The amino acid sequence of harpin (HrpZ2_Ps) was retrieved from the NCBI GenBank: OQ338148.1, in the FASTA format. The physiochemical properties of harpin were analyzed using the ProtParam tool (https://web.expasy.org/protparam/). Hydropathicity or hydrophobicity, isoelectric point (pI), instability index, and molecular weight of the harpin sequence were analyzed using the Grand Average of Hydropathicity (GRAVY) web server. Intrinsic disorder was evaluated with the PrDOS server (https://prdos.hgc.jp/cgi-bin/top.cgi) (Ishida and Kinoshita, 2007).

Determination of protein sequence and structure is fundamental to understanding its biological function. Currently, there is no experimentally concluded tertiary structure available for any harpin in the Protein Data Bank (PDB). Consequently, the tertiary structure of the HrpZ2_Ps protein (GenBank ID: WDY61303.1) was modeled by utilizing the I-TASSER server (https://zhanggroup.org/I-TASSER/) (Zheng et al., 2025). Tools such as VERIFY 3D (Colovos and Yeates, 1993), ERRAT (Bowie et al., 1991), and PROCHECK (Laskowski et al., 1996) of the SAVES v.6.0 server (https://saves.mbi.ucla.edu) were used to analyze the Ramachandran plot and validate the computationally predicted 3D structure. Z-score was calculated using the ProSA-web server (https://prosa.services.came.sbg.ac.at/prosa.php).

Conserved domain (CD) analysis was performed using NCBI’s CD search tool (https://www.ncbi.nlm.nih.gov). Harpin domains were further detected and annotated with the PSIPRED server (https://bioinf.cs.ucl.ac.uk/psipred) (Cozzetto et al., 2016), which infers functional domains from the predicted secondary structure. The transmembrane helix packing and orientations were evaluated via the PSIPRED Workbench using the MEMPACK α-helical transmembrane protein structure prediction server (Nugent et al., 2011). Helix and loop regions were predicted with MEMSAT-SVM algorithms, and their orientations were cross-validated with the TMHMM web server and the Kyte–Doolittle hydropathy profiling method of the PSIPRED Workbench (Nugent and Jones, 2009).

The evolutionary history was inferred using the maximum likelihood (ML) method, and the phylogenetic tree was constructed using MEGA11.0 software (https://www.megasoftware.net/older_versions). Bootstrap analysis with 1,000 replicates was performed to ensure statistical robustness and reliability of the branching patterns. The phylogenetic analysis was done among 23 harpin proteins derived from different bacterial genera, such as Xanthomonas, Pseudomonas, Ralstonia, and Erwinia. The phylogenetic tree was visualized on the iTOL online tool (https://itol.embl.de/shared/2PuqbCYlUfiSH) (Letunic and Bork, 2024).

The subcellular localization of HrpZ2_Ps was predicted using DeepLocPro - 1.0 (https://services.healthtech.dtu.dk/services/DeepLocPro-1.0/). The server predicted the subcellular localizations using the prokaryotic predictive model. It provides the prediction probability for different subcellular compartments like the outer membrane, periplasmic space, cell wall and surface, and cytoplasmic membranes based on the amino acid sequence of the signal sequence of the protein.

Hydrogen peroxide (H₂O₂) is a key signal molecule in the plant defense pathway, and its tissue accumulation can be visualized histochemically with 3,3′-diaminobenzidine dyes (DAB). The purified harpin protein was syringe-infiltrated into the interveinal region of the abaxial side of fully extended tobacco leaves using 1 mL of a sterile needleless syringe (final harpin concentration of 30 μg/mL), and H₂O₂ detection was performed following Daudi and O'Brien (2012) with some modifications. The third to fourth leaves were used for the infiltration. Sodium phosphate buffer (10 mM, pH 7.5) served as the negative control. For the detection of H₂O₂ accumulation, a 1-mg/mL DAB (Himedia, RM2735) solution was prepared in 10 mM of sodium phosphate (Na₂HPO₄) buffer with 0.05% (v/v) Tween 20, adjusted to pH 3.8 with conc. HCl. The harpin-treated leaves were harvested at 6, 12, and 24 hours post-infiltration (hpi) and soaked in DAB-HCl solution (pH 3.8) for 10 h at room temperature in dark conditions followed by boiling in bleaching solution (ethanol:acetic acid:glycerol in a 3:1:1 v/v/v ratio) for 15 min. Chlorophyll was fully bleached and the red-brown precipitate resulting from DAB’s reaction with H₂O₂ was visualized under a light microscope and photographed. Each experiment was repeated three times, with six leaves from different plants per replicate. ROS signals were assessed in harpin-treated tobacco leaf tissue by submerging in 10 µM of 2′,7′-dichlorofluorescin diacetate (DCFDA, Sigma-Aldrich, D6883-50MG) prepared in 10 mM of sodium phosphate buffer (pH 7.5) and incubated in the dark for 20 min at room temperature. Tissues were then gently washed with the same buffer and mounted on a glass slide, and ROS signals were observed using a fluorescence microscope (Nikon, Japan).

