Plant Expression of Trans-Encapsidated Chimeric Viral Vaccines with Animal RNA Replicons: An Update.

In this protocol, we outline how to produce a chimeric viral vaccine in a biosafety level 1 (BSL1) environment. An animal viral vector RNA encapsidated with tobacco mosaic virus (TMV) coat protein can be fully assembled in planta. Agrobacterium cultures containing each component are inoculated together into tobacco leaves and the self-assembled hybrid chimeric viral vaccine is harvested 4 days later and purified with a simple PEG precipitation. The viral RNA delivery vector is derived from the BSL1 insect virus, Flock House virus (FHV), and replicates in human and animal cells but does not spread systemically. A polyethylene glycol purification protocol is also provided to collect and purify these vaccines for immunological tests. In this update, we also provide a protocol for in trans co-inoculation of a modified FHV protein A, which significantly increased the yield of in planta chimeric viral vaccine.

A putative role for the tobacco mosaic virus in smokers' resistance to COVID-19.

Reports from various countries suggest that tobacco smoking might protect from SARS-CoV-2 infection, since the prevalence of smoking in COVID-19 hospitalized patients is lower than in the respective general population. Apart from nicotine or other chemicals contained in tobacco smoke, we propose that a single-stranded RNA virus that infects tobacco leaves, tobacco mosaic virus (TMV), might be implicated in this effect. TMV, though non-pathogenic, is found in smokers' airways, and stimulates adaptive and innate immunity, with release of specific antibodies and interferons. The latter may have preventive and/or therapeutic effects against COVID-19. If confirmed by epidemiological and interventional studies, this might lead to the use of TMV as an immunological adjuvant against SARS-CoV-2 infection and COVID-19 disease.

Complexes Formed via Bioconjugation of Genetically Modified TMV Particles with Conserved Influenza Antigen: Synthesis and Characterization.

Recently we obtained complexes between genetically modified Tobacco Mosaic Virus (TMV) particles and proteins carrying conserved influenza antigen such as M2e epitope. Viral vector TMV-N-lys based on TMV-U1 genome was constructed by insertion of chemically active lysine into the exposed N-terminal part of the coat protein. Nicotiana benthamiana plants were agroinjected and TMV-N-lys virions were purified from non-inoculated leaves. Preparation was analyzed by SDS-PAGE/Coomassie staining; main protein with electrophoretic mobility of 21 kDa was detected. Electron microscopy confirmed the stability of modified particles. Chemical conjugation of TMV-N-lys virions and target influenza antigen M2e expressed in E. coli was performed using 5 mM 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide and 1 mM N-hydroxysuccinimide. The efficiency of chemical conjugation was confirmed by Western blotting. For additional characterization we used conventional electron microscopy. The diameter of the complexes did not differ significantly from the initial TMV-N-lys virions, but complexes formed highly organized and extensive network with dense "grains" on the surface. Dynamic light scattering demonstrated that the single peaks, reflecting the complexes TMV-N-lys/DHFR-M2e were significantly shifted relative to the control TMV-N-lys virions. The indirect enzyme-linked immunosorbent assay with TMV- and DHFR-M2e-specific antibodies showed that the complexes retain stability during overnight adsorption. Thus, the results allow using these complexes for immunization of animals with the subsequent preparation of a candidate universal vaccine against the influenza virus.

Characterization of Rhodopseudomonas palustris population dynamics on tobacco phyllosphere and induction of plant resistance to Tobacco mosaic virus.

Although many biocontrol bacteria can be used to improve plant tolerance to stresses and to promote plant growth, the hostile environmental conditions on plant phyllosphere and the limited knowledge on bacterial colonization on plant phyllosphere minimized the beneficial effects produced by the biocontrol bacteria. Rhodopseudomonas palustris strain GJ-22 is known as a phyllosphere biocontrol agent. In this paper, we described detailed processes of strain GJ-22 colony establishment at various colonization stages. Four different types of bacterial colonies, Type 1, scattered single cells; Type 2, small cell clusters; Type 3, small cell aggregates; and Type 4, large cell aggregates, were observed in the course of bacterial colonization. We categorized bacterial colonization into four phases, which were, Phase I: bacterial colony exists as Type 1 and cell population reduced quickly; Phase II: Type 1 evolved into Type 2 and cell population remained steady; Phase III: Type 3 arose and replaced Type 2, and cell population expanded slowly; and Phase IV: Type 3 matured into Type 4 and cell population increased quickly. We have shown that the preferable location sites of bacterial aggregates on leaf phyllosphere are grooves between plant epidermal cells. Analyses of expressions of plant defence-related genes showed that, starting from Phase III, bacterial cells in the Type 3 and Type 4 colonies produced unidentified signals to induce host defence against Tobacco mosaic virus infection. In addition, we determined the crucial role of aggregates formation of GJ-22 cell on plant phyllosphere in terms of bacterial cell stress tolerance and ISR (induced systemic resistance) priming. To our knowledge, this is the first report focused on the colonization process of a phyllosphere biocontrol agent and gave a clear description on the morphological shift of bacterial colony on phyllosphere.

