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

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. NIFC1, NIFC2, and NIFC3 are zoosporogenesis-induced and grouped within 4 kb, and 20 kb away resides a sporulation-induced form, NIFS. To test the function of the NIFC family, plasmids expressing full-length hairpin constructs of NIFC1 or NIFC2 were stably transformed into P. infestans. This triggered silencing of the cognate gene in about one-third of transformants, and all three NIFC genes were usually cosilenced. However, NIFS escaped silencing despite its high sequence similarity to the NIFC genes. Silencing of the three NIFC 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 NIFC cluster and were not associated with increased DNA methylation. A plasmid expressing a short hairpin RNA having sequence similarity only to NIFC1 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.Strategies for testing the function of genes in eukaryotes often involve transcriptional or posttranscriptional gene silencing methods (TGS and PTGS, respectively), which exploit natural RNA regulation systems of the cell. For example, the canonical RNA interference (RNAi) pathway of PTGS involves introducing or generating within cells double-stranded RNA (dsRNA). This is processed into 20-to 25-nucleotide (nt) small interfering RNAs (siRNAs) by Dicer, which join the RISC complex to direct the cleavage of cognate mRNA through the "slicing" action of Argonaute (28,43). In contrast, TGS is induced traditionally by introducing extra copies of a gene, which repress both the transgene and the native locus through chromatin remodeling, histone modification, and/or DNA methylation (53). However, distinctions between TGS and PTGS are blurry since PTGS may also lead to chromatin alterations, and dsRNA can also trigger TGS if targeted to a promoter (2,23,29). Moreover, in Schizosaccharomyces pombe a single Argonaute protein is required for both PTGS and TGS (41). More knowledge about this machinery is needed to both advance gene silencing methods and understand how it participates in the normal regulation of the transcriptome.Only a few reports exist of the use of silencing methods to test gene function in oomycetes, which are fungus-like eukaryotes that include significant plant and animal pathogens. Genes ...

bZIP Transcription Factors in the Oomycete Phytophthora infestans with Novel DNA-Binding Domains Are Involved in Defense against Oxidative Stress

“…In the case of the latter, a single construct was made that was expected to silence PITG_09190, PITG_09198, PITG_09199, PITG_09200, and PITG_09201, since these exhibit about 96% sequence identity. Two prior studies in P. infestans have shown that single constructs can silence closely related genes (50,51).…”

RNA silencing in fungi

“…Although gene silencing is functional in P. infestans (van West et al, 1999;Latijnhouwers and Govers, 2003;Blanco and Judelson, 2005;Judelson and Tani, 2007) and has been shown to also work in Phytophthora parasitica (Gaulin et al, 2002), the mechanism is poorly understood and to obtain stably silenced strains has been difficult. To facilitate gene function analysis, a different method has been developed based on the delivery of in vitro synthesized dsRNA into protoplasts allowing transient silencing of a gene of interest (Whisson et al, 2005).…”

Optimization of transgene-mediated silencing in Phytophthora infestans and its association with small-interfering RNAs

“…The target of those studies was inf1, which encodes a 10-kDa secreted protein that is easily detected in broth cultures. Application of the method to other Phytophthora genes have subsequently been reported (Blanco and Judelson, 2005;Gaulin et al, 2002;Judelson and Tani, 2007;Latijnhouwers and Govers, 2003;Latijnhouwers et al, 2004). However, these studies reported wide variation in the frequency of silenced transformants, sometimes being very low (<5%).…”

“…However, it is now recognized that the PTGS and TGS pathways may overlap and double-stranded RNA can also trigger TGS (Bernstein and Allis, 2005;Matzke and Birchler, 2005). The nature of silencing reported so far in stable transformants of Phytophthora is TGS, based on nuclear run-on assays (Judelson and Tani, 2007;van West et al, 1999). The PTGS machinery also appears to be present based on transient RNAi experiments (Whisson et al, 2005).…”