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BMC Plant Biology

, 18:357 | Cite as

Enhanced Nicotiana benthamiana immune responses caused by heterologous plant genes from Pinellia ternata

  • Hafiz Muhammad Khalid Abbas
  • Jingshu Xiang
  • Zahoor Ahmad
  • Lilin Wang
  • Wubei DongEmail author
Open Access
Research article
Part of the following topical collections:
  1. Plant-biotic interactions

Abstract

Background

Pinellia ternata is a Chinese traditional medicinal herb, used to cure diseases including insomnia, eclampsia and cervical carcinoma, for hundreds of years. Non-self-recognition in multicellular organisms can initiate the innate immunity to avoid the invasion of pathogens. A design for pathogen independent, heterosis based, fresh resistance can be generated in F1 hybrid was proposed.

Results

By library functional screening, we found that P. ternata genes, named as ptHR375 and ptHR941, were identified with the potential to trigger a hypersensitive response in Nicotiana benthamiana. Significant induction of ROS and Callose deposition in N. benthamiana leaves along with activation of pathogenesis-related genes viz.; PR-1a, PR-5, PDF1.2, NPR1, PAL, RBOHB and ERF1 and antioxidant enzymes was observed. After transformation into N. benthamiana, expression of pathogenesis related genes was significantly up-regulated to generate high level of resistance against Phytophthora capsici without affecting the normal seed germination and morphological characters of the transformed N. benthamiana. UPLC-QTOF-MS analysis of ptHR375 transformed N. benthamiana revealed the induction of Oxytetracycline, Cuelure, Allantoin, Diethylstilbestrol and 1,2-Benzisothiazol-3(2H)-one as bioactive compounds. Here we also proved that F1 hybrids, produced by crossing of the ptHR375 and ptHR941 transformed and non-transformed N. benthamiana, show significant high levels of PR-gene expressions and pathogen resistance.

Conclusions

Heterologous plant genes can activate disease resistance in another plant species and furthermore, by generating F1 hybrids, fresh pathogen independent plant immunity can be obtained. It is also concluded that ptHR375 and ptHR941 play their role in SA and JA/ET defense pathways to activate the resistance against invading pathogens.

Keywords

Durable disease resistance Hypersensitive response Nicotiana benthamiana; non-self-recognition Pathogenesis related genes Pinellia ternata 

Abbreviations

hpi

Hours post infiltration

HR

Hypersensitive response

POD

Peroxidase

PPO

Poly phenol oxidase

PR-genes

Pathogenesis related genes

ptHR

Pinellia ternata hypersensitive response

qRT-PCR

Quantitative reverse transcription PCR

ROS

Reactive oxygen species

SOD

Superoxide dismutase

Background

Non-self-recognition is one of the most important phenomena to initiate the innate immunity in multicellular organisms. It helps to prevent the invasion of pathogens and to maintain the genetic polymorphism of organisms [1]. All kinds of organisms have the ability to recognize foreign DNA, RNA or proteins of invading pathogens. Non-self-recognition is the event of early stages of parasites’ invasion and plays an important role to overcome the infection by pathogens, and it occurs among all organisms as basic characteristics of the cell to cell interaction [2, 3]. In plants, induced disease resistances are triggered by pathogen infection, which is dependent on the non-self-recognition. As a result of non-self-recognition, phytoalexins and other cell wall strengthening materials are produced around the infection site to inhibit the penetration of pathogen [4].

It is considered that the mutual interactions between host and pathogen are the major drivers for their co-evolution. A pathogen can be more virulent in the presence of a variety of hosts instead of single kind of host and vice versa. The way of pathogen attack is the key factor to determine the mechanism of host defense [5]. The activation of defense response in plants includes sudden death of plant cells known as hypersensitive response (HR) [6], a burst of reactive oxygen species (ROS) [7], activation of defense-related genes [8] and production of antimicrobial compounds e.g. phytoalexins [9]. In the non-inoculated portion of plants, plants have the ability to develop systemic resistance in addition to local resistance. This kind of resistance usually divided into two groups: systemic acquired resistance (SAR) and induced systemic resistance (ISR) [10]. Accumulation of salicylic acid (SA) and induction of pathogenesis-related (PR) genes are involved in the establishment of SAR. Moreover, SAR is long lasting and have potential to inhibit the broad spectrum of pathogens such as fungi, bacteria, nematodes and viruses [11]. While on the other hand, ISR is dependent on the plant roots colonization by plant growth promoting bacteria and Jasmonic acid (JA) and ethylene (ET) signaling [12].

For the development of resistant crops, understanding with basic mechanism of host-pathogen interaction is necessary. It is divided into two types, incompatible and compatible interactions leading to accelerating the resistance and susceptibility to certain diseases, respectively. Plant breeders use incompatible interaction as a basic tool for the development of resistant cultivars in the sustainable agricultural system. The activation of incompatible interaction is mainly dependent on the presence of two-layered plant immune systems to protect them against a variety of pathogens. The first layer, pathogen-associated molecular patterns (PAMP) trigger immunity, is activated upon the recognition of the pathogen by PRR (pattern recognition receptors), while the second layer, effector trigger immunity (ETI), is activated by the pathogen effectors (Avr) recognized by R-genes of the host plant [13]. As this R-gene recognizes the specific Avr gene and then initiate the immune response, so it is also known as R-gene mediated immunity [14, 15]. As the R-gene of the host is a basic selection force on corresponding pathogen Avr gene which results in the modification of Avr gene to overcome the effect of old R gene of the host. This host-pathogen evolution can explain that how one cultivar loses its resistance in field [16, 17]. By keeping in view, it is the basic need of time to develop some new and unique strategies to introduce long-term and broad-spectrum resistance against potential pathogens.

