Genome-wide analysis of R2R3-MYB transcription factors in poplar and functional validation of PagMYB147 in defense against Melampsora magnusiana

Main conclusion Transcription of PagMYB147 was induced in poplar infected by Melampsora magnusiana, and a decline in its expression levels increases the host's susceptibility, whereas its overexpression promotes resistance to rust disease. Abstract Poplars are valuable tree species with diverse industrial and silvicultural applications. The R2R3-MYB subfamily of transcription factors plays a crucial role in response to biotic stresses. However, the functional studies on poplar R2R3-MYB genes in resistance to leaf rust disease are still insufficient. We identified 191 putative R2R3-MYB genes in the Populus trichocarpa genome. A phylogenetic analysis grouped poplar R2R3-MYBs and Arabidopsis R2R3-MYBs into 33 subgroups. We detected 12 tandem duplication events and 148 segmental duplication events, with the latter likely being the main contributor to the expansion of poplar R2R3-MYB genes. The promoter regions of these genes contained numerous cis-acting regulatory elements associated with response to stress and phytohormones. Analyses of RNA-Seq data identified a multiple R2R3-MYB genes response to Melampsora magnusiana (Mmag). Among them, PagMYB147 was significantly up-regulated under Mmag inoculation, salicylic acid (SA) and methyl jasmonate (MeJA) treatment, and its encoded product was primarily localized to the cell nucleus. Silencing of PagMYB147 exacerbated the severity of Mmag infection, likely because of decreased reactive oxygen species (ROS) production and phenylalanine ammonia-lyase (PAL) enzyme activity, and up-regulation of genes related to ROS scavenging and down-regulation of genes related to PAL, SA and JA signaling pathway. In contrast, plants overexpressing PagMYB147 showed the opposite ROS accumulation, PAL enzyme activity, SA and JA-related gene expressions, and improved Mmag resistance. Our findings suggest that PagMYB147 acts as a positive regulatory factor, affecting resistance in poplar to Mmag by its involvement in the regulation of ROS homeostasis, SA and JA signaling pathway. Supplementary Information The online version contains supplementary material available at 10.1007/s00425-024-04458-3.


Introduction
The MYB family of transcription factors (TFs) is a widely distributed and functionally important group of TFs found in eukaryotes.Members of this family are characterized by the presence of a highly conserved MYB domain, which plays a crucial role in DNA binding and the regulation of gene expression.The MYB domain typically consists of one to four incompletely repeating amino acid sequence repeats (R), each containing 52 amino acids and forming three alpha helices.Each R sequence, along with the helix-turn-helix motif, contains conserved amino acid sequences that enable the MYB TFs to bind to specific DNA sequences with high specificity.In plants, the MYB family can be classified into four subfamilies based on the number of MYB repeats: R1-MYB, R2R3-MYB, R1R2R3-MYB, and R1R2R2R1/2-MYB (Dubos et al. 2010).
R2R3-MYB is the largest subfamily within the MYB family, and its members significantly affect plant growth, development, and secondary metabolism (Chopy et al. 2023;Matías-Hernández et al. 2017;Wang et al. 2019a, b, c).R2R3-MYB TFs also play a significant role in plant disease resistance.For instance, tomato SlMYB49 inhibits the production of reactive oxygen species (ROS), thereby enhancing resistance against Phytophthora infestans (Cui et al. 2018).Arabidopsis AtMYB96 enhances resistance to Pseudomonas syringae infection by regulating genes associated with the salicylic acid (SA) pathway (Seo and Park 2010).Wheat TaMYB391 and TaMYB29 strengthen resistance to Puccinia striiformis f. sp.tritici by modulating the balance of ROS and the SA pathway (Zhu et al. 2021;Hawku et al. 2022).In both, Arabidopsis and cotton, MYB36 promotes the activation of the PR1 gene in the SA pathway, thereby enhancing resistance against Verticillium dahliae (Liu et al. 2021).Together, the results of those studies show that R2R3-MYB TFs, through their involvement in regulating plant-pathogen interactions and activating defense responses, enhance plants' resistance to pathogens.
Poplar is a widely distributed deciduous tree with multiple ecological and economic values and was chosen as a model species for studying woody plants for its simple genetic characteristics, robust reproductive capacity, and small genome size (Eberl et al. 2018).However, poplar suffers from heavy rust disease caused by the obligate biotrophic pathogen Melampsora, which may result in premature leaf abscission in young seedlings, and up to 50% reduction in biomass (Polle et al. 2013;Wan et al. 2013).R2R3-MYB TFs regulate the response of poplar to pathogenic fungi.For example, MYB115 increases resistance to Dothiorella gregaria by regulating the biosynthesis of proanthocyanidins (Wang et al. 2017).Overexpression of PalMYB90 and PalbHLH1 in poplar enhances resistance to Botrytis cinerea and Dothiorella gregaria by regulating the flavonoid pathway and ROS pathway (Bai et al. 2020).Overexpression of MYB134 enhances proanthocyanidins accumulation, thereby increasing resistance to Melampsora larici-populina infection in poplar (Ullah et al. 2017).Additionally, RNAseq analysis identified many R2R3-MYB genes responsive to M. larici-populina in poplar, but their functions have not been validated (Chen et al. 2022).Although a total of 192, 196, or 207 R2R3-MYB TFs have been identified across various genome versions, it is challenging to locate the IDs for some of these genes in the latest database version (Wilkins et al. 2008;Zhao et al. 2020;Yang et al. 2021).To date, there haven't been reports on the identification and characterization of the R2R3-MYB family in the latest version of the poplar genome and its response to rust fungus.
In this study, we performed a comprehensive analysis of the R2R3-MYB TF subfamily in poplar.The molecular features, chromosomal localization, phylogenetic relationships, gene structure, conserved motifs, synteny, cis-acting elements and transcriptional profiles of PtrMYB genes were determined.We identified a significantly up-regulated gene during M. magnusiana (Mmag) inoculation, PagMYB147, and determined its transcription patterns in response to Mmag, salicylic acid (SA), and methyl jasmonate (MeJA).To gain deeper insights into the role of PagMYB147 in rust resistance, we conducted experiments using virus-induced gene silencing (VIGS) and overexpressed methods in poplar.The changes in certain physiologic and biochemical traits, as well as the transcript levels of defense-related genes, were analyzed after Mmag inoculation.These findings provide valuable insights into the roles of poplar R2R3-MYB TFs in the response to biotic stress.

