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BpEIL1 negatively regulates resistance to Rhizoctonia solani and Alternaria alternata in birch
2022 Volume 97 Issue 2 Pages 81-91
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2022 Volume 97 Issue 2 Pages 81-91
Pathogen attacks affect tree health, causing considerable economic losses as well as serious damage to the surrounding environment. Understanding the disease resistance mechanisms of trees is important for tree breeding. In previous studies on birch (Betula platyphylla × B. pendula), we identified a lesion mimic mutant called lmd. We found that reduced expression of BpEIL1 was responsible for the phenotype in lmd. Following cloning, we acquired several BpEIL1 overexpression and suppression lines in birch. In this study, we cloned the BpEIL1 promoter and found that BpEIL1 was primarily expressed in leaves, particularly in veins. We further studied the traits of transgenic lines and the function of BpEIL1 in disease resistance in birch using the BpEIL1 overexpression line OE9, the suppression line SE13 and the non-transgenic line NT. We found that hydrogen peroxide accumulated in SE13 leaves. Ascorbate peroxidase and catalase activity significantly increased in SE13. SE13 was more resistant to the fungal pathogens Alternaria alternata and Rhizoctonia solani than were the OE9 and NT lines. RNA-seq indicated that pathways related to signal transduction, disease resistance and plant immunity were enriched in SE13. BpEIL1 is thus a negative regulatory transcription factor for disease resistance in birch. This study provides a reference for disease resistance of birch and other trees.
Plants can suffer from attacks by different pathogens in the natural environment, including bacteria, viruses, fungi, oomycetes and nematodes (Islam et al., 2020). However, 70–80% of plant diseases are caused by fungi. Long-term coevolution has allowed plants to develop specific pathogen responses, including pathogen recognition, signal transduction, production of related primary and secondary metabolites, pathogenesis-related protein (PR) expression, the hypersensitive response and acquired immunity (Gao et al., 2019). Reactive oxygen species (ROS) are oxygen-containing molecules with a strong oxidative capacity and mostly contain O2·−, ·OH, 1O2 and H2O2 (Del Río, 2015). Bacterial, viral and fungal attacks can induce ROS production and accumulation. ROS are one of the earliest responses to pathogen attacks in plants and are toxic to pathogens. They are also important for plant signaling to activate the immune response and induce PR expression to defend against infection (Qi et al., 2017). Plant hormones such as salicylic acid (SA), jasmonic acid (JA), abscisic acid and ethylene are also involved in plant pathogen resistance (Zhou and Zhang, 2020).
Ethylene is an important plant hormone that is activated during pathogen–plant interaction (Helliwell et al., 2016). An increased amount of ethylene is released by a plant following a pathogen attack (Liu et al., 2017a). EIN3/EILs are important transcription factors in ethylene signal transduction, and can induce ethylene response genes and act as connections between ethylene signaling and SA, JA, cytokinin, auxin and gibberellin (He et al., 2017; Munné-Bosch et al., 2018). EIN3/EILs are also involved in chloroplast development, cold resistance, salt stress, ion balance, iron metabolism and root hair growth (Peng et al., 2014; Liu et al., 2017b; Robison et al., 2019; García et al., 2021; Xiao et al., 2021). Therefore, it is clear that EIN3/EILs are important nodal transcription factors in the whole transcriptional regulatory network in plants, and have the functions of integrating signaling and regulating gene expression networks. EIN3/EILs are also related to disease resistance in plants. Suppression of EIL1 expression enhances resistance to Puccinia striiformis f. sp. tritici in wheat (Duan et al., 2013). In Arabidopsis, EIN3/EIL1 directly binds to the SID2 promoter through the inhibition of SA synthesis to negatively regulate pathogen-associated molecular pattern (PAMP)-triggered immunity (Chen et al., 2009). EIN3/EIL1 regulates disease resistance in Arabidopsis by directly regulating FLS2 expression and activating components of the MAP pathway including MEKK-MKK4/5-MPK3/6 and MEKK1-MKK1/2-MPK4, followed by defense gene activation via WRKY transcription factors (containing the WRKYGQK domain) (Boutrot et al., 2010). In rice, OsEIL1 directly binds to the OsrbohA/OsrbohB and OsOPR4 promoters to positively regulate pathogen resistance (Yang et al., 2017).