Heterosis, also known as hybrid vigor, is the phenomenon which describes the vigor of F1 heterozygous hybrid more than that of their homozygous parents. It can explain the increased plant biomass, yield, fertility, development potential and disease resistance [18]. For the better understanding of heterosis, three different genetic hypothesis, dominance, overdominance and epistasis, have been proposed [18, 19]. Heterosis has been widely used to enhance the yield and productivity of plants [20, 21, 22].

In our study, we proposed that pathogen triggered plant immunity is a specific form and result of non-self-recognition. We also proposed that pathogen infection is a driving force for the formation and evolvement of non-self-recognition. In the evolution of species formation, without pathogen, it might not have non-self-recognition, furthermore, some species boundary might be blur. Non-self-recognition might be used to develop pathogen independent plant resistance. Based on these hypotheses, we designed this research and identified some genes from P. ternata, which can initiate the local and systemic defense responses in N. benthamiana through the mechanism of non-self-recognition. For further characterization, these genes were cloned and transferred into N. benthamiana, and found that they can increase tobacco basal resistances.

Methods

Plant materials and pathogen cultures

P. ternata was collected from Wuhan Botanical Garden, Wuhan, Hubei Province, P. R. China and grown in a controlled growth chamber at 20–26 °C and 14 h/10 h of light/dark conditions. N. benthamiana and Lycopersicon esculentum seeds were germinated on nutrient soil and transplanted individually in pots under controlled growth conditions of 24–28 °C and 16 h/8 h light and dark intervals. Gossypium hirsutum was grown on nutrient soil under controlled conditions at 24–28 °C and 16 h/8 h light and dark intervals. Pectobacterium carotovora was maintained on Luria-Bertani (LB) medium (10 g tryptone, 5 g yeast extract, 10 g NaCl per liter distilled water) at 28 °C in the dark. Phytophthora capsici (LT263), procured from the Key Lab of Crop Disease Monitoring and Safety Control in Hubei Province, Huazhong Agricultural University, Wuhan 430,070, China, was maintained on V8 (100 ml V8 juice, 1 g CaCO3 per liter distilled water) media at 25 °C under dark conditions.

Construction of P. ternata cDNA library

P. ternata was inoculated with P. carotovora for the construction of cDNA library. The tuber of P. ternata was cleaned and soaked into P. carotovora liquid, which was incubated at 28 °C for 24 h before use, for 10 min. Leaf samples were collected at different time intervals (24, 36 and 48 h), frozen in liquid nitrogen and stored at − 80 °C.

Total RNA from collected samples was extracted with the Trizol reagent. After that, the mRNA was purified from total RNA using PolyATtract® mRNA isolation systems (Promega). The cDNA library was created using PrimeScript™ double strand cDNA synthesis kit (TaKaRa) with specific Oligo dT primer (containing Xba I cleavage site). Three pairs of adaptor containing Sac I cleavage sites were added to cDNA library. Then the library was digested with Xba I and Sac I enzyme at the same time. The short fragments were removed by AxyPrep PCR cleanup kit (AxyGEN). Afterwards, the product was ligated into pTRV2Ex vector [23] (Xba I and Sac I enzymes were used to digest vector in advance). The ligation product was subsequently transformed into Trans DH5α E. coli cells. Plasmids of E. coli cells were extracted using EasyPure® Plasmid MiniPrep Kit (TransGen) and then transformed into A. tumefaciens strain EHA105. Individual colonies were picked with toothpicks and incubated at 28 °C for overnight. Colony PCR was performed using primers GF (5’-TACAGGTTACTGAATCACTTGCGCTA-3′) and GR (5’-CCGTAGTTTAATGTCTTCGGGACA-3′) to confirm the cDNA library quality and then saved at − 80 °C.

Functional screening and sequence analysis of cDNA library

For functional screening of cDNA library of P. ternata, Agrobacterium containing pTRV1, pTRV2Ex empty vector, pTRV2Ex::target gene, respectively, were grown overnight at 28 °C in LB containing kanamycin (50 mg/L) and rifampicin (50 mg/L). Agrobacterium cells were collected by centrifugation at 4000 rpm for 10 min, re-suspended in MMA solution (10 mmol/L MgCl2, 10 mmol/L MES, 20 g/L sucrose, 100 mmol/L acetosyringone, PH = 5.6), adjusted to final OD600 = 0.8–1.0 and left at room temperature for 3 h without shaking. The pTRV1 solution and target gene solution were mixed 1:1, and pressure-infiltrated into the leaf of N. benthamiana plant using a 1 mL syringe without a needle. Avr4-Cf4 was used as positive control while MMA buffer and empty vector were used as negative control. Symptom variation of injected leaves was observed and recorded every day.

Colonies repeatedly showing HR symptoms on plants were sequenced by Wuhan AuGCT (http://wh.augct.com/) using GR primer. NCBI (National Center for Biotechnology Information) BLAST search was performed to determine the homologs of the colonies.

