| Harpins are multifunctional proteins produced by Gram-negative plant pathogenic bacteria HrpNEa is the first-characterized, well-studied harpin secreted by Erwinia amylovora. Harpins’multiple functions, especially in eliciting plant defense responses, were also elucidated initially by studies using HrpNEa as a paradigm. Early studies demonstrated that the external application of HrpNEa was able to induce resistance in a variety of plant species, and that the induced resistance effectively protected plants from attacks by insect herbivores. In response to the phloem-feeding stress, plants defend themselves by using the phloem-based defense mechanism. This mechanism involves the biosynthesis of β-1,3-glucan callose and subsequent closure of sieve pores and coagulation on sieve plates. Amain purpose of this study was to elucidate the elements which play roles in HrpNEa-induced resistance to M. persicae in Arabidopsis and the relationship among elements.1. Thirty-seven transcription factor genes differentially respond to a harpin protein and affect resistance to the green peach aphid in ArabidopsisThe harpin protein HrpNEa induces Arabidopsis resistance to the green peach aphid by activating the ethylene signaling pathway and by recruiting EIN2, an essential regulator of ethylene signaling, into defense response in the plant. Here we investigated37ethylene-inducible Arabidopsis transcription factor genes for effects on the activation of ethylene signaling and insect defense. Twenty-eight of the37genes responded to both ethylene and HrpNEa, either promoted or inhibited in transcription, and18genes were promoted not only by ethylene but also by HrpNEa. In response to HrpNEa,22genes increased in transcription levels with AtMYB44being the most inducible, six genes had decreased transcript levels, and nine remained unchanged. When Arabidopsis mutants defected at the37genes were surveyed,24mutants 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. Comparing aphid-susceptible mutants showed a greater susceptibility in atmybl5, atmyb38, and atmyb44, which were generated previously by T-DNA insertion into the exon region of AtMYB15and the promoter regions of AtMYB38and AtMYB44. The atmyb44mutant was most susceptible to aphid infestation and most compromised in induced resistance. Resistance accompanied the expression of PDF1.2, an ethylene signaling marker gene that requires EIN2for transcription, in wild type but not in atmyb15, atmyb38, and atmyb44, suggesting a disruption of ethylene signaling in the mutants. However, only atmyb44incurred an abrogation in induced EIN2expression, suggesting a close relationship between AtMYB44and EIN2.2. HrpNEa-induced deterrent effects on phloem feeding of the green peach aphid myzus persicae requires ATGSL5and ATMYB44genes in arabidopsis thalianaIn Arabidopsis thaliana(Arabidopsis) treated with the harpin protein HrpNEa, resistance to the green peach aphid Myzus persicae, a generalist phloem-feeding insect, develops with induced expression of the AtMYB44gene. Special GLUCAN SYNTHASE-LIKE (GSL) genes and β-1,3-glucan callose play an important role in plant defense responses to attacks by phloem-feeding insects. Here we report that AtGLS5and AtMYB44are both required for HrpNEa-induced repression of M. persicae feeding from the phloem of Arabidopsis leaves. In24-hour successive surveys on large-scale aphid populations, the proportion of feeding aphids was much smaller in HrpNEa-treated plants than in control plants, and aphids preferred to feed from37atgsl mutants tested rather than the wild-type plant. The atgsl mutants were generated previously by mutagenesis in twelve identified AtGSL genes (AtGSLl through AtGSL12); in the24-hour survey, both atgsl5and atgsl6performed to tolerate aphid feeding while atgsl5was the most tolerant. Consistently, atgsl5was also most inhibitive to the deterrent effect of HrpNEa on the phloem-feeding activity of aphids monitored by the electrical penetration graph technique. Theses results suggested an important role of the AtGSL5gene in the effect of HrpNEa. In response to HrpNEa, AtGSL5expression and callose deposition were induced in the wild-type plant but not in atgsl5. In response to HrpNEa, moreover, the AtMYB44gene known as required for repression of aphid reproduction on the plant was also required for repression of the phloem-feeding activity. Little amounts of the AtGSL5transcript and callose deposition were detected in the atmyb44mutant as in atgsl5. Both mutants performed similarly in tolerating the phloem-feeding activity and impairing the deterrent effect of HrpNEa, suggesting that AtGSL5and AtMYB44both contributed to the effect.3. Harpin-induced expression and transgenic overexpression of the phloem protein gene ATPP2-A1in arabidopsis repress phloem feeding of the green peach aphid myzus persicaeTreatment 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 protein2(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-Al in resistance to M. persicae when the plant was treated with HrpNEa and after the plant was transformed with AtPP2-Al.The 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-al/E/142, the plant mutant that had a defect in the AtPP2-Al gene, the most HrpNEa-responsive of30AtPP2genes. In PP2OETAt (AtPP2-Al-overexpression transgenic Arabidopsis thaliana) plants, abundant amounts of the AtPP2-A1gene 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 for24hours, 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.The repression in phloem-feeding activities of M. persicae as a result of AtPP2-Al overexpression, and as a deterrent effect of HrpNEa treatment in WT Arabidopsis rather than the atpp2-alVE/142mutant suggest that AtPP2-Al 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-Al is related to colonization of the plant.4. AtMYB44regulates resistance to the green peach aphid and diamondback moth by activating EIN2-affected defenses in arabidopsis Recently we show that the transactivator AtMYB44regulates transcription of EIN2, a gene essential for ethylene signaling and insect resistance, in Arabidopsis thaliana (Arabidopsis). To link the transactivation with insect resistance, we investigated the wild-type and atmyb44mutant plants, genetically Complemented atmyb44(Catmyb44), and AtMYB44-Overexpression Transgenic Arabidopsis (MYB44OTA). We found that AtMYB44played a critical role in Arabidopsis resistance to the phloem-feeding generalist green peach aphid and leaf-chewing generalist caterpillar diamondback moth. Resistance levels were consistent with amounts of the AtMYB44protein either induced by insect infestations in Catmyb44or constitutively produced in MYB44OTA. In both cases, AtMYB44bound the EIN2promoter coincidently with EIN2expression at a greater extent in MYB44OTA than in Catmyb44. Both events, however, did not occur in the hybrid MYB44OTA ein2-1, generated by crossing MYB44OTA and EIN2-deficient Arabidopsis mutant ein2-1. In the different plant genotypes, only MYB44OTA constitutively displayed phloem-based defenses specific to phloem-feeding insects and robust expression of genes involved in biosynthesis of glucosinolates known to be the deterrent of both phloem-feeding and leaf-chewing insects. Phloem-based defenses and glucosinolate-related gene expression were not detected in ein2-1and MYB44OTA ein2-1. These results establish a genetic connection between the regulatory role of AtMYB44in EIN2expression and development of Arabidopsis resistance to insects. |