Trees generally have a long lifespan and can experience an array of pathogen attacks at any developmental stage. As trees generally have a larger stature compared to herbaceous plants, they can be harder to work with, and extensive studies on the molecular mechanisms of disease resistance are therefore lacking. Although some studies have demonstrated similar molecular mechanisms underlying disease resistance in trees and herbaceous plants, there will likely be differences due to diversity in physiological and biochemical features, as well as morphological and anatomical structures, and to the reduced developmental and evolutionary rates of trees (Naidoo et al., 2014). It is therefore imperative to investigate how trees withstand pathogen infection. Birch is a deciduous pioneer tree primarily used to make paper and is widespread in natural forests due to its high growth rate (Chen et al., 2021). However, birch seedlings are vulnerable to pathogens, including damping-off disease that can result in 10–30% death (Meng, 2020).
In previous studies, we found a T-DNA insertion mutant with a lesion mimic and premature senescence phenotype called lmd in birch (Betula platyphylla × B. pendula). The lmd mutant shows resistance to a fungal pathogen, Alternaria alternata. We further found that the T-DNA insertion site was in the BpEIL1 promoter in lmd, which leads to a reduced BpEIL1 expression level (Li et al., 2017). We then cloned BpEIL1 and acquired several overexpression and suppression lines via Agrobacterium-mediated transformation in birch. The suppression lines showed a similar phenotype to the lmd mutant (Li et al., 2019). In the present study, in order to investigate the tissue-specific expression of BpEIL1 in birch, we cloned the BpEIL1 promoter into the pCAMBIA 1300-35S-Gus (Supplementary Fig. S1) vector and developed a transgenic line via Agrobacterium-mediated infiltration. Using the overexpression line OE9, the suppression line SE13, and a birch non-transgenic line (NT), we investigated levels of resistance to Rhizoctonia solani and A. alternata to further explore the function of BpEIL1 in disease resistance in birch. Finally, we performed RNA-seq on the SE13 and NT lines to explore the gene expression profile of the BpEIL1 suppression line. This study provides a reference for breeding and the development of disease-resistant lines in birch and other trees.
We cloned the promoter of BpEIL1, constructed a pBpEIL1::GUS expression vector, and used it to transform the zygotic embryo of birch. In total, we obtained six transgenic lines (Fig. 1A). GUS staining showed that the BpEIL1 gene was expressed strongly in leaves, but weakly in roots and stems. There was also less expression in the developing leaves. However, the entire mature leaf showed staining, particularly the veins (Fig. 1B). These results demonstrate that the BpEIL1 gene is primarily expressed in mature leaves in birch. To monitor the response of BpEIL1 to pathogens, we infected birch seedlings (NT line) with A. alternata or R. solani, and then examined the expression of BpEIL1 at different infection times through RT-qPCR. We found that the expression of BpEIL1 decreased with increasing infection time for both A. alternata and R. solani, indicating a negative regulation of BpEIL1 on disease resistance in birch (Fig. 1C).
Tissue-specific expression of BpEIL1. (A) PCR of transgenic lines. M: DL2000 DNA Marker; 1: recombinant plasmid; 2: non-transgenic line; 3: water; 4–9: transgenic lines. (B) GUS staining of the non-transgenic (NT) and transgenic (pBpEIL1::GUS) lines. (C) Response of BpEIL1 to A. alternata and R. solani. Data in (C) are presented as mean ± standard deviation.