Quantification of H₂O₂ was done using the FOX (ferrous oxidation in xylenol orange) method as per Jiang et al. (1990) with few modifications. The absorbance (560 nm) of a colored complex formed due to the oxidation of Fe²⁺ to Fe³⁺ by H₂O₂ and binding of the resulting Fe³⁺ to xylenol orange was measured. Briefly, the FOX reagent (250 μM of ferrous ammonium sulfate (NH₄)₂Fe(SO₄)₂.6H₂O, 100 μM of xylenol orange, 25 mM of H₂SO₄, and 100 mM of sorbitol) was prepared by taking approximately 80 mL of 25 mM of H₂SO₄, 9.8 mg of ammonium ferrous sulfate, 7.6 mg of xylenol orange, and 1.82 g of sorbitol, mixed thoroughly until the sorbitol got completely dissolved, and the volume was increased to 100 mL with 25 mM of H₂SO₄. At 0, 3, 6, 9, 12, 24, 48, and 72 hpi, 0.5 g of leaf tissue was ground in liquid nitrogen and suspended in 5 mL of ice-cold 0.1% trichloroacetic acid (TCA). The supernatant was separated by centrifugation at 18,894 rcf for 25 min at 4°C, and 0.5 mL of the supernatant was mixed with 0.5 mL of the FOX reagent and incubated in the dark for 30 min at room temperature. Absorbance was measured at 560 nm, using H₂O₂ as standard. Results were expressed in μM H₂O₂ per gram fresh weight (μM/g FW).

Where:

C = concentration from the standard curve (μM),

V = extraction volume (mL),

W = tissue weight (g)

For the detection of dead cells in harpin-treated tobacco leaves, trypan blue staining was performed as described by Koch and Slusarenko (1990). The leaves were detached at 24, 48, and 72 hpi and boiled in lactophenol trypan blue solution containing lactic acid (10 mL), glycerol (10 mL), trypan blue (7.6 mg), and phenol (10 mL) mixed in 10 mL of distilled water. Full (uncut) leaves were boiled for approximately 1 min in the staining solution and then decolorized by using a clearing solution (mixing 3 parts of ethanol (96%) with 1 part of acetic acid) (for a 100-mL solution: 75 mL of ethanol (96%) and 25 mL of glacial acetic acid). The leaves were stained at room temperature for 3–4 h; the solution was replaced three times until the tissue became transparent. The destained leaves were rinsed with distilled water, followed by mounting in 60% glycerol and covered with a coverslip and carefully photographed using a light microscope (Magnus Opto Systems India Pvt. Ltd., Noida, India).

Callose accumulation was detected as per Luna et al. (2011) with some minor modifications. Tobacco plants at the six- to eight-leaf stage were syringe-infiltrated onto the abaxial surface without a hypodermic needle. At 24 hpi, infiltrated leaf tissue was excised followed by chlorophyll removal, and the samples were completely dehydrated in 96% ethanol by three successive immersions. Specimens were briefly rinsed with distilled water, then equilibrated for 30 min in 10 mM of sodium phosphate buffer (pH 7.5) at room temperature. Tissues were stained with 0.05% aniline blue for 1 h in the dark at room temperature, mounted in 50% glycerol, and covered with a coverslip. Imaging was performed on a confocal laser scanning microscope (Zeiss, LSM780, Oberkochen, Germany) using 405 nm excitation and 530 nm emission wavelength. Buffer alone (10 mM of sodium phosphate, pH 7.5) served as a control. Callose intensity was quantified (using ImageJ software) by measuring the blue pixels on the image and the total pixels covering the plant material. Values were normalized to the control (set as unity). Data represent means ± SD from at least three independent experiments.

