Characterization, expression profiling, and functional analysis of a Populus trichocarpa defensin gene and its potential as an anti-Agrobacterium rooting medium additive

Wei, Hui; Movahedi, Ali; Xu, Chen; Sun, Weibo; Li, Lingling; Li, Dawei; Zhuge, Qiang

doi:10.1038/s41598-019-51762-0

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Published: 25 October 2019

Characterization, expression profiling, and functional analysis of a Populus trichocarpa defensin gene and its potential as an anti-Agrobacterium rooting medium additive

Hui Wei¹^na1,
Ali Movahedi ORCID: orcid.org/0000-0001-5062-504X¹^na1,
Chen Xu^1,2^na1,
Weibo Sun¹^na1,
Lingling Li¹,
Dawei Li¹ &
…
Qiang Zhuge¹

Scientific Reports volume 9, Article number: 15359 (2019) Cite this article

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Abstract

The diverse antimicrobial properties of defensins have attracted wide scientific interest in recent years. Also, antimicrobial peptides (AMPs), including cecropins, histatins, defensins, and cathelicidins, have recently become an antimicrobial research hotspot for their broad-spectrum antibacterial and antifungal activities. In addition, defensins play important roles in plant growth, development, and physiological metabolism, and demonstrate tissue specificity and regulation in response to pathogen attack or abiotic stress. In this study, we performed molecular cloning, characterization, expression profiling, and functional analysis of a defensin from Populus trichocarpa. The PtDef protein was highly expressed in the prokaryotic Escherichia coli system as a fusion protein (TrxA–PtDef). The purified protein exhibited strong antibacterial and antifungal functions. We then applied PtDef to rooting culture medium as an alternative exogenous additive to cefotaxime. PtDef expression levels increased significantly following both biotic and abiotic treatment. The degree of leaf damage observed in wild-type (WT) and transgenic poplars indicates that transgenic poplars that overexpress the PtDef gene gain enhanced disease resistance to Septotis populiperda. To further study the salicylic acid (SA) and jasmonic acid (JA) signal transduction pathways, SA- and JA-related and pathogenesis-related genes were analyzed using quantitative reverse-transcription polymerase chain reaction; there were significant differences in these pathways between transgenic and WT poplars. The defensin from Populus trichocarpa showed significant activity of anti-bacteria and anti-fungi. According to the results of qRT-PCR and physiological relevant indicators, the applied PtDef to rooting culture medium was chosen as an alternative exogenous additive to cefotaxime. Overexpressing the PtDef gene in poplar improve the disease resistance to Septotis populiperda.

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Introduction

Antimicrobial peptides (AMPs), including cecropins, histatins, defensins, and cathelicidins, have recently become an antimicrobial research hotspot for their broad-spectrum antibacterial and antifungal activities^1,2. Studies have shown that cecropins isolated from silkworm moth hemolymph are positively charged peptides with a helix–bend–helix structure, which exhibit a broad spectrum of activity against both gram-negative and gram-positive bacteria^3,4. Histatins isolated from primate saliva have been demonstrated to have antifungal activity⁵. Cathelicidin with a C-terminal peptide has the ability to resist group A streptococcus, and cathelicidin production appears to be mainly restricted to mammals^6,7. Defensins have been isolated from a wide range of organisms^8,9,10,11, and different plant defensins have shown low amino acid sequence similarities, except in conservative cysteine (Cys) residues^10,11,12,13. Despite the large variability among amino acid sequences, many plant defensins exhibit similarities in three-dimensional (3D) structure, including three-stranded reverse-parallel β-sheets and an α-helix bound to β-sheets¹³. Defensins exhibit antimicrobial activity against fungi, gram-positive bacteria, and gram-negative bacteria; however, their activity is stronger against fungi than against bacteria^14,15,16,17. Besides antimicrobial activity, defensins have also been shown to exhibit anti-human immunodeficiency virus (HIV) effects¹⁸. Defensin binds to viral coat proteins, causing the viruses to lose their biological activity. Human and mouse defensins can significantly inhibit or even kill enveloped viruses, herpes viruses, and the vesicular stomatitis virus¹⁹. Defensins have been shown to influence antitumor cell activity and may play an important role in tumor immunity²⁰. Defensins also enhance acquired immunity by activating cellular and humoral immunity to kill and eliminate microorganisms²¹ through the attraction of immature dendritic cells and memory cells to inflammatory sites. Defensins can aggregate T cells and monocytes on the mucosal surface. At low concentrations, defensins do not kill microorganisms, but can play an immune surveillance role²². Plant defensins were discovered much later than mammalian defensins; however, after more than a decade of research, it has been confirmed that plant defensins are present in all plants, within various organs and tissues. Defensins play important roles in plant growth, development, and physiological metabolism, and demonstrate tissue specificity and regulation in response to pathogen attack or abiotic stress²³. However, defensins from Populus trichocarpa have not yet been characterized.

Plants living in natural environments are vulnerable to various diseases due to their lack of mobility and a somatic immune system. Therefore, chemical fungicides are widely applied to improve the economic benefits of plant production. The use of chemical reagents improves plant biomass over a limited period of time, but also causes serious long-term problems. These problems can include increasing the risk of pathogen drug resistance, directly threatening the quality and safety of plant products, as well as polluting soil, rivers, lakes, and air to some extent. Therefore, improving plant resistance to pathogens as an alternative to chemical treatment has become a focus of research, with many promising directions for transgenic work. AtPDF1.1 overexpression in Arabidopsis thaliana has been shown to result in decreased sensitivity to the non-host pathogen Cercospora beticola²⁴. In addition, heterologous overexpression of a defensin from Brassica juncea was shown to significantly increase resistance to Fusarium moniliforme in Nicotiana tabacum²⁵. Transferring the DmAMP gene from Dahlia merckii to Oryza sativa by transgenic technology promoted resistance to Magnaporthe oryzae and Rhizoctonia solani²⁶. Overexpression of defensin from Orychophragmus in Brassica napus reduced damage to plants caused by Sclerotinia sclerotiorum²⁷. The expression of NmDef from Nicotiana megalosiphon demonstrated that the NmDef02 gene could improve resistance to Phytophthora infestans and Alternaria solani in Solanum²⁸. A heterologous functional assay of defensin from Solanum lycopersicum showed that the transgenic SlDef gene significantly improved resistance to Botrytis cinerea in tomato²⁹. However, it has not been confirmed whether the overexpression of defensin from P. trichocarpa (PtDef) promotes pathogen resistance in poplar species.

Salicylic acid (SA) and jasmonic acid (JA) play important roles in plant defense signal transduction, and their signaling pathways are closely related to plant resistance^30,31. SA is an endogenous signal molecule that activates plant hypersensitive response (HR) and systemic resistance (SAR). SA can induce disease resistance, the expression of many disease-related proteins³², and abiotic stress resistance in plants. The response mechanism of SA to disease has been well studied in Arabidopsis. Non-expressor of pathogenesis-related gene 1 (NPR1) is the dominant regulator of acquired resistance in plants. Maintaining low SA concentrations in plant cells promotes NPR1 accumulation, thus activating the TGA transcription factors (WRKYs and Transcription factor TGAs) to activate the expression of plant resistance genes³³. In the absence of SA, TGA2 and TGA5 can interact with NIMIN1, TOPLESS, the CBNAC–SNI1 complex, and other components to inhibit PATHOGENESIS RELATED 1 (PR-1) promoter transcription³⁴. NPR3 and NPR4 are also SA signal receptors; SA has higher affinity with NPR4 than with NPR3. When the SA concentration in plants is low, a combination of SA and NPR4 inhibits the degradation of NPR1 to maintain plant resistance. When plants are invaded by pathogens, different concentrations of SA accumulate around the infection site. A high SA concentration at the infection site binds NPR3, thus promoting NPR1 degradation, leading to plant cell death at the infection site and hindering the spread of pathogen infection³⁵. JA, the key signal component of induced systemic resistance (ISR), accumulates rapidly and abundantly when plant tissues are invaded by pathogens. Studies have demonstrated that the coronatine insensitive 1/jasmonate ZIM-domain (COI1/JAZ) transcription factor signaling cascade pathway, an inhibitor of the JA pathway, is a key link in inducing plant pathogen resistance by JA and its derivatives. Jasmonoyl–L-isoleucine conjugates of JA and isoleucine form in plants under the influence of catalytic enzymes. JAZ proteins are thus degraded, relieving transcription factor inhibition and initiating JA response gene transcription³⁶. SA and JA signal transduction mediation is known as the basic signal pathway for plant defense. Cross-talk between pathways induced by SA and JA and other signal transduction pathways can form a complex signal transduction network, allowing plants to respond quickly to different stimuli and speeding up defensive responses³⁷.

In this study, we conducted molecular cloning, characterization, and functional analysis of the plant defensin PtDef from P. trichocarpa. Based on an analysis of its inhibition zone, we determined that the PtDef protein has antibacterial and antifungal activities. Defensins inhibited the growth and activity of Agrobacterium tumefaciens strain EHA105 to a greater extent when applied as an exogenous additive to rooting culture. Overexpression of PtDef resulted in field resistance to Septotis populiperda in transgenic poplars. Finally, we preliminarily explored the mechanism for enhanced disease resistance through PtDef gene overexpression, and determined that the SA and JA signal transduction pathways may be involved in the pathogen resistance response of poplar.

