丝状真菌中的RNA干扰及其应用技术

RNA干扰是真核生物中相对保守的一种基因特录后表达调控机制,它通过双链RNA介导细胞内mRNA发生特异性降解或翻译抑制,从而调控靶基因的表达.对丝状真菌中RNA压制和减数分裂沉默等现象的研究表明,与动、植物一样,丝状真菌中也存在RNA干扰现象.通过对RNA压制缺失突变株和减教分裂沉默缺失突变株的一系列分子生物学研究,获得了与之密切相关的一系列蛋白,而这些蛋白在结构和功能上与动、植物RNA干扰途径的蛋白高度相似,这些结果证明了丝状真菌中的RNA存在干扰现象.RNA干扰技术作为丝状真菌分子生物学研究或遗传改造的工具具有特殊的意义,因为丝状真菌具有多核和发生非同源重组频率高的特点,难以用基因敲除手段进行改造.系统地介绍丝状真菌中的RNA干扰途径以及使用RNA干扰对真菌进行遗传改造的方法.     

Coexpression of Escherichia coli RNase III in silkworm cells improves the efficiency of RNA interference induced by long hairpin dsRNAs

Abstract Long hairpin dsRNA transcribed from chromosomal DNA can induce RNA interference in Bombyx mori cells, although its gene silencing efficiency is lower than that of exogenously introduced double‐stranded RNAs (dsRNAs). To solve this problem, we monitored the nuclear cytoplasmic translocation of the transcribed hairpin dsRNA and analyzed the processing efficiency into mature small interfering RNA (siRNA). Northern blot analysis revealed that the transcribed hairpin dsRNAs were spliced and transported into the cytoplasm, but were not effectively diced into siRNAs. Interestingly, RNAi with hairpin dsRNAs from genome‐integrated IR transgene was stimulated by the coexpression of Escherichia coli RNase III, although this exogenous enzyme seemed to bring about nonspecific cleavage of cellular mRNA.     

RNA interference-mediated gene knockdown within specific cell types

In plants, RNA interference (RNAi)-induced gene silencing can spread from the initiation site to nearby cells. The silencing signal moves from cell-to-cell through plasmodesmata and, over long distances, through the phloem. In this study, we employed a nuclear-localized GFP fusion protein to visualize the pattern of gene silencing induced by three different transgenes expressing double-stranded RNA (dsRNA) in Arabidopsis root tips. In all cases, we found that dsRNA-induced silencing did not spread from the silencing initiation site to adjacent cells. In the first set of experiments, in a transgenic background expressing nuclear-localized GFP within a contiguous cell layer that included endodermis, cortex/endodermis (joint) initial (CEI) cells and the quiescent center (QC) cells, expression of the marker gene was silenced specifically in the QC cells without affecting gene expression in the adjacent CEI and endodermal cells. The next two sets of experiments examined the knockdown of two endogenous genes. We observed that silencing was completely restricted to the QC and endodermal cells within which the dsRNA transgenes were expressed. Overall, these results accentuate one important aspect of RNAi-induced gene silencing, that it can be cell autonomous, and demonstrated the feasibility of selective gene knockdown within specific cell types.     

Transgene Induced Co-Suppression during Vegetative Growth in Cryptococcus neoformans