Staining for ROS accumulation and callose deposition

The 4–5-weeks old N. benthamiana plants were used to study ROS accumulation and callose deposition. Leaves of tobacco plants were infiltrated with ptHR genes and empty vector as control. At the start of symptoms development, leaves were collected to detect ROS and callose. ROS staining was performed by using DAB kit (CWBIO). Briefly, leaves samples were incubated with DAB solution mixture (1 mL reagent A and 50 μl reagent B) in dark for 1 h and then boiled in 95% ethanol for 30 min to remove chlorophyll. The ROS accumulates were observed under Nikon eclipse 55i optical microscope. This experiment was repeated thrice with three replicates.

Callose staining was performed according to the method described by [24]. In brief, tobacco leaves were treated with Buffer I (90 mmol/L Na2HPO4, 5 mmol/L citric acid and 1% glutaraldehyde, pH 7.4) for overnight. After that leaves were washed with ddH2O and 95% ethyl alcohol was used to soak leaves, then put into boiling water to clear chlorophyll. Sequentially transparent leaves were washed with Buffer II (50% ethyl alcohol, 67 mmol/L Na2HPO4, pH 12.0) and then staining was performed for 1 h in dark at room temperature with staining buffer (0.1% aniline blue, 67 mmol/L Na2HPO4, pH 12.0). Callose deposits were observed under Nikon eclipse 80i ultraviolet epifluorescence.

Analysis of antioxidants activity

Leaves of 4–5 weeks old tobacco plants were infiltrated with ptHR genes and empty vector as control. Samples were collected at different time intervals (0, 24, 48, 72, 96, 120, 144 and 168 h) after infiltration, frozen in liquid nitrogen and stored at − 80 °C. Polyphenol oxidase (PPO), Peroxidase (POD) and Superoxide dismutase (SOD) were measured according to the methods of [25, 26, 27], respectively.

Analysis of the relative expression of pathogenesis-related genes induced by ptHR genes

To analyze the relative expression of pathogenesis-related genes induced by ptHR genes in N. benthamiana, leaves of N. benthamiana were infiltrated with ptHR genes and empty vector as control. Samples were collected at different time intervals (0, 24, 48, 72, 96 and 120 h), frozen in liquid nitrogen and saved at − 80 °C for further use. Total RNA was extracted with the Trizole reagent. cDNA was synthesized by using HiFiScript Quick gDNA Removal cDNA kit (CWBIO) and concentrations were adjusted to be equal. Real-time quantitative PCR (RT-qPCR) was performed to study the relative expression of several pathogenesis-related genes, by using SYBR® Premix Ex TaqTMII (TliRNaseH Plus) (TaKaRa Clontech). The specific primers (Additional file 1: Table S1) were used from [7, 8]. EF-1α was used as reference gene. PCR mixture was processed by using CFX96™ Real-Time PCR Detection System (BIO-RAD), under the following program: 95 °C for 30 s followed by 40 cycles of 95 °C for 5 s and 60 °C for 30 s. A melting curve was established from 65 °C to 95 °C. Three replicates were used for amplification from each treatment and also for control. The 2-∆∆CT method [28] was used to quantify the relative expression of defense-related genes.

Construction of pCAMBIA3301 expression vector

ptHR375 and ptHR941 were cloned into pCAMBIA3301 binary expression vector [29] between Bgl II-BstE II enzyme sites. TF primer, which contains Bgl II enzyme site (5’-ACTGGAAGATCTTACAGGTTACTGAATCACTTGCGCTA-3′) and TR primer (5′- ATAGATGGTNACCCCGTAGTTTAATGTCTTCGGGAC-3′), which contains BstE II enzyme site were used to amplify ptHR genes in pTRV2 Ex vector. Double digestion was subsequently performed on amplified genes. The product was ligated into corresponding sites of the pCAMBIA3301 vector in place of the GUS-containing region. The correct insertions of ptHR genes were confirmed by DNA sequence analysis. Finally, the constructs were introduced into A. tumefaciens EHA105 strain.

Generation of transgenic plants expressing ptHR genes

Transgenic N. benthamiana plants expressing ptHR375 and ptHR941 genes were obtained by using Agrobacterium-mediated leaf disc transformation method [30]. Transgenic T1 N. benthamiana seeds were selected on MS media containing bialaphos (3 mg/L), verified by PCR and sequence analysis.

Controlled pollination to develop F1 hybrid

Female flowers were emasculated at stage 11 [31] of flower development to avoid self-pollination, and hand pollination was performed with pollens collected from male flowers at anthesis. After artificial pollination, flowers were labeled accordingly and covered with 5 cm soda drinking straw enclosed at one end. About 60 h after pollination, corolla fell down together with protective soda drinking straw. After 20–25 days of pollination, seeds were collected and sun-dried [32].

Engineered defense responses protect tobacco plant against fungal pathogen

Leaves were detached from 3 to 4 weeks old T3 generation of transformed N. benthamiana and F1 hybrid plants, and placed in a petri dish lined with a tissue moistened with sterile water. Detached leaves from non-transformed N. benthamiana were used as a control. Leaves were inoculated with P. capsici by using the method of [33]. The diameter of lesions was measured at 48 h after inoculation and inhibition ratio was calculated with the formula: [(control lesion diameter – treatment lesion diameter) / (control lesion diameter – pathogen disk diameter)] × 100.

Penalty test of ptHR genes on N. benthamiana

To observe the effect of ptHR375 and ptHR941 genes on seed germination and morphological characters, 30 seeds of each, non-transformed and transformed N. benthamiana were grown on petri plates lined with moistened filter paper. After 10 days, percent germination was calculated by using the formula: (No of seeds germinated/total No of seeds sown) × 100. Morphological characters were measured after 15 days and averages were calculated.

