Functional genomics in the rice blastfungus, Magnapathe oryzae using an RNA siencing approach

我们开发了一个RNA沉默载体pSilent-DuAll(PSD1)，它具有一个聚合的双Pol II启动子系统，为丝状真菌功能基因组学研究提供了一个高通量的平台。在pSD1系统中，目的基因被设计为与增强型绿色荧光蛋白(EGFP)RNA转录成嵌合RNA。这使我们能够使用GFP荧光作为基因沉默的指示器来有效地筛选得到的转化子。对EGFP基因的模型研究表明，基于pSD1的载体通过RNAi途径诱导基因沉默，但效率略低于发夹EGFP RNA表达载体。为了证明pSD1系统在阐明稻瘟病菌基因功能方面的适用性，该载体针对37个钙信号相关基因进行了基因沉默，这些基因几乎包括基因组中所有已知的钙信号蛋白。对沉默的转化子的表型分析表明，在所检测的37个基因中，至少有26个基因、35个基因和15个基因分别与菌丝生长、产孢量和致病性有关。这些发现包括一些新的发现，如PmCL-、SPFL-和Neo1-like Ca^&lt；+&gt；2泵、钙网蛋白和钙化肌动蛋白重链是真菌致病所必需的。

We developed an RNA silencing vector, pSilent-Duall (pSD1), with a convergent dual Pol II promoter system that provides a high-throughput platform for functional genomics research in filamentous fungi. In the pSD1 system, the target gene was designed to be transcribed as a chimeric RNA with enhanced green fluorescent protein (eGFP) RNA. This enabled us to efficiently screen the resulting transformants using GFP fluorescence as an indicator of gene silencing. A model study with the eGFP gene showed that pSD1-based vectors induced gene silencing via the RNAi pathway with slightly lower efficiency than did hairpin eGFP RNA-expressing vectors. To demonstrate the applicability of the pSD1 system for elucidating gene function in the rice blast fungus Magnaporthe oryzae, 37 calcium signaling-related genes that include almost all known calcium signaling proteins in the genome were targeted for gene silencing by the vector. Phenotypic analyses of the silenced transformants showed that at least 26, 35, and 15 of the 37 genes examined were involved in hyphal growth, sporulation and pathogenicity, respectively, in M oryzae. These included several novel findings such as that Pmcl-, Spfl- and Neo1-like Ca^<+>2 pumps, calreticulin, and calpactin heavy chain were essential for fungal pathogenicity.