Activation of Zoosporogenesis-Specific Genes in Phytophthora infestans Involves a 7-Nucleotide Promoter Motif and Cold-Induced Membrane Rigidity

Abstract

Infections of plants by the oomycete Phytophthora infestans typically result from zoospores, which develop from sporangia at cold temperatures. To help understand the relevant cold-induced signaling pathway, factors regulating the transcription of the zoosporogenesis-specific NIF (nuclear LIM-interactor-interacting factor) gene family were examined. Sequences required for inducing PinifC3 were identified by analyzing truncated and mutated promoters using the ␤-glucuronidase reporter in stable transformants. A 7-nucleotide (nt) sequence located 139 bases upstream of the major transcription start point (GGACGAG) proved essential for the induction of PinifC3 when sporangia were shifted from ambient to cold temperatures. The motif, named the cold box, also conferred cold inducibility to a promoter normally activated only during sexual development. An identical motif was detected in the two other zoosporogenesis-specific NIF genes from P. infestans and three Phytophthora sojae orthologues, and a closely related sequence was found in Phytophthora ramorum orthologues. The 7-nt motif was also found in the promoters of other zoosporogenesis-induced genes. The presence of a cold box-interacting protein in nuclear extracts of P. infestans sporangia was demonstrated using electrophoretic mobility shift assays. Furthermore, zoospore release and cold box-regulated transcription were stimulated by the membrane rigidizer dimethyl sulfoxide and inhibited by the membrane fluidizer benzyl alcohol. The data therefore delineate a pathway in which sporangia perceive cold temperatures through membrane rigidity, which activates signals that drive both zoosporogenesis and cold-box-mediated transcription.

Architecture of the Sporulation-Specific Cdc14 Promoter from the Oomycete Phytophthora infestans

“…Site-directed mutagenesis involved a combined PCR method (40) in which A was changed to C, C to A, G to T, and T to G. To mutate block M1, for example, PCR was performed using primer pairs 868f/M1r and M1f/868r, and then the two reaction mixtures were combined and amplified with 868f and 868r, digested with ApaI and ClaI, and cloned into pOGUS. This strategy was also used to mutate blocks M2 to M7 by using primer pairs 868f/M2r and M2f/868r, 868f/M3r and 868r/M3f, 868f/M4r and 868r/M4f, 868f/M5r and 868r/M5f, 868f/M6r and 868r/ M6f, and 868f/M7r and 868r/M7f, respectively.…”

Transgene-Induced Silencing of the Zoosporogenesis-Specific NIFC Gene Cluster of Phytophthora infestans Involves Chromatin Alterations

“…Therefore, the boundary of chromatin alterations is approximately 400 nt upstream of the start codon. This equals 305 nt upstream of the transcriptional start point and 166 nt upstream of the "cold box" transcription factor binding site required for cold-induced NIFC expression (45).…”

Dynamics and Innovations within Oomycete Genomes: Insights into Biology, Pathology, and Evolution

“…The same principle likely underlies the expansion of the genepoor regions of oomycete genomes, as these more than the genedense regions can tolerate transposon-mediated expansion. The small size of most intergenic regions can also be helpful for identifying regulatory motifs through mutagenesis or bioinformatics (1,69,81,93). For example, by searching just upstream of open reading frames for overrepresented motifs, over 100 putative transcription factor binding sites were able to be identified from Phytophthora, of which many were verified by functional assays (69).…”