Overexpression of the PdpapERF109 gene enhances resistance of Populus davidiana × P. alba var. pyramidalis to Fusarium oxysporum infection

Jian Diao · Mingrui Li · Ping Zhang · Cheng Zong ·Wei Ma · Ling Ma

Abstract The key transcription factor gene PdPapERF109 was cloned from Populus davidiana × P. alba var. pyramidalis (Pdpap), and after overexpression of PdPapERF109 in transformants, the gene functions in the resistance response to Fusarium oxysporum infection. Compared with the wild Pdpap, after inoculation with F. oxysporum, the physiological and biochemical characteristics, including relative fresh weight, peroxidase activity, and the percentage of electrolyte leakage showed that, after overexpression of the PdPapERF109 gene, the transformants grew well and displayed signif icant resistance to F. oxysporum infection. By comparing the reactive oxygen species scavenging capacity of Pdpap plants after pathogen infection, the PdPapERF109-overexpressing plants had significantly better reactive oxygen species scavenging ability than the wild plants.Comprehensive analysis of plant morphology and various physiological and biochemical parameters showed that the overexpression of the PdpapERF109 gene significantly improved the resistance of Pdpap plants to F. oxysporum root rot. Therefore, increasing the expression of the homologous ERF109 gene can be an eff ective strategy to increase disease resistance in hybrid poplars.

Keywords ERF · Fusarium oxysporum tolerance · Gene over-expression · Genetic transformation · Transcription factor

Introduction

Populusis a genus widely distributed around the world, and many species are highly adaptable to various environmental conditions. As a model species for woody plant research(Ning et al. 2018),Populushas the advantage of having a smaller genome (480 Mb) (Cossu et al. 2012), and that effi -cient genetic transformation systems are available for different species (Maheshwari and Kovalchuk 2016). A reason whyPopulus davidiana×P. albavar.pyramidalisLouche(PdPap) is suitable as an urban species is that it has a male vegetative breeding system (Yao et al. 2018). After blooming in spring, no catkins are formed so it can be used as a choice of urban tree species in north China (Yao et al.2018). Studies have conf irmed that the hybrid tolerates low temperatures and drought (Qin et al. 2005; Zhao et al. 2015).However, due to deteriorating environmental conditions in recent years, the hybrid is facing increasingly challenges to growth and development from pathogenic microorganisms (Yu et al. 2019), temperature extremes (Zhao et al.2015), moisture extremes or def icits (Qu et al. 2014) and ion concentrations (Lu et al. 2004; Chen et al. 2020) that can result in diff erent degrees of damage. Among the many hazards, root rot is one of the most serious and devastating diseases and has attracted widespread attention (Moncrief 2010 ). Once the disease breaks out, it will cause large-scale root rot and death (Ikeda et al. 2005). Xie et al ( 1993) was the f irst to report on the damage status, symptom characteristics, and infection pathways ofFusarium oxysporuminPopulus×euamerica(Dode) Guinier that occurred in the Dongting Lake area. Reports note thatF. oxysporumhas been used for the infection ofPopulus trichocarpa(Zadworna et al. 2014) and Pdpap (Guo et al. 2020).

Fusarium oxysporumis one of the main pathogenic species of theFusariumgenus, and infects many types of plants,eventually causing root rot and plant wilt (Puhalla 2011). In addition to being parasitic on the host plant,F. oxysporumalso survives in soil and in the air, and after 10 years of survival, it may still be strongly pathogenic (Wu et al. 2009).The f irst physical barrier to be breached byF. oxysporumin plant infection is the plant cell wall (Huang 2001). The degradation of the cell wall is achieved by secreting cellulase,pectinase, and β-glucosidase enzymes (Li and Zhang 2010).The pectin produced after degradation of the cell wall blocks the ducts of the host plant (Florendo et al. 2003), which hinders the absorption of water and eventually causes the plant to wilt and die (Miedes et al. 2013).Fusariumacid(5-butyl-2-picolinic acid) secreted byFusariumenhances the permeability of the root cell membranes causing serious damage (Wang and Ng 1999). In addition, the acid will also reduce the active oxygen content in mitochondria,ultimately hindering the water absorbing function of plant roots and inhibiting plant growth (Lu et al. 2004). In addition, the presence of pedigree-specif ic regions makes theF.oxysporumgenome highly susceptible to horizontal transfer(Bootsma et al. 2000), resulting in the formation of novel specialized types and physiological races of the pathogen(K?ppl and Carr 2003), givingF. oxysporuma wide host range and extremely strong pathogenicity (Ma et al. 2013).

