Poplar miR1447 Is a Negative Regulator of Disease Resistance Through the SA‐Dependent Pathway

ABSTRACT

Although microRNA1447 (miR1447) regulates poplar responses to abiotic stress and pest infestation, how miR1447 regulates poplar immunity against pathogens and its underlying molecular mechanisms remain to be elucidated. Here, we revealed that miR1447 functioned as a negative regulator of poplar disease resistance against fungal and bacterial pathogens using overexpression (OE) and short tandem target mimic (STTM) poplar lines of miR1447. Moreover, we demonstrated that PopTCTP contributed to poplar immunity as a target of miR1447 through integrative analysis of overexpression and RNAi lines, degradomes, transient co‐expression assay and GFP fluorescence report system, and found that PopTCTP interacted with dnaJ A6. Further molecular and genetic analyses revealed that the promoters of miR1447 and PopTCTP were responsive to exogenous salicylic acid (SA) treatment. We showed that the negative regulatory role of miR1447 in SA signalling and poplar resistance was weakened with exogenous SA treatment. Notably, the miR1447‐PopTCTP module contributed to PTI in poplar triggered by flg22 and associated with crosstalk between PTI and ETI via regulating MAPK signalling and scavenging ROS. Taken together, these findings unveil a novel pathway by which the miR1447‐PopTCTP‐SA signalling mediates disease resistance to diverse pathogens in poplar, offering promising genetic targets for tree breeding of disease resistance.

1. Introduction

Poplar is one of the most widely distributed and adaptable tree species worldwide, and it is also used as a model tree species for studying the molecular mechanisms and genetic engineering breeding (Kim et al. 2015; Ning et al. 2018). However, poplars are frequently subjected to numerous diseases, such as cankers, anthracnose and rust. Among them, poplar canker caused by Cytospora chrysosperma is a devastating fungal disease worldwide (Fan, Bezerra, et al. 2020), and an emerging poplar canker caused by the gram‐negative bacterium Lonsdalea populi results in enormous losses in poplar plantations (Li and He 2019; Xiao et al. 2021). Anthracnose, a typical poplar foliage disease, is generated by the hemibiotrophic ascomycete fungus Colletotrichum gloeosporioides (He et al. 2017). However, the underlying mechanism of poplar disease resistance remains largely unknown.

MicroRNAs (miRNAs), a class of noncoding RNAs with 20–24 nucleotides (nts) in length, are ubiquitous in plants and play crucial roles in defences against biotic stresses (Jones‐Rhoades and Bartel 2004; Lu et al. 2008). MiRNAs act as essential regulators by suppressing the expression of target genes via DNA methylation, mRNA cleavage, or translational inhibition through the binding to their target DNA or RNA sites in plants (Fabian et al. 2010; Feng et al. 2022; Voinnet 2009). The conserved Ptc‐miR472a exerts a key role in plant immunity to C. gloeosporum and C. chrysosperma (Su et al. 2018). Another conserved miRNA, miR159, has been reported to play important roles in poplar response to C. gloeosporum, C. chrysosperma and L. populi (Yang, Fu, et al. 2023). These results indicated a regulatory role for conserved miRNAs in poplar defence against pathogens. However, so far, there is still limited information on whether the non‐conserved miRNAs contribute to poplar disease resistance. Intriguingly, miR1447 was proved to be absent from the Arabidopsis genome and regulated the poplar response to different abiotic stresses (Bao et al. 2019; Lu et al. 2008). Additionally, sRNA library analysis of the resistant host P. tomentosa and the preferred host Populus × euramericana ‘74/76’ (poplar 107) infected with the beetle Anoplophora glabripennis revealed that miR1447 may play crucial regulatory roles in the poplar response to pest infestation (Wang et al. 2025). Taken together, investigations of miR1447 were mostly focused on poplar responses to abiotic stress and pest resistance using omics techniques. However, the biological function of miR1447 and its molecular mechanism in poplar defence against pathogens remain poorly understood.

The phytohormone SA and its signalling have been widely reported to participate in plant immunity against pathogens (Chhana Ullah 2023; Glazebrook 2005). MiRNAs could regulate key components of hormone signalling pathways and hormone homeostasis (Pagano et al. 2021). The miR7125‐MdARF1 module enhanced the resistance of apple to C. gloeosporioides by promoting lignin synthesis in response to SA signalling (Boccara et al. 2014). Both overexpression and knockdown of miR160 led to auxin‐SA antagonism, potentially resulting in an enhanced susceptibility phenotype (Natarajan et al. 2018). Overexpression of Arabidopsis miR167 induced SA‐dependent defence against Pseudomonas syringae by modulating its targets ARF6 and ARF8 (Caruana et al. 2020). The Arabidopsis miR773 target mimicry lines showed stronger activation of SA signalling (NPR1 and PR1), consistent with enhanced disease resistance (Salvador‐Guirao et al. 2018). The negative regulation of target genes and their downstream gene expressions by miR396a‐5p and miR396a‐3p was critical for tomato abiotic stresses by affecting SA or JA signalling pathways (Chen et al. 2017). However, the relationship between miR1447 and SA signalling in poplar defence against pathogens has not been uncovered.

In this study, we examined the roles of miR1447 in poplar resistance to pathogens. We found that miR1447 negatively regulated the poplar response to diverse pathogens, including fungal and bacterial pathogens, such as C. chrysosperma , C. gloeosporioides and L. populi . The results presented here supported the core role of miR1447 in controlling poplar disease resistance by regulating the SA pathway and ROS scavenging. In addition, PopTCTP was a direct target of miR1447 and interacted with chaperone protein dnaJ A6. Moreover, we found that the miR1447‐PopTCTP module was responsive to SA and participated in PTI and PTI‐ETI crosstalk. Collectively, our findings define the miR1447‐PopTCTP module as a previously uncharacterised regulator involved in poplar pathogen resistance via SA‐mediated pathways.

2. Results

2.1. Characterisation and Expression of miR1447

We first examined the tissue‐specific expression pattern of miR1447 in poplar and found that the expression of miR1447 was significantly higher in stems than in other tissues, suggesting that miR1447 mainly exerted its biological functions in stems (Figure 1a). To explore whether miR1447 plays a role in poplar defence against stem‐damaging pathogens, we assessed miR1447 expression levels among poplar varieties, including Populus deltoides ‘Zhonglin 2025’ (highly susceptible), poplar 107 (moderately resistant) and Populus tomentosa (highly resistant), inoculated with L. populi at 6 days post‐inoculation (dpi) (Yu et al. 2024). The results showed that miR1447 accumulation greatly decreased in infected poplar compared with mock‐inoculated poplar, indicating that miR1447 regulated poplar resistance to L. populi (Figure 1b). Furthermore, the expression level of miR1447 in the transcriptome database of 107 poplar inoculated with L. populi implied the potentially regulatory role of miR1447 in the poplar response to L. populi (Figure 1c) (Yang, Yu, et al. 2024). These results showed the potential role of miR1447 in poplar disease resistance.

FIGURE 1.

Expression profiles of miR1447 in poplar. (a) The tissue‐specific expression pattern of miR1447 in 1‐month‐old 84 K tissue‐cultured poplar by qRT‐PCR. YL: Young leaves, OL: Old leaves, S: Stems, R: Roots. Data represent mean ± SEM, n = 3 technical replicates. (b) Expression analysis of miR1447 in different poplar varieties inoculated with L. populi at 6 dpi based on miRNA profiles. 107: Populus × euramericana ‘74/76’, 2025: Populus deltoides ‘Zhonglin 2025’. CK: Control; IN: Inoculation. n = 3 mixed samples. (c) Expression analysis of miR1447 based on the transcriptome database of 107 poplar inoculated with L. populi . CK: Control; IN1: 1 dpi, IN3: 3 dpi, IN6: 6 dpi. dpi, days post‐infection. Data represent mean ± SEM, n = 3 biological replicates. (d) Overexpression of miR1447 by qRT‐PCR with 1‐month‐old tissue‐cultured transgenic lines. 7O1‐7O4: MiR1447 overexpression lines. Data represent mean ± SEM, n = 3 technical replicates. (e) STTM of miR1447 by qRT‐PCR with 1‐month‐old tissue‐cultured transgenic lines. 7S1‐7S4: MiR1447 STTM lines. Data represent mean ± SEM, n = 3 technical replicates. *p ≤ 0.05, **p ≤ 0.01, unpaired t‐test.

2.2. Poplar Resistance to Various Pathogens Is Compromised by Overexpression of miR1447 and Is Enhanced by STTM of miR1447

To further elucidate the biological function of miR1447, the OE and STTM lines of miR1447 were obtained in 84 K poplar (Figure 1d,e). The susceptibility analysis of miR1447 transgenic lines to L. populi revealed that the OE lines exhibited more severe disease symptoms and longer lesion lengths, whereas the opposite was observed in the STTM lines, indicating that miR1447 negatively regulated the poplar response to L. populi (Figure 2a,b). Furthermore, the detailed regulatory role of miR1447 in the disease progression was investigated. There were no obvious changes in plants at 0 dpi. The xylem of infected plants was red at 1 dpi, and the stress symptoms of miR1447‐OE lines were more pronounced than those of WT, whereas miR1447‐STTM lines showed the opposite symptoms. The white, odorous bacterial pus overflowed from the inoculation points in susceptible transgenic lines at 4 dpi, whereas the WT and resistant transgenic lines exhibited typical canker symptoms at 9 dpi. Compared to WT, the miR1447‐OE lines exhibited more severe symptoms during the disease course, whereas the opposite was observed in the miR1447‐STTM lines (Figure S1).

