A large-scale complex haploinsufficiency-based genetic interaction screen in Candida albicans: analysis of the RAM network during morphogenesis.
A large-scale complex haploinsufficiency-based genetic interaction screen in Candida albicans: analysis of the RAM network during morphogenesis.
复制标题
DOI：
10.1371/journal.pgen.1002058
复制
发表时间：
2011-04
期刊：
影响因子：
4.5
通讯作者：
Krysan DJ
中科院分区：
文献类型：
--
作者：
Bharucha N;Chabrier-Rosello Y;Xu T;Johnson C;Sobczynski S;Song Q;Dobry CJ;Eckwahl MJ;Anderson CP;Benjamin AJ;Kumar A;Krysan DJ
人类真菌病原体白色念珠菌的酵母和丝状形式之间的形态发生转变受到多种信号通路的调节。然而，这些途径如何相互作用以协调形态发生，还没有得到很好的表征。为了解决这一问题，并确定在表达过程中与Ace 2和形态发生（RAM）途径相互作用的基因，我们报告了第一个大规模的遗传相互作用筛选C。白色念珠菌我们的筛选策略是基于复杂单倍不足（CHI）的概念。对RAM途径关键蛋白激酶CBK 1的杂合突变体（cbk 1 Δ/CBK 1）进行转座子介导的插入突变。筛选所得双杂合突变体（6，528个独立菌株）在蜘蛛培养基（SM）上的降低的表达。从441个突变体中，139个转座子插入位点被测序，产生41个独特的CBK 1相互作用基因。该基因集富含Ace 2的转录靶点，并且引人注目地富含cAMP依赖性蛋白激酶A（PKA）途径，表明这两种途径之间存在相互作用。进一步的分析表明，RAM和PKA途径共调节一组共同的基因在形态发生和PKA途径的过度激活可能会弥补RAM途径功能的丧失。我们的数据还表明，PKA调节的转录因子Efg 1主要定位于酵母相细胞，而RAM途径调节的转录因子Ace 2定位于丝状细胞的子细胞核，这表明Efg 1和Ace 2调节一组共同的基因在不同的阶段形态发生。综上所述，我们的观察表明，基于CHI的筛选是一个有用的方法，遗传互作分析在C。这两种途径在不同的表达阶段调节一组共同的基因。 白色念珠菌是人类真菌感染的最常见原因。作为一种没有典型性周期的二倍体酵母，许多用于模式酵母Saccharomycescerevisiae中大规模遗传互作研究的遗传学方法不能应用于C.白色念珠菌。遗传互作研究已被证明是分析复杂生物过程的有力遗传工具。在这里，我们证明了C.可以产生在两个不同基因中含有杂合突变的白色念珠菌菌株，并用于研究念珠菌中的遗传相互作用。大规模的白色念珠菌。双杂合突变体显示出比具有单杂合突变的菌株更严重的表型，表明通过称为复杂单倍不足（CHI）的现象的遗传相互作用。我们将此方法应用于对C.白色念珠菌从酵母到丝状生长。在与RAM途径的关键信号激酶CBK 1相互作用的基因中，是RAM途径和蛋白激酶A途径的转录靶点。进一步的分析支持了一个模型，其中这两种途径共同调节一组共同的基因在不同阶段的表达。
The morphogenetic transition between yeast and filamentous forms of the human fungal pathogen Candida albicans is regulated by a variety of signaling pathways. How these pathways interact to orchestrate morphogenesis, however, has not been as well characterized. To address this question and to identify genes that interact with the Regulation of Ace2 and Morphogenesis (RAM) pathway during filamentation, we report the first large-scale genetic interaction screen in C. albicans. Our strategy for this screen was based on the concept of complex haploinsufficiency (CHI). A heterozygous mutant of CBK1 (cbk1Δ/CBK1), a key RAM pathway protein kinase, was subjected to transposon-mediated, insertional mutagenesis. The resulting double heterozygous mutants (6,528 independent strains) were screened for decreased filamentation on Spider Medium (SM). From the 441 mutants showing altered filamentation, 139 transposon insertion sites were sequenced, yielding 41 unique CBK1-interacting genes. This gene set was enriched in transcriptional targets of Ace2 and, strikingly, the cAMP-dependent protein kinase A (PKA) pathway, suggesting an interaction between these two pathways. Further analysis indicates that the RAM and PKA pathways co-regulate a common set of genes during morphogenesis and that hyper-activation of the PKA pathway may compensate for loss of RAM pathway function. Our data also indicate that the PKA–regulated transcription factor Efg1 primarily localizes to yeast phase cells while the RAM–pathway regulated transcription factor Ace2 localizes to daughter nuclei of filamentous cells, suggesting that Efg1 and Ace2 regulate a common set of genes at separate stages of morphogenesis. Taken together, our observations indicate that CHI–based screening is a useful approach to genetic interaction analysis in C. albicans and support a model in which these two pathways regulate a common set of genes at different stages of filamentation. Candida albicans is the most common cause of fungal infections in humans. As a diploid yeast without a classical sexual cycle, many genetic approaches developed for large-scale genetic interaction studies in the model yeast Saccharomyces cerevisiae cannot be applied to C. albicans. Genetic interaction studies have proven to be powerful genetic tools for the analysis of complex biological processes. Here, we demonstrate that libraries of C. albicans strains containing heterozygous mutations in two different genes can be generated and used to study genetic interactions in C. albicans on a large scale. Double heterozygous mutants that show more severe phenotypes than strains with single heterozygous mutations are indicative of genetic interactions through a phenomenon referred to as complex haploinsufficiency (CHI). We applied this approach to the study of the RAM (Regulation of Ace2 and Morphogenesis) signaling network during the morphogenetic transition of C. albicans from yeast to filamentous growth. Among the genes that interacted with CBK1, the key signaling kinase of the RAM pathway, were transcriptional targets of the RAM pathway and the protein kinase A pathway. Further analysis supports a model in which these two pathways co-regulate a common set of genes at different stages of filamentation.
登录
查看更多内容
影响因子：
64.5
作者：
Lo, HJ;Kohler, JR;Fink, GR
通讯作者：
Fink, GR
影响因子：
3
作者：
Cloutier, M;Castilla, R;Cantore, ML
通讯作者：
Cantore, ML
影响因子：
4.5
作者：
Homann OR;Dea J;Noble SM;Johnson AD
通讯作者：
Johnson AD
影响因子：
14.9
作者：
Biery, MC;Stewart, FJ;Craig, NL
通讯作者：
Craig, NL
影响因子：
10.5
作者：
Kumar, A;Agarwal, S;Snyder, M
通讯作者：
Snyder, M