Claims:We claim:
1. A biopesticide composition for plants comprising stable harpinPss-loaded chitosan nanoparticles, wherein the composition is a sustained release composition, wherein the chitosan nanoparticles improve the bioavailability of the harpin by releasing the harpin steadily over an extended period of time, wherein the composition exhibits improved induction of disease resistance in plants as compared with native harpin.
2. The biopesticide composition as claimed in claim 1, wherein the harpin is harpinPss.
3. The biopesticide composition as claimed in claim 1, wherein the composition further comprises of at least one of an agriculturally acceptable carrier, diluent and adjuvant.
4. The biopesticide composition as claimed in claim 1, wherein the plant is tomato.
5. The biopesticide composition as claimed in claim 1, wherein the composition has a shelf life of up to 90 days.
6. The biopesticide composition as claimed in claim 1, wherein the size of the harpin-loaded chitosan nanoparticles is in the range of about 120±50 nm.
7. The biopesticide composition as claimed in claim 1, wherein the ratio of the harpin to the chitosan is about 0.1:1, the composition thereof exhibiting synergistic effect.
8. The biopesticide composition as claimed in claim 1, wherein the composition is in the form of powder, granules, liquid, semi-solid, suspension, emulsion or spray.
9. A method for making a biopesticide composition for plants comprising:
a) Preparing chitosan nanoparticles; and
b) Mixing harpin with the chitosan nanoparticles to form stable harpin-loaded chitosan nanoparticles, wherein the biopesticide composition is a sustained release composition comprising the harpin-loaded chitosan nanoparticles, wherein the chitosan nanoparticles improve the bioavailability of the harpin by releasing the harpin steadily over an extended period of time, wherein the composition exhibits improved induction of disease resistance in plants as compared with native harpin.
10. A method for application of a biopesticide composition to plants comprising:
a) Providing a sustained release biopesticide composition, wherein the composition comprises of stable harpin-loaded chitosan nanoparticles, and
b) Contacting at least a part of a plant with an effective amount of the biopesticide composition, wherein the chitosan nanoparticles improve the bioavailability of the harpin by releasing the harpin steadily over an extended period of time, the composition thereby exhibiting improved induction of disease resistance in plants as compared with native harpin. , Description:TECHNICAL FIELD
[001] The present invention generally relates to biopesticide compositions for application in plants. More particularly, the present invention relates to sustained release biopesticide compositions comprising of stable harpinPss loaded chitosan nanoparticles for improved induction of disease resistance in plants and methods thereof.
BACKGROUND
[002] Numerous Gram-negative phytopathogenic bacteria harbor a gene cluster (HRP, for hypersensitive reaction and pathogenicity) that controls pathogenicity in susceptible plants and the ability to elicit the hypersensitive reaction (HR) in nonhost plants or resistant cultivars of host plants (Lindgren et al., 1986). Proteinaceous elicitors of the HR known as harpin have been isolated from plant pathogenic Gram negative bacteria Erwinia amylovora (harpinEa) (Wei et al., 1992) and Pseudomonas syringae pv. syringae (harpinPss) (He et al., 1993). It was reported that harpin binds with receptors on the plant cell membrane (Lee et al., 2001), initiates an intracellular signaling cascade that results in the activation of plant non-specific defense responses by enhancing phenylalanine ammonia lyase (PAL), peroxidase activity, and inducing PR genes expression, cell programmed death, ions flux across membranes, the effect level is according to the harpin dosage. The HR elicited by harpins result in three kinds of effects, the first is enhancing plant disease-resistant ability by inducing cell programmed death, activating signaling pathways associated with PAL, jasmonic acid, salicylic and PR genes expression, etc. The PR genes expression products possess the activities of chitinase, peroxidase, lysozyme, and aggression pathogen directly.
[003] Chitosan, poly[ß-(1–4)-linked-2-amino-2-deoxy-d-glucose], is a N-1deacetylated product of chitin, which is a major component of arthropod and crustacean shells, such as lobsters, crabs, shrimps, and cuttlefishes. In addition, chitosan has many significant biological and chemical properties, such as biodegradable, biocompatible, bioactive, and polycationic properties. There are at least four methods for the preparation of chitosan nanoparticles as ionotropic gelation, microemulsion, emulsification solvent diffusion, and polyelectrolyte complex. Among these methods, ionotropic gelation was most efficient due to simple and eco-friendly approach and has an excellent capacity for the association of proteins.
[004] Advances in the field of nanotechnology and nanofabrication have brought an immediate impact on the field of drug delivery. However, the implementation of nanotechnology is less popular in agriculture. Recently, nano-formulations of pesticides and herbicides have been developed with added advantage of increased efficacy, specificity, reduced toxicity and environment risk during field application. In the same line, biological colloidal carriers of nano-dimension such as chitosan has emerged as a promising option for improving the delivery of macromolecules such as peptides, proteins, oligonucleotides and plasmids across biological surfaces.
[005] In the light of above mentioned discussion, there exists a need for the development of alternatives to the use of conventional ecologically hazardous chemicals for the control of plant diseases. Though foliar spray application of harpin is widely employed, the technique requires high dosage of harpin and target sustained delivery is not possible. Nanoparticles usage can minimize the dose of harpin and sustained target delivery of harpin in plants can be obtained.
[006] The present invention discloses biopesticide compositions comprising stable harpin-loaded chitosan nanoparticles that overcome the above mentioned disadvantages. The composition is a sustained release composition, and the chitosan nanoparticles improve the bioavailability of the harpin by releasing the harpin steadily over an extended period of time and this reduces the dose of harpin. The composition also exhibits improved induction of disease resistance in plants as compared with conventional harpin biotic pesticides. Another advantage is that chitosan serving as a wall material has disease-resistant and induced resistance effects leading to synergistic effect of the composition. The present invention further discloses methods for making the composition and methods for application of the composition as well.