FIGURE 2.

miR1447 modulates dual disease resistance in Populus against fungal and bacterial pathogens. (a) The phenotype of 1‐year‐old miR1447 transgenic lines detached twigs inoculated with L. populi at 8 dpi. Scale bars: 1 cm. (b) The lesion length of 1‐year‐old miR1447 transgenic lines inoculated with L. populi at 8 dpi. Data represent mean ± SEM, n = 4 biological replicates. (c) The infected stem phenotype of miR1447 transgenic lines inoculated with C. chrysosperma at 8 dpi. Scale bars: 1 cm. (d) Lesion length of infected stems in 1‐year‐old miR1447 transgenic lines inoculated with C. chrysosperma at 8 dpi. Data represent mean ± SEM, n = 3 biological replicates. (e) The phenotype of 2‐month‐old miR1447 transgenic lines inoculated with C. gloeosporioides at 7 dpi. Scale bars: 2 cm. Data represent mean ± SEM, n = 3 biological replicates. (f) The representative pictures of F _v/F _m of 2‐month‐old miR1447 transgenic lines inoculated with C. gloeosporioides at 7 dpi. (g) Cell death in 2‐month‐old miR1447 transgenic lines was analysed by trypan blue staining after inoculation with C. gloeosporioides at 7 dpi. Scale bars: 20 μm. (h) H₂O₂ accumulation in 2‐month‐old miR1447 transgenic lines was analysed by DAB staining after inoculation with C. gloeosporioides at 7 dpi. Scale bars: 20 μm. (i) Lesion area of 2‐month‐old miR1447 transgenic lines inoculated with C. gloeosporioides at 7 dpi. The bars represent the mean ± SEM, n = 3 biological replicates. (j) Infection level of 2‐month‐old miR1447 transgenic lines inoculated with C. gloeosporioides at 7 dpi. Level 1: F _v/F _m > 0.7, Level 2: F _v/F _m 0.5–0.7, Level 3: F _v/F _m < 0.5. (k) F _v/F _m of in 2‐month‐old miR1447 transgenic lines inoculated with C. gloeosporioides at 7 dpi. The bars represent the mean ± SD, n = 3 biological replicates.

To further investigate the effect of miR1447 on plant resistance to poplar canker fungus, we performed C. chrysosperma inoculation assays with miR1447 transgenic lines. The infected plants produced typical canker symptoms (Figure 2c). The lesion lengths in miR1447‐OE lines were significantly greater than those in WT, whereas miR1447‐STTM lines showed reduced lesion lengths compared to WT at 8 dpi (Figure 2d).

Additionally, to check the function of miR1447 in poplar response to the anthracnose fungus, the miR1447 transgenic lines were inoculated with C. gloeosporioides. Overexpressing miR1447 resulted in larger leaf lesions, whereas suppressing its expression led to smaller leaf lesions compared to the WT (Figure 2e,i). The percentage of infection level further indicated that miR1447‐OE lines showed increased disease symptoms compared with WT, and that miR1447‐STTM lines showed decreased disease symptoms compared with WT (Figure 2f,j). The observation of cell death verified more spots and necrosis in miR1447‐OE lines than in the WT, whereas the opposite was observed in miR1447‐STTM lines (Figure 2g). Histochemical observation showed that the brown colour was deeper in miR1447‐OE leaves than in WT leaves and miR1447‐STTM leaves, indicating more H₂O₂ accumulation in miR1447‐OE leaves (Figure 2h). The F _v/F _m analysis showed that miR1447 negatively regulated the potential photosynthetic capacity of poplars under C. gloeosporioides infection (Figure 2k). Combinedly, these findings showed that miR1447 is a negative regulator of poplar resistance to diverse pathogens.

2.3. miR1447 Regulates the Transcript Levels of Disease Resistance‐Related Genes

To explore the molecular mechanism underlying miR1447 mediated regulation of poplar disease resistance, we analysed the gene expression profiles in miR1447 transgenic lines. The KEGG enrichment analysis of miR1447 transgenic lines identified differential expression in the mitogen‐activated protein kinase (MAPK) signalling pathway, flavonoid biosynthesis and phenylpropanoid biosynthesis pathways (Figure S2). The results revealed that miR1447 was involved in the plant response to pathogens through these disease resistance‐related pathways. MAPKs are key signal regulators of PTI and ETI, and MPK3/6 are essential for pre‐PTI‐mediated ETI suppression (Wang, Wei, et al. 2023). The analysis of DEGs revealed that miR1447 positively regulated MPK3 expression and negatively regulated MPK6 (Figure S3a). We next checked whether MPK3/6 contributed to plant immunity; their phosphorylation status was detected. As expected, the results showed that the infection triggered the changes in the phosphorylation levels of plant MAPKs (Figure S3b). Taken together, these findings suggest a regulatory role of miR1447 in the PTI/ETI signalling.

Reactive oxygen species (ROS) are involved in plant defence against pathogens (Gao et al. 2021). Antioxidant enzymes eliminate ROS to protect plants against pathogens (Wei et al. 2020). The antioxidant enzymes, including superoxide dismutase gene (SOD), peroxidase (POD), ascorbate peroxidase (APX), catalase (CAT), glutathione peroxidase (GPX) and glutathione S‐transferase (GST), showed upregulated expression in miR1447‐OE lines and downregulated expression in miR1447‐STTM lines, indicating that the overexpression of miR1447 might enhance the ROS scavenging of poplars (Figure S4a,b). Furthermore, the exogenous application of ascorbic acid to scavenge ROS reduced lesion symptoms, but it did not alter resistance phenotypes in miR1447 OE or STTM lines (Figure S4c). Intriguingly, antioxidant enzyme expression was downregulated in SA‐treated miR1447‐OE lines and upregulated in SA‐treated miR1447‐STTM lines, suggesting that SA is involved in miR1447 regulation of ROS scavenging in poplars (Figure S4d,e).

2.4. miR1447 Is Involved in Disease Resistance Modulation Through SA Signalling

SA is a major defence hormone involved in plant innate immunity, and SA signalling plays a crucial role in plant defence (Glazebrook 2005; Zhou and Zhang 2020). To gain insights into the potential regulatory roles of miR1447, its cis‐acting elements were further identified. The results showed that the binding sites were related to hormone response, MYB and MYC, and light responsiveness (Figure S5a). SA responsiveness is instrumental for the biotic stress response in plants (Ullah et al. 2022). We sought to check whether cis‐acting SA responsiveness elements were involved in poplar defence by responding to SA (Figure S5a). We found that transient overexpression of the cis‐elements of miR1447 in N. benthamiana leaves reduced GUS activity in SA‐treated leaves (Figure S5b,c).

Given that nonexpression of pathogen‐related gene‐1 (NPR1) is the master regulator controlling the SA‐mediated gene expression response that induces pathogenesis‐related (PR) genes (Yang, Li, et al. 2023), we subsequently measured the transcript levels of NPR1 and PR1 in miR1447 OE and STTM lines affected by C. gloeosporioides. The results showed that miR1447 negatively regulated the early expression (0 h, 24 h) of NPR1 and the basal level expression (0 h) of PR1, indicating that miR1447 served as a negative regulator of SA signalling in the poplar response to pathogen (Figure 3a,b). To further validate the involvement of SA signalling downstream in miR1447 regulating plant disease resistance, the miR1447 transgenic lines were treated with tenoxicam, which inhibited NPR1‐mediated SA‐dependent plant immunity. The results showed that the exogenous application of tenoxicam increased the susceptibility of miR1447 transgenic lines compared to the control (Figure 3c). The expression trend of NPR1 under tenoxicam treatment was generally consistent with that without treatment (Figure 3d). Combinedly, these results indicated that miR1447 contributes to the poplar response to C. gloeosporioides by regulating SA signalling.

FIGURE 3.

The salicylic acid and defence signalling analysis in miR1447 transgenic lines. (a, b) The dynamic expression of SA marker genes in 45‐day‐old miR1447 transgenic lines with C. gloeosporioides inoculation by qRT‐PCR. Data represent mean ± SEM, n = 3 technical replicates. (c) The phenotype of tenoxicam‐treated 1‐month‐old miR1447 tissue‐cultured transgenic lines infected with C. gloeosporioides. Scale bars: 0.5 cm. (d) The expression levels of NPR1 in tenoxicam‐treated 1‐month‐old miR1447 tissue‐cultured transgenic lines infected with C. gloeosporioides by qRT‐PCR. Data represent mean ± SEM, n = 3 technical replicates. (e) The expression levels of miR1447 based on transcriptome database of SA‐treated 1‐month‐old 84 K tissue‐cultured poplar infected with C. gloeosporioides. Data represent mean ± SEM, n = 3 biological replicates. (f) The phenotype of SA‐treated 1‐month‐old miR1447 tissue‐cultured transgenic lines infected with C. gloeosporioides at 2 dpi. (g) The heatmap of SA biosynthesis genes based on transcriptome database of SA‐treated 1‐month‐old miR1447 tissue‐cultured transgenic lines infected with C. gloeosporioides. Scale bars: 0.5 cm. (h) The endogenous SA content in SA‐treated 1‐month‐old miR1447 tissue‐cultured transgenic lines infected with C. gloeosporioides. Data represent mean ± SEM, n = 3 biological replicates. (i, j) The dynamic expression of SA marker genes based on transcriptome database of SA‐treated 1‐month‐old miR1447 tissue‐cultured transgenic lines infected with C. gloeosporioides. Data represent mean ± SEM, n = 3 biological replicates. ICS, isochorismate synthase; PAL, phenylalanine ammonia lyase. NPR1: Non‐expression of pathogens related gene‐1. PR1: Pathogenesis‐related 1 gene. *p ≤ 0.05, **p ≤ 0.01, unpaired t‐test.

