Small RNA molecules of about 20 to 30 nucleotides function in gene regulation and genomic defense via conserved eukaryotic RNA interference (RNAi)-related pathways. The RNAi machinery consists of three core components: Dicer, Argonaute, and RNA-dependent RNA polymerase. In fungi, the RNAi-related pathways have three major functions: genomic defense, heterochromatin formation, and gene regulation. Studies of Schizosaccharomyces pombe and Neurospora, and other fungi have uncovered surprisingly diverse small RNA biogenesis pathways, suggesting that fungi utilize RNAi-related pathways in various cellular processes to adapt to different environmental conditions. These studies also provided important insights into how RNAi functions in eukaryotic systems in general. In this review, we will discuss our current understanding of the fungal RNAi-related pathways and their functions, with a focus on filamentous fungi. We will also discuss how RNAi can be used as a tool in fungal research.

). † These authors contributed equally to this work. SummaryThe S-locus F-box (SLF/SFB) protein, recently identified as the pollen determinant of S-RNase-based selfincompatibility (SI) in Solanaceae, Scrophulariaceae and Rosaceae, has been proposed to serve as the subunit of an SCF (SKP1-CUL1-F-box) ubiquitin ligase and to target its pistil counterpart S-RNase during the SI response. However, the underlying mechanism is still in dispute, and the putative SLF-binding SKP1-equivalent protein remains unknown. Here, we report the identification of AhSSK1, Antirrhinum hispanicum SLF-interacting SKP1-like1, using a yeast two-hybrid screen against a pollen cDNA library. GST pull-down assays confirmed the SSK1-SLF interaction, and showed that AhSSK1 could connect AhSLF to a CUL1-like protein. AhSSK1, despite having a similar secondary structure to other SKP1-like proteins, appeared quite distinctive in sequence and unique in a phylogenetic analysis, in which no SSK1 ortholog could be predicted in the sequenced genomes of Arabidopsis and rice. Thus, our results suggest that the pollen-specific SSK1 could be recruited exclusively as the adaptor of putative SCF SLF in those plants with S-RNase-based SI, providing an important clue to dissecting the function of the pollen determinant.

The Neurospora RNA-dependent RNA polymerase QDE-1 is an RNA polymerase that can use both RNA and DNA as templates, suggesting a new mechanism for small RNA production.

As a model system in classical plant genetics, the genus Antirrhinum has been well studied, especially in gametophytic self-incompatibility, flower development biology, and transposon-induced mutation. In contrast to the advances in genetic and molecular studies, little is known about Antirrhinum cytogenetics. In this study, we isolated two tandem repetitive sequences, CentA1 and CentA2, from the centromeric regions of Antirrhinum chromosomes. A standard karyotype has been established by anchoring these centromeric repeats on meiotic pachytene chromosome using FISH. An ideogram based on the DAPIstaining pattern of pachytene chromosomes was developed to depict the distribution of heterochromatin in the Antirrhinum majus genome. To integrate the genetic and chromosomal maps, we selected one or two molecular markers from each linkage group to screen an Antirrhinum transformation-competent artificial chromosome (TAC) library. These genetically anchored TAC clones were labeled as FISH probes to hybridize to pachytene chromosomes of A. majus. As a result, the relationship between chromosomes and the linkage groups (LGs) in Antirrhinum has been established.

These authors contributed equally to this work. SUMMARYRapid actin turnover is essential for numerous actin-based processes. However, how it is precisely regulated remains poorly understood. Actin-interacting protein 1 (AIP1) has been shown to be an important factor by acting coordinately with actin-depolymerizing factor (ADF)/cofilin in promoting actin depolymerization, the rate-limiting factor in actin turnover. However, the molecular mechanism by which AIP1 promotes actin turnover remains largely unknown in plants. Here, we provide a demonstration that AIP1 promotes actin turnover, which is required for optimal growth of rice plants. Specific down-regulation of OsAIP1 increased the level of filamentous actin and reduced actin turnover, whereas over-expression of OsAIP1 induced fragmentation and depolymerization of actin filaments and enhanced actin turnover. In vitro biochemical characterization showed that, although OsAIP1 alone does not affect actin dynamics, it enhances ADF-mediated actin depolymerization. It also caps the filament barbed end in the presence of ADF, but the capping activity is not required for their coordinated action. Real-time visualization of single filament dynamics showed that OsAIP1 enhanced ADF-mediated severing and dissociation of pointed end subunits. Consistent with this, the filament severing frequency and subunit off-rate were enhanced in OsAIP1 over-expressors but decreased in RNAi protoplasts. Importantly, OsAIP1 acts coordinately with ADF and profilin to induce massive net actin depolymerization, indicating that AIP1 plays a major role in the turnover of actin, which is required to optimize F-actin levels in plants.