Results

PtDef molecular cloning and sequence analysis

Using PtDef sequence information (GenBank accession no. ABK93231.1), we designed specific primers and obtained the full length of PtDef (614 bp) by rapid amplification of cDNA ends. The PtDef gene had an ORF of 228 bp, encoding a PtDef polypeptide composed of 75 amino acids (Fig. S1A). The predicted Mw of the PtDef protein was 8.59 kD and its pI was 9.27. We found lower conservatism in the N-terminal than the C-terminal; both had eight conserved Cys structures (Fig. S1B), which contribute to the stability of the overall defensin spatial structure by allowing monomer proteins to form four disulfide bonds. This conservative Cys structure also exists in other species; their structural role appears to be universal and selected for in the evolutionary process. Indeed, the deduced PtDef amino acid sequence showed a high degree of homology with defensins from other plant species sequences, e.g., Ziziphus jujuba (XP_015879652.1, 78.67% identity) and Vitis vinifera (XP_002272913.2, 77.63% identity). A defensin phylogenetic tree was then constructed using the CLUSTAL W software and deduced amino acid sequences of PtDef and defensins from 19 other species. The defensin results classified 19 species into a single group, with a second group comprising only Theobroma cacao (GenBank accession no. EOY04853.1). The larger group was divided into four subgroups, suggesting that it may comprise multiple branches derived from an earlier ancestor (Fig. S1C). We then used the SWISS-MODEL server (http://www.expasy.org/swissmod/SWISS-MODEL.html) to construct the tertiary structures of the PtDef protein and Arabidopsis AtDEF proteins, and found that they were similar, consisting of three reverse-parallel β-pleated sheets and an α-helix (Fig. S1D,E).

Prokaryotic expression, antibacterial activity, and antifungal activity assays

Using our PET-32a plasmid and PtDef gene restriction enzyme site analysis results, the ORF of the PtDef gene was inserted into the prokaryotic PET-32a plasmid between the NotI and BamHI restriction sites (Fig. 1A). IPTG was added into the bacterial solution as an inducer, and 12% SDS–PAGE results revealed specific protein bands at about 27 kDa after induced expression, which was consistent with the theoretical calculation of protein size; however, no corresponding protein bands were detected at the corresponding location in the non-induced bacterial solution (Fig. 1B).

The target protein was detected in the supernatant and precipitation under induction conditions of 220 rpm and 37 °C for 4 h (Fig. 1D). The supernatant fusion protein was bound to Ni-IDA resin using 20 mmol/L imidazole, and the target protein was eluted using 250 mmol/L imidazole (Fig. 1C,D). To further explore whether the expressed product was a target protein, we performed Western blot analysis of the expressed product using mouse anti-6 × His antibody, with horseradish peroxidase-labeled sheep anti-mouse as the second antibody. The results showed that the expressed fusion protein was indeed the target protein (Fig. 1E).

Following successful expression, enterokinase was released from the fusion protein to release the target protein. Affinity chromatography was then used to isolate and purify the PtDef protein after enzymatic digestion and the affinity between Trx-6 × His and Ni²⁺ was used to remove the Trx-labeled protein through its binding with His TrapTMHP. The effluent of affinity chromatography contained the PtDef protein. Complete digestion was confirmed by 4–20% gradient gels (Fig. 1F).

The minimum inhibitory concentration (MIC) and minimum bactericidal concentration were then determined and used to evaluate the antibacterial activity of the purified PtDef protein. The purified PtDef protein showed antimicrobial activity against E. coli K12D31 and A. tumefaciens EHA105; as the PtDef protein concentration increased, the number of bacteria decreased (Fig. 2A,C). These results showed that the MIC values of E. coli K12D31 and A. tumefaciens EHA105 differed, demonstrating that, although the PtDef protein was resistant to both bacteria, its sensitivity to each bacterial species differed, with much stronger activity against A. tumefaciens EHA105 than against E. coli K12D31. We then added 0–96 μg of purified PtDef protein to E. coli K12D31 cultures, and incubated the samples for 8 h. Compared with the control group, different concentrations of purified PtDef protein yielded different bacteriostatic growth effects; when ≥24 μg of purified PtDef protein was added, E. coli K12D31 growth was clearly inhibited (Fig. 2B). The same experiment was repeated with A. tumefaciens EHA105 for 16 h of incubation. Compared with the control group, bacterial growth was dose-dependent; A. tumefaciens EHA105 growth was clearly inhibited by exposure to ≥12 μg of purified PtDef protein (Fig. 2C). The inhibitory effect of the PtDef protein on A. tumefaciens EHA105 was greater than that on E. coli K12D31 (Fig. 2).

To evaluate the antifungal activity of the purified PtDef protein, we applied treatments of 40, 20, 10, 5, 2.5, 1.25, and 0.625 μg/mL of protein in an agar well diffusion assay of six fungal species: Aspergillus niger, Alternaria Nees, Mucor corymbifer, Marssonina populi, Rhizopus sp., and Neurospora crassa. All treatments were performed in triplicate. We used the Scan 1200 (Interscience Laboratories Inc., Woburn, MA, USA) automatic colony counter to measure the diameters of bacteriostatic circles. Purified PtDef protein showed significant antifungal activity, with different inhibitory effects observed for different fungi at the same concentrations of purified PtDef protein. The greatest inhibitory effect was observed in M. populi, with a MIC diameter of 16 mm, whereas the MIC diameter of Rhizopus sp. was 10 mm (Figs 3 and S2).

Evaluation of the purified PtDef protein as an analog of cefotaxime

We grew transgenic poplars in two rooting medium treatments, one containing 200 mg/L cefotaxime and the other containing 100 mg/L purified PtDef protein, and observed rooting and growth over a 30-day period. After 30 days, A. tumefaciens EHA105 was inhibited significantly when the purified PtDef protein as antibiotic analog, but the A. tumefaciens EHA105 was detected in MS medium containing the cefotaxime (Fig. S3). Thus, the purified PtDef protein acted as an effective antibiotic analog for cefotaxime to inhibit A. tumefaciens EHA105 growth.

We compared MDA, Pro, POD, and SOD contents in leaves grown within each treatment; there were no significant differences in these components among treatments, suggesting that purified PtDef can be used as a substitute for cefotaxime in rooting culture medium to inhibit the growth of A. tumefaciens EHA105 in transgenic poplar (Fig. 4A).

Poplar leaves and roots were grown for 30 days in rooting medium (negative control), rooting medium with cefotaxime (positive control), and rooting medium with purified PtDef protein, and then removed. Genes encoding ROS-scavenging enzymes and those related to ROS generation were detected by qRT-PCR. The transcriptional levels of these genes increased significantly in the cefotaxime and purified PtDef treatments, indicating that these treatments simulated exogenous stress, which negatively affected poplar physiology (Fig. 4B,C). GST, RbohA, and RbohB expression were significantly lower in leaves from the purified PtDef treatment than in those from the cefotaxime treatment (Fig. 4B). CAT, GST, RbohA, and RbohB expression were significantly lower in roots from the purified PtDef treatment than in leaves from the cefotaxime treatment (Fig. 4C). We conclude that less toxicity is produced by the exogenous addition of purified PtDef than that of cefotaxime. These results demonstrate that the purified PtDef protein can be used as a biological antibiotic for plant tissue and tissue organ culture.

Tissue-specific expression of PtDef in response to abiotic and biotic stress

We used qRT-PCR to investigate PtDef expression in different tissues, including mature leaves, young leaves, petioles, roots, and the upper and lower regions of stems. PtDef expression was highest in leaves, and higher in young leaves than in mature leaves, probably because young leaves are rich in nutrients and susceptible to disease invasion. PtDef expression was also high in roots, perhaps because they are in direct contact with soil and easily invaded by soil diseases during plant growth (Fig. 5A). Thus, PtDef plays an important role in above- and below-ground poplar development.

With the extension of 200-μM ABA treatment duration, PtDef expression increased, reaching maximum expression at 12 h (Fig. 5B). When subjected to 200-mM NaCl treatment, PtDef expression decreased during the first 6 h, perhaps because the high NaCl concentration disrupted normal plant growth and required a minimum reaction time for its defense system to respond. However, after 8 h of NaCl treatment, PtDef expression reached a maximum (Fig. 5C). In the 10% PEG 6000 treatment, PtDef expression was significantly upregulated from days 2 to 7, with a peak appearing on day 7 (Fig. 5D). Under cold treatment, PtDef expression first increased, then decreased, and finally reached a maximum on day 7 (Fig. 5E). PtDef expression was upregulated during the first 48 h of 2-mM H₂O₂ treatment, reaching a maximum at 24 h (Fig. 5F). Under Septotis populiperda treatment, the highest expression level occurred at 72 h, followed by a decrease from 6 to 24 h (Fig. 5G), suggesting that PtDef expression was adjusted to alleviate plant damage following S. populiperda invasion. PtDef expression did not change significantly within the first 8 h of 200-μM JA treatment, but decreased significantly after 12 h, followed by a significant increase at 24 and 48 h (Fig. 5H). In the 200-μM SA treatment, PtDef expression reached a maximum (≥10 times higher than the control) at 3 h of induction (Fig. 5I). These results indicate that PtDef responds to a variety of biotic and abiotic stresses and can be used as a candidate gene for stress tolerance.