To further study the relationship between miR1447 in modulating plant disease resistance and SA signalling, we measured endogenous SA levels and performed RNA‐seq analysis of SA‐treated miR1447 transgenic plants following pathogen inoculation. miR1447 presented a downward expression trend in SA‐treated WT plants infected with C. gloeosporioides (Figure 3e). Notably, exogenous SA application enhanced C. gloeosporioides resistance in miR1447‐OE lines and reduced it in miR1447‐STTM lines, indicating that miR1447 improved disease resistance in response to SA (Figure 3f). Interestingly, among two distinct routes for synthesising SA, including the isochorismate synthase (ICS) pathway and phenylalanine ammonia lyase (PAL) pathway, there were no obvious changes in ICS genes between OE vs. WT and STTM vs. WT, suggesting that poplar harboured ICS that were transcriptionally insensitive to SA (Figure 3g), however, the PAL genes were upregulated in the miR1447‐OE lines but downregulated in miR1447‐STTM at 0 h, and there was no obvious trend in the expression of PAL genes at 24 and 48 h, suggesting that the SA biosynthesis change in SA‐treated miR1447 transgenic poplar lines was mainly based on the PAL pathway under C. gloeosporioides infection (Figure 3g). The determination of endogenous SA content in SA‐treated miR1447 transgenic lines infected with C. gloeosporioides further revealed an increase in miR1447‐OE endogenous SA levels compared with WT at 0 h and a decrease in miR1447‐STTM lines (Figure 3h). The endogenous SA content of miR1447 transgenic lines was significantly lower than that of WT at 24 h, and no significant changes were observed at 48 h (Figure 3h). Correspondingly, the differential expression of PAL genes supported the analysis of endogenous SA content in SA‐treated miR1447 transgenic lines infected with C. gloeosporioides (Figure 3g,h). The expression of SA signalling genes (NPR1 and PR1) was dynamically changed in SA‐treated miR1447 transgenic lines, which were upregulated in miR1447‐OE lines, whereas downregulated in miR1447‐STTM lines (Figure 3i,j). Taken together, these results indicated that exogenous SA and SA signalling contributed to C. gloeosporioides resistance in miR1447 transgenic lines.

In‐depth analysis of transcriptome GO enrichment revealed the molecular mechanism by which SA treatment in miR1447‐OE lines enhanced disease resistance (Figure S6). The metal ion response, cutin and cell wall development, especially photosynthetic terms, were upregulated in SA‐treated miR1447‐STTM lines, which were mainly involved in plant growth and development. The disease resistance‐related terms were downregulated, including methyl salicylate esterase (MeSA) activity, methyl jasmonate (MeJA) esterase activity, systemic acquired resistance (SAR), and SA metabolic process. The transcriptome KEGG enrichment analysis showed that SA‐treated miR1447‐OE lines positively regulated plant disease resistance by upregulating phenylpropanoid biosynthesis, flavonoid biosynthesis and biosynthesis of various secondary metabolites to directly defend against the pathogen (Figure S7). Galactose metabolism, the MAPK signalling pathway, plant hormone signal transduction and other pathways were downregulated to reduce energy consumption. The SA‐treated miR1447‐STTM lines with upregulated phenylpropanoid and flavonoid biosynthesis and other pathways prevent pathogen invasion at early stages (0–24 h), but the upregulated pathways were mainly photosynthesis pathways at late stages (24–48 h). Additionally, phenylpropanoid biosynthesis and starch and sucrose metabolism were downregulated in SA‐treated miR1447‐STTM lines. The results revealed that SA‐treated miR1447‐OE lines exhibited direct and active disease resistance mechanisms, whereas SA‐treated miR1447‐STTM lines exhibited indirect defence mechanisms.

2.5. PopTCTP Is a Direct Target of miR1447 and Contributes to Disease Resistance via SA Signalling

To identify the target mRNA of miR1447, we performed the degradome analysis of both 84 K poplar and 107 poplar bark infected with L. populi . A total of 21 target genes were detected (Table S1). PopTCTP (Pop_G10G022958) was detected in two different degradomes, and the cut position was shown to be complementary to the 10th and 11th nts of miR1447 (Figure 4a). To confirm the miR1447/PopTCTP pairs, a transient co‐expression assay was thereafter undertaken with N. benthamiana leaves. As expected, the expression of PopTCTP was significantly downregulated when coexpressed with miR1447 in tobacco, revealing that miR1447 cleaved PopTCTP and led to its degradation, supporting the endogenous downregulation of PopTCTP by miR1447 (Figure 4b). Additionally, PopTCTP was significantly downregulated in miR1447‐OE lines and upregulated in miR1447‐STTM lines, indicating that the expression pattern of PopTCTP in plants was opposite to that of miR1447 (Figure 4c). Furthermore, the PopTCTP‐GFP construct with the pCAMBIA2300 vector was transiently cotransformed into tobacco leaves, GFP fluorescence was observed (Figure 4d). As expected, the PopTCTP‐GFP fluorescence was weakened when PopTCTP‐GFP was cotransformed with the 35S::miR1447‐OE strain (Figure 4d). Additionally, coinjection of PopTCTP‐GFP and 35S::miR1447‐STTM enhanced the signal intensity of GFP fluorescence (Figure 4d). These analyses demonstrated that miR1447 targeted PopTCTP with its cleavage complementarity in PopTCTP.

FIGURE 4.

The target gene of miR1447. (a) miR1447 targeted PopTCTP by degradome analysis of 4‐week‐old tissue‐cultured 84 K poplar and 3‐year‐old 107 poplar bark infected with L. populi . (b) Validation of the predicted miR1447/PopTCTP pairs using the transient co‐expression assay in tobacco by qRT‐PCR. Data represent mean ± SEM, n = 4 technical replicates. (c) The relative expression level of PopTCTP in 1‐month‐old miR1447 tissue‐cultured transgenic lines by qRT‐PCR. Data represent mean ± SEM, n = 4 technical replicates. WT: Wild type; 7O1: MiR1447 overexpression lines; 7S1: MiR1447 STTM lines. (d) The targeting relationship between miR1447 and PopTCTP was validated using a tobacco GFP reporter system. TCTP+vector: Coinjection equal volumes of the PopTCTP‐eGFP and pCAMBIA2300‐35S‐OCS vector, TCTP+7O1: Coinjection equal volumes of the miR1447‐OE and PopTCTP‐eGFP, TCTP+7S2: Coinjection equal volumes of the miR1447‐STTM and PopTCTP‐eGFP. Scale bars: 100 μm. (e) The tissue‐specific expression pattern of PopTCTP in 1‐month‐old 84 K tissue‐cultured poplar by qRT‐PCR. L1: Apical bud and young leaves, L3‐L9: The 3rd to 9th leaves from apical bud downwards, S1: Upper part of the stem, S2: Middle part of the stem, S3: Lower part of the stem, Ph: Phloem, Xy: Xylem, Rt: Root. Data represent mean ± SEM, n = 3 technical replicates. (f) The subcellular localisation identification of PopTCTP protein. 35S::GFP represents an empty vector representing GFP expression driven by the 35S promoter; 35S::TCTP‐GFP represents TCTP‐GFP fusion protein driven by the 35S promoter. 35S::GFP scale bars: 25 μm, 35S::TCTP‐GFP scale bars: 10 μm. (g) The expression of the subcellular localisation fusion protein PopTCTP‐GFP was confirmed using western blot. CK: Blank control. GFP: Empty vector. TCTP‐GFP: TCTP‐GFP fusion protein. (h) The phylogenetic analysis and conserved domain of TCTP gene family in 84 K poplar. Maximum Likelihood method (ML) trees were constructed using MEGA 7.0 with bootstrap analysis (1000 replicates). *p ≤ 0.05, **p ≤ 0.01, unpaired t‐test.

The tissue‐specific expression pattern analysis revealed that PopTCTP had the highest expression level in the terminal bud, followed by higher expression levels in the stem, especially in the upper part of the stem, and relatively lower expression levels in the leaves and roots (Figure 4e). The subcellular localisation analysis in tomato found that TCTP was observed in the nucleus and cytoplasm (Bruckner et al. 2017). In the rubber tree, HbTCTP was localised in the membrane, cytoplasm and nucleus (Deng et al. 2016). In this investigation, we validated that clear fluorescence signals of PopTCTP fused with GFP were observed in both the nucleus and the cytoplasm (Figure 4f). Furthermore, the western blot analysis further confirmed the expression of the PopTCTP‐GFP fusion protein, indicating that PopTCTP exerted biological functions in the nucleus and cytoplasm (Figure 4g). Phylogenetic analyses showed that PopTCTP belongs to the TCTP gene family, and PopTCTP has the closest genetic relationship with Pop_A10G001121 (Figure 4h). Further analysis based on protein sequences showed that all members of TCTP in 84 K poplar and AtTCTP1 possessed a TCTP domain, indicating the conserved role of TCTP in plants (Figure 4h). The promoter analysis showed that PopTCTP contained multiple cis‐acting regulatory elements, including MYB and MYC recognition sites, light and low‐temperature responsiveness, hormone responsive elements such as SA responsiveness elements, abscisic acid responsive elements and gibberellin‐responsive elements (Figure S8a). Moreover, the promoter of PopTCTP was transiently overexpressed in SA‐treated N. benthamiana (Figure S8b). The results showed that GUS activity was higher in SA‐treated leaves than in those under normal conditions, indicating the response of PopTCTP to exogenous SA (Figure S8c).