UPLC-QTOF-MS analysis of transformed N. benthamiana

For the analysis of different metabolites, the samples were prepared according to the method described by [34]. For the LC-MS analysis, Waters Ultra Performance Liquid Chromatography system was used according to the method previously described by [35]. Non-transformed N. benthamiana was used as a control. Ribitol (50 μg/mL), Sigma Aldrich, USA (Cat#A9790) was used as internal standard. The raw data were processed by using the MZmine2 software.

Results

Screening of P. ternata genes inducing hypersensitive responses in N. benthamiana

Complementary DNA reverse transcripts from total RNA of P. ternata were ligated into a binary pTRV expression vector and transformed into A. tumefaciens EHA105 strain. A total of 1268 cDNA colonies were selected, cultured and saved at − 80 °C. To check the quality of this library, 100 random cDNA colonies were selected to perform colony PCR, and it was observed that 95% of the recombinant colonies inserts ranged from 100 to 1000 bp and seldom repeated (Additional file 1: Figure S1). Each individual A. tumefeciens colony, 1268 colonies in total, was infiltrated into N. benthamiana, Lycopersicon esculentum and Gossypium hirsutum leaves with a 1 mL needleless syringe. Typical HR symptoms were observed around infiltration sites at the 48 h of post infiltration as compared to empty vector and buffer which were used as negative control (Fig. 1a-h, Additional file 1: Figure S2a-h). In total, 49 cDNA colonies can repeatedly induce typical HR symptoms, which indicates that 3.86% (49/1268) of P. ternata genes can cause non-self recognition in N. benthamiana.
Fig. 1

Hypersensitive response, ROS burst and callose deposition induced by ptHR genes in N. benthamiana leaves. N. benthamiana leaves were infiltrated with buffer, EHA105 Agrobacterium strain, empty vector, pTRV2-Avr4 and pTRV2-Cf4 as positive control, and ptHR genes. Pictures were taken at 48 hpi. a-h HR induced by ptHR genes in N. benthamiana leaves treated with a buffer, b Agrobacterium EHA105 strain, c pTRV empty vector, d pTRV1 as a control, e pTRV2-Avr4 and pTRV2-Cf4 as a positive control, f ptHR941, g ptHR375, and h ptHR293. i-l ROS accumulation induced by ptHR genes in N. benthamiana leaves treated with, i ptHR941 cloned in pTRV Ex vector, j pTRV empty vector, k ptHR941 cloned in pCAMBIA3301 vector, l pCAMBIA3301 empty vector. m-p Callose deposition induced by ptHR genes in N. benthamiana leaves treated with, m ptHR941 cloned in pTRV Ex vector, n pTRV empty vector, o ptHR941 cloned in pCAMBIA3301 vector, p pCAMBIA3301 empty vector. N benthamiana leaves were infiltrated with ptHR genes and empty vector (pTRV and pCAMBIA3301). Each experiment was repeated three times, and each time the same results were observed

After the transformation of ptHR genes into a pCAMBIA3301 vector, Agrobacterium-mediated transient expression was performed to confirm the typical HR symptoms on N. benthamiana and L. esculentum leaves. It was clearly observed that these genes induced HR symptoms on N. benthamiana and L. esculentum (Additional file 1: Figure S2i-o). P. ternate leaves were also injected (Additional file 1: Figure S2p-q) with these genes to verify the non-self-recognition, and it was found that these genes couldn’t activate the defense mechanism of parent source. During our initial screening, ptHR375 and ptHR941 showed very promising and consistent results related to our objective, so we selected these 2 genes for further experiments.

Homology analysis of P. ternata genes

Forty-nine different colonies, repeatedly inducing HR symptoms on N. benthamiana leaves, were selected for sequencing. NCBI analysis of sequences revealed that 25 sequences showed homology to known proteins with identity from 70 to 96% (Additional file 1: Table S2), 5 sequences shared homology with transcription factors (Additional file 1: Table S3), and remaining 19 shared no homology to known proteins considered to be novel (Additional file 1: Table S4). These newly identified genes were named as ptHR (Pinella ternata hypersensitive response) genes.

ptHR genes induced ROS accumulation and callose deposition in N. benthamiana leaves

ROS burst and callose deposition, both are the early events of plant defense mechanism against invading pathogens and resist their penetration. ROS accumulation and callose deposition, both experiments were performed with pTRV and pCAMBIA3301 constructs. pTRV and pCAMBIA3301 empty vectors were used as controls for comparison. As compared to empty vector, it was observed clearly from the microscopic images that leaves treated with ptHR genes induced ROS accumulation (Fig. 1i-l) and callose deposition in N. benthamiana leaves (Fig. 1m-p).

ptHR genes enhanced antioxidant activities in N. benthamiana

Antioxidants are the elements which can protect plants against a variety of pathogens, the plants which possess a level of resistance often have a certain amount of these elements. SOD, POD and PPO were measured from 0 to 168 h after infiltration of ptHR genes. In the case of POD, it was observed that its activity stimulated at 24 h after treatment, peaked at 96 h for ptHR941, and at 120 h for ptHR375, and then declined gradually (Fig. 2a). While in the case of PPO, maximum activity was observed at 48 h for ptHR941 and at 72 h for ptHR375, and then started to decline down gradually (Fig. 2b). Similar case was observed with SOD where maximum activity was observed at 120 h for ptHR941 and at 144 h for ptHR375 (Fig. 2c), while control was lesser than the treatments.
Fig. 2