H2O2 is an important signal for plant defense induction. Based on the RT-qPCR results (Fig. 2A), we chose the highest overexpression line, OE9, the strongest RNAi suppression line, SE13, and NT to detect H2O2 accumulation and antioxidase activity (Fig. 2B). The results showed that H2O2 accumulated in SE13 (Fig. 2C, 2D). Superoxide dismutase (SOD) is an important antioxidant enzyme that can scavenge ROS. Peroxidase (POD), catalase (CAT) and ascorbate peroxidase (APX) can hydrolyze H2O2 during high H2O2 accumulation. Here, we found that CAT and APX activity were significantly higher in SE13 than in NT and OE9 (Fig. 2E, 2F). POD activity in both NT and SE13 was significantly higher than in OE9 but showed no difference between them (Fig. 2G). For SOD activity, there were no differences among NT, OE9 and SE13 (Fig. 2H). Among the three tested lines, APX activity in SE13 was highest, followed by NT and then OE9. Similarly, BpEIL1 expression in SE13 was the lowest of all three lines, followed by NT and then OE9. APX activity and BpEIL1 expression showed a negative correlation.
Relative BpEIL1 gene expression, H2O2 accumulation and antioxidase activity in NT and transgenic lines. (A) Relative gene expression of four overexpression (OE) and five suppression (SE) lines compared to NT. (B) NT, OE9 and SE13 lines were chosen for this experiment. Hypersensitive response-like cell death was visible on older SE13 leaves (arrows). (C) DAB staining result. (D) H2O2 content of different lines. (E), (F), (G) and (H) show CAT, APX, POD and SOD activity, respectively. Data in (A), (D), (E), (F), (G) and (H) are presented as mean ± standard deviation. Different letters above the error bars indicate a significant difference (ANOVA, P < 0.05). FW, fresh weight.
In a previous study, we identified the lmd mutant (the promoter of BpEIL1 was disrupted) that showed resistance to A. alternata. In the current work, we tested the resistance of the BpEIL1 overexpression line OE9, the RNAi suppression line SE13 and NT to A. alternata to verify the function of BpEIL1 in birch disease resistance. We evenly sprayed a spore suspension of A. alternata onto birch seedlings. After six days, we found leaves on OE9 and NT with blight symptoms on the leaf margin (Fig. 3A). The stem apices of NT and OE9 withered (Fig. 3B). SE13, however, showed a normal phenotype until 20 days after the inoculation. The proportion of blight leaves in all leaves for each plant was identified to assess the severity of the disease seven days after spraying. We found 61.9% blight leaves in OE9, 48.4% blight leaves in NT and no blight leaves in SE13, indicating that SE13 was more resistant to A. alternata (Fig. 3C). In an in vitro assay, we found that A. alternata growth on SE13 leaves was negligible, but spread quickly in OE9 and NT (Fig. 3D). The infection diameters were 2.88 cm, 2.08 cm and 0 cm on OE9, NT and SE13, respectively (Fig. 3E). Both types of analysis demonstrated that SE13 was more resistant to A. alternata than OE9 and NT.
BpEIL1 negatively regulates resistance to A. alternata in birch. (A, B) Six days after inoculation. Leaves (A) and stem apices (B) on OE9 and NT were withered (arrows in (A)), but absolutely normal on SE13. (C) The proportion of blight leaves in all leaves for each plant. (D) Seven days after inoculation in an in vitro assay. (E) Diameter of infection areas. Data in (C) and (E) are mean ± standard deviation.