BRIEF SUMMARY
[007] The following presents a simplified summary of the disclosure in order to provide a basic understanding to the reader. This summary is not an extensive overview of the disclosure and it does not identify key/critical elements of the invention or delineate the scope of the invention. Its sole purpose is to present some concepts disclosed herein in a simplified form as a prelude to the more detailed description that is presented later.
[008] An exemplary embodiment of the present disclosure is directed towards a biopesticide composition for plants, which comprises of stable harpin-loaded chitosan nanoparticles. The composition is a sustained release composition, and the chitosan nanoparticles improve the bioavailability of the harpin by releasing the harpin steadily over an extended period of time. The composition also exhibits improved induction of disease resistance in plants as compared with native harpin.
[009] Yet other exemplary embodiments of the present disclosure are directed towards methods for making the biopesticide composition for plants comprising:
a) Preparing chitosan nanoparticles; and
b) Mixing harpin with the chitosan nanoparticles to form stable harpin-loaded chitosan nanoparticles. In a particular embodiment, the chitosan nanoparticles are prepared by ionotropic gelation method.
[010] Yet other exemplary embodiments of the present disclosure are directed towards methods for application of a biopesticide composition to plants comprising:
a) Providing a sustained release biopesticide composition comprising stable harpin-loaded chitosan nanoparticles; and
b) Contacting a part of the plant with an effective amount of the biopesticide composition.
[011] It is an object of the present invention to disclose sustained release biopesticide compositions comprising harpin-loaded chitosan nanoparticles, wherein the chitosan nanoparticles improve the bioavailability of harpin by releasing harpin steadily over an extended period of time.
[012] It is another object of the present invention to disclose biopesticide compositions that require lesser dose of harpin than conventional harpin biotic pesticides.
[013] It is another object of the present invention to disclose biopesticide compositions comprising harpin-loaded chitosan nanoparticles, wherein other than the active component harpin, the wall material chitosan itself has disease-resistant and induced resistance effects leading to synergistic effect of the composition.
[014] It is another object of the present invention to disclose biopesticide compositions that exhibit improved induction of disease resistance in plants as compared with conventional harpin biotic pesticides.
[015] It is another object of the present invention to disclose methods for making biopesticide compositions that are simpler and easier and demonstrate high encapsulation efficiency. The obtained harpin encapsulated monodispersed nanoparticles have no adhesion, good glomeration, round surface, and are protected from external interference.
BRIEF DESCRIPTION OF DRAWINGS
[016] Other objects and advantages of the present invention will become apparent to those skilled in the art upon reading the following detailed description of the preferred embodiments, in conjunction with the accompanying drawings, wherein like reference numerals have been used to designate like elements, and wherein:
[017] Fig. 1A and 1B represent Field Emission Scanning Electron Microscopy of CSNPs and H-CSNPs. (A) CSNPs were prepared with 5:1 ratio of CS and TPP at a pH of 5.5. (B) H-CSNPs prepared at 0.1:1 ratio of harpinPss: CS. The chitosan and TPP were taken as 1 mg mL-1. Image mag: 25 KX.
[018] Fig. 2A-2E represent FTIR Spectra of: (A) CS, (B) TPP, (C) CSNPs, (D) harpinPss and (E) H-CSNPs. Interaction of CS and TPP resulted C-N stretch (1021 cm-1) and O-H bend (904 cm-1), absorption band (1563 cm-1) of protonated amino groups observed in CSNPs spectrum and many characteristic peaks of harpinPss shifted when it was entrapped in the CSNPs, suggesting strong interaction with CSNPs.
[019] Fig.3A and 3B represent stability of the NPs during a period of 90 days. Morphological stability of (A) CSNPs and (B) H-CSNPs was observed under FE-SEM after storage for 90 days and (C) change in zeta potential. SEM micrograph of CSNPs and H-CSNPs showing slight aggregation after storage of 90 days and decrease in zeta potential was observed. SEM image mag: 25 KX.
[020] Fig. 4A-4C represent Encapsulation efficiency of (A) CSNPs loaded with various concentrations of harpinPss, (B) H-CSNPs stored for different time periods and (C) SDS-PAGE, lane 1-H-CSNPs pellet (150 µg mL-1) , lane 2-H-CSNPs supernatant, lane 3-HarpinPss (150 µg mL-1) and lane 4-protein marker. Error bars represent the standard deviation of the mean from three independent experiments.
[021] Fig. 5 represents effect of storage of H-CSNPs on subsequent harpinPss release profile. The cumulative release of harpinPss from CSNPs stored for different time periods (30, 60 and 90 days) were compared with freshly prepared NPs. Particle preparation conditions: CS & TPP concentration = 1.0 mg/ml, harpinPss concentration = 0.1 mg mL-1. Release medium T=37±1 ?C, pH 7.0.
[022] Fig. 6A-6F represent time sequential FE-SEM images of H-CSNPs during harpinPss release studies. (A) 0 h (B) 6 h (C) 12 h (D) 24 h (E) 72 h and (F) 96 h. Particle preparation conditions: CS and TPP concentration = 1.0 mg mL-1, harpinPss concentration = 0.1 mg mL-1, CS to TPP mass ratio = 5:1, release medium T=37±1?C, pH 7.4. Mag: 25 KX.
[023] Fig. 7 represents microscopic detection of H2O2 accumulation and cell death together (DAB + Evans blue). (A) control, (B) harpinPss, (C) H-CSNPs and (D) CSNPs treated leaves 2 days after infiltration. H2O2 accumulation detected with DAB (brown spots) and cell death with evans blue (blue spots). H2O2 bursts and cell death colocalize in cells of tomato leaves infiltrated with harpinPss and H-CSNPs, and the accumulation of H2O2 precedes cell death.