Analysis of antioxidant activities in N. benthamiana induced by ptHR genes. a POD activity under influence of ptHR941 and ptHR375, b PPO activity under influence of ptHR941 and ptHR375, and c SOD activity under influence of ptHR941 and ptHR375. Results are mean values from three independent experiments. Vertical bars indicate SD

ptHR genes activated the expression of pathogenesis-related genes in N. benthamiana

To further study the mechanism of ptHR gene induced plant resistance, we analyzed the relative expression levels of pathogenesis-related genes, from SA and JA/ET pathways, of N. benthamiana infiltrated with ptHR941 and ptHR375 compared to empty vector (Fig. 3, Additional file 1: Figure S3). From the results, it was observed that ptHR genes were playing a significant role for the enhancement of plant defense by up-regulating the expression level of PR-genes. In the case of N. benthamiana plants infiltrated with ptHR941, it was observed that PR-1a and PR-5 showed maximum up-regulation at 48 h and 72 h of post-infiltration, respectively, (Fig. 3a-b), and then started to decline. While at 24 h of post- infiltration, PDF1.2, NPR1, PAL, RBOHB and ERF1 were showing maximum up-regulation (Fig. 3c, d, e, f and g). From the present results we speculated that ptHR941 and ptHR375 works in SA and JA/ET pathways to contribute in resistance mechanism.
Fig. 3

Relative expression levels of pathogenesis-related genes in N. benthamiana. N. benthamiana leaves were infiltrated with ptHR941 and an empty vector as control for RT-qPCR analysis. Leaves infiltrated with empty vector were used as control for relative quantification of gene expression. EF-1α was used as internal control. a-g Relative expression levels of a PR-1a vs control, b PR-5 vs control, c PDF1.2 vs control, d NPR1 vs control, e PAL vs control, f RBOHB vs control, and g ERF1 vs control. Significance was determined by t-test: *P < 0.05, **P < 0.01. Results are the mean values from three independent experiments. Vertical bars indicate SD

Generation of transgenic tobacco plants expressing ptHR375 and ptHR941

By using the Agrobacterium-mediated leaf disc transformation method, positive transgenic N. benthamiana plants were obtained (Fig. 4). A typical phenotypic variation was observed on leaves of transgenes as compared to non-transgene plants (Fig. 4b-c). From these variation, positivity of target gene integration was assumed, but for further confirmation, after the extraction of genomic DNA, PCR was performed with gene-specific primers and found that plants with variations all were positive and containing ptHR genes. Seeds were collected from positive plants and screened on MS media against Bialaphos antibiotic for the development of next generations.
Fig. 4

Typical phenotypic response of ptHR gene transformed N. benthamiana. a Non-transformed N. benthamiana plant, (b) and (c) transformed N. benthamiana plants with lesions caused by non-self-recognition of ptHR genes in T0 generation, and (d) T1 generation of transformed N. benthamiana. Lesions were disappeared in the T1 generation and with normal morphology

N. benthamiana expressing ptHR genes confers increased transcriptome levels of pathogenesis-related genes

To study the resistance level, transcriptome levels of PR-genes were quantified in 12 independent transformed N. benthamiana lines relative to non-transformed N. benthamiana. For the confirmation of resistance level either it is inherited to the next generation or not, PR-gene expression level was measured up to T3 generation (Fig. 5). From the results it was observed that, in case of ptHR941 and ptHR375 transformed N. benthamiana, PR-genes were showing significant upregulation relative to their non-transformed N. benthamiana control (Fig. 5a and c). Significant expression of ptHR941 and ptHR375 genes was also observed in transformed N. benthamiana (Additional file 1: Figure S4). F1 hybrid was generated by performing the cross of T3 generations of transformed N. benthamina with non-transformed N. benthamina, and then PR-genes expression level was measured. It was observed from the results that all considered PR-genes in F1 hybrid showed significant upregulation relative to transformed (T3) N. benthamiana control (Fig. 5b and d), while the expression level of ptHR genes in F1 hybrid was non-significantly down regulated as compared to transformed (T3) N. benthamiana (Fig. 5e). From these results, it is considered that, in view of the phenomenon of heterosis, the F1 hybrid has obtained the considerable level of pathogen independent resistance by enhancing its PR-gene transcriptome levels.
Fig. 5

Relative expression levels of pathogenesis-related genes in transformed and F1 hybrid N. benthamiana. Leaves were sampled from transformed and F1 hybrid N. benthamiana to extract total RNA for RT-qPCR analysis. Non-transformed and transformed (T3) N. benthamiana were used as control for relative quantification of gene expression. EF-1α was used as internal control. a Relative expression of PR-genes in ptHR941 transformed N. benthamiana compared with non-transformed N. benthamiana control, b Relative expression of PR-genes in ptHR941-F1 hybrid compared with transformed (T3) N. benthamiana control, c Relative expression of PR-genes in ptHR375 transformed N. benthamiana compared with non-transformed N. benthamiana control, d Relative expression of PR-genes in ptHR375-F1 hybrid compared with transformed (T3) N. benthamiana control, and e Relative expression of ptHR genes in F1 hybrid compared with transformed (T3) N. benthamiana control. Results are the mean values from three independent experiments. Vertical bars indicate SD. Significance was determined by t-test: *P < 0.05, **P < 0.01

