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Protein-based plant immune inducers are special compounds that can induce plant defense responses. They are mainly derived from pathogenic microorganisms, biocontrol microorganisms, host plants, and host-pathogen interaction processes. Protein elicitors improve plant resistance by triggering plant pathogen-associated molecular patterns to trigger immunity and effectors to trigger immune responses that frequently involve reactive oxygen species, Ca2+, salicylic acid, jasmonic acid, gibberellin, and ethylene cascade signaling pathways. They can bolster plant resistance against bacterial, fungal, and viral diseases, as well as environmental stress. Here we summarize the sources, mechanisms of action, and current applications of protein-based elicitors, identify existing problems, and outline future development trends. We propose that future research should focus on improving the persistence and stability of these elicitors, exploring the combined use of multiple elicitors or their combination with other agents, and their application in plant breeding., aiming for developing green biopesticides using protein-based elicitors. [ABSTRACT FROM AUTHOR]

Hpa1 (a type of harpin) is involved in T3SS (Type III Secretion System) assembly in the infection mechanism by Xanthomonas Oryzae pv. oryzae (Xoo). Hpa1 interacts with the plasma membrane components of plants thereby assisting effector proteins toward the cytoplasm, wherein effectors execute their pathological functions. Independently, harpins also induce hypersensitive response and systemic acquired resistance in plants. However, lack of knowledge regarding the plant–harpin interaction mechanism constrains the pathway of its agricultural application. Although an in vitro study proved that Hpa1 protein can interact with OsPIP1;3, a rice aquaporin, the structural basis of the interaction is yet to be discovered. The presented work is the first of its kind where an in silico approach is used for the PPI (protein–protein interaction) of harpin protein. The study discovered participation of Hpa1 N-terminal amino acids at the interface. Besides, MD simulation studies were performed to assess the stability. RMSD values were 0.35 ± 0.049, 0.73 ± 0.11, and 0.50 ± 0.065 nm for OsPIP1;3, Hpa1, and Hpa1-OsPIP1;3 complex, respectively. Additionally, Residue-wise fluctuations have also been studied post-MDS. Taken together, these findings not only give a solid foundation for a deeper knowledge of various interacting target molecules with Harpin protein orthologs but also bring a new avenue for the structural–functional relationship study of harpin proteins. [ABSTRACT FROM AUTHOR]

Phytopathogens entry into plant cell is restricted by several chemical substances which hamper the natural ecosystem and are expensive. Induction of defense responses in plants by elicitors is a better alternative approach to combat phytopathogens. Controlled and steady release of elicitors from nanoparticles core will improve the bioavailability and alleviate the repeated application of molecules that triggers the hypersensitive response (HR) in plants. Here we reported the efficacy of chitosan-harpin as nanoparticles in eliciting defense responses in tobacco wherein harpin (30 ng/mL) loaded chitosan nanoparticles (H-CSNPs) were prepared by ionotropic gelation method using chitosan and sodium tripolyphosphate (TPP). The nanoparticles were spherical and possess a size of about ∼160 nm with a zeta potential of 30.2 ± 0.7 mV. The FT-IR and XRD studies confirmed the formation of chitosan nanoparticles. The encapsulation efficiency of harpin was 85% and steady release was observed up to 4 h in releasing kinetics. Topical application of H-CSNPs resulted histopathological evidences which coincided with the induction HR specific marker genes viz., pathogenises related proteins and induction of defense enzymes. The harpin at nanogram concentrations with chitosan improved the bioavailability than the harpin or chitosan alone in the tobacco plant.

Red clover, a perennial forage legume, is globally significant because of its high nutritional value and nitrogen fixation capacity. However, one of the disadvantages of red clover is its sensitivity to salinity conditions. The aim of this study was to investigate the effect of the PopW pre-treatment on physiological and molecular responses of red clover under salinity stress conditions. Different salinity levels (0, 30, 60, 90, 120, 150, 180, 210 mM NaCl) were applied to the red clover plants. The results indicated that the PopW pre-treated plants had lower yield/quality losses, and better physiological state than the non-pretreated ones under salinity stress. The PopW pre-treatment enabled to maintain K, Na, and Ca contents in red clover plants under all salinity stress levels, compared to non-pretreated ones (p < 0.01). Moreover, after salinity stress treatment, the increased levels of GR, SOD, crude protein rate, IAA, GA, ABA, JA, stress-related genes and decreased levels of leaf senescence genes were determined in the PopW pre-treated plants, compared to non-pretreated ones (p < 0.01). The findings indicate that endogenous hormone content changes and modulation of stress-related and leaf senescence genes played significant preventive roles against salt stress conditions in the PopW pre-treated red clover plants. The results of this study suggest that the PopW pre-treatment could be utilized for marginal areas having salinity problems in red clover. [ABSTRACT FROM AUTHOR]

In interactions between pathogens and plants, pathogens secrete many molecules that facilitate plant infection, and some of these compounds are recognized by plant pattern recognition receptors (PRRs), which induce immune responses. Molecules in both pathogens and plants that trigger immune responses in plants are termed elicitors. On the basis of their chemical content, elicitors can be classified into carbohydrates, lipopeptides, proteinaceous compounds and other types. Although many studies have focused on the involvement of elicitors in plants, especially on pathophysiological changes induced by elicitors in plants and the mechanisms mediating these changes, there is a lack of up-to-date reviews on the characteristics and functions of proteinaceous elicitors. In this mini-review, we provide an overview of the up-to-date knowledge on several important families of pathogenic proteinaceous elicitors (i.e., harpins, necrosis- and ethylene-inducing peptide 1 (nep1)-like proteins (NLPs) and elicitins), focusing mainly on their structures, characteristics and effects on plants, specifically on their roles in plant immune responses. A solid understanding of elicitors may be helpful to decrease the use of agrochemicals in agriculture and gardening, generate more resistant germplasms and increase crop yields. [ABSTRACT FROM AUTHOR]

Pathogen-associated molecular patterns, PAMPs, are a diverse group of molecules associated with pathogenic microbes and are known to activate immune response and in some cases enhance growth in plants. Two PAMPs, harpin and flg22, have shown these affects in various plant species. PAMPs are known to activate basal immunity, the ethylene signaling pathway, alter gene expression and change plant composition. Pretreatment with harpin enhanced hemp seedling resistance to Pythium aphanidermatum, while flg22 failed to induce the defense mechanism towards P. aphanidermatum. In the absence of the pathogen, both harpin and flg22 enhanced seedling growth when compared to the water control. Ethylene is a hormone involved in both plant defense signaling and growth. Both harpin and flg22 pretreatment induced certain ethylene responsive genes but not all the genes examined, indicating that harpin and flg22 act differently in ethylene and potentially defense signaling. In addition, both harpin and flg22 induced CsFRK1 and CsPR1, two marker genes for plant innate immunity. Both PAMPs can enhance growth but likely induce different defense signaling pathways. [ABSTRACT FROM AUTHOR]