Rhizoctonia solani is a non-sporogenic fungus that causes damping-off in plants. We prepared a mycelial suspension of R. solani and injected it into the soil near the roots of birch seedlings. After five days, the basal leaves of NT and OE9 turned yellow, while the SE13 seedlings looked normal and lacked any pathogen symptoms. After six days, we found that OE9 seedlings were damping off, and the stem of NT seedlings began to brown, but the SE13 seedlings looked normal, lacking any pathogen-related symptoms (Fig. 4A, 4B). After twelve days, most seedlings of NT and OE9 wilted, but the basal leaves of SE13 turned yellow and none fell off (Fig. 4C). Stereomicroscope observation showed that OE9 and NT seedlings were infected by R. solani, but SE13 seedlings were not (Fig. 4D, 4E, 4F). The infected seedlings collapsed because of the decayed stem. The incidence rates of leaves turning yellow and disease occurrence were 100, 100 and 50% in NT, OE9 and SE13, respectively, nine days after infection (Fig. 4G). Overall, SE13 seedlings showed more resistance to R. solani than NT or OE9, while OE9 seedlings showed the highest sensitivity. RT-qPCR results demonstrated that several PRs, including PR1, PR1a, PR1b and PR5, were up-regulated in SE13 (Fig. 4H, 4I, 4J, 4K).
BpEIL1 negatively regulates resistance to R. solani in birch. (A), (B) and (C) show NT, OE9 and SE13 lines at six, eight and twelve days after inoculation. (D), (E) and (F) show stereomicroscope views of NT, OE9 and SE13. Arrows in (D) and (E) show decayed sites on the stem. (G) Disease incidence rates in NT, OE9 and SE13 on the indicated days after inoculation. (H), (I), (J) and (K) show relative expression of PR1, PR1a, PR1b and PR5, respectively; data are presented as mean ± standard deviation.
To investigate the gene expression profile of the BpEIL1 RNAi transgenic line, we performed RNA-seq for the NT and SE13 lines. In total, we obtained 40.72 Gb of clean data and 84.43–86.76% of these data for each sample were mapped to the reference genome. The Q30 of each sample was above 93.52%. We identified 1,737 differentially expressed genes (DEGs) between NT and SE13 lines of which 945 were up-regulated and 792 down-regulated (fold change ≥ 2 and FDR < 0.05 as standard).
We then performed GO and KEGG analyses. For the category ‘Biological Process’, defense response, immune response, innate immune response and immune system process were enriched in SE13 compared to NT, indicating an enhanced resistance of the SE13 line (Fig. 5A). For ‘Cellular Component’, items associated with membrane systems including integral component of membrane, plasma membrane, intrinsic component of plastid outer membrane, integral component of plastid outer membrane, intrinsic component of chloroplast outer membrane, membrane and plasma membrane region were enriched (Fig. 5B). For ‘Molecular Function’, the most enriched items were ADP binding, kinase activity, primary amine oxidase activity, protein kinase activity, protein serine/threonine kinase activity, quinone binding, heme binding, UDP-glycosyltransferase activity and copper ion binding (Fig. 5C). The most enriched KEGG pathways were plant–pathogen interaction, phenylpropanoid biosynthesis, beta-alanine metabolism, flavone and flavonol biosynthesis, tropane, piperidine and pyridine alkaloid biosynthesis, zeatin biosynthesis and MAPK signaling pathway–plant (Fig. 5D).
GO and KEGG analysis of enriched DEGs between NT and SE13 for (A) Biological Process, (B) Cellular Component, (C) Molecular Function and (D) KEGG pathways.
To verify the reliability of DEG assignations from RNA-seq, we chose 15 unigenes from DEGs at random to perform RT-qPCR. Correlation analysis showed a strong positive correlation between RT-qPCR and RNA-seq results (R2 = 0.823), indicating that the RNA-seq data were reliable (Fig. 6A). At the same time, we found 80 disease resistance- or immune-related unigenes in the up-regulated DEGs cluster. We selected 10 plant immune-related genes according to the literature (Bentham et al., 2020; Leng et al., 2021), and performed RT-qPCR to confirm. We found that these plant immune-related genes were indeed up-regulated, indicating an enhanced immunity of the SE13 line compared to the NT line (Fig. 6B).
RNA-seq verification. (A) Correlation analysis of 15 randomly chosen DEGs showed a strong positive correlation between RT-qPCR and RNA-seq data. (B) RT-qPCR analysis of the relative expression in NT and SE13 of 10 plant immune-related unigenes.
