Heterologous WRKY and NAC transcription factors triggered resistance in Nicotiana benthamiana

2.1 Plant and pathogen

P. ternata was grown under controlled conditions at 22–26 °C and 14/10 h of light and dark intervals. N. benthamiana, Lycopersicon esculentum and Gossypium hirsutum were grown under 25–28 °C and 15/9 h of light and dark conditions. Pectobacterium carotovora and Botrytis cinerea (B05.10) were maintained on LB and PDA at 28 °C and 20 °C, respectively.

2.2 Construction of cDNA library

For P. ternata cDNA library construction, tubers of P. ternata were infected with P. carotovora. Leaf samples collection was performed at 24, 36 and 48 h intervals directly in liquid nitrogen and preserved at −80 °C. cDNA library was constructed according to previous method (Wu et al., 2020). Colony PCR was performed with specific TF primers (Table S1). Clones were saved at −80 °C.

2.3 Analysis of cDNA library

Agrobacterium cells containing pTRV₁, pTRV₂ and pTRV₂: target gene were grown on LB (50 mg/L kanamycin and rifampicin) at 28 °C for overnight. Agrobacterium cells were centrifuged for 10 min at 4000 rpm and re-suspended in infilteration solution (10 mmol/L MES, 20 g/L sucrose, 10 mmol/L MgCl₂, 100 mmol/L acetosyringone, PH = 5.6) at OD₆₀₀ = 0.8–1.0. For functional screening, 1 mL needless syringe was used to infilterate N. benthamiana, Lycopersicon esculentum and Gossypium hirsutum leaves with pTRV₁ and pTRV₂:target gene solutions (1:1 ratio). pTRV empty vector was used as control. HR symptoms were observed daily. Clones constantly showing HR symptoms were sequenced from Wuhan AuGCT (http://wh.augct.com/) with TF (reverse) primer. NCBI blast search was used for homology analysis and phylogenetic tree was constructed using MEGA X (Kumar et al., 2018).

2.4 Microscopy for ROS burst and callose deposition

N. benthamiana leaves injected with ptHR293, ptHR759 and pTRV empty vector were collected at the start of HR development. DAB kit (CWBIO) was used according to manufacturer instructions for ROS staining and amasses were detected under an optical microscope (Nikon Eclipse 55i), while callose deposits were stained according to previous protocol (Gómez-Gómez et al., 1999) and detected under ultraviolet epifluorescence (Nikon eclipse 80i).

2.5 Determination of antioxidant enzymes activity

N. benthamiana leaves injected with ptHR293, ptHR759 and pTRV empty vector were collected at 0, 24, 48, 72, 96, 120, 144 and 168 h intervals and preserved at −80 °C. Peroxidase (POD), superoxide dismutase (SOD) and polyphenol oxidase (PPO) were determined using previous protocols (Beauchamp and Fridovich, 1971; Mayer et al., 1966; Putter, 1974), respectively.

2.6 Determination of transcriptome level of PR-genes

Leaf samples were collected as mentioned above. Total RNA was extracted and cDNA was synthesized with HiFiScript Quick gDNA Removal cDNA kit (CWBIO). qRT-PCR was performed with SYBR® Premix Ex TaqTMII (TliRNaseH Plus) (TaKaRa Clontech). PR-genes specific primers are given in Table S1 (Muhammad et al., 2018). EF-1α served as an indigenous control. 2^−ΔΔCT method (Livak and Schmittgen, 2001) was used for quantification.

2.7 Generation of ptHR293 and ptHR759 transformed N. benthamiana

Transgenic plants were obtained using previous method (Horsch et al., 1985). Positive seeds were screened on MS media (3 mg/L bialaphos), confirmed with PCR and sequence analysis. Hybridization was performed (Brito et al., 2015) to generate F₁ hybrids.

2.8 Resistance assay

Leaves from 3 to 4 weeks old transformed and hybrid generation were used. Non-transformed leaves were used as a control. B. cinerea was inoculated according to previous method (Viaud et al., 2006) and data were recorded at 48 hpi. Following formula determined percent inhibition$$\left[\left(\mathit{control}lesiondiameter-treatmentlesiondiameter\right)/\left(\mathit{control}lesiondiameter-pathogendiskdiameter\right)\right]\times 100$$

2.9 Penalty-assay for ptHR293 and ptHR759 transformed N. Benthamiana

For the determination of ptHR293 and ptHR759 effect on transformed N. benthamiana, assay was conducted according to previous method (Muhammad et al., 2018).

2.10 UPLC-QTOF-MS/MS analysis and amino acid interaction

For the detection of induced bioactive compounds, samples were processed according to previous protocol (Hu et al., 2017). Non-transformed N. benthamiana was considered for comparison. Ribitol (50 µg/mL) was used as an internal standard and MZmine2 was used for raw data processing.