Plant defense elicitors (PDEs) are chemicals that stimulate plant defenses against pathogens and herbivores. Previous work shows that PDEs acibenzolar‐S‐methyl (ASM) and harpinαβ protein (harpin) can induce the pathogenesis‐related gene PR‐1 in plants and suppress herbivorous arthropods. In this study, we tested the potential for these PDEs to induce PR‐1 in pear, Pyrus communis L. (Rosaceae) orchards and suppress pear psylla, Cacopsylla pyricola (Förster) (Hemiptera: Psyllidae), and spider mites, Tetranychus spp. (Acari: Tetranychidae). In 2017, we compared densities of each pest on mature pear trees following a single application of either an ASM product (Actigard; Syngenta), a harpin product (Employ; Plant Health Care), or no PDE treatment in four commercial and two research center orchards. In 2018, we sampled pear psylla and used qPCR to assess PR‐1 induction in pear leaf samples before and after PDE treatments at one commercial orchard. Neither PDE treatment showed evidence of pest suppression in either year, and no differences in PR‐1 expression were detected. Potted greenhouse trees treated with ASM in 2019 showed higher PR‐1 expression relative to untreated trees, verifying that our procedures can detect induction and suggesting that a single PDE application was sufficient to induce PR‐1 in potted but not mature pear trees. We conclude that plant defense elicitors may contribute to pear pest suppression in some contexts, but effects are unlikely to be strong or consistent. Our results highlight the need for field experiments to advance plant defense elicitor knowledge towards effective field applications. [ABSTRACT FROM AUTHOR]

Abstract Background Harpins are proteins secreted by the type III secretion system of Gram-negative bacteria during pathogen–plant interactions that can act as elicitors, stimulating defense and plant growth in many types of non-host plants. Harpin-treated plants have higher resistance, quality and yields and, therefore, harpin proteins may potentially have many valuable agricultural applications. Harpins are characterized by high thermal stability at 100 °C. However, it is unknown whether harpins are still active at temperatures above 100 °C or whether different temperatures affect the activity of the harpin protein in different ways. The mechanism responsible for the heat stability of harpins is also unknown. Results We identified a novel harpin, HpaXpm, from the cassava blight bacteria Xanthomonas phaseoli pv. manihotis HNHK. The predicted secondary structure and 3-D structure indicated that the HpaXpm protein has two β-strand domains and two major α-helical domains located at the N- and C-terminal regions, respectively. A phylogenetic tree generated using the maximum likelihood method grouped HpaXpm in clade I of the Hpa1 group along with harpins produced by other Xanthomonas spp. (i.e., HpaG-Xag, HpaG-Xcm, Hpa1-Xac, and Hpa1Xm). Phenotypic assays showed that HpaXpm induced the hypersensitive response (HR), defense responses, and growth promotion in non-host plants more effectively than Hp1Xoo (X. oryzae pv. oryzae). Quantitative real-time PCR analysis indicated that HpaXpm proteins subjected to heat treatments at 100 °C, 150 °C, or 200 °C were still able to stimulate the expression of function-related genes (i.e., the HR marker genes Hin1 and Hsr203J, the defense-related gene NPR1, and the plant growth enhancement-related gene NtEXP6); however, the ability of heat-treated HpaXpm to induce HR was different at different temperatures. Conclusions These findings add a new member to the harpin family. HpaXpm is heat-stable up to 200 °C and is able to stimulate powerful beneficial biological functions that could potentially be more valuable for agricultural applications than those stimulated by Hpa1Xoo. We hypothesize that the extreme heat resistance of HpaXpm is because the structure of harpin is very stable and, therefore, the HpaXpm structure is less affected by temperature.

The conserved oligomeric Golgi (COG) complex, functioning in retrograde trafficking, is a universal structure present among eukaryotes that maintains the correct Golgi structure and function. The COG complex is composed of eight subunits coalescing into two sub-complexes. COGs1–4 compose Sub-complex A. COGs5–8 compose Sub-complex B. The observation that COG interacts with the syntaxins, suppressors of the erd2-deletion 5 (Sed5p), is noteworthy because Sed5p also interacts with Sec17p [alpha soluble NSF attachment protein (α-SNAP)]. The α-SNAP gene is located within the major Heterodera glycines [soybean cyst nematode (SCN)] resistance locus (rhg1) and functions in resistance. The study presented here provides a functional analysis of the Glycine max COG complex. The analysis has identified two paralogs of each COG gene. Functional transgenic studies demonstrate at least one paralog of each COG gene family functions in G. max during H. glycines resistance. Furthermore, treatment of G. max with the bacterial effector harpin, known to function in effector triggered immunity (ETI), leads to the induced transcription of at least one member of each COG gene family that has a role in H. glycines resistance. In some instances, altered COG gene expression changes the relative transcript abundance of syntaxin 31. These results indicate that the G. max COG complex functions through processes involving ETI leading to H. glycines resistance.

Pathogen-associated molecular patterns, PAMPs, are a diverse group of molecules associated with pathogenic microbes and are known to activate immune response and in some cases enhance growth in plants. Two PAMPs, harpin and flg22, have shown these affects in various plant species. PAMPs are known to activate basal immunity, the ethylene signaling pathway, alter gene expression and change plant composition. Pretreatment with harpin enhanced hemp seedling resistance to Pythium aphanidermatum, while flg22 failed to induce the defense mechanism towards P. aphanidermatum. In the absence of the pathogen, both harpin and flg22 enhanced seedling growth when compared to the water control. Ethylene is a hormone involved in both plant defense signaling and growth. Both harpin and flg22 pretreatment induced certain ethylene responsive genes but not all the genes examined, indicating that harpin and flg22 act differently in ethylene and potentially defense signaling. In addition, both harpin and flg22 induced CsFRK1 and CsPR1, two marker genes for plant innate immunity. Both PAMPs can enhance growth but likely induce different defense signaling pathways.

The conserved oligomeric Golgi (COG) complex, functioning in retrograde trafficking, is a universal structure present among eukaryotes that maintains the correct Golgi structure and function. The COG complex is composed of eight subunits coalescing into two sub-complexes. COGs1–4 compose Sub-complex A. COGs5–8 compose Sub-complex B. The observation that COG interacts with the syntaxins, suppressors of the erd2-deletion 5 (Sed5p), is noteworthy because Sed5p also interacts with Sec17p [alpha soluble NSF attachment protein (α-SNAP)]. The α-SNAP gene is located within the major Heterodera glycines [soybean cyst nematode (SCN)] resistance locus (rhg1) and functions in resistance. The study presented here provides a functional analysis of the Glycine max COG complex. The analysis has identified two paralogs of each COG gene. Functional transgenic studies demonstrate at least one paralog of each COG gene family functions in G. max during H. glycines resistance. Furthermore, treatment of G. max with the bacterial effector harpin, known to function in effector triggered immunity (ETI), leads to the induced transcription of at least one member of each COG gene family that has a role in H. glycines resistance. In some instances, altered COG gene expression changes the relative transcript abundance of syntaxin 31. These results indicate that the G. max COG complex functions through processes involving ETI leading to H. glycines resistance. [ABSTRACT FROM AUTHOR]