SummarySelf-incompatibility (SI) is a genetic mechanism to prevent self-fertilization that is found in many species of flowering plants. Molecular studies have demonstrated that the S-RNase and SLF/SFB genes encoded by the single polymorphic S locus, which control the pollen and pistil functions of SI in three distantly related families, the Solanaceae, Scrophulariaceae and Rosaceae, are organized in a haplotype-specific manner. Previous work suggested that the haplotype structure of the two genes is probably maintained by recombination suppression at the S locus. To examine features associated with this suppression, we first mapped the S locus of Antirrhinum hispanicum, a member of the Scrophulariaceae, to a highly heterochromatic region close to the distal end of the short arm of chromosome 8. Both leptotene chromosome and DNA fiber fluorescence in situ hybridization analyses showed an obvious haplotype specificity of the Antirrhinum S locus that is consistent with its haplotype structure. A chromosome inversion was also detected around this region between A. majus and A. hispanicum. These results revealed that DNA sequence polymorphism and a heterochromatic location are associated with the S locus. Possible roles of these features in maintenance of the haplotype specificity involved in both self and non-self recognition are discussed.

Most plant and animal microRNAs (miRNAs) are transcribed by RNA polymerase II. We previously discovered miRNA–like small RNAs (milRNAs) in the filamentous fungus Neurospora crassa and uncovered at least four different pathways for milRNA production. To understand the evolutionary origin of milRNAs, we determined the roles of polymerases II and III (Pol II and Pol III) in milRNA transcription. Our results show that Pol III is responsible for the transcription of the major milRNAs produced in this organism. The inhibition of Pol III activity by an inhibitor or by gene silencing abolishes the production of most abundant milRNAs and pri–milRNAs. In addition, Pol III associates with these milRNA producing loci. Even though silencing of Pol II does not affect the synthesis of the most abundant milRNAs, Pol II or both Pol II and Pol III are associated with some milRNA–producing loci, suggesting a regulatory interaction between the two polymerases for some milRNA transcription. Furthermore, we show that one of the Pol III–transcribed milRNAs is derived from a tRNA precursor, and its biogenesis requires RNase Z, which cleaves the tRNA moiety to generate pre–milRNA. Our study identifies the transcriptional machinery responsible for the synthesis of fungal milRNAs and sheds light on the evolutionary origin of eukaryotic small RNAs.

Catalase is an important enzyme found in nearly all aerobic organisms and plays an essential role in protecting cells from oxidative damage by catalyzing the degradation of hydrogen peroxide into water and oxygen. In filamentous fungus Neurospora crassa, the expression levels of catalases are rigorously regulated by morphogenetic transition during growth and development in cells. Our study revealed that catalase-3 transcription is positively regulated by histone acetyltransferase GCN5 and the cross-pathway control gene cpc-1, as the cat-3 expression level is significantly decreased in gcn5 and cpc-1 (j-5) mutants. Moreover, gcn5 and cpc-1 (j-5) mutants could not respond to HO treatment due to the inadequate cat-3 transcription, while wild-type strains showed high expression levels of catalase upon HO treatment. The global H3 acetylation and the acetylation of H3 at cat-3 locus dramatically decreased in gcn5 under normal or oxidative stress conditions. Meanwhile, the expression of CAT-3 is reduced in gcn5, the catalytically dead mutant, suggesting that the catalytic activity of GCN5 functions in regulation of cat-3 transcription. In addition, GCN5 cannot acetylate histone H3 efficiently at cat-3 locus in cpc-1 (j-5) mutant strains under normal or oxidative stress conditions. Furthermore, ChIP assays data revealed that the CPC1/GCN4 can directly target the cat-3 promoter region, which may recruit GCN5 to modify the histone acetylation of this region. These results disclosed a distinctive function of CPC1/GCN4 in the regulatory pathway of cat-3 transcription, which is mediated by GCN5-dependent acetylation.