PAL activity was assessed following the method outlined by Cheng and Breen (1991). PAL activity was measured by conversion of L-phenylalanine to trans-cinnamic acid. The harpin-treated leaf was detached after different intervals, including 2, 4, 12, 24, and 48 hpi. A total of 0.5 g of the leaf tissue was ground in pre-cooled mortar and pestle using liquid-N2 and resuspended into 5 mL of boric acid extraction buffer consisting of 5 mM of β-mercaptoethanol, 0.05 M of boric acid, 1 mM of EDTA, and 0.05 g of polyvinylpyrrolidone. The supernatant was collected after centrifugation at 18,894 rcf for 20 min at 4°C. The reaction mixture contained 500 μL of 50 mM boric acid buffer (pH 8.5), 200 μL of 50 mM L-phenylalanine (substrate), 200 μL of crude enzyme extract, and 100 μL of distilled water and incubated at 37°C for 1 h. The reaction was stopped by adding 100 μL of TCA (10%). The absorbance (290 nm) was recorded using a spectrophotometer. The trans-cinnamic acid molar extinction coefficient (174,000 M⁻¹ cm⁻¹) was used to calculate PAL activity. The enzyme activity was conveyed as the μM of trans-cinnamic acid formed per minute per gram of fresh weight of leaf (μM/min/g FW).

Calculation:

Where: 17,400 = Extinction coefficient of trans-cinnamic acid (M⁻¹ cm⁻¹)

The enzymatic activity of PPO was examined by following the method reported by Chen et al. (2022) with a few modifications. Catechol, a PPO substrate, was added exogenously based on the protocol. The harpin-infiltrated leaf samples were collected at different time intervals (i.e., 3, 6, 12, 24, 48, and 72 h). The leaf sample (0.5 g) was ground and made into a fine powder using a mortar and pestle and resuspended in a 3-mL extraction buffer [sodium phosphate buffer (100 mM) and 0.1 mL of Triton X-100 (0.1% v/v), pH 6]. The homogenate was transferred to a pre-chilled tube, incubated for 30 min at 4°C, followed by centrifugation at 18,894 rcf for 30 min at 4°C. The supernatant was used as the crude enzyme extract. The reaction mixture contained 1.9 mL of reaction buffer containing 10 mM of catechol solution and 0.1 mL of enzyme extract supernatant. The absorbance at 420 nm was measured every 15 s for 5 min (for a blank, reaction buffer containing 10 mM of catechol solution). One unit (U) of PPO activity was defined as the amount of enzyme that causes an increase in absorbance of 0.001 per minute under the assay conditions. PPO activity was determined using the formula:

Where: ΔA₄₂₀/min = change in absorbance per minute, Total volume = total reaction volume (2 mL), Dilution factor = any dilution of the enzyme extract, ϵ = molar extinction coefficient for quinone formation (24,300 M⁻¹ cm⁻¹ for benzoquione), d = path length (1 cm), Volume of enzyme = volume of extract used (0.1 mL), and Sample weight = fresh weight of leaf tissue (g).

The amino acid sequence of harpin retrieved from the NCBI was used for the determination of different physicochemical properties of harpin using the ProtParam tool of the ExPASy server (Table 1) (Gasteiger et al., 2005). The theoretical pI and molecular weight of harpin were 4.93 and 34.5 kDa, respectively. The aliphatic index was 85.99, and the instability index was 35.97 with a grand average of hydrophobicity (GRAVY) of −0.214. The ProtParam tool also computed the extinction coefficient (Table 1). The sequence contains 19 positively charged residues such as Arg (R) and Lys (K) and 42 negatively charged residues such as Glu (D) and Asp (E).

The predicted harpin protein comprised 50.29% (172 residues) alpha helices, 48.53% (166 residues) random coils, and only 1.16% (4 residues) β-sheets. The representative predicted secondary structure of the harpin protein is shown in Figure 1A. A total of 42% (144 aa residues) of the total structure was predicted to be of intrinsically disordered regions (Figure 1B) (Jones, 1999).

A 3D model of harpin was generated using the I-TASSER web server and validated with the VERIFY 3D, ERRAT, and PROCHECK tools of the SAVES server, including the Ramachandran plot analysis (Figure 2). The plot showed that 90.1% of harpin residues reside in the most favored regions, 8.9% are in additionally allowed regions, 0.4% are in generously allowed regions, while 0.7% are in disallowed areas. The sequence comprises 282 non-proline/non-glycine residues and 46 glycine and 12 proline residues. The 3D model of harpin was visualized using the BIOVIA Discovery Studio.