Transcription factors are closely related to the plant stress response (Sakuma et al. 2002). The process by which transcription factors enhance the ability of plants to resist stress is mainly a result of the increased expression of transcription factor genes in response to stress (Ali et al. 2013), and this can then further regulate the expression of downstream functional genes (Stein et al. 2001; Hou et al. 2009). Zeng and Mu ( 2016) have shown that transcription factors play a vital role in plant stress resistance, and the expression of this stress resistance function is usually coordinated under the common regulation of many transcription factors (Sun et al.2009). In addition, the adaptation of plants to adverse environmental conditions can be achieved by generating secondary metabolites (Stangarlin et al. 2011). When their growth status is disturbed, secondary metabolites will increase the tolerance of plants to stress (Makkar et al. 2007). Studies have shown that different types of transcription factors can jointly participate in the regulation of plant secondary metabolite biosynthesis through synergistic eff ects (Wasternack and Strnad 2019), which then respond to an adverse environment.

The largest transcription factor family is the APETALA2/ethylene-responsive factor (AP2/ERF) (Xue and Loveridge 2010), which eff ectively responds to biotic or abiotic stresses during the process of adaptation, thereby mobilizing various resistance response genes (Zorrilla et al. 2014). Ethylene response factors (ERFs) are a family of transcription factors unique to plants (Qiu et al. 2012), and named becauseERFgenes can respond to ethylene stress to a certain extent(Mizoi et al. 2012). Diff erent types of ERF transcription factors have diff erent gene expression regulation. For example, ERF transcription factors can simultaneously activate or inhibit expression of genes related to disease control (Fujimoto et al. 2000). In addition, since there is no GCC-box element in the promoter of genes involved in the regulation of maturation and senescence (Jin et al. 2010), ERF transcription factors work together with other types of cis-acting elements (Takagi et al. 2020). Based on the results of published research, there are several ways in which ERF transcription factors function in the process of stress resistance:(1) through direct regulation of PR gene expression; (2)via indirect regulation of key gene expression in hormone synthesis pathways (Mader and Cameron 2006); and, (3)in combination with the promoters of key genes to regulate gene expression and the expression of secondary metabolites(Kagaya et al. 1999). The ERF transcription factor family plays a powerful role in many diverse life activities, including positive responses to biotic/abiotic stresses, growth,and metabolite regulation (Mizoi et al. 2012). ERF member genes respond to stresses, including pathogenic microorganism infection (Shi et al. 2015), temperature extremes (Dossa et al. 2016), ion concentrations, and mechanical damage(Licausi et al. 2013). At the same time, the ERF family, as an important regulatory center of the responses to various types of stress, can comprehensively integrate hormone signals such as jasmonic acid and ethylene (Müller and Bosch 2015). In addition, the heterologous expression of the ERF family member genes can produce diff erent degrees of resistance to various stresses through the activation of resistance pathways (Yi et al. 2004).

Based on the results of previous research on transcriptome data (Diao et al. 2021), the transcription level of Pdpap infected withF.oxysporumis well understood. The transcriptome data was screened with the standard of log2(Fold Change) ＞ 4 andPadj ＜ 0.05. Based on this, genes were obtained in Pdpap, whose transcription levels continued to increase after infection (Supplementary Table S1). Among these genes, there are three typical transcription factors(ERF109, ERF6, MYB41). Among them,ERF109is basically at the highest level and therefore an in-depth analysis was carried out onERF109.

The 809 bp cDNA of thePdPapERF109gene was isolated from Pdpap. The recombinant vector pBI121-PdPapERF109for the over-expression ofPdPapERF109was constructed under the control of the CaMV 35S promoter,and it was successfully transformed into Pdpap. In addition,evidence based on the analysis of molecular, morphological, and physiological data showed that thePdPapERF109-overexpressing transformants have enhanced resistance toF.oxysporumCFCC86068. The objectives of this study were to determine the function of thePdPapERF109gene in resistance toF. oxysporuminfection using physiological and biochemical methods to provide a contribution to improving plant disease resistance in hybrid poplars.