[024] Fig. 8 represents bioassay of harpin/H-CSNPs for resistance against Rhizoctonia solani in tomato. Leaves treated with (A) buffer, (B) harpinPss, (C) H-CSNPs and (D) CSNPs. Detached tomato leaves were inoculated with Rhizoctonia solani. Leaves were wounded prior to inoculation. Photographs were taken seven days post-inoculation. The severity of disease reduced in harpinPss and H-CSNPs treated leaves.
[025] Fig. 9A and 9B represent biochemical responses of tomato during elicitor treatment. (A) Peroxidase (POD), (B) Phenylalanine ammonia lyase (PAL) activity (nkat/mg protein) in a time course after treatment of tomato leaves with CSNPs, harpinPss and H-CSNPs. Error bars represent the standard deviation of the mean from three independent experiments.
[026] Fig. 10 represents functional categories of differentially expressed transcripts during treatment with harpinPss, CSNPs, and H-CSNPs. Assigned functional categories of differentially expressed transcripts using cut-off statistical parameter P <0.05 with log2 expression value of = 2 and = -2. Differentially expressed transcripts in harpinPss, CSNP and H-CSNP treated tomato leaves compared to mock-inoculated tomato leaves.
[027] Fig. 11A-11B represent subcellular localization of GFP-HrpZ fusion proteins and GH-RCSNPs. (A) The subcellular localization of N-terminal GFP fusion construct was examined in toamto leaves. Fluorescence in the green channel represents the GFP signal; fluorescence in the red channel represents the chloroplasts autofluorescence. (B) Subcellular localization of N-terminal GFP fusion construct and rhodamine labeled CSNPs. Fluorescence in the green channel represents the GFP signal; fluorescence in the red channel represents the rhodamine signal and fluorescence in the blue channel represents chloroplasts autofluorescence.

DETAILED DESCRIPTION
[028] It is to be understood that the present disclosure is not limited in its application to the details of construction and the arrangement of components set forth in the following description or illustrated in the drawings. The present disclosure is capable of other embodiments and of being practiced or of being carried out in various ways. In addition, it is to be understood that the phraseology and terminology used herein is for the purpose of description and should not be regarded as limiting.

[029] The use of “including”, “comprising” or “having” and variations thereof herein is meant to encompass the items listed thereafter and equivalents thereof as well as additional items. The terms “a” and “an” herein do not denote a limitation of quantity, but rather denote the presence of at least one of the referenced item. Further, the use of terms “first”, “second”, and “third”, and the like, herein do not denote any order, quantity, or importance, but rather are used to distinguish one element from another.
[030] According to different non limiting exemplary embodiments of the present disclosure, biopesticide compositions comprising stable harpin-loaded chitosan nanoparticles for improved induction of disease resistance in plants and methods thereof are disclosed.
[031] According to a non limiting exemplary embodiment of the present disclosure, a biopesticide composition for plants, which comprises of stable harpin-loaded chitosan nanoparticles, is disclosed. The composition is a sustained release composition, and the chitosan nanoparticles improve the bioavailability of the harpin by releasing harpin steadily over an extended period of time. In a particular embodiment, harpin was sustainably released from about 24 h to about a week though it might vary depending of the amount of harpin loaded within the nanoparticles, the cross linking agent and other critical factors without limiting the scope of the present disclosure. The composition also exhibits improved induction of disease resistance in plants as compared with native harpin. The term “native harpin” used herein includes conventional harpin biotic pesticides as well. The present composition is suitable for all plants without limiting the scope of the present disclosure and in particular suitable for vegetable crops. The present composition in general is effective against all plant pathogens known in the art without limiting the scope of the present disclosure. In a particular embodiment, the composition has plant growth enhancing properties as well.
[032] In accordance with various non limiting exemplary embodiments of the present disclosure, the composition further comprises of agriculturally acceptable carriers, diluents and/or carriers. The composition can further comprise other additives that are known in the art for efficient application onto plants without limiting the scope of the present disclosure.
[033] According to a non limiting exemplary embodiment of the present disclosure, a method for making a biopesticide composition for plants is disclosed and the method comprises of the following steps:
a) Preparing chitosan nanoparticlesby ionotropic gelation method; and
b) Mixing harpin with the chitosan nanoparticles to form stable harpin-loaded chitosan nanoparticles.
[034] In accordance with a non limiting exemplary embodiment of the present disclosure, a method for application of the biopesticide composition to plants comprising of the following steps is disclosed:
a) Providing a sustained release biopesticide composition, and
b) Contacting a part of the plant with an effective amount of the biopesticide composition.
[035] The biopesticide composition could be applied in the form of powder, granules, liquid, semi-solid, suspension, emulsion, spray or any other suitable form that is known in the art without limiting the scope of the present disclosure. In a particular embodiment, the biopesticide composition is a lyophilized powder comprising harpin-loaded chitosan nanoparticles.

[036] In a particular embodiment, a spray solution with a composition of about 100 µg of harpin loaded into the nanoparticles per ml of suitable buffer was used. About 5 ml of the solution was sprayed onto 4 to 6 weeks old tomato plants which was found to be an effective dose. This is just an example and the effective dose would vary depending on the type of crop, its age, the part of the plant to be treated, the pathogen to be treated and other critical factors and a person skilled in the art would be able to tailor the dose accordingly.

