Particular Signaling Interactions In Harpin-stimulated Plant Defense And Growth

Plants can activate different signaling pathways to defend themselves in response tobiotic and abiotic stimuli. Basal defense pathways leading to non-specific resistance todifferent categories of pathogens are mediated by phytohormones salicylic acid (SA),jasmonic acid (JA) and ethylene. However, these pathways also play an important role ininsect resistance, plant growth and development in response to stimulations by exogenoussignals. Abscisic acid (ABA), another phytohormone, can be an endogenous signal toinitiate adaptive responses when plants are challenged by drought and salt stress. ABA isrequired, however, not only as a stress hormone but also under non-stress conditions foroptimal development because it fine-tunes plant growth through a regulatory circuit thatincludes the phytohormone ethylene. So it is ubiquitous for these pathways to crosstalkwith each other, depending on which signaling components are recruited into the pathwayin response to different stimuli. Such signaling components are the keys to link differentpathways and construct an efficient signaling network for plant to confront challengesencountered. Studies involved in the M.Sc. project are aimed to determine the signalingpathways and their crosstalk in plants responding to harpins that can induce pathogendefense, plant growth enhancement, insect resistance and drought tolerance in plants.Dissection of harpin signaling mechanism in plantsHarpins are bacterial proteins that can enhance plant growth and defense againstpathogens and insects. To elaborate whether harpins perform the diverse functions incoordination with the activation of specific promoters that contain particular elements, wecloned pathogen-inducible plant promoters PPP1, PPP2, and PPP3 from tobacco andinvestigated their responses to harpin_Xoo or its truncated fragments DEG, DIR, and DPR(domains for enhancing plant growth, insect resistance and pathogen resistance). PPP1 contains an internal repeat composed of two tandem 111 bp fragments; 111 bp in the repeatwas deleted in PPP2. PPP3 contains a bacteria-inducible element; PPP1 and PPP2additionally contain TAC-1 and Eli boxes inducible correspondingly by salicylic acid (SA)and elicitors. Function of cloned PPPs was confirmed based on their activation intransgenic Arabidopsis plants by Ralstonia solanacearum (Ralston) or SA. Harpin_Xoo, DEG,DIR, or DPR activated PPP1 and PPP2 but not PPP3, consistent with the presence of Eliboxes in promoters. PPP1 was ca. 3-fold more active than PPP2, suggesting that theinternal repeat affects levels of the promoter activation.Harpin_Xoo encode by the hpaG_Xoo gene of Xathomonas oryzae pv. oryzae, is a memberof the harpin group of proteins that induce pathogen resistance and hypersensitive cell death(HCD) in plants. We elaborated whether both processes are correlated inhpaG_Xoo-expressing tobacco (HARTOB) plants, which produced harpin_Xoo intracellularly.Resistance to fungal, bacterial, and viral pathogen increased in HARTOB, in correlationwith the expression of hpaG_Xoo, the gene NPR1 that regulates several resistance pathways,and defense genes GST1, Chia5, PR-1a, and PR-1b that are mediated by different signals.However, reactive oxygen intermediate burst, the expression of HCD marker genes hsr203and hinl, and cell death did not occur spontaneously in HARTOB, though they did inuntransformed and HARTOB plants treated exogenously with harpinxoo. Thus, thetransgenic expression of harpin_Xoo confers nonspecific pathogen defense in the absence ofHCD.Dissection of Harpin Signaling in PGE, IR and DTEthylene (ET) signal transduction may regulate plant growth and defense, dependingon which components are recruited into the pathway in response to different stimuli. Wereport here that the ET pathway controls both insect resistance (IR) and plant growthenhancement (PGE) in Arabidopsis (Arabidopsis thaliana) plants responding to harpin, aprotein produced by a plant pathogenic bacterium. PGE may result from spraying plant topswith harpin or by soaking seeds in harpin solution; the latter especially enhances rootgrowth. Plants