Materials and methods

Materials

Wild Pdpap seedlings were cultured on 0.5 × MS (Murashige and Skoog) medium supplemented with 0.01 mg/mL 1-naphthaleneacetic acid (NAA). For stable gene transformation,leaves from 1-month-old plants were transferred to diff erentiation medium that contained 0.5 mg/mL 6-benzylaminopurine (6-BA), 0.1 mg/mL NAA, and 0.02 mg/mL thidiazuron(TDZ). The tissue cultures were grown in a tissue culture room (Zhang et al. 2009). The seedlings obtained by redifferentiation are transferred to 0.5 × MS medium. In order to simulate growth under natural conditions, the seedlings were transplanted to artif icial soil and cultivated in an controlled environment at 22 ± 2 °C with a light intensity of 400 lx/m 2 /s, a photoperiod of 16 h light and 8 h dark, and a relative humidity of 65-75% (Lu et al. 2011).

The strain ofF. oxysporumCFCC86068 was obtained from the China Forestry Culture Collection Center. The preactivatedF. oxysporumwas transferred to PDA medium and cultured in dark for 7 days. A 9 mm-diameter borer was used to punch holes at the edge of the hyphae and transferred to a new PDA medium. After culturing at 28 °C for 14 days,spores were washed f ive times with sterile water, f iltered through eight layers of gauze and collected into a 50 mL centrifuge tube. Spore density was calculated using a hemocytometer and stored at room temperature for later use.

RNA extraction and cDNA transformation

Total RNA was extracted from young leaves using a TaKaRa MiniBEST Plant RNA Extraction kit (Takara, Dalian,China), and cDNA was synthesized using PrimeScript?RT reagent Kit with gDNA Eraser (Takara, Dalian, China).

Evolutionary analysis of the predicted PdPapERF109 protein

To fully understand the evolutionary relationships of the PdPapERF109 protein, 16 gene sequences that encode similar ERF proteins were obtained from the NCBI GenBank database ( https:// www. ncbi. nlm. nih. gov/ ) (Stoesser et al.2014). Using MEGA 5.1 software (Tamura et al. 2011), a neighbor joining (NJ) phylogenetic tree was constructed and the conf idence of the branches was estimated using 1,000 iterations of bootstrap resampling and the Poisson model.In addition, to further verify the evolutionary relationships between the ERF proteins, we constructed a second phylogenetic tree using the maximum likelihood (ML) method.

Diff erential expression of PdPapERF109 gene

To clarify the role ofPdpapERF109gene in the process responds to pathogen infection, 6-month-old wild Pdpap were treated withF.oxysporumat diff erent stages, and the treatment times were at the beginning, then at 6, 12, 24, and 48 h, respectively. The RNA from each culture was subjected to reverse transcription to obtain f irst-strand cDNAs. The expression ofPdPapERF109was analyzed by reverse transcription quantitative real-time PCR (qRT-PCR) performed on a Stratagene Mx3000P real-time PCR system (Agilent Technologies, Santa Clara, USA) using the 2 × SYBR Green qPCR Master Mix kit (Bimake, Shanghai, China). The amplif ication curve was generated after analyzing the raw data. Cycle threshold (Ct) value 0.01 was calculated based on the f luorescence threshold.PdpapactinandPdpapEF1-αwere used as reference genes to normalize the gene expression (Guo et al. 2021a). The primer sequences for amplifying the reference genes andPdPapERF109are in Supplementary Table S2, and the components of the qRT-PCR assays are in Supplementary Table S3. The qRT-PCR amplif ication conditions were: an initial denaturation of 94 °C for 30 s followed by 44 cycles of 94 °C for 12 s, 58 °C for 30 s,and 72 °C for 45 s, and 79 °C for 1 s. The reaction specif icity was determined by performing a melting-curve analysis from 55 ℃ to 99 ℃, with f luorescence readings taken every 0.5 °C for 1 s. The 2 -△△Ct method (Livak and Schmittgen 2001) was used to calculate the relative expression levels of the target genes, def ined as:

where “Ct-control” represents the average Ctvalue of reference genes, “1” represents the untreated group, and “2” represents the treated group. Three biological replicates were performed for each assay.

To determine the spatial expression prof iles of thePdPapERF109gene, six tissue types were sampled: roots, whole stems, leaves, and the base, middle, and top sections of the stem (～ 5 cm in length). RNA was extracted from these tissues from 6-month-old wild seedlings. The RNA from each tissue was subjected to reverse transcription to obtain f irststrand cDNAs. The expression pattern ofPdpapERF109gene was analyzed according to the qRT-PCR method described above.