To investigate the regulatory role of PopTCTP in the poplar response to the stem canker pathogen, its expression was analysed based on the transcriptome and proteome database of poplar affected by L. populi (Yang, Yu, et al. 2024). PopTCTP showed a continuous downward expression trend (Figure 5a). The protein expression level of PopTCTP presented an upward trend at 0–3 dpi and a downward trend at 3–6 dpi (Figure 5b). To further understand whether PopTCTP was involved in the poplar response to foliage pathogens, its expression level was analysed in poplar infected with C. gloeosporioides. The transcription abundance of PopTCTP was significantly decreased during the time course from 12 h post inoculation to 12 dpi, indicating that PopTCTP modulated the defence of poplar against fungal stress (Figure 5c). The expression level of PopTCTP presented an upward trend in SA‐treated WT plants infected with C. gloeosporioides, which was opposite to that of miR1447 (Figures 3e and 5d).

FIGURE 5.

PopTCTP regulates poplar resistance against pathogens. (a) The expression of PopTCTP in the complete transcriptome of poplar inoculated with L. populi . Data represent mean ± SEM, n = 3 biological replicates. (b) The expression of PopTCTP in the proteome of poplar inoculated with L. populi . (c) The expression level of PopTCTP in wild type poplar inoculated with C. gloeosporioides by qRT‐PCR. Data represent mean ± SEM, n = 3 technical replicates. (d) The expression levels of PopTCTP based on transcriptome database of SA‐treated 1‐month‐old wild type tissue‐cultured poplar infected with C. gloeosporioides. Data represent mean ± SEM, n = 3 biological replicates. (e) The synonymic mutation of complementary nucleotide sequences between PopTCTP and miR1447. (f) Schematic diagram of PopTCTP‐RNAi carrier. (g) The interference sequences in PopTCTP. (h, i) Identification expression of PopTCTP with 1‐month‐old tissue‐cultured overexpression lines by qRT‐PCR. Data represent mean ± SEM, n = 3 biological replicates. (j, k) Identification expression of PopTCTP with 1‐month‐old tissue‐cultured RNAi lines by qRT‐PCR. Data represent mean ± SEM, n = 3 biological replicates. CK: Control. IN: Inoculation, IN1: 1dpi, IN3: 3dpi, IN6: 6dpi. dpi: Days post inoculation. 84 K: Wild type. p2300: Transgenic lines of pCAMBIA2300‐35S‐OCS empty carrier. 8OX: PopTCTP overexpressing lines, X represents the number of different overexpressing lines. 8iY: PopTCTP RNAi lines, where Y represents the number of different RNAi lines. *p ≤ 0.05, **p ≤ 0.01, unpaired t‐test.

To investigate how PopTCTP regulates poplar resistance to pathogens, we obtained OE lines with synonymous amino acid mutations in complementary sequences of miR1447 and RNAi lines of PopTCTP (Figure 5e–k). Moreover, we analysed the DGE profiles of PopTCTP transgenic lines. The KEGG enrichment analysis showed that PopTCTP may participate in plant disease resistance by regulating plant‐pathogen interactions, secondary metabolite (including diterpenoid and isoquinoline alkaloid) biosynthesis, phenylpropanoid biosynthesis, glutathione metabolism, limonene and pinene degradation, phenylalanine metabolism and the MAPK signalling pathway (Figure S9). Furthermore, the expression of the MAPK signalling pathway‐related genes MPK3/6 was affected by PopTCTP (Figure S10a). Additionally, the phosphorylation status of MPK3/6 was detected, and the results indicated that pathogen infection triggered changes in the phosphorylation levels of MAPKs in PopTCTP transgenic lines (Figure S10b). Intriguingly, PopTCTP exhibited opposite regulatory effects on MPK3 and MPK6 expression compared with miR1447 (Figures S3a and S10a). These results revealed a potential three‐level regulatory network of the miR1447‐PopTCTP‐MAPK pathway in plant immune regulation.

2.6. The miR1447‐ PopTCTP Module Is Involved in Poplar Growth and flg22‐Triggered PTI

To uncover the regulatory role of miR1447 in poplar growth, we analysed the photosynthetic indexes of miR1447 transgenic lines (Figure 6a). Compared to WT, the net photosynthetic rate, stomatal conductance, and transpiration rate of miR1447‐OE lines were slightly lower, whereas they were higher in STTM lines (Figure 6b–d). Additionally, inhibition of miR1447 expression increased the instantaneous leaf water use efficiency (WUE) (Figure 6e). Overexpression of miR1447 decreased the potential photosynthetic capacity; however, the suppression of miR1447 through STTM obtained the opposite result (Figure 2k). The KEGG enrichment analysis of miR1447 transgenic lines revealed the differential expression of starch and sucrose metabolism and photosynthesis‐antenna proteins (Figure S2). Furthermore, gene expression analysis of the photosynthetic machinery in miR1447 transgenic lines revealed that multiple photosynthesis‐related genes were differentially expressed (Figure S3c). Comprehensively, both photosynthetic indexes and gene expression profiles indicated that miR1447 may negatively regulate the photosynthetic capacity of poplar. The growth indexes determination of PopTCTP transgenic lines showed that PopTCTP‐OE lines increased the plant height and ground diameter, whereas the PopTCTP‐RNAi lines showed the opposite phenotype, suggesting that PopTCTP may positively regulate the growth of poplars (Figure 6f,g). Additionally, the height and ground diameter of the pCAMBIA2300‐35S‐OCS (empty carrier) transgenic lines showed no significant difference compared to WT. The analysis of photosynthetic machinery in PopTCTP transgenic lines revealed that multiple photosynthesis‐related genes were differentially expressed (Figure S10c). Intriguingly, the expression trend of genes encoding protein PHOTOSYSTEM I ASSEMBLY 2, photosynthetic NDH subunit of subcomplex B 5, and phototropic‐responsive NPH3 family protein was opposite in miR1447 and PopTCTP transgenic lines (Figures S3c and S10c).

FIGURE 6.

miR1447‐PopTCTP module is involved in poplar growth and flg22‐triggered PTI. (a) The phenotype of miR1447 transgenic lines. Scale bars: 2 cm. (b–e) The transpiration and photosynthetic activity of 5‐week‐old miR1447 transgenic lines. Data represent mean ± SEM, n = 3 biological replicates. (f) The high of 8‐week‐old PopTCTP transgenic lines. Data represent mean ± SEM, n = 3 biological replicates. (g) The ground diameter of 8‐week‐old PopTCTP transgenic lines. Data represent mean ± SEM, n = 3 biological replicates. (h) Identification expression of miR1447 in 4‐week‐old tissue‐cultured wild type poplar sprayed with flg22 by qRT‐PCR. Data represent mean ± SEM, n = 3 technical replicates. (i) Identification expression of PopTCTP in 4‐week‐old tissue‐cultured wild type poplar sprayed with flg22 by qRT‐PCR. Data represent mean ± SEM, n = 3 technical replicates. WT: Wild type, 7O1 and 7O2: MiR1447 overexpression lines, 7S1 and 7S2: MiR1447 STTM lines, p2300: Transgenic lines of pCAMBIA2300‐35S‐OCS empty carrier, 8O2 and 8O7: PopTCTP overexpression lines, 8i5: PopTCTP‐RNAi lines. Mock: No treatment, CK: Water treatment as a control, flg22: 10 μM flg22 solution treatment, h: Hour post treatment. *p ≤ 0.05, unpaired t‐test.

PTI is a broad‐spectrum immune response in plants that serves as the first line of defence in the plant innate immune system (Jones and Dangl 2006). Additionally, the PTI triggered by flg22 is one of the most common model systems for investigating plant‐bacterial pathogen interactions (Wang, Wei, et al. 2023). To explore the regulatory role of the miR1447‐PopTCTP module in PTI, we analysed the expression of miR1447/PopTCTP in WT plants treated with flg22. As a result, the expression of miR1447 was upregulated at 1 h treatment of flg22 compared with CK, and significantly increased at 6 h, whereas the expression was downregulated at 3 h (Figure 6h). The expression of PopTCTP was downregulated in WT plants treated with flg22 at 1 h compared with CK and significantly decreased at 3 h and then increased at 6 h (Figure 6i). The results revealed that the miR1447‐PopTCTP module might regulate flg22‐triggered PTI in poplar.

2.7. The miR1447‐ PopTCTP Module May Regulate the Interaction Protein dnaJ A6 of PopTCTP

To explore the role of PopTCTP in plant immunity, the PopTCTP transgenic lines were inoculated with C. gloeosporioides. The PopTCTP OE lines showed more severe symptoms than the control, and the RNAi lines showed the opposite, suggesting that PopTCTP may negatively regulate poplar resistance to C. gloeosporioides (Figure 7a). Furthermore, the interaction proteins of PopTCTP were screened with the 84 K poplar cDNA prey library. The enrichment analysis of the PopTCTP interaction protein showed that the fatty acid biosynthetic process and acyl‐[acyl‐carrier‐protein] hydrolase activity had the most annotations (Figure S11a). KEGG enrichment analysis revealed that the PopTCTP interaction proteins were mainly enriched in energy metabolism and lipid metabolism (Figure S11b). Moreover, the protein–protein interaction between PopTCTP and chaperone protein dnaJ A6 was validated in the Y2H assay (Figure 7b). To understand the impact of miR1447 and PopTCTP on dnaJ A6, the expression level of dnaJ A6 was analysed in the miR1447 and PopTCTP lines. Upregulation of dnaJ A6 expression was observed in miR1447‐OE lines and PopTCTP‐RNAi lines, whereas the expression level of dnaJ A6 was downregulated in miR1447‐STTM lines and PopTCTP‐OE lines. Thus, we proposed that miR1447 positively regulated the expression of dnaJ A6, whereas PopTCTP exerted an opposite regulatory effect (Figure 7c,d).

