Integrated transcriptomic and transgenic analyses reveal potential mechanisms of poplar resistance to Alternaria alternata infection

Abstract

Background: Populus davidiana × P. bollena is a species of poplar from northeastern China that is characterized by cold resistance and fast growth but now suffers from pathogen infections. Leaf blight caused by Alternaria alternata has become a common poplar disease that causes serious economic impacts, but the molecular mechanisms of resistance to A. alternata in P. davidiana × P. bollena are still unclear.

Results: In this study, the transcriptomic response of P. davidiana × P. bollena to A. alternata infection was determined via RNA-Seq. Twelve cDNA libraries were generated from RNA isolated from three biological replicates at four time points (0, 2, 3, and 4 d post inoculation), and a total of 5,930 differentially expressed genes (DEGs) were detected (| log₂ fold change |≥ 1 and FDR values < 0.05). Functional analysis revealed that the DEGs were mainly enriched for the "plant hormone signal transduction" pathway, followed by the "phenylpropanoid biosynthesis" pathway. In addition, DEGs that encode defense-related proteins and are related to ROS metabolism were also identified. Numerous transcription factors, such as the bHLH, WRKY and MYB families, were also induced by A. alternata infection. Among these DEGs, those related to JA biosynthesis and JA signal transduction were consistently activated. Therefore, the lipoxygenase gene PdbLOX2, which is involved in JA biosynthesis, was selected for functional characterization. Overexpression of PdbLOX2 enhanced the resistance of P. davidiana × P. bollena to A. alternata, whereas silencing this gene enhanced susceptibility to A. alternata infection.

Conclusions: These results provide new insight into the molecular mechanisms of poplar resistance to A. alternata infection and provide candidate genes for breeding resistant cultivars using genetic engineering.

Keywords: Alternaria alternate; Defense response; Lipoxygenase gene; Poplar; RNA-Seq.

Fig. 1

Changes in phenotype and biochemical characteristics during infection. A Changes in symptoms in P. davidiana × P. bollena leaves after infection with A. alternata. Images show the disease blotches at 0, 2, 3, and 4 DPI, with enlarged views of portions of infected tissue in the inset. B Lesion areas after infection. C Percentage disease index at 2, 3, and 4 DPI. D-F Contents of SA (D), JA (E) and H₂O₂ (F) in P. davidiana × P. bollena during different stages of infection. G-K Activities of CAT, SOD, POD, PPO and PAL during infection. The error bar represents the standard deviation of triplicate assays. Asterisks show significant differences between the inoculated and noninoculated leaves (**P < 0.01 and *P < 0. 05)

Fig. 2

DEGs between samples and functional annotation. A Numbers of DEGs between two samples (i.e., 0 vs. 2 DPI, 0 vs. 3 DPI, 0 vs. 4 DPI). DEGs are shown in red (upregulated) and gray (downregulated). B Venn diagram of the DEGs in poplar leaves after inoculation with A. alternata. C GO functional enrichment analysis of DEGs. D KEGG pathway enrichment analysis of DEGs. E Identification of differentially expressed transcription factors

Fig. 3

Expression patterns of genes related to the phenylpropane biosynthetic pathway during A. alternata infection. A The synthesis of lignin. B The synthesis of anthocyanin. The log₂ fold change was colored using Cluster 3.0 (red for upregulated, green for downregulated), each horizontal row represents a DEG with its gene ID, and the vertical columns represent 2, 3, and 4 DPI from left to right

Fig. 4

Heat maps of DEGs related to ROS signaling and pathogenesis-related proteins. The log₂ fold change was colored using Cluster 3.0 (red for upregulated, green for downregulated), each horizontal row represents a DEG with its gene ID, and the vertical columns represent 2, 3, and 4 DPI from left to right

Fig. 5

Expression patterns of genes related to JA/ET signaling during A. alternata infection. The log₂ fold change was colored using Cluster 3.0 (red for upregulated, green for downregulated), each horizontal row represents a DEG with its gene ID, and the vertical columns represent 2, 3, and 4 DPI from left to right

Fig. 6

PdbLOX2 enhance the resistance to A. alternata of poplar. A Leaves of WT and transgenic poplar inoculated with A. alternata plugs for 4 DPI and 8 DPI. B Relative expression level of PdbLOX2 during infection in WT and transgenic plants. C Lesion area of leaves. D Percentage disease index at 4 and 8 DPI. The error bar represents the standard deviation of triplicate assays. Asterisks show significant differences compared with the corresponding controls. (**P < 0.01 and *P < 0. 05)

Fig. 7

PdbLOX2 involved in JA and ROS signals. A Activities of LOX in WT and transformed plants. B JA content in WT and over expressed plants. C Contents of H₂O₂ in WT and transformed plants. D Relative expression levels of JA biosynthetic and JA signaling pathway genes. E Activities of enzymes related to ROS scavenging. The error bar represents the standard deviation of triplicate assays. Asterisks show significant differences compared with the corresponding controls (**P < 0.01 and *P < 0. 05)

Fig. 8

Potential mechanism of poplar resistance to A. alternata