Similarly, the same method was used to analyze the expression level ofPdPapERF109in transformants. After 2 months, thePdPapERF109-overexpressing shoots grown in soil were treated with 20 mLF. oxysporum(1 × 10 5 cfu/mL)by perfusion into the root. The infection time was at 0 h, and 6, 12, 24, and 48 h; each experimental group had three replicates. The transformants were frozen in liquid nitrogen, and total RNA extracted and reverse transcribed to obtain cDNA.The expression level ofPdPapERF109was determined by qRT-PCR using cDNA as a template from the wild material and transformants for the diff erent pathogen infection times.The primer sequences are given in Supplementary Table S2.

Cloning of the PdPapERF109 gene and vector construction

PCR cloned thePdPapERF109gene using primer sequences(Supplementary Table S4). The components of the PCR amplif ications are shown in Supplementary Table S5. The amplif ication program for PCR was: 94 °C for 3 min followed by 30 cycles of 94 °C for 30 s, 58 °C for 30 s, and 72 °C for 1.5 min, followed by a f inal extension step of 72 °C for 20 min.

The DNA fragment and the pBI121 overexpression vector were digested with the restriction endonucleasesXbaI andXmaI, and thePdPapERF109gene was inserted into pBI121 under control of the caulif lower mosaic virus (CaMV) 35S promoter. The pBI121-PdPapERF109recombinant vector was transferred intoAgrobacterium tumefaciensGV3101 by heat shock transformation (Holsters et al. 1978), and thenAgrobacterium-mediated transformation was used to introduce the recombinant vector into the Pdpap genome (Guo et al. 2018).

Generating recognized transformant Pdpap plants

Acceptable transformant Pdpap lines were obtained by the leaf disc method (Julia et al. 2017). After cutting the young leaves from 2-month-old Pdpap plants, discs ～ 1 cm diameter were co-cultured with theAgrobacteriumstrain harboring the pBI121-PdPapERF109recombinant vector and transferred onto MS diff erentiation medium containing 50 mg/L kanamycin and 200 mg/L cephalosporin for shoot regeneration. The kanamycin-resistant shoots were rooted on 0.5 × MS medium containing 50 mg/L kanamycin for root regeneration. After selection by kanamycin, acceptable transformants were used for subsequent analysis.

Molecular detection of acceptable transformant Pdpap plants

A DNA extraction kit (Tiangen, Beijing) was used to extract genomic DNA from the acceptablePdPapERF109-overexpressing transformed Pdpap plants. PCR detection was performed on DNA extracted from the plants. The pBI121-PdPapERF109plasmid as the positive control, wild Pdpap and water as the negative controls. For molecular detection of recombinant vectors, primer pairs pBI121-F were used in the pBI121 vector/PdPapERF109-R in the gene sequence,andPdPapERF109-F in the gene sequence/pBI121-R in the vector. The PCR primer sequences are given in Supplementary Table S4.

Growth and physiological index measurements

To test resistance toF. oxysporumunder natural conditions,the wild-type and overexpressing transformant OE4 and OE5 lines were inoculated withF. oxysporumand grown in the artif icial chamber for 10 days, after which the growth of the plants was assessed. The experiment had three biological replicates. During the experiment, water was added every 3 days to keep the soil moist to avoid the inf luence of drought.

The survival rates of wild and transformants inoculated withF.oxysporumat zero, 5, 10, 15, and 20 days were counted according to Sun et al. ( 2018). Disease levels were classif ied according to the following criteria: 0 (no symptoms), 1 (1-25% wilted leaves), 2 (26-50% wilted leaves), 3(51-75% wilted leaves), and 4 (76-100% wilted leaves) (Xu et al. 2014 ). The experiment had ten samples. Refer to the following equation (Sun et al. 2018) for calculation:

The 2-month-old wild material andPdPapERF109-overexpressing transformant Pdpap plantlets in the same growth state were inoculated withF. oxysporumand sampled at zero, 5, 10, 15, and 20 days as described above.The value of pathogen inoculation of the wild Pdpap plants at day zero was set to 1. The growth status of each infected plantlet was observed, and fresh weight and root lengths measured. The experiment had three replicates.

The 2-month-oldPdPapERF109-overexpressing transformants and wild Pdpap plants at similar growth stages were inoculated withF. oxysporumand sampled at 0 h, and at 6, 12, 24, 48 h, and 96 h. The inoculated plants were frozen in liquid nitrogen immediately after sampling. Using the methods of Cheng et al. ( 2020), the samples were used to determine various physiological indicators, including peroxidase (POD) activity, catalase(CAT) activity (Góth 1991), H2O2content, malondialdehyde (MDA) content, and the percentage of electrolyte leakage (Nguyen et al. 2016). The experiment had three replicates.

