Coordinate Regulation Of Defense Responses And Growth Alternation By Four Hormone Signals In Plants Responding To Harpin Proteins

The activation of defense responses in plants is initiated by host recognition of pathogen-encoded molecules called elicitors, e.g., microbial proteins, small peptides, and oligosaccharides, etc. The interaction of pathogen elicitors with host receptors (many of which may be encoded by R genes) likely activates a signal transduction cascade that may involve protein phosphorylation, ion fluxes, reactive oxygen species (ROS), and other signaling events. Subsequent transcriptional and/or posttranslational activation of transcription factors eventually leads to the induction of plant defense genes. In addition to eliciting primary defense responses, pathogen signals may be amplified through the generation of secondary plant signal molecules such as SA. Both primary pathogen elicitors and secondary endogenous signals may activate a diverse array of plant protectant and defense genes. So it is ubiquitous for these pathways to crosstalk with each other, depending on which signaling components are recruited into the pathway in response to different stimuli. Such signaling components are the keys to link different pathways and construct an efficient signaling network for plant to confront challenges encountered.1. Identification of specific fragments of HpaG_Xooc, 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. Identification of beneficial and deleterious regions might facilitate the advantageous use of harpin-related proteins on crops without causing negative effects like cell death. HpaG_Xooc 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 that contains two copies of the glycine-rich motif (GRM), a characteristic of harpins, and a cysteine, which is absent in other harpins. Here we report the identification and testing of nine functional fragments of HpaGxooc using PCR-based mutagenesis. These specific proteins named hpaG_1-105, hpaG_1-94, hpaG_1-61, hpaG_1-47, hpaG_7-61, hpaG_62-137, hpaG_10-42, hpaG_95-137 and hpaG_84-94 which span the indicated amino acid residues of the HpaG sequence, which caused different responses following their application to Nicotiana tabaccum (tobacco) and Oryza sativa (rice). Cell death levels were close when induced by HapG_1-94, HpaG_95-137, HpaG_84-94, and HpaG_Xooc, over 63% greater in response to HpaG_1-105, HpaG_1-16, HpaG_1-47, or HpaG_7-62, and AGRM, but 2-fold greater when induced by HpaG_62-137 compared to HpaG_Xooc- Noticeably, HpaG_10-12 was over 90% less active than other proteins in the effect. Fragments HpaG_62-137 and HpaG_10-42 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, HpaG_Xooc.When the 10 variants of HpaG_Xooc were used to induce resistance to Xanthomonas oryzae pv. oryzae, which causes bacterial leaf blight of rice, HpaG_1-61 and HpaG_7-61 were much more active than others except HpaG_10-42, whereas HpaG_10-42 was most effective, HpaG_62-137 surprisingly provided the lowest level of disease resistance. Of the nine fragments and full-length HpaG_Xooc tested to induce the resistance to Magnaporthe grisea, which causes rice blast, blast symptoms shown as tissue necrosis appeared on several sites of leaves treated with EVP, but were limited on HpaG_Xooc-treated leaves. On leaves treated with HpaG_62-137, infection signs were found but necrosis was not evident, while even infection signs were not evident on leaves treated with HpaG_10-42 So HpaG_10-42 was consistent in impeding pathogenicity by the bacterial and fungal pathogens.We studied rice root growth by soaking seeds separately in solutions of cell-free preparations of the 12 proteins. Compared to control, all the proteins tested supported better growth of roots as observed at 6 dpt. When compared with HpaG_Xooc, HpaG_1-105, HpaG_1-62, HpaG_1-47, HpaG_62-137, and HpaG_84-94 were less active; in contrast,â–³GRM, HpaG_1-94, HpaG_7-61, and HpaG_10-42 increased root length by 15%, 55%, 20%, and 48%, respectively. Clearly, HpaG_1-94 and HpaG_10-42 were robust in promoting rice growth.Because HpaG_1-94, HpaG_10-42, HpaG_62-137, and AGRM markedly preponderated over HpaG_Xooc to affect plants in assays with cell-free protein preparations, purified forms of these proteins were tested for bioactivities to corroborate results shown above. We obtained the same results in testing both cell-free and purified proteins. Because HpaG_62-137 and HpaG_10-42 tested versus other proteins provided higher levels of HCD and all the defined beneficial effects, respectively, both variants were compared to HpaG_Xooc in activating plant signaling events associated with HCD and associated with plant defense and growth.In leaves treated with HpaG_62-137, hsr203 and hin1 both were induced to accumulate transcripts since 6 hpt and expression leaves were increased with time. When compared at 48 hpt, markedly greater transcripts of both genes were detected in leaves treated with HpaG_62-137 than HpaG_Xooc. We studied responses of NPR1 and PR1 to HpaG_10-42 tested in comparison with HpaG_Xooc, both genes were expressed at levels increased with time after treatment with HpaG_10-42, as monitored at intervals in 48 hpt. The low levels of expression in 6 hpt suggested that both genes were induced for