FIGURE 7.

The interaction protein analysis of PopTCTP. (a) The phenotype of 1‐month‐old PopTCTP tissue‐cultured transgenic lines infected with C. gloeosporioides. Scale bars: 0.5 cm. (b) The dnaJ A6 is identified as the interaction protein of PopTCTP by Y2H. (c) The expression level of dnaJ A6 based on the transcriptome database of miR1447 transgenic lines. n = 3 mixed samples. (d) The expression level of dnaJ A6 based on the transcriptome database of PopTCTP transgenic lines. Data represent mean ± SEM, n = 3 biological replicates. (e) Identification expression of dnaJ A6 in 1‐month‐old PopTCTP overexpression tissue‐cultured lines infected with C. gloeosporioides by qRT‐PCR. Data represent mean ± SEM, n = 3 technical replicates. (f) Identification expression of dnaJ A6 in 1‐month‐old PopTCTP RNAi tissue‐cultured lines infected with C. gloeosporioides by qRT‐PCR. Data represent mean ± SEM, n = 3 technical replicates. (g) Expression analysis of dnaJ A6 based on transcriptome database of poplar inoculated with L. populi . CK: Control, IN: Inoculation, IN1: 1 dpi, IN3: 3 dpi, IN6: 6 dpi. Data represent mean ± SEM, n = 3 biological replicates. (h) The expression levels of dnaJ A6 based on the transcriptome database of SA‐treated 1‐month‐old wild type tissue‐cultured poplar infected with C. gloeosporioides. Data represent mean ± SEM, n = 3 biological replicates. WT: Wild type, 7O1 and 7O2: MiR1447 overexpression lines, 7S1 and 7S2: MiR1447‐STTM lines, 8O2 and 8O7: PopTCTP overexpression lines, 8i2 and 8i5: PopTCTP‐RNAi lines. Scale bars: 0.5 cm. *p ≤ 0.05, **p ≤ 0.01, unpaired t‐test.

To further explore the relationship between PopTCTP and dnaJ A6, the expression level of dnaJ A6 in PopTCTP transgenic lines infected with C. gloeosporioides was investigated. The dnaJ A6 was downregulated in PopTCTP‐OE lines upon the infection, but upregulated in PopTCTP‐RNAi lines (Figure 7e,f). To understand the role of dnaJ A6 in plant immunity, its expression level was investigated in the gene expression profiles of poplar defence against L. populi and C. gloeosporioides. The expression of dnaJ A6 showed a gradual downward trend in poplar inoculated with L. populi (Figure 7g), suggesting that dnaJ A6 should participate in plant defence against bacterial pathogens. The expression level of dnaJ A6 presented an upward trend in SA‐treated WT plants infected with C. gloeosporioides, which is consistent with the expression trend of PopTCTP (Figures 5d and 7h). These results showed that dnaJ A6, the interaction protein of PopTCTP, may be regulated by the miR1447‐PopTCTP module and was involved in the poplar response to diverse pathogens (Figure 8).

FIGURE 8.

Hypothetical model of how miR1447 mediated plant immunity in poplar. MiR1447 is involved in poplar resistance by regulating SA pathway. MiR1447 negatively regulates the expression of NPR1 and PR1, and overexpression of miR1447 compromises poplar resistance. After exogenous SA treatment, miR1447 positively regulates PAL‐mediated SA biosynthesis in the poplar with the pathogen infection. Moreover, miR1447 exerts biological functions by cleaving its target gene PopTCTP. The promoters of miR1447 and PopTCTP, containing the TCA element, are responsive to exogenous SA treatment. The miR1447‐PopTCTP module regulates the expression of chaperone protein dnaJ A6, which is a potential interaction protein of PopTCTP. Arrows represent positive regulation, and blocked arrows represent negative regulation. Scissors represent cleaving.

3. Discussion

As a pivotal defence hormone, the accumulation of SA triggers SAR upon pathogen invasion, which plays a crucial role in plant immunity (Wang et al. 2020). Lower GUS staining was observed in SA‐treated transgenic Arabidopsis leaves overexpressing the miR472a promoter, indicating that miR472a was downregulated in response to pathogens (Su et al. 2018). Overexpression of miR159a induced changes in SA‐related genes, suggesting an important role for miR159a in poplar responses to pathogens (Yang, Fu, et al. 2023). Here, cis‐acting element analysis of the promoter and GUS staining investigation showed that the miR1447‐PopTCTP module responded to SA (Figure 8, Figures S5 and S8). The application of exogenous SA reversed the expression trend of miR1447/PopTCTP in poplar inoculated with C. gloeosporioides, revealing the SA responsiveness of the miR1447‐PopTCTP module in plant‐pathogen interactions (Figure 3e, Figure 5d). Furthermore, miR1447‐OE lines showed increased disease resistance with exogenous SA treatment (Figure 3f), which was similar to the investigation that the miR7125‐MdARF1 module enhanced the resistance of apple to C. gloeosporioides by promoting lignin synthesis in response to SA signalling (Liu et al. 2024).

The PAL/BSH pathway (rather than the ICS pathway) has been shown to be the main pathway for SA biosynthesis in most plants outside the Brassicaceae species (Liu et al. 2025). Here, SA biosynthesis was mainly via the PAL pathway, not the ICS pathway in 84 K poplar (Figure 8), consistent with the previous report (Chhana Ullah 2023). In summary, both exogenous and endogenous SA contributed to the increase in the resistance of the miR1447‐OE line to C. gloeosporioides (Figure 3). Investigations have shown that the SA receptor NPR1 and induced PR genes play a profound role in plant immunity (Liu et al. 2020). The miR472‐RDR6 silencing pathway modulates the defence response of Arabidopsis by regulating the transcript level of the SA‐dependent marker gene PR1 (Boccara et al. 2014). The expression analysis of NPR1 and PR1 in miR1447 transgenic lines inoculated with C. gloeosporioides revealed that miR1447 was involved in plant‐pathogen interactions by regulating SA signalling pathway genes (Figure 3a,b,i,j). The miR1447‐PopTCTP module was involved in plant defence against pathogens by responding to the SA signal (Figure 8). AGO1a, a target of miR168, is under the control of the SA signalling pathway, and its phosphorylation by MAP kinases is required for miRNA‐mediated resistance of rice against Xanthomonas oryzae pv. oryzae infection (Singh et al. 2024). In this study, miR1447 regulated poplar disease resistance through the SA‐dependent pathway, the expression of MPK3/6 and the phosphorylation status of MAPKs, suggesting miR1447‐SA‐MAPK crosstalk in poplar defence (Figure 8, Figure S3a,b).

Plants have evolved different layers of immune responses to defend against evolving pathogens (Jones and Dangl 2006; Wang et al. 2019). A previous study found that miR472a regulated flg22‐triggered PTI (Su et al. 2018). In this study, the dynamic expression analysis indicated that the miR1447‐PopTCTP module was involved in flg22‐triggered PTI (Figure 6h,i). Previous studies found that miRNAs were required for plant immunity by regulating MAPK signalling (Wang et al. 2017; Yang, Fu, et al. 2023). The differential expression of MAPK signalling pathway genes in the transcriptomes of miR1447/PopTCTP transgenic poplar plants showed that the miR1447‐PopTCTP module is involved in PTI‐ETI crosstalk by regulating MAPK signalling (Figures S2 and S9). The investigation found that the absence of MPK3 and MPK6 significantly reduces pre‐PTI‐mediated ETI suppression (Wang, Wei, et al. 2023). MPK3 and MPK6 might be involved in miR159a‐modulated poplar resistance against pathogens (Yang, Fu, et al. 2023). Indeed, the gene expression profile analysis showed that miR1447 and PopTCTP exhibited opposite regulatory patterns on the expression of MPK3 and MPK6 (Figures S3a and S10a). Phosphorylation status of MAPKs in miR1447/PopTCTP transgenic lines further validated the involvement of miR1447‐PopTCTP in PTI‐ETI crosstalk (Figures S3b and S10b).

ROS function can be induced by PTI and ETI as key defence and signalling molecules (Yuan et al. 2021). The detection of ROS accumulation in miR472a transgenic lines at 0 and 24 hpi showed lower ROS accumulation in the control and the susceptible miR472a OE plants than in the resistant miR472a STTM plants, suggesting that the susceptible lines exhibited stronger ROS scavenging ability at the early pathogen inoculation stage (Su et al. 2018). Similarly, in this investigation, the miR1447‐OE lines were susceptible to pathogens and showed enhanced basal ROS scavenging in poplars, whereas the opposite was observed in the miR1447‐STTM lines (Figure S4a,b). Additionally, plants strictly regulate ROS homeostasis to prevent the toxic effects of high ROS concentrations on plant cells (Chu et al. 2022). The susceptible transgenic lines with increased susceptibility accumulated more ROS at the late stage of pathogen inoculation, as supported by investigations of miR159a in poplar and miR482c in tomato (Hong et al. 2019; Yang, Fu, et al. 2023). Notably, the DAB staining experiment demonstrated that the relatively high accumulation of ROS caused more severe toxic effects on plant cells in susceptible miR1447‐OE lines at the late inoculation stage (Figure 2h). In addition, the peroxisome pathway was enriched in the gene expression profile of miR1447‐OE lines, indicating that miR1447 modulated plant immunity by regulating ROS homeostasis (Figure S2). Additionally, scavenging ROS reduced lesion symptoms in miR1447 transgenic lines by exogenous application of ascorbic acid (Figure S4c). Combined together, the miR1447‐TCTP module may be involved in PTI‐ETI crosstalk by dynamically regulating ROS scavenging.