Examples:
Materials and plants
[037] Water-soluble chitosan (MW: < 200 kDa with 90% degree of deacetylation) was a gift from Mahtani Chitosan (India). Sodium tripolyphosphate (TPP) was obtained from Loba chemie Pvt. Ltd and BSA from Himeida (India). Molecular biology kits and enzymes were procured from Sigma-Aldrich (USA), Qiagen (Germany), MBI Fermentas (Germany) and Takara bio (Japan). All other chemicals used in the work were of analytical grade and obtained from Sigma-Aldrich (USA), GE health care (USA), Promega Life Science (USA), Fermentas (Germany), Himedia (India), and Qualigens fine chemicals (India). Tomato (Lycopersicon esculentum cv. Arka vikas) plants were grown in soil in a growth chamber with a 16 h photoperiod at 350 lE/m2 light intensity at 24°C and at constant (70%) humidity.
Harpin expression and purification
[038] The hrpZ gene (1.02 kb) encoding full length harpinwas cloned under Nde I and Xho I sites of pET 28a vector.E. coli BL 21 (DE3) cells transformed with pET28a-hrpZ were grown in Luria Bertani broth containing Kanamycin (50 µg/mL) to OD 600~0.5 and induced with 0.5mM isopropyl thiogalactoside. Harpin was purified using Ni-NTA column. Concentration of harpin was determined using BCA protein assay.
Preparation of CSNPs and harpin-loaded CSNPs
[039] CSNPs were prepared as per the method of Zhang et al. (2004) with slight modification in concentrations of chitosan, acetic acid, and TPP. Chitosan solution was prepared by dissolving 0.1% (w/v) chitosan (MW: < 200 kDa, DD 90%), in 1% acetic aqueous solution. Chitosan solution pH was adjusted to 5.5 using 10N NaOH, to 5ml of chitosan 1ml of 1% TPP (w/v), a cross linking agent was added drop by drop with continuous stirring on magnetic stirrer at 1200 rpm in order to avoid agglomeration of nascent nanoparticles. This resulted in an opalescent solution indicating the formation of CSNPs. The CSNPs were separated from the suspension by centrifuging at 20,000g for 30 min, washed twice in milliQ water, and re-suspended in same. The CS/TPP (5/1) ratio leads to efficient cross-linking of amino groups producing the most compact particle structure. At this ratio, CSNPs with size less than 100 nm are produced. Harpin-loaded CSNPs (H-CSNPs) were prepared by mixing Harpin: Chitosan weight ratios as 0.1:1, i.e.100 µg/ml.
2.4. Physicochemical characterization and stability of nanoparticles
2.4.1.Physicochemical characterization of CSNPs:
[040] The particle size and surface morphology of the prepared NPs were estimated using FE-SEM. To determine the functional groups involved in reduction and stabilization between the polymer and the protein molecules, FTIR samples were prepared by grinding 1 to 2 mg of lyophilized NPs suspension along with 100 mg KBr and pelletized using hydraulic press at 20,000 prf.

2.4.2. Stability of nanoparticles
[041] The physicochemical stability of the nanoparticles was evaluated using measurements of size, zetapotential, encapsulation efficiency and invitro release of harpin with the nanoparticles stored for a period of 90 days. The samples were analyzed in triplicate at 26 ± 2°C.
[042] The entrapment efficiency of harpin within the CSNPs was determined by pelletizing the sample at 20,000g for 20 min; the amount of free harpin in the supernatant was analyzed with UV-Vis spectrophotometer at 562 nm using the micro BCA protein assay and resolved on 12% SDS-PAGE. The supernatant of non-loaded CSNP suspension was used as a blank. Entrapment efficiency [EE] was calculated based on the ratio of amount of harpin present in the NPs to the amount of protein used in the loading process.
[043] In vitro release profile of harpin from CSNPs was measured by direct dispersion method. Initially free harpin in the supernatant was removed by centrifugation at 30,000 g for 40 min and pellet was transferred to another tube containing phosphate buffer saline (pH 7.4) and spectrophotometrically assayed for release of harpin at regular interval of 24 h up to 120 h at 562 nm.
2.5. Defense Responses
2.5.1. Harpin induced cell death in tomato leaves; Staining Methods
[044] Purified recombinant harpin used to evaluate the HR inducing ability and compared with H-CSNPs. Five to six-leaf stage greenhouse-grown tomato (Solanum lycopersicum ‘Moneymaker’) leaves were infiltrated with 20 µg of recombinant harpinPss /H-CSNPs using hypodermic syringe. Buffer infiltration alone served as negative control and the plants were maintained at 28°C. After 48 h the HR due to H-CSNPs was compared with the HR induced by harpinPss alone.
[045] Simultaneous detection of H2O2 accumulation and cell death in one tomato leaf specimen was accomplished by a combination of DAB (3, 3’-diaminobenzidine tetrahydrochloridedihydrate) and Evans blue staining methods as described by Pogany (Pogany et al., 2009). Intact leaves were infiltrated with 2.5 mM DAB solution (dissolved in 2.5 mM TRIS-phosphate buffer, pH 7.8). DAB-infiltrated leaves were incubated in light for 2 h and vacuum infiltrated for a second time with 1.3 mM Evans blue (Fluka) solution (0.125 g of Evans blue dissolved in 100 mL of distilled water). After 15 min of incubation in the dark, leaves were decolorized by soaking in a clear solution (80% ethanol and 20% chloroform) containing 0.15% trichloroacetic acid for 24 h.
2.5.2. Bioassay of harpin treated tomato for disease resistance
[046] Third leaf from the top of treated (Harpin, H-CSNPs, CSNPs, ?Harpin and ?H-CSNPs) and control tomato plants was excised, sterilized with 70 % alcohol for 10-20 sec, washed with sterile water for 2-3 times and placed on moist sterile Whatman no 3 paper in petri plates under sterile conditions in dark. Fungal (Rizoctonia Solani) plugs of 5 mm diameter were cut from culture growing on PDA plates and placed upside down on the leaf. The plates were sealed and incubated at 30 °C. Lesion areas were determined after 5 days of infection.