Comparative transcriptome profiling uncovers a Lilium regale NAC transcription factor, LrNAC35, contributing to defence response against cucumber mosaic virus and tobacco mosaic virus.

Cucumber mosaic virus (CMV) is a highly prevalent viral pathogen causing substantial damage to the bulb and cut-flower production of Lilium spp. Here, we performed an Illumina RNA sequencing (RNA-Seq) study on the leaf tissues of a virus-resistant species Lilium regale inoculated with mock control and CMV. A total of 1346 differentially expressed genes (DEGs) were identified in the leaves of L. regale upon CMV inoculation, which contained 34 up-regulated and 40 down-regulated DEGs that encode putative transcription factors (TFs). One up-regulated TF, LrNAC35, belonging to the NAM/ATAF/CUC (NAC) superfamily, was selected for further functional characterization. Aside from CMV, lily mottle virus and lily symptomless virus infections provoked a striking increase in LrNAC35 transcripts in both resistant and susceptible Lilium species. The treatments with low temperature and several stress-related hormones activated LrNAC35 expression, contrary to its reduced expression under salt stress. Ectopic overexpression of LrNAC35 in petunia (Petunia hybrida) resulted in reduced susceptibility to CMV and Tobacco mosaic virus infections, and enhanced accumulation of lignin in the cell walls. Four lignin biosynthetic genes, including PhC4H, Ph4CL, PhHCT and PhCCR, were found to be up-regulated in CMV-infected petunia lines overexpressing LrNAC35. In vivo promoter-binding tests showed that LrNAC35 specifically regulated the expression of Ph4CL. Taken together, our results suggest a positive role of transcriptome-derived LrNAC35 in transcriptional modulation of host defence against viral attack.

TurboID-based proximity labeling reveals that UBR7 is a regulator of N NLR immune receptor-mediated immunity.

Nucleotide-binding leucine-rich repeat (NLR) immune receptors play a critical role in defence against pathogens in plants and animals. However, we know very little about NLR-interacting proteins and the mechanisms that regulate NLR levels. Here, we used proximity labeling (PL) to identify the proteome proximal to N, which is an NLR that confers resistance to Tobacco mosaic virus (TMV). Evaluation of different PL methods indicated that TurboID-based PL provides more efficient levels of biotinylation than BioID and BioID2 in plants. TurboID-based PL of N followed by quantitative proteomic analysis and genetic screening revealed multiple regulators of N-mediated immunity. Interestingly, a putative E3 ubiquitin ligase, UBR7, directly interacts with the TIR domain of N. UBR7 downregulation leads to an increased amount of N protein and enhanced TMV resistance. TMV-p50 effector disrupts the N-UBR7 interaction and relieves negative regulation of N. These findings demonstrate the utility of TurboID-based PL in plants and the N-interacting proteins we identified enhance our understanding of the mechanisms underlying NLR regulation.

Induced Resistance Mechanism of Novel Curcumin Analogs Bearing a Quinazoline Moiety to Plant Virus.

Plant immune activators can protect crops from plant virus pathogens by activating intrinsic immune mechanisms in plants and are widely used in agricultural production. In our previous work, we found that curcumin analogs exhibit excellent biological activity against plant viruses, especially protective activity. Inspired by these results, the active substructure of pentadienone and quinazoline were spliced to obtain curcumin analogs as potential exogenously induced resistant molecule. Bioassay results showed that compound A13 exhibited excellent protective activity for tobacco to against Tobacco mosaic virus (TMV) at 500 µg/mL, with a value of 70.4 ± 2.6% compared with control treatments, which was better than that of the plant immune activator chitosan oligosaccharide (49.0 ± 5.9%). The protective activity is due to compound A13 inducing tobacco resistance to TMV, which was related to defense-related enzymes, defense-related genes, and photosynthesis. This was confirmed by the up-regulated expression of proteins that mediate stress responses and oxidative phosphorylation.

