Abstract
The small cellulose-binding-domain protein CBD1 is tightly bound to the cellulosic cell wall of the plant pathogenic stramenopile Phytophthora infestans. Transgene expression of the protein in potato plants also demonstrated binding to plant cell walls. A study was undertaken using 47 isolates of P. infestans from a worldwide collection, along with 17 other Phytophthora species and a related pathogen Plasmopara halstedii, to determine if the critical cell wall protein is subject to amino acid variability. Within the amino acid sequence of the secreted portion of CBD 1, encoded by the P. infestans isolates, 30 were identical with each other, and with P. mirabilis. Four isolates had one amino acid difference, each in a different location, while one isolate had two amino acid substitutions. The remaining 13 isolates had five amino acid changes that were each in identical locations (D17/G, D31/G, I32/S, T43/A, and G50/A), suggesting a single origin. Comparison of P. infestans CBD1 with other Phytophthora species identified extensive amino acid variation among the 60 amino acids at the amino terminus of the protein, and a high level of conservation from G61, where the critical cellulose-binding domain sequences begin, to the end of the protein (L110). While the region needed to bind to cellulose is conserved, the region that is available to interact with other cell wall components is subject to considerable variation, a feature that is evident even in the related genus Plasmopara. Specific changes can be used in determining intra- and inter-species relatedness. Application of this information allowed for the design of species-specific primers for PCR detection of P. infestans and P. sojae, by combining primers from the highly conserved and variable regions of the CBD1 gene.
Highlights
Cellulose-binding domains (CBD), found within the larger class of protein structural patterns known as carbohydrate binding modules (CBM), facilitate binding to target substrates
We have focused on CBD1 and compared conservation of sequence in a worldwide collection of P. infestans (USDA-ARS, Beltsville, MD)
The highest degree of similarity within the Phytophthora infestans CBD 1 amino acid sequence was centered on the carboxyl half of the protein where the specific amino acids involved in cellulose binding are located
Summary
Cellulose-binding domains (CBD), found within the larger class of protein structural patterns known as carbohydrate binding modules (CBM), facilitate binding to target substrates. In addition to aiding the binding of enzymes, the CBDs can have the ability to dissociate cellulose microfibrils independently, as shown with CBDs produced in protein expression systems [9, 15, 34]. Phytophthora species are hemibiotrophic plant pathogens that generally lack a CBD on their cellulolytic enzymes [6, 7, 32]; they are one of the few organisms that produce CBDs as discrete proteins [12, 18, 28]. The Phytophthora CBD-containing proteins generally harbor the binding domains at the carboxy terminal region of the protein, while the function of the amino half of the proteins remains undefined.
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