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Copyright of Chinese Journal of Biochemistry & Molecular Biology is the property of Editorial Office of Chinese Journal of Biochemistry & Molecular Biology and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use. This abstract may be abridged. No warranty is given about the accuracy of the copy. Users should refer to the original published version of the material for the full abstract. (Copyright applies to all Abstracts.)

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]

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]

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]

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]

Copyright of Journal of Agricultural Faculty of Gaziosmanpasa University / Gazi Osman Pasa Üniversitesi Ziraat Fakültesi Dergisi is the property of Gaziosmanpasa University, Agricultural Faculty and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use. This abstract may be abridged. No warranty is given about the accuracy of the copy. Users should refer to the original published version of the material for the full abstract. (Copyright applies to all Abstracts.)

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]

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]

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 non-pathogenic 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 homolog 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. [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]

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]

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]

Harpin protein is an elicitor of hypersensitive response in plants. When applied exogenously, harpin is claimed to control plant diseases, increase plant growth and yield, and reduce postharvest fruit decay. The plant responses to harpin application have been inconsistent. This study was intended to determine the effects of harpin on plant growth, gas exchange, control of Phytophtora blight, fruit yield, and control of postharvest fruit decay in bell pepper (Capsicum annum L.). When applied only before planting, harpin increased marketable yield of bell pepper, but when applied both before and after planting, it reduced marketable and total yields. Harpin had no beneficial effect on plant growth, leaf mineral nutrient content, leaf gas exchange, incidence of Phytophtora blight in the field, and incidence of softrot of fruit after harvest. Harpin had little or no positive effect on most plant attributes in bell pepper in the field or after harvest. [ABSTRACT FROM AUTHOR]

Recent advances in the understanding of plant signaling pathways have opened the way for using elicitor-induced plant resistance as a tactic for protecting plants against arthropod pests. Four common elicitors of induced responses in tomato, Lycopersicon esculentum Mill. (Solanaceae), were evaluated with regard to phytotoxicity, induction of plant defensive proteins, and effects on population growth and fecundity of a common pest, the green peach aphid, Myzus persicae (Sulzer) (Homoptera: Aphididae). Ethephon and methyl jasmonate (MJ) treatments caused varying degrees of phytotoxicity. Ethephon caused pronounced changes in plant growth form and severe, dose-dependent negative impacts on plant growth and flowering. Effects with MJ were milder, but still caused temporary inhibition of development, leading to smaller plants and delayed flowering. The commercial elicitors benzothiadiazole (BTH) and harpin did not cause detectable phytotoxicity. The highest doses of ethephon and MJ significantly increased leaf peroxidase (POD) levels but only MJ treatments significantly increased polyphenol oxidase (PPO) levels. BTH and harpin had no detectable effects on POD and PPO. Populations of green peach aphids grew significantly more slowly on plants treated with BTH or MJ than on control plants or plants treated with harpin or ethephon. Slowed aphid population growth on BTH-treated plants was due to significant reductions in aphid fecundity, although this was independent of changes in time to onset of reproduction or time to death. Aphid fecundity was also reduced on MJ-treated plants relative to controls, but this difference was not statistically significant, suggesting that other mechanisms are involved in slowing aphid population growth on MJ-treated plants. Growth of aphid populations on plants treated with a MJ–BTH mixture was reduced almost as much as with treatments of MJ alone, suggesting that antagonism between JA-dependant and SA-dependent plant signaling pathways is only mild with regard to induced defenses against aphids. [ABSTRACT FROM AUTHOR]

