Hansong Dong

Harpin-elicited hypersensitive cell death and pathogen resistance require the NDR1 and EDS1 genes

Abstract Plants sprayed with harpin, a bacterial protein that induces hypersensitive cell death (HCD), develop systemic acquired resistance (SAR) without macroscopic necrosis. HCD sometimes accompanies the development of resistance conferred by resistance ( R ) genes. In Arabidopsis , some R genes require one or both of the signalling components NDR1 and EDS1 for function. This study addresses whether HCD, NDR1 and EDS1 are required for induction of SAR by harpin. When Arabidopsis and tobacco leaves were sprayed with harpin, microscopic hypersensitive response (micro-HR) lesions developed. Systemic expression of PR genes and the development of resistance were accompanied by micro-HR, except in the ndr 1-1 mutant, in which harpin induced micro-HR without the development of resistance or expression of the PR -1 gene. Cell death and resistance did not occur following treatment with harpin in plants that could not accumulate salicylic acid. Harpin also failed to induce resistance in Arabidopsis eds 1-1 mutants. Therefore, harpin-induced resistance seems to develop concomitantly with cell death and resistance requires NDR 1 and EDS 1.

Harpin-induced expression and transgenic overexpression of the phloem protein gene AtPP2-A1 in Arabidopsis repress phloem feeding of the green peach aphid Myzus persicae

BackgroundTreatment of plants with HrpNEa, a protein of harpin group produced by Gram-negative plant pathogenic bacteria, induces plant resistance to insect herbivores, including the green peach aphid Myzus persicae, a generalist phloem-feeding insect. Under attacks by phloem-feeding insects, plants defend themselves using the phloem-based defense mechanism, which is supposed to involve the phloem protein 2 (PP2), one of the most abundant proteins in the phloem sap. The purpose of this study was to obtain genetic evidence for the function of the Arabidopsis thaliana (Arabidopsis) PP2-encoding gene AtPP2-A1 in resistance to M. persicae when the plant was treated with HrpNEa and after the plant was transformed with AtPP2-A1.ResultsThe electrical penetration graph technique was used to visualize the phloem-feeding activities of apterous agamic M. persicae females on leaves of Arabidopsis plants treated with HrpNEa and an inactive protein control, respectively. A repression of phloem feeding was induced by HrpNEa in wild-type (WT) Arabidopsis but not in atpp2-a1/E/142, the plant mutant that had a defect in the AtPP2-A1 gene, the most HrpNEa-responsive of 30 AtPP2 genes. In WT rather than atpp2-a1/E/142, the deterrent effect of HrpNEa treatment on the phloem-feeding activity accompanied an enhancement of AtPP2-A1 expression. In PP2OETAt (AtPP2-A1-overexpression transgenic Arabidopsis thaliana) plants, abundant amounts of the AtPP2-A1 gene transcript were detected in different organs, including leaves, stems, calyces, and petals. All these organs had a deterrent effect on the phloem-feeding activity compared with the same organs of the transgenic control plant. When a large-scale aphid population was monitored for 24 hours, there was a significant decrease in the number of aphids that colonized leaves of HrpNEa-treated WT and PP2OETAt plants, respectively, compared with control plants.ConclusionsThe repression in phloem-feeding activities of M. persicae as a result of AtPP2-A1 overexpression, and as a deterrent effect of HrpNEa treatment in WT Arabidopsis rather than the atpp2-a1/E/142 mutant suggest that AtPP2-A1 plays a role in plant resistance to the insect, particularly at the phloem-feeding stage. The accompanied change of aphid population in leaf colonies suggests that the function of AtPP2-A1 is related to colonization of the plant.

The Internal Glycine-Rich Motif and Cysteine Suppress Several Effects of the HpaGXooc Protein in Plants