transcription. Especially, the expression of PR1 seemed dependent of induction because little transcripts were detectable as constitutive expression determined immediately after treatment. Comparison indicated that HpaG_10-42 was markedly stronger than HpaGxooc in the induction of NPR1 and PR1 expression analyzed at 48 hpt. HpaG_10-42 was more active than HpaG_Xooc in inducing expression of several genes that regulate rice defence and growth processes, which may explain the greater beneficial effects observed. We found also that specific fragments are more effective in activating certain signaling pathways than HpaG_Xooc. Overall, our results suggest that a particular fragment of HpaG_Xooc, HpaG_10-42, holds promise for practical agricultural use to enhance disease resistance and growth of rice.2. Plant growth and expansin gene expression regulated by ethylene and gibberellin in response to a bacterial typeâ…¢effectorExpansin proteins (EXPs) act to loose cell walls and modulate growth of the cell and plant under mediation by some hormones. It is not clear whether a hormone is specific and distinct hormones interact to regulate EXP activity. Here we report plant growth enhancement (PGE) and expansin gene (EXP) expression in response to HrpN_Ea, a bacterial type-â…¢effector, and roles of ethylene (ET) and gibbrellin (GA) in both responses.Arabidopsis root growth at 15 dpt and weight of 20-d seedlings revealed 89% and 67% increases following soaking seeds and spaying treatment by HrpN_Ea vs. EVP. Treating tomato seeds with a HrpN_Ea solution promoted seed germination and subsequent growth of roots and plants; Plants grew better evidently in treatment with HrpN_Ea vs. EVP, as observed at 12 and 30 dpt; Similarly, 46% and 52% increases were found in height and weight of 12-d tomato plants. The percentages were 32% and 50% for height and weight of 40-d tobacco plants, 56% and 48% for height and weight of 12-d rice plants treating with a HrpN_Ea solution. These results suggest that HrpN_Ea similarly stimulates growth of the different plants. Besides, HrpN_Ea treatment resulted in not only increase in leaf size but also production of 2-4 more leaves per plants compared to control. For example, Arabidopsis and tobacco plants had 2-3 and 3-4 additional leaves in treatment with HrpN_Ea vs. EVP when plants were surveyed at 30 and 40 dpt. Total nitrogen content was increased by 28%, 36.4%, 28.9% and 25.4%, respectively, in Arabidopsis, tomato, tobacco and rice plants following treatment with HrpN_Ea in contrast to EVP. The increase was consistent with PGE levels in the plants. Levels of chlorophylls a and b, total levels either, increased evidently in the fours plants responding to HrpN_Ea vs. EVP, as determined at 10 d after treated imbibed seeds. Moreover, marked increases were seen in proportion of chlorophyll a to b. Therefore, HrpN_Ea intensifies the biosynthetic and productive pathways through affecting the basic physiological responses.We conducted RT-PCR protocols using EF1αas a standard to test EXP expression in Arabidopsis, tomato and tobacco plants following treatment with HrpN_Ea. Expression of AtEXP7 in Arabidopsis leaves and stems was not evident but showed high levels in roots. In contrast, LeEXP5 preferred to express in stems but not leaves and roots, whereas the other 8 EXPs, including AtEXP10, LeEXP2 and AtEXP2, were expressed greater in leaves compared to roots and stems. The induced expression of AtEXP 16 and NtEXP1 was quite similar to that of AtEXP10 and NtEXP2, respectively. AtEXP10, LeEXP2 and NtEXP2 were leaf-specific in expression accumulated transcripts at lower levels in stems and roots vs. leaves. When expressed in the preferential organs, patterns and time course of expression varied depending on genes. AtEXP2, LeEXP18 and NtEXP2 exhibited a similar expression pattern; they increased transcripts gradually with time in 72 or 96 hpt; the other EXPs maintained high expression levels from 6 hpt through 72 hpt.To relate an ET signal with EXPs and PGE, we studied a transcriptional coincidence of ET-response genes with EXPs and PGE. ETR1 was induced by the protein and increased levels with time. EIN2 was constitutive and was enhanced quickly at 6 hpt with HrpNEa but not change evidently thereafter. The PDF1.2 and PR-3b were expressed after induction, suggesting an activation of the pathway. Consistently, LeETR1 and NtETR1, which encode ET receptor homologs in tomato and tobacco, showed to be induced by HrpN_Ea applied to 20-d plants. We found that the Arabidopsis etr1-1 mutant markedly compromised AtEXP10 expression, compared to WT, in 20-d seedlings sprayed with a HrpN_Ea solution. PGE in tomato, tobacco and rice was impaired markedly by 1-MCP present in HrpN_Ea treatment, a ethylene sensitivity inhibitor. Growth of plants subsequent to soaking seeds in a solution of HrpN_Ea and 1-MCP was evidently decreased relative to that in treatment with only HrpN_Ea. Therefore, ET sensing is critical to the induction of EXP expression and PGE by HrpN_Ea in the four plants.We compared HrpN_Ea and GA₃ in the effects on OsEXPs, which increase expression in rice plants treated with GA₃, an important form of GA. When applied to 20-d rice plants, HrpN_Ea acted similarly as did GA₃ in inducing expression of OsEXP2, OsEXP4 and 