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Eukaryotic Cell doi:10.1128/EC.00311-06
Copyright (c) 2007, American Society for Microbiology and/or the Listed Authors/Institutions. All Rights Reserved.

Transgene-induced silencing of the zoosporogenesis-specific PiNIFC gene cluster of Phytophthora infestans involves chromatin alterations

Department of Plant Pathology, University of California, Riverside, CA 92521 USA

^* To whom correspondence should be addressed. Email: howard.judelson{at}ucr.edu.

	Abstract

Clustered within the genome of the oomycete phytopathogen Phytophthora infestans are four genes encoding spore-specific nuclear LIM interactor-interacting factors (NIF proteins, a type of transcriptional regulator) that are moderately conserved in DNA sequence. PiNIFC1, PiNIFC2, and PiNIFC3 are zoosporogenesis-induced and grouped within 4 kb, and 20 kb away resides a sporulation-induced form, PiNIFS. To test the function of the PiNIFC family, plasmids expressing full-length hairpin constructs of PiNIFC1 or PiNIFC2 were stably transformed into P. infestans. This triggered silencing of the cognate gene in about one-third of transformants, and all three PiNIFC genes were usually co-silenced. However, PiNIFS escaped silencing despite its high sequence similarity to the PiNIFC genes. Silencing of the three PiNIFC genes impaired zoospore cyst germination by 60%, but did not affect other aspects of the life cycle. Silencing was transcriptional based on nuclear run-on assays, and associated with tighter chromatin packing based on nuclease accessibility experiments. The chromatin alterations extended a few hundred nucleotides beyond the boundaries of the transcribed region of the PiNIFC cluster, and were not associated with increased DNA methylation. A plasmid expressing a short hairpin RNA having sequence similarity only to PiNIFC1 silenced both that gene and an adjacent member of the gene cluster, likely due to the expansion of a heterochromatic domain from the targeted locus. These data help illuminate the mechanism of silencing in Phytophthora, and suggest that caution should be used when interpreting silencing experiments involving closely-spaced genes.