Background: Harpins are proteins secreted by the type III secretion system of Gram-negative bacteria during pathogen–plant interactions that can act as elicitors, stimulating defense and plant growth in many types of non-host plants. Harpin-treated plants have higher resistance, quality and yields and, therefore, harpin proteins may potentially have many valuable agricultural applications. Harpins are characterized by high thermal stability at 100 °C. However, it is unknown whether harpins are still active at temperatures above 100 °C or whether different temperatures affect the activity of the harpin protein in different ways. The mechanism responsible for the heat stability of harpins is also unknown. Results: We identified a novel harpin, HpaXpm, from the cassava blight bacteria Xanthomonas phaseoli pv. manihotis HNHK. The predicted secondary structure and 3-D structure indicated that the HpaXpm protein has two β-strand domains and two major α-helical domains located at the N- and C-terminal regions, respectively. A phylogenetic tree generated using the maximum likelihood method grouped HpaXpm in clade I of the Hpa1 group along with harpins produced by other Xanthomonas spp. (i.e., HpaG-Xag, HpaG-Xcm, Hpa1-Xac, and Hpa1Xm). Phenotypic assays showed that HpaXpm induced the hypersensitive response (HR), defense responses, and growth promotion in non-host plants more effectively than Hp1Xoo (X. oryzae pv. oryzae). Quantitative real-time PCR analysis indicated that HpaXpm proteins subjected to heat treatments at 100 °C, 150 °C, or 200 °C were still able to stimulate the expression of function-related genes (i.e., the HR marker genes Hin1 and Hsr203J, the defense-related gene NPR1, and the plant growth enhancement-related gene NtEXP6); however, the ability of heat-treated HpaXpm to induce HR was different at different temperatures. Conclusions: These findings add a new member to the harpin family. HpaXpm is heat-stable up to 200 °C and is able to stimulate powerful beneficial biological functions that could potentially be more valuable for agricultural applications than those stimulated by Hpa1Xoo. We hypothesize that the extreme heat resistance of HpaXpm is because the structure of harpin is very stable and, therefore, the HpaXpm structure is less affected by temperature. [ABSTRACT FROM AUTHOR]

Phytophthora root and stem rot (PRR) caused by an oomycete pathogen Phytophthora sojae is one of the most devastating and widespread diseases throughout soybean-producing regions worldwide. The diversity and variability of P. sojae races make effective control of the pathogen challenging. Here, we introduced an elicitor of plant defense response, the harpinXooc-encoding hrf2 gene from the rice bacterial pathogen Xanthomonas oryzae pv. oryzicola into soybean and evaluated resistance to P. sojae infection. Molecular analysis confirmed the integration and expression of hrf2 in the transgenic soybean. After inoculation with P. sojae, non-transformed control (NC) plants exhibited typical PRR symptoms, including necrotic and wilting leaves, and plant death, whereas most of the transgenic plants showed slightly chlorotic leaves and developed normally. Through T3 to T5 generations, the transgenic events displayed milder disease symptoms and had higher survival rates compared to NC plants, indicating enhanced and stable resistance to P. sojae infection, whereas without P. sojae inoculation, no significant differences in agronomic traits were observed between the transgenic and non-transformed plants. Moreover, after inoculation with P. sojae, significant upregulation of a set of plant defense-related genes, including salicylic acid- and jasmonic acid-dependent and hypersensitive response-related genes was observed in the transgenic plants. Our results indicate that hrf2 expression in transgenic soybean significantly enhanced resistance to P. sojae by eliciting multiple defense responses mediated by different signaling pathways. The potential functional role of the hrf2 gene in plant defense against P. sojae and other pathogens makes it a promising tool for broadening disease resistance in soybean. [ABSTRACT FROM AUTHOR]

Soybean ( Glycine max ) infection by the charcoal rot (CR) ascomycete Macrophomina phaseolina is enhanced by the soybean cyst nematode (SCN) Heterodera glycines . We hypothesized that G. max genetic lines impairing infection by M. phaseolina would also limit H. glycines parasitism, leading to resistance. As a part of this M. phaseolina resistance process, the genetic line would express defense genes already proven to impair nematode parasitism. Using G. max [DT97-4290/PI 642055] , exhibiting partial resistance to M. phaseolina , experiments show the genetic line also impairs H. glycines parasitism. Furthermore, comparative studies show G. max [DT97-4290/PI 642055] exhibits induced expression of the effector triggered immunity (ETI) gene NON-RACE SPECIFIC DISEASE RESISTANCE 1/HARPIN INDUCED1 (NDR1/HIN1) that functions in defense to H. glycines as compared to the H. glycines and M. phaseolina susceptible line G. max [Williams 82/PI 518671] . Other defense genes that are induced in G. max [DT97-4290/PI 642055] include the pathogen associated molecular pattern (PAMP) triggered immunity (PTI) genes ENHANCED DISEASE SUSCEPTIBILITY1 (EDS1), NONEXPRESSOR OF PR1 (NPR1) and TGA2. These observations link G. max defense processes that impede H. glycines parasitism to also potentially function toward impairing M. phaseolina pathogenicity. Testing this hypothesis, G. max [Williams 82/PI 518671] genetically engineered to experimentally induce GmNDR1-1, EDS1-2, NPR1-2 and TGA2-1 expression leads to impaired M. phaseolina pathogenicity. In contrast, G. max [DT97-4290/PI 642055] engineered to experimentally suppress the expression of GmNDR1-1, EDS1-2, NPR1-2 and TGA2-1 by RNA interference (RNAi) enhances M. phaseolina pathogenicity. The results show components of PTI and ETI impair both nematode and M. phaseolina pathogenicity. [ABSTRACT FROM AUTHOR]

Summary: Ferredoxins, the major distributors for electrons to various acceptor systems in plastids, contribute to redox regulation and antioxidant defence in plants. However, their function in plant immunity is not fully understood. In this study, we show that the expression of the major leaf ferredoxin gene Fd2 is suppressed by Pseudomonas syringae pv. tomato (Pst) DC3000 infection, and that knockout of Fd2 (Fd2‐KO) in Arabidopsis increases the plant's susceptibility to both Pst DC3000 and Golovinomyces cichoracearum. On Pst DC3000 infection, the Fd2‐KO mutant accumulates increased levels of jasmonic acid and displays compromised salicylic acid‐related immune responses. Fd2‐KO also shows defects in the accumulation of reactive oxygen species induced by pathogen‐associated molecular pattern‐triggered immunity. However, Fd2‐KO shows enhanced R‐protein‐mediated resistance to Pst DC3000/AvrRpt2 infection, suggesting that Fd2 plays a negative role in effector‐triggered immunity. Furthermore, Fd2 interacts with FIBRILLIN4 (FIB4), a harpin‐binding protein localized in chloroplasts. Interestingly, Fd2, but not FIB4, localizes to stromules that extend from chloroplasts. Taken together, our results demonstrate that Fd2 plays an important role in plant immunity. [ABSTRACT FROM AUTHOR]

The bacterial effector harpin induces the transcription of the Arabidopsis thaliana NON-RACE SPECIFIC DISEASE RESISTANCE 1 / HARPIN INDUCED1 ( NDR1 / HIN1 ) coiled-coil nucleotide binding leucine rich repeat (CC-NB-LRR) defense signaling gene. In Glycine max , Gm-NDR1-1 transcripts have been detected within root cells undergoing a natural resistant reaction to parasitism by the syncytium-forming nematode Heterodera glycines , functioning in the defense response. Expressing Gm-NDR1-1 in Gossypium hirsutum leads to resistance to Meloidogyne incognita parasitism. In experiments presented here, the heterologous expression of Gm-NDR1-1 in G. hirsutum impairs Rotylenchulus reniformis parasitism. These results are consistent with the hypothesis that Gm-NDR1-1 expression functions broadly in generating a defense response. To examine a possible relationship with harpin, G. max plants topically treated with harpin result in induction of the transcription of Gm-NDR1-1. The result indicates the topical treatment of plants with harpin, itself, may lead to impaired nematode parasitism. Topical harpin treatments are shown to impair G. max parasitism by H. glycines , M. incognita and R. reniformis and G. hirsutum parasitism by M. incognita and R. reniformis . How harpin could function in defense has been examined in experiments showing it also induces transcription of G. max homologs of the proven defense genes ENHANCED DISEASE SUSCEPTIBILITY1 ( EDS1 ), TGA2, galactinol synthase, reticuline oxidase, xyloglucan endotransglycosylase/hydrolase, alpha soluble N-ethylmaleimide-sensitive fusion protein (α-SNAP) and serine hydroxymethyltransferase (SHMT). In contrast, other defense genes are not directly transcriptionally activated by harpin. The results indicate harpin induces pathogen associated molecular pattern (PAMP) triggered immunity (PTI) and effector-triggered immunity (ETI) defense processes in the root, activating defense to parasitic nematodes. [ABSTRACT FROM AUTHOR]

