Max Planck Institute, Köln, Germany

Background and objectives
Phytophthora infestans is the causal agent of the most important disease of potato, the late blight disease which brings about world-wide substantial crop losses every year. The molecular mechanisms enabling infection of the host plant and the factors and genes determining pathogenicity of this oomycete are largely unknown. Therefore, we made efforts to identify and characterize genes of P. infestans potentially related to the infection process. Taking advantage of in vitro produced structures of P. infestans [1], a PCR-based subtractive hybridization method was employed to clone genes that are activated just before the onset of the actual infection process during germination of cysts and formation of appressoria and, hence, may participate in establishing pathogenicity.

Results and conclusions
Two partial cDNAs were identified that belong to a polymorphic gene family of 6-7 members that is transiently expressed during the prepenetration phase in germinating cysts and appressoria. A genomic region of 24 kb was found carrying a cluster of three genes of this family. The most striking feature of the deduced proteins is an internal octapeptide repeat with the consensus sequence TTYAPTEE. Since the proteins belonging to this P. infestans gene family share this structural motif, we named them CAR (cyst germination-specific acidic repeat proteins). The CAR90 gene was sequenced in its entire length. It has a single open reading frame of 4470 nt coding for 1489 amino acids. The deduced CAR90 protein exhibits a typical eucaryotic signal peptide indicating secretion. Due to the high content of glutamic acid residues (EE) the CAR90 protein is extremely acidic (net charge: -322, calculated pI: 3.27). The calculated molecular weight of the mature protein is 162 kDa. The well conserved octapeptide is 120 times tandemly arranged. As investigated by Western blot experiments, germlings of P. infestans synthesize the CAR proteins only under starvation conditions; if nutrients, preferentially a carbon source, are available, the proteins do not occur. By immunocytological staining the CAR proteins were localized at the surface of germlings. The structural motif of tandemly repeated oligopeptides is well known from a prominent class of proteins, the mucins of mammals. The P. infestans CAR proteins show 51% partial sequence homology with some subfamilies of mucins exhibiting an octapeptide repeat with the consensus sequence STTSAPTT. Thus, compared to the CAR protein octapeptide, five out of eight amino acids are identical and at the same position. Human mucins are macromolecular acidic glycoconjugates constituting the mucus secreted by various epithelia. Important physiological functions are ascribed to mucins, as protection from pathogenic microorganisms, desiccation, mechanical and chemical damage, pH changes and proteolysis. Because of their homology the CAR proteins may have similar functions significant for pathogenicity of P. infestans. As a mucous cover they may protect the germling from desiccation, physical damage and adverse effects of plant defense mechanisms. In addition, CAR proteins may participate in adhesion to the leaf surface which is essential for the succesful penetration of host cells.

1. Krämer R, Freytag S, Schmelzer E, 1997. European Journal of Plant Pathology 103, 43-53.