Identification of R2R3-MYB TFs in P. trichocarpa
The P. trichocarpa genome sequencing project was accessed from the Phytozome database (https:// phyto zome-next.jgi.doe.gov/).The MYB protein sequence from A. thaliana was obtained from the TAIR database (http:// www.arabi dopsis.org).We used two methods to maximize the identification of R2R3-MYBs.The first method involved using A. thaliana R2R3-MYBs protein sequences as search queries in a BLASTp search to identify R2R3-MYBs in the P. trichocarpa genome.The second method leveraged a hidden Markov model of MYB DNA-binding domain (PF00249) downloaded from the Pfam database (http:// pfam.xfam.org/) to identify MYB candidate genes in the P. trichocarpa genome (El-Gebali et al. 2018).Subsequently, SMART (http:// smart.embl-heide lberg.de/) was used to scrutinize the conserved structures of all potential proteins with a threshold e value < 0.0001 to identify all the MYB candidate genes in the P. trichocarpa genome.The location of each gene was determined by TBtools, and then genes were named according to their position on the P. trichocarpa chromosomes (Chen et al. 2020).The characteristics of the putative proteins, including their isoelectric point, molecular weight, grand average hydrolysis (GRAVY), and instability index were predicted using Prot-Param (https:// web.expasy.org/ protp aram/) and TBtools (Chen et al. 2020).Signal peptides and phosphorylation sites were predicted using tools at the SignalP 5.0 server and NetPhos 3.1 server (http:// www.cbs.dtu.dk/ servi ces/ NetPh os/), respectively.Plant-mPLoc (http:// www.csbio.sjtu.edu.cn/ bioinf/ plant-multi/) was used to predict the subcellular localization of the identified gene products.

Phylogenetic and synteny analyses of R2R3-MYBs in P. trichocarpa
The genomic coordinates of P. trichocarpa R2R3-MYBs were retrieved from the genome database, and a visual representation of the data was created using TBtools (Chen et al. 2020).Using the neighbor-joining (NJ) method of MEGA7 and 1000 bootstrap values, a phylogenetic tree was constructed from the protein sequences of R2R3-MYBs in P. trichocarpa and A. thaliana.The generated tree was visualized using iTOL (https:// itol.embl.de/) (Letunic and Bork 2019).Gene duplication events were detected using TBtools, BLASTP, and the Multiple Collinearity Scan (MCScan) toolkit (Zhao et al. 2020).Tandem duplication events were indicated by the presence of two genes located within the same chromosomal segment and not separated by more than five genes within a 100-kb region (Li et al. 2022).Finally, a synteny map of R2R3-MYBs in P. trichocarpa, A. thaliana (dicots), and Oryza sativa (a monocot), was generated using the Dual Synteny Plotter software (Chen et al. 2020).

