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.

Increased expression of the calmodulin gene of the late blight fungus Phytophthora infestans during pathogenesis on potato.

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.

Characterization of two putative pathogenicity genes of the fungal tomato pathogen Cladosporium fulvum.

The fungus Cladosporium fulvum is a biotrophic pathogen of tomato. On susceptible tomato plants, the fungus grows abundantly in the extracellular spaces between the mesophyll cells. The mechanism by which C. fulvum is able to establish and maintain basic compatibility on its one and only host species, the tomato, is unknown. The isolation and characterization of pathogenicity factors and the corresponding genes will provide insight into the mechanism by which C. fulvum colonizes its host. Two putative pathogenicity genes of C. fulvum encoding proteins, which occur abundantly in the extracellular space of infected tomato leaves, were isolated and characterized (ecp1 and ecp2). The DNA sequences of these ecp genes (encoding extracellular protein) do not share homology to any sequence present in the DNA databases. The ecp genes are highly expressed in planta but not in vitro, suggesting that they play a significant role in pathogenesis.