Marssonina brunnea causes a major disease that limits poplar growth. Lignin and lignan play essential roles in protecting plants from various biological stresses. Dirigent (DIR) proteins are thought to control the stereoselective coupling of coniferyl alcohol in the formation of lignan and lignin. DIR family members have been well studied in several plant species, but no previous detailed genome-wide analysis has been carried out in forest trees, such as poplar. We identified 40 PtDIR genes in Populus trichocarpa and classified them into three subgroups (DIR-a, DIR-b/d, and DIR-e) based on phylogenetic analyses. These genes are distributed on 11 poplar chromosomes, and 80% of PtDIRs (32/40) are intronless. The cis-element analysis inferred that PtDIRs possess many types of biological and abiotic stress-response cis-elements. We also analyzed intra- and inter-specific collinearity, which provided deep insights into the evolutionary characteristics of the poplar DIR genes. Analyses of the protein tertiary structure and critical amino acid residues showed that PtDIR7–10 and PtDIR13–16, which belong to the DIR-a subfamily, might be involved in the regio- and stereo-selectivity of bimolecular phenoxy radical coupling in poplars. Quantitative reverse transcription polymerase chain reaction (RT-qPCR) analysis revealed different expression patterns for the PtDIR genes of P. trichocarpa and the PeDIR genes of ‘Nanlin 895’ in various tissues. Additionally, we analyzed responses of PeDIRs to M. brunnea and different phytohormone treatments (abscisic acid, salicylic acid, methyl jasmonate, and ethylene) in ‘Nanlin 895’. The results showed that at least 18 genes responded strongly to M. brunnea, and these PeDIRs also showed significant responses to phytohormones. These results suggest that DIR genes are involved in the poplar defense response against M. brunnea, and this study will provide fundamental insights for future research on poplar DIR genes.

Poplar is one of the most widely used tree in afforestation projects. However, it is susceptible to abiotic and biotic stress. CCR4-associated factor 1 (CAF1) is a major member of CCR4-NOT, and it is mainly involved in transcriptional regulation and mRNA degradation in eukaryotes. However, there are no studies on the molecular phylogeny and expression of the CAF1 gene in poplar. In this study, a total of 19 PtCAF1 genes were identified in the Populus trichocarpa genome. Phylogenetic analysis of the PtCAF1 gene family was performed with two closely related species (Arabidopsis thaliana and Oryza sativa) to investigate the evolution of the PtCAF1 gene. The tissue expression of the PtCAF1 gene showed that 19 PtCAF1 genes were present in different tissues of poplar. Additionally, the analysis of the expression of the PtCAF1 gene showed that the CAF1 family was up-regulated to various degrees under biotic and abiotic stresses and participated in the poplar stress response. The results of our study provide a deeper understanding of the structure and function of the PtCAF1 gene and may contribute to our understanding of the molecular basis of stress tolerance in poplar.

Increasing areas of artificial afforestation and poplar monoculture in China have led to serious problems with insect pests. The development of genetic engineering technology, such as transgenic modification with Bacillus thuringiensis (Bt) genes, provides novel solutions to the pest problem. We generated a Bt-Cry1Ah1 gene incorporating codon optimization and transferred it into Populus deltoides × P. euramericana cv “Nanlin895” using an Agrobacterium-mediated method. The resulting Bt-Cry1Ah1 transgenic poplars were planted in the field with permission from the State Forestry Administration in 2017. Field and laboratory studies were conducted in Jiangsu, China, to investigate the effects of these transgenic poplars expressing the Cry1Ah1 protein on target and non-target pests and their parasitic natural enemy. Target pests included Hyphantria cunea (Lepidoptera, Arctiidae), Micromelalopha troglodyta (Lepidoptera, Notodontidae), and Clostera anachoreta (Lepidoptera, Notodontidae). Plagiodera versicolora (Coleoptera, Chrysomelidae) served as the non-target pest. Laboratory trials showed that the six transgenic poplar lines exhibited resistance against the target insects. The corrected mortality rates of the target pest larvae fed leaves from the six lines were as high as 87.0%, significantly higher than that of the control. However, the corrected mortality rate of the non-target pest larvae was markedly lower and did not differ significantly from that of the control. Field experiments showed that transgenic poplar exhibited resistance against H. cunea and M. troglodyta. Field mortality rates were slightly higher than laboratory mortality rates. In addition, we investigated Chouioia cunea (Hymenoptera, Eulophidae) as a parasitoid of H. cunea pupae that had been fed transgenic poplar leaves. The emergence time, parasitism rate, and abundance of C. cunea did not differ significantly from those of the control. Therefore, Bt-Cry1Ah1 transgenic poplar can be used to effectively control damage by target insect pests without negatively affecting non-target insects and parasitoids.