The mechanisms by which plants resist pathogens can be divided into two types: physical and physiological resistance. Physical resistance primarily consists of cornification, development of wax, lignification and other related physical structures (Vanholme et al., 2019; Arya et al., 2021). When pathogens pass these barriers, they will encounter physiological resistance such as enhanced plant respiration, antimicrobial substance production and immune system activation (Saur and Hückelhoven, 2021). Physiological resistance is more complex and important. Plants attacked by incompatible pathogens can induce rapid cell death, which is a resistance response known as the hypersensitive response (HR). This local programmed cell death (PCD) can limit pathogen spread and is a signal for plants to activate their defense response (Pontier et al., 1998). Lesion mimic mutants (LMMs) usually show an HR-like phenotype including PCD, ROS accumulation, expression of disease resistance genes, and pathogen resistance without any pathogen attack (Ma et al., 2019; Tu et al., 2020). LMMs are usually divided into two classes, the initiation class and the propagation class, depending on whether or not the cell death was limited. In a previous study, we identified an LMM in birch called lmd. Further experiments showed that the lesion mimic phenotype was due to the down-regulation of BpEIL1 (Li et al., 2017). We constructed transgenic BpEIL1 lines, including the overexpression OE9 and suppression SE13 lines. We found HR-like cell death on the leaves of SE13, but none on the OE9 or non-transformed NT leaves. The HR-like cell death of SE13 belongs to the initiation class, as is the case for the lmd mutant. Although there are many genes related to lesion mimic formation, most LMMs show ROS burst and disease resistance enhancement (Bruggeman et al., 2015). DAB staining and H2O2 content measurement were performed to examine the accumulation of H2O2 in OE9, SE13 and NT. We also measured the antioxidant enzyme activity of POD, SOD, APX and CAT, which showed that H2O2 accumulated in SE13 leaves. APX and CAT activity were significantly higher in SE13 than in OE9 and NT. The activity of APX was lowest in OE9 among three different lines and was negatively correlated with BpEIL1 expression. Most LMMs show resistance to different pathogens. In this study, we found that SE13 was more resistant to both A. alternata and R. solani. We conclude that the down-regulated BpEIL1 gene induced the lesion mimic phenotype in birch.
Ethylene is related to the HR in plants. For example, ethylene application increases the production of H2O2 and anaphylaxis induced by camptothecin in tomatoes (de Jong et al., 2002). EIN3 is an important transcription factor in ethylene signal transduction. It also plays an important role in many life processes and is a node for different plant hormone signaling pathways (Zhang et al., 2016). BpEIL1 belongs to the EIN3 superfamily. In this study, the down-regulated BpEIL1 gene-induced lesion mimic phenotype may not be due to ethylene signal transduction, but rather to SA signal transduction. We found that SA accumulated in SE13 leaves and that NPR1 (non-expressor of pathogenesis-related genes 1), an important regulatory gene in SA signaling, was highly up-regulated in SE13 (Supplementary Fig. S2). PR genes that are downstream of the SA signal were also up-regulated. SA accumulation can activate systemic acquired resistance, which can make plants resistant to many pathogens (Ding and Ding, 2020). Similarly, SE13 showed higher resistance to the two pathogens used in this study. We conclude that BpEIL1 induced the lesion mimic phenotype through SA signaling, although the molecular mechanisms should be further investigated.