Acquisition and identification of transgenic poplar plants

Transgenic poplar plants were screened and integration of the PtDef gene with poplar DNA was detected by PCR. Using 35S as a forward primer and the downstream of the PtDef gene as the reverse primer, a specific band was amplified in transgenic poplar, but not in the WT (Fig. S4A). We performed PCR using upstream and downstream primers of the PtDef gene, and produced a band >250 bp in the WT, perhaps due to introns in the poplar genome, whereas bands for the targeted gene appeared in the transgenic lines (Fig. S4B). We then performed qPCR to detect PtDef expression, and found that expression was significantly higher in the transgenic lines than in the WT, indicating that PtDef was successfully transcribed into the poplar genome and could transcribe intact mRNA (Fig. S4C).

Growth differences between WT and transgenic poplars

Phenotypic analysis revealed no significant difference in growth, height, width, or leaf number between 4-month-old WT and transgenic poplars (Fig. 6A). qRT-PCR analysis showed lower expression of IAA-related genes, including IAA1, IAA2, IAA6, IAA18, and IAA19, in the transgenic lines Def1–1 and Def1–4 than in WT poplars (Fig. 6C). The expression of ABA-related genes, including 9-cis-epoxycarotenoid dioxygenase 1 (NCED1), NCED3, NCED5 and zeaxanthin epoxidase 1 (ZEP1), ZEP2, and ZEP3, was significantly higher in transgenic lines that in WT poplars (Fig. 6C). Expression levels of GA-related genes were also higher in transgenic poplar lines (Fig. 6C).

The standard curves for IAA, GA, and ABA based on HPLC–MS/MS reference standard analysis are shown in Figs S5–7. IAA content was significantly lower (35–46%) in leaves from the transgenic lines than in those from WT poplars (Fig. 6D,G). PtDef gene overexpression in poplars significantly increased the ABA content of leaves (Fig. 6E,G). GA4 content was 2–3.5 times higher in transgenic lines than in WT poplars (Fig. 6F,G). Thus, although there was no difference in phenotype between transgenic and WT poplars within 4 months of growth, molecular and biochemical levels differed greatly between the poplar groups.

To further analyze differences in growth among poplar groups, we compared various photosynthesis parameters. Gs was lower and Pn slightly lower in transgenic lines than in WT poplars (Fig. 6B), whereas Tr and Ci were higher in transgenic lines than in WT poplars. Despite the phenotypic similarity among poplar groups, transgenic and WT poplars differed in molecular-level physiological indicators. Greater changes were observed in transgenic poplars than WT; thus, we expect phenotype to vary with the growth environment.

JA-, SA-, LTP-, and PR-related gene expression analysis and comparison of JA and SA contents between WT and transgenic poplars

Among the JA-related genes examined, the transcript levels of LOC XM_002318978.3, AOC XM_024605953.1, and HPL XM_002321100.3 were significantly higher in transgenic lines than in WT poplars. However, the expression levels of LOC XM_002314512.3 and AOC XM_002305915.3 were significantly lower in transgenic lines (Fig. 7A). Among the SA-related genes, ICS GQ260071.1 expression was significantly higher in transgenic lines, but there was no difference in PAL expression between poplar groups (Fig. 7A).

PR and nsLTP gene expression were compared between the transgenic and WT poplars using qRT-PCR analysis. The expression levels of all nsLTP genes were significantly higher in transgenic lines than in WT except nsLTP XM_002312093.3 (Fig. 7B). Expression levels of five of the six PR genes were higher in transgenic poplars, and that of one PR gene (XM_002313896.3) was lower (Fig. 7C).

The JA and SA standard curves prepared according to the HPLC–MS/MS results are shown in Figs S8 and S9. JA content was significantly lower in leaves of transgenic poplars than in WT leaves, which contained 1.7–2.7 times more JA (Fig. 7D,F). Overexpression of the PtDef gene significantly increased the SA content of leaves (Fig. 7D,F). These results suggest that PtDef overexpression affects the expression of JA- and SA-related genes, ultimately increasing JA and SA contents in poplar, which can subsequently affect plant disease resistance.

PtDef overexpression enhances S. populiperda tolerance in transgenic poplars

Figure 8A shows the extent of S. populiperda spread on poplar leaves with time. Plaques were significantly smaller on leaves from transgenic lines (Def1–4 and Def1–6) than on those from WT plants on days 2, 3, and 4 (Fig. 8B), indicating that PtDef expression can improve poplar resistance to S. populiperda. We compared the degree of leaf damage in WT and Def1–4 transgenic poplar by microscopy. By day 4, many WT leaf cells had been destroyed, and the loss of integrity caused cellular fluid to flow out (Fig. 8C). However, in leaves from the Def1–4 line, cells remained closely arranged and intact, with no efflux of cellular fluid. We then examined S. populiperda morphology within a plaque by microscopy. By day 2, S. populiperda on WT leaves had produced conidia, whereas that on transgenic poplar remained in the mycelial state, producing conidia only on day 3 (Fig. 8C).

The S. populiperda inhibition zone was larger in leaves of the transgenic line than in those of WT (Fig. 8D), as S. populiperda grew more slowly near the pore where the PtDef gene was added. This result indicated that the transgenic protein inhibited S. populiperda growth better than WT endogenous proteins. Crude extracts of two transgenic lines (Def1–4 and Def1–6) and WT were added to PDA medium to cultivate and quantify S. populiperda growth for 7 days. We observed that S. populiperda extracted from WT grew faster, entirely covering the PDA medium by day 3. However, S. populiperda extracted from the transgenic lines grew slowly, covering the entire medium only at day 7 (Fig. 8E). These results demonstrate that PtDef overexpression in poplar significantly improves resistance to S. populiperda.

Expression levels of genes related to JA and SA synthesis and PR genes following inoculation in transgenic and WT poplar

The expression of LOX XM_002318978.3, a gene related to JA synthesis, first decreased and then increased following inoculation, and that of LOX XM_002318978.3 changed greatly in WT poplars; however, the expression of LOX XM_002318978.3 changed less in transgenic poplars (Fig. 9A). The expression of LOX XM_002314512.3 was upregulated 3 times in transgenic poplars, reaching a maximum at 12 h post-inoculation; in WT, its expression changed little post-inoculation, then increased to a peak at 72 h (Fig. 9B). The expression of AOC XM_002305915.3 was upregulated about 15 times in the WT, reaching a peak at 48 h after inoculation, but was upregulated only 9 times in transgenic poplars (Fig. 9C). AOC XM_024605953.1 expression reached a peak 1 h after inoculation in transgenic poplars, with a ratio of about 3.5, whereas it reached an expression peak 48 h after inoculation in WT, with a ratio of about 2.7 (Fig. 9D). The expression of HPL XM_002321100.3 reached a maximum 1 h after inoculation in transgenic poplars, and then decreased, whereas it reached a lower maximum 12 h after inoculation in WT poplar (Fig. 9E). These different expression levels of JA-related genes after inoculation in transgenic and WT poplars may indicate different JA content between poplar groups. JA forms a conjugate with isoleucine that can be catalyzed by an enzyme. When JAZ proteins are degraded, transcription factor inhibition is relieved and JA response gene transcription is initiated. This process may explain the improved disease resistance observed in transgenic lines.

Transcript levels of ICS and PAL, which are genes related to SA synthesis, were detected by qRT-PCR after inoculation. ICS expression in transgenic and WT poplars decreased after inoculation, reaching a minimum at 48 h (Fig. 9F). PAL expression reached a maximum 12 h after inoculation in WT poplar, with an upregulation rate of about 6. PAL expression in transgenic poplars reached a maximum at 24 h after inoculation, with an upregulation rate of about 2.5. The range of PAL expression was smaller in transgenic poplars than in WT (Fig. 9G). Maintaining low SA concentration in plant cells promotes NPR1 accumulation and activates TGA transcription factors (WRKYs and TGACG-SEQUENCE-SPECIFIC BINDING PROTEINs) to activate the expression of plant resistance genes³³.

We then analyzed PR gene expression using qRT-PCR. The expression of PR XM_006379094.2 did not change significantly in either WT or transgenic poplars within 24 h after inoculation. At 48 h, PR XM_006379094.2 expression was 2.4 times higher in transgenic than in WT poplars; at 72 h, it reached a maximum in WT poplars, which differed significantly from that observed in transgenic poplars (Fig. 9H). PR XM_024582168.1 was upregulated after inoculation, with maximum expression observed in both transgenic and WT poplars at 72 h; these expression peaks were 120 and 108 times that of the control, respectively. However, PR XM_024582168.1 expression was 4 times higher in transgenic than in WT poplars at 48 h (Fig. 9I). PR XM_002324451.3 expression was downregulated within 72 h in WT poplars, but within 1–24 h in transgenic poplars, and returned to the original level at 48 h, and was again downregulated (Fig. 9J). The expression levels of PR XM_002319041.3 and PR XM_006386317.2 decreased within 72 h in both transgenic and WT poplars (Fig. 9K,L). PR XM_002313896.3 expression decreased and then increased in transgenic poplars, becoming 34% higher than that in WT poplars at 48 h, and then reaching a maximum at 72 h (Fig. 9M). These changes in the expression of PR genes and those related to SA synthesis after inoculation suggest that the SA signaling pathway may be involved in the poplar induction response to Septotis populiperda.