It has been widely reported that miRNA‐mRNA modules can simultaneously participate in plant growth and defence responses (Shen et al. 2024). The miR8171‐MFAP1 module in rice simultaneously enhanced plant immunity and yield (Li et al. 2022). The miR319‐TCP module regulates trichome development and improves the defence of P. tomentosa against insects (Fan, Ran, et al. 2020). MiR1885 can dynamically regulate innate immunity, plant growth and the plant response to viral infections in Brassica plants (Cui et al. 2020). In this investigation, miR1447 may negatively regulate the basal level photosynthetic capacity (Figure 6b) and the potential photosynthetic capacity of plants under pathogen stress (Figure 2k). Additionally, miR1447 negatively regulated different pathogen resistance in plants (Figure 2a,c,e). The SAR, phenylpropanoid and flavonoid biosynthesis pathways promote plant defence against pathogen colonisation (Du et al. 2021; Yang et al. 2022). The DEG analysis of SA‐treated miR1447 transgenic lines infected with C. gloeosporioides demonstrated that the enhanced resistance of miR1447‐OE lines responds to pathogen stress by upregulating defence‐related genes, including SAR, phenylpropanoid and flavonoid biosynthesis pathway genes, and downregulating photosynthetic pathway genes (Figures S6 and S7). However, the declined resistance of miR1447‐STTM lines exhibited the opposite pattern (Figures S6 and S7).

TCTP is highly conserved across eukaryotes (Berkowitz et al. 2008; Chitpatima et al. 1988). Recent advancements in TCTP have supported that TCTP alters plant growth and pathogen responses in crops and forestry trees (Branco and Masle 2019; Bruckner et al. 2017; Deng et al. 2016; Meng et al. 2018; Qin et al. 2011; Yang et al. 2019). RNAi silencing of TCTP results in retarded growth in Arabidopsis and cabbage (Berkowitz et al. 2008; Cao et al. 2010). The overexpression of TCTP increased growth and altered photosynthesis in tomato (de Carvalho et al. 2017). The transient overexpression of StTCTP increased sprout diameter in potato (Xoconostle‐Morán et al. 2023). Similarly, PopTCTP positively regulating the growth of poplar was validated by overexpression and RNAi transgenic poplar lines (Figure 6f,g). These investigations revealed that the PopTCTP homologue in Arabidopsis, crops and vegetables exhibits similar functions in positively regulating plant growth, indicating the conserved role of TCTP in annual and perennial plants. Additionally, TCTP functions in plant defence by interacting with other proteins. The CsTCTP proteins from cucumber not only promoted plant growth and development but modulated the resistance to G. cichoracearum. Furthermore, CsTCTP interacted with CsRab11A and promoted activation of the target of rapamycin in response to Podosphaera xanthii (Chen, Zhou, et al. 2024). TCTP, as the phosphorylation target of TaCIPK23, interacted with TaCIPK23 to positively regulate wheat resistance to Puccinia triticina (Ma et al. 2024). The interaction between NbTCTP and Nb14‐3‐3 h was involved in plant defence against Potato Virus Y infections (Fang et al. 2024).

Here, the expression analysis of PopTCTP in poplars infected with L. populi and C. gloeosporioides revealed the regulatory role of PopTCTP in poplar resistance against bacterial and fungal pathogens (Figure 5a–d). Furthermore, the inoculation investigation demonstrated PopTCTP negatively regulated poplar response to C. gloeosporioides (Figure 7a). In addition, the RNA‐seq data of PopTCTP transgenic lines revealed that the expression levels of plant defence‐related pathways, such as the ‘MAPK signaling pathway’, ‘plant‐pathogen interaction’ and ‘phenylpropanoid biosynthesis’ were significantly enriched. The photosynthesis‐related pathways ‘glutathione metabolism’ and ‘alanine, aspartate and glutamate metabolism’ were also significantly enriched (Figure S9). Previous investigation identified the DnaJ protein, OsDjA6, which negatively regulates rice basal resistance to M. oryzae by regulating burst of ROS, affecting the SA pathway (Zhong et al. 2018). In this study, the Y2H substantiated the interaction between PopTCTP and chaperone protein dnaJ A6 (Figure 7b). The gene expression analysis revealed that PopTCTP may negatively regulate dnaJ A6 in poplar immunity under normal conditions or C. gloeosporioides stress (Figures 7d–f and 8). Moreover, the expression of dnaJ A6 in poplar response to L. populi and C. gloeosporioides suggested dnaJ A6 is involved in poplar immunity (Figure 7g,h). Overall, these results suggested that the miR1447‐PopTCTP module likely modulated the trade‐off between plant growth and defence responses.

4. Materials and Methods

4.1. Plant and Pathogen Materials and Plant Growth Conditions

Poplar propagule tissue‐cultured plants of 84 K ( Populus alba × Populus glandulosa), provided by the State Key Laboratory of Tree Genetics and Breeding, Beijing Forestry University, were used for gene cloning and transformation. Poplars were grown in a greenhouse under 25°C ± 1°C with 70% relative humidity and 16/8 h photoperiod, or grown in the nursery of Beijing Forestry University in Beijing, China. The pathogens Lonsdalea populi, Colletotrichum gloeosporioides and Cytospora chrysosperma were provided by the State Key Laboratory of Efficient Production of Forest Resources, Beijing Forestry University.

4.2. Identification of miR1447 Target Gene

The degradome sequencing of 4‐week‐old tissue‐cultured plants of 84 K and 3‐year‐old 107 poplar bark infected with L. populi (mixed samples at 0, 1, 3 and 6 days post inoculation) were performed based on the 84 K poplar genome (ftp://ftp.cngb.org/pub/CNSA/CNP0000339/CNS0047055/CNA0003521/) and P. trichocarpa genome (https://phytozome‐next.jgi.doe.gov/pz/portal.html) respectively at LC‐Bio Technologies, China (Table S1). The sequence of miR1447 precursor was obtained from miRBase (http://www.mirbase.org/). Furthermore, 478 base pairs (bp) of miR1447 genomic DNA (Potri.013G147100) were amplified with gene‐specific primers designed online (https://www.ncbi.nlm.nih.gov/tools/primer‐blast/) (Table S2). The candidate target gene CDS (PopTCTP, Pop_G10G022958) was cloned from 84 K poplar. MiR1447 and its target gene (PopTCTP) were ligated into the pCAMBIA2300‐35S‐OCS vector and transformed into Agrobacterium. The transient co‐expression assays of the miRNA‐target pair were performed in N. benthamiana leaves as described previously, using an equal amount of Agrobacterium culture containing miR1447 and the target gene (Cao et al. 2016). The leaves were collected at 48–60 h after the infiltration for the qRT‐PCR experiment with tobacco 18S rRNA used as the reference gene (Table S2). The 35S::miR1447‐OE, 35S::miR1447‐STTM and 35S::PopTCTP‐eGFP were transformed into Agrobacterium. The N. benthamiana leaves were injected with Agrobacterium solution (OD₆₀₀ = 0.8). The transient co‐expression assays with N. benthamiana were performed by coinjecting equal volumes of the PopTCTP‐eGFP and pCAMBIA2300‐35S‐OCS vector, the miR1447‐OE and PopTCTP‐eGFP, the miR1447‐STTM and PopTCTP‐eGFP, respectively. After 48 h of infiltration, the GFP fluorescence of PopTCTP protein was observed with laser confocal fluorescence microscopy (TCS SP8, Leica) (Wang, Li, et al. 2023).

4.3. Promoter Analysis

The promoter, with a length of 2000 bp upstream of the miR1447 or PopTCTP transcription start site, was obtained from P. trichocarpa and the 84 K poplar genome, respectively (Han et al. 2022; Zhang et al. 2024). The promoter cis‐acting element was analysed by plantCARE (http://bioinformatics.psb.ugent.be/webtools/plantcare/html/). The promoter of miR1447 (1024 bp) and PopTCTP (1100 bp) was cloned with ClonExpressII One Step Cloning Kit (Vazyme, China) based on the P. trichocarpa genome and 84 K poplar genome respectively. The promoter of miR1447 or PopTCTP was ligated to the pBI121‐35S‐eGFP‐NOS expression vector. The Agrobacterium‐mediated transient transgenic N. benthamiana of miR1447 or PopTCTP promoter were cultured under a 12/12 h photoperiod at 25°C. The transient transgenic N. benthamiana plants were cultured for 24 h and then sprayed with 500 μM SA solution (MACKLIN) for 6 h, followed by overnight incubation in GUS solution (Coolaber) (Yang et al. 2022). The chlorophyll was cleared with anhydrous ethanol and 75% ethanol for the GUS activity observation (Su et al. 2018).

