Integrating multiregulatory analysis reveals the negative regulatory function of miR482a in the response of poplar to canker pathogen infection.

Canker disease caused by the bacterium Lonsdalea populi is one of the most destructive diseases affecting poplar stems. However, the detailed stress response mechanisms of poplar have not been widely characterized. To explore the diverse regulatory RNA landscape and the function of key regulators in poplar subjected to L. populi stress, we integrated time-course experiment with mock-inoculation (CK) and inoculation (IN) with L. populi at the first, third, and sixth day (IN1, IN3, IN6) on Populus × euramericana cv. '74/76' (107), small RNA-seq, whole transcriptome-wide analysis, degradome analysis and transgenic experiments. A total of 98 differentially expressed (DE) miRNA, 17 974 DEmRNA, and 807 DElncRNA were identified in poplar infected by L. populi, presenting dynamic changes over the infection course. Regulatory networks among RNAs were further constructed. Notably, a network centered on ptc-miR482a in CK-vs-IN3 contained most DEGs. We show that miR482a and miR1448 are located in one transcript as a polycistron. Overexpression of pre-miR482a-miR1448 (OX482-1448) and pre-miR482a (OX482) increased poplar susceptibility to canker pathogen with reduced accumulation of reactive oxygen species, while the suppression of miR482a (STTM482) conferred poplar disease resistance. PHA7 was validated as the target of miR482a with degradome sequencing and tobacco transient co-transformation, its expression being downregulated in OX482-1448 and OX482 lines. Additionally, a series of phasiRNAs were triggered by miR482a targeting PHA7, forming regulatory cascades with more RLP, NBS-LRR, and PK genes, further verifying the defense function of miR482a. These findings provide insights for understanding the roles of ncRNAs and regulatory networks involved in poplar immunity.

MiRNAs profiles among three poplar varieties provide insights into different molecular responses in resistance to newly emerging bacterial pathogen.

Canker caused by Lonsdalea populi has seriously reduced the economic and ecological benefits of poplar. MicroRNAs play vital roles in the response of plants to biotic stress. However, there is little research about the regulatory mechanism of miRNAs among different tree varieties upon pathogen infection. To dissect miRNAs involved in L. populi resistance, three poplar varieties, 2025 (susceptible), 107 (moderately resistant) and Populus. tomentosa cv 'henan' (resistant) were selected to elucidate the expression profiles of miRNAs using small RNA-seq. A total of 227 miRNAs were identified from all varieties. Intriguingly, miR160, miR169, miR171 and miR482b-5p were only identified in the resistant variety P. tomentosa upon pathogen infection, and these miRNAs might be important candidates for future investigation to improve the tolerance of poplar to L. populi. Among all identified miRNAs, 174 were differentially expressed in all varieties. Functional annotation analysis indicated that an array of miRNAs, including miR482, miR472, miR169, miR481, and miR172, should be involved in disease resistance and phytohormone signal transduction. Furthermore, correlation analysis of small RNA-seq and RNA-seq identified a handful of L. populi-responsive miRNAs and target genes, which exhibited that miR159 and miR172 played key roles in resistant variety P. tomentosa by targeting MYB and ERF, while miR6462c-5p and miR828 were related to the susceptibility of 2025 by targeting MYB. The comprehensive integration analysis in this research provides new insights into the regulatory pathways involved in the defence response of poplar to L. populi and offers crucial candidate miRNAs-target genes modules for poplar resistance improvement.

Integrated transcriptomic and metabolomic profiles reveal adaptive responses of three poplar varieties against the bacterial pathogen Lonsdalea populi.

Different poplar varieties vary in their tolerance to certain pathogens. However, knowledge about molecular regulation and critical responses of resistant poplars during pathogen infection remains scarce. To investigate adaptive responses to canker disease caused by the bacterium Lonsdalea populi, we screened three poplar varieties with contrasting tolerance, including Populus deltoides. 'Zhonglin 2025' (2025), Populus × Euramericana. '74/76' (107) and Populus tomentosa cv 'henan' (P. tomentosa). Transcriptomic analysis revealed significant changes in the expression levels of defence-related genes in different poplar varieties in response to infection, which reshaped the PTI and ETI processes. Intriguingly, photosynthesis-related genes were found to be highly expressed in the resistant variety, whereas the opposite was observed in the susceptible variety. Susceptible poplars maintained the activation of defence-related genes during early period of onset, which restricted the expression of photosynthesis-related and auxin signal-related genes. Furthermore, combined with metabolomic analysis, differences in the content of antibacterial substances and key differentially expressed genes in phenylpropane and flavonoid biosynthesis pathways were identified. Delayed induction of catechin in the susceptible variety and it's in vitro antibacterial activity were considered to be one of the important reasons for the differences in resistance to L. populi compared with the resistant variety, which is of practical interest for tree breeding. Moreover, the trade-off between growth and defence observed among the three poplar varieties during infection provides new insights into the multilevel regulatory circuits in tree-pathogen interactions.

miR159a modulates poplar resistance against different fungi and bacteria.

Trees are inevitably attacked by different kinds of pathogens in their life. However, little is known about the regulatory factors in poplar response to different pathogen infections. MicroRNA159 (miR159) is a highly conserved microRNA (miRNA) in plants and regulates plant development and stress responses. Here, transgenic poplar overexpressing pto-miR159a (OX-159) showed antagonistic regulation mode to poplar stem disease caused by fungi Cytospora chrysosperma and bacteria Lonsdalea populi. OX-159 lines exhibited a higher susceptibility after inoculation with bacterium L. populi, whereas enhanced disease resistance to necrotrophic fungi C. chrysosperma compared with wild-type (WT) poplars. Intriguingly, further disease assay found that OX159 line rendered the poplar susceptible to hemi-biotrophic fungi Colletotrichum gloeosporioide, exhibiting larger necrosis and lower ROS accumulation than WT lines. Transcriptome analyses revealed that more down-regulated differentially expressed genes with disease-resistant domains in OX-159 line compared with WT line. Moreover, the central mediator NPR1 of salicylic acid (SA) pathway showed a decrease in expression level, while jasmonic acid/ethylene (JA/ET) signal pathway marker genes ERF, as well as PR3, MPK3, and MPK6 genes showed an increase level in OX159-2 and OX159-5 compared with WT lines. Further spatio-temporal expression analysis revealed JA/ET signaling was involved in the dynamic response process to C. gloeosporioides in WT and OX159 lines. These results demonstrate that overexpression of pto-miR159a resulted in the crosstalk changes of the downstream hub genes, thereby controlling the disease resistance of poplars, which provides clues for understanding pto-miR159a role in coordinating poplar-pathogen interactions.