2.5.3. Treatment of Tomato Plants for enzyme assays
[047] Four week-old tomato plants were sprayed with harpin (100 µg/ml) and H-CSNPs (10 µg/ml) to observe the efficiency of CSNPs in delivering harpin to elicit the defense responses in tomato. After treatment with H-CSNPs, tomato leaf samples were collected at regular time intervals of 24 h up to 96 h. The leaf samples were immediately frozen in liquid.N2 and stored in -800C. The induced defense response was assessed by assay of defense-related enzymes. All the experiments were performed in triplicates.
Preparation of plant extract
[048] All steps were carried out at 4°C. Leaf tissue (0.5 g) was crushed in liquid nitrogen and extracted with 2 mL of 100 mM homogenization buffer (100 mM potassium phosphate pH 7, 0.5 mM EDTA, 0.1 mM PMSF and 2% PVP). The suspension was homogenized for 1 min and centrifuged at 10000 g for 30 min. The supernatant was used as plant extract for assay of defense related enzymes.
2.5.4. Peroxidase (POD)
[049] Peroxidase activity was determined in the supernatant using an assay system consisting of 5 mM guaiacol, 50 mM sodium acetate buffer (pH 7.0), 0.03% hydrogen peroxide (H2O2) and the enzyme extract (0.1 mL) with a final volume of 1mL (Chance and Maehly, 1955). Oxidation of guaiacol was measured by the increase in absorbance at 470 nm. One unit of peroxidase activity represented the amount of enzyme catalyzing the oxidation of 1 ??mol of guaiacol in 1min.
2.5.5. Phenylalanine ammonia lyase (PAL) Activity Assay in Tomato leaves
[050] PAL was assayed directly in the supernatant of crude enzyme extracted. 200 mM Tris-HCl (pH 7.0) was used as assay buffer. 20mM L-phenylalanine was used as substrate of the enzyme in the assay. The reaction was carried out for 60min at 37°C and the increase in ??290 nm was recorded (Sainders and McClure, 1975). The rate of formation t-cinnamic acid was taken as a measure of enzyme activity followed by increase in absorbance at ??290nm. The PAL activity was expressed as nkat/mg protein.
2.6. Experimental design and GeneChip analysis for transciptome study
[051] All samples were collected in two independently repeated experiments at 24, 48 and 72 h post inoculation (hpi). For each sample, leaf material was harvested from three plants inoculated with harpin (100µg/ml), H-CSNPs and CSNPs pooled separately, and flash frozen in liquid nitrogen. Leaves collected from mock-inoculated plants were used as the reference sample to which all other samples were compared. RNA isolation Total RNA was isolated from 100 mg of the frozen leaves using NucleoSpin RNA plant kit (Machery Nagel, Duren, Germany).
[052] Only genes with high levels of significance (P < 0.05) and a minimum absolute value of log2>2 were systematically considered in this study, to minimize the false positive as up- or down regulated. Expression profiles from each time point were clustered based on their similarity in expression pattern using a hierarchical average linkage clustering algorithm and Pearson correlation distance.
[053] Differentially expressed transcripts were annotated using the BLAST hit from the non-redundant database of NCBI http://www.ncbi.nlm.nih.gov) against Solanum tuberosum total genome. For GO, we used potato gene model for each probe set.

ACCESSION NUMBER
The NCBI Gene Expression Omnibus accession number for the transcriptome data reported in this paper is GEO: GSE100594.
2.7. Labeling of Harpin and CSNPs to track nanoparticles:
2.7.1. Amplification and cloning of HarpinPssN terminal GFP fusion:
[054] Overlap extension/fusion PCR was performed to generate HarpZ and GFP fusion chimeras. Plasmid templates pHrpZ-pET28a (+) and pGFP-pET28a (+) with appropriate combinations of primers were used to generate harpin fusion chimeras. Based on the fusion of auxiliary domains to either ‘N’ or ‘C’ termini of the HrpZ gene, the chimeras were designated as GFP+HrpZ and HrpZ+GFP. The amplicons (GFP+HrpZ and HrpZ+GFP of 1.75 kb) were double-digested and ligated to Eco R I &Xho I sites of pET-28a (+) expression vector. All the ligation reactions were performed at 16°C for 16 h, using T4 DNA ligase. Highly efficient competent cells of Escherichia coli Rosetta-gami II (DE3) were used for transformation. Positive clones were selected on appropriate antibiotic plates and confirmed by both double digestion and sequencing
2.7.2. Heterologous expression and purification:
[055] E. coli Rosetta-gami II (DE3) cells containing appropriate HrpZ and GFP fusion chimeras were used for over expression of protein. The purified protein in 50 mM sodium phosphate, pH 7.4, was used for localization studies.
2.7.3. Labeling of chitosan with rhodamine-123:
[056] Five mL of 0.1% chitosan solution was incubated for 1 h with 25 µL of sterile rhodamine-123 (1 mg/mL) and the pH of solution adjusted to 5.5 using 10N NaOH. Later NPs were prepared by drop wise addition of 0.1% (w/v) TPP into it till the solution became opalescent.
[057] GFP-HrpZ loaded RCSNPs (GH-RCSNPs) were prepared by taking GFP-HrpZ: CS weight ratios as 0.1:1, i.e.100 µg/ml. The preparation of nanoparticles was similar with an addition of GFP-HrpZ with rhodamine labeled chitosan.
2.7.4. Sub-cellular localization of nanoparticles and proteins:
[058] Four week-old tomato plants were infiltrated with GFP, GFP-hrpZ and GFP-hrpZ RCSNPs (GH-RCSNPs). After 24 h the abaxial side of leaf was directly visualized under a laser scanning confocal microscope (ZEISS LSM 880). The localization of GFP fusions was visualized with sequential imaging of GFP at 488 nm excitation and 505–525 nm emission and chloroplast auto fluorescence at 633 nm excitation and 660 nm emission.