Antioxidant capability test

Nitroblue tetrazolium chloride (NBT) can detect superoxide activity in plant tissues (Kumar et al. 2013). In order to assess the damage to the leaves by ROS due toF. oxysporuminfection, the third to sixth leaves of seedlings were sampled 3 days after infection and immediately stained with NBT (Zhou et al. 2020). Dark blue staining indicates that the cells were more damaged by ROS,which means that the antioxidant capacity of the cells was relatively lower.

Statistical analysis

The data were analysed with the Statistical Software Package for Social Science (SPSS) version 17.0. Using the Student’st-test to compare the data,P＜ 0.05 was considered signif icantly diff erent. Signif icant diff erences (P＜ 0.05) are indicated by diff erent lowercase letters.

Results

Phylogenetic analysis

An 809 bp cDNA fragment of thePdPapERF109gene was cloned from RNA extracted from wild seedlings. ERF protein sequences likePdPapERF109were downloaded and compared. A NJ phylogenetic tree was constructed to show the evolutionary relationships among the sequences.Bootstrap resampling tested the statistical support of the branches. The NJ analysis is shown in Fig. 1 A. To test the reliability of the NJ model in detail, the JTT + G + F model was used to build a ML phylogenetic tree using the same data (Fig. 1 B). A comparison of the two phylogenetic trees showed that the two models had good consistency, and thatPdPapERF109shared 100% identify withERF109fromP. trichocarpa(XM_006384349.2).The PdPapERF109 shared high sequence homology with similar proteins from other species, such asP. trichocarpa(100%, LOC18097788, XM_006384349.2),Lactuca sativaL. (92.2%, LOC111890860, XM_023886942.1),Raphanus sativusL. (91.5%, LOC108816386, XM_018588961.1),Dendrobium catenatumLind. (91.1%, LOC110108677,XM_020839427.2),Pistacia veraL. (90.4%,LOC116118480, XM_031404463.1),Cynara cardunculusvar.Scolymus(90.2%, LOC112529749, XM_025141136.1),Cucurbita peposubsp.Pepo(86.7%, LOC111810595,XM_023697325.1),Populus albavar.pyramidalis(85.8%, MK955879.1),Populus euphratica(84.8%,LOC105108020, XM_011002160.1),Ricinus communisL.(80.3%, LOC8275304, XM_002518721.3),Manihot esculentaCrantz (76.3%, LOC110624639, XM_021769854.1)andEucalyptus grandisHill (76.2%, LOC104455366,XM_010070166.1).

Fig. 1 Dendrogram of PdPapERF109 and high homology proteins from other species. A NJ-phylogenetic tree; B ML-phylogenetic tree.Dendrogram was constructed by MEGA5 with neighbor method and Poisson model. PdPapERF109 was marked with red frame. It showed similar evolutionary relationship under two algorithms

Analysis of diff erential expression of the PdPapERF109 gene

After Pdpap seedlings were infected byF.oxysporum, the expression of thePdPapERF109gene was signif icantly induced (Fig. 2 A). With an extension of infection time, the expression level continued to increase and reached the highest at 48 h after infection.

To determine the spatial expression prof iles of thePdPapERF109gene, qRT-PCR was used to analyze the expression in diff erent tissues. From the results of this analysis,the expression level is lowest in the leaves and highest in the stems (Fig. 2 B). Expression was also compared in diff erent parts of the same tissue, and was lowest in the top part of the stem and highest in the bottom (Fig. 2 C).

Molecular detection in putative transformants overexpressing PdPapERF109

Fig. 2 A Analysis of expression patterns of PapapERF109 gene under diff erent stages of F. oxysporum infection; B tissue diff erential expression of PdPapERF109 in diff erent tissues of Pdpap; C tissue diff erential expression of PdPapERF109 in diff erent parts of the same tissue. T0-T4: Infection times were 0, 6, 12, 24 and 48 h. Signif icant diff erences ( P ＜ 0.05) are indicated by lowercase letters. Error bars represented standard deviation of three independent replicates

