WGCNA分析应用——真菌与宿主互作的分子机制--菌根共生

Dual RNA-seq reveals large-scale non-conserved genotype × genotype-specific genetic reprograming and molecular crosstalk in the mycorrhizal symbiosis9.493

. 2019 May; 13(5): 1226–1238.

Published online 2019 Jan 15. doi: 10.1038/s41396-018-0342-3

Abstract

Arbuscular mycorrhizal fungi (AMF) impact plant growth and are a major driver of plant diversity and productivity. We quantified the contribution of intra-specific genetic variability in cassava (Manihot esculenta) and Rhizophagus irregularis to gene reprogramming in symbioses using dual RNA-sequencing. A large number of cassava genes exhibited altered transcriptional responses to the fungus but transcription of most of these plant genes (72%) responded in a different direction or magnitude depending on the plant genotype. Two AMF isolates displayed large differences in their transcription, but the direction and magnitude of the transcriptional responses for a large number of these genes was also strongly influenced by the genotype of the plant host. This indicates that unlike the highly conserved plant genes necessary for the symbiosis establishment, most of the plant and fungal gene transcriptional responses are not conserved and are greatly influenced by plant and fungal genetic differences, even at the within-species level. The transcriptional variability detected allowed us to identify an extensive gene network showing the interplay in plant–fungal reprogramming in the symbiosis. Key genes illustrated that the two organisms jointly program their cytoskeleton organization during growth of the fungus inside roots. Our study reveals that plant and fungal genetic variation has a strong role in shaping the genetic reprograming in response to symbiosis, indicating considerable genotype × genotype interactions in the mycorrhizal symbiosis. Such variation needs to be considered in order to understand the molecular mechanisms between AMF and their plant hosts in natural communities.

Subject terms: Fungal ecology, Microbial ecology


1.主要亮点内容：WGCNA还可以用来分析两个物种之间的关联性？

2.数据

2.1样本：作者设计了双因素实验，总共15个样本

  • 木薯品种：CM6438-14, COL2215, BRA337, CM4574-7, CM523-7 ，每个品种三株。

  • 真菌因素：三组，DAOM17978，B1，mock(对照)，每一组处理包含五个木薯品种

说的有点绕，来个一图看懂数据实验设计：

2.2测序数据

  • 选取每一株植物的几个细根须(several fine root samples)

  • 提取RNA

  • 建库

  • 上机测序

  • 针对测序数据分别比对真菌参考基因组和木薯物种参考基因组得到两份表达数据用于后续分析。

2.3表型性状

用到了以下性状：

  1. Fungal colonization

  2. Aboveground plant dry weight (g)

  3. Fine roots plant dry weight (g)

  4. Tuberized roots plant dry weight (g)

  5. Total plant dry weight (g)

  6. Belowground plant dry weight (g)

  7. Height (cm)

数据如下（由于篇幅有限，截取部分样本展示，完整数据在文章Supplementary data2）：

3.结果

这篇文章主要用WGCNA做了：

1.挖掘出与植物基因和性状相关的模块，总共识别出了9个模块。

注意点：这里作者用来做WGCNA分析的基因是前面做差异表达分析的并集。

we used the pool of cassava genes that were significantly differentially expressed between the two AMF treatments and the nonmycorrhizal treatment to build cassava gene modules.   

2.挖掘出与真菌基因和性状相关的模块，总共识别出了20个模块

注意点：与前面用差异基因并集不一样，这里采用的是全部的真菌相关基因。

Because AMF can only be grown in presence of the host plant, we could not select AMF genes that were only involved in the symbiosis. In order to obtain a set of fungal transcripts in non-symbiotically growing fungal material would require germinating spores and sequencing their transcriptome. This would have to be done in a different environment to which these experiments were conducted and would not serve as an appropriate control for detection of true symbiotically-upregulated genes. We, therefore, selected all the R. irregularis genes in the dataset in order to build R. irregularis gene modules.   

由于作者比较关心真菌与木薯之间的共生分子机制，因此特别关注了fungal colonization这个性状识别出的两个显著相关的真菌基因模块，F_black（cor=-0.54,pvalue=0.002）和F_royalblue(cor=-0.59,pvalue=0.0006)；木薯基因中P_magenta模块（cor=-0.49,pvalue=0.006）。然后，根据GS值，挑选出模块中的‘’key gene“，每个模块中的key gene以及相关的功能展示如下。

其中，我们可以看到F_black模块中的a fungal chitin synthase（真菌产生的几丁质合成酶）和p_magenta模块中a plant sucrose synthase （植物的蔗糖合酶）功能相关基因与the fungal colonization 关联，而植物糖的运输正是真菌共生体内碳水化合物分配的重要环节。

3.利用WGCNA中的相关性方法将植物模块和真菌模块联系起来来挖掘菌根互作的共生机制。原文如下：   

We associated the modules between the two organisms by measuring the Pearson correlation between cassava module eigengenes and R. irregularis module eigengenes using the default WGCNA ‘relating modules to external information’ analysis.   

这个就是全文用WGCNA的一个主要的亮点了，借用了WGCNA的功能将了两个物种之间的模块关联起来。图中红色的点表示真菌基因模块，绿色的点表示木薯基因模块，边表示这两个模块之间有显著关联。

随后，作者将两个物种中有关联并且与表型性状有显著关系的子网提出出来进行了生物学意义的解读。如下图，我们可以看到真菌相关的模块和木薯相关的木块紧密的联系在一起，在这些紧密链接的模块中，我们看到了与细胞骨架的形成（cytoskeleton formation），细胞组织（cell organization ），肌动蛋白和肌球蛋白基因（actin and myosin genes）功能相关的key gene。

对于发现的这些生物学过程，arbuscule formation inside the plant cells已经得到了广泛的研究, 而关于cellular organization 和 cytoskeleton在共生的晚期阶段的变化还很少有研究。

总结：

技术点：作者选用相关基因进行WGCNA的时候，是根据自己实际情况做了筛选，比如用于分析真菌模块的基因和用于分析木薯模块的基因选取策略就不一样。

生物学角度：作者用一套测序数据比对两个参考基因组得到两个物种相关的表达数据，然后分别进行WGCNA模块挖掘，最后分析了两个物种模块之间的关联性，以此揭示了真菌与宿主之间的一个分子生物学的共生机制。

转自生信人

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