorium formation on an artificial surface the genes are highly expressed. Expression studies in mycelium grown in vitro revealed that during nutrient starvation the expression of the ipiB and ipiO genes is induced. For ipiO gene expression, carbon deprivation appeared to be sufficient. The ipiO gene promoters contain a sequence motif that functions as a glucose repression element in yeast and this motif might be involved in the regulation of ipiO gene expression.

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.

Procedures were identified for manipulating the expression of genes in the oomycete fungus, Phytophthora infestans. The activities of five putative promoter sequences, derived from the 5' regions of oomycete genes, were measured in transient assays performed in protoplasts and in stable transformants. The sequences tested were from the ham34 and hsp70 genes of Bremia lactucae, the actin-encoding genes of P. infestans and P. megasperma, and a polyubiquitin-encoding gene of P. infestans. Experiments using the GUS reporter gene (encoding beta-glucuronidase) demonstrated that each 5' fragment had promoter activity, but that their activities varied over a greater than tenfold range. Major variation was revealed in the level of transgene expression in individual transformants containing the same promoter::GUS or promoter::lacZ fusion. The level of expression was not simply related to the number of genes present, suggesting that position effects were also influencing expression. Fusions between the ham34 promoter, and full-length and partial GUS genes in the antisense orientation blocked the expression of GUS in protoplasts and in stable transformants.

In order to isolate in planta-induced genes encoding putative pathogenicity factors of the late blight fungus Phytophthora infestans, a genomic library was differentially screened. For the differential hybridization, labeled first-strand cDNA synthesized on mRNA isolated from P. infestans-infected potato leaves and on mRNA isolated from the fungus grown in vitro were used as probes. This screening resulted in the isolation of the P. infestans calmodulin gene. The gene, designated calA, contains an open reading frame of 447 base pairs without introns and is unique in the P. infestans genome. The predicted amino acid sequence is 89.9-94.6% identical to calmodulins from higher eukaryotes, whereas the identity to calmodulins of higher fungi is significantly less (60.8-85.1%). Expression studies revealed that the P. infestans calA gene is constitutively expressed in in vitro grown mycelium. However, during pathogenesis on potato the level of P. infestans calmodulin mRNA is increased approximately fivefold.

An in planta induced gene of Phytophthora infestans (the causal organism of potato late blight) was selected from a genomic library by differential hybridization using labelled cDNA derived from poly(A)+ RNA of P. infestans grown in vitro and labelled cDNA made from potato-P. infestans interaction poly(A)+ RNA as probes. Sequence analysis showed that the gene codes for ubiquitin, a highly conserved protein which plays an important role in several cellular processes. The structure of the polyubiquitin gene (designated ubi3R) is consistent with the structure of other known polyubiquitin genes. It consists of three repeats in a head-to-tail arrangement without intervening sequences, each encoding a ubiquitin unit of 76 amino acids. The last ubiquitin unit is followed by an extra asparagine residue at the carboxy-terminal end. Northern and Southern blot analyses revealed that the polyubiquitin gene is a member of a multigene family, all genes of which show induced expression in planta.