0sEXP16. However,HrpN_Ea seemed to function slower and less effectively than GA₃; gene expression became evident earlier and expression levels also were greater in response to GA₃ vs. HrpN_Ea. In particular, OsEXP2 and OSEXP16 were expressed at high extents successively since 6 hpt with GA₃ but increased expression levels gradually with time since 12 hpt with HrpN_Ea. OsEXP4 markedly accumulated transcript at 12 hpt with HrpN_Ea or GA₃ and thereafter remained a moderate level of expression. We addressed if EXP expression and PGE require a GA signal by determining effects of the GA synthesis inhibitor pp333 on EXPs and PGE. The expression of OsEXP4 was greatly compromised when pp333 was present in HrpN_Ea treatment. In WT tomato, the expression of LeEXP2 induced by the application of HrpN_Ea was inhibited by pp333 supplied to HrpN_Ea treatment. NtEXP2 showed evident constitutive expression and was enhanced markedly to increase expression level by HrpN_Ea applied alone; pp333 eliminated the effect. In consistence, pp333 evidently impaired the effect of HrpN_Ea in promoting growth of the three plants. Based on these results, a GA signal is required for the induction of EXP expression and PGE.3. Cloning and function analysis of tobacco cDNA involved in constitutive expression of systemic acquired resistance.Tobacco constitutive expresser of SAR1-1 and ces2-1 mutants have been generated from the variety NC89. The three genotypes varied greatly in growth and resistance to Alternaria alternata. Primers used for rapid amplification of cDNA 5'- and 3'- ends (5'-RACE and 3'-RACE) were designed on the basis of the mRNA differential display fragment. 5- terminal fragment cloned by 5'-RACE and 3'- terminal fragment amplified by 3'-RACE were pieced together integrally and the complete sequence was validated by homologous Blast and DNAStar software.The newly identified cDNA fragment was 759 bp in length which included the complete ORF or CDS of CES1 from 88 bp to 459 bp coding 123 amino acid (aa), and nominated CES1. Beside the ORF region 372 bp in length, 87 bp upstream form initiation codon ATG and 300 bp downstream from stop codon TGA were also amplified by 5'-RACE and 3'-RACE, respectively.The online blast result indicated that CES1 shared 89% identities with the auxin-repressed protein gene (APR1) in a 123 aa region. The predicted protein contained a Glycosaminoglycan attachment site, two Protein kinase C phosphorylation sites, two Casein kinaseâ…¡phosphorylation sites and three N-myristoylation sites. A sequence similarity search in public database showed that the ARP gene has homologs in various higher plants including monocots and dicots. The deduced amino acid sequences are highly conserved among these homologs (up to 89% identity).The semi-quantitative RT-PCR assay showed the development and defense-related genes were strongly induced by treated with 2,4-D and hrpN_Ea and the plants were enhanced to resistance to Alternaria alternate. Transient expression of the CES1-GFP protein in onion epidermal cell showed that CES1 was localized in cell nuclei. These indicated that the CES1 gene positively regulates SAR but negatively regulates growth by regulating associating gene and activating different signaling pathway.4. Ethylene and abscisic acid signaling synergizes to regulate the phloem-related defense and insect repellency in plants responding to HrpN_EaPlant phloem-related defense (PRD) functions against attacks by sap-sucking insects. Known signaling pathways are implicated in PRD induction by biotic elicitors, such as HrpN_Ea (a bacterial type-â…¢protein), but how the pathways interact to regulate PRD is unclear. Here we show that abscisic acid (ABA) and ethylene (ET) signaling synergism modulated by transcription factor(s) controls HrpNEa-induced PRD and insect repellency in Arabidopsis and we have screened 13 transcription factors up-regulated by HrpN_Ea. Aphid repellency and depressed phloem-feeding activities were attributed to PRD configuration by phloem protein and callose deposition in the phloem. Aphid Repellency Is Affected by Feeding Activity and Both Events Require Plant EIN2/ABI2 Synergism in Response to HrpN_Ea.5. Summary remarksResults obtained from studies described above have provided us with further understanding on action mechanism of defense and growth in plants responding to Harpin proteins. First, we identify and test nine functional fragments of HpaG_Xooc, and these proteins have different effects on Nicotiana tabaccum (tobacco) and Oryza sativa (rice). Among them, HpaG_62-137 and HpaG_10-42 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, HpaG_Xooc Second, plant growth enhancement was induced by HrpN_Ea concomitantly with induced expression of EXPs gene, a cell wall loose protein, and both inductions require GA and ET in plants responding to HrpN_Ea. The results show that inhibiting Arabidopsis, tomato and tobacco plants to synthesize GA or sense ET compromised EXP expression and PGE phenotypes. Third, abscisic acid and ethylene signaling controls HrpNEa-induced PRD and insect repellency in Arabidopsis, and aphid repellency requires plant EIN2/ABI2 synergism in response to HrpN_Ea. Moreover, we have screened 13 transcription factors based on their gene transcripts up-regulated by HrpN_Ea.