The mapping of amino acid sequences of the newly identified harpin (HrpZ2_Ps) from P. syringae showed that it belongs to the harpin superfamily (Figure 3A). Proteins in this family play various roles, such as eliciting HR in non-host plants and forming a cation-permeable pore, which plays an essential role in ion conduction. A signal peptide is present at the N-terminal (from 1 to 23 residues) of the protein as detected by the MEMSAT-SVM method, whereas another region (from 323 to 338 residues) at the C-terminal forms a pore-lining helix, while the residues from 24 to 322 form an extracellular loop confirmed by the Kyte–Doolittle method in the TMHMM web server (Figure 3B).

Choi et al. (2013) presented the first domain-based classification of harpins, predicting the secondary structure domain architectures using the PSIPRED web server and grouping harpins into five classes based on the structural features and similarity. In the present study, we performed a parallel domain analysis of our harpin (HrpZ2_Ps) alongside representatives of each of the five different groups of harpins defined by Choi et al. (2013), incorporating additional input parameters such as the intrinsically disordered regions (IDRs) and residue composition (number of glycines, cystines, and leucines) to refine comparisons (Figure 4).

The subcellular localizations of our harpin (HrpZ2_Ps) predicted using the DeepLocPro-1.0 server is graphically represented in Figure 5 (Moreno et al., 2024). The graph of harpin is leveled with sorting signal importance, which represents the contribution of each amino acid residue of harpin protein to the prediction of its subcellular localization and the peaks of amino acid residues important for corresponding the protein to its predicted location. The subcellular location of the harpin was primarily predicted as extracellular with a high probability (0.9915), showing a strong likelihood that the protein is located outside the cell. At the same time, alternative localizations were also predicted with very low probability. The alternative potential localizations are outer membrane (0.006) and periplasmic space (0.0023), while cell wall and surface, cytoplasmic membrane, and cytoplasmic are predicted to be very low with 0.0001 probability.

Phylogenetic analysis of different harpins showed sequence clustering, which indicates a strong genus-specific grouping, with harpins from the same or closely related species forming tight clades. For instance, the HrpN protein from E. amylovora formed an independent clade (highlighted in red), reflecting a conserved evolutionary lineage distinct from other harpins. Similarly, harpins from P. syringae pathovars showed broader distributions across several subbranches (highlighted in green). The new harpin (HrpZ2_Ps) is marked with a red circle in the picture (Figure 6).

Purified harpin protein was infiltrated into the abaxial (lower) surface of the leaves of several non-host plants through a sterile needleless syringe. A clear, visible HR was observed in the localized area of dead cells where the infiltration was done, while buffer treatment showed no such effect (Figures 8A, B). It is reported that elicitor proteins of a critical concentration and above can only induce visible macroscopic HR lesions, and below that concentration, it may fail to induce such visible HR or may induce microscopic lesions (micro-HR) (Peng et al., 2003). This critical concentration may vary from harpin to harpin, plant species variants, methods of application, etc. The results from the infiltration experiments with serial dilutions of HrpZ2_Ps revealed that the minimum concentration of the harpin elicitor required to elicit a visible HR was 30 µg/mL (Figure 8E).

To know whether the harpin can elicit HR in various non-host plants, we tested 15 different non-host plants belonging to eight different angiosperm families, including Solanaceae (Solanum lycopersicum, Capsicum annuum, and Solanum melongena), Brassicaceae (Brassica rapa and Brassica sp.), Cucurbitaceae (Cucumis sativus and Momordica charantia), Caricaceae (Carica papaya), Fabaceae (Glycine max), Poaceae (Zea mays), Anacardiaceae (Mangifera indica), and Musaceae (Musa sp.). Sodium phosphate buffer (10 mM), pH 7.5, was used as a control. We observed that the onset and intensity of HR varied among the tested plants from different angiosperm families (Figure 8B). Among them, M. charantia (Cucurbitaceae) exhibited the fastest HR response comparative to other plants with harpin infiltration. In contrast, plants such as Musa spp. (Musaceae), C. papaya (Caricaceae), and M. indica (Anacardiaceae) exhibited a delayed development of macroscopic HR. The bar graph (Figure 8D) represents the relative HR (cell death) development areas induced by infiltration with the harpin protein. The above non-host plants are categorized as easy, moderate, or difficult on the basis of the ease of infiltration of harpin (Figure 8C).