3. Results
3.1. Preparation and characterization of CSNPs
[059] Monodispersed CSNPs were successfully prepared using ionic gelation method. Addition of TPP to CS solution at pH 5.5 was facilitated electrostatic interaction between amino groups of chitosan and anionic TPP. The CS/TPP (5/1) ratio leads to efficient cross-linking of amino groups producing the most compact particle structure. The size of the spherical CSNPs was in the range of 80±50 nm (Fig. 1A). Later the harpin was entrapped within the nanoparticles by cross linking with the TPP. At pH of 5.5, harpin (pI=8.6) is positively charged and electrostatic interactions between the positively charged harpin and negatively charged TPP caused greater entrapment. The size of the H-CSNPs was in the range of 120±50 nm (Fig. 1B). The increase in size of the H-CSNPs could be due to the encapsulation of harpin in the CSNPs. Having a sufficient zeta potential is extremely important for the role of nanoparticles as carriers for drugs or proteins; the nanoparticles must be capable of ionically holding active molecules or biomolecules. Nanoparticles with Zeta Potential values greater than +25 mV or less than -25 mV typically have high degrees of stability. Zeta potential of CSNPs and H-CSNPs showed +32mV and +49mV, respectively.
[060] Referring to the FTIR spectra represented in figures 2A - 2E, several characteristic absorbances were identified in the FT-IR spectrum of pure CS (Fig. 2A). At 3452 cm¯1, the characteristic peak of the hydroxyl group (OH) was recorded, overlapped with N–H stretch. At 1645 cm¯1 the characteristic peaks of CS with high intensity that corresponds to the vibration of amide I. At 1384cm¯1 the C–N stretch was recorded and finally at 1093 cm¯1 the peak of C–O stretch group appeared. In the spectrum of CSNPs prepared with TPP (without any protein), the amino group absorption shifted at 1640 cm¯1, which is an indication that these groups interacted with TPP creating ionic bonds (Fig. 2C ). Furthermore, the absorption band at 1563 cm¯1 that was assigned to the protonated amino groups of CS at pH 5.5. The phosphate absorption band at 1212 cm¯1 in TPP shifted to 1210 cm¯1 in CSNPs, an indication of conjugation with CS. At 1021 cm¯1 and 904 cm¯1 characteristic peaks of C-N stretch and O-H bend, respectively appeared in CSNPs. Many characteristic peaks of harpin shifted to different wave numbers when it was entrapped in the NPs (Fig. 2E), suggesting strong interaction between harpin and NPs matrix. Some other differences were also recorded at the wave numbers 1649 cm¯1 that shifted to 1666 cm¯1 and at 1454 cm¯1 which shifted to 1413 cm¯1. Absorption band of harpin at 1398 cm¯1assigned to C-N stretch shifted to 1345 cm¯1 and 1063 cm¯1 of C-O stretch shifted to 1095 cm¯1 in H-CSNPs.
3.2. Stability of nanoparticles:
3.2.1. Size and Zeta potential
[061] Stability of the nanoparticles was evaluated over a period of 90 days. It was observed that there was an increase in size with slight agglomeration (Fig. 3A and 3B). The agglomeration of NPs is due to decrease in charge with time which enables enhanced interactions between the particles after storage of CSNPs for 90 days.
3.2.2. Entrapment efficiency and In vitro drug release studies
[062] Entrapment efficiency of the NPs increased with increase in the concentration of the protein up to an optimum level (150 µg ml-1) that later decreased with increase in protein concentration (Fig. 4A). Highest encapsulation efficiency of CSNPs was 90%. From the electrophoretic analysis of the protein, a small amount of protein was detected in the supernatant i.e. free protein fraction. However, maximum protein entrapped within the NPs could be detected in the pellet fraction of bound protein (Fig. 4C). To determine the shelf life of CSNPs, the encapsulation efficiency was calculated for H-CSNPs for every 30 days, till 3 months. The encapsulation efficiency of CSNPs decreased sharply after 30 days but remained moderately stable thereafter up to 90 days (Fig. 4B).
[063] The in vitro release of harpinPss from the CSNPs occurred in two phases. In the first phase, 30% of harpinPss was burst released (Fig. 5), while in the second phase, it was slowly released up to 120 h, resulting in a cumulative harpinPss release. A sequential time frame of SEM images of H-CSNPs during the release confirmed that the H-CSNPs maintained their shape, size and integral structure up to 6 h (Fig. 6). The H-CSNPs had an increased particle size due to swelling after 6 h, and by 12 h the NPs lost shape, a process that progressed till 96 h. At 96 h, there was a distinctive pattern of the structural disintegration of NPs into fractions of smaller particles, with the loss of compact structure.
3.3. Defense Responses
3.3.1. Micro- and macroscopic changes in tomato leaves following harpin and H-CSNPs treatments
[064] The composition comprising harpin-CSNPs has synergistic effects as seen by the following examples. H-CSNPs were infiltrated into tomato leaves to see whether there was difference in their ability to induce cell death due to the encapsulation of harpinPss in CSNPs. In the leaves infiltrated with H-CSNPs, cell death symptoms developed faster. The size of the necrotic lesion was larger than in the harpinPss, followed by CSNPs treatment. There was no cell death in control leaves. Simultaneous detection of H2O2 and cell death revealed that ROS accumulation and cell death were enhanced in the H-CSNPs infiltration, compared with with harpinPss treatment. It was evident that cellular H2O2 accumulation and occurrence of cell death colocalized, and that H2O2 accumulation preceded cell death (Fig. 7).