Materials and Methods

Bacterial strains, plants, fungi, vectors, adipose cells, and enzyme reagents

E. coli TOP10 strains were used for subcloning and E. coli BL21 (DE3) was used as the expression strain. A PET-32a plasmid containing BamHI and NotI restriction sites was purchased from Life Sensors (Malvern, PA, USA) for use as a prokaryotic expression vector, and PGWB9 (accession number AB289772) was used to construct the plant expression vector. Leaf disks from Populus deltoides × Populus euramericana ‘Nanlin895’ seedlings were prepared for transformation and precultured in 1/2 Murashige and Skoog (MS) medium. A. tumefaciens strain EHA105 was purchased from the Solarbio Bioengineering Institute (Beijing, China) and used to mediate poplar transformation. Aspergillus niger, Alternaria Nees, Mucor corymbifer, Marssonina populi, Rhizopus sp., and Neurospora crassa were obtained from the Institute of Microbiology at the Chinese Academy of Sciences. S. populiperda cells were kindly provided by Professors Zhu, respectively, at Nanjing Forestry University. BamHI and NotI restriction enzymes and T4 DNA ligase were purchased from Roche (Basel, Switzerland). A Gateway homologous recombination kit was purchased from Invitrogen (Carlsbad, CA, USA). All other reagents were purchased from the Nanjing Jiancheng Bioengineering Institute (Nanjing, China).

RNA isolation, reverse-transcription (RT)-PCR and open reading frame (ORF) cloning

Total RNA was extracted using a plant-specific RNA kit (Invitrogen) according to the manufacturer’s instructions. First-strand cDNA was synthesized from RNA isolated from P. trichocarpa using M-MLV reverse transcriptase. Primers (Supplement Table 1) were designed and synthesized for defensin ORF cloning, the PCR system includes 2 µL forward and reverse primers, 2.0 µL cDNA as template, 5.0 µL 10× PCR buffer (Mg²⁺ plus), 1 µL 10 mM dNTPs, 0.5 µL rTaq DNA polymerase (Takara, Japan) and the ddH₂O was to a constant volume up to 50 µL. Also, the PCR reaction was performed as follows: 95 °C for 10 min, 35 cycles of 95 °C for 30 s, 58 °C for 30 s, and 72 °C for 30 s and, finally, 72 °C for 10 min. Also the PtDef PCR product was purified (Takara, Japan) and inserted into the PEASY-T3 plasmid (TransGen Biotech, China). Subsequently, the positive clones were sequenced using the M13 primers. The nucleotide sequence, deduced amino acid sequence, and ORF of defensin from P. trichocarpa were analyzed using the ExPASy online package (http://www.expasy.org/translate/). We then calculated the molecular weight (Mw) of defensin and predicted its isoelectric point (pI) using the ExPASy pI/Mw tool (http://www.expasy.org/tools/pi_tool.html).

Construction and expression of the recombinant plasmid

Primers with BamHI and NotI restriction sites were designed and synthesized (Supplement Table 1), the procedure of PCR was performed as below description and the PtDef product fused BamHI and NotI restriction sites obtain. Then, the PtDef PCR product and PET-32a plasmid were ligated together using the same sticky end after double digestion, and the recombinant plasmid mixture was transformed into E. coli TOP10 cells. Subsequently, PCR was used to identify the positive clones and the positive clones were sequenced using the T7 promoter primers.

The recombinant PET-32a-PtDef plasmid was transformed into E. coli BL21 (DE3). Positive colonies cultured in 1 L of lysogeny broth (LB) medium containing ampicillin were induced with 1.0 mM isopropyl-β-d-1-thiogalactopyranoside (IPTG) followed by incubation at 25 °C for 8 h. The uninduced solution, induced solution, supernatant, and sediment were analyzed using 12% sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE). Western blotting was performed according to the manufacturer’s instructions (Zhongshan Biotechnique, Beijing, China).

The fusion label thioredoxin (Trx) can help proteins fold correctly during the process of protein formation, to improve the expression of soluble proteins³⁸. Therefore, thioredoxin fusion is among the most commonly used recombinant protein expression systems. TrxA–PtDef fusion protein purification was conducted using a nickel–nitrilotriacetic acid (Ni–NTA)-chelating column. Purified TrxA–PtDef fusion protein was incubated in the presence of an enterokinase (10 U) at 25 °C for 16 h. The solution was reapplied to the Ni column containing Ni–iminodiacetic acid (IDA) resin to remove His-tagged TrxA. The elution liquid and washing solution were analyzed using 4–20% gradient gels (Mini-R/PROTEAN TGX Gels; Bio-Rad, Hercules, CA, USA).

Antibacterial activity and antifungal sensitivity assays

We performed an agar well diffusion assay to analyze the antibacterial activity of the purified PtDef protein. We analyzed the activity of the PtDef protein against E. coli K12D31 and A. tumefaciens EHA105 using the dilution method and configured seven gradient PtDef protein solutions. The microbial growth curve method was used to analyze the antibacterial activity of the PtDef protein, with the following steps: first, the cells were activated and cultured overnight; 80 μL fresh agrobacteria or bacteria solution was then added to 3 mL of LB growth medium on the next day. When the optical density at 600 nm (OD₆₀₀) reached 0.6, we added 100 μL of purified PtDef solution at a concentration gradient (1.5–96 μg) to 3 mL LB growth medium (three replicates per group). Finally, the OD₆₀₀ value was determined at the experimental point. We used 3 mL of LB growth medium without purified PtDef protein as a negative control (three replicates per group). We assessed antibacterial activity against A. tumefaciens EHA105 incubated at 28 °C and 220 rpm by determining the OD₆₀₀ value after 2, 4, 6, 8, 10, 12, 14, and 16 h. We assessed the growth of E. coli K12D31 incubated at 37 °C and 220 rpm by measuring the OD₆₀₀ after 1, 2, 3, 4, 5, 6, 7, and 8 h.

We analyzed antifungal activity by incubating six fungal species (Aspergillus niger, Alternaria Nees, Mucor corymbifer, Marssonina populi, Rhizopus sp., and Neurospora crassa) on potato dextrose agar (PDA) medium. We then dissolved 120 μL of purified PtDef protein in 0.9% NaCl (pH 7.0) to final concentrations of 40, 20, 10, 5, 2.5, 1.25, and 0.625 μg/mL and added these solutions to PDA medium plates (three replicates per experiment). As a negative control, we added 0.9% NaCl (pH 7.0) to the plates (three replicates per experiment). All plates were incubated at 23 °C for 72 h until mycelial growth was observed as pink, crescent-shaped samples. Agar diffusion was performed to determine antifungal activity³⁹.

Purified PtDef protein as an alternative to cefotaxime in the formation of transgenic poplars

Several methods have been used to inhibit the growth of A. tumefaciens EHA105 in transgenic operations; examples include cleaning leaves with MS liquid medium containing 200 mg/L cefotaxime and adding 200 mg/L cefotaxime during rooting medium screening. Generally, if the growth of A. tumefaciens EHA105 can be effectively suppressed during rooting medium screening, the success rate of transgenic procedures will be improved. The results of our defensin characterization research indicate that purified PtDef protein also substantially inhibits A. tumefaciens EHA105 growth. Therefore, we used defensin instead of cefotaxime for the transgenic processes in this study. Wild-type (WT) poplars (Populus deltoides × Populus euramericana ‘Nanlin895’) were selected for transgenic analysis using an efficient Agrobacterium-mediated transformation system⁴⁰. In this experiment, 100 mg/L purified PtDef protein and 200 mg/L cefotaxime were added to the growth culture medium.

A. tumefaciens growth in rooting medium can lead to an increase in the production of reactive oxygen species (ROS), in turn causing oxidative damage to plant cells. Plant protective mechanisms include the production of secondary metabolites and enzymatic scavengers, including proline (Pro), superoxide dismutase (SOD), peroxidase (POD), malondialdehyde (MDA), ascorbate peroxidase (APX), catalase (CAT), and glutathione S-transferase (GST), which eliminate excessive ROS to maintain the reactive balance in plants cells. Therefore, the application of cefotaxime to rooting medium to inhibit A. tumefaciens growth also limits ROS accumulation. To investigate whether purified PtDef protein could similarly influence ROS accumulation, we established three treatment groups: rooting medium containing 100 mg/L purified PtDef protein as the experimental group, pure rooting medium as a negative control, and rooting medium containing 200 mg/L cefotaxime as a positive control. We compared the MDA, Pro, POD, and SOD contents of leaves grown within each treatment, and used a microplate reader (Bio-Rad) to detect activity levels of

PtDef expression in various tissues in response to abiotic stress

To investigate PtDef transcription in different tissues, total RNA was isolated from mature leaves, young leaves, petioles, roots, and the upper and lower regions of stems of P. trichocarpa. P. trichocarpa seedlings treated with 200 mM NaCl, 200 μm abscisic acid (ABA), 200 μm SA, 200 μm JA, and 2 mM H₂O₂ were sampled at 0, 2, 4, 6, 8, 12, 24, and 48 h, and those subjected to 4 °C cold stress and 10% PEG 6000 were collected after 1, 2, 3, 4, 5, 6, and 7 days. In addition, a group of P. trichocarpa seedlings were wounded with S. populiperda and sampled at 0, 1, 3, 6, 9, 12, 24, 48, and 72 h. Following these treatments, leaves were immediately subjected to RNA extraction using a plant-specific RNA kit (Invitrogen). The housekeeping gene actin (XM_006370951.1) was used as an internal control for each reaction^41,42,43,44. Triplicate measurements were used to determine the mean values for each parameter.