Engineered defense responses protect tobacco plant against fungal pathogen

Resistance acquired by transformed N. benthamiana in response to ptHR375 and ptHR941 was measured against fungal pathogen, P. capsici, in 12 independent lines. From the results, it was found that ptHR375 and ptHR941 played an important and significant role to inhibit the infection of P. capsici (Fig. 6). Results revealed that leaves of transformed N. benthamiana and F1 hybrids significantly restricted the growth of P. capsici as compared to non-transformed N. benthamiana.
Fig. 6

Resistant functions of ptHR375 and ptHR941 in transformed N. benthamiana and F1 hybrids. a Non-transformed N. benthamiana leaves (lesion diameter is 36 ± 0.086 mm) as control, b ptHR375 transformed N. benthamiana, c ptHR941 transformed N. benthamiana, d ptHR375 F1 hybrid, e ptHR941 F1 hybrid and f percent inhibition against P. capsici compared with control. Results are the mean values from three independent experiments. Vertical bars indicate SD. Significance was determined by t-test: **P < 0.01

N. benthamiana expressing ptHR375 and ptHR941 showed normal morphology

Seed germination, and morphological characters were measured to observe the penalty of ptHR375 and ptHR941 on transformed N. benthamiana. Results have shown that no significant differences were present between non-transformed and transformed N. benthamiana (Fig. 7). From these results, we can conclude that ptHR genes have increased the plant resistance level without affecting its normal germination and morphological characters. No significant penalty was caused by ptHR genes in transformed N. benthamiana.
Fig. 7

Germination and morphological characters of non-transformed and transformed N. benthamiana. a Average root and shoot lengths of transformed N. benthamiana as compared to non-transformed, b Germination rate of transformed N. benthamiana as compared to non-transformed, and c Growth of transformed and non-transformed N. benthamiana in soil pots under greenhouse conditions. Results are the mean values from three independent experiments. Vertical bars indicate SD

Induced bioactive compounds in ptHR375 transformed N. benthamiana

For the analysis of induced metabolites profile, ptHR375 transformed N. benthamiana was considered and compared with non-transformed N. benthamiana. UPLC-QTOF-MS results revealed that a number of specific bioactive compounds were detected in ptHR375 transformed N. benthamiana (Table 1, Additional file 1: Figure S5 and Table S5). Among these detected compounds, the contents of Cuelure were present consistently at high amount, and can be considered as the biomarker for ptHR375 because of its easy detection.
Table 1

List of induced bioactive compounds present in ptHR375 transformed N. benthamiana

Compound

Content (μg ribitol equivalent/g of dry weight)

Functions

Reference

Oxytetracycline

235.07 ± 18.19

1) Broad spectrum antibiotic against wide range of bacteria.

2) Used to treat infections caused by Chlamydia and Mycoplasma.

[67]

Cuelure

1776.7 ± 140.53

1) Used for the detection, monitoring and control of fruit fly.

2) Enhance the sex attraction in male fruit flies.

[68, 69]

Allantoin

627.1463 ± 49.27

1) Activates the antioxidant mechanisms in Arabidopsis to increase the tolerance against heavy metals.

[70, 71, 72]

Diethylstilbestrol

44.149 ± 3.71

1) Damages the DNA of spermatogonial stem cells and used for the pest management by inducing infertility in male rates.

[73, 74]

1,2-Benzisothiazol-3(2H)-one

795.9675 ± 61.61

1) Derivatives of this compound exhibits antibacterial activities against Staphylococcus aureus-ATCC 7000699, and S. aureus-ATCC 29213, and antifungal activities against Candida albicans, Cryptococcus neoformans, Sporothrix schenckii, and C. parapsilosis.

[75, 76]

Discussion

In plant resistance breeding, a bottleneck is that the absence and easy to be overcome of plant resistance genes. In this study, we attempted to study the non-self-recognition mechanism between two different plant species, P. ternata and N. benthamiana, for the development of a strategy to solve the bottleneck of plant resistance breeding. For this purpose, we identified unique DNA sequences from P. ternata, which can repeatedly induce non-self-recognition reaction and enhance the basal resistance level of N. benthamiana.

P. ternata is one of the famous Chinese traditional medicinal herb used to treat a number of diseases. It is widely studied for the extraction of its extracts and identification of its compounds [36]. Until now, there is no study available related to its potential use for the inhibition of phytopathogens. So for this purpose, cDNA library was constructed [37] and screened by using transient expression system. It was found that specific genes from P. ternata, can alter the response of N. benthamiana plant towards invading pathogens.

Reactive oxygen species are involved in plant defense mechanisms and also play roles in the plant developmental process. Oxidative burst is the early event of plant defense where it generates localized ROS to inhibit the spreading of the pathogen. ROS burst is associated with pathogen/microbes associated molecular patterns (PAMPs/MAMPs) and cause HR [38, 39]. Plant cell wall is the first barrier to the entry of pathogen, and it is modified by the formation of papillae at the site of interaction with invading pathogens. Formation of papillae is the earliest plant defense response at the cellular level, and chemical analysis indicated that callose is the main component of this structure [40, 41, 42, 43]. In our study, it was found that ptHR genes can activate the ROS burst and callose deposition as early events of a defense response in N. benthamiana. This activation of early events of plant defense system indicates that ptHR375 and ptHR941 have the potential to inhibit the penetration of potential pathogens and help to maintain the plant vigor against adverse environment.

