Quantifying the effect of pine wood nematode invasion on host Pinus massoniana based on multi-omics
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摘要:
目的 探究松材线虫入侵对寄主马尾松的影响。 方法 以4年生马尾松（Pinus massoniana）为试材，以皮接法接入5 000条松材线虫的马尾松为处理组[PWN（+）组]，以接入无菌水的马尾松为对照组[PWN（−）组]，基于多组学（表型组学、转录组学、宏基因组学、代谢组学）量化松材线虫入侵马尾松14 d后的变化。 结果 与PWN(−)组相比，PWN（+）组活性氧（ROS）含量和H2O2含量分别显著上升3.2倍和1.7倍（P＜0.05）；编码应激反应通路的c60547.graph_c0、c82953.graph_c0在PWN（+）组中的表达水平显著高于PWN（−）组（P＜0.05），萜类生物合成途径通路的c64867.graph_c0、c68789.graph_c0及合胞体形成通路的c81022.graph_c0在PWN（+）组中的表达水平显著低于PWN（−）组（P＜0.05）；PWN（+）组的微生物多样性显著低于PWN（−）组（P＜0.05），肉座菌目（Hypocreales）是其体内的优势微生物，在生物信息数据库KEGG的功能注释和丰度信息主要集中在复制修复通路、DNA复制通路（PATH:ko03030）、DNA复制蛋白通路（BR:ko03032）；代谢组上、下调的差异代谢物分别有365和351个，PWN（+）组中根皮素、熊去氧胆酸、羧苄青霉素等物质的含量会增加以抵御松材线虫的侵染，差异显著的代谢物显著富集于ABC转运蛋白通路、花生四烯酸代谢通路、类黄酮生物合成通路、甘油磷脂代谢通路。 结论 当松材线虫入侵马尾松时，寄主会启动一系列复杂的防御反应。这些反应并非孤立存在，而是通过多种机制协同作用，共同应对松材线虫的侵染。上述结果有助于从多组学角度阐明松材线虫入侵对马尾松的影响，并为松材线虫病害诱导的森林衰退和寄主植物马尾松之间的相互作用提供基础参考。 Abstract: :Objective In order to explore the effect of pine wood nematode invasion on the host Pinus massoniana. Method 4-year old Masson pine was used as test material, Masson pine injected with pine wood nematode (PWN(+)) was used as treatment group, and Masson pine injected with ddH2O (PWN(−)) was used as control group in this study. The changes of pine wood nematode invaded Masson pine at 14 days post-inoculation (dpi) were quantified based on multi-omics [phenomics, transcriptome, microbiome and metabolome]. Result The results showed that compared to the PWN(−) group, the PWN(+) group exhibited a significantly increase in reactive oxygen species (ROS) contented by 3.2-fold and hydrogen peroxide (H2O2) contented by 1.7-fold (P＜0.05). The expression levels of c60547.graph_c0 and c82953.graph_c0, unigenes encoding stress responsive pathways, were significantly higher in the PWN(+) group than in the PWN(−) group (P＜0.05). On the other hand, the expression levels of c64867.graph_c0, c68789.graph_c0, terpenoid biosynthesis pathways, and c81022.graph_c0, syncytium formation, were significantly lower in the PWN(+) group compared to the PWN(−) group (P＜0.05). The microbial diversity in the PWN(+) group was significantly lower than in the PWN(−) group (P＜0.05). The order Hypocreales was the dominant microorganism in the PWN(+) group. Functional annotations and abundance information from the bioinformatics database KEGG mainly focused on the replication and repair pathways, DNA replication pathway (PATH:ko03030), and DNA replication protein pathway (BR:ko03032). In the metabolome, there were 365 and 351 differentially regulated metabolites in the upregulated and downregulated categories, respectively. In the PWN(+) group, substances like Phloretin, Ursodiol, and carbenpenicillin increased in content to resist the invasion of PWN. The significantly different metabolites were notably enriched in the ABC transporters pathway. transporters, Arachidonic acid metabolism, Flavonoid biosynthesis, and Glycerophospholipid metabolism. Conclusion When the pine wood nematode invades Masson pine, the host initiates a series of complex defensive responses. These reactions do not exist in isolation, but work together through various mechanisms to cope with the infection of pine wood nematode. These results contribute to a multi-omic understanding of the impact of PWN invasion on Masson pine and provide a foundational reference for exploring the interactions between forest decline induced by PWN infestation and its host plant, Masson pine. -
图 1 松材线虫接种效果的验证
以PWN作为阳性对照，以ddH2O作为阴性对照。
Figure 1. Validation of PWN inoculation efficiency
PWN was used as a positive control and ddH2O was used as a negative control.