To learn more about the gene expression profile of SE13, we performed RNA-seq. For KEGG enrichment analysis, in the 20 most highly enriched pathways, 110 genes were enriched in the plant–pathogen interaction (ko04626) pathway, 47 genes were enriched in the MAPK signaling pathway–plant (ko04016) pathway and 11 genes were enriched in the ascorbate and aldarate metabolism (ko00053) pathway. For GO enrichment analysis, items related to plant defense, plant immunity and kinase activity were enriched. The above results indicate that plant defense is enhanced in SE13. Ascorbic acid (AsA) plays an important role in plant growth and development and the stress response. AsA is an electron donor for APX to eliminate H2O2 generated during stress. At the same time, AsA can be oxidized to monodehydroascorbic acid (MDHA). Some MDHA is reduced to AsA by monodehydroascorbate reductase (MDHAR) and some is dismutated at low pH to dehydroascorbic acid (DHA). DHA is reduced to AsA by dehydroascorbate reductase and L-glutathione and H2O2 can be finally eliminated (Arrigoni and Tullio, 2002). We found that H2O2 accumulated in SE13 leaves and APX activity was significantly increased. DEGs were enriched in the ascorbate metabolism pathway. The above results show that H2O2 accumulation induced changes in related pathways.
Mitogen-activated protein kinase (MAPK) plays an important role in perceiving signals and transporting extracellular signals into cells (Meng and Zhang, 2013; Zhang et al., 2018). The MAPK pathway, which is conserved in eukaryotes, contains three levels of kinase models, including MAP kinase kinase kinase (MKKK), MAP kinase kinase and MAPK (Neupane et al., 2019). MAPK transfers phosphate from ATP to serine and threonine residues of its substrates, and MKKK is a key enzyme to activate this process (Benhamman et al., 2017). MKKK-led cascade reactions are important for disease resistance responses in plants. In this study, ~10% of genes in the KEGG pathway MAPK signaling pathway–plant were up- or down-regulated. Among the 47 DEGs, 28 were up-regulated and 19 down-regulated, including LRR receptor-like serine/threonine-protein kinase, leucine-rich repeat receptor-like protein kinase, rust resistance kinase, serine/threonine-protein kinase, G-type lectin S-receptor-like serine/threonine-protein kinase and cysteine-rich receptor-like protein kinase. These changes in protein kinase expression indicate altered signal transduction in SE13.
In this study, we found that BpEIL1 was primarily expressed in leaves, particularly in veins. H2O2 accumulated in leaves of the BpEIL1 suppression line SE13. The activities of APX and CAT significantly increased in SE13. SE13 was more resistant to both A. alternata and R. solani than were the BpEIL1 overexpression line OE9 and the non-transgenic line NT. RNA-seq indicated that pathways related to signal transduction, disease resistance and plant immunity were enriched. We thus conclude that BpEIL1 is a negative regulatory transcription factor for disease resistance in birch.
Birch (Betula platyphylla × B. pendula) seeds were collected in the Birch Breeding Base of Northeast Forestry University in Harbin, China. The BpEIL1 overexpression line OE9, the RNAi line SE13, NT and Alternaria alternata were provided by Prof. Jing Jiang. Escherichia coli DH5α and Agrobacterium tumefaciens EHA105 strains were bought from Harbin Xinkerui Business, China. Rhizoctonia solani was provided by Jinyu Qi. Birch seedlings growing in culture vessels were cultured in a tissue culture laboratory at 28 ℃, with a 16 h light (1,500 lux)/8 h dark photoperiod. Seedlings growing in soil were cultured in a greenhouse at 28 ℃, with a 16 h light (10,000 lux)/8 h dark photoperiod. Seedlings infected with pathogens were cultured in an illumination incubator at 28 ℃, with a 16 h light (10,000 lux)/8 h dark photoperiod, and 60–80% relative humidity.
Vector construction and genetic transformationThe 3,000-bp region upstream of the ATG start codon of the BpEIL1 gene was designated as its promoter. The primers used for PCR were BpEIL1-P-F and BpEIL1-P-R (Supplementary Table S1). DNA was extracted from birch samples using a Plant DNA Isolation Reagent (Takara, Japan). Using the above primers, we amplified the 2,860-bp sequence from the birch genome. SacI and SalI restriction enzymes were used to insert the sequence into the 1300-35S-Gus vector, and EHA105 cells were transformed with the recombinant vector by electroporation. Birch seeds were sterilized with 30% hydrogen peroxide and cut in half longitudinally, and then soaked in Agrobacterium (OD600 = 0.3) for 2–3 min. The cut surfaces were placed on the surface of Woody Plant Medium (WPM) (supplemented with 0.02 mg/l NAA and 0.8 mg/l 6BA). Hygromycin (50 mg/l) was added to the medium to screen the transformants as the transferred DNA (T-DNA) carried an hptII reporter gene. The resistant callus developed after 15 days. Different transformants were then transferred to a new medium (WPM + 0.02 mg/l NAA + 0.8 mg/l 6BA + 0.5 mg/l GA3 + 50 mg/l hygromycin) and, after 30 days, we acquired different transgenic lines.