Assessment of structurally modified plant virus as a novel adjuvant in toxicity studies.

Spherical particles (SPs) generated by thermally denatured tobacco mosaic virus (TMV) coat protein can act as an adjuvant, as they are able to enhance the magnitude and longevity of immune responses to different antigens. Here, the toxicity of TMV SPs was assessed prior to it being offered as a universal safe adjuvant for the development of vaccine candidates. The evaluation included nonclinical studies of a local tolerance following the single administration of TMV SPs, and of the local and systemic effects following repeated administrations of TMV SPs. These were conducted in mice, rats and rabbits. General health status, haematology and blood chemistry parameters were monitored on a regular basis. Also, reproductive and development toxicity were studied. No significant signs of toxicity were detected following single or repeated administrations of the adjuvant (TMV SPs). The absence of toxicological effects following the injection of TMV SPs is promising for the further development of recombinant vaccine candidates with TMV SPs as an adjuvant.

A DREPP protein interacted with PeaT1 from Alternaria tenuissima and is involved in elicitor-induced disease resistance in Nicotiana plants.

PeaT1 is a proteinaceous elicitor from fungal pathogen Alternaria tenuissima. Our previous research revealed that this elicitor could induce defense response and enhance disease resistance in various plants including Nicotiana plants. However, immune activation mechanisms whereby PeaT1 elicits defense response remain unclear. In this study, the association between elicitor protein PeaT1 and the plasma membrane was assessed using the FITC (Fluorescein isothiocyanate) labeling method. A PeaT1-interacting protein was isolated via 125I-PeaT1 cross-linking and Far Western blot analyses, and designated PtBP1 (PeaT1 Binding Protein 1). From the data of Mass spectrometry (MS) and bioinformatics analysis, the 22 kDa plasma membrane protein PtBP1 was inferred to be a member of DREPP (developmentally regulated plasma membrane polypeptide) family that is induced in plants under stress conditions and might get involved in downstream signaling. For further verification of this association, Far Western blot, co-immunoprecipitation and bimolecular fluorescence complementation (BiFC) analyses were performed, showing PtBP1 could bind with PeaT1 in vitro and in vivo. Virus-induced gene silencing (VIGS) analysis exhibited that PtBP1 silencing in Nicotiana benthamiana attenuated tobacco mosaic virus (TMV) resistance compared to the tobacco rattle virus (TRV) control after PeaT1 treatment.

Chimeric Virus as a Source of the Potato Leafroll Virus Antigen.

Large quantities of potato leafroll virus (PLRV) antigen are difficult to obtain because this virus accumulates in plants at a low titer. To overcome this problem, we constructed a binary vector containing chimeric cDNA, in which the coat protein (CP) gene of the crucifer infecting tobacco mosaic virus (crTMV) was substituted for the coat protein gene of PLRV. The PLRV movement protein (MP) gene, which overlaps completely with the CP gene, was doubly mutated to eliminate priming of the PLRV MP translation from ATG codons with no changes to the amino acid sequence of the CP. The untranslated long intergenic region located upstream of the CP gene was removed from the construct. Transcribed powerful tobamovirus polymerase of the produced vector synthesized PLRV CP gene that was, in turn, translated into the protein. CP PLRV packed RNAs from the helical crTMV in spherical virions. Morphology, size and antigenic specificities of the wild-type and chimeric virus were similar. The yield of isolated chimera was about three orders higher than the yield of native PLRV. The genetic manipulations facilitated the generation of antibodies against the chimeric virus, which recognize the wild-type PLRV.

Plant Expression of Trans-Encapsidated Viral Nanoparticle Vaccines with Animal RNA Replicons.

In this protocol, we outline how to produce a live viral nanoparticle vaccine in a biosafety level 1 (BSL1) environment. An animal viral vector RNA encapsidated with tobacco mosaic virus (TMV) coat protein can be fully assembled in planta. Agrobacterium cultures containing each component are inoculated together into tobacco leaves and the self-assembled hybrid nanoparticle vaccine is harvested 4 days later and purified with a simple PEG precipitation. The viral RNA delivery vector is derived from the BSL1 insect virus, Flock House virus (FHV), and replicates in human and animal cells but does not spread systemically. A polyethylene glycol purification protocol is also provided to collect and purify these vaccines for immunological tests.