Overexpression plasmid construction and transformation

Coding sequences of the PtDef gene were inserted into the pGWB9 vector using the homologous recombination method according to the manufacturer’s instructions (Invitrogen); the primers are listed in Supplement Table 1. We used the freeze–thaw method⁴⁵ to introduce the recombinant plasmid pGWB9-PtDef into A. tumefaciens EHA105. Genetic transformation of the PtDef gene can be achieved by manipulation as previously described⁴⁰; however, we modified this process for the present study, adding the PtDef protein as an exogenous additive to rooting culture medium to inhibit the growth of A. tumefaciens EHA105. We conducted RT-PCR and qRT-PCR analyses, and collected the putative transgenic lines to confirm that the PtDef gene had been successfully inserted into chromosomes and was stably expressed in cells. Regenerated shoots from the transgenic lines were recovered and transferred to half-strength MS rooting medium.

Transplantation and growth comparison of transgenic and WT poplars

WT and transgenic poplars were obtained during the same growth period from subculture on half-strength MS medium and transferred to a mixture of equal quantities of soil, sterilized peat, and perlite (7:2:1) in a greenhouse. WT and transgenic poplar were cultured under long-day conditions (16-h light/8-h darkness). 23 °C and 74% humidity were kept constant during transplantation and plants were watered daily.

Plant hormones are small, simple molecular organic compounds with diverse and complex physiological effects. Plant hormones influence cell division, elongation, and differentiation to regulate plant germination, rooting, flowering, fruiting, sex determination, dormancy, and abscission, among other processes. To explore differences in the growth of WT and transgenic poplars, expression levels of auxin (IAA), gibberellin (GA), and ABA-related genes were measured using qRT-PCR analysis. To calculate standard curves for ABA, GA, and IAA standard solutions, we conducted high-performance liquid chromatography–tandem mass spectrometry (HPLC–MS/MS; Agilent 1290 HPLC system, Agilent Technologies, Santa Clara, CA, USA; QTRAP 6500 MS/MS system, SCIEX, Framingham, MA, USA). These curves were then used to compare the content of these phytohormones between transgenic and WT poplars. A concentration gradient of 0.1, 0.2, 0.5, 2, 5, 20, 50, and 200 ng/mL was used to prepare standard solutions of IAA, ABA, and GA with methanol (containing 0.1% formic acid). Points negatively affecting linearity were removed from the plot of the scalar equation. Liquid phase conditions were as follows: Poroshell 120 SB-C18 reversed-phase column (2.1 mm × 150 mm, 2.7 μm; Agilent); column temperature 30 °C; injection volume 2 L; mobile phase A:B = (methanol/0.1% formic acid):(water/0.1% formic acid); elution gradient 0–1 min: A = 20%, 1–9 min: A increased to 80%, 9–10 min: A = 80%, 10–10.1 min: A decreased to 20%, N, A = 20%). The mass spectrum conditions were as follows: curtain gas 15 psi, spray voltage 4500 V, atomizing gas pressure 65 psi, auxiliary gas pressure 70 psi, and atomizing temperature 204 °C.

To further explore quantitative differences between WT and transgenic poplars, we calculated photosynthesis-related indices, including net photosynthesis (Pn), intercellular CO₂ concentration (Ci), transpiration (Tr), and stomatal conductance (Gs) using a portable photosynthetic apparatus (Li6400; LI-COR Biosciences, Lincoln, NE, USA).

JA, SA, pathogenesis-related (PR), and lipid-transfer protein (LTP) gene expression and JA and SA content in transgenic and WT poplars

SA and JA are important endogenous signaling molecules that regulate plant disease resistance signaling pathways. Plants activate the expression of defense-related genes to improve resistance to biotic stress⁴⁶. SA can induce plant resistance to pathogens and induce the expression of plant PR genes, further inducing SAR⁴⁷. Non-specific LTPs (nsLTPs), which are also categorized as PR proteins, can inhibit the growth of pathogenic fungi and induce systemic resistance^48,49. JA plays an important role as a plant hormone and signal compound in the processes of plant growth and development, particularly against biotic and abiotic stresses. JA can accumulate rapidly when plant tissues are attacked by pathogenic bacteria or insects; therefore, it is a key signaling molecule for ISR⁵⁰.

Isochorismate synthase (ICS) and phenylalanine ammonia lyase (PAL) are important genes in the plant SA synthesis pathway; they are responsible for the conversion of branched acid and phenylalanine into isobranched acid and trans-cinnamic acid, respectively. Lipoxygenase (LOX), allene oxide cyclase (AOC), and fatty acid hydroperoxide lyase (HPL) are the corresponding genes for JA synthesis. Therefore, in this study, we determined JA and SA contents using an HPLC–MS/MS system. We also used qRT-PCR to detect transcript levels of the following PRs and LTPs: PR XM_006379094.2, PR XM_024582168.1, PR XM_002324451.3, PR XM_002319041.3, PR XM_006386317.2, PR XM_002313896.3, LTP XM_002312093.3, LTP XM_002314714.3, LTP XM_002322032.3, LTP XM_002315155.3, and LTP EF144767.1.

HPLC–MS/MS analysis was performed to create standard curves for the JA and SA contents in transgenic and WT poplars, using the same method and concentration gradient used to prepare the standard curves for phytohormones.

Pathogen inoculation and disease response assay

S. populiperda was selected to analyze poplar disease response. Pathogens grown at 23 °C on PDA medium were activated for 1 week. Leaves from 4-month-old transgenic and WT poplars were punctured using a 5-mL syringe needle and inoculated with a small amount of PDA containing the pathogen or PDA as a negative control. Pathogenic soft rot symptoms were evaluated periodically. The maximal lesion size (diameter, mm) on leaves was recorded at 24–72 h as previously described⁵¹, and the degree of leaf damage was assessed in WT and transgenic poplars. The time of conidia appearance was determined in both groups by microscopy. Each experiment was repeated at least three times.

We performed in vitro assays of antifungal activity as described by Yuan et al.⁵². Leaves sampled from transgenic and WT poplars were used to extract total proteins according to the manufacturer’s instructions (Nanjing Jiancheng Bioengineering Institute). Total proteins were dissolved in 0.9% NaCl (pH 7.0) to a final concentration of 200 μg/mL and added to PDA medium plates (three replicates per experiment). We used 0.9% NaCl (pH 7.0) as a negative control (three replicates per experiment). All plates were incubated at 23 °C for 72 h until mycelial growth was observed. Extraction was performed on transgenic and WT poplar leaves using 70% acetone in liquid nitrogen. The crude extract was centrifuged and treated with ultrasound for 30 min; the residue was then re-extracted twice and the supernatant was mixed with chloroform. The same dose of crude extract was added to PDA medium, inoculated with S. populiperda, and cultured at 23 °C for 4 days. S. populiperda growth was then observed.

Discussion

Like animals, plants have an intrinsic defense system. When plants are infected by external pathogens, their delicate immune defense systems are activated. This reaction is generally referred to as systemic induction of resistance in plants and confers broad-spectrum resistance to a range of unrelated pathogens. A large number of resistance compounds are synthesized in plants; antibacterial proteins including defensins are also induced to prevent further propagation and expansion of pathogenic bacteria^3,4,11,53,54. Plant defensins are a class of cationic, alkaline, Cys-rich polypeptides with small Mw (about 5 kD, consisting of 45–54 amino acids) and a complex 3D structure. There are eight conservative Cys structures within the primary structure of plant defensins, forming four pairs of intrachain disulfide bonds. These four pairs of disulfide bonds are usually matched by Cys1–Cys8, Cys2–Cys5, Cys3–Cys6, and Cys4–Cys7. The other amino acid sequences are not conservative, but their advanced structures are very similar^55,56. For example, the solution structure of a defensin (RsAFP1) isolated from Raphanus sativus was elucidated by nuclear magnetic resonance; it consists of three reverse-parallel β-sheets (Lys2 Arg6, His33 Tyr38, and His43 Pro50) and an α-helix (Asn18 Leu28), which form a very stable β–β structure with the help of four disulfide bonds⁵⁷. The solution structures of defensins in Pisum sativum and Vigna radiata have also been elucidated. The basic structure of PsDef is an α-helix and three reverse-parallel β-sheets^58,59. In this study, the predicted Mw of the PtDef protein (8.59 kD) was greater than that of the identified defensin protein. Through multiple sequence alignment, the N-terminal of defensins was relatively conservative aside from the eight conserved Cys structures. We also separately predicted the 3D structures of defensin proteins from Arabidopsis and Populus trichocarpa, and determined that the defensin consists of three reverse-parallel β-pleated sheets and an α-helix. These findings further demonstrate the conservativeness of defensins within their 3D structure.