Analysis of R2R3-MYBs conserved motifs, gene structure, and cis-acting elements upstream of R2R3-MYB genes in P. trichocarpa
The online software MEME (https:// meme-suite.org/ meme/ tools/ meme) was used to find conserved motifs in P. trichocarpa R2R3-MYBs (Bailey et al. 2009).TBtools was used to analyze and show intron/exon structures (Chen et al. 2020).The PlantCARE database (http:// bioin forma tics.psb.ugent.be/ webto ols/ plant care/ html/) was used to identify cis-acting elements associated with stress and phytohormone responsiveness in the promoter region (2 kb upstream) of each R2R3-MYB gene.The distribution of these elements was visualized with TBtools (Chen et al. 2020).

Plant materials and treatments
One-month-old sterile seedlings of the 84 K poplar (P.alba × P.glandulosa) were transplanted into commercial substance soil and cultivated in a greenhouse.For one month, the conditions were strictly controlled at 25 °C, 50%-60% relative humidity, and a 16-h light/8-h dark photoperiod.Uniformly sized seedlings were selected, and the leaves were washed softly with sterile water before inoculation.A suspension of fresh Mmag uredospore (1 × 10 5 spores/mL), or water as the control, was sprayed on abaxial leaves with a leaf plastochron index (LPI) of 5-10 ( Ullah et al. 2018).The plants were then placed in a light-proof container and kept in the dark for 24 h, and then returned to the same growth conditions as described above.Leaf samples were collected and immediately frozen in liquid nitrogen at 0, 24, and 144 h post-inoculation (hpi).The experiment was designed with three independent biologic replicates, and at least three leaf samples were collected at each time point.Each sample was subjected to RNA-Seq sequencing to pick up the putative MYB related to rust resistance.All rust infestation experiments were conducted using the same inoculation and sampling procedures unless otherwise noted.

Expression analysis of poplar R2R3-MYB genes
To analyze the transcriptional profiles of R2R3-MYB genes in 84 K poplar, transcriptome data (unpublished data) obtained from plants after inoculation with Mmag were obtained, and gene transcript levels were calculated using fragments per kilobase of transcript per million fragments mapped (FPKM) values.A gene clustering heatmap was generated using the TBtools HeatMap tool.In addition, differentially expressed genes (DEGs) were identified by selecting those with a Log 2 |foldchange|≥ 1, and these DEGs under infection conditions were compared to control samples (Chen et al. 2020).For validation, fourteen genes were selected for quantitative real-time polymerase chain reaction (qRT-PCR) analysis.The transcript levels of these genes were determined by qRT-PCR using 2 × SYBR Green qPCR Master Mix (US Everbright, Suzhou, China) on a CFX-96 system (Bio-Rad, Hercules, CA, USA).Total RNA was extracted using the E.Z.N.A.® Plant RNA Kit (OMEGA, Norcross, GA, USA), and cDNA was synthesized using UEIris RT mix with DNase (US Everbright).The relative gene expression levels were determined using the 2 −ΔΔCt method and normalized to that of the endogenous reference actin gene (Livak and Schmittgen 2001).The primer sequences, which were designed with Primerquest (https:// sg.idtdna.com/ pages/ tools/ prime rquest), are listed in Table S1.

Cloning and characterization of PagMYB147 in 84 K poplar
We selected MYB147 and designed primers based on the PtrMYB147 (Potri.015G046200)sequence.Initially, RNA was extracted from the leaves of 84 K poplar plants, cDNA was synthesized, and PCR amplification was subsequently performed.The product was purified, ligated into the pMD-19 T vector and sequenced.The subcellular localization was analyzed by inserting the open reading frame sequence of the PagMYB147 gene, without the termination codon, into the EGFP vector (Miaoling Biology, Wuhan, China) under the control of the CaMV35S promoter.The resulting fusion plasmid was then transformed into competent cells of Agrobacterium tumefaciens GV3101 (Weidi Biotechnology, Shanghai, China), which were then used to introduce the gene construct into one-month-old Nicotiana benthamiana seedlings for transient expression.The inner epidermis of tobacco specimens was injected with A. tumefaciens buffer containing 35S-EGFP and 35S-PagMYB147-EGFP.The plants were then incubated in darkness for 24 h.After 48 h, a confocal laser scanning microscope (Olympus, Tokyo, Japan) was used to observe the fluorescence of the heterologously expressed protein in the leaves.The primer sequences used for gene cloning and vector construction are described in Table S1.To determine the transcriptional profile of PagMYB147, qRT-PCR was conducted on untreated roots, stems, young and mature leaves, as well as leaves of 84 K poplar plants that had been treated with Mmag, SA (5 mM) and MeJA (1 mM).