Intranasal delivery of a protein subunit vaccine using a Tobacco Mosaic Virus platform protects against pneumonic plague.

Yersinia pestis, one of history's deadliest pathogens, has killed millions over the course of human history. It has attributes that make it an ideal choice to produce mass casualties and is a prime candidate for use as a biological weapon. When aerosolized, Y. pestis causes pneumonic plague, a pneumonia that is 100% lethal if not promptly treated with effective antibiotics. Currently, there is no FDA approved plague vaccine. The current lead vaccine candidate, a parenterally administered protein subunit vaccine comprised of the Y. pestis virulence factors, F1 and LcrV, demonstrated variable levels of protection in primate pneumonic plague models. As the most likely mode of exposure in biological attack with Y. pestis is by aerosol, this raises a question of whether this parenteral vaccine will adequately protect humans against pneumonic plague. In the present study we evaluated two distinct mucosal delivery platforms for the intranasal (IN) administration of LcrV and F1 vaccine proteins, a live bacterial vector, Lactobacillus plantarum, and a Tobacco Mosaic Virus (TMV) based delivery platform. IN administration of L. plantarum expressing LcrV, or TMV-conjugated to LcrV and F1 (TMV-LcrV+TMV-F1) resulted in the similar induction of high titers of IgG antibodies and evidence of proinflammatory cytokine secretion. However, only the TMV-conjugate delivery platform protected against subsequent lethal challenge with Y. pestis. TMV-LcrV+TMV-F1 co-vaccinated mice had no discernable morbidity and no mortality, while mice vaccinated with L. plantarum expressing LcrV or rLcrV+rF1 without TMV succumbed to infection or were only partially protected. Thus, TMV is a suitable mucosal delivery platform for an F1-LcrV subunit vaccine that induces complete protection against pneumonic infection with a lethal dose of Y. pestis in mice.

Chitosan oligosaccharide induces resistance to Tobacco mosaic virus in Arabidopsis via the salicylic acid-mediated signalling pathway.

Chitosan is one of the most abundant carbohydrate biopolymers in the world, and chitosan oligosaccharide (COS), which is prepared from chitosan, is a plant immunity regulator. The present study aimed to validate the effect of COS on inducing resistance to tobacco mosaic virus (TMV) in Arabidopsis and to investigate the potential defence-related signalling pathways involved. Optimal conditions for the induction of TMV resistance in Arabidopsis were COS pretreatment at 50 mg/L for 1 day prior to inoculation with TMV. Multilevel indices, including phenotype data, and TMV coat protein expression, revealed that COS induced TMV resistance in wild-type and jasmonic acid pathway- deficient (jar1) Arabidopsis plants, but not in salicylic acid pathway deficient (NahG) Arabidopsis plants. Quantitative-PCR and analysis of phytohormone levels confirmed that COS pretreatment enhanced the expression of the defence-related gene PR1, which is a marker of salicylic acid signalling pathway, and increased the amount of salicylic acid in WT and jar1, but not in NahG plants. Taken together, these results confirm that COS induces TMV resistance in Arabidopsis via activation of the salicylic acid signalling pathway.

A Novel Protein Elicitor (PeBA1) from Bacillus amyloliquefaciens NC6 Induces Systemic Resistance in Tobacco.

Here we reported a novel protein elicitor from Bacillus amyloliquefaciens NC6 induced systemic resistance (ISR) in tobacco. The purification was executed by ion-exchange chromatography, native-page extraction and HPLC, and the amino acid sequence was identified by mass spectrometry. This recombinant elicitor protein, expressed in Escherichia coli by an E1 expression vector, had good thermal stability, and the elicitor caused a clearly defined hypersensitive response (HR) necrosis in tobacco leaves. It could also trigger early defence events, including generation of reactive oxygen species (H2O2 and O2 (-)) and phenolic-compound accumulation. Quantitative real-time PCR (Q-RT-PCR) results indicated that several plant defence genes, including the salicylic acid (SA)-responsive PR1a, PR1b, PR5, and phenylalanine ammonia lyase (PAL), as well as the jasmonic acid (JA)-responsive PDF1.2 and CORONATINE INSENSITIVE 1 (COI1), were all up-regulated. Moreover, infiltration conferred systemic resistance against a broad spectrum of pathogens, including Tobacco mosaic virus (TMV) and the fungal pathogen Botrytis cinerea.

