Gene Regulatory Machinery and Proteomics of Sexual Reproduction in Phytophthora infestans

Mating is important for the survival of Phytophthora infestans, the causal agent of potato and tomato late blight. The mating process increases population fitness and produces thick-walled oospores that can survive harsh environmental conditions. To better understand the mechanisms that regulate of sexual reproduction in P. infestans, the promoters of two mating-induced genes, PITG_002525 and PITG_00483, were studied in order to identify their cis-regulatory elements. PITG_02525 encodes an elicitin-like protein, and PITG_00483 encodes a PUMILIO (Pum) protein. The promoter of PITG_02525 was sequentially deleted and fused with a beta-glucuronidase reporter gene to identify its regulatory motifs. The full length promoter was found to be active in young and mature, and male and female, structures. The deletion analysis identified a potential repressor element and an activator. Similar studies of the pum promoter showed expression both during oosporogenesis and sporulation. One promoter element was found to be essential for the expression of the gene in both stages. Fusing this element with a minimal promoter indicated that it was sufficient for gene expression during sporulation, but not in mating. The RNA binding domain (RBD) of Pum then was used as a model to develop a system for P. infestans protein interaction studies. Expression of the RBD with a tandem affinity purification tag designed with P. infestans codon bias, allowed the successful purification of the Pum RBD with a potential binding partner. This system is adaptable for future studies of other protein interactions. A MudPIT global proteomics comparison of oospores with nonsporulating hyphae (NSH) was then conducted to profile the protein composition of oospores and identify important proteins for germination. It was proposed that the proteins detected in dormant oospores were synthesized during early oosporogenesis and stored for germination. Our study identified proteins that are more abundant in oospores belonging to several functional groups. These included enzymes involved in carbohydrate and fatty acid oxidation to produce energy, and anti-oxidant and anti-stress proteins that may protect the germ tubes, and secreted proteins that could be involved in host infection. This revealed that P. infestans has a very well designed life cycle