ABSTRACT HpaG(Xooc), produced by Xanthomonas oryzae pv. oryzicola, is a member of harpin group of proteins that stimulate plant growth, hypersensitive cell death (HCD), and pathogen defense. The protein contains two copies of the glycine-rich motif (GRM), a characteristic of harpins, and a cysteine, which is absent in other harpins. Genetic modification generated the pro-tein mutants HpaG(Xooc)MG (MG) by deleting GRMs and HpaG(Xooc)C47T (C47T) by replacing cysteine with threonine. When applied to tobacco plants, C47T and MG were 1.2- and 1.7-fold stronger, respectively, than HpaG(Xooc) in inducing HCD, which occurred consistently with expression of the marker genes hin1 and hsr203. The proteins markedly alleviated infection of tobacco by Tobacco mosaic virus and Arabidopsis and tomato by Pseudomonas syringae. Treating tobacco plants with HpaG(Xooc), C47T, and MG decreased the viral infection by 58, 81, and 92%, respectively. In Arabidopsis and tomato plants treated with HpaG(Xooc), C47T, or MG, P. syringae multiplication was inhibited; bacterial population multiplied in 5 days in these plants were ca. 160-, 1,260-, or 15,860-fold smaller than that in control plants. So pathogen defense was induced in both plants. Defense-related genes Chia5, NPR1, and PR-1a were expressed consistently with resistance. In response to HpaG(Xooc), C47T, and MG, aerial parts and roots of tomato plants increased growth by 15 and 53%, 25 and 77%, and 46 and 106%, relative to controls. The expansin gene, EXP2, involved in the cell expansion and plant growth was expressed coordinately with plant growth promotion. These results suggest that the presence of GRM and cysteine in HpaG(Xooc) represses the effects of the protein in plants.

An h-Type Thioredoxin Functions in Tobacco Defense Responses to Two Species of Viruses and an Abiotic Oxidative Stress

Various thioredoxin (Trx) proteins have been identified in plants. However, many of the physiological roles played by these proteins remain to be elucidated. We cloned a TRXh-like gene predicted to encode an h-type Trx in tobacco (Nicotiana tabacum) and designated it NtTRXh3, based on the biochemical activity of the NtTRXh3 protein. Overexpression of NtTRXh3 conferred resistance to Tobacco mosaic virus and Cucumber mosaic virus, both of which showed reduced multiplication and pathogenicity in NtTRXh3-overexpressing plants compared with controls. NtTRXh3 overexpression also enhanced tobacco resistance to oxidative stress induced by paraquat, an herbicide that inhibits the production of reducing equivalents by chloroplasts. The NtTRXh3 protein localized exclusively to chloroplasts in coordination with the maintenance of cellular reducing conditions, which accompanied an elevation in the glutathione/glutathione disulfide couple ratio. NtTRXh3 gene expression and NtTRXh3 protein production were necessary for these defensive responses, because they were all arrested when NtTRXh3 was silenced and the production of NtTRXh3 protein was abrogated. These results suggest that NtTRXh3 is involved in the resistance of tobacco to virus infection and abiotic oxidative stress.

Transcription Factor AtMYB44 Regulates Induced Expression of the ETHYLENE INSENSITIVE2 Gene in Arabidopsis Responding to a Harpin Protein

AtMYB44 is a transcription factor that functions in association with the ethylene-signaling pathway in Arabidopsis thaliana. The pathway depends on ETHYLENE INSENSITIVE2 (EIN2), an essential component of ethylene signaling, to regulate defense responses in the plant following treatment with HrpN(Ea), a harpin protein from a bacterial plant pathogen. Here, we show that AtMYB44 regulates induced expression of the EIN2 gene in HrpN(Ea)-treated Arabidopsis plants. A HrpN(Ea) and ethylene-responsive fragment of the AtMYB44 promoter is sufficient to support coordinate expression of AtMYB44 and EIN2 in specific transgenic Arabidopsis. In the plant, the AtMYB44 protein localizes to nuclei and binds the EIN2 promoter; the HrpN(Ea) treatment promotes AtMYB44 production, binding activity, and transcription of AtMYB44 and EIN2. AtMYB44 overexpression results in increased production of the AtMYB44 protein and the occurrence of AtMYB44-EIN2 interaction under all genetic backgrounds of wild-type Arabidopsis and the etr1-1, ein2-1, ein3-1, and ein5-1 mutants, which have defects in the ethylene receptor ETR1 and the signal regulators EIN2, EIN3, and EIN5. However, AtMYB44 overexpression leads to enhanced EIN2 expression only under backgrounds of wild type, ein3-1, and ein5-1 but not etr1-1 and ein2-1, suggesting that ethylene perception is necessary to the regulation of EIN2 transcription by AtMYB44.

Thirty-seven transcription factor genes differentially respond to a harpin protein and affect resistance to the green peach aphid in Arabidopsis.