treated similarly develop resistance to the green peach aphid (Myzuspersicae). The salicylic acid pathway, although activated by harpin, does not lead to PGEand IR. By contrast, PGE and IR are induced in both wild-type plants and genotypes thathave defects in salicylic acid signaling. In response to harpin, levels of jasmonic acid (JA)decrease, and the COI1 gene, which is indispensable for JA signal transduction, is not expressed in wild-type plants. However, PGE and IR are stimulated in the JA-resistantmutant jarl-1. In the wild type, PGE and IR develop coincidently with increases in ETlevels and the expression of several genes essential for ET signaling. The ET receptor geneETR1 is required because both phenotypes are arrested in the etrl-1 mutant. Consistently,inhibition of ET perception nullifies the induction of both PGE and IR. The signaltransducer EIN2 is required for IR, and E1N5 is required for PGE because IR and PGE areimpaired correspondingly in the ein2-1 and ein5-1 mutants. Therefore, harpin activates ETsignaling while conscribing EIN2 and EIN5 to confer IR and PGE, respectively.Harpin not only stimulates plant growth and resistance to pathogens and insects, butalso activates abscisic acid (ABA) signalling to induce drought tolerance (DT) inArabidopsis thaliana L. plants grown with water stress. Spraying wild-type plants withHrpN-promoted stomatal closure decreased leaf transpiration rate, increased moisture andproline levels in leaves, and alleviated extents of damage to cell membranes and plantdrought symptoms caused by water deficiency. In plants treated with HrpN, ABA levelsincreased; expression of several ABA-signalling regulatory genes and the importanteffector gene rd29B was induced or enhanced. Induced expression of rd29B, promotion ofstomatal closure, and reduction in drought severity were observed in the abil-1 mutant,which has a defect in the phosphatase ABI1, after HrpN was applied. In contrast, HrpNfailed to induce these responses in the abi2-1 mutant, which is impaired in the phosphataseABI2. Inhibiting wild-type plants to synthesize ABA eliminated the role of HrpN inpromoting stomatal closure and reducing drought severity. Moreover, resistance toPseudomonas syringae developed in abi2-1 as in wild-type plants following treatment withHrpN. Thus, an ABI2-dependent ABA signalling pathway is responsible for the inductionof DT but does not affect pathogen defence under the circumstances of this study.Crosstalk between ethylene signaling and ABA signaling in harpininduced PGE, IR and DTIn Arabidopsis thaliana (Arabidopsis) plants responding to harpin, a protein producedby a plant pathogenic bacterium, abscisic acid (ABA) and ethylene (ET) are stimulated tomediate drought tolerance (DT), plant growth enhancement (PGE), and insect resistance(IR). These effects involve synergistic and differential actions by both hormones based onpharmacological studies with wild-type (WT) plant, ET-resistant (etr) or ET-insensitive (ein)and ABA-insensitive (abi) mutants of Arabidopsis. Soaking WT seeds in a solution of harpin promoted germination and subsequent root growth. The effects were impaired byetrl-1 and abi2-1, and by inhibiting WT seeds to synthesize or sense either of ABA and ET.Thus, signaling by both hormones is required. When harpin was applied to the aerial partsof WT plants, plant growth increased and multiplication of aphids colonized on the plantsdecreased. Signaling by ET rather than ABA was critical because PGE and IR werenullified in etrl-1 rather than abi2-1 and by inhibiting WT plants to synthesize or sense ET,instead of ABA. In plants growing with drought stress, signaling by ABA in contrast to ETregulated harpin-induced DT responses. The responses similarly occurred in WT and etrl-1but were abolished by abi2-1 and by inhibiting WT to synthesize ABA rather than ET. Inparallel assays, ein2-1 lost IR and ein5-1 compromised the promotion of germination andgrowth, but abil-1 showed no effect in all the cases. These results suggest that theharpin-stimulated signaling pathways function differentially according to the immediaterequirements for plants to confront challenges encountered.