OmicsNet was used to process the genes and concomitant metabolites data. The reactant pairs module was discotinued and reaction classes were kept available (Wang et al., 2013). The number of active compounds was enlisted to draw structure transformation maps to show relationships between bioactive cmpounds and amino acids. Data were also compared with organism specific genome-based expansion data. Overall outcome was in the form of correlation of amino acids with target bioactive compounds in which each amino acid also carries representative gene numbers. Universally colored lines connecting circles (amino acids and compounds) were linked to BlastKOALA identifiers (Kanehisa et al., 2016) in pathway maps.

2.11 Statistical analysis

For significance analysis between control and treatment, student t-test was conducted by SPSS 25 at p ≤ 0.05 and p ≤ 0.01.

3.1 Induced hypersensitive response in N. benthamiana leaves

cDNA was ligated into expression vector and transformed into A. tumefaciens. Selected clones (1268) were saved at −80 °C for further investigation. Randomly 100 clones were selected for quality analysis, which revealed about 95% clones ranged from 100 to 1000 bp. Leaves of N. benthamiana, G. hirsutum and L. esculentum were infiltrated with each clone and typical HR reaction was observed around the infiltration site at 48 h of post infiltration (hpi), while no symptoms were observed for empty vector (Fig. 1A, B; Fig. S1A–D). Induction of typical HR reaction by 49/1268 clones, indicated that about 3.86% of P. ternata genes could be potential candidates for future studies. Further, P. ternata leaves were also infiltrated with 49 cDNA clones but no such HR reaction was observed (Fig. S1E and F).

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Fig. 1

HR symptoms, ROS accumulation and callose deposits. (A–B) HR symptoms on leaves infiltrated by (A) control and (B) ptHR293. (C–D) ROS accumulates in leaves infiltrated by (C) control and (D) ptHR293. (E–F) Callose deposition in leaves infiltrated by (E) control and (F) ptHR293.

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3.2 Sequence analysis of P. ternata cDNA clones

Homology analysis indicated that 25 clones shared homology with reported proteins, while 5 clones were the homolog of transcription factors and remaining 19 were not found to share homology. After homology analysis, sequences were named as ptHR (P. ternata hypersensitive response) genes. According to phylogenetic tree analysis, ptHR genes were divided into 3 clades (Fig. S2). Following initial screening and homology analysis (Table S2), ptHR293 (MN458409) and ptHR759 (MN458410) were selected for further experiments.

3.3 Activated ROS burst and callose deposition

Oxidative burst and callose deposition play a vital role in plant defense mechanism to avoid the spreading of pathogen. Infiltration of ptHR293 and ptHR759 in N. benthamiana leaves induced these two early mechanisms as revealed by microscopic images for ROS and callose (Fig. 1C–F).

3.4 Enhanced antioxidant enzymes activity in N. benthamiana

POD, PPO and SOD were measured from 0 to 168 hpi. The maximum activities of POD, PPO and SOD were recorded 72 hpi after ptHR293 and ptHR759 infiltration as compared to control, which started to decline with the progressive time period (Fig. 2A (a), B (a) and C (a)). These antioxidant enzymes were also considered in ptHR293 and ptHR759 transformed N. benthamiana. Significantly higher activity of these enzymes was recorded in transgenic N. bethamiana compared with non-transgenic N. bnethamiana (Fig. 2A (b), B (b) and C (b)).

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Fig. 2

Kinetics of POD, PPO and SOD activities. (A) Kinetics of POD activity against (a) ptHR293 and ptHR759, and (b) in transformed N. benthamiana compared with non-transformed control, (B) Kinetics of PPO activity against (a) ptHR293 and ptHR759, and (b) in transformed N. benthamiana compared with non-transformed control, (C) Kinetics of SOD activity against (a) ptHR293 and ptHR759, and (b) in transformed N. benthamiana compared with non-transformed control. Data represent mean ± SD.

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3.5 Activated PR-genes expression in N. benthamiana

ptHR293, ptHR759 and an empty vector as a control were infiltrated in N. benthamiana leaves to quantify the expression of PR-genes in SA and JA/ET mediated defense mechanism at different time intervals (Fig. 3; Fig. S3). Results revealed that PR-5 expression was maximum at 24 hpi, while PR-1a, PDF1.2, RBOHB and ERF1 were showing maximum expression at 48 hpi. The relative expression level of PAL was maximum at 72 hpi and then started to decline down. While, in case of NPR1, a continuous increase in expression was observed until 120 hpi. These results indicated that ptHR293 and ptHR759 have potential to enhance resistance in N. benthamiana.