Serum albumin 'camouflage' of plant virus based nanoparticles prevents their antibody recognition and enhances pharmacokinetics.

Plant virus-based nanoparticles (VNPs) are a novel class of nanocarriers with unique potential for biomedical applications. VNPs have many advantageous properties such as ease of manufacture and high degree of quality control. Their biocompatibility and biodegradability make them an attractive alternative to synthetic nanoparticles (NPs). Nevertheless, as with synthetic NPs, to be successful in drug delivery or imaging, the carriers need to overcome several biological barriers including innate immune recognition. Plasma opsonization can tag (V)NPs for clearance by the mononuclear phagocyte system (MPS), resulting in shortened circulation half lives and non-specific sequestration in non-targeted organs. PEG coatings have been traditionally used to 'shield' nanocarriers from immune surveillance. However, due to broad use of PEG in cosmetics and other industries, the prevalence of anti-PEG antibodies has been reported, which may limit the utility of PEGylation in nanomedicine. Alternative strategies are needed to tailor the in vivo properties of (plant virus-based) nanocarriers. We demonstrate the use of serum albumin (SA) as a viable alternative. SA conjugation to tobacco mosaic virus (TMV)-based nanocarriers results in a 'camouflage' effect more effective than PEG coatings. SA-'camouflaged' TMV particles exhibit decreased antibody recognition, as well as enhanced pharmacokinetics in a Balb/C mouse model. Therefore, SA-coatings may provide an alternative and improved coating technique to yield (plant virus-based) NPs with improved in vivo properties enhancing drug delivery and molecular imaging.

iTRAQ protein profile analysis provides integrated insight into mechanisms of tolerance to TMV in tobacco (Nicotiana tabacum).

UNLABELLED: To further investigate the mechanism of the plant tolerance to tobacco mosaic virus (TMV) infection, tobacco NC89 (N) hypersensitive to TMV and its natural mutant Yuyan8 (Y) with tolerance to TMV were employed for differential accumulation proteome analysis. There were 260 specifically accumulated proteins in Yuyan8 after 24 h inoculation (Yd), and the accumulations of 285 proteins inherent in Y have changed after TMV infection. Equally, there were 183 specifically accumulated proteins in NC89 after 24 h inoculation (Nd), and 132 proteins inherent in N have changed after TMV infection. These differential proteins were respectively enriched in two pathways, of which photosynthesis pathway was the common pathway in two varieties. In photoreaction system, the accumulations of differential proteins, especially D1 protein, were not decreased in Yd compared to Nd. The results indicated that maintaining the stability of D1 protein and reasonable utilization of the energy was the essential for tolerance to TMV infection. It was also revealed that 14-3-3 protein and PR4 was specific expressed, and the expression of LRR was enhanced in Yd, suggesting that regulation of defense protein mediated by 14-3-3 protein quickly activated resistance system and enhanced the plant tolerance to TMV infection. SIGNIFICANCE: This is the first work that the molecular basis of tobacco tolerance was discussed basic on proteomic investigation performed on wild type and its natural mutant. Our results lay the foundation for development of molecular breeding and further proteome research in tobacco.

Tobacco mosaic virus efficiently targets DC uptake, activation and antigen-specific T cell responses in vivo.

Over the past 20 years, dendritic cells (DCs) have been utilized to activate immune responses capable of eliminating cancer cells. Currently, ex vivo DC priming has been the mainstay of DC cancer immunotherapies. However, cell-based treatment modalities are inherently flawed due to a lack of standardization, specialized facilities and personnel, and cost. Therefore, direct modes of DC manipulation, circumventing the need for ex vivo culture, must be investigated. To facilitate the development of next-generation, in vivo targeted DC vaccines, we characterized the DC interaction and activation potential of the Tobacco Mosaic virus (TMV), a plant virus that enjoys a relative ease of production and the ability to deliver protein payloads via surface conjugation. In this study we show that TMV is readily taken up by mouse bone marrow-derived DCs, in vitro. Footpad injection of fluorophore-labeled TMV reveals preferential uptake by draining lymph node resident DCs in vivo. Uptake leads to activation, as measured by the upregulation of key DC surface markers. When peptide antigen-conjugated TMV is injected into the footpad of mice, DC-mediated uptake and activation leads to robust antigen-specific CD8(+) T cell responses, as measured by antigen-specific tetramer analysis. Remarkably, TMV priming induced a greater magnitude T cell response than Adenovirus (Ad) priming. Finally, TMV is capable of boosting either Ad-induced or TMV-induced antigen-specific T cell responses, demonstrating that TMV, uniquely, does not induce neutralizing self-immunity. Overall, this study elucidates the in vivo DC delivery and activation properties of TMV and indicates its potential as a vaccine vector in stand alone or prime-boost strategies.