3.3.2. Disease inhibition enhanced by harpin after encapsulated in CSNPs
[065] To study the role of harpinPss and H-CSNPs in reducing the severity of disease caused by Rhizoctonia solani in tomato, we performed detached leaf bioassays. When tomato leaves treated with harpinPss and H-CSNPs were inoculated with R. solani, plants were highly tolerant to the fungus. In H-CSNPs treated leaves the severity of infection was less. In contrast, plants treated with buffer were highly susceptible to the fungus (Figure. 8). Rhizoctonia growth ceased in H-CSNPs treated tomato leaves. Upon extended incubation, the fungus failed to colonize and infect tomato leaves. In contrast, the fungus completely colonized and macerated leaf tissue during the same time in control leaves.
3.3.3. Time Course Studies of biochemical responses
[066] Enzymes like peroxidase and PAL were evaluated to compare the effect of harpin, when loaded in the NPs, in inducing the defense response in tomato. POD activity increased sharply and peak was observed at 24h in case of all three treatments. However, harpin could increase POD activity maximum at 24h followed by CSNPs and H-CSNPs (Fig. 9A). POD activity registered a sharp declining trend after 24h in case of harpin, almost reaching the activity level of control plants at 96h. However, sustain increased level of POD activity was observed for CSNPs and H-CSNPs up to 48h and 72h respectively. Like POD, PAL activity also increased sharply at 24h for harpin and gradually decreased up to 96h (Fig. 9B). But in case of CSNPs and H-CSNPs the activity reached maximum at 48h and 72h respectively and then found decreasing.
3.4. Transcriptional changes in tomato during elicitors, nanoparticles and elicitor loaded nanoparticles treatment
[067] We studied the transcriptome changes in tomato against treatment with harpin, CSNPs and H-CSNPs. We used the GeneChip ® Tomato Genome Array (Agilent) to measure and compare the difference in transcript accumulation (44,000 probes) between treated- and control tomato leaves at 24, 48 and 72 hpi. Two independent replications of the experiment were conducted. All GeneChip data were analyzed using GeneSpring GX v12 software.
[068] Tomato genes that were differentially regulated (more than two-fold change with a P = 0.05) upon all three treatments were identified at 24, 48 and 72 hpi, using samples harvested at 0 hpi as reference samples, to which the samples from all other time points were compared. In the harpin treatment, the number of up-regulated genes was 634, 388 and 263 at 24, 48 and 72 hpi, respectively, while the number of down-regulated genes was 441, 248 and 542 at 24, 48 and 72 hpi, respectively. In CSNP treatment of tomato, the number of genes up-regulated was 476, 582 and 375 at 24, 48 and 72 hpi, respectively. The number of genes down-regulated was 362, 673 and 389 at 24, 48 and 72 hpi, respectively. In H-CSNP treatment of tomato, the number of genes up-regulated was 862, 277 and 209 at 24, 48 and 72 hpi, respectively. The number of genes down-regulated was 764, 288 and 242 genes at 24, 48 and 72 hpi, respectively. Only genes with high levels of significance (P < 0.05) and a minimum absolute value of log2 > 2 were systematically considered, to minimize the false positive as up- or down-regulated.
[069] We performed an orthology prediction (majorly with Solanum tuberosum) for all gene probe sets that can be probed by the GeneChip and assigned gene ontology annotation to all genes identified as differentially regulated transcripts. These were used to identify the major differentially regulated biological processes. The genes with significant expression changes were observed were tentatively arranged in six functional groups (Fig. 10) implicated in defense response (I), signal transduction (II), transport (III), transcription (IV), Photosynthesis and Housekeeping (V) and Aromatics biosynthesis (VI).
3.5. Confocal laser scanning microscopy
[070] To locate the targeting site of harpin and CSNPs in plant tissue, hrpZ was translationally fused with GFP and encapsulated into RCSNPs. GFP alone was considered as a control. From the confocal data it was found that GFP alone is targeting to cytoplasm. Whereas a strong GFP signal was prominently found at chloroplast in GFP-HrpZ treated leaves, which was confirmed by detecting the chloroplast auto fluorescence (Fig 11a). Overlay with chloroplast indicated the strong and specific localization of harpinPss in chloroplast. In case of GH-CSNPs treated leaves GFP and Rhodamine fluorescence could be visible at chloroplast along with the plasma membrane (Fig 11b) indicating localization of harpin and CSNPs in chloroplasts.
4. Discussion
[071] Harpins induce the systemic acquired resistance, as an “immune system booster”. EDEN Bioscience developed the commercial product Messenger® using Harpin. Though the mechanism by which the harpins affect plants has not been studied thoroughly, most researchers agree with the following process: Harpin binds with receptors on the plant cell membrane (Lee et al., 2001), initiates an intracellular signaling cascade that results in the activation of non-specific defense responses such as enhancing phenylalanine ammonia lyase (PAL), peroxidase activity, and inducing pathogenesis-related (PR) genes expression, cell programmed death, ions flux across membranes etc. The effect of harpin was often proportionate to concentration.
