Multiomically Quantified Effects of Nematode Invasion on Pinus massoniana
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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依据。 Abstract:Objective Effects of pine wood nematode (PWN) invasion on Pinus massoniana were quantified based on multiomics. Method Among 4-year-old Masson pine plants, a treatment, PWN(+), by inoculating 5,000 Bursaphelenchus xylophilus and a control, PWN(−), with the injection of sterile distilled water into the tree trunks were subjected to a 14d post-inoculation followed by a quantified study including phenomics, transcriptome, microbiome, and metabolome. Result The pine trees in the PWN(+) group significantly increased over control on the contents of the reactive oxygen species (ROS) by 3.2-fold and hydrogen peroxide (H2O2) by 1.7-fold (P<0.05), on the expressions of c60547.graph_c0 and c82953.graph_c0 in the unigenes encoding stress responsive pathways (p<0.05), as well as on the expressions of c64867.graph_c0 and c68789.graph_c0 in the terpenoid biosynthesis pathways; and significantly lower on the c81022.graph_c0 in syncytium formation (p<0.05) and the microbial diversity (p<0.05). Hypocreales was the dominant order of microorganisms in the PWN(+) group. The functional annotations and abundance information from the KEGG database were mainly related to the replication and repair pathways, DNA replication pathway (PATH:ko03030), and DNA replication protein pathway (BR:ko03032). There were 365 upregulated and 351 downregulated differentially regulated metabolites in the metabolome. Metabolites like phloretin, ursodiol, and carbenpenicillin were notably enriched in the ABC transporters pathway, arachidonic acid metabolism, flavonoid biosynthesis, and glycerophospholipid metabolism in the PWN-infected pine wood to resist the invasion. Conclusion After PWN was inoculated in Masson pine, the host responded with a series of complex defensive reactions initiated by various mechanisms. Through multiomics, some such interactions between the species were unveiled providing clues for combating forestry declination by the infestation. -
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 functions of microflora in P. massoniana under varied conditions
Diversity of microbial communities in different sample groups differed in Shannon (a), Simpson (b), and Invsimpson (c) indexes; data with different lowercase letters represented significant differences at P<0.05; d: clustering histogram of samples at order level; e: functional prediction of microorganisms at KEGG level 3.
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. Metabolites in P. massoniana before and after PWN inoculation
(a) OPLS-DA plots for different treatments; X-axis: predicted principal component score of 1st principal component; Y-axis: orthogonal principal component score with varied scatter shapes and colors indicating different groups. (b): differential metabolite screening volcano plot between different groups; each dot represents a metabolite; X-axis: change by fold of each substance in the group; Y-axis: P-value of t-test; scatter color represents final screening results with red representing significantly differentially upregulated metabolites, green representing significantly differentially downregulated metabolites, , and gray representing non-significantly differentially differentiated metabolites. (c): VIP scatter plots of differentially differentiated metabolites in different groups; top 20 differential metabolites based on VIP values are displayed; X-axis: VIP values calculated by OPLS-DA model; color represent expression patterns of metabolites. (d): X-axis: Impact of each pathway; Y-axis: pathway name with bubble color representing P-value of enrichment analysis (the redder the color, the more significant enrichment), and dot size representing number of differential metabolites enriched in pathway. (e): Hierarchical cluster analysis heat map of all differential metabolites of groups. X-axis: different samples; Y-axis: differential metabolites for all combinations with color patches at different locations representing relative expression levels of metabolites at corresponding locations.
表 1 特异引物
Table 1. Specific primers
引物名称
Primer name正向引物
Forward primer(5′-3′)反向引物
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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