Most plant defensins function to inhibit fungal growth, with half-maximal inhibitory concentration (IC₅₀) values depending on the fungal species and the source of the plant defensin. For instance, the defensin from mustard (WT1) was expressed in Nicotiana benthamiana, and the protein purified by affinity chromatography. The IC₅₀ of WT1 against Magnaporthe grisea and Botrytis cinerea was 5 and 20 μg/mL, respectively. Meanwhile, the IC₅₀ of RsAFP2, a defensin from radish seeds, against M. grisea was 0.1 μmol/L⁹. Besides their antifungal function, plant defensins also exhibit antibacterial activity. WT1 showed no significant inhibitory effect on Pseudomonas aeruginosa at a concentration of 10 μg/mL; however, when the WT1 concentration reached 20 μg/mL, it displayed a weak inhibitory effect on bacterial growth. In the current study, we first cloned the full-length genome of a defensin from P. trichocarpa, and then successfully obtained defensin proteins using a prokaryotic expression system and solubilizing tag (TrxA) on the PET-32a vector. Through bacteriostasis circle analysis, we found that PtDef inhibited the growth of two bacterial species and six tested fungi. PtDef inhibited the activity of A. tumefaciens EHA105 more strongly than that of E. coli K12D31. Antifungal activity also varied among target species; PtDef exhibited the highest inhibitory effect against Marssonina populi, and the lowest against Rhizopus sp. Antibiotics are widely used in transgenic processes, and their significant antibacterial effects can improve the efficiency of transgenic operations in plants. However, their use increases antibiotic resistance in some bacteria. In recent years, the search for suitable analogs to replace antibiotics has become a focus of research. Defensin is an ideal alternative to antibiotics due to its unique antibacterial mechanism and the inability of bacteria to develop resistance. In this study, PtDef strongly inhibited the growth of A. tumefaciens EHA105. To prevent the growth of agrobacteria, exogenous cefotaxime is typically applied to the rooting medium during the genetic modification process; therefore, we wondered whether PtDef could serve as an alternative to cefotaxime in the genetic modification process. We found that PtDef exhibited better anti-Agrobacterium activity than cefotaxime, and could reduce the need for medium replacement. Moreover, quantitative analysis showed that PtDef was less toxic than cefotaxime. Based on these results, the PtDef protein can be used instead of cefotaxime to inhibit A. tumefaciens EHA105 growth during the genetic modification process.

Previous studies have shown that defensins influence plant growth. For example, RsAFP2, MsDef2, and MsDef1 have been reported to have negative effects on plant growth^60,61, whereas MtDef has been shown to have no effect on plant growth⁶². In the current study, when PtDef was overexpressed in poplar, there was no difference in phenotype between 4-month-old transgenic and WT poplars. However, molecular detection revealed significant differences between poplar groups. The expression of ABA- and GA-related genes was significantly higher in transgenic lines, but that of IAA-related genes was lower in transgenic lines. Plants lack the ability of active avoidance; however, they have evolved complex defense mechanisms in response to infection by disease. In recent years, research interest in the cloning, expression, and function of defensin has grown. Through in-depth studies of the mechanisms of plant-acquired resistance, researchers have found that defensins play an important role in plant resistance to pathogens. For example, overexpression of the defensin gene in transgenic Oryza sativa led to its improved resistance to M. grisea⁶³. Overexpression of the defensin gene in transgenic potato, led to increased resistance to B. cinerea⁶⁴. A defensin gene from Medicago sativa was transferred into a tomato genome, resulting in good resistance to Fusarium wilt⁶⁵. The radish seed defensin gene Rs2AFP2 was transferred into tobacco and tomato, significantly enhancing their resistance to Alternaria longipes⁹. Overexpression of the alfAFP peptide, isolated from seeds of M. sativa, in transgenic potato plants provided significant resistance to Verticillium dahlia⁶⁶. Overexpression of RsAFP2 in rice showed resistance to Magnaporthe oryzae and Rhizoctonia solani²⁶. Overexpression of mustard defensins in tobacco also led to resistance to Fusarium moniliform²⁵. However, transgenic plants incorporating defensin genes generally do not attain complete resistance to pathogenic fungi. To enhance resistance, other resistance genes can be transferred into plant cells in combination with the defensin, creating a synergistic effect⁶⁷. Overexpression of two different AMPs from Bombyx mori in Arabidopsis enhanced its disease resistance⁶⁸. An accumulation of antimicrobial genes in potato transgenic plants increased their resistance to bacterial and fungal pathogens⁶⁹. In the current study, following inoculation with S. populiperda, the rate of disease occurrence was slower in transgenic lines than in WT poplars. For example, at 4 days post-inoculation, cells in leaves of the transgenic lines were able to maintain their integrity, whereas leaf cells of WT poplars were loosely aggregated and leaked cellular fluid. WT poplar leaves also developed conidia on day 2 post-inoculation, whereas the transgenic lines produced conidia one day later. Total protein and crude extracts from the transgenic lines also slowed the growth of S. populiperda in vitro.

JA and SA are the main hormones involved in plant resistance to disease invasion³¹. SA is an endogenous signaling molecule that activates plant HR and SAR, and induces the expression of many disease-related proteins³². Increased levels of endogenous SA enhance PR gene expression and plant resistance; exogenous SA application can stimulate the transcription of PR genes, increasing disease resistance⁷⁰. We analyzed the expression of SA-related genes (PAL and ICS) and found that overexpression of the PtDef gene significantly increased that of the ICS gene. Based on the results of HPLC–MS/MS analysis, we found that SA content was significantly higher in PtDef transgenic poplar lines than in WT poplars. This result indicates that endogenous SA content increases in transgenic poplars, thereby inducing the expression of PR and LTP genes to enhance disease resistance in poplars. LOX, AOC, and HPL genes are related to JA synthesis; when plants are attacked by disease, genes that encode JA biosynthetase are upregulated⁷¹. In this study, the overexpression of defensin genes in poplar altered the expression of genes related to JA and SA synthesis. Two LOX genes and two AOC genes were detected in WT poplars and transgenic lines. One LOX gene (XM_002318978.3) and one AOC gene (XM_024605953.1) were up-regulated in transgenic lines, but those of another LOX gene (XM_002314512.3) and another AOC gene (XM_002305915.3) were downregulated. The expression of HPL gene XM_002321100.3 was significantly higher in transgenic lines.

HPLC–MS/MS also showed that overexpression of the PtDef gene improved JA content in poplars. JA is a key endogenous growth regulator of the plant defense response to pathogenic microorganisms, and plays an important role in signal transduction of the system defense response to increase plant disease resistance. We observed a synergistic reaction⁷² between the SA signaling pathway and the JA signaling pathway in poplar; both of these pathways can induce resistance to pathogens, and the combination of SAR and ISR improves the plant’s ability to control disease⁷³. Hormone signaling molecules can form a complex defense system in the face of pathogen infection, including the distribution and effective regulation of components, to enhance the efficiency of the plant defense response. We also examined changes in the expression of PR genes and SA- and JA-related genes after inoculation with pathogens, and observed significant differences in expression levels between transgenic and WT poplars. This result indicates that the SA and JA signal transduction pathway in poplar displays synergistic and antagonistic effects similar to those in Arabidopsis^72,74. Our study of the SA and JA signal transduction pathways in poplars represents a preliminary exploration; further study is required to elucidate their interaction.

Conclusion

In conclusion, we cloned a full-length gene encoding PtDef, and investigated its expression patterns in different tissues of P. trichocarpa and under different stresses. We also purified the PtDef protein based on prokaryotic expression and purification technology; purified PtDef inhibited the growth of E. coli K12D31, A. tumefaciens EHA105, and six tested fungal species. Purified PtDef was a suitable alternative to cefotaxime as an exogenous additive to rooting culture medium, as shown by its inhibition of A. tumefaciens EHA105 production in rooting culture medium. Such an application would improve the efficiency of obtaining transgenic plants. Overexpression of PtDef significantly increased poplar resistance to S. populiperda based on phenotype analysis and the expression of PR and LTP genes, as well as JA- and SA-related genes. Finally, we performed a preliminary exploration of the SA and JA signaling pathways for enhanced resistance in transgenic poplars, and observed synergistic or antagonistic effects, which together regulate disease resistance in poplars.