After thePdPapERF109overexpression vector was transformed into wild Pdpap, f ive putative transformant lines were obtained. DNA was extracted from leaves of the WT and the f ive putative transformant Pdpap plants, and PCR was performed using primer pairs pBI121-F/PdPapERF109-R andPdPapERF109-F/pBI121-R. After PCR amplif ication,each line gave DNA fragments amplif ied by both pairs of primers (Fig. 3 A). Bands in lanes 1-1 to 5-2 are the same size as bands in lanes “+1” and “+2”. A single amplif ied fragment was detected in each lane, while there were no amplif ied bands in lanes “- -1”, “- -2” and “w-1”,“w-2”. The gel electrophoresis results show that the size of the amplif ied target bands from the f ive putative transformant lines are as expected. The recombinant plasmid (positive control) showed that the gene can be amplif ied only in the transformants, but not in the wild material, indicating that the pBI121-PdPapERF109vector successfully transformed Pdpap, and the f ive putative transformants contained thePdPapERF109gene under the expression of the constitutive CaMV 35S promoter.

Expression analysis of PdPapERF109 in transformants

As shown in Fig. 4, after inoculation withF. oxysporum,wild Pdpap seedlings had wilted and died, but plants of thePdPapERF109-overexpression transformants (OE4 and OE5) grew well. There was a slight water loss in leaf blades and petioles of the transformants. The results show that overexpression of thePdPapERF109gene in Pdpap plants results in a strong resistance againstF. oxysporuminfection compared to wild plants.

Fig. 3 A Molecular detection in overexpressing putative transformant Pdpap of PdPapERF109. M: DL 2000 Marker; 1-5: Five overexpressing putative transformant lines were used as amplif ication templates; +: Positive control using PBI121- PdPapERF109 plasmid as template; -: Negative control with water as template; W: Negative control with wild-type Pdpap as template; -1: PCR detection of putative transformant lines using PBI121-F and PdPapERF109-R as primers; -2: PCR detection of putative transformant lines with PdPapERF109-F and PBI121-R as primers. B: Expression level analysis of PdPapERF109 in transformants. WT: Wild-type Pdpap. OE1-OE5:Transformant lines. Error bars represented standard deviation of three independent replicates. Signif icant diff erences ( P ＜ 0.05) were indicated by diff erent lowercase letters

Fig. 4 The growth status of wild Pdpap and overexpressing transformants OE4 and OE5 lines infected by F. oxysporum and grown for 10 days. CK: Wild Pdpap, OE4, OE5: Transformant lines

The survival rate showed that, after diff erent infections, the transformants (OE4, OE5) were greater than that of the wild material (Fig. 5 A). The fresh weights and root lengths of wild and transformants inoculated at zero (initial step), and at 5,10, 15, and 20 days were determined, and the values at zero were set to 1. The fresh weights and root lengths are shown in Fig. 5 B and C, respectively, and values of the two transformants were consistent with the wild Pdpap plants at D0.In contrast, afterF. oxysporuminfection, the transformants were superior to the wild plants under the same conditions,indicating that they grow better after infection. These results show that ectopic expression ofPdPapERF109can enhance resistance toF. oxysporumduring early growth stages in Pdpap plants.

Physiological analysis of transformants infected with F.oxysporum

The results of H2O2contents are shown in Fig. 6 A. Under normal conditions, the levels in the wild plants were 1.0 ± 0.0 times more than the transformants (OE4, OE5). In plants infected withF. oxysporum, the relative diff erences were 1.3 ± 0.1, 1.3 ± 0.0, 1.3 ± 0.1, 1.2 ± 0.1, and 1.3 ± 0.0 times than those of the transformants after 6, 12, 24, 48,and 96 h. At T0, the H2O2content of the two transformants was basically the same as in the wild Pdpap. However, afterF. oxysporuminfection, H2O2levels in thePdPapERF109-overexpressing lines were lower than in the wild plants under the same treatments.

The results of POD activity measurements are shown in Fig. 6 B. The experiment had three biological replicates.Under normal conditions, POD activity in the transformants was 1.0 ± 0.0 times greater than in the wild materials. The relative diff erences in transformants were 1.2 ± 0.0,1.3 ± 0.0, 1.3 ± 0.1, 1.2 ± 0.1, and 1.5 ± 0.0 times that of the wild Pdpap at 6, 12, 24, 48, and 96 h after infection. From these results, it is shown that at T0, POD activity of the two transformants is basically the same as in the wild plants.After infection, POD activity in the overexpression lines was higher than in wild plants at the same infection times.