4.4. Vector Construction and Plant Transformation

The miR1447 genomic DNA from P. tomentosa TC1521, ligated to the pCAMBIA2300‐35S‐OCS vector, was used for overexpression transformation. The miR1447‐STTM sequence was designed based on the previous report (Tang et al. 2012). Synonymous mutations of the PopTCTP CDS sequence harbouring the miR1447 complementary sites were performed, especially the triplet codon at the cleavage site, which formed the mPopTCTP sequence as the PopTCTP overexpression sequence. A 3× Flag protein tag was added before the mutant mPopTCTP sequence. The RNA interference sequence of PopTCTP was designed with siDirect (http://sidirect2.rnai.jp/). The PDK intron sequence of the intermediate vector pHANNIBAL‐35S‐OCS was inserted between the forward and reverse interference sequences to ultimately form the PopTCTP‐RNAi sequence. The miR1447‐STTM, mPopTCTP and PopTCTP‐RNAi sequences were synthesised in Sangon Biotech, China. The modified plasmid pCAMBIA2300‐35S‐OCS was used as the final expression vector. The constructs were stably transformed into 84 K poplar clones using the Agrobacterium GV3101‐mediated leaf disc transformation technique. For transformation, Agrobacterium was cultured at 28°C, 200 rpm/min to OD₆₀₀ = 0.6–0.8 in liquid YEB with 50 mg/L kanamycin and 20 mg/L rifampicin. The transgenic lines were cultured on MS medium with 0.5 mg/L 6‐BA (benzylaminopurine) and 0.05 mg/L NAA (Sigma‐Aldrich), and 1/2 MS medium with 0.5 mg/L IBA and 0.05 mg/L NAA, and were screened by 50 mg/L kana and 200 mg/L termetine.

4.5. qRT‐PCR Expression Analysis

MiRNA was prepared according to a plant miRNA extraction kit (BioTeke, China). Total RNA was isolated from plants following the manufacturer's instructions (E.Z.N.A. Plant RNA Kit, omega BIO‐TEK, USA). For qRT‐PCR analysis, first‐strand cDNAs of miRNA and total RNA were synthesised using Mir‐X miRNA First‐Strand Synthesis kit and PrimeScript RT Master Mix (Takara, Japan), respectively. qRT‐PCR reactions were performed using TB Green Premix Ex Taq Kit (Takara, Japan). 5.8S rRNA, U6, 18S rRNA and ubiquitin were used as internal controls (Table S2).

4.6. Flg22 Peptide Treatment and Pathogen Inoculation

The 10 μM flagellin‐derived peptide flg22 solution (Phyto Technology Laboratories, China) was prepared by dissolving the lyophilised peptide in sterile distilled water (Cheng et al. 2020; Zipfel et al. 2004). The 4‐week‐old tissue‐cultured 84 K plants were sprayed with a 10 μM flg22 solution with sterile distilled water as a control. Fully expanded leaves were collected at 0 h (without flg22 treatment), 1, 3 and 6 h. Three different plants of each genotype were combined for miRNA isolation.

The hemibiotrophic fungus C. gloeosporioides was cultured in potato dextrose broth (PDB) at 28°C. For the C. gloeosporioides inoculation, detached leaves of 2‐month‐old miR1447 transgenic lines were inoculated with 50 μL conidium suspension (2 × 10⁶ conidia mL⁻¹) at each inoculation site, and leaves inoculated with PDB were used as a control. The infected leaves were incubated in a humidified chamber and cultured in a 16/8 h photoperiod growth chamber at 28°C. The C. chrysosperma was cultured on potato dextrose agar (PDA) at 25°C. For the C. chrysosperma inoculation, detached leaves of 2‐month‐old transgenic poplar lines were inoculated with mycelial plugs (d = 4 mm) at each inoculation site, and leaves inoculated with PDA were used as a control. The leaves were incubated in a humidified chamber and cultured in a 16/8 h photoperiod growth chamber at 25°C. The 15‐cm‐long twigs (d = 0.6–1.0 cm) were cut from 1‐year‐old transgenic poplar lines for C. chrysosperma and L. populi inoculation. The twigs inoculated with C. chrysosperma were cultured in the dark, humidified chamber at 25°C (Li et al. 2025). The twigs inoculated with L. populi were performed as described previously (Yang, Ming, et al. 2024) and cultured in a 16/8 h photoperiod growth chamber at 28°C with 90% relative humidity. The lesion area and lesion diameter were measured using ImageJ. The 3‐year‐old miR1447 transgenic lines and WT were infected with L. populi under in vivo conditions. And the phenotype of infected poplars was observed at 0, 1, 4 and 9 dpi. Each experiment was performed with at least three replicates.

4.7. Assays of H₂O₂ and Cell Death

The H₂O₂ produced in the inoculated leaves of poplar was detected using 3,3′‐diaminobenzidine‐HCl (DAB) staining. For H₂O₂ staining, leaves were incubated in the freshly prepared 10 mg/mL DAB solution (pH = 3.8) overnight at 25°C. To test whether scavenger application alters resistance phenotypes of miR1447 transgenic lines. The C. gloeosporioides was sprayed on the miR1447 transgenic tissue‐cultured plants (7O1, 7S2) for 24 h, followed by the 10 mM ascorbic acid (Solarbio LIFE SCIENCES, China) solution spray treatment every 12 h for 1 day (Hamel et al. 2024). The phenotype of infected poplars was observed at 72 h post the treatment.

To detect cell death in inoculated leaves by trypan blue staining, leaves were immersed in a 2.5 g/L trypan blue solution overnight at 25°C. The stained leaves were treated in 95% alcohol for 8 h at 25°C to decolor the Chl. The decolorised leaves were cut into 1 cm² and photographed with a microscope (MDi8).

4.8. Determination of the Expression of SA Marker Genes

The leaves of 45‐day‐old potted poplar plants of miR1447‐OE lines, miR1447‐STTM lines and the wild type line were inoculated with C. gloeosporioides in vivo. The expression of SA marker genes (NPR1 and PR1) in miR1447 transgenic lines was analysed by qRT‐PCR experiment at 0 h (mock‐inoculated), 12, 24, 48 and 72 h post inoculation.

4.9. Pharmacological Inhibition Assays

The miR1447 transgenic lines (7O1, 7S2) were sprayed with C. gloeosporioides at 12 h post 100 μM tenoxicam (AbMole) treatment. The infected leaves were sampled at 0 (mock‐inoculated), 12, 24 and 48 h for qRT‐PCR analysis of NPR1 expression (Ishihama et al. 2021). The phenotype of infected poplars was observed and photographed at 72 h.

4.10. Photosynthetic Parameters and Plant Phenotype Measurement

Photosynthetic parameters of the 4‐6th leaves of 5‐week‐old transgenic lines in the greenhouse were measured with a portable photosynthesis system (Li‐6800, LI‐COR). To evaluate the severity of symptoms of inoculated leaves, high‐resolution images were obtained using the Plant Explorer system. The condition of infected leaves was classified as level 1 (healthy, shown in green), level 2 (slightly damaged, shown in yellow) and level 3 (severely damaged, shown in red) based on the F _v/F _m (maximum quantum yield) value (Li et al. 2019). Additionally, the efficiency of photosystem II (F _v/F _m) was assessed under pathogen stress (Baker 2008).

4.11. Subcellular Localisation of PopTCTP

The CDS of PopTCTP was cloned into the pBI121‐eGFP vector to generate the pBI121‐35S‐PopTCTP‐eGFP‐NOS construct. The N. benthamiana leaves were injected with Agrobacterium solution with OD₆₀₀ = 0.8–1.0. N. benthamiana injected with Agrobacterium transformed with 35S::PopTCTP‐eGFP was the experimental group, and the Agrobacterium transformed with 35S::pBI121‐eGFP was used as the control. After 48 h of injection, the subcellular localisation of the labelled proteins was observed with laser confocal fluorescence microscopy (TCS SP8, Leica). The infiltrated N. benthamiana leaves were used for western blot analysis to confirm the expression of the PopTCTP‐eGFP fusion protein (Rao et al. 2014).

4.12. Gene Family Analysis of PopTCTP

The gene family protein sequences were obtained according to the genome of 84 K poplar. Phylogenetic and molecular evolution were analysed with the Maximum Likelihood method based on the JTT matrix‐based model in MEGA 7. TCTP1 (AT3G16640) of Arabidopsis thaliana was used as the outgroup. The conserved domains were obtained online through NCBI Batch CDD (Conserved Domain Database) (https://www.ncbi.nlm.nih.gov/Structure/bwrpsb/bwrpsb.cgi) based on protein sequences, and visualised in TBtools (Chen et al. 2023).

4.13. Protein–Protein Interaction

The full‐length CDS of PopTCTP were fused to the pGBKT7 vector (PopTCTP‐BD) and used as a bait vector to screen against the library of 84 K leaf cDNAs fused to pGADT7 (Chen, Tan, et al. 2024). The positive interactions of PopTCTP were acquired by screening the 84 K poplar cDNA prey library through the mating method (oebiotech, China). To confirm the interaction between PopTCTP and chaperone protein dnaJ A6, the bait and prey plasmids were cotransformed into the Y2H Gold yeast strain (Fan, Ran, et al. 2020). Yeast colonies were cultured in SD/‐Leu/‐Trp/X‐α‐gal, SD/‐Leu/‐Trp/‐His/X‐α‐gal, and SD/‐Leu/‐Trp/‐His/‐Ade/X‐α‐gal/AbA media for 3–5 days to test the growth of yeast.

The 1‐month‐old PopTCTP transgenic lines (8O2, 8O7, 8i2, 8i5) were sprayed with C. gloeosporioides. The infected leaves were collected at 0, 24 and 48 h of treatment and the expression level of dnaJ A6 analysed by qRT‐PCR experiment (Table S2). The phenotype of infected poplars was observed and photographed at 72 h.