The harpin protein HrpNEa induces Arabidopsis resistance to the green peach aphid by activating the ethylene signalling pathway and by recruiting EIN2, an essential regulator of ethylene signalling, for a defence response in the plant. We investigated 37 ethylene-inducible Arabidopsis transcription factor genes for their effects on the activation of ethylene signalling and insect defence. Twenty-eight of the 37 genes responded to both ethylene and HrpNEa, and showed either increased or inhibited transcription, while 18 genes showed increased transcription not only by ethylene but also by HrpNEa. In response to HrpNEa, transcription levels of 22 genes increased, with AtMYB44 being the most inducible, six genes had decreased transcript levels, and nine remained unchanged. When Arabidopsis mutants previously generated by mutagenicity at the 37 genes were surveyed, 24 mutants were similar to the wild type plant while four mutants were more resistant and nine mutants were more susceptible than wild type to aphid infestation. Aphid-susceptible mutants showed a greater susceptibility for atmyb15, atmyb38 and atmyb44, which were generated previously by T-DNA insertion into the exon region of AtMYB15 and the promoter regions of AtMYB38 and AtMYB44. The atmyb44 mutant was the most susceptible to aphid infestation and most compromised in induced resistance. Resistance accompanied the expression of PDF1.2, an ethylene signalling marker gene that requires EIN2 for transcription in wild type but not in atmyb15, atmyb38, and atmyb44, suggesting a disruption of ethylene signalling in the mutants. However, only atmyb44 incurred an abrogation in induced EIN2 expression, suggesting a close relationship between AtMYB44 and EIN2.

RAP2.6L overexpression delays waterlogging induced premature senescence by increasing stomatal closure more than antioxidant enzyme activity

Waterlogging usually results from overuse or poor management of irrigation water and is a serious constraint due to its damaging effects. RAP2.6L (At5g13330) overexpression enhances plant resistance to jasmonic acid, salicylic acid, abscisic acid (ABA) and ethylene in Arabidopsis thaliana. However, it is not known whether RAP2.6L overexpression in vivo improves plant tolerance to waterlogging stress. In this study, the RAP2.6L transcript was induced by waterlogging or an ABA treatment, which was reduced after pretreatment with an ABA biosynthesis inhibitor tungstate. Water loss and membrane leakage were reduced in RAP2.6L overexpression plants under waterlogging stress. Time course analyses of ABA content and production of hydrogen peroxide (H2O2) showed that increased ABA precedes the increase of H2O2. It is also followed by a marked increase in the antioxidant enzyme activities. Increased ABA promoted stomatal closure and made leaves exhibit a delayed waterlogging induced premature senescence. Furthermore, RAP2.6L overexpression caused significant increases in the transcripts of antioxidant enzyme genes APX1 (ascorbate peroxidase 1) and FSD1 (Fe-superoxide dismutase 1), the ABA biosynthesis gene ABA1 (ABA deficient 1) and signaling gene ABH1 (ABA-hypersensitive 1) and the waterlogging responsive gene ADH1 (alcohol dehydrogenase 1), while the transcript of ABI1 (ABA insensitive 1) was decreased. ABA inhibits seed germination and seedling growth and phenotype analysis showed that the integration of abi1-1 mutation into the RAP2.6L overexpression lines reduces ABA sensitivity. These suggest that RAP2.6L overexpression delays waterlogging induced premature senescence and might function through ABI1-mediated ABA signaling pathway.

Nicotiana tabacum TTG1 contributes to ParA1-induced signalling and cell death in leaf trichomes.

Leaf trichomes serve as a physical barrier and can also secrete antimicrobial compounds to protect plants from attacks by insects and pathogens. Besides the use of the physical and chemical mechanisms, leaf trichomes might also support plant responses by communicating the extrinsic cues to plant intrinsic signalling pathways. Here we report a role of leaf trichomes in tobacco (Nicotiana tabacum) hypersensitive cell death (HCD) induced by ParA1, an elicitin protein from a plant-pathogenic oomycete. After localized treatment with ParA1, reactive oxygen species were produced first in the leaf trichomes and then in mesophylls. Reactive oxygen species are a group of intracellular signals that are crucial for HCD to develop and for cells to undergo cell death subsequent to chromatin condensation, a hallmark of HCD. These events were impaired when the production of hydrogen peroxide (H2O2) was inhibited by catalase or a NADPH-oxidase inhibitor applied to trichomes, suggesting the importance of H2O2 in the pathway of HCD signal transduction from the trichomes to mesophylls. This pathway was no longer activated when leaf trichomes were treated with C51S, a ParA1 mutant protein defective in its interaction with N. tabacum TTG1 (NtTTG1), which is a trichome protein that binds ParA1, rather than C51S, in vitro and in trichome cells. The ParA1-NtTTG1 interaction and the HCD pathway were also abrogated when NtTTG1 was silenced in the trichomes. These observations suggest that NtTTG1 plays an essential role in HCD signal transduction from leaf trichomes to mesophylls.