This investigation aimed to examine the possible role of foliar Messenger (a bio-product based on harpin protein) application in reducing the Potato virus X (PVX) development on tomato plants treated with 3 different doses of Messenger (M1: 0.5g/l, M2: 1g/l, M3: 0.25g/l). For that reason, seventy-two hours post treatment (hpt) a group of tomato plants was inoculated with PVX. Besides, another group of tomato plants were inoculated 24 hours afterwards to check the effect of plant age on PVX infection. Plants treated with M1 at 72h prior to the PVX inoculation showed the maximum recovery as an increase in chlorophyll content (25.78±0.67), plant height (31±4.58) cm and leaf area (361.07±76.15) cm². However, M1-enhancement in plant height (27±0.4) cm and leaf area (289.83±45.04) cm² of the non-inoculated tomato plants was lower than that in the inoculated ones. This indicated that M1 induction was most distinct after viral inoculation with higher growth of tomato plants than before introducing the virus. Furthermore, mature plant resistance against PVX was developed with as short plant age increase as to 24 hours. [ABSTRACT FROM AUTHOR]

The targeting of bacterial type III secretion systems (T3SSs), which are critical virulence factors in most Gram-negative pathogens, is regarded as an alternative strategy for the development of novel anti-microbial drugs. Xanthomonas oryzae pv. oryzae ( Xoo) and X. oryzae pv. oryzicola ( Xoc) are two of the most important bacterial pathogens on rice, which cause leaf blight and leaf streak diseases, respectively. To identify potential anti-virulence drugs against these two pathogens, we screened a library of plant phenolic compounds and derivatives for their effects on the Xoo T3SS. Ten of 56 compounds significantly inhibited the promoter activity of a harpin gene, hpa1. These inhibitors were further tested for their impact on the hypersensitive response (HR) caused by Xoo on non-host tobacco plants. The results showed that pretreatment of Xoo with TS006 ( o-coumaric acid, OCA), TS010, TS015 and TS018 resulted in significantly attenuated HR without affecting bacterial growth or survival. In addition, Cya translocation assays demonstrated that the translocation of two T3 effectors was suppressed by the four inhibitors. Quantitative reverse transcription-polymerase chain reaction (qRT-PCR) analysis showed that mRNA levels of representative genes in the hrp ( hypersensitive response and pathogenicity) cluster, as well as the regulatory genes hrpG and hrpX, were reduced by treatment with the four inhibitors, suggesting that expression of the Xoo T3SS was suppressed. The expression of other virulence factors was not suppressed, which indicated possible T3SS-specific inhibition. Finally, we demonstrated that these inhibitors reduced the disease symptoms of Xoo and Xoc on the rice cultivar ( Oryza sativa) IR24 to varying extents. [ABSTRACT FROM AUTHOR]

Programmed cell death (PCD) is considered as a hallmark of strain-specific immunity. In contrast, generic basal immunity is thought to act without PCD. This classical bifurcation has been questioned during recent years. Likewise, the role of jasmonate signalling for these two modes of innate immunity has remained ambiguous. We have addressed both questions using two closely related grapevine cell lines (V. rupestris , V. vinifera cv. 'Pinot Noir') that contrast in their cell-death response to the bacterial elicitor harpin and the hormonal trigger methyl jasmonate (MeJA). We follow different cellular (loss of membrane integrity, mortality), molecular (induction of transcripts for phytoalexin synthesis and for metacaspases), as well as metabolic (sphingolipid profiles) responses to the two triggers in the two cell lines. The role of NADPH oxidases and induction of transcripts for the class-II metacaspases MC5 differ qualitatively between the two cell lines. We tested a possible role of sphingolipid metabolism but can rule this out. We propose a model, where V. rupestris , originating from co-evolution with several biotrophic pathogens, readily activates a hypersensitive cell death in response to harpin, while the context of MeJA-induced cell death in 'Pinot Noir' might not be related to immunity at all. We propose that the underlying signalling is modular, recruiting metacaspases differently depending on upstream signalling. • 1st time to report the elicitors harpin and MeJA induce mortality in an inverted pattern in grapevine. • Based on the above two induced death modes, histochemistry fluorescence assay and relative quantitative study further revealed harpin and MeJA induce a different mode of cell death. • V. rupestris originating from co-evolution with several biotrophic pathogens, readily activates a hypersensitive cell death in response to harpin. [ABSTRACT FROM AUTHOR]

Fruits are one of the most important sources of polyphenols for humans, whether they are consumed fresh or as processed products. To improve the phenolic content of fruits, a novel field of interest is based on results obtained using elicitors, agrochemicals which were primarily designed to improve resistance to plant pathogens. Although elicitors do not kill pathogens, they trigger plant defense mechanisms, one of which is to increase the levels of phenolic compounds. Therefore, their application not only allows us to control plant disease but also to increase the phenolic content of plant foodstuffs. Pre- or post-harvest application of the most commonly used elicitors to several fruits is discussed in this review.

O objetivo deste trabalho foi avaliar o efeito dos eliciadores acibenzolar-S-metil e proteína harpina, aplicados em pós-colheita, na indução de resistência sistêmica à podridão-parda em pêssegos. O delineamento experimental foi inteiramente casualizado, com quatro repetições, em parcelas subdivididas - com e sem ferimentos provocados aos frutos -, e os tratamentos foram constituídos por: acibenzolar-S-metil (50 mg do i.a. L-1), dois produtos comerciais com proteína harpina (80 mg do i.a. L-1) e uma testemunha (água destilada). Os frutos foram pulverizados, individualmente, com 1 mL de solução aquosa com os tratamentos e, após 12 horas, efetuou-se a inoculação com Monilinia fructicola (0,2 mL da suspensão com concentração de 10(5) esporos mL-1, em cada lado do fruto). Após 60 horas da inoculação, avaliaram-se: a área lesionada, a esporulação e o percentual de controle. Determinaram-se os teores de proteínas totais, açúcares redutores e totais, fenóis, além da atividade da enzima fenilalanina amônia-liase (FAL). Os eliciadores induziram resistência dos frutos a M. fructicola, com redução do desenvolvimento do fungo. O uso dos eliciadores aumentou os teores dos parâmetros bioquímicos avaliados e a atividade da FAL, que esteve relacionada à redução da área lesionada em pêssegos. Os indutores podem contribuir para o manejo integrado da podridão-parda em pêssegos, em pós-colheita.The objective of this work was to evaluate the effects of acibenzolar-S-methyl and harpin protein elicitors, applied in post-harvest, in systemic resistance induction to brown-rot, on peaches. A completely randomized design was utilized, with four replicates, in a split plot scheme - injured or noninjured peaches -, and treatments were constituted of : acibenzolar-S-methyl (50 mg of a.i. L-1), two commercial products with harpin protein (80 mg of a.i. L-1); and control (distilled water). Peaches were sprayed individually with 1 mL of aqueous solution containing the treatments and, after 12 hours, the inoculation of Monilinia fructicola was carried out (0.2 mL of spore suspension with 10(5) spores mL-1, in each side of the fruit). Sixty hours past inoculation, the size of lesions, esporulation and percentage of control were evaluated. The contents of total protein, total phenol, total and reducing sugar, besides activity of phenylalanine ammonia-lyase (PAL), were determined. The elicitors induce resistance to M. fructicola, with reduction in fungal development. Elicitor's increased the evaluated biochemical parameters and the activity of PAL, which was related to the size reduction of lesions in peaches. The elicitors could be used in integrated management of brown-rot, in post-harvest applications.