Systemic acquired resistance (SAR) is a process whereby a plant that successfully resists a pathogen becomes highly resistant to subsequent infection not only by the original pathogen but also by a wide variety of pathogens. Most SAR research has focused on resistance in leaves, so much less is known about the effectiveness of foliar applications of SAR compounds in the protection of plant roots and associated microorganisms in soil. This study was conducted to determine if foliar SAR‐inducing applications (BTH or harpin) negatively impact the potato root system beneficial rhizosphere microbial populations and activity or influence pathogenic nematode populations. Foliar applications of benzo (1,2,3) thiadiazole‐7‐carbothioic acid S‐methyl ester (BTH) and the microbial protein harpin applied in various combinations, timings, and rates showed no effects on microbial biomass, culturable bacteria, Pseudomonas populations, or N‐mineralization potentials over 2 years. No stimulatory or inhibitory effects on major bacterial populations were observed, indicating that SAR induction does not have a negative effect on general microbial populations or activities. BTH and harpin both reduced the numbers of lesion nematodes ( Pratylenchus spp.) by potato harvest. BTH reduced root knot nematodes, Meloidogyne chitwoodi at the end of the season. In addition, BTH and high‐dose harpin (applied at the 4× rate) reduced the nematode infection index in comparison to the control. The SAR elicitors increased the population densities of nontarget free‐living nematodes in the soil compared to the control. Potato yields were not affected by plant elicitors but BTH and harpin both reduced the number of culled potatoes 26% compared to the control. Future studies are designed to determine if these plant elicitors have any direct effect on rhizosphere diversity or if plants with active defense pathways alter carbon flow and root exudates into the soil. [ABSTRACT FROM AUTHOR]

It is reported here that salicylic acid (SA) is rapidly taken up by Arabidopsis cells, and its uptake is accompanied by media alkalization and cytosolic acidification, and it is inhibited by the ionophore nigericin, suggesting that its import is linked with that of H+ and driven by a proton gradient. Such import and accumulation declined sharply within a narrow physiological pH range (pH 5.7–6.1), corresponding to a reduction in the [H+] of the media from 1.99 μmol l−1 to 0.79 μmol l−1. Following the initial uptake, SA was exported back into the media as free SA against a continued [H+]-dependent import. Since the uptake and accumulation of SA declines sharply within a narrow pH range and cell wall alkalization is an early response during incompatible plant/pathogen interactions, the bacterial elicitor harpinPss was used to investigate how SA transport may be modulated during defence responses. Harpin induced a rapid and sustained alkalization of the cell suspension media, reaching the critical pH (pH 5.9–6.1) at which SA import is inhibited at c. 60 min. Such media alkalization corresponded with a reduction in the SA associated with cells co-treated with harpin, and an inhibition of SA uptake in cells pretreated with harpin. Scavengers of ROS, or compounds which generate H2O2 or NO had little effect on the import or net export of SA, suggesting that media alkalization induced by harpin is sufficient to modulate the kinetics of SA transport. [ABSTRACT FROM PUBLISHER]

Harpin fromPseudomonas syringaepv.phaseolicola(HrpZ) elicits a rapid cell death response in tobacco plants. Multiple signaling components, including mitogen-activated protein kinase (MAPK), reactive oxygen species (ROS) and salicylic acid (SA), have been reported to be involved in this cell death process, but the interaction between these molecules is poorly understood. Here we show through utilizing plants manipulated in SIPK expression levels that lack of SIPK results in increased sensitivity to harpin with concomitant accumulation of higher levels of ROS. Conversely, SIPK-overexpressing plants show reduced sensitivity to harpin relative to wild-type plants, and display reduced ROS accumulation. Harpin-induced cell death was found to be conditional on the ability of the plant to accumulate SA, whereas harpin induction of MAPK activation and ROS accumulation are not. However, harpin-induced ROS accumulation is required for activation of SIPK and wound-induced protein kinase. Transcriptional profiling revealed that suppression of SIPK signaling also affects early expression of a range of pathogen- and stress-responsive genes during harpin challenge. [ABSTRACT FROM AUTHOR]

