Identification Of Functional Fragments Of The HpaG Protein And A Transcriptional Regulatory Pathway In Control Of HrpN_Ea-Induced Aphids Resistance

Harpin proteins that produced by Gram-negative plant pathogenic bacteria can induce multiple benificial responses such as disease resistance, insect resistance, enhanced plant growth and enhanced tolerance to environment adversity like drought. As a multiple eilicitor, harpin is perspective in studying signalling of plant development and plant defense.Hence, how harpin is recognized by plant? How to induce different effects, which signalling pathway takes part in the effects of harpin? And how these effects come true? These questions are important to be answered. Our study aimed at the signalling of harpin in enhanced plant growth,disease resistance and insect resistance as well as its transcription regulation.1. Identification of specific fragments of HpaGXooc, a harpin from Xanthomonas oryzae pv. oryzicola, that induce disease resistance and enhance growth in riceHarpin proteins from plant pathogenic bacteria can stimulate hypersensitive cell death (HCD) and pathogen defence, and they can enhance growth in plants. Two of these diverse activities clearly are beneficial and may depend on particular functional regions of the proteins. HpaGXooc, produced by Xanthomonas oryzae pv. oryzicola, the pathogen that causes bacterial streak of rice, is a 137-amino acid harpin as a member of Harpin group of proteins. Here we report the identification and testing of nine functional fragments of HpaGXooc using PCR-based mutagenesis. These specific proteins caused different responses following their application to Nicotiana tabaccum (tobacco) and Oryza sativa (rice). Noticeably, HpaG10-42 was over 90% less active than other proteins in inducing HCD, but its effet in inducing the resistance to Xanthomonas oryzae pv. oryzae and Magnaporthe grisea was greatest. HpaG1-94 was most active in growth enhancement. Cell death level was 2-fold greater when induced by HpaG62-137 compared to HpaGXooc. Fragments HpaG62-137 and HpaG10-42 induced more intense HCD and did not cause evident cell death in tobacco, respectively, but both stimulated stronger defence responses and enhanced more growth in rice than the parent protein, HpaGxooc.2. Studies on effects of HpaG10-42, a specific fragment of HpaGxooc from Xanthomonas oryzae pv. oryzicola, in inducing disease resistance and enhancing growth in riceIn the previous study, we selected a fragment of HpaGXooc called HpaG10-42, which did not cause evident cell death in tobacco but both stimulated stronger defence responses and enhanced more growth in rice than the parent protein, HpaGXooc.Now we studying the effects of HpaG10-42 in the field, in controlling rice blast, sheath blight and leaf blight, as compared to HpaGXooc.The results showed that plants were sprayed once in nursery and three times in the field at vegetative growth V6 and V11 stages (collar formation on leaves 6 and 11 on main stem, respectively) and the reproductive stage R2 (collar formation on flag leaf) with 6,6,6,6μg/ml HpaG10-42, can deduce the severrity index of leaf blight and blast, as a result, the production was also enhanced obviously, as compaired to HpaGxooc·In the case of single ues of 6μg/ml HpaG10-42 in the four stages, its effect is better than using 12μg/ml HpaGxooc, but is worse than the 4-times appliction; HpaG10-42 was compared to chemicals in control of plant diseases; the effects of HpaG10-42 on nine different rice varieties was observed but were different; and HpaG10-42 effects was confirmed by large-scale experimentation.3. HrpNEa induces repellency and the inhibition of aphids feeding requiring EIN2-mediated plant phloem-related defense (PRD) in ArabidopsisHrpNEa is a multiple functional elicitor produced by Erwinia amylovora. In the previous study, we found that HrpNEa induces insect resistance though EIN2, but the repellency and the inhibition of feeding were not studied.In this study, we found HrpNEa can induce repellency. We used Rodanmin B as a marker and EPG to study the aphids feeding activities in Arabidopsis, and we found it identical to repellency. HrpNEa can promote the level of ethylene, and the inhibitor of the ethylene biosythesis can inhibit the effect of HrpNEa. HrpNEa can induce repellency in ein5-1 as in Col-0 rather than in ein2-1, so the effect of HrpNEa to induce repellency required EIN2. The in situ hybridization result showed the the plant phloem-related protein AtPP2 genes can be induced to express in the phloem, and its modle was the same as that of PRD, while PRD functions against attacks by sap-sucking insects. HrpNEa can induce the expression of AtPP2 in Col-0 but not in ein2-1. The expression of AtPP2 is always companied by callose deposition. Our results show that callose deposition is also induced by HrpNEa in Col-0 rather than in ein2-1. In all, our studyt shows that HrpNEa can induce repellency and PRD, and EIN2 mediated this progress.4. Screening of hrpNEa-responsive transcription factors in Arabidopsis defenses against the green peach aphidWe screen transcription factor genes that are HrpNEa-responsive by RT-PCR. Selected 37 TF genes were investigated for expression in WT Arabidopsis following treatment with HrpNEa, which was used in contrast to EVP. Twenty-one genes increased in plants 12 h post treatment (hpt) with HrpNEa, compared with EVP. As confirmed by Northern blot analysis,12 of 13 selected genes accumulated great amounts of transcripts in 12 hpt with HrpNEa-To determine transcript levels, the 9 genes were subjected to real time RT-PCR protocols. In 12 hpt, transcripts of AtMYB44 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. In the atmyb44 mutant, the repellency was least, and aphids multiplication were not repressed in atmyb44 responding to HrpNEa, indicating that the effect of HrpNEa was abolished, indicating AtMYB44 was a possible regulator of insect repellency. Finally, we get a selected transcription factor AtMYB44, which may paticipate the induced insect resistance in Arabidopsis.5. AtMYB44 Targets EIN2 to Regulate Arabidopsis Resistance against the Green Peach AphidAtMYB44 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. HrpNEa can promote the ethylene content in Col-0, and induce repellency and repress production. In atmyb44, can also promote the ethylene content, but the effect of inducing repellency and repressing production were lost. Through genetic, molecular, and pharmacological studies, we show that HrpNEa induce Arabidopsis resistance against the green peach aphid required AtMYB44. This effect is arrested in atmyb44. EIN2 can be induced by HrpNEa in Col-0 but not in AtMYB44 indicating the expression of EIN2 required AtMYB44. 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 atmyb44-complemented plants, moreover, AtMYB44 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.6. Concluding remarks1. We identify and test nine functional fragments of HpaGxooc, and these proteins have different effects on Nicotiana tabaccum (tobacco) and Oryza sativa (rice). Among them, HpaG62-137 induced more intense HCD, HpaG10-42 did not cause evident cell death in tobacco, but both stimulated stronger defence responses and enhanced more growth in rice than the parent protein, HpaGXooc.We used a selected fragment of HpaG10-42 in the field, studying the effects of HpaG10-42 in controlling rice blast,sheath blight and leaf blight, as compared to HpaGxooc.The results show that HpaG10-42 can induce disease resistance and finally increase the production as chemicals.2. EIN2 mediated HrpNEa-induced plant phloem-related defense which functions against attacks by sap-sucking insects.3. We selected transcription factors that are responsive to HrpNEa, and AtMYB44 is a candidate, which may also take part in the effect of HrpNEa in regulating insect resistance in Arabidopsis. Our results showed that AtMYB44 controls Arabidopsis resistance to the insect by activating the expression of EIN2, providing a mechanism of EIN2 transcription regulation.