Influence of high hydrostatic pressure on epitope mapping of tobacco mosaic virus coat protein.

In this study, we investigated the effect of high hydrostatic pressure (HHP) on tobacco mosaic virus (TMV), a model virus in immunology and one of the most studied viruses to date. Exposure to HHP significantly altered the recognition epitopes when compared to sera from mice immunized with native virus. These alterations were studied further by combining HHP with urea or low temperature and then inoculating the altered virions into Balb-C mice. The antibody titers and cross-reactivity of the resulting sera were determined by ELISA. The antigenicity of the viral particles was maintained, as assessed by using polyclonal antibodies against native virus. The antigenicity of canonical epitopes was maintained, although binding intensities varied among the treatments. The patterns of recognition determined by epitope mapping were cross checked with the prediction algorithms for the TMVcp amino acid sequence to infer which alterations had occurred. These findings suggest that different cleavage sites were exposed after the treatments and this was confirmed by epitope mapping using sera from mice immunized with virus previously exposed to HHP.

Complexes assembled from TMV-derived spherical particles and entire virions of heterogeneous nature.

Previously, we described some structural features of spherical particles (SPs) generated by thermal remodelling of the tobacco mosaic virus. The SPs represent a universal platform that could bind various proteins. Here, we report that entire isometric virions of heterogeneous nature bind non-specifically to the SPs. Formaldehyde (FA) was used for covalent binding of a virus to the SPs surface for stabilizing the SP-virus complexes. Transmission and high resolution scanning electron microscopy showed that the SPs surface was covered with virus particles. The architecture of SP-virion complexes was examined by immunologic methods. Mean diameters of SPs and SP-human enterovirus C and SP-cauliflower mosaic virus (CaMV) compositions were determined by nanoparticle tracking analysis (NTA) in liquid. Significantly, neither free SPs nor individual virions were detected by NTA in either FA-crosslinked or FA-untreated compositions. Entirely, all virions were bound to the SPs surface and the SP sites within the SP-CaMV complexes were inaccessible for anti-SP antibodies. Likewise, the SPs immunogenicity within the FA-treated SPs-CaMV compositions was negligible. Apparently, the SP antigenic sites were hidden and masked by virions within the compositions. Previously, we reported that the SPs exhibited adjuvant activity when foreign proteins/epitopes were mixed with or crosslinked to SPs. We found that immunogenicity of entire CaMV crosslinked to SP was rather low which could be due to the above-mentioned masking of the SPs booster. Contrastingly, immunogenicity of the FA-untreated compositions increased significantly, presumably, due to partial release of virions and unmasking of some SPs-buster sites after animals immunization.

Mutational analysis of the Verticillium dahliae protein elicitor PevD1 identifies distinctive regions responsible for hypersensitive response and systemic acquired resistance in tobacco.

In our previous study, PevD1 was characterized as a novel protein elicitor produced by Verticillium dahliae inducing hypersensitive response (HR) and systemic acquired resistance (SAR) in tobacco plants; however, the detailed mechanisms of PevD1's elicitor activity remain unclear. In this study, five mutant fragments of PevD1 were generated by polymerase chain reaction-based mutagenesis and the truncated proteins expressed in Escherichia coli were used to test their elicitor activities. Biological activity analysis showed that the N-terminal and C-terminal of PevD1 had distinct influence on HR and SAR elicitation. Fragment PevD1ΔN98, which spans the C-terminal 57 amino acids of PevD1, was critical for the induction of HR in tobacco plants. In contrast, fragment PevD1ΔC57, the N-terminal of 98 amino acids of PevD1, retained the ability to induce SAR against tobacco mosaic virus (TMV) but not induction of HR, suggesting that the induction of HR is not essential for SAR mediated by PevD1. Our results indicated that fragment PevD1ΔC57 could be a candidate peptide for plant protection against pathogens without causing negative effects.