RNA silencing in the model mycorrhizal fungus Laccaria bicolor: gene knock-down of nitrate reductase results in inhibition of symbiosis with Populus
RNA silencing in the model mycorrhizal fungus Laccaria bicolor: gene knock-down of nitrate reductase results in inhibition of symbiosis with Populus
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DOI：
10.1111/j.1462-2920.2009.01912.x
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发表时间：
2009-07-01
影响因子：
5.1
通讯作者：
Pardo, Alejandro G.
中科院分区：
文献类型：
--
作者：
Kemppainen, Minna;Duplessis, Sebastien;Pardo, Alejandro G.
菌根共生是自然界的一种规律，可能在植物和真菌的进化中起着至关重要的作用。外生菌根是温带和北方森林中典型的树根和土壤真菌之间的相互作用。对参与发育和代谢过程的基因进行功能分析，如N营养，对于理解这种互惠共生的个体发育很重要。通过农杆菌介导的基因转移，在模式菌根真菌二色漆树中实现了RNA沉默。利用启动子控制的双链RNA和部分编码序列的Laccaria硝酸还原酶基因的表达，导致了以硝酸盐为氮源的真菌转基因菌株在高浓度可利用碳源的条件下生长受到强烈影响。这种表型与靶基因mRNA水平的明显降低有关，这种影响不是由硝酸还原酶基因座上T-DNA的同源重组引起的。用发夹序列转化导致沉默的转基因和硝酸还原酶编码基因的特异性CpG甲基化。目的基因的甲基化仅限于沉默触发序列，并不代表双核细胞中的整个基因组DNA，这表明伴随RNA沉默的表观遗传变化只影响转化的核。与野生型相比，杨树与强沉默真菌菌株的菌化实验表明，在菌化条件下(C饥饿)和硝酸盐作为氮源时，杨树的共生受到系统的抑制。这种抑制真菌生长的作用被只被真菌利用的有机氮源逆转。这些观察将表明，植物可能能够监测和检测潜在共生体的营养状况，避免建立不令人满意的相互作用。当真菌伴侣的代谢谱不合适，共生结构的互惠不能保证时，由植物进行的可能的控制机制将抑制共生。我们的结果是首次报道了真菌基因表达的改变会损害真菌的菌丝化。此外，这项工作首次证明了菌根真菌中的RNA沉默，并清楚地表明基因敲除是菌根研究中进一步功能基因组学研究的有力工具。
P>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.