图 2 有无接种松材线虫对寄主马尾松ROS（a）和H2O2（b）含量的影响
不同小写字母代表差异显著（P＜0.05）。
Figure 2. Effects of PWN inoculation on ROS(a) and H2O2(b) contents in P. massoniana
Different lowercase letters represent significant difference(P＜0.05).
图 3 松材线虫入侵前后马尾松体内抗性基因的变化
Figure 3. Changes of resistance genes in P. massoniana before and after PWNinvasion
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图 4 不同处理条件下马尾松微生物群的多样性和结构功能
不同处理组微生物群落的多样性Shannon（a）、Simpson（b）、Invsimpson（c）指数差异，不同小写字母代表差异显著（P＜0.05）；（d）代表样本在目水平上的聚类柱状图；（e）样本微生物在KEGG level3水平上具有显著性差异的功能预测。
Figure 4. Diversity and structural function of microflora of P. massoniana under different treatment conditions
The diversity of microbial communities in different treatment groups was different in Shannon (a), Simpson (b) and Invsimpson (c) indexes, and different lowercase letters represented significant differences (P＜0.05) ; (d)which represents the clustering histogram of the samples at the order level; (e) the functional prediction of the microorganisms at the KEGG level3 level.
图 5 松材线虫入侵前后诱导的马尾松代谢物组成
（a） 不同处理的OPLS-DA图。X轴：第一主成分的预测主成分得分，Y轴：正交主成分得分，散点形状和颜色表示不同的实验分组；（b）不同处理间差异代谢物筛选火山图。每个点代表一个代谢物，X轴：该组对比各物质的倍数变化，Y轴：t-检验的P-value，散点颜色代表最终的筛选结果，红色、绿色和灰色分别表示显著差异上调的代谢物、显著差异下调的代谢物和非显著差异的代谢物；（c）不同处理的差异代谢物VIP散点图。显示基于VIP值的前20种差异代谢物，X轴：OPLS-DA模型计算的VIP值，颜色代表不同代谢物的表达模式；(d)不同处理的代谢通路分析气泡图。X轴：每条通路的Impact，Y轴：通路名称，气泡颜色表示富集分析的P-value，颜色越红则富集程度越显著，点的大小代表富集到该通路的差异代谢物的个数；（e）关于不同处理的所有差异代谢物的层次聚类分析热图。X轴：不同样本，Y轴：所有组合的差异代谢物，不同位置的色块代表对应位置代谢物的相对表达量。
Figure 5. Metabolite composition of P. massoniana induced before and after invasion of pine wood nematode
(a) OPLS-DA plots for different treatments. X-axis: predicted principal component score of the first principal component, Y-axis: orthogonal principal component score, scatter shape and color indicate different experimental groupings. (b) differential metabolite screening volcano plot between different treatments. Each dot represents a metabolite, X-axis: fold change of each substance in this group, Y-axis: P-value of t-test, scatter color represents the final screening results, red, green and gray represent significantly differentially upregulated metabolites, significantly differentially downregulated metabolites and non-significantly differentially differentiated metabolites. (c) VIP scatter plots of differentially differentiated metabolites under different treatments. The top 20 differential metabolites based on VIP values are displayed, X-axis: VIP values calculated by OPLS-DA model, and the colors represent the expression patterns of different metabolites;(d) X-axis: Impact of each pathway, Y-axis: pathway name, bubble color represents the P-value of enrichment analysis, the redder the color, the more significant enrichment, and the size of the dot represents the number of differential metabolites enriched to the pathway; (e) Hierarchical cluster analysis heat map of all differential metabolites of different treatments. X-axis: different samples, Y-axis: differential metabolites for all combinations, and the color patches at different locations represent the relative expression levels of metabolites at the corresponding locations.
表 1 特异引物
Table 1. Specific primers
引物名称
Primer name正向引物（5′-3′）
Forward primer（5′-3′）反向引物（5′-3′）
Reverse primer（5′-3′）Internal control AACGTCATTTCTAGCCGCCA TCAGCCCTACAAACCCCTCT Bx-cathepsin TTGCATTCTACGGCCAGTCC ACTGACTTTCGATGGCTCCG c60547.graph_c0 TAAATTCCAAGTGCCCCGCA ACCGTGATACACATTTCAGA c64867.graph_c0 AAATCGTGTGTGTCCCTGCA GGTTGCAATGATAACGGCCC c68789.graph_c0 CGCCCGAATCTCTGCACTTA TCGATGGTCTTGGTGATGGC c81022.graph_c0 TTGGCTGTACAGATTCCCGT ACCTATGGATGTCTGCTCCA c82953.graph_c0 ACTGTTAACCTGGCTCACGG CTACGCAAATTCACCGCCAC 
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