Expression patterns of the poplar NF-Y gene family in response to Alternaria alternata and hormone treatment and the role of PdbNF-YA11 in disease resistance

Abstract

Plant nuclear factor-Y (NF-Y) transcription factors (TFs) are key regulators of growth and stress resistance. However, the role of NF-Y TFs in poplar in response to biotic stress is still unclear. In this study, we cloned 26 PdbNF-Y encoding genes in the hybrid poplar P. davidiana × P. bollena, including 12 PdbNF-YAs, six PdbNF-YBs, and eight PdbNF-YCs. Their physical and chemical parameters, conserved domains, and phylogeny were subsequently analyzed. The protein-protein interaction (PPI) network showed that the three PdbNF-Y subunits may interact with NF-Y proteins belonging to two other subfamilies and other TFs. Tissue expression analysis revealed that PdbNF-Ys exhibited three distinct expression patterns in three tissues. Cis-elements related to stress-responsiveness were found in the promoters of PdbNF-Ys, and most PdbNF-Ys were shown to be differentially expressed under Alternaria alternata and hormone treatments. Compared with the PdbNF-YB and PdbNF-YC subfamilies, more PdbNF-YAs were significantly induced under the two treatments. Moreover, loss- and gain-of-function analyses showed that PdbNF-YA11 plays a positive role in poplar resistance to A. alternata. Additionally, RT‒qPCR analyses showed that overexpression and silencing PdbNF-YA11 altered the transcript levels of JA-related genes, including LOX, AOS, AOC, COI, JAZ, ORCA, and MYC, suggesting that PdbNF-YA11-mediated disease resistance is related to activation of the JA pathway. Our findings will contribute to functional analysis of NF-Y genes in woody plants, especially their roles in response to biotic stress.

Keywords: JA signal; NF-Y transcription factor; disease resistance; expression pattern; poplar.

FIGURE 1

Multiple alignments of the PdbNF-Y family. (A) Multiple alignments of the PdbNF-YA conserved domains. (B) Multiple alignments of the PdbNF-YB conserved domains. (C) Multiple alignments of the PdbNF-YC conserved domains. The logo of the conserved domains was visualized in WebLogo.

FIGURE 2

Phylogenetic analysis of the NF-Y TFs in poplar and Arabidopsis. All NF-Y TFs were clustered into three clades (NF-YA, NF-YB, and NF-YC).

FIGURE 3

Phylogenetic relationships, gene structures, and motif compositions of the PdbNF-Y genes. (A) Unrooted NJ tree. (B) Exon/intron organization. Exons are shown as yellow boxes, and introns are shown as black lines. (C) Schematic representation of conserved MEME motifs of full-length NF-Y proteins. Colored boxes indicate different conserved motifs, while the black lines represent sequences with no MEME motifs detected.

FIGURE 4

Heatmap of cis-elements in the promoter regions of the PdbNF-Ys. Colored bars indicate the numbers of cis-elements.

FIGURE 5

Protein–protein interaction analysis of PdbNF-Ys and other transcription factors. Protein–protein interaction network of PdbNF-Ys and other transcription factors. Colored circles indicate different transcription factors (TFs).

FIGURE 6

Expression profiles of PdbNF-Ys in three poplar tissues. X-axis: different PdbNF-Ys and Y-axis: expression level = log₂ (fold change in expression).

FIGURE 7

Heatmap of PdbNF-Ys expression under biotic stresses. The log₂ fold change was colored using Cluster 3.0 (red for upregulated and green for downregulated). Each horizontal row represents a gene, and the vertical columns represent time points.

FIGURE 8

PdbNF-YA11 enhances resistance to A. alternata in poplar. (A) Leaves of WT and transgenic poplar inoculated with A. alternata plugs for 4 dpi. (B) Relative expression levels of PdbNF-YA11 during infection in WT and transgenic plants. (C) Lesion areas of leaves. (D) Percentage disease index at 4 dpi. The error bars represent the standard deviation of triplicate assays. Asterisks show significant differences between inoculated and noninoculated leaves (**p < 0.01 and *p < 0.05).

FIGURE 9

PdbNF-YA11 regulates JA signaling. (A) JA contents in WT and transformed plants. (B)–(I) Relative expression levels of the JA biosynthetic and JA signaling pathway genes. The error bars represent the standard deviation of triplicate assays. Asterisks show significant differences between inoculated and noninoculated leaves (**p < 0.01 and *p < 0.05).