Combinative effects of a bacterial type-III effector and a biocontrol bacterium on rice growth and disease resistance

Expression of HpaGXoo, a bacterial type-III effector, in transgenic plants induces disease resistance. Resistance also can be elicited by biocontrol bacteria. In both cases, plant growth is often promoted. Here we address whether biocontrol bacteria and HpaGXoo can act together to provide better results in crop improvement. We studied effects ofPseudomonas cepacia on the rice variety R109 and the hpaGXoo-expressing rice line HER1. Compared to R109, HER1 showed increased growth, grain yield, and defense responses toward diseases and salinity stress. Colonization of roots byP. cepacia caused 20% and 13% increase, in contrast to controls, in root growth of R109 and HER1. Growth of leaves and stems also increased in R109 but that of HER 1 was inhibited. WhenP. cepacia colonization was subsequent to plant inoculation withRhizoctonia solani, a pathogen that causes sheath blight, the disease was less severe than controls in both R109 and HER1; HER1, nevertheless, was more resistant, suggesting thatP.cepacia and HpaGXoo cooperate in inducing disease resistance. Several genes that critically regulate growth and defense behaved differentially in HER1 and R109 while responding toP. cepacia. In R109 leaves, theOsARF1 gene, which regulates plant growth, was expressed in consistence with growth promotion byP. cepacia. Inversely,OsARF1 expression was coincident with inhibition in growth of HER1 leaves. In both plants, the expression ofOsEXP1, which encodes an expansin protein involved in plant growth, was concomitant with growth promotion in leaves instead of roots, in response toP. cepacia. We also studiedOsMAPK, a gene that encodes a mitogen-activated protein kinase and controls defense responses toward salinity and infection by pathogens in rice. In response toP. cepacia, an early expression ofOsMAPK was coincident with R109 resistance to the disease, while HER1 expressed the gene similarly whetherP. cepacia was present or not. Evidently,P. cepacia and GXoo-gene mediated resistance may act differently in rice growth and resistance. Whereas combinative effectsof P. cepacia and HpaGXoo in disease resistance have a great potential in agricultural use, it is interesting to study mechanisms that underlie interactions involving biocontrol bacteria, type-III effectors and pathogens.

Productivity and biochemical properties of green tea in response to full-length and functional fragments of HpaG Xooc, a harpin protein from the bacterial rice leaf streak pathogen Xanthomonas oryzae pv. oryzicola.

Harpin proteins from plant pathogenic bacteria can stimulate hypersensitive cell death (HCD), drought tolerance, defence responses against pathogens and insects in plants, as well as enhance plant growth. Recently, we identified nine functional fragments of HpaGXooc, a harpin protein from Xanthomonas oryzae pv. oryzicola, the pathogen that causes bacterial leaf streak in rice. Fragments HpaG1–94, HpaG10–42, and HpaG62–138, which contain the HpaGXooc regions of the amino acid sequence as indicated by the number spans, exceed the parent protein in promoting growth, pathogen defence and HCD in plants. Here we report improved productivity and biochemical properties of green tea (Camellia sinensis) in response to the fragments tested in comparison with HpaGXooc and an inactive protein control. Field tests suggested that the four proteins markedly increased the growth and yield of green tea, and increased the leaf content of tea catechols, a group of compounds that have relevance in the prevention and treatment of human diseases. In particular, HpaG1–94 was more active than HpaGXooc in expediting the growth of juvenile buds and leaves used as green tea material and increased the catechol content of processed teas. When tea shrubs were treated with HpaHXooc and HpaG1–94 compared with a control, green tea yields were over 55% and 39% greater, and leaf catechols were increased by more than 64% and 72%, respectively. The expression of three homologues of the expansin genes, which regulate plant cell growth, and the CsCHS gene encoding a tea chalcone synthase, which critically regulates the biosynthesis of catechols, were induced in germinal leaves of tea plants following treatment with HpaG1–94 or HpaGXooc. Higher levels of gene expression were induced by the application of HpaG1–94 than HpaGXooc. Our results suggest that the harpin protein, especially the functional fragment HpaG1–94, can be used to effectively increase the yield and improve the biochemical properties of green tea, a drink with medicinal properties.