References

Koczulla, A. R. & Bals, R. Antimicrobial peptides: current status and therapeutic potential. Drugs. 63, 389–406 (2003).
Article CAS PubMed Google Scholar
Keymanesh, K., Soltani, S. & Sardari, S. Application of antimicrobial peptides in agriculture and food industry. World J Microb Biotechnol. 25, 933–944 (2009).
Article Google Scholar
Hultmark, D., Steiner, H., Rasmuson, T. & Boman, H. G. Insect immunity Purification and properties of three inducible bactericidal proteins from hemolymph of immunized pupae of Hyalophora cecropia. Eur J Biochem. 106, 7–16 (1980).
Article CAS PubMed Google Scholar
Cociancich, S., Bulet, P., Hetru, C. & Hoffmann, J. A. The inducible antibacterial peptides of insects. Parasitol Today. 10, 132–139 (1994).
Article CAS PubMed Google Scholar
Helmerhorst, E. J. et al. Synthetic histatin analogues with broad-spectrum antimicrobial activity. Biochem J. 326, 39–45 (1997).
Article CAS PubMed PubMed Central Google Scholar
Gallo, R. L. et al. Identification of CRAMP, a cathelin-related antimicrobial peptide expressed in the embryonic and adult mouse. J Biol Chem. 272, 13088–13093 (1997).
Article CAS PubMed Google Scholar
Dorschner, R. A. et al. Cutaneous injury induces the release of cathelicidin anti-microbial peptides active against group A streptococcus. J Investig Dermatol. 117, 91–97 (2001).
Article CAS PubMed Google Scholar
Broekaert, F. R., Terras, B. P., Cammue, R. W. & Osborn, R. W. Plant defensins: novel antimicrobial peptides as components of the host defense system. Plant Phys. 108, 1353–1358 (1995).
Article CAS Google Scholar
Terras, F. R. G. et al. Small cysteine-rich antifungal proteins from radish: their role in host defense. Plant Cell. 7, 573–588 (1995).
CAS PubMed PubMed Central Google Scholar
Thomma, B. P., Cammue, B. P. A. & Thevissen, K. Plant defensins. Planta. 216, 193–202 (2002).
Article CAS PubMed Google Scholar
Lay, F. T. & Anderson, M. A. Defensins—components of the innate immune system in plants. Curr Protein Pept Sci. 6, 85–101 (2005).
Article CAS PubMed Google Scholar
Van, der, W. N. L. & Anderson, M. A. Plant defensins: common fold, multiple functions. Fungal Biol Rev. 26, 121–131 (2013).
Article Google Scholar
Lacerda, A. F., Vasconcelos, E. A., Pelegrini, P. B., Grossi, de & Sa, M. F. Antifungal defensins and their role in plant defense. Front Microbiol. 5, 116 (2014).
Article PubMed PubMed Central Google Scholar
Aerts, A. M., François, I. E. J. A., Cammue, B. P. A. & Thevissen, K. The mode of antifungal action of plant, insect and human defensins. Cell Mol Life Sci. 65, 2069–2079 (2008).
Article CAS PubMed Google Scholar
Games, P. D. et al. Isolation, characterization and cloning of a cDNA encoding a new antifungal defensin from Phaseolus vulgaris L. seeds. Peptides. 29, 2090–2100 (2008).
Article CAS PubMed Google Scholar
Medeiros, L. N. et al. Backbone dynamics of the antifungal PsD1 pea defensin and its correlation with membrane interaction by NMR spectroscopy. Biochem Biophy Acta. 2, 105–113 (2010).
Article CAS Google Scholar
Mello, E. O. et al. Antifungal activity of PvD1 defensin involves plasma membrane permeabilization, inhibition of medium acidification and induction of ROS in fungi cells. Curr Microbiol. 62, 1209–1217 (2011).
Article CAS PubMed Google Scholar
Bharucha, J. P. et al. Human beta-defensin 2 and 3 inhibit HIV in primary macrophages: implications for HIV infection of the CNS. J Neurobiol. 19, S13–S14 (2013).
Google Scholar
Bals, R., Goldman, M. J. & Wilson, J. M. Mouse beta-defensin 1 is a salt-sensitive antimicrobial peptide present in epithelia of the lung and urogenital tract. Infect Immun. 66, 1225–1232 (1998).
CAS PubMed PubMed Central Google Scholar
Mizukawa, N. et al. Levels of human defensin-1, an antimicrobial peptide, in saliva of patients with oral inflammation. Oral Surg Oral Med Oral Pathol Oral Radiol Endod. 87, 539–543 (1999).
Article CAS PubMed Google Scholar
Yang, D. et al. β-defensins: linking innate and adaptive immunity through dendritic and T cell CCR6. Science. 286, 525–528 (1999).
Article CAS PubMed Google Scholar
Zhang, G. et al. Cloning and characterization of the gene for a new epithelial β-defensin: genomic structure, chromosomal localization, and evidence for its constitutive expression. J Biolog Chem. 274, 24031–24037 (1999).
Article CAS Google Scholar
Davis, A., Renner, K., Sprague, C., Dye, L. & Mutch, D. Integrated Weed Management: “One Year’s Seeding”. Michigan State Univ Extens Bull. E-2931, 112 (2005).
Google Scholar
De, Coninck, B. et al. Arabidopsis thaliana plant defensin AtPDF1.1 is involved in the plant response to biotic stress. New Phytol. 187, 1075–1088 (2010).
Article PubMed CAS Google Scholar
Anuradha, T. S., Divya, K., Jami, S. K. & Kirti, P. B. Transgenic tobacco and peanut plants expressing a mustard defensin show resistance to fungal pathogens. Plant Cell Reports. 27, 1777–1786 (2008).
Article CAS Google Scholar
Jha, S., Tank, H. G., Prasad, B. D. & Chattoo, B. B. Expression of Dm-AMP1 in rice confers resistance to Magnaporthe oryzae and Rhizoctonia solani. Transgenic Res. 18, 59–69 (2009).
Article CAS PubMed Google Scholar
Wu, J. et al. A plant defensin gene from Orychophragmus violaceus can improve Brassica napus resistance to Sclerotinia sclerotiorum. African J Biotech. 8 (2009).
Portieles, R. et al. NmDef02, a novel antimicrobial gene isolated from Nicotiana megalosiphon confers high-level pathogen resistance under greenhouse and field conditions. Plant Biotech J. 8, 678–690 (2010).
Article CAS Google Scholar
Stotz, H. U., Spence, B. & Wang, Y. A defensin from tomato with dual function in defense and development. Plant Mol Biol. 71, 131e143 (2009).
Article CAS Google Scholar
Von, D. et al. Tuning the herbivore-induced ethylene burst: the role of transcript accumulation and ethylene perception in Nicotiana attenuata. Plant J. 51, 293–307 (2010).
Google Scholar
Coll, N. S., Epple, P. & Dangl, J. L. Programmed cell death in the plant immune system. Cell Death Differ. 18, 1247–1256 (2011).
Article CAS PubMed PubMed Central Google Scholar
Bari, R. & Jones, J. D. G. Role of plant hormones in plant defence responses. Plant Mol Biol. 69, 473–488 (2009).
Article CAS PubMed Google Scholar
Fu, Z. Q. et al. NPR3 and NPR4 are receptors for the immune signal salicylic acid in plants. Nature. 486, 3153 (2012).
Article CAS Google Scholar
Seyfferth, C. & Tsuda, K. Salicylic acid signal transduction: the initiation of biosynthesis, perception and transcriptional reprogramming. Front Plant Sci. 5, 697 (2014).
Article PubMed PubMed Central Google Scholar
Michel, G., Tonon, T., Scornet, D., Cock, J. M. & Kloareg, B. Central and storage carbon metabolism of the brown alga Ectocarpus siliculosus: insights into the origin and evolution of storage carbohydrates in eukaryotes. New Phytol. 188, 67–81 (2010).
Article CAS PubMed Google Scholar
Yan, C. & Xie, D. Jasmonate in plant defence: sentinel or double agent? Plant Biotech J. 13, 1233–1240 (2015).
Article Google Scholar
Lorenzo, O. & Solano, R. Molecular players regulating the jasmonate signalling network. Curr Opinion Plant Biol. 8, 532–540 (2005).
Article CAS Google Scholar
Zhou, L., Zhao, Z., Li, B., Cai, Y. & Zhang, S. TrxA mediating fusion expression of antimicrobial peptide CM4 from multiple joined genes in Escherichia coli. Protein Expre Purif. 64, 225–230 (2009).
Article CAS Google Scholar
Lehrer, R. I., Rosenman, M., Harwig, S. L., Jackson, R. & Eisenhauer, P. Ultrasensitive assays for endogenous antimicrobial polypeptides. J Immunol Methods. 137, 167–73 (1991).
Article CAS PubMed Google Scholar
Movahedi, A. et al. Expression of the chickpea CarNAC3 gene enhances salinity and drought tolerance in transgenic poplars. Plant Cell Tissue Organ Cult. 120, 141–154 (2015).
Article CAS Google Scholar
Zhang, J. et al. Genome-wide analysis of the populus hsp90 gene family reveals differential expression patterns, localization, and heat stress responses. BMC Genomics. 14 (2013).
Article CAS PubMed PubMed Central Google Scholar
Movahedi, A. et al. Functional analyses of PtRDM1 gene overexpression in poplars and evaluation of its effect on DNA methylation and response to salt stress. Plant physiology and biochemistry. 127, 64–73 (2018).
Article CAS PubMed Google Scholar
Movahedi, A., Zhang, J., Yin, T. & Zhuge, Q. Functional analysis of two orthologous NAC genes, CarNAC3, and CarNAC6 from Cicer arietinum, involved in abiotic stresses in poplar. Plant molecular biology reporter. 33, 1539–1551 (2015).
Article CAS Google Scholar
Movahedi, A. et al. Functional Analyses of PtROS1-RNAi in Poplars and Evaluation of Its Effect on DNA Methylation. Journal of Plant Biology. 61, 227–240 (2018).
Article CAS Google Scholar
Holsters, M. et al. Transfection and transformation of Agrobacterium tumefaciens. Mol Gen Genet. 163, 181e187 (1978).
Google Scholar
Anderson, J. P., Badruzsaufari, E., Schenk, P. M., Manners, J. M. & Desmond, O. J. Antagonistic interaction between abscisic acid and jasmonate–ethylene signaling pathways modulates defense gene expression and disease resistance in Arabidopsis. Plant Cell. 16, 3460–3479 (2004).
Article CAS PubMed PubMed Central Google Scholar
Leon-Reyes, A. et al. Ethylene modulates the role of NONEXPRESSOR OF PATHOGENESIS-RELATED GENES1 in cross talk between salicylate and jasmonate signaling. Plant Phys. 149, 1797 (2009).
Article CAS Google Scholar
Kirubakaran, S. I., Begum, S. M., Ulaganathan, K. & Sakthivel, N. Characterization of a new antifungal lipid transfer protein from wheat. Plant Phys Biochem. 46, 918–927 (2008).
Article CAS Google Scholar
Lee, S. B. et al. Disruption of glycosylphosphatidylinositol-anchored lipid transfer protein gene altered cuticular lipid composition, increased plastoglobules, and enhanced susceptibility to infection by the fungal pathogen Alternaria brassicicola. Plant Phys. 150, 42–54 (2009).
Article CAS Google Scholar
Wasternack, C. & Kombrink, E. Jasmonates: structural requirements for lipid-derived signals active in plant stress responses and development. ACS Chem Biol. 5, 63–77 (2010).
Article CAS PubMed Google Scholar
Li, C. W. et al. Tomato RAV transcription factor is a pivotal modulator involved in the AP2/EREBP-mediated defense pathway. Plant Physiology. 156, 213–227 (2011).
Article CAS PubMed PubMed Central Google Scholar
Yuan, L. et al. Molecular cloning and characterization of PtrLAR3, a gene encoding leucoanthocyanidin reductase from Populus trichocarpa, and its constitutive expression enhances fungal resistance in transgenic plants. J Exp Bot. 63, 2513–2524 (2012).
Article CAS PubMed PubMed Central Google Scholar
Boman, H. G. & Hultmark, D. Cell-free immunity in insects. Ann Rev Microbiol. 41, 103–126 (1987).
Article CAS Google Scholar
Kanost, M. R. K. J. et al. Insect haemolymph proteins. Adv Insect Phys. 22, 299–396 (1990).
Article Google Scholar
Cornet, B. et al. Refined three-dimensional solution structure of insect defensin A. Structure. 3, 435–448 (1995).
Article CAS PubMed Google Scholar
García-Olmedo, F., Molina, A., Alamillo, J. M. & Rodríguez-Palenzuéla, P. Plant defense peptides. Peptide Sci. 47, 479–491 (2015).
Article Google Scholar
Fant, F., Vranken, W., Broekaert, W. & Borremans, F. Determination of the three-dimensional solution structure of Raphanus sativus Antifungal Protein 1 by 1H NMR. J Mol Biol. 279, 257–270 (1998).
Article CAS PubMed Google Scholar
Almeida, M. S., Cabral, K., Kurtenbach, E., Almeida, F. C. & Valente, A. P. Solution structure of Pisum sativum defensin 1 by high-resolution NMR: plant defensins, identical backbone with different mechanisms of action. J Mol Biol. 315, 749–757 (2002).
Article CAS PubMed Google Scholar
Liu, Y. J. et al. Solution structure of the plant defensin VrD1 from mung bean and its possible role in insecticidal activity against bruchids. Proteins. 63, 777–786 (2006).
Article CAS PubMed Google Scholar
Allen, A. et al. Plant defensins and virally encoded fungal toxin KP4 inhibit plant root growth. Planta. 227, 331–339 (2008).
Article CAS PubMed Google Scholar
Lay, F. T. et al. The C-terminal propeptide of a plant defensin confers cytoprotective and subcellular targeting functions. BMC Plant Biol. 14, 41 (2014).
Article PubMed PubMed Central CAS Google Scholar
Gomes, V. M. & Carvalho, A. D. O. Plant defensins and defensin-like peptides—biological activities and biotechnological applications. Curr Pharm Des. 17, 4270–4293 (2011).
Article PubMed Google Scholar
Kanzaki, H. et al. Overexpression of the wasabi defensin gene confers enhanced resistance to blast fungus (Magnaporthe grisea) in transgenic rice. Theor Appl Gen. 105, 809–814 (2002).
Article CAS Google Scholar
Khan, R. S., Nishihara, M., Yamamura, S., Nakamura, I. & Mii, M. Transgenic potatoes expressing wasabi defensin peptide confer partial resistance to gray mold (Botrytis cinerea). Plant Biotech. 23, 179–183 (2006).
Article Google Scholar
Abdalla, N. A., Shah, D. M., Abbas, D. & Madkour, M. Stable integration and expression of a plant defensin in tomato confers resistance to fusarium wilt. GM Crops. 1, 344–350 (2010).
Article Google Scholar
Gao, A. G. et al. Fungal pathogen protection in potato by expression of a plant defensin peptide. Nature Biotechnology. 18, 1307–1310 (2000).
Article CAS PubMed Google Scholar
Ntui, V. O. et al. Stable integration and expression of wasabi defensin gene in “Egusi” melon (Colocynthis citrullus l.) confers resistance to Fusarium wilt and Alternaria leaf spot. Plant Cell Reports. 29, 943–54 (2010).
Article CAS PubMed Google Scholar
Xing, H., Lawrence, C. B., Chambers, O., Davies, H. M. & Li, E. Q. Q. Increased pathogen resistance and yield in transgenic plants expressing combinations of the modified antimicrobial peptides based on indolicidin and magainin. Planta. 223, 1024–1032 (2006).
Article CAS PubMed Google Scholar
Rivero, M. et al. Stacking of antimicrobial genes in potato transgenic plants confers increased resistance to bacterial and fungal pathogens. J Biotech. 157, 334–343 (2012).
Article CAS Google Scholar
Dong, J., Chen, C. & Chen, Z. Expression profiles of the Arabidopsis WRKY gene superfamily during plant defense response. Plant Mol Biol. 51, 21–37 (2003).
Article CAS PubMed Google Scholar
Glazebrook, J. Contrasting mechanisms of defense against biotrophic and necrotrophic pathogens. Ann Rev Phytopathol. 43, 205–227 (2005).
Article CAS Google Scholar
Mur, L. A. J., Kenton, P., Atzorn, R., Miersch, O. & Wasternack, C. Outcomes of concentration-specific interactions between salicylate and jasmonate signaling include synergy, antagonism, and oxidative stress leading to cell death. Plant Phys. 140, 249–262 (2006).
Article CAS Google Scholar
Coupe, S. A., Watson, L. M., Ryan, D. J., Pinkney, T. T. & Eaton, J. R. Molecular analysis of programmed cell death during senescence in Arabidopsis thaliana and Brassica oleracea: cloning broccoli LSD1, Bax inhibitor and serine palmitoyltransferase homologues. J Exp Bot. 55, 59–68 (2004).
Article CAS PubMed Google Scholar
Koornneef, A. et al. Kinetics of salicylate-mediated suppression of jasmonate signaling reveal a role for redox modulation. Plant Phys. 147, 1358–1368 (2008).
Article CAS Google Scholar