The type III secretion system (T3SS) of plant and animal bacterial pathogens directs the secretion and injection of proteins into host cells. Some homologous genes of T3SS were found also in nonpathogenic bacteria, but the organization of its machinery and basic function are still unknown. In this study, we identified a T3SS gene cluster from the plant growth-promoting Pseudomonas fluorescens 2P24 and isolated the corresponding T3SS apparatus. The T3SS gene cluster of strain 2P24 is similar organizationally to that of pathogenic P. syringae, except that it lacks the regulator hrpR and the hrpK1 and hrpH genes, which are involved in translocation of proteins. Electron microscopy revealed that the T3SS supramolecular structure of strain 2P24 was comprised of two distinctive substructures: a long extracellular, filamentous pilus and a membrane-embedded base. We show that strain 2P24 deploys a harpin homologue protein, RspZ1, to elicit a hypersensitive response when infiltrated into Nicotiana tabacum cv. xanthi leaves with protein that is partially purified, and by complementing the hrpZ1 mutation of pHIR11. The T3SS of strain 2P24 retained ability to secrete effectors, whereas its effector translocation activity appeared to be excessively lost. Mutation of the rscC gene from 2P24 T3SS abolished the secretion of effectors, but the general biocontrol properties were unaffected. Remarkably, strain 2P24 induced functional MAMP-triggered immunity that included a burst of reactive oxygen species, strong suppression of challenge cell death, and disease expansion, while it was not associated with the secretion functional T3SS.

Harpin proteins encoded by hrp genes are bacterial protein elicitors that can stimulate hypersensitive response (HR) in non-host plants. HR-related pathogen resistance involves a complex form of programmed cell death (PCD). It is increasingly viewed as a key component of the hypersensitive disease response of plants. Currently, the evidence of harpin proteins-induced PCD is deficient though it exhibits phenotypic parallels with HR, and the mechanism of harpin proteins-induced PCD is not well understood. In this study, we demonstrate that harpin protein from Xanthomonas oryzae pv. oryzae of rice bacterial blight expressed and isolated from bacterial cells acted as an agent to induce PCD in infiltrated tobacco plants. Treatment of tobacco leaves with harpin induced typical PCD-related morphological and biochemical changes including cell shrinkage and nuclear DNA degradation. We further analyzed the expression of several genes in signal transduction pathway of PCD in tobacco plants by real-time qRT-PCR analysis using EF- 1α as an endogenous control. Our results showed that the expression of NtDAD1 was down-regulated and the expression of BI- 1, tpa1 and aox1 was up-regulated following the infiltration of harpin into tobacco leaves. Our data suggest that harpin can induce PCD with the coordination of PCD-related genes in infiltrated tobacco leaves, providing evidence to further investigate the signal transduction pathways of HR and PCD. [ABSTRACT FROM AUTHOR]

MADS-box transcription factor genes are well known for their role in floral organ and seed development. In this study, a novel MADS-box-containing gene, designated NbMADS1, was isolated from leaves of Nicotiana benthamiana. The full-length cDNA was 666 bp and encoded a putative polypeptide of 221 aa with a mass of 24.3 kDa. To assess the role of NbMADS1 in the defence response to bacterial harpinXoo, an elicitor of the hypersensitive response, a loss-of-function experiment was performed in N. benthamiana plants using virus-induced gene silencing. Analyses of electrolyte leakage revealed more extensive cell death in the control plants than in NbMADS1-silenced plants. The NbMADS1-silenced plants showed impaired harpinXoo-induced stomatal closure, decreased harpinXoo-induced production of hydrogen peroxide (H2O2) and nitric oxide (NO) in guard cells, and reduced harpinXoo-induced resistance to Phytophthora nicotianae. The compromised stomatal closure observed in the NbMADS1-silenced plants was inhibited by the application of H2O2 and sodium nitroprusside (an NO donor). Taken together, these results demonstrate that the NbMADS1-H2O2-NO pathway mediates multiple harpinXoo-triggered responses, including stomatal closure, hypersensitive cell death, and defence-related gene expression, suggesting that NbMADS1 plays an important role in regulating the response to harpinXoo in N. benthamiana plants. [ABSTRACT FROM AUTHOR]

MADS-box transcription factor genes are well known for their role in floral organ and seed development. In this study, a novel MADS-box-containing gene, designated NbMADS1, was isolated from leaves of Nicotiana benthamiana. The full-length cDNA was 666 bp and encoded a putative polypeptide of 221 aa with a mass of 24.3 kDa. To assess the role of NbMADS1 in the defence response to bacterial harpinXoo, an elicitor of the hypersensitive response, a loss-of-function experiment was performed in N. benthamiana plants using virus-induced gene silencing. Analyses of electrolyte leakage revealed more extensive cell death in the control plants than in NbMADS1-silenced plants. The NbMADS1-silenced plants showed impaired harpinXoo-induced stomatal closure, decreased harpinXoo-induced production of hydrogen peroxide (H2O2) and nitric oxide (NO) in guard cells, and reduced harpinXoo-induced resistance to Phytophthora nicotianae. The compromised stomatal closure observed in the NbMADS1-silenced plants was inhibited by the application of H2O2 and sodium nitroprusside (an NO donor). Taken together, these results demonstrate that the NbMADS1-H2O2-NO pathway mediates multiple harpinXoo-triggered responses, including stomatal closure, hypersensitive cell death, and defence-related gene expression, suggesting that NbMADS1 plays an important role in regulating the response to harpinXoo in N. benthamiana plants. [ABSTRACT FROM AUTHOR]

Plant ferredoxin-like protein ( PFLP) is a photosynthesis-type ferredoxin ( Fd) found in sweet pepper. It contains an iron-sulphur cluster that receives and delivers electrons between enzymes involved in many fundamental metabolic processes. It has been demonstrated that transgenic plants overexpressing PFLP show a high resistance to many bacterial pathogens, although the mechanism remains unclear. In this investigation, the PFLP gene was transferred into Arabidopsis and its defective derivatives, such as npr1 ( nonexpresser of pathogenesis-related gene 1) and eds1 ( enhanced disease susceptibility 1) mutants and NAHG-transgenic plants. These transgenic plants were then infected with the soft-rot bacterial pathogen Pectobacterium carotovorum subsp. carotovorum ( Erwinia carotovora ssp. carotovora, ECC) to investigate the mechanism behind PFLP-mediated resistance. The results revealed that, instead of showing soft-rot symptoms, ECC activated hypersensitive response ( HR)-associated events, such as the accumulation of hydrogen peroxide ( H2O2), electrical conductivity leakage and expression of the HR marker genes ( ATHSR2 and ATHSR3) in PFLP-transgenic Arabidopsis. This PFLP-mediated resistance could be abolished by inhibitors, such as diphenylene iodonium ( DPI), 1- l- trans-epoxysuccinyl-leucylamido-(4-guanidino)-butane ( E64) and benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone (z- VAD-fmk), but not by myriocin and fumonisin. The PFLP-transgenic plants were resistant to ECC, but not to its harpin mutant strain ECCAC5082. In the npr1 mutant and NAHG-transgenic Arabidopsis, but not in the eds1 mutant, overexpression of the PFLP gene increased resistance to ECC. Based on these results, we suggest that transgenic Arabidopsis contains high levels of ectopic PFLP; this may lead to the recognition of the harpin and to the activation of the HR and other resistance mechanisms, and is dependent on the protease-mediated pathway. [ABSTRACT FROM AUTHOR]

