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Abstract
Three putative exo-1,3-β-glucanase genes (Piexo1, Piexo2, Piexo3), one endo-1,3-β-glucanase gene (Piendo1) and one endo-1,3;1,4-β-glucanase gene (Piendo2) were cloned and characterized from the plant pathogenic oomycete Phytophthora infestans. The genes have no introns and are single copy, except for Piendo1 that is present as two copies. The 1,3-β-glucanases are differentially expressed in the different developmental stages of the pathogen, and are also expressed during infection of potatoes.
Nucleotide sequence analysis of the glucanase promoter regions showed that they had 56–81% similarity to a highly conserved 16nt core sequence hypothesized to be the transcriptional start site in oomycete genes. Primer extension analyses of Piexo1, Piexo3 and Piendo1 showed that their transcriptional start sites mapped to nucleotides within the 16nt core sequence. Subsequent sequence comparison of the 16nt core sequences of the glucanases and several published oomycete promoters revealed the presence of a putative core promoter element, the initiator (Inr). The putative oomycete Inr has a consensus sequence (YCA+1TTYY, where Y = C or T) similar to the Inr consensus sequence of mammals, Trichomonas and Drosophila.
The function of the 16nt core sequence, containing the putative oomycete Inr, was further investigated by creating deletions and mutations in a vector containing the 993 bp promoter region of Piexo1 fused to the β-glucuronidase gene. The function of the 16nt core sequence in Piendo1 (1 kb promoter fragment) was also evaluated to confirm some of the trends seen in the promoter of Piexo1. The 16nt core region and flanking nucleotides in Piexo1 were also further investigated in electrophoretic mobility shift assays (EMSA) using 31–36 bp double strand oligonucleotides and P. infestans whole-cell extract proteins. Altogether the transient expression analyses and EMSA studies showed that oomycetes contain a 19nt core sequence that includes the previously identified 16nt core sequence, which is composed of the Inr and a flanking promoter region (FPR) at positions +6 to +15. The Inr and FPR seem to function cooperatively, since both have to be present for transcription as well as protein binding to take place.