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Acknowledgements

We thank Prof. Tsuyoshi Nakagawa (Research Institute of Molecular Genetics Shimane University) for providing the pGWB9 plasmid. We gratefully thank Prof Lihua Zhu (Nanjing Forestry University) for providing Septotis populiperda. We also thank Prof. Kees Venema (EEZ, CSIC, and Granada, Spain) for suggestion on 3D structure analysis. This work was supported by the National Key Program on Transgenic Research (2018ZX08020002), the National Science Foundation of China (31570650) and the Priority Academic Program Development of Jiangsu Higher Education Institutions.

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These authors contributed equally: Hui Wei, Ali Movahedi, Chen Xu and Weibo Sun.

Authors and Affiliations

Co-Innovation Center for Sustainable Forestry in Southern China, Key Laboratory of Forest Genetics & Biotechnology, Ministry of Education, College of Biology and the Environment, Nanjing Forestry University, Nanjing, 210037, China
Hui Wei, Ali Movahedi, Chen Xu, Weibo Sun, Lingling Li, Dawei Li & Qiang Zhuge
Jiangsu Provincial Key Construction Laboratory of Special Biomass Resource Utilization, Nanjing Xiaozhuang University, Nanjing, 211171, China
Chen Xu

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Hui Wei
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Ali Movahedi
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Chen Xu
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Weibo Sun
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Lingling Li
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Dawei Li
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Qiang Zhuge
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Contributions

H.W. and A.M. have written the manuscript and designed experiments. H.W., C.X., W.S., L.L. and D.L. have carried out all experiments. A.M. and Q.Z. supervised this manuscript. Q.Z. has funded this manuscript.

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Correspondence to Qiang Zhuge.

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Wei, H., Movahedi, A., Xu, C. et al. Characterization, expression profiling, and functional analysis of a Populus trichocarpa defensin gene and its potential as an anti-Agrobacterium rooting medium additive. Sci Rep 9, 15359 (2019). https://doi.org/10.1038/s41598-019-51762-0

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Received: 09 April 2019
Accepted: 25 September 2019
Published: 25 October 2019
DOI: https://doi.org/10.1038/s41598-019-51762-0

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