To assess ROS accumulation in the harpin-treated leaves, H₂O₂ levels were recorded at several time points after infiltration of harpin into tobacco leaves. Harpin triggered rapid H₂O₂ accumulation in harpin-infiltrated leaves (Figure 9). The level of H₂O₂ started increasing by 6 h after infiltration and continued up to 24 h. On the other hand, the level of H₂O₂ remained at lower levels consistently in the control leaves. The macroscopic HR was also visible at the site of infiltration after 24 h. The in vivo H₂O₂ accumulation was also spotted as a brown precipitate using the DAB staining method in the harpin-treated tobacco leaves (Figure 9A), whereas no such brown precipitate was found in the control samples. All these results indicate accumulation of H₂O₂ in the harpin-treated tobacco leaves. ROS generated by harpin was also evident and visualized 6, 12, and 24 hpi, while the most prominent was observed in a 24-hpi leaf using the ROS-sensitive fluorescent dye DCFDA. This fluorescent dye detects a broad range of ROS responses, such as generation of H₂O₂ and superoxide anion O₂⁻. Tobacco leaves infiltrated with purified harpin showed a strong H₂O₂-associated ROS burst, as indicated by increased DCFDA fluorescence, relative to buffer-infiltrated controls. In parallel, DAB staining showed a more intense reddish-brown precipitate in harpin-treated tissues, further confirming elevated H₂O₂ accumulation.

After infiltration of the purified harpin, a rapid burst of ROS was observed by determining the levels of H₂O₂ at various time points. ROS was quantified on tobacco leaves using the FOX reagent method, and it was found that the first peak was visible at 6 hpi (7.55 μM/FW) and then fluctuated till 12 hpi (10.03 μM/FW). The highest peak was observed at 24 hpi (11.85 μM/FW). After that, the level of H₂O₂ started declining at 48 hpi (5.55 μM/FW) and at 72 hpi (2.75 μM/FW) in harpin-infiltrated leaves. However, the level of H₂O₂ was consistently lower in the leaves of control plants. All these results indicate that harpin (HrpZ_Ps) induced the accumulation of H₂O₂ in N. tabacum leaves (Figure 9B).

Localized plant cell death is a well-recognized defense response that restricts nutrient availability to the invading pathogens and limits their spread. To detect the harpin (HrpZ_Ps)-induced plant cell death at the site of infiltration, tobacco leaves were infiltrated with the harpin and subsequently stained with trypan blue (Figure 10A). Harpin treatment clearly induced localized cell death at and around the infiltration site. This defense response is indicative of a hypersensitive reaction of the plant to restrict the pathogen invasion and prevent systemic infection.

Callose is an essential polysaccharide that plays a crucial role in the various processes of plant development and in defense signaling. Callose accumulation was estimated by aniline blue staining of leaf samples followed by observation under confocal microscopy. The images of aniline blue staining of the leaves after infiltration are shown in Figure 10B. Quantitative analysis of the callose deposition was done using the number of callose deposits per mm² leaf tissue as shown in Figure 10B. The results show that there were distinctive arrangements of callose accumulation subject on the time point that was being observed. While there was no callose deposition observed in the control leaves, the results suggested that harpin induced callose deposition in the infiltrated leaves.

Harpin also elicits defense-related enzymes, notably PAL and PPO, in the infiltrated leaves of tobacco. PPO activity was considerably increased in harpin-treated leaves compared to the control. Results show that the PPO activity began to rise by 6 h after infiltration and continued increasing through 24 h. The activity of PPO in harpin-infiltrated leaves was higher than the control leaves, with maximum activity observed at 24 hpi (329.21 U/g), after which the level of activity started to decline at 48 and 72 hpi (Figure 11A). Overall, PPO activity remained higher in harpin-treated samples than in control leaves.

Our results show another key enzyme, i.e., PAL, was also regulated in response to harpin protein in tobacco leaves. PAL activity was moderate initially (21.12 U/g) through 4 hpi, peaked at 24 hpi (63.36 U/g), and declined by 48 hpi (Figure 11B). Overall, PAL activity in harpin-treated leaves was higher than the control.