Construction of virus-induced gene silencing (VIGS) and overexpression vectors and genetic transformation of poplar
The PagMYB147 gene was amplified using specific primers by PCR, and then integrated into the pTRV2 vector.Then, pTRV2-PagMYB147 was introduced into A. tumefaciens GV3101, and the pTRV2 empty vector was introduced into A. tumefaciens as the control.The VIGS experiments were conducted in accordance with the protocol outlined by Shen et al. (2015).The transcript levels of PagMYB147 were detected by qRT-PCR in transformed with A. tumefaciens harboring pTRV2-PagMYB147 and pTRV2.Silenced plants displaying significantly reduced PagMYB147 levels were selected for disease resistance testing.
The PagMYB147 sequence was cloned into the pCAMBIA-1300 vector.The recombinant plasmid was then transformed into A. tumefaciens GV3101.
Subsequently, the transformed A. tumefaciens was used for leaf disk transformation into the 84 K poplar (He et al. 2018).Initially, transgenic lines were selected based on their resistance to hygromycin, and then the presence of the transgene was confirmed by PCR confirmation using specific primers (Table S1).The relative transcript level of PagMYB147 in the transgenic lines was further examined using qRT-PCR.One-month-old transgenic seedlings of the 84 K poplar were transplanted according to the method described in the 'plant material and treatment' section, and were subsequently subjected to rust resistance testing.

Analysis of disease resistance, physiological parameters, and defense gene expression
To validate the disease resistance function of PagMYB147, PagMYB147-silenced plants, PagMYB147-overexpressing (OE) plants and wild-type (WT) plants were inoculated with Mmag.Three independent biologic replicates were prepared and analyzed for each treatment.Leaves were collected at 0, 24, and 48 hpi, and then immediately frozen in liquid nitrogen, and subsequently, stored in an ultralow temperature freezer until further analysis.Phenotypic observations and evaluation of uredinial density (number of uredinia/cm 2 ) were conducted 10 d post-inoculation (dpi).Using qRT-PCR to determine the transcription level of the Mmag ITS gene to determine the biomass of the rust fungus, with ELF1α as reference genes (Table S1) (Zheng et al. 2021).The contents of H 2 O 2 , along with the activities of superoxide dismutase (SOD, EC:1.15.1.1),catalase (CAT, EC 1.11.1.6),and phenylalanine ammonialyase (PAL, EC 4.3.1.5)were determined using previously described methods (Li et al. 2015b).The relative transcript levels of PtrSOD1, PtrCAT1, PtrPAL1, PtrPR5, and PtrDefensin were also determined by qRT-PCR using the primers specified in Table S1.The PlantCARE program was used to analyze the MYB, MYB binding sites (MBS) and MYB recognition elements (MRE) within the upstream 2 kb region of these genes, which were considered to contain complete promoters (Lescot 2002).

Statistical analysis
All data were shown as means with standard deviations (SD), calculated from three independent biologic replicates.Statistical analyses were conducted using SPSS version 26.0 software (IBM Corp., Chicago, Ill., USA).To detect significant differences among groups, a one-way analysis of variance (ANOVA) was conducted, followed by Duncan's multiple range test, with a significance threshold value of P < 0.05.

Identification of R2R3-MYB genes in the P. trichocarpa genome and physical properties of their putative products
In total, 191 R2R3-MYB genes were identified in the P. trichocarpa genome.These genes were named PtrMYB001 to PtrMYB190.1 based on their chromosomal locations.Comparison of the amino acid sequences of the R2 and R3 MYB repeats revealed three conserved tryptophan (Trp, W) residues in the R2 repeat.The second and third W residues were conserved in the R3 repeat (Fig. 1a), whereas the first could be replaced by phenylalanine (Phe, F), isoleucine (Ile, I), or leucine (Leu, L) (Fig. 1b).The lengths and physical traits of the putative P. trichocarpa R2R3-MYBs are summarized in Table S2.The R2R3-MYBs had an average length of 321 amino acids, ranging from 180 (PtrMYB035) to 568 (PtrMYB042).The molecular weights of the putative proteins ranged from 20.52 kDa (PtrMYB035) to 62.68 kDa (PtrMYB002), and the isoelectric points ranged from 4.72 (PtrMYB136) to 9.74 (PtrMYB079).Of the 191 R2R3-MYBs, 104 had an isoelectric point below 7.0 and 87 had an isoelectric point above 7.0.All R2R3-MYBs were hydrophilic, according to predicted GRAVY scores, which ranged from − 1.067 (PtrMYB119) to − 0.282 (PtrMYB175).The putative R2R3-MYB proteins had multiple phosphorylation sites (Fig. S1), were primarily located in the nucleus, and lacked signal peptides.Of the 191 putative R2R3-MYBs, 186 were categorized as unstable (instability index > 40).The instability index of the putative R2R3-MYBs ranged from 34.25 (PtrMYB007) to 72.65 (PtrMYB147).
Next, we investigated the chromosomal distribution of R2R3-MYB genes in P. trichocarpa (Fig. 3a).Of the 191 R2R3-MYB genes, 189 were widely dispersed among the 19 chromosomes of P. trichocarpa, and two were located on the scaffold.Notably, chromosome 1 had the highest number of genes, with 20 R2R3-MYB genes, while chromosome 16 had the lowest number, with only one gene.The number of genes on other chromosomes ranged from six to 15 (Fig. 3b).In total, we identified 160 pairs of gene duplicates across 15 chromosomes, consisting of tandem duplicates and segmental duplicates or whole-genome duplication (WGD) duplicates.The distribution of these duplicates was uneven (Table S4).Twelve tandem duplication events involving 19 P. trichocarpa R2R3-MYBs genes were identified on chromosomes 3, 11, 13, 17, and 19.Although most of the tandem repeats comprised duplicated or triplicate genes, one notable tandem repeat on chromosome 17 harbored five genes (Fig. 3a).Moreover, 148 segmental duplication events were observed across 19 chromosomes, involving 158 R2R3-MYB genes (Fig. 4; Table S4).These results firmly establish the possibility that segmental duplication has had an important effect on the evolutionary process of the R2R3-MYB family in P. trichocarpa.