Catalase activity is shown in Fig. 6 C. Under normal conditions, CAT activity in the OE4 and OE5 transformants was 1.0 ± 0.0 times that of the wild material. After infection byF. oxysporum, the relative diff erences in transformants were 1.1 ± 0.0, 1.1 ± 0.0, 1.2 ± 0.0, 1.1 ± 0.0, and 1.2 ± 0.0 times that in wild Pdpap plants at 6, 12, 24, 48, and 96 h. At T0, CAT activity of the two transformants is similar to that of wild plants. However, afterF. oxysporuminfection, CAT activity in the two overexpression lines was higher than in wild Pdpap at the same infection times.

Fig. 5 Morphological characteristics of PdPapERF109 overexpression transformants. A: Survival rate of transformants (OE4 and OE5)and wild Pdpap infected with F. oxysporum. B: Relative fresh weight of wild Pdpap and transformants treated with F. oxysporum; C: Root lengths of wild Pdpap and transformants treated with F. oxysporum.D0-D4: Infection times were 0, 5, 10, 15 and 20 days. The error bars represent standard deviation of three independent replicates. Signif icant diff erences ( P ＜ 0.05) are indicated by diff erent lowercase letters

Malondialdehyde (MDA) contents of leaves from wild and transformants were measured (Fig. 6 D). Under normal conditions, MDA levels of the transformants were 1.0 ± 0.0 times that of the wild plants. Conversely, the wild plants were 1.2 ± 0.0, 1.3 ± 0.1, 1.3 ± 0.0, 1.4 ± 0.1, and 1.3 ± 0.0 times higher than those of the transformants at 6, 12, 24,48, and 96 h after infection. At T0, MDA contents of the transformants were essentially the same as that of the wild plants. Following infection, the overexpressing lines had lower malondialdehyde levels than the wild Pdpap plants at the same infection times.

The percentage of electrolyte leakage is shown in Fig. 6 E.Under normal conditions, electrolyte leakage in wild plants was 1.1 ± 0.0 times that of the transformants. After infection,the wild plants were 1.4 ± 0.1, 1.4 ± 0.0, 1.4 ± 0.1, 1.4 ± 0.0,and 1.2 ± 0.0 times higher than that of overexpressing lines at 6, 12, 24, 48, and 96 h. At T0, the percentage of electrolyte leakage in the two transformants was basically the same as in wild plants. After infection, the percentage of leakage in thePdPapERF109-overexpressing lines was lower than in the wild Pdpap at the same infection times.

Histochemical staining

Infected leaves were stained with nitroblue tetrazolium (NBT)to assess the antioxidant capacity of the transformants (Fig. 7).The staining of the leaves fromPdPapERF109-overexpressing transformants after infection (Fig. 7 C) is slightly darker than in the uninfected wild controls (Fig. 7 A), but the overall appearance is similar. In addition, the diff erences in color compared with leaves of infected wild plants (Fig. 7 B) are obvious. These results indicate that in plants infected withF. oxysporum, the cells in the transformants show less damage, indicating that they have a greater ability to remove reactive oxygen species(ROS), including O 2- and H2O2, thereby reducing cell damage and enhancing plant tolerance. This is consistent with POD and CAT activity measurements, and further shows that the overexpression ofPdPapERF109in Pdpap plants reduces the accumulation of intracellular ROS. Overexpression eff ectively enhances the antioxidant capacity of the plants, thus improving the resistance of Pdpap toF. oxysporuminfection.

Fig. 6 Physiological characteristic analysis of wild Pdpap and PdPapERF109 overexpression transformants infected with F. oxysporum.Two transformants and WT were used as biological replicates for physiological analysis. A Result of H 2 O 2 content measurement; B Result of POD activity measurement; C result of catalase activity; D Result of malondialdehyde content; E Percentage of electrolyte leakage. T0-T5: Infection times were 0, 6, 12, 24, 48, and 96 h. Error bars represent standard deviation of three independent replicates. Signif icant diff erences ( P ＜ 0.05) are indicated by diff erent lowercase letters

Discussion

Transcription factors play an important role in abiotic and biological stress responses (Akhtar et al. 2012). TF families such as WRKY, AP2/ERF, NAC, bZIP, and MYB (Pu et al.2019) affect stress tolerance by regulating downstream response genes (Joshi et al. 2016). Members of the ERF family play a critical role in the responses to stresses, including pathogenic microorganism infection (Guo et al. 2016),ion concentration damage (Yang et al. 2018), excess water or drought (Labbo et al. 2018), and temperature stress (Licausi et al. 2013). At the same time, in the process of defending against stress, the regulation of jasmonic acid, abscisic acid,and ethylene pathways will be integrated (Müller and Bosch 2015 ). The results of Yi et al. ( 2004) showed that ERF family genes produce resistance responses to various pathogens during heterologous expression of plants, which can promote the activation of related resistance pathways, thereby eff ectively reducing the damage to plants exposed to stress.