[072] The bioavailability of harpin would be low in the normal spray application because it can hardly pass through epidermis or penetrate the cell. Even when harpin enters mesophyll through wounds on leaves by infiltration, there would still be difficulties on pervasion. We hypothesized that nanoparticles could help harpinPss pervade through epidermis or penetrate the cell wall and then release harpin continuously in situ. Such pervasion will extend the duration of harpin effect due to its bioavailability over a longer duration. Our data suggests that the encapsulation of harpinPss resulted in a prolonged effect and enhanced bioavailability and thus will avoid the need for repeated application. Improved bioavailability of harpin (from Pseudomonas syringae pv. tomato) was shown in tobacco using PLGA (poly D, L-lactide-co-glycolide)-based NPs with a change of PAL activity and PR-5dB expression. But the use of CSNPs would be significant to develop better nanoscale protein elicitor pesticide. CSNPs produced using various techniques can be employed to deliver pesticides and fertilizers, as well as genes. The ionic interaction between CS and TPP was dependent on the solution pH, owing to the variation in ionization degree of CS and TPP. The pH of CS solution may affect protein interaction and encapsulation. The CS molecular chain is fully extended in solution at pH 5.5 because of electrostatic expulsion between amine groups present along the molecular chain. It was evident that the increase in CS/TPP ratio resulted in the generation of NPs with smaller size. Furthermore, an optimum CS/TPP ratio of 5/1 (w/w), was observed to give a maximum yield of mono-disperse NPs. The CS/TPP 5/1 weight ratio lead to the most efficient cross-linking of amino groups producing the most compact particle structure (Zhang et al., 2004). In the present study, the particle size of the H-CSNPs was affected by loading of harpin (Fig. 1). The nature of interactions between the protein and CS or TPP was established with FT-IR spectroscopy since the physicochemical interactions like the formation of hydrogen bonds between the drugs and CS or TPP, will automatically lead to frequency shifts in absorption peaks (Papadimitriou et al., 2008). Shifts of characteristic peaks of amino groups in the FT-IR spectra of CSNPs indicated that these groups interacted with TPP, creating ionic bonds (Fig. 2). Many characteristic peaks of harpinPss shifted to different wave numbers when it was entrapped in the NPs, suggesting a strong interaction between harpinPss and NPs matrix.
[073] The CSNPs synthesized by ionotropic gelation lose their integrity in aqueous media. Most protein release profiles from CSNPs exhibit an initial burst release, apparently from the particle surface, followed by a sustained release driven by diffusion of drug through the polymer wall and polymer erosion. The burst is more likely a consequence of rapid surface desorption of large amounts of protein molecules from a huge specific surface area provided by large numbers of particles at nanoscale (Gan and Wang, 2007). We observed a similar pattern of harpinPss release from the NPs in a biphasic pattern, characterized by an initial burst release period followed by a period of slower release. The initial fast release might be the result of the rapid desorption of harpinPss located on the surface of the NPs. After the burst release, the rate of release changed to sustained diffusion through the matrix. This is in agreement with a study of CSNPs containing lysozyme that was attributed to the increased porosity of the NPs generated during drug dissolution.
[074] Harpin proteins from different sources are reported to protect crop plants by inducing defense responses. The infiltration or spray of harpin protein on to the leaves of non-host plants triggers disease resistance–associated responses, such as HR, transcript accumulation of pathogenesis-related (PR) protein genes, and SAR (Dong et al., 1999). Many approaches have been followed to induce resistance against certain plant viruses using proteinaceous and non-proteinaceous molecules. Cucumber plants treated with harpin protein exogenously were protected from bacterial, fungal and viral infection (Strobel et al., 1996). Harpin-induced resistance protected the cucumber plants from anthracnose, bacterial angular leaf spot and tobacco necrosis virus infections. Transgenic tobacco plants expressing harpin gene remarkably reduced the severity of TMV infection (Peng et al 2004). We have, therefore, treated the tomato plants with harpin/H-CSNPs and evaluated the response for resistance against a fungal pathogen. Pre-treatment with harpin to inoculation revealed that harpin-induced resistance against and is enhanced by encapsulation of harpin in CSNPs. The plants that are treated with H-CSNPs, resisted to Rizoctona Solani infection to a longer period (4-5 weeks) when compared with harpin.
[075] To gain insight into, that how tomato plant responds to H-CSNPs application and how this response leads to resistance, we have analysed the whole transcriptome of tomato induced by harpin, H-CSNPs, CSNPs. Surprisingly, combinational treatment of H-CSNPs resulted alteration of comparatively large number of transcripts among other treatments. Harpin or H-CSNPs treatment of tomato plants resulted in massive changes in gene expression, causing transcript accumulation or depletion. Down-regulation was most prominent for genes related to photosynthesis and transport. The down-regulation of these genes may be related to the senescence-promoting activity of JA. JA is known to inhibit the biosynthesis of photosynthetic pigments and photosynthetic activity.
[076] The penetration of NPs through the plant cell wall also provides great opportunities to explore the potential of using nanomaterials as vehicles for the delivery of DNA and proteins into plant cells as is done routinely in mammalian cells (Du et al., 2014). Early attempts using the mesoporous silica nanoparticles system demonstrated DNA and chemical transport into isolated plant cells and intact leaves. The reality that nanoparticles passed through the epidermal cell wall opens up the possible ways of nanotechnology application for agricultural production. Known special characteristics of the epidermic outer cell wall, specifically its considerable thickness, and the presence of protective waxes, a possible particle penetration point could be through the stomata. Interestingly, water-suspended 43 nm hydrophilic particles have been described as infrequently penetrating Vicia faba leaves through stomatal pores (Eichert et al., 2008). In this study we have shown H-CSNPs entry into plant cells and localization in sub cellular organelle, i.e. chloroplast. Further studies for a better understanding of nanoparticles-plant cell interaction will lead to the development of novel tools for Agricultural development.
[077] Although the present disclosure has been described in terms of certain preferred embodiments and illustrations thereof, other embodiments and modifications to preferred embodiments may be possible that are within the principles and spirit of the invention. The above descriptions and figures are therefore to be regarded as illustrative and not restrictive.

[078] Thus the scope of the present disclosure is defined by the appended claims and includes both combinations and sub combinations of the various features described herein above as well as variations and modifications thereof, which would occur to persons skilled in the art upon reading the foregoing description.

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