Motifs and gene structures of R2R3-MYB genes in P. trichocarpa
Ten conserved motifs were detected in the 191 P. trichocarpa R2R3-MYB genes.These motifs had amino acid lengths ranging from 11 to 50 (Table S7).All of the R2R3-MYBs contained one significantly conserved motif (motif 3), and 188 out of the 191 putative proteins contained two highly conserved motifs (motif 2 and motif 3) (Fig. 6a).These motifs represent critical repetitive components of R2R3-MYBs.These findings demonstrate that all R2R3-MYBs contained the characteristic SANT structural domains that are typical of this gene family (Fig. 6b).A detailed analysis of gene exon/intron structures revealed significant diversity among the 191 genes.Specifically, 11 R2R3-MYB genes (nine distributed between PtrMYB025 and PtrMYB138, as well as PtrMYB163 and PtrMYB048) lacked introns, whereas the remaining 180 genes had varying numbers of introns (ranging from one to 11) (Fig. 6c).These genes within an identical subgroup exhibited a high level of conservation despite differences in the number of introns and exons.

Cis-elements in promoter regions and transcriptional profiles of poplar R2R3-MYB genes
Analysis of the promoter region of P. trichocarpa R2R3-MYB genes identified numerous elements involved in responsiveness to various stresses and phytohormones (Fig. S2).These included elements that are known to be involved in responsiveness to stress, defense, phytohormones (abscisic acid (ABA), SA, MeJA), and drought.Of the 191 genes, around 152 had at least one stress-responsive cis-element in their promoter regions, which highlighted the importance of the P. trichocarpa R2R3-MYB family in biotic stress responses.In addition, 93, 134, and 152 R2R3-MYB genes had cis-elements involved in responsiveness to SA, MeJA, and ABA, respectively, in their promoter regions.
To investigate how poplar R2R3-MYB genes respond to rust fungus, we analyzed RNA-Seq data from samples at 24 hpi and 144 hpi with inoculated Mmag (Fig. 7a).A heatmap was constructed, revealing 27 DEGs at 24 hpi, with 9 up-regulated and 18 down-regulated genes.Similarly, at 144 hpi, there were 26 DEGs, with 9 up-regulated and 17 down-regulated genes.Comparison of DEGs among different time points identified 24 genes consistently responsive to Mmag at both 24 hpi and 144 hpi, consisting of 8 up-regulated and 16 down-regulated genes.To validate RNA-Seq data, we selected 14 genes with fold changes greater than 3 (six up-regulated and eight down-regulated) for qRT-PCR validation.The qRT-PCR results were consistent with those detected from the transcriptome data, validating the reliability of the transcriptome data (Fig. 7b).Of particular interest was MYB147, which exhibited a significant increase in its transcript levels in response to Mmag at both 24 hpi and 144 hpi.At 24 hpi, MYB147 expression was up-regulated by 4.59-fold, showing the most substantial increase.Similarly, at 144 hpi, its expression was increased by 3.83-fold.As a result, we selected MYB147 as the primary candidate for further functional analyses to better understand poplar's response to rust fungus.

