Roles Of Ethylene, Abscisic Acid, And AtMYB44 In HrpN_Ea-induced Arabidopsis Growth And Resistance To The Green Peach Aphid

In Arabidopsis thaliana (Arabidopsis) responding to HrpNEa, a harpin protein produced by a plant pathogenic bacterium, abscisic acid (ABA) and ethylene are stimulated to mediate drought tolerance, plant growth enhancement, and insect resistance, i.e., Arabidopsis resistance to the green peach aphid (GPA). These effects involve synergistic and differential actions by both hormones based on pharmacological studies with wild-type (WT) plant, ethylene-resistant(etr) or ethylene-insensitive (ein) and ABA-insensitive (abi) mutants of Arabidopsis. This study was aimed at characterizations of:(â…°) the relationship between ABA signaling and ethylene signaling pathways activated by HrpNEa; (â…±) the possibility that any types of transcription factors could be recruited into HrpNEa-activated ethylene signaling to control Arabidopsis resistance against GPA, and (â…²) the possibility that the phloem-related defense (PRD) plays a role in plant resistance.The Induction of ABA-Mediated Drought Tolerance is Independent of Ethylene Signaling in Arabidopsis Plants Responding to HrpNEa When HrpNEa was applied to the aerial parts of WT plants, multiplication of aphids colonized on the plants decreased. Signaling by ethylene rather than ABA was critical because resistance to GPA were nullified in etr 1-1 rather than abi2-1 and by inhibiting WT plants to synthesize or sense ethylene, instead of ABA. In plants growing with drought stress, signaling by ABA in contrast to ethylene regulated HrpNEa-induced DT responses. The responses similarly occurred in WT and etrl-1 but were abolished by abi2-l and by inhibiting WT to synthesize ABA rather than ethylene. In parallel assays, ein2-1 lost IR, but abil-1 showed no effect in the cases. These results suggest that the HrpNEa-stimulated signaling pathways function differentially according to the immediate requirements for plants to confront challenges encountered.Screening of HrpNEa-Responsive Transcription Factors Involved in Arabidopsis Resistance to GPA Totally 37 transcription factors were investigated. RT-PCR was conducted to determine the expression of 37 transcription factor genes in HrpNEa-treated WT plants. Selected 37 TF genes were investigated for expression in WT Arabidopsis following treatment with HrpNEa, which was used in contrast to EVP, a specific control containing inactive proteins but no HrpNEa. Twenty-one genes increased,5 genes decreased, and else 11 genes did not evidently change amounts of transcripts in plants 6 and 12 h post treatment (hpt) with HrpNEa, compared to 0 hpt with HrpNEa and 12 hpt with EVP. As confirmed by RNA gel blot analysis,12 of 13 selected genes accumulated great amounts of transcripts in 12 hpt with HrpNEa, they are AtMYB51, AtMYB38, AtMYB108, AtMYB15, AtMYB30, AtMYB44, AtERF11, RAP2.12, F15H21.12, K13N2.14, AtHB-7and AtC3HC4, but the expression of MLN21.9 was repressed. To determine transcript levels, the 9 genes were subjected to real time PCR protocols. In 12 hpt, transcripts of AtMYB44 and other 8 genes expressed in HrpNEa-treated plants were greater than those in EVP-treated plants. To correlate the expression of TF genes with Arabidopsis resistance to the green peach aphid, the plant mutants disrupted at the 37 TF genes were tested for variations in supporting the insect to colonize plant leaves and multiply on leaves. First, Repellency rate was scored to identify possible regulators of insect repellency. A lower repellency rate means a preference of aphids. To determine if 37 transcription factor genes are required for HrpNEa to induce plant resistance against aphids, mutants and WT were compared in supporting aphids to colonize leaves and multiply on leaves of plants 5 d posttreatment (dpt) with EVP or HrpNEa. Wingless agamic females of GPA were placed on leaves of EVP-treated and HrpNEa-treated plants of WT and mutants, respectively. Repellency was scored 24 h later. In the atmyb44 mutant, the repellency% was least, indicating that the effect of HrpNEa was abolished and AtMYB44 was an possible regulator of insect repellency. Second, to study consistency of mutant effects on reproduction rates, multiplication of the insect on mutants were tested 5 d posttreatment (dpt). In the selected 9 mutants, aphids multiplication were repressed except atmyb44 responding to HrpNEa. In all, we get a selected transcription factor AtMYB44 which is responsive to HrpNEa, and it may take part in the effect of HrpNEa in regulating insect resistance in Arabidopsis.AtMYB44 controls Arabidopsis resistance to GPA by targeting ethylene signaling regulator ein2AtMYB44 is a transcription factor responsive to ethylene and implicated in Arabidopsis defense response. Plant defense is often subject to ethylene signaling that recruits EIN2 as a central regulator. Previously we have shown that EIN2 plays a critical role during the development of insect defense in Arabidopsis treated with HrpNEa, a harpin protein produced by bacterial plant pathogen. Regulatory targets of AtMYB44 and regulation of EIN2 activation have not been characterized. Through genetic, molecular, and pharmacological studies, we show that AtMYB44, EIN2, and ethylene are required for HrpNEa to induce Arabidopsis resistance against the green peach aphid. In wild-type plant, the application of HrpNEa impairs reproduction of aphids colonized on leaves. This effect is arrested in atmyb44, an Arabidopsis mutant disrupted at AtMYB44 promoter, and can be retrieved by complementing atmyb44 with AtMYB44 coding sequence fused to a HrpNEa/ethylene-responsive fragment of the promoter. Constitutive AtMYB44 expression also enables atmyb44 to defy aphid reproduction. In wild-type and atmyb44-complemented plants, extrinsic ethylene duplicates the function of intrinsic ethylene to induce resistance and the expression of AtMYB44 and EIN2. In atmy44-complemented plants, moreover, AtMYB44 protein localizes to nuclei and specifically binds EIN2 promoter. HrpNEa treatment promotes AtMYB44 production and the binding activity as well as EIN2 expression. When atmyb44-complemented and wild-type plants are subjected to aphid infestation, AtMYB44 and EIN2 expression is induced and plants become resistant to secondary infestation. Therefore, AtMYB44 controls Arabidopsis resistance to the insect by activating the expression of EIN2. This regulatory pathway functions as an important mechanism that protects plants from insect herbivores.PRD Plays a Role in HrpNEa-Induced Arabidopsis resistance to GPA PRD in plants functions against attacks by sap-sucking insects. Known signaling pathways are implicated in PRD induction by biotic elicitors, such as the HrpNEa protein, but how the pathways interact to regulate PRD is unclear. In WT Arabidopsis, HrpNEa-induced PRD against GPA included the production of phloem proteins and callose deposition. The induction of PRD contributed to plant resistance shown to cause GPA repellency and inhibition of the insect reproduction. The abi2-1 and ein2-1 mutants failed to display both PRD responses and failed to develop resistance to GPA, suggesting the importance of ABI2 and EIN2 in the induction of PRD.