Transgene induced co-suppression during vegetative growth in Cryptococcus neoformans.
Transgene induced co-suppression during vegetative growth in Cryptococcus neoformans.
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DOI：
10.1371/journal.pgen.1002885
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发表时间：
2012
期刊：
影响因子：
4.5
通讯作者：
Heitman J
中科院分区：
文献类型：
--
作者：
Wang X;Wang P;Sun S;Darwiche S;Idnurm A;Heitman J
将 DNA 序列引入基因组通常会导致植物、真菌、苍蝇、线虫和哺乳动物等多种生物体中同源依赖性基因沉默。我们之前在新型隐球菌中表明，重复转基因阵列可以在交配期间以高频率诱导基因沉默（〜50％），但在营养生长期间以低得多的频率诱导基因沉默（〜0.2％）。在这里，我们报告了由引入 cpa1::ADE2 转基因引发的强大的无性共抑制现象。 cpa1::ADE2 转基因的多个拷贝异位整合到基因组中，导致编码环孢菌素 A 靶蛋白亲环蛋白 A 的内源性 CPA1 和 CPA2 基因沉默。鉴于在沉默的分离株中检测到 CPA1 衍生的反义 siRNA，并且沉默需要 RNAi 成分（Rdp1、Ago1 和 Dcr2），我们假设 RNAi 途径其中 siRNA 作为反式因子，沉默 CPA1 和 CPA2 基因。 CPA1 和 CPA2 基因的沉默效率与转基因拷贝数相关，并且在存在 > 25 个转基因拷贝时达到~90%。我们将这种转基因沉默现象称为无性共抑制，以区别于相关的性诱导沉默（SIS）过程。我们进一步表明，复制蛋白 A (RPA)（一种单链 DNA 结合复合物）是转基因沉默所必需的，这表明 RPA 可能在异常 RNA 产生中发挥与粗糙脉孢菌抑制作用类似的作用。有趣的是，我们还观察到 ADE2 基因的沉默发生频率比 CPA1/2 基因低得多，即使它存在于相同的转基因阵列中，这表明除了拷贝数之外还有其他因素影响沉默。综上所述，我们的结果表明，转基因诱导的共抑制过程在新型隐球菌营养生长过程中起作用，其与抑制具有共同的机制特征。基因转移方法的发展允许在无数真核生物中产生转基因系。通常，转基因 DNA 会整合到基因组中并作为可遗传的孟德尔性状传播。然而，引入的转基因在某些情况下不表达（沉默）。此外，转基因还可引发与其具有序列同源性的内源基因的沉默。这种现象首先在植物中观察到并命名为共抑制。在真菌中，记录最充分的共抑制现象发生在丝状真菌粗糙脉孢菌的营养组织中，被称为抑制。在这里，我们报告了一种强大的无性共抑制途径，该途径在致病真菌新型隐球菌中发挥作用，并与抑制作用共享分子成分。与先前在新型隐球菌中发现的性诱导沉默（SIS）现象相比，该现象在交配期间（~50%）有效地沉默基因，但在营养生长期间则不然（~0.2%），无性共抑制在营养生长期间有效地抑制转基因表达，并且还可能沉默转座子和其他重复序列。
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 development of gene transfer methods allows the production of transgenic lines in myriad eukaryotes. Frequently, transgenic DNA is integrated into the genome and transmitted as a heritable Mendelian trait. However, the introduced transgenes are in some cases not expressed (silenced). In addition, transgenes can also provoke silencing of endogenous genes with which they share sequence homology. This phenomenon was first observed in plants and named co-suppression. In fungi the best-documented co-suppression phenomenon occurs in vegetative tissue of the filamentous fungus Neurospora crassa and is termed quelling. Here we report a robust asexual co-suppression pathway that operates in the pathogenic fungus Cryptococcus neoformans and shares molecular components with quelling. Compared with the sex induced silencing (SIS) phenomenon previously discovered in C. neoformans, which efficiently silences genes during mating (∼50%) but not during vegetative growth (∼0.2%), asexual co-suppression operates efficiently during vegetative growth to suppress transgene expression and may also silence transposons and other repetitive sequences.
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影响因子：
3
作者：
Janbon, Guilhem;Maeng, Shinae;Yang, Dong-Hoon;Ko, Young-Joon;Jung, Kwang-Woo;Moyrand, Frederique;Floyd, Anna;Heitman, Joseph;Bahn, Yong-Sun
通讯作者：
Bahn, Yong-Sun
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3.3
作者：
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作者：
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通讯作者：
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作者：
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通讯作者：
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影响因子：
64.8
作者：
Cogoni, C;Macino, G
通讯作者：
Macino, G