WRKY is an important complex family of transcription factors involved in plant immune responses. Among them, WRKY70 plays an important role in the process of the plant defense response to the invasion of pathogens. However, the defense mechanism of PsnWRKY70 is not clear in Populus nigra. In this study, we showed that PsnWRKY70-overexpression lines (OE) had fewer leaf blight symptoms than PsnWRKY70-repressing lines (RE). PsnWRKY70 activated MAP kinase cascade genes (PsnM2K4, PsnMPK3, PsnM3K18), calcium channel proteins-related genes (PsnCNG3, PsnCNGC1, PsnCNG4), and calcium-dependent protein kinases genes (PsnCDPKL, PsnCDPKW, PsnCDPKS, PsnCDPKQ). Furthermore, 129 genes of PsnWRKY70 putative genome-wide direct targets (DTGs) were identified by using transcriptome (RNA-seq) and DNA affinity purification sequencing (DAP-seq). PsnWRKY70 directly binds to the promoters of homologous genes and LRR domain proteins to promote the expression of WRKY6, WRKY18, WRKY22, and WRKY22–1, LRR domain proteins LRR8, LRR-RLK, ADR1-like 2, NB-ARC, etc. Our study suggests that PsnWRKY70 enhances the resistance of A. alternata in poplar by activating genes in both pathogen-associated molecular pattern-triggered immunity (PTI) and effector-triggered immunity (ETI).

Plant polyploidization changes its leaf morphology and leaf development patterns. Understanding changes in leaf morphology and development patterns is a prerequisite and key to studying leaf development in polyploid plants. In this study, we quantified and analyzed the differences in leaf morphology, leaf growth polarity, and leaf size between diploid and tetraploid birches (Betula pendula subsp. pendula), and preliminarily investigated genes involved in leaf growth and development in birch. The results showed significant changes in leaf morphology in tetraploid birches, especially the basal part of the leaf. In addition, the proximal growth rate of tetraploid leaves was altered. The changed proximal growth rate did not affect the growth polarity pattern of tetraploid leaves. The leaf area of tetraploid was significantly larger than that of diploid birch. The difference in leaf size was mainly due to differences in their growth rates in the middle and late stages of leaf development. Increased cell expansion capacity was the major reason for the enormous leaves of tetraploid birch; however, cell proliferation did not contribute to the larger tetraploid leaf. The gene expression of ATHB12 was associated with cell size and leaf area, and may be a critical gene affecting the leaf size in diploid and tetraploid birches. The results will provide valuable insights into plant polyploid leaf development and a theoretical basis for later investigations into the molecular mechanisms underlying the gigantism of tetraploid birch leaves.

Poplar is an important afforestation and ornamental tree species in Northeast China. The distribution area of saline-alkali land is approximately 765 hm² in Northeast China. The breeding of saline-alkali-resistant transgenic trees could be an effective method of afforestation in saline-alkali land. WRKY transcription factors play a crucial role in abiotic stress. In this study, we analyzed the genetic stability of the two-year-old PsnWRKY70 transgenic poplars. The results showed that PsnWRKY70 of transgenic poplars had been expressed stably and normally at the mRNA level. The gene interference expression (RE) lines had no significant effect on the growth of PsnWRKY70 under NaHCO₃ stress, and the alkali damage index of RE lines was significantly lower than that of WT and overexpression (OE) lines at day 15 under NaHCO₃ stress. POD activity was significantly higher in RE lines than in WT. The MDA content of the RE line was lower than that of the WT line. Transcriptome analysis showed that RE lines up-regulated genes enriched in cell wall organization or biogenesis pathway-related genes such as EXPA8, EXPA4, EXPA3, EXPA1, EXPB3, EXP10, PME53, PME34, PME36, XTH9, XTH6, XTH23, CESA1, CESA3, CES9; FLA11, FLA16 and FLA7 genes. These genes play an important role in NaHCO₃ stress. Our study showed that the interference expression of the PsnWRKY70 gene can enhance the tolerance of NaHCO₃ in poplar.

WRKY transcription factors have been implicated in the regulation of disease resistance associated with plant immune responses, which has crucial implications for defense responses against stress in plants. The role played by the PsnWRKY70 gene of Populus ( Populus simonii × P. nigra ) in triggering the mechanism between the phyllosphere microbiome and plant defense against foliar pathogens remains unclear. Molecular ecological network analysis demonstrated that the stability and complexity of the phyllosphere bacterial community of Populus were influenced by Alternaria alternata infection. Specifically, compared to the wild-type line, the PsnWRKY70 -overexpressing (OE) line had a higher average clustering coefficient and modularity. Furthermore, metabolomic analysis revealed that 19 differential metabolites were significantly enriched in the leaves of the OE line. Among these metabolites, coumarin compounds, such as fraxetin-8-O-glucoside (fraxin) and scopoletin-7-O-glucoside (scopolin), significantly promoted the proliferation of the genera Methylobacterium and Achromobacter with resistance to A. alternata . Additionally, these genera also served as connectors in the molecular ecological network of the phyllosphere microbiome of the OE line. Thus, we concluded that the PsnWRKY70 gene enhanced the stability, complexity, and core taxa cooperation of the phyllosphere microbial network in Populus and regulated the biosynthesis of fraxin and scopolin to recruit beneficial bacteria controlling A. alternata infection. These findings provide valuable insights into the ability of resistant plant genotypes to drive the assembly of the phyllosphere microbiome, advancing our understanding of defense against pathogens using the biocontrol phyllosphere microbial community. IMPORTANCE: Poplar leaf blight caused by Alternaria alternata , a common disease in Northeast China, can cause abnormal abscission of poplar leaves and even lead to plant death in severe cases. WRKY transcription factors have been implicated in the regulation of disease resistance associated with plant immune responses to secondary metabolism via a complicated gene network. However, little is known about how the metabolites regulated by the PsnWRKY70 gene trigger changes in the phyllosphere microbiome, leading to increased resistance to foliar pathogens. Here, the PsnWRKY70 overexpressing line of Populus ( Populus simonii × P. nigra ) exhibited increased coumarin synthesis in the leaves, triggering changes in microbial species central in phyllosphere microbial networks and leading to increased resistance to A. alternata infection. This study provides insights into the role of the PsnWRKY70 gene in triggering the resistance mechanism to A . alternata in Populus .