DNA extraction and PCRDNA was extracted using Plant DNA Isolation Reagent (Takara). Primers used to amplify the GUS gene were GUS-F and GUS-R (Supplementary Table S1). The PCR program was as follows: 95 ℃ denaturation for 3 min, followed by 35 cycles of 95 ℃ for 30 s, 58 ℃ for 30 s and 72 ℃ for 30 s, with a final elongation at 72 ℃ for 7 min. The predicted PCR product was 494 bp.
GUS stainingBirch seedlings (6–7 cm height) were dipped into a 5-bromo-4-chloro-3-indolyl β-D-glucuronic acid cyclohexylammonium salt (X-Gluc) (0.5 mg/ml) solution, put into a sealed glass container, and the air was then removed with a vacuum pump for 30 min. After staining overnight at 37 ℃, the X-Gluc solution was discarded and the stained seedlings were submerged in 95% ethanol to remove the chlorophyll.
DAB stainingFresh mature leaves from birch (50–60 cm height) were harvested for 3,3-diaminobenzidine (DAB) staining. Leaves were soaked overnight in 1 mg/ml (pH 3.8) DAB at 28 ℃ in the dark and then illuminated (incandescent lamp, 2,000 lux) for 1 h, after which the stained leaves were transferred to 95% ethanol overnight to elute the chlorophyll.
RNA extraction and RT-qPCRRNA was extracted from mature leaves of birch (50–60 cm height) using a Plant RNA Extract kit (Bio Teke, China). Total RNA (1 μg) of each sample was used to synthesize cDNA with ReverTra Ace qPCR RT Master Mix (Toyobo, Japan). Quantitative real-time PCR (RT-qPCR) was run on a 7500 Real-Time PCR System (ABI, USA) using SYBR Green (Toyobo). 18S rRNA, actin and ubiquitin were used as reference genes (Gang et al., 2019). For verification of RNA-seq, we chose 15 unigenes at random from DEGs for quantification. Four pathogenesis-related genes (PR1, PR1a, PR1b and PR5) and 10 immune-related genes were also quantified using RT-qPCR. Primers used in these experiments are shown in Supplementary Table S1. RT-qPCR relative expression levels were calculated using the 2−ΔΔCT method. Quantification of each unigene was repeated three times. The thermocycler parameters used during RT-qPCR were as follows: 95 ℃ denaturation for 30 s, followed by 45 cycles of 95 ℃ for 15 s and 58 ℃ for 45 s, and finally 95 ℃ for 15 s, 58 ℃ for 1 min and 95 ℃ for 15 s.