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Fig. 3

Transcription level of PR-genes against ptHR293. Relative expression of (A) PR-1a, (B) PR-5, (C) PDF1.2, (D) NPR1, (E) PAL, (F) RBOHB and (G) ERF1 compared with empty vector. Data represent mean ± SD.

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3.6 Enhanced PR-genes expression in ptHR293 and ptHR759 transformed plants

ptHR293 and ptHR759 transformed plants were attained using Agrobacterium-mediated transformation technique. Seeds from transformed N. benthamiana were collected to grow the next generation for the confirmation of transformation stability. From the analysis of the transcriptome level of PR-genes, it was found that PDF1.2, RBOHB, ERF1, NPR1, PR-1a and PAL were showing the high relative expression level in ptHR293 transformed N. benthamiana (Fig. 4A), while in case of ptHR759 transformed N. benthamiana, NPR1, ERF1, PR-1a, PAL and PDF1.2 were expressing high relative to non-transformed control (Fig. 4B). Analysis of ptHR293 + ptHR759-F₁ hybrid revealed the high relative expression of RBOHB, PR-5, PDF1.2, PR-1a, PAL, NPR1, and ERF1 (Fig. 4C). Relative expression level of ptHR293 and ptHR759 was down regulated in T₃ generation, while significantly upregulated in F₁ generation (Fig. 4D). From results, it was speculated that ptHR293 and ptHR759 are the contributors of SA and JA/ET signaling pathways.

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Fig. 4

Influence of ptHR293 and ptHR759 on transcriptome level of PR-genes. (A) Relative expression of PR-genes in ptHR293 transformed plants as compared to control, (B) Relative expression of PR-genes in ptHR759 transformed plants as compared to control, (C) Relative expression of PR-genes in ptHR293 + ptHR759-F₁ plants as compared to control, (D) Relative expression of ptHR293 and ptHR759 in transformed T₃ and F₁ N. benthamiana compared with respective control. Data represent mean ± SD.

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3.7 Enhanced resistance response of transformed N. benthamiana

The uniformed size leaves were detached from T₃ transformed, F₁ hybrid and non-transformed N. benthamiana and inoculated with B. cinerea. Percent inhibition was determined and found that ptHR293 and ptHR759 transformed N. benthamiana, and ptHR293 + ptHR759-F₁ hybrid confers significant resistance against B. cinerea compared to control (Fig. 5A–E).

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Fig. 5

Resistance analysis against B. cinerea. (A) Non-transformed control leaves (lesion diameter 24.3 ± 0.130 mm), (B) ptHR293 transformed leaves, (C) ptHR759 transformed leaves, (D) ptHR293 + ptHR759-F₁ leaves, and (E) percent inhibition against B. cinerea as compare to control.

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3.8 ptHR293 and ptHR759 transformed N. benthamiana exhibited normal morphology

To determine the effect of ptHR293 and ptHR759 on transformed plants, seed germination, root length and shoot length were observed. Results showed that growth of transformed plants was as normal as non-transformed. There was no significant difference for seed germination, and root and shoot length, between transformed and non-transformed plants (Fig. 6A and B). The findings revealed that ptHR293 and ptHR759 enhanced the plant basic resistance without influencing its normal growth parameters.

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Fig. 6

Penalty analysis of ptHR293 and ptHR759 transformed N. benthamiana. (A) Germination rate of transformed seeds compared with non-transformed. (B) Average root and shoot lengths of transformed plants in comparison with non-transformed. Data represent mean ± SD.

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3.9 Detected bioactive compounds in transformed N. benthamiana and their interaction

In order to observe the perturbations in plant metabolomics inside transformed N. benthamiana, UPLC-QTOF-MS was conducted and compounds were identified on the basis of mass spectra using NIST compound library. Results indicated the production of several compounds elicited by ptHR293 transformation. These compounds were not found in non-transformed control (Table 1; Fig. S4; Table S3). Among these compounds, 3,4-Dichloromaleimide was considered as biomarker for ptHR293 because of its consistent presence and detection.

ptHR293 showed positive interaction with different amino acids viz; L-Serine, Cholin, Glycine, Glycerol and several other amino acids (Fig. 7A), while in case of ptHR759, negative interaction was observed with these amino acids (Fig. 7B). From results, we can conclude that amino acids showing positive interaction with ptHR293 were responsible for the generation of bioactive compounds (Table 1) detected in ptHR293 transformed plants. Negative interaction of ptHR759 with amino acids can be considered as main reason for the generation of no bioactive compounds in ptHR759 transformed plants.

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Fig. 7

Interaction of ptHR genes with amino acids. Red circle represent positive interaction and green circle represent negative interaction. Tagged red and green circles are surrounded with already reported genes. (A) Interaction of ptHR293 with amino acids and, (B) Interaction of ptHR759 with amino acids.

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