Characterization of PagMYB147
PagMYB147 had a coding sequence length of 999 bp, encoding 332 amino acids, which was characterized by its instability and hydrophilicity.Through laser confocal microscopy observations, it was determined that the green fluorescence signal of PagMYB147 was primarily concentrated in the nucleus of tobacco leaf epidermal cells (Fig. 8a), indicating that PagMYB147 is localized in the cell nucleus.We then conducted qRT-PCR analyses to investigate the patterns of PagMYB147 transcription in different 84 K poplar tissues.The transcript levels of PagMYB147 were higher in the young leaves than in the mature leaves, stems, and roots (Fig. 8b).To investigate the role of PagMYB147 in poplar defense, we analyzed the expression levels of PagMYB147 by qRT-PCR within 144 h of inoculation with Mmag and 24 h of treatment with SA and MeJA.The findings revealed that the expression of PagMYB147 was induced by Mmag, and its expression levels gradually increased from 0 to 24 hpi.At 24 hpi, the expression of PagMYB147 reached its peak, being 4.12 Fig. 3 Chromosomal locations.a Chromosome distribution of P. trichocarpa R2R3-MYB genes.b Numbers of P. trichocarpa R2R3-MYB genes on each chromosome times higher than at 0 hpi.Subsequently, the expression levels gradually declined.By 144 hpi, the expression of PagMYB147 increased once again (Fig. 8c).After treating seedlings with SA (Fig. 8d) and MeJA (Fig. 8e), the transcript levels of PagMYB147 reached the highest level at 4 h post-treatment (hpt) and 8 hpt, with increases of 2.57 and 3.44 times, respectively.Our findings indicate the involvement of PagMYB147 in poplar resistance against Mmag and diverse hormone signal pathways.

Rust resistance analyses of PagMYB147-silenced and -OE plants
In PagMYB147-silenced 84K poplar plants, the transcript level of PagMYB147 was significantly reduced compared with those in the controls (the empty TRV2 and WT) (Fig. S3).PagMYB147-OE plants were detected by PCR analysis using specific primers.As shown in Fig. S4a specific bands were present in the transgenic poplar lines 1-3 and 5-7, while no such positive band was detected in the WT plants.Subsequently, the relative transcript levels of PagMYB147 in the selected six transgenic lines were detected using qRT-PCR.The three transgenic lines with the highest expression levels (OE 1, OE 5, and OE 7) were used in subsequent experiments (Fig. S4b).To investigate the rust resistance of PagMYB147-silenced and OE plants, all plants were inoculated with Mmag to assess disease phenotypes and uredial density.The results demonstrated that PagMYB147-OE plants exhibited significantly enhanced resistance to Mmag inoculation compared to both WT and silenced plants (Fig. 9a).OE plants developed lower uredinia density with 20-27 uredinia/cm 2 , whereas silenced plants exhibited a significant (P < 0.05) increase in uredinia density with 68 uredinia/cm 2 (Fig. 9b).Additionally, the biomass of Mmag, as indicated by the transcription level of the ITS gene, was measured in PagMYB147-silenced and OE plants at 10 d post-inoculation (dpi) using qRT-PCR.The results revealed that, after Mmag inoculation, the relative expression levels of ITS in PagMYB147-OE plants significantly (P < 0.05) decreased to 0.43-0.57times that of WT plants.In contrast, the relative expression level of ITS in PagMYB147-silenced plants significantly (P < 0.05) increased to 2.20 times that of WT plants (Fig. 9c).These findings indicate that PagMYB147 positively contributes to poplar resistance to rust fungus Mmag.

Analyses of physiologic traits
To investigate the effects of PagMYB147-silenced and OE on the physiologic status of leaves, the H 2 O 2 content, CAT activity, SOD activity, and PAL activity of the leaves were also analyzed.Before inoculation, there were no significant changes in the H 2 O 2 content and enzyme activities of CAT, SOD, and PAL in all plants (Fig. 10a-d).However, at 24 hpi and 48 hpi, the OE plants exhibited a significant (P < 0.05) increase in H 2 O 2 content compared to WT plants, while the silenced plants showed a noteworthy decrease.Moreover, at the same time points, the OE plants showed significantly (P < 0.05) reduced CAT and SOD activities compared to WT plants, whereas the silenced plants displayed a significant (P < 0.05) increase.
In addition, at 24 hpi and 48 hpi, the OE plants exhibited significantly (P < 0.05) increased PAL activity compared to WT plants, while the silenced plants displayed a notable decrease.Overall, these findings suggest that PagMYB147 is involved in the regulation of ROS levels and defense enzyme activities.