4.14. Transcriptome Analysis and SA Content Determination

The leaves of 1‐year‐old miR1447 OE lines (7O1, 7O2), STTM lines (7S1, 7S2) and WT were sampled for RNA‐seq (Beijing Genomics Institute, China). The clean reads were aligned to the 84 K poplar genome with HISAT (v2.1.0, ‐‐dta ‐‐phred64 unstranded ‐‐new‐summary ‐x index ‐1 read_r1 ‐2 read_r2 (PE)) and Bowtie2 (v2.2.5, ‐q ‐‐phred64 ‐‐sensitive ‐‐dpad 0 ‐‐gbar 99 999 999 ‐‐mp 1,1 ‐‐np 1 ‐‐score‐min L,0,‐0.1 ‐p 16 ‐k 200) (Langmead and Salzberg 2012). The expression level of genes and transcripts were calculated using RSEM (Li and Dewey 2011). Differentially expressed gene (DEG) analysis was performed using DEGseq (Wang et al. 2010). DEGs (Fold Change ≥ 1 and Adjusted p value ≤ 0.05) were annotated to the Gene Ontology (GO) (http://www.blast2go.com/b2ghome) and Kyoto Encyclopedia of Genes and Genomes (KEGG) database (http://www.genome.jp/kegg) by blastx software (http://blast.ncbi.nlm.nih.gov/Blast.cgi).

The 1‐month‐old PopTCTP OE lines (8O2) and RNAi lines (8i5) tissue‐cultured poplar plants were sampled for RNA‐seq (BIOMARKER TECHNOLOGIES, China). The clean reads were aligned to the 84 K poplar genome with HISAT (v2.0.4, ‐‐dta ‐p 6 ‐‐max‐intronlen 5000000) and StringTie (Pertea et al. 2015). The DEG analysis was performed with DESeq2 (v1.30.1, default: test = ‘Wald’, fitType = ‘parametric’). Genes with Fold Change ≥ 2 and FDR < 0.01 were defined as DEGs.

1‐month‐old miR1447 transgenic tissue‐cultured plants (7O1, 7S2) were sprayed with C. gloeosporioides after 12 h of 500 μM SA solution (MACKLIN) spray treatment. The infected leaves were sampled at 0, 24 and 48 h of treatment (with lesion) for RNA‐seq (Majorbio Bio‐pharm Technology, China) (Liu et al. 2024). DEG (|log₂FC| > 1.5, P value < 0.05) was used for GO and KEGG analyses. 1‐month‐old miR1447 transgenic tissue‐cultured plants (7O1, 7S2) with the same treatment were used for the endogenous SA content detection through LC–MS/MS (METWARE, China).

4.15. Western Blot Analysis of MAPK Phosphorylation

The miR1447 (7O1, 7S2) and PopTCTP (8O2, 8O7, 8i2, 8i5) transgenic poplar lines were sprayed with C. gloeosporioides. The infected leaves were collected at 0, 24 and 48 h of treatment for protein extraction with the MAPK inhibitor (Coolaber, China). The protein samples were separated in 10% SDS‐PAGE gels and transferred to a PVDF membrane using a semi‐dry transfer method with transfer buffer. Phosphorylation of MAPKs was detected using p44/42 MAPK (Erk1/2) Rabbit Monoclonal Antibody (Cell Signalling TECHNOLOGY) and HRP‐conjugated Goat anti‐Rabbit IgG (H + L) (ABclonal, China). Hybridising bands were visualised using the Super ECL Plus reagent (BIORIGIN) in a Gel Imaging System (Aplegen, USA) (Fernandes et al. 2023).

4.16. Statistical Analysis

Statistics and analysis of variance were performed with experimental data using GraphPad Prism software. The multiple comparisons were performed based on the t‐test to calculate P‐values (asterisks denote significant differences: *p ≤ 0.05, **p ≤ 0.01).

Author Contributions

Yanwei Wang and Yonglin Wang designed this study; Dandan Xiao, Yanwei Wang and Yonglin Wang wrote the manuscript; Kaijing Rong performed the inoculation and investigation of SA‐treated poplar; Yicheng Li analysed the transcriptome data of SA‐treated poplar; Ke Zhou obtained the miR1447 transgenic lines; Tiantian Fu, Chun Wang, Ruen Yu, Xiaoqian Yang and Yuzhang Yang revised the manuscript; Yuanyuan Wang supervised the inoculation experiment. All authors have read and approved the manuscript.

Funding

This work was supported by National Key Research and Development Program of China, 2022YFD1401000; National Natural Science Foundation of China, 32371577, 32071504; Fundamental Research Funds for the Central Universities, QNTD202510.

Conflicts of Interest

The authors declare no conflicts of interest.

Supporting information

Figure S1: The phenotype of 3‐year‐old miR1447 transgenic lines inoculated with L. populi under in vivo conditions. WT: wild type, 7O1 and 7O2: miR1447 overexpression lines, 7S1 and 7S2: miR1447 STTM lines, d: days post‐infection. Scale bars: 2 cm.

Figure S2: The KEGG enrichment analysis based on transcriptome database of 1‐month‐old miR1447 tissue‐cultured transgenic lines. WT: wild type, 7O1 and 7O2: miR1447 overexpression lines, 7S1 and 7S2: miR1447 STTM lines.

Figure S3: The miR1447 regualtaed MAPKs and photosynthetic genes expression in poplar. (a) The expression analysis of MPK3 and MPK6 based on transcriptome database of miR1447 transgenic lines. (b) The phosphorylation status of MAPKs identified by western blot analysis. (c) The gene expression analysis of photosynthetic machinery based on transcriptome database of miR1447 transgenic lines. Pop_A13G082608 annotation: PHOTOSYSTEM I ASSEMBLY 2, Pop_A10G047253 annotation: photosynthetic NDH subunit of subcomplex B 5, Pop_A05G073419 annotation: phototropic‐responsive NPH3 family protein. WT: wild type. 7O1: miR1447 overexpression line. 7S1 and 7S2: miR1447 STTM line. MPK3/6: mitogen‐activated protein kinase 3/6.

Figure S4: The miR1447 involvement of antioxidant enzymes genes in poplar. (a‐b) The expression analysis of antioxidant enzymes genes based on transcriptome database of 1‐month‐old miR1447 transgenic lines. WT: wild type. 7O1: miR1447 overexpression line. 7S1 and 7S2: miR1447 STTM line. (c) The phenotype of ascorbic acid treated 1‐month‐old miR1447 tissue‐cultured transgenic lines infected with C. gloeosporioides. Scale bars: 0.5 cm. (d‐e): The expression analysis of antioxidant enzymes genes based on transcriptome database of SA‐treated 1‐month‐old miR1447 transgenic lines. WT: SA‐treated wild type. 7O1: SA‐treated miR1447 overexpression line. 7S2: SA‐treated miR1447 STTM line.

Figure S5: The SA responsiveness of miR1447. (a) The cis‐acting elements of miR1447 promoter sequence. (b) The cloning of miR1447 promoter sequence containing TCA‐element. (c) MiR1447 promoter responds to SA treatment. CK: the transient transgenic N. benthamiana of empty vector. miR1447: the transient transgenic N. benthamiana of miR1447 promoter. CK‐SA: the empty vector transient transgenic N. benthamiana treated with salicylic acid. miR1447‐SA: the miR1447 promoter transient transgenic N. benthamiana treated with salicylic acid.

Figure S6: The GO enrichment analysis of SA‐treated miR1447 transgenic lines infected with C. gloeosporioides. WT: wild type. 7O1: miR1447 overexpression line. 7S2: miR1447 STTM line.

Figure S7: The KEGG enrichment analysis of SA‐treated miR1447 transgenic lines infected with C. gloeosporioides. WT: wild type. 7O1: miR1447 overexpression line. 7S2: miR1447 STTM line.

Figure S8: The SA responsiveness of PopTCTP. (a) The cis‐acting elements of PopTCTP promoter sequence. (b) The cloning of miR1447 promoter sequence containing TCA‐element. (c) PopTCTP promoter responds to SA treatment. CK: the transient transgenic N. benthamiana of empty vector. PopTCTP: the transient transgenic N. benthamiana of PopTCTP promoter. CK‐SA: the empty vector transient transgenic N. benthamiana treated with salicylic acid. PopTCTP‐SA: the PopTCTP promoter transient transgenic N. benthamiana treated with salicylic acid.

Figure S9: The KEGG enrichment analysis of PopTCTP transgenic lines. (a) WT_vs_OE KEGG enrichment analysis. (b) WT_vs_RNAi KEGG enrichment analysis.

Figure S10: The PopTCTP regualtaed MAPKs and photosynthetic genes expression in poplar. (a) The expression analysis of MPK3 and MPK6 based on transcriptome database of PopTCTP transgenic lines. (b) The phosphorylation status of MAPKs identified by western blot analysis. (c) The gene expression analysis of photosynthetic machinery based on transcriptome database of PopTCTP transgenic lines. Pop_A13G082608 annotation: PHOTOSYSTEM I ASSEMBLY 2, Pop_A10G047253 annotation: photosynthetic NDH subunit of subcomplex B 5, Pop_A05G073419 annotation: phototropic‐responsive NPH3 family protein. WT: wild type. 8O2 and 8O7: PopTCTP overexpression line. 8i2 and 8i5: PopTCTP RNAi line. MPK3/6: mitogen‐activated protein kinase 3/6.

Figure S11: The GO term classification (a), and KEGG enrichment (b) analysis of PopTCTP interaction protein.

Table S1: The miR1447 target genes identified through degradome analysis.

Table S2: Primers used in this investigation.

Data Availability Statement

Data will be made available on request.