Fig. 7 Results of superoxide staining by NBT. A Leaves of wild PdPap without treatment; B Leaves of wild PdPap with F. oxysporum;C Leaves of overexpressing transformants with F. oxysporum. Staining intensities of WT without treatment and transformants infected by F. oxysporum are similar, but greatly increased in WT treated by F.oxysporum

Previous studies have shown that thePdPapERF109gene plays a positive role in various biological and abiotic stresses. There are several reports on the resistance functions of thePdPapERF109gene in salt stress (Bahieldin et al. 2018), low temperature stress (Wang et al. 2019) and drought resistance (Shi et al. 2015), and these studies have established a foundation for our early prediction and subsequent verif ication experiments. Research has shown that the JA-responsive ethylene response factor 109 (ERF109)mediates crosstalk between JA signaling and auxin biosynthesis to regulate lateral root formation inArabidopsis(Cai et al. 2014). This indicates that thePdPapERF109gene may regulate the formation of lateral roots in Pdpap to achieve an eff ective response after infection byF. oxysporum.

In this study, diff erential expression levels forPdPpapERF109in recognized transformants may be due to different copy numbers and sites of insertion. The overexpression of the gene plays a signif icant role in the growth and physiological state of plants after pathogen infection. The results indicate that thePdPapERF109gene plays a role in signaling pathways under stress conditions.

One of the important mechanisms of plant disease resistance is the change and increase in the activities of defenserelated enzymes (Gong et al. 2010). By measuring the malondialdehyde content and the percentage of electrolyte leakage, the relative degree of damage to the plant cell membranes can be quantif ied. The determination of physiological indexes such as H2O2 contents, CAT levels, and POD activity can provide a detailed understanding of the ability of thePdPapERF109-overexpressing transformants to remove ROS from the cells and their antioxidant capacity. Studies have shown that diff erent rapeseed varieties express diff erent degrees of resistance to black shank disease, and these resistance capabilities are ref lected in a signif icant increase in the level of POD activity (Peng et al. 2015).Trichoderma aculeatusresists tomato wilt caused byF. oxysporumby increasing the activity of diff erent defense enzymes, thereby improving the disease resistance of tomatoes (Patel and Saraf 2017). In this study, we measured the physiological indicators of thePdPapERF109-overexpressing transformants, including H2O2, POD, CAT, MDA, and the percentage of electrolyte leakage. The purpose of these experiments was to evaluate the resistance of Pdpap plants through the changes in the activities of defense-related enzymes.

NBT staining showed that the overexpression ofPdPapERF109eff ectively reduced the accumulation of ROS in plants infected withF. oxysporum.

According to previous research, key transcription factors can positively regulate the expression levels of SOD and POD genes, thereby enhancing their activity for removing ROS (Zhao et al. 2019; Guo et al. 2021b). It is also believed that under stress conditions, key transcription factors promote the production of POD and SOD through physiological or biochemical reactions, thereby promoting the removal of ROS (Cheng et al. 2020). Based on the previous reports, it is speculated that under normal conditions, thePdPapERF109gene will not directly increase POD and CAT contents in the overexpressing transformants. Similarly, the content of ROS did not change signif icantly in Pdpap. However, in response toF. oxysporuminfection, expression of thePdPapERF109gene can promote the production of POD and CAT through the action of the PdpapERF109 transcription factor on gene expression, thereby promoting the removal of ROS. Therefore, under normal conditions, there is no signif icant differences in physiological changes between wild plants and transformants. Our data indicates that thePdPapERF109gene may play an important role in ROS clearance to protect the plant from external stresses. In addition, the molecular mechanism of gene regulation and the gene network related toPdPapERF109expression in response toF. oxysporuminfection requires further study.

Conclusion

In this study, we cloned a transcription factor gene,PdpapERF109, from Pdpap. Subsequently, overexpressing transgenic lines were obtained in Pdpap. The results of morphological and physiological analyses showed that overexpressing transgenic lines were signif icantly resistant toF.oxysporumcompared with WT. Further studies showed thatPdpapERF109could enhance the ROS scavenging ability of Pdpap under stress. Therefore, we concluded thatPdpapERF109has an important regulatory role during the stress response toF. oxysporum.