Lonsdalea quercina N-5-1 is a bacterial pathogen that causes poplar bark cankers. It has been isolated from the branch of Populus × euramericana cv. '74/76' in Henan, China. Previous studies have revealed that the Type III secretion system (T3SS) acts as an essential pathogenic factor in L. quercina N-5-1. HrpW is a putative effector of T3SS in strain N-5-1, which has a typical harpin domain at the amino terminal and a pectate lyase (Pel) domain at its carboxyl terminal. Genetic evidence had shown that, compared to the wild-type and the complementary strain, the hrpW mutation causes a small but significant reduction in virulence when inoculated on the poplar branches. The amino terminal domain of HrpW was found to trigger tobacco hypersensitive response, but the carboxyl terminal domain of HrpW was not. Unlike most HrpW homologs in other bacteria, the carboxyl terminal domain of HrpW of strain N-5-1 exhibited detectable pectate lyase activity. Site-direction mutations (W104A, W171M) further demonstrated that two tryptophan residues were essential to its pectate lyase activity. The results of the present work suggest that HrpW in L. quercina N-5-1 possesses pectate lyase activity and acts as a nonessential but important pathogenic factor in poplar bark canker disease. [ABSTRACT FROM AUTHOR]

Harpin proteins stimulate hypersensitive response (HR) in plants. However, the mechanism by which HR is regulated is not clear. The role of the auxin, indole-3-acetic acid (IAA), in the control of harpin-stimulated HR was investigated. IAA was used to inhibit HR that was stimulated by purified fusion harpin protein in tobacco. Semi-quantitative PCR and qRT-PCR were employed to detect the expression of HR related genes. IAA at 100 μM reversed harpin-induced HR which was inhibited by 500 μM 2,3,5-triiodobenzoic acid (TIBA). Semi-quantitative PCR and qRT-PCR showed the combined application of 100 μM IAA and harpin protein from Xanthomonas oryzae enhanced the expression of HR marker gene, hsr203J, but weakened the expression of the disease-defense gene, chia5. TIBA also decreased the expression of hsr203J but increased the expression of chia5. Thus, the auxin can reverse harpin-induced HR. [ABSTRACT FROM AUTHOR]

Bacterial canker of tomato caused by Clavibacter michiganensis subsp. michiganensis produces considerable economic losses in many countries because effective control measures are lacking. In this study, the effectiveness of some chemicals, a plant growth regulator (Prohexadione-Ca (PC)), two plant activators (hydrogen peroxide (HP)) and harpin protein (Hrp), fungicides, maneb+copper (MC), copper compounds (copper sulfate pentahydrate (CSP) copper hydroxide (CH) and copper oxychloride (CO)) and an antibiotic, streptomycin sulphate on the pathogen were assessed in vitro and in vivo on two tomato cultivars, Newton and Orient. In Hrp and HP treated plants, reduction in disease severity (about 45%) was correlated with suppression of bacterial growth during the time course of infection. The activities of POX, GPX and PAL enzymes were determined at 5th day as markers of resistance and applications of Hrp, PC and HP induced a progressive and significant increase of both enzymes in locally treated tissues. Only PC treatments have decreased plant heights and the effectiveness of PC in reduction of plant heigths was up to 40%. Among copper compounds, CSP and CO combination with Maneb were the most effective on the disease severity (about 50%). In addition, CSP combination with Hrp, HP and PC significantly limited the pathogen population in vitro and they showed considerable effects on the disease symptoms compared with CSP alone. We conclude that Hrp, HP and PC may be useful in controlling external symptoms of this disease in greenhouses, and is environmentally friendly, reducing the amount of copper applied to crops. [ABSTRACT FROM AUTHOR]

Highlights: [•] Postharvest treatments with BTH, BABA, INA, SA, ASA, sodium silicate and harpin were tested. [•] INA at 0.03mM, SA at 0.25mM, BABA at 0.3mM and BTH at 0.9mM were selected in in vivo primary screenings. [•] SSi at 1000mM was an effective treatment, but residues in the fruit rind might be phytotoxic. [•] Preventive or curative dips at 20°C for 60 or 150s had no effect or only reduced mold development very slightly. [ABSTRACT FROM AUTHOR]

Harpin proteins secreted by phytopathogenic bacteria have been shown to activate the plant defense pathway, which involves transduction of a hydrogen peroxide (HO) signal generated in the apoplast. However, the way in which harpins are recognized in the pathway and what role the apoplastic HO plays in plant defenses are unclear. Here, we examine whether the cellular localization of Hpa1, a harpin protein produced by the rice bacterial leaf blight pathogen, impacts HO production and pathogen resistance in Arabidopsis thaliana. Transformation with the hpa1 gene and hpa1 fused to an apoplastic localization signal ( shpa1) generated h pa1- and sh pa1- expressing transgenic A . t haliana (HETAt and SHETAt) plants, respectively. Hpa1 was associated with the apoplast in SHETAt plants but localized inside the cell in HETAt plants. In addition, Hpa1 localization accompanied HO accumulation in both the apoplast and cytoplasm of SHETAt plants but only in the cytoplasm of HETAt plants. Apoplastic HO production via nicotinamide adenine dinucleotide phosphate (NADPH) oxidase (NOX) located in the plasma membrane is a common feature of plant defenses. In SHETAt plants, HO was generated in apoplasts in a NOX-dependent manner but accumulated to a greater extent in the cytoplasm than in the apoplast. After being applied to the wild-type plant, Hpa1 localized to apoplasts and stimulated HO production as in SHETAt plants. In both plants, inhibiting apoplastic HO generation abrogated both cytoplasmic HO accumulation and plant resistance to bacterial pathogens. These results suggest the possibility that the apoplastic HO is subject to a cytoplasmic translocation for participation in the pathogen defense. [ABSTRACT FROM AUTHOR]