RNA-seq and data analysisTotal RNA of mature leaves of NT and SE13 lines (50–60 cm height) was extracted using a Trizol kit (Takara). Three biological replicates were performed for each line. Total RNA quality and quantity were analyzed using a Nanodrop 2000 instrument (Thermo Fisher Scientific, USA) and an Agilent 2100 Bioanalyzer (Agilent, USA). mRNA of each sample was then enriched using oligo (dT) magnetic adsorption and fragmented. The mRNA fragments were taken as templates for first-strand cDNA synthesis using random hexamers and reverse transcriptase (Promega, USA). Second-strand cDNA was synthesized using DNA polymerase I and RNase H, and then purified using AMPure XP beads (Beckman Coulter, USA). cDNA fragments of a suitable length (300–500 bp) were obtained by AMPure XP beads and amplified by PCR for constructing the final cDNA libraries. RNA-seq was performed by Biomarker Company (Beijing, China) using an Illumina HiSeq 4000 sequencing platform. Low-quality data were filtered from the raw data using fastp to ensure data quality. TopHat2 was used for reads mapping. Betula pendula subsp. pendula (v1.4c) (https://genomevolution.org/coge/GenomeInfo.pl?gid=35080) was used as a reference genome (Salojärvi et al., 2017). The expected number of fragments per kilobase of transcript per million mapped fragments was used as a measure of standardized gene expression. Differentially expressed genes were identified with False Discovery Rate < 0.01 and Fold Change ≥ 2 using EBSeq (Leng et al., 2013). Gene function was annotated based on the following databases: NCBI NR (non-redundant protein sequences from GenPept, Swissprot, PIR, PDF, PDB and NCBI RefSeq), GO (Gene Ontology), Pfam (Protein family) and KO (KEGG Ortholog database) (Kanehisa and Goto, 2000).
Protective enzyme activity assayCatalase (CAT), ascorbate peroxidase (APX), superoxide dismutase (SOD) and peroxidase (POD) activity in the mature leaves were determined using CAT (A007-1), APX (A123-1), SOD (A001-1) and POD assay kits (A084-3) (Nanjing Jiancheng Technology Company, China). Three independent biological replicates were performed for each sample with each enzyme.
Alternaria alternata culture and plant infectionHyphae of A. alternata were inoculated on PDA medium (300 g/l potato, 20 g/l glucose, 20 g/l agar) and cultured at 25 ℃ for seven days in the dark. Spores were eluted by injecting 5–10 ml sterile water into the test tube. The eluted suspension was then filtered through gauze, and sterile water was used to dilute the spore suspension to 105/ml. The suspension was then evenly sprayed with a sprinkling can onto birch seedlings (30–50 cm high). The infected plants were first cultured in an incubator at 26 ℃ and 80% relative humidity in the dark for 24 h and then at 28 ℃ and 60% relative humidity with a 16 h light (1,500 lux)/8 h dark photoperiod for seven days. For the in vitro experiment, sterile filter paper (6 mm diameter) was dipped into the spore suspension described above, and then put on the adaxial surface of the tested leaves. The inoculated leaves were cultured in Petri dishes with wet filter paper at 26 ℃ in the dark for seven days. The incidence of infection was evaluated as the proportion of blight leaves in all leaves of each plant at seven days after spraying. Each experiment was performed for 10 independent biological replicates.
Rhizoctonia solani culture and plant infectionHyphae of R. solani were inoculated on PDA medium as above and cultured at 25 ℃ for seven days in the dark. Hyphae (0.05 g) were immersed in 1 ml of sterile water and blended by a vortex mixer five times for 1 min each time. The suspension was added to 19 ml of sterile water. One milliliter of the suspension was injected into the soil near the root of birch seedlings growing in culture vessels. The infected plants were cultured at 28 ℃ with a 16 h light (1,500 lux)/8 h dark photoperiod, and were observed under a stereomicroscope six days after inoculation.
Funding: This study was supported by the following foundations: 1. National Natural Science Foundation of China (NSFC) (Grant No. 31800558); 2. Mudanjiang Normal University (MNUB201908).
Conflicts of interest: The authors declare that they have no competing financial interests.
Availability of data and material: Not applicable.
Code availability: Not applicable.
Authors’ contributions: Ranhong Li designed the experiments, analyzed RNA-sequencing data and wrote the manuscript. Jingjing Sun and Xiaomeng Ning performed the experiments. Dan Liu and Xin Chen analyzed the data and made the figures. All authors read and approved the final manuscript.
Ethics approval: Not applicable.
Consent to participate: Not applicable.
Consent for publication: This work has not been published before. Its publication has been approved by all co-authors.