Antioxidants are the proteinaceous compounds which can alter the internal environment of plant to eliminate the invading pathogen. Some of these antioxidants/enzymes are selected to study their behavior in response to ptHR375 and ptHR941 genes. POD prevents the entry of the pathogen into the plant tissue by making it hard, as it is involved in the formation of lignins from monolignols by the process of polymerization [44, 45]. Quinones are the toxic compounds for a variety of microorganisms. PPO plays a significant role in the oxidation of phenolic compounds into Quinones and contributes a part of the resistance against pathogens [6]. SOD is an important enzyme which plays a pivotal role in plant defense mechanisms. It is responsible to protect the plant from ROS burst which causes oxidative damages [46]. In the present study, it has been found that N. benthamiana shows an increased level of these antioxidants after the infitration with ptHR genes.

Many studies have shown that many invading pathogens can trigger biochemical pathways associated with the expression of pathogenesis-related proteins, such as SA pathway marker genes PR-1a and PR-5 [47, 48], JA/ET pathway marker gene PDF1.2 [49] and ET pathway marker gene ERF1 [8]. In plants, induction of PR-1, PR-2, PR-5 and PR-8 is the basic characteristic of SAR, as in TMV-infected tobacco leaf tissue, PR-1 accounts for the 1% of total leaf protein [50]. Expression of PDF1.2 is induced by the pathogen attack locally at the site of infection as well as systemically in other non-infected plant parts [51]. PDF1.2 has also been reported to encode a small protein with antifungal potential [52]. Studies suggested that ERF1 enhance the resistance against eyespot disease caused by Rhizoctonia cerealis in wheat, by the inducing the PR genes from ET pathway [53]. It is documented that the cloning of ERF1-V from wild species of wheat, Haynaldia villosa, can induce the high level resistance against powdery mildew and also improve abiotic stress tolerance [54]. NPR1, involved in the cross functioning of SA- and JA-dependent defense pathways, is a key regulator of systemic acquired resistance (SAR) to activate the expression of other pathogenesis-related genes [7, 55]. NPR1 also interacts with the TGA transcription factor members that can bind to the activator sequence-1 (as-1) or the elements which are identified in promoters of PR-1 genes [56]. PAL is the first enzyme in phenylpropanoid biosynthetic pathway which is involved in plant defense by producing antimicrobial compounds like phytoalexins, lignins and other phenolic compounds to create barriers to pathogens. PAL gene is known to be involved in the synthesis of these antimicrobial compounds by providing precursors [7, 57, 58]. It is already reported that ROS burst, especially H2O2 stimulated by RBOHB (respiratory burst oxidase homolog) gene, plays an important role in plant defense against biotic and abiotic stresses [59, 60]. It is also reported for the death induction in cells infected with fungus and instantaneously inhibits the free salicylic acid and ethylene to avoid the death in neighboring cells [61]. In this study, positive expression of these genes was observed to induce the resistance in N. benthamiana. So it is considered that might be, the mechanism of plant defense response induced by ptHR375 and ptHR941 is regulated by SA and JA/ET signal pathways together but still, further investigation is required to make exact signaling pathways clear.

Positively transformed tobacco plants were obtained by using Agrobacterium-mediated leaf disc transformation method. For the selection of positive plants, 3 mg/L bialaphos antibiotic was used as a selection marker, and also gene-specific primers were used for the detection of gene integration after the extraction of genomic DNA. From our results, at T0 stage necrotic lesions were observed on initial leaves of transformed plants while at the same time these lesions were not present on non-transformed leaves, later on, these lesions did not appear on T1, T2 and T3 generations. From 1268 genes, we selected ptHR375 and ptHR941 two genes, and these two genes can trigger severe responses in N. benthamiana with necrotic lesions appeared at the T0 stage. However, the transformed tobacco plants can adapt, to some extent, to these two genes and the necrotic lesions disappeared at the T1 generation. At the T1 generation, although the necrotic lesions disappeared, the resistance level was still significantly increased comparing with non-transformed plants. Why the necrotic lesions disappeared but the foreign transformed ptHR genes still play a role in the T1 generation? The enhanced PR gene expression level (and other evidences like secondary metabolites and resistance against P. capsici) at the T1 generation may provide a clue but for underlying mechanism it still needs further researches. It was also assumed that at T0 stage when ptHR375 and ptHR941 genes were not in stable interaction with tobacco genome, development of necrotic lesions was a sign of positive gene integration which was later confirmed by PCR with gene-specific primers.

In plant-microbe interactions, the apoplastic space is the place where many biochemical interactions occur, including the interaction between attacking enzymes and inhibitors of host plant [62, 63]. In plants, hypersensitive response (localized infection) can produce strong defense signals to activate the defense response of the whole plant against upcoming invading pathogens [64]. NPR1 is a key regulator of systemic acquired resistance (SAR) to protect plants against a variety of pathogens [55]. For the present study, we have used a fungal pathogens, P. capsici [33, 63], and found that ptHR375 and ptHR941 have potential to inhibit the growth of pathogen and create pathogen independent durable resistance which can be inherited generation after generation. Decreased infestation area of pathogen in transformed N. benthamiana lines, which were showing high PR genes expression also indicated an obvious correlation between ptHR and PR genes expression and pathogen resistance.