Expression analyses of defense-related genes
To investigate the molecular regulatory role of Pag-MYB147, we evaluated the transcription levels of defense genes, including PtrPAL1, ROS scavenging genes (Ptr-CAT1 and PtrSOD1), and SA/MeJA signaling pathway genes (PtrPR5 and PtrDefensin).After Mmag inoculation, compared to the WT plants, the expression levels of PtrCAT1 and PtrSOD1 were significantly (P < 0.05) decreased in the OE plants but significantly (P < 0.05) increased in the silenced plants (Fig. 11a-b).In addition, the transcription levels of PtrPAL1, PtrPR5, and PtrDefensin were significantly (P < 0.05) increased in all plants after inoculation Mmag, but significantly (P < 0.05) slow in the silenced plants (Fig. 11c-e).Taken together, these findings indicate that PagMYB147 participates in poplar response to leaf rust fungus by directly or indirectly regulating the expression of ROS scavenging and certain defense-related genes.

Discussion
The role of R2R3-MYB TFs is crucial in plants responses to biotic stresses (Seo and Park 2010;Mabuchi et al. 2018;Zhu et al. 2018).Previous studies identified 192 R2R3-MYB poplar genes from the Joint Genome Institute (JGI) Ptri 1.1 version database, and 196 or 207 R2R3-MYB poplar genes from the Ptri 3.0 version database (Wilkins et al. 2008;Zhao et al. 2020;Yang et al. 2021).With the advances in genomic information, we can now more accurately identify members of gene families.In our study, we identified 191 R2R3-MYB genes in the P. trichocarpa genome, categorizing them into 33 subgroups.The primary mechanisms driving gene family expansion are tandem and segmental duplications (Li et al. 2022).Specifically, we detected 12 tandem repeats on five chromosomes and 148 segmental repeats from 158 genes on 19 chromosomes in the P. trichocarpa genome.These findings highlight the significance of segmental duplications in the R2R3-MYB gene family expansion in P. trichocarpa.Moreover, our results showed that duplicated TFs have equivalent structures and motif compositions, as reported elsewhere (Zhao et al. 2020;Zhu et al. 2023).We found that 51 R2R3-MYBs in P. trichocarpa exhibit synteny with both O. sativa and A. thaliana, indicating that they had possibly evolved before the monocot-dicot split, as previously reported (Li et al. 2022;Wang et al. 2022).Consequently, our results support the notion that expansion of the P. trichocarpa R2R3-MYB gene subfamily may potentially result in the functional divergence of these genes, thereby facilitating plant adaptation to diverse environmental conditions.
R2R3-MYB genes have been shown to respond to the invasion of various pathogens in different plant species (Liu et al. 2015(Liu et al. , 2021;;Wang et al. 2017;Zhu et al. 2022;Ding et al. 2023).After inoculation with M. larici-populina E4, 34 R2R3-MYB genes showed significant differences in expression between tolerant and intolerant poplars (Chen et al. 2022).In this study, we identified 27 R2R3-MYB DEGs at 24 hpi and 26 R2R3-MYB DEGs at 144 hpi in poplar in response to Mmag, indicating the widespread involvement of R2R3-MYBs in response to rust infection.Among these, the MYB147 gene was the most up-regulated in response to Mmag.Its silencing led to increased susceptibility, while overexpression enhanced resistance, suggesting a positive regulatory role in Mmag resistance.This is supported by studies showing increased resistance to the overexpression of GhMYB108, TaMYB391 and MYB115 (Cheng et al. 2016;James et al. 2017;Hawku et al. 2022).Plants generate ROS, including H 2 O 2 , to limit pathogen spread when attacked (Fichman et al. 2020).Overexpression of PagMYB147 resulted in higher H 2 O 2 levels post-Mmag inoculation, whereas silenced plants exhibited lower levels.Furthermore, plants possess a complex enzymatic antioxidant defense system, including CAT and SOD enzymes, which play a key role in regulating ROS homeostasis (Li et al. 2015a, b;Mabuchi et al. 2018).We observed that the activities of CAT and SOD were lower in PagMYB147-OE plants but higher in PagMYB147-silenced plants compared to WT  2014) also confirmed that the enhancement of resistance against Alternaria alternata in SpMYB-OE transgenic plants attributed to PAL accumulation.Moreover, R2R3-MYB TFs such as PacMYBA and MdMYB73 have been identified as regulators of genes associated with SA and JA signaling pathways, thereby regulating resistance to pathogens (Shen et al. 2017;Gu et al. 2020).Overexpression of VqMYB154 not only enhances Arabidopsis resistance to Uncinula necator but also upregulates genes linked to the SA and JA/ET signaling pathways (Jiang et al. 2021).In this study, PagMYB147 was found to improve plant resistance to rust fungus by positively modulating genes associated with the SA and JA pathways, indicating a beneficial role of PagMYB147 in enhancing rust resistance in poplar through collaboration with SA and JA signaling pathways.
R2R3-MYB TFs regulate the expression of defenserelated genes by binding to specific cis-acting elements in the promoter region (Cheng et al. 2016;Liu et al. 2021).Our findings suggest that PagMYB147 affects the transcription of five defense genes (PtrSOD1, PtrCAT1, PtrPAL1, PtrPR5, and PtrDefensin).We hypothesize that PagMYB147 may interact with cis-acting elements  S8).Consequently, we speculate that PagMYB147 could regulate rust resistance by modulating the expression levels of these genes, directly or indirectly.However, further studies are needed to confirm whether PagMYB147 exerts its function through binding to these targets.Based on our data, we have proposed a hypothetical model to elucidate the function of PagMYB147 (Fig. 12).Our future research will focus on identifying the specific target genes regulated by PagMYB147, validating its function through gene editing, and investigating the associated signaling pathways.These investigations aim to provide deeper insights into the molecular signaling network of PagMYB147 in response to biotic stress.