To investigate the role of the proteinaceous elicitor, harpin, on host and nonhost plants, we isolated the harpin-coding gene, hrpZ, from Pseudomonas syringae pvs. pisi, glycinea, tabaci and tomato. Effects of the recombinant harpin proteins on pea plants were analyzed and compared with the effects of the corresponding bacterial treatment. After inoculation of pea with pea pathogen P. syringae pv. pisi, the bacterial population increased and the accumulation of PAL-mRNA and pisatin was inhibited. The nonpathogenic pathovars, glycinea, tabaci and tomato induced both defense responses in pea. However, none of the harpins induced the hypersensitive reaction or accumulation of PAL-mRNA and pisatin in pea. Harpins from P. syringae pvs. glycinea, tomato and pisi did induce these defense responses in tobacco, however, suggesting that externally applied harpins either are not recognized or are nonfunctional in pea plants. [ABSTRACT FROM AUTHOR]

To elucidate the role of harpins produced by Pseudomonas syringae, the corresponding hrpZ gene was isolated from P. s. pv. tabaci. The sequence information revealed that this gene carries a serious mutation with 326 bp lacking in the central region and potentially encodes only 140 N-terminal amino acids because of a frame shift. The investigation of biological properties using recombinant harpin indicated harpinpsta was incapable of inducing HR in both host and nonhost plants. Based on an immunoblot analysis to detect harpin from P. s. pathovars in hrp-inducing medium, the truncated harpinpsta was neither expressed nor secreted into the culture medium. These results suggest that harpin is not the sole determinant of the host-parasite specificity in P. s. pv. tabaci. [ABSTRACT FROM AUTHOR]

Methyl jasmonate inhibited the harpin-induced defense responses such as cell death, H2O2 generation and gene expression encoding phenylalanine ammonia-lyase in tobacco suspension cultured BY-2 cells. These results suggest that MeJA may act as an endogenous suppressor for plant defense response including hypersensitive reaction. [ABSTRACT FROM PUBLISHER]

Elicitation ofArabidopsis thaliana(L.) Heynh. suspension cultures with the bacterial protein harpin (fromPseudomonas syringaepv.syringae) induced the activation of two kinases of 39 and 44?kDa, as demonstrated by in-gel kinase assays using myelin basic protein (MBP) as a substrate. Both these kinases appeared to be tyrosine-phosphorylated upon activation, as demonstrated by treatment with tyrosine phosphatase and immunoprecipitation using an anti-phosphotyrosine monoclonal antibody. An inhibitor of mammalian mitogen-activated protein kinase (MAPK) activation, PD98059, inhibited harpin-induced MBPK activation, but did not inhibit the activity of these kinases. PD98059 also inhibited harpin-induced programmed cell death and defence gene expression, suggesting the involvement of harpin-induced MAPKs in defence responses inArabidopsis thaliana. [ABSTRACT FROM AUTHOR]

Using harpin protein gene from apple fire blight pathogen Erwinia amylovora and potato prp1-1 promoter as main DNA elements, the feasibility of using pathogen infection-induced hypersensitive response was explored as a new strategy of engineering fungal disease resistance. Three plant transformation vectors were constructed and 68 transgenic potato plants were produced through Agrobacterium mediated transformation method. Southern, Northern and Western blot analysis demonstrated the insertion, transcription and protein expression of harpin protein gene in transgenic plants. Disease resistance test using a complex race of Phytophthora infestans as challenging pathogen showed that both constitutive and pathogen infection-induced expression of harpin protein gene in transgenic potato reduced the lesion growth rate of fungus. Among plants where harpin protein gene expression was induced only by fungus infection, two plants were found to be highly resistant to P. infestans infection. Fungal hyphae were not produced on total of 30 inoculated leaves from the two resistant plants and necrotic lesion was limited to inoculation area. The results highlighted that engineered hypersensitive response in plants was a very promising approach to produce fungal disease resistant genotype. [ABSTRACT FROM AUTHOR]