Introduction of DNA sequences into the genome often results in homology-dependent gene silencing in organisms as diverse as plants, fungi, flies, nematodes, and mammals. We previously showed in Cryptococcus neoformans that a repeat transgene array can induce gene silencing at a high frequency during mating (～50%), but at a much lower frequency during vegetative growth (～0.2%). Here we report a robust asexual co-suppression phenomenon triggered by the introduction of a cpa1::ADE2 transgene. Multiple copies of the cpa1::ADE2 transgene were ectopically integrated into the genome, leading to silencing of the endogenous CPA1 and CPA2 genes encoding the cyclosporine A target protein cyclophilin A. Given that CPA1-derived antisense siRNAs were detected in the silenced isolates, and that RNAi components (Rdp1, Ago1, and Dcr2) are required for silencing, we hypothesize that an RNAi pathway is involved, in which siRNAs function as trans factors to silence both the CPA1 and the CPA2 genes. The silencing efficiency of the CPA1 and CPA2 genes is correlated with the transgene copy number and reached ～90% in the presence of >25 copies of the transgene. We term this transgene silencing phenomenon asexual co-suppression to distinguish it from the related sex-induced silencing (SIS) process. We further show that replication protein A (RPA), a single-stranded DNA binding complex, is required for transgene silencing, suggesting that RPA might play a similar role in aberrant RNA production as observed for quelling in Neurospora crassa. Interestingly, we also observed that silencing of the ADE2 gene occurred at a much lower frequency than the CPA1/2 genes even though it is present in the same transgene array, suggesting that factors in addition to copy number influence silencing. Taken together, our results illustrate that a transgene induced co-suppression process operates during C. neoformans vegetative growth that shares mechanistic features with quelling.     

The role of RNA editing by ADARs in RNAi

Adenosine deaminases that act on RNA (ADARs) are RNA-editing enzymes that deaminate adenosines to create inosines in double-stranded RNA (dsRNA). Here we demonstrate that ADARs are not required for RNA interference (RNAi) and that they do not antagonize the pathway to a detectable level when RNAi is initiated by injecting dsRNA. We find, however, that transgenes expressed in the somatic tissues of wild-type animals are silenced in strains with deletions in the two genes encoding ADARs, adr-1 and adr-2. Transgene-induced gene silencing in adr-1;adr-2 mutants depends on genes required for RNAi, suggesting that a dsRNA intermediate is involved. In wild-type animals we detect edited dsRNA corresponding to transgenes, and we propose that editing of this dsRNA prevents somatic transgenes from initiating RNAi in wild-type animals.     

RNA silencing in the model mycorrhizal fungus Laccaria bicolor: gene knock-down of nitrate reductase results in inhibition of symbiosis with Populus

Mycorrhizal symbioses are a rule in nature and may have been crucial in plant and fungal evolution. Ectomycorrhizas are mutualistic interactions between tree roots and soil fungi typical of temperate and boreal forests. The functional analysis of genes involved in developmental and metabolic processes, such as N nutrition, is important to understand the ontogeny of this mutualistic symbiosis. RNA silencing was accomplished in the model mycorrhizal fungus Laccaria bicolor by Agrobacterium‐mediated gene transfer. Promoter‐directed expression of double‐stranded RNA with a partial coding sequence of the Laccaria nitrate reductase gene resulted in fungal transgenic strains strongly affected in growth with nitrate as N source in a medium with high concentration of an utilizable C source. The phenotype correlated with a clear reduction of the target gene mRNA level and this effect was not caused by homologous recombination of the T‐DNA in the nitrate reductase locus. Transformation with the hairpin sequence resulted in specific CpG methylation of both the silenced transgene and the nitrate reductase encoding gene. The methylation in the target gene was restricted to the silencing trigger sequence and did not represent the entire genomic DNA in the dikaryon suggesting that the epigenetic changes accompanying RNA silencing affected only the transformed nucleus. Mycorrhization experiments of Populus with strongly silenced fungal strains revealed a systematic inhibition of symbiosis under mycorrhization conditions (C starvation) and nitrate as N source compared with the wild type. This inhibition of mycorrhization was reversed by an organic N source only utilizable by the fungus. These observations would indicate that the plant may be capable of monitoring and detecting the nutritional status of a potential symbiont avoiding the establishment of an unsatisfactory interaction. A probable control mechanism conducted by the plant would inhibit symbiosis when the metabolic profile of the fungal partner is not proper and mutual benefit from the symbiotic structure cannot be assured. Our results are the first report showing that the alteration of expression of a fungal gene impairs mycorrhization. Moreover, this work is the first demonstration of RNA silencing in mycorrhizal fungi and clearly shows that gene knock‐down is a powerful tool for further functional genomic studies in mycorrhizal research.