Conclusion
In total, 191 R2R3-MYB TFs were identified in the P. trichocarpa genome and were classified into 33 subgroups.Twelve tandem duplication events and 148 segmental duplication events were detected.The promoter sequences contain numerous cis-regulatory elements associated with responsiveness to plant stress and phytohormones.Through RNA-Seq data analysis, we identified multiple R2R3-MYB genes responsive to Mmag.Among them, nucleus-located PagMYB147 was the most significantly up-regulated R2R3-MYB gene by Mmag infection.Silencing of PagMYB147 in poplar reduced ROS accumulation, and down-regulate SA and JA-related genes, thereby reducing resistance against Mmag.Conversely, overexpression of PagMYB147 showed the opposite ROS accumulation, PAL enzymes activity, SA and JA-related gene expressions, and thus improved Mmag resistance.All our results suggested that PagMYB147 positively regulates poplar resistance against Mmag through ROS homeostasis, SA and JA signaling pathway.

Fig. 1
Fig. 1The sequence logos of P. trichocarpa R2 a and R3 b MYB domains.The arrow indicates typically conserved tryptophan (Trp, W) and the star indicates the first W residues can be replaced by phenylalanine (Phe, F), isoleucine (Ile, I) and leucine (Leu, L)

Fig. 2
Fig. 2 Phylogenetic relationship between A. thaliana and P. trichocarpa R2R3 MYB TFs.Subgroups are marked with a different background color.This annotation comes from genetic function in A. thaliana

Fig. 4
Fig. 4 Duplication events between R2R3-MYB members in P. trichocarpa.Red lines between chromosomes and gray lines in the background indicate segmental duplicate gene pairs and all synteny

Fig. 7
Fig. 7 Expression analysis of poplar R2R3-MYB genes in response to Mmag. a Heatmap and hierarchical clustering of R2R3-MYB gene expression in 84K poplar leaves after inoculation with Mmag 0 hpi, 24 hpi, 144 hpi.b Expression analysis of fourteen poplar R2R3-MYB genes in response to Mmag.The data are expressed as the means ± SD of three biologic replicates.Different lowercase letters indicate significant differences (P < 0.05)

Fig. 8
Fig. 8 Characterization of PagMYB147.a Subcellular localization of PagMYB147.35S-EGFP was taken as the control and the scale bars were equal to 20 μm.b Analysis of tissue-specific expression patterns of PagMYB147.c-e Expression patterns of PagMYB147 in 84K pop-

Fig. 9
Fig. 9 Phenotypes of PagMYB147 -silenced and -overexpressing plants after inoculation with Mmag. a Phenotypes of PagMYB147 -silenced and -overexpressing plants inoculated with Mmag for 10 d, yellow spots were uredinia.b Uredial density statistics.c Relative transcript levels of the Mmag ITS gene 10 d after inoculation.WT:

Fig. 10
Fig. 10 Physiologic and biochemical analysis of Mmag before and after inoculation in the WT, PagMYB147-silenced, and -overexpressing poplar plants.a H 2 O 2 content.b CAT activity.c SOD activity.d PAL activity.All data are presented as the means ± SD of three biologic replicates.Different letters above the bars indicate significant differences (P < 0.05)

Fig. 11
Fig. 11 Relative expression levels of defense-related genes in the WT, PagMYB147-silenced, and -overexpressing poplar plants before and after inoculation.a-e Relative transcript levels of PtrCAT1, PtrSOD1, PtrPAL1, PtrPR5, and PtrDefensin.All data are presented as the means ± SD of three biologic replicates.Different letters above the bars indicate significant differences (P < 0.05)

Fig. 12
Fig. 12 Hypothetical model of PagMYB147 regulation in rust resistance.The solid line denotes potential direct or indirect regulatory effects.The dotted line indicates this mode of regulation has been already confirmed