The rapid production and accumulation of active oxygen species (AOS), the oxidative burst, has been shown to occur in a variety of plant/pathogen systems. In particular, two species, hydrogen peroxide (H2O2) and the superoxide radical anion O2•- have received considerable attention. H2O2 and O2•-, while acting directly as antimicrobial agents, may also serve as second messengers or catalysts in plants to activate a more diverse set of defense responses. Some of the better studied downstream responses promoted by AOS are (1) the cross-linking of cell wall proteins, (2) the induction of defense-related genes, (3) the stimulation of phytoalexin biosynthesis and (4) promotion of the hypersensitive response (HR). A useful model for studying the oxidative burst in plants is the neutrophil NADPH oxidase complex, the primary source of AOS production in mammals. Several of the subunits of the neutrophil NADPH oxidase complex have been immunologically identified in plants. Furthermore, many of the components known to be involved in the signal transduction pathway in neutrophils have also been found to play a role in the oxidative burst in plants. Just as various ligands activate the oxidase complex in neutrophils, several ligands (elicitors or pathogens) also lead to induction of the oxidative burst in plant cells. The similarities between the neutrophil and plant oxidative bursts will be elaborated in this review. Following stimulation with elicitors, different signal transduction pathways are activated in plants, depending on the source of elicitor used. While the identities and chronologies of the major intermediates in these pathways remain largely unknown, there is strong evidence at least for participation of phospholipases, H+/K+ exchange, Ca2+ influxes, protein kinases and phosphatases, and GTP binding proteins. In an effort to integrate these various signaling events into a single scheme, we have constructed a hypothetical model that proposes how different elicitors might induce the oxidative burst in the same cell by different pathways. [ABSTRACT FROM AUTHOR]

Harpins are proteins secreted by the type-three secretion system of phytopathogenic bacteria. They are known to induce a hypersensitive response (HR) in non-host plant leaf tissue. Erwinia amylovora, the fire blight pathogen of pear and apple trees, secretes two different harpins, HrpNea and HrpWea. In the present study, we showed that an Erwinia amylovora hrpWea mutant induces stronger electrolyte leakages in Arabidopsis thaliana foliar disks than the wild-type strain, thus suggesting that HrpWea could function as a HR negative modulator. We confirmed this result by using purified HrpWea and HrpNea. HrpWea has dual effects depending on its concentration. At 200 nM, HrpWea, like HrpNea, provoked the classical defense response -- active oxygen species (AOS) production and cell death. However, at 0.2 nM, HrpWea inhibited cell death and AOS production provoked by HrpNea. HrpWea probably inhibits HrpNea-induced cell death by preventing anion channel inhibition, confirming that anion channel regulation is a determinant feature of the plant response to harpins. Collectively our data show that the HrpWea harpin can act antagonistically to the classical HrpNea harpin by suppressing plant defense mechanisms. [ABSTRACT FROM AUTHOR]

Following the infiltration of heterologous pathovars of Pseudomonas syringae two localized responses develop in a parallel manner in tobacco leaves: an early form of induced resistance (EIR) and the hypersensitive reaction (HR). The EIR inhibits the metabolic activity of infiltrated bacteria and the leaf tissue remains symptomless whereas HR also inhibits bacteria, but the leaf tissue shows confluent necrosis or necrosis of individual plant cells, depending on the inoculum concentration. After infiltration of a heterologous strain the rapid development of HR usually masks the effects of EIR which is developing at the same time. The aim of this study was to characterize the conditions in which EIR occurs in the absence of HR following the infiltration of heterologous pathovars of Pseudomonas syringae pathovars. A study was made of numerous experimental interactions of heterologous bacteria/tobacco leaves in which the HR induction time of the bacteria and/or the EIR response of the plant were modified. When (a) the HR induction time was extended by chloramphenicol treatment of bacterial cells or by using transposon mutants (Tn5), or (b) the time needed for the development of EIR was shortened (at 30°C or in young leaves) the heterologous strains were unable to induce visible HR. But when the EIR was suppressed in tobacco leaves by pretreatment with cycloheximide or thermal shock (50°C for 13 s) the HR appeared. Comparison of results of numerous experiments revealed that solely EIR was present if its development time was shorter than the induction period of HR. This interpretation was confirmed by the infiltration of partially purified harpinPss into tobacco leaves, where it caused plant cell death in the presence of EIR. [ABSTRACT FROM AUTHOR]