Heterosis is a phenomena which has been used to explain the improvement of certain characters in F1 hybrids of different crops [20]. In our experiments, transformed N. benthamiana was crossed with non-transformed N. benthamiana to generate F1 hybrid. From the analysis of F1 hybrid, it was observed that expression of PR-genes is significantly up-regulated. There is also an elevated basal resistance level as it was evaluated against P. capsici. So from our findings, it can be concluded that heterosis works well to promote F1 hybrid disease resistance as it has also been used to boost up the yield potential of different crops [18, 20, 65, 66]. Here when generated F1 hybrids, we took ptHR gene transformed N. benthamiana (T3 generation) as the paternal line and non-transformed N. benthamiana as the maternal line. The function of ptHR gene in the F1 hybrids is more or less like that in the ptHR gene transformed T0 generation. This can explain our result that the fresh F1 hybrids have fresh disease resistance. Moreover, the resistance generated by this strategy is not related to any specific pathogen, and it has a broad spectrom resistance.This finding may change the mode of plant resistance breeding because the F1 hybrids produced by using this strategy can be used to develop different and durable resistant cultivars to manage field crop diseases.

Secondary metabolites profile of ptHR375 transformed N. benthamiana revealed the presence of some bioactive compounds which were not detected in non-transformed N. benthamiana. These bioactive compounds are reported for their biological activities as Oxytetracycline reported to treat Chlamydia and Mycoplasma infection [67]. Cuelure was reported for the monitoring of fruit fly [68, 69] and Allantoin have the potential to activate the plant system against heavy metals [70, 71, 72]. For the pest management, Diethylstilbestrol was used because it can make male rates infertile [73, 74], and the derivatives of 1,2-Benzisothiazol-3(2H)-one are strong antibacterial and antifungal agents [75, 76]. It can be considered that induction of these bioactive compounds also confers role to contribute resistance in ptHR375 transformed N. benthamiana.

From this research, we can conclude that non-self-recognition between two different plant species is an important mechanism which works to initiate the immune responses against pathogens. As these ptHR genes from P. ternata also have the ability to initiate HR symptoms on other important crops like L. esculentum and G. hirsutum (Additional file 1: Figure S2), so in future, these genes can be used to create resistance in other economically important crops. Here we tried to prove that when heterologous genes from P. ternata were introduced into N. bentamiana plant, its basal resistance level was increased up to several folds. This concept has opened a new window of plant-microbe interaction research which can be helpful to develop a new genetic system to introduce new potential resistant cultivars for the betterment of sustainable agriculture. In the course of crop domestication and improvement, genetic diversity was reduced, and plant resistance was lost. Here introducing one kind of ptHR genes into modern crop plant, the plant may get into a prime state, and the loss of natural resistances may get compensation. In the next researches, based on these ptHR genes, we will evaluate, to what extent, the lost natural resistances from wild to cultivated crops can get compensated.

Conclusion

Two ptHR genes, ptHR375 and ptHR941 were identified from P. ternata with the potential to trigger HR. Both genes are involved in different resistance pathways. ptHR375 involved in the activation of JA/ET pathway, while ptHR941 also in the SA pathway. These heterologous plant genes can activate disease resistance in N. benthamiana and furthermore, by generating F1 hybrids, fresh pathogen independent plant resistance can be obtained. Feasibility of this hybridization design may help to improve the breeding strategies for the development of durable resistance in economically important crops.

Notes

Acknowledgements

We would like to thank Kangkang Wan, Lun Zhou, Jiaojiao Huan and Min Wang for vector constructions and helpful discussions. This work was supported by the National Major Project for Transgenic Organism Breeding (2011ZX08003-001 and 2016ZX08003-001) and the Hubei Provincial Technology Innovation Program (2016ABA093).

Funding

This work was supported by the National Major Project for Transgenic Organism Breeding (2011ZX08003–001 and 2016ZX08003–001) and the Hubei Provincial Technology Innovation Program (2016ABA093).

Author contributions

HMKA, JX and ZA performed the transformation, HR analysis, UPLC-QTOF-MS analysis, cross and resistance tests. JX and LW performed the library construction and screening. HMKA and WD designed the experiments, analyzed the data and wrote the paper. All authors read and approved the final manuscript.

Conflict of interest

The authors declare that they have no conflict of interest.

Availability of data and materials

The data sets generated and analyzed during this study are available from corresponding author on reasonable request.

Ethics approval and consent to participate

Not applicable.

Consent for publication

Not applicable.

Publisher’s Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Supplementary material

12870_2018_1598_MOESM1_ESM.pdf (1 mb)
Additional file 1: Table S1. Primers used for qRT-PCR. Figure S1. Agarose gel of cDNA inserts. M, 100 bp marker. Figure S2. Hypersensitive response induced by ptHR genes on L. esculentum, G. hirustum, N. benthamiana and P. ternata leaves. Table S2. NCBI blast results showing homology with known sequences. Table S3. NCBI blast results showing homology with transcription factors. Table S4. NCBI blast results showing no homology with known sequences. Figure S3. Relative expression levels of pathogenesis-related genes in N. benthamiana. Figure S4. Relative expression levels of ptHR941 and ptHR375 genes in transformed N. benthamiana. Figure S5. Mass spectra for the induced bioactive compounds detected in ptHR375 transformed N. benthamiana. Table S5. List of induced bioactive compounds present in ptHR375 transformed N. benthamiana. (PDF 1033 kb)

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Authors and Affiliations

  1. 1.Department of Plant Pathology, College of Plant Science and Technology and the Key Lab of Crop Disease Monitoring & Safety Control in Hubei ProvinceHuazhong Agricultural UniversityWuhanChina

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