Abscisic Acid/Ethylene Signaling Interaction In Plant Growth And Defense Responses Toward Harpins And Plant Proteins Hypothetically Interacting With HpaG_Xoo

Harpins are glycine-rich,protease-sensitive,heat-stable,acidic proteins produced by Gram-negative plant pathogenic bacterial,and are required for induction of the hypersensitive response(HR) or hypersensitive cell death(HCD) in nonhost plants of bacteria.Application of harpins to many plants can enhance plant growth,induce resistance to pathogens,insects and drough.These effects have been observed in plants treated with HrpN_Ea from Eriwinia amylovora,HrpZ_Pss from Pseudomonas syringae pv.syringae, HrpZ_Psph from P.syringae pv.phaseolicola,and HpaG from Xanthomonas oryzae.How harpins perform these diverse functions has not been clear completely.1.Root growth of Arabidopsis thaliana is regulated by ABA and ethylene signaling interaction in response to HrpN_EaApplying HrpN_Ea to plants can stimulate ethylene and abscisic acid to induce plant growth and drought tolerance,respectively.Here we report that both hormones cooperate to mediate the role of HrpN_Ea in promoting root growth of Arabidopsis thaliana(Arabidopsis). Root growth was promoted coordinately with elevation in levels of ABA and ethylene subsequent to soaking seeds of wild-type Arabidopsis in a solution of HrpN_Ea.These responses were arrested by inhibiting wild-type seeds to sense ethylene or synthesize either of ABA and ethylene.Consistently,HrpN_Ea effects on roots were nullified in ethylene-insensitive etr1-1 and ein5-1 mutants,and ABA-insensitive mutant abi2-1 of Arabidopsis.Our results establish a mechanistic connection between enhancement of root growth and signaling by ABA and ethylene in response to HrpN_Ea.However,ethylene signaling was working in the absence of ABA signaling to promote plant growth when HrpN_Ea was applied to leaves,indicating different signaling mechanisms in leaves from roots.2.Transgenic expression of HpaG_Xoo enhances plant growth and confers plant resistance to bacteriaHpaG_Xoo,encoded by the hpaG_Xoo gene of X.oryzae pv.oryzae,is a member of harpin group of proteins.Like others harpins,HpaG_Xoo induces HR and others various effects in the plant.Here we show that expression of the hpaG_Xoo gene in transgenic Arabidopsis enhances plant growth and confers pathogen defense without HCD.The hpaG_Xoo gene was inserted into the transformation vector pBI121 between the 35S promoter and uidA(GUS) gene.Transformation was conducted by flower vacuum infiltration with recombinant Agrobacterium tumefaciens.Integration of hpaG_Xoo gene into Arabidopsis chromosomes were determined by polymerase chain reaction(PCR).The expression of hpaG_Xoo gene in hpaG_Xoo-expressing transgenic Arabidopsis(HATA1) plants was detected by RT-PCR and assays for activity of HpaG_Xoo isolated from transgenic plants.T3 plants of HATA1 lines and transgenic lines containing the vector only were tested,together with the parent (ecotype Col-0) plants,for expression of defense-related genes,cell death and resistance to pathogen bacteria.Resistance to Pseudomonas syringae pv.tomato(DC3000) was enhanced at various levels in HATA1 lines.Genes NPR1,PR-1,PR3b,which are involved in pathogen defense,were expressed to various levels in HATA1 plants tested.However, cell death was not observed in HATA1 plants.Based on these data,we concluded that expression of HpaG_Xoo in transgenic Arabidopsis plants enhances plant growth and induces expression of defense-related genes and confers nonspecific resistance to pathogenic bacteria in the absence of HCD.In addition,plant growth and resistance to bacteria expression can be induced in transgenic plants expressing HpaG_Xoo constructed with or without a signal peptide in the transformation unit.3.HpaG affects grain yield of rice in extensive grower plantingsBased on works in our lab,HpaG_10-42 is most active among the nine fragments, generated