Salt stress is a major constraint for many crop plants, such as the moderately salt-sensitive economically important fruit crop grapevine. Plants have evolved different strategies for protection against salinity and drought. Jasmonate signalling is a central element of both biotic and abiotic stress responses. To discriminate stress quality, there must be cross-talk with parallel signal chains. Using two grapevine cell lines differing in salt tolerance, the response of jasmonate ZIM/tify-domain (JAZ/TIFY) proteins (negative regulators of jasmonate signalling), a marker for salt adaptation Na+/H+ EXCHANGER (NHX1), and markers for biotic defence STILBENE SYNTHASE (StSy) and RESVERATROL SYNTHASE (RS) were analysed. It is shown that salt stress signalling shares several events with biotic defence including activity of a gadolinium-sensitive calcium influx channel (monitored by apoplastic alkalinization) and transient induction of JAZ/TIFY transcripts. Exogenous jasmonate can rescue growth in the salt-sensitive cell line. Suppression of jasmonate signalling by phenidone or aspirin blocks the induction of JAZ/TIFY transcripts. The rapid induction of RS and StSy characteristic for biotic defence in grapevine is strongly delayed in response to salt stress. In the salt-tolerant line, NHX1 is induced and the formation of reactive oxygen species, monitored as stress markers in the sensitive cell line, is suppressed. The data are discussed in terms of a model where salt stress signalling acts as a default pathway whose readout is modulated by a parallel signal chain triggered by biotic factors downstream of jasmonate signalling. [ABSTRACT FROM PUBLISHER]

Purpose – The aim of this research is to determine the effects of pre-harvest harpin (H) and modified atmosphere packaging (MAP) on the storage and fruit quality of cherry tomato (Lycopersicon esculentum Mill. cvs. "Alona" and "Cluster"). Design/methodology/approach – H was used at 50 g 100 l-1 concentration. First application was done on the seedlings in 3-4 leaf stages and the other four applications were repeated at 20-day intervals. Then, treated and untreated fruits were stored in plastic film materials with various O2 and CO2 permeabilities. Findings – Weight loss was significantly higher in tomatoes stored under normal atmosphere (NA) compared to MAP. Initial total soluble solids of 3-4 percent increased to 7-9 percent in without H (WH)+NA. Firmness was measured as 0.44 ("Alona") and 1.20 kg ("Cluster") in H+NA and 0.17 ("Alona") and 0.30 kg ("Cluster") in WH+NA at the end of storage. The acidity and ascorbic acid contents of tomatoes decreased during storage. Between H and WH treatments the levels of lycopene and β-carotene were different significantly. At the end of the study, H gave successful results in both cultivars. This success was even higher especially when the H was combined with MAP. This combination slowed down the changes in TSS, firmness values. Therefore, H+50μ PE (polyethylene) treatment was effective with regard to delaying the maturity along the storage and fruit quality in cherry tomatoes. Originality/value – He is a plant activator. It is the first bacterial hypersensitive response (HR)-elicitor characterized. Effectiveness of H is evaluated on quality changes in treated cherry tomato fruits. [ABSTRACT FROM AUTHOR]

Harpin proteins are well known as eliciters that induce multiple responses in plants, such as systemic acquired resistance, hypersensitive response, enhancement of growth, resistance to the green peach aphid, and tolerance to drought. Overexpression of Harpin-encoding genes enhances plant resistance to diseases in tobacco, rice, rape, and cotton; however, it is not yet known whether the expression of Harpin-encoding genes in vivo improves plant tolerance to abiotic stresses. The results of this study showed that overexpression of a Harpin-encoding gene hrf1 in rice increased drought tolerance through abscisic acid (ABA) signalling. hrf1- overexpression induces an increase in ABA content and promotes stomatal closure in rice. The hrf1 transgenic rice lines exhibited a significant increase in water retention ability, levels of free proline and soluble sugars, tolerance to oxidative stress, reactive oxygen species-scavenging ability, and expression levels of four stress-related genes, OsLEA3-1, OsP5CS, Mn-SOD, and NM_001074345, under drought stress. The study confirmed that hrf1 conferred enhanced tolerance to drought stress on transgenic crops. These results suggest that Harpins may offer new opportunities for generating drought resistance in other crops. [ABSTRACT FROM PUBLISHER]

Bacterial soft rot caused by Pectobacterium carotovorum subsp . carotovorum is a serious plant disease in Zantedeschia spp. (also called calla lily). In this study, two independent genes (a N-acyl homoserine lactonase gene attM from Agrobacterium tumefaciens and a hypersensitive response and pathogenicity gene hrf1 from Xanthomonas oryzae pv . oryzae), transcribed by a strong and constitutive Escherichia coli promoter P, respectively, were cloned into plasmid pUC19, and was transformed into E. coli, creating strain JM109/pPHA. The result of sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) assay showed that both genes ( hrf1 and attM) were successfully expressed in one plasmid system in strain JM109/pPHA. The expressed Harpin (Hrf1) and AttM protein had the ability of inducing hypersensitive response (HR) in nonhost tobacco and degrading the N-acyl homoserine lactones (AHLs) produced by P. carotovorum subsp . carotovorum, respectively, whereas Harpin and AttM protein did not seem to interfere with the normal growth of this pathogen. In planta, strain JM109/pPHA could significantly reduce the soft rot disease severity on dormant tubers (control efficiency: 92.8%) or potted plants (control efficiency: 92.4%) of calla lily. We have first demonstrated the both biocontrol effects of Harpin and AttM proteins (also described as Quorum interference) on the bacterial soft rot disease of calla lily, caused by P. carotovorum subsp . carotovorum. This work provided a potential way to control this serious plant disease. [ABSTRACT FROM AUTHOR]

The cytoskeleton undergoes dramatic reorganization during plant defence. This response is generally interpreted as part of the cellular repolarization establishing physical barriers against the invading pathogen. To gain insight into the functional significance of cytoskeletal responses for defence, two Vitis cell cultures that differ in their microtubular dynamics were used, and the cytoskeletal response to the elicitor Harpin in parallel to alkalinization of the medium as a fast response, and the activation of defence-related genes were followed. In one cell line derived from the grapevine cultivar ‘Pinot Noir’, microtubules contained mostly tyrosinylated α-tubulin, indicating high microtubular turnover, whereas in another cell line derived from the wild grapevine V. rupestris, the α-tubulin was strongly detyrosinated, indicating low microtubular turnover. The cortical microtubules were disrupted and actin filaments were bundled in both cell lines, but the responses were elevated in V. rupestris as compared with V. vinifera cv. ‘Pinot Noir’. The cytoskeletal responsiveness correlated with elicitor-induced alkalinization and the expression of defence genes. Using resveratrol synthase and stilbene synthase as examples, it could be shown that pharmacological manipulation of microtubules could induce gene expression in the absence of elicitor. These findings are discussed with respect to a role for microtubules as positive regulators of defence-induced gene expression. [ABSTRACT FROM PUBLISHER]

Abstract: Harpin protein induces disease resistance in diverse plant species. Transcriptome study of tomato (Solanum lycopersicum) genome indicated that harpin activated cellular events of transcription regulation, signaling transduction, stress response, membrane transporting, photosynthesis and cell wall biosynthesis. Among the harpin-induced genes, the expression of tomato ethylene-response factor 5 (designated as SlERF5) was induced to the highest level. The amino acid sequence of SlERF5 was closely related to that of CaEREBP-C4, NtERF4 and NsERF4. Overexpression of SlERF5 in Arabidopsis thaliana activated a large number of genes involved in signaling pathways of disease resistance, redox system, abiotic response and protein phosphorylation. Seedlings of transgenic plants exhibited hypersensitive response to exogenous ABA and increased tolerance to salt stress. In summary, the results of this study suggest that SlERF5 is a transcription activator for genes involved in the responses to ABA, biotic and abiotic stress. Thus, harpin may exert its effect on plant growth and defense response through activation of SlERF5 transcription factors. [Copyright &y& Elsevier]