from truncating the X.oryzae pv.oryzicola HpaG_Xooc protein,in enhancing growth and inducing disease resistance in rice.Here we show evidence that HpaG_10-42 significantly exceeds HpaG_Xooc to increase grain yield of rice under grower plantings based on 9 rice varieties growing at 3 locations.Application procedures were established by testing 18 combinations of two proteins doses with treating time according to rice growth stages.HpaG_10-42 was applied to nursery seedling 10 d before transplant,late turning-green stage,late tillering stage and early heading stage of indica and japonica rice varieties using different concentration arrays.Orthogonal experimental analysis on grain yield show that the optimized concentration array of HpaG_10-42 is 6μg/ml array.HpaG_10-42 used at the minimal dosage was significantly greater than HpaG_Xooc used at higher concentrations applied at the four stages of rice growth in the effects on yield of rice.HpaG_10-42 enhances gain yield higher than local agronomic measures,including use of chemicals and HpaG_Xooc. In addition,effect of HpaG_10-42 on 9 varieties of rice is different,and have no related to type of rice.Our results provide an example for effective use of beneficial fragments derived from pathogen metabolites to increase yield in the staple food crop.4.Expression and antibody preparation of HpaG_Xoo proteinToward cellular localization of HpaG_Xoo in plants,an hpaG_Xoo expression vector was made to involve a his-tag,which facilitates protein purification by affinity chromatography. In the study,hpaG_Xoo gene was amplified by PCR from JXOâ…¢gDNA,and PCR product was 420 bp.Purified hpaG_Xoo fragment was ligased to pET30a(+) vector,creating recombinant plasmid pET30a(+)::hpaG_Xoo,which contains a His-tag-encoding sequence. Recombinant HpaG_Xoo protein was produced and subjected to sodium dodecyl sulphate polyacrylamide gel electrophoresis(SDS-PAGE),which revealed the protein as 21.6 kD in size.After infiltrated into tobacco leaves,HpaG_Xoo caused typical HR.Recombinant HpaG_Xoo protein was purified through His Trap HP columns(pre-charged with Ni²⁺).The yield was 2-4 mg/L culture and the concentration of purified proteins was 0.5-1.0 mg/ml. The HpaG_Xoo-His fusion protein also caused typical HR on tobacco.Therefore,we made an effective construction and produced active HpaG_Xoo.The polyclonal antibody against HpaG_Xoo was produced with New Zealand White Rabbit.The titer of the polyclonal antibody against HpaG_Xoo was greater than 1:16,000.The anti-HpaG_Xoo polyclonal antibody reacted with HpaG_Xoo protein in the analysis of ELISA and Western blot hybridization analysis.Moreover,Western blot hybridization analysis also revealed specific presence of HpaG_Xoo in HATA1 lines.5.Screening of proteins that interact with HpaG_Xoo using yeast two-hybridTo explore plant proteins that could interact with HpaG_Xoo,a yeast two-hybrid system was used to screen Arabidopsis cDNA library.The bait and library plasmid were cotransformed into the yeast strain Y190.Six positive clones were obtained by the selection of autotrophic phenotype andβ-galactosidase assay.Sequencing and Genbank blasting comparison demonstrated that these six clones were distinct in sequences and encoded parts of Arabidopsis riboflavin synthase(RS),tonoplast intrinsic protein 2(TIP2),fasciculin-like arabinogalactan-protein(FLAS),plasma membrane intrinsic protein(PIP1),nuclear transport factor 2(NTF2),or carbonic anhydrase 2(CA2) proteins.Moreover,similarity of the cloned sequences is 100%compared with the corresponding homologues reported previously.In conclusion,plant responses to harpins involve regulation by ABA and ethylene signaling pathways.Plant proteins interacting with HpaG may act in the pathways by different mechanisms.Characterization of how HpaG_Xoo and its interacting factors function will shed light on signaling regulation of diverse responses of plants to harpins.