Abstract: Protection of crops against bacterial disease is an important issue in agricultural production. One of the strategies to lead plants become resistant against bacterial pathogens is employing a transgene, like plant ferredoxin-like protein (PFLP). PFLP is a photosynthetic type ferredoxin isolated from sweet pepper and contains a signal peptide for targeting towards chloroplasts. Our previous reports indicated that transgenic plants with this protein are more resistant against bacterial pathogens. However, this heterologous protein was visualized not only inside the chloroplasts, but also in the cytoplasm. In this article, we moved to study its heterologous expression in Arabidopsis by expressing the protein in chloroplast, apoplast and cytoplasm. This work was achieved by engineering a chloroplast target (CPF), an apoplast target (ESF), and cytoplasm target (DF) plants. The expression and subcellular localization of PFLP were analyzed by Western blot and immuno-staining by confocal microscopy, respectively. We tested the ability of the transgenic Arabidopsis for resistance to two Ralstonia solanacearum strains and their ability to increase the hypersensitive response (HR) triggered by harpin (HrpZ) from Pseudomonas syringae. The DF and ESF plants conferred resistance against bacterial wilt strains and increased HR by harpin, but no resistance found in the CPF plants. In addition, we determined the level of reduced ascorbate in all transgenic plants and further analyzed the expression of two NADPH-oxidase genes (AtrbohD and AtrbohF) in ESF plant. Among the transgenic Arabidopsis plants, ESF plants confer the highest resistance to bacterial pathogens and followed by DF plants. We concluded that PFLP enhances disease resistance in Arabidopsis when expressed in the apoplast or in cytoplasm but not when targeted into the chloroplast. This study provides a strategy for molecular breeding to improve resistance of crops against bacterial pathogens. [Copyright &y& Elsevier]

Pear psylla Cacopsylla pyri (L.) is among the most serious pests in pear orchards. Field studies were conducted in 2005 and 2006 on pear trees (Pyrus communis L.) to assess the effect of kaolin-based particle film, spirodiclofen acaricide, harpin protein, and an organic biostimulant on C. pyri infestations. Pear psylla nymph densities were effectively suppressed over the entire season by kaolin particle film and spirodiclofen treatments compared with the untreated control. Harpin protein protected pear trees almost as well as the spirodiclofen acaricide, while the organic biostimulant provided no protection. Kaolin particle film, spirodiclofen, and harpin protein-sprayed pear trees significantly outperformed their unsprayed counterparts in terms of fruit load. Based on the results of this study, it seems that kaolin particle film, spirodiclofen, and harpin protein could be used as alternatives to broad-spectrum insecticides for suppressing pear psylla infestations. [ABSTRACT FROM AUTHOR]

Abstract: Harpins can induce systemic acquired resistance (SAR) pathway on scores of non-host plant, provide protection against a range of pathogens. In this study, we demonstrated that applied recombinant HarpinZ Pseudomonas syringae pv. tomato (rHrpZ) on tobacco with three kinds of methods: infiltrating from micro-pore into leaf; injecting into petiole, and spraying on leaf, there is great difference in assimilation of protein because of the poor osmosis of tobacco leaves, and with multi-application of rHrpZ, the stimulation effect decreased. We prepared poly d,l-lactide-co-glycolide nanoparticles containing rHrpZ (rHrpZ PLGA NPs). To study the drug effect, we analyzed the change ratio of phenylalanine ammonia lyase (PAL) activity and PR-5dB gene expression after administration of rHrpZ and rHrpZ PLGA NPs on tobacco leaves. The results show that rHrpZ could elicit a rapid and transient increase in both PAL activity and PR-5dB expression, but the effect decreased after multi-application. While sprayed rHrpZ PLGA NPs on leaves, both the change ratio of PAL activity and PR-5dB expression were comparatively smooth and durable. Our study suggested that rHrpZ NPs could help protein enter leaf epidermis and cell wall, release rHrpZ in situ continuously, and enhance the bioavailability of rHrpZ. [Copyright &y& Elsevier]

Abstract: Transgenic Citrus sinensis (L.) Osb. cv. Hamlin plants expressing the hrpN gene were obtained by Agrobacterium tumefaciens (Smith and Towns) Conn-mediated transformation. hrpN encodes a harpin protein, which elicits the hypersensitive response and systemic acquired resistance in plants. The gene construct consisted of gst1, a pathogen-inducible promoter, a signal peptide for protein secretion to the apoplast, the selection genes nptII or aacC1 and the Nos terminator. The function of gst1 in citrus was evaluated in transgenic C. sinensis cv. Valencia harboring the reporter gene uidA (gus) driven by this promoter. Histochemical analysis for gus revealed that gst1 is activated in citrus leaves by both wounding and inoculation with Xanthomonas axonopodis Starr and Garces pv. citri (Hasse) Vauterin et al. Genetic transformation was confirmed by Southern blot hybridization in eight cv. Hamlin acclimatized plants. RT-PCR confirmed hrpN gene expression in seven cv. Hamlin transgenic lines before pathogen inoculation. Some hrpN transgenic lines showed severe leaf curling and abnormal growth. Six hrpN transgenic lines were propagated and evaluated for susceptibility to X. axonopodis pv. citri. RT-PCR confirmed gene expression in all six hrpN transgenic lines after pathogen inoculation. Several of the hrpN transgenic lines showed reduction in susceptibility to citrus canker as compared with non-transgenic plants. One hrpN transgenic line exhibited normal vegetative development and displayed very high resistance to the pathogen, estimated as up to 79% reduction in disease severity. This is the first report of genetic transformation of citrus using a pathogen-inducible promoter and the hrpN gene. Further evaluations of the transgenic plants under field conditions are planned. Nevertheless, the evidence to date suggests that the hrpN gene reduces the susceptibility of citrus plants to the canker disease. [Copyright &y& Elsevier]

Abstract: The tremendous increase in crop yields associated with the ‘green’ revolution has been possible in part by the discovery and utilization of chemicals for pest control. However, concerns over the potential impact of pesticides on human health and the environment has led to the introduction of new pesticide registration procedures, such as the Food Quality Protection Act in the United States. These new regulations have reduced the number of synthetic pesticides available in agriculture. Therefore, the current paradigm of relying almost exclusively on chemicals for pest control may need to be reconsidered. New pesticides, including natural product-based pesticides are being discovered and developed to replace the compounds lost due to the new registration requirements. This review covers the historical use of natural products in agricultural practices, the impact of natural products on the development of new pesticides, and the future prospects for natural products-based pest management. [Copyright &y& Elsevier]

Abstract: Harpins constitute one group of effector proteins which elicit a hypersensitive response in nonhost plants, but the subcellular localization and tissue distribution of harpin receptors are still controversial. Antigen mimicry by anti-idiotypic antibodies is employed as a reliable strategy to probe receptors that are present in very low concentrations in the organism. In this study, a monoclonal anti-idiotypic antibody (Ab2), named 6B2, was elicited by F(ab′)2 fragments digested from the purified polyclonal antibody specific for HarpinXoo (Ab1). 6B2 competed with HarpinXoo for binding to Ab1 and the total protein extracted from tobacco leaves indicated its anti-idiotypic character and internal image property. The relevance of antigen mimicry was further confirmed by eliciting a third generation antibody (Ab3), which was shown not only to bind to Ab2 competing with Ab1 but also to react with the original antigen, HarpinXoo. Taken together, these results demonstrate functional and biochemical mimicry of HarpinXoo by Ab2 and suggest that 6B2 can be a useful tool in probing its receptor in nonhost plants and other downstream studies. [Copyright &y& Elsevier]