High levels of GUS expression were evident in response to each of the three treatments

These combinations may be capable of activating specific pathways to differing degrees to achieve pathogen appropriate defense responses. Such stimulators of the plant’s inherent immune responses are likely to be superior to conventional biocidal pesticides that are harmful to both environment and consumers . Knoth et al., previously identified two different synthetic elicitors, DCA and CMP442 – thiazolidine-4-carboxylic acid. Both elicitors induce defense in Arabidopsis against Hpa and DCA also against P. syringae. The modes of action of these compounds are distinct; although both act on the SA-dependent branch of the defense network. CMP442 appears to interfere with defense-signaling processes upstream from SA perception, while DCA activates signaling steps downstream from SA . This screening system focused on compounds targeting components of the SA-dependent sector of the defense network and yielded many candidate synthetic elicitors . To widen the spectrum of functionally distinct synthetic elicitors a screening system was needed that allows for the identification of synthetic elicitors interfering with the JA- and ET-dependent parts of the defense signaling network. A set of five genes was identified in microarray experiments that exhibit SA-independent upregulation in response to infection with Hpa . Four of these genes were members of the PDF family including JA-pathway marker gene PDF1.2b. Because PDF genes are known to response to JA and ET, but not SA,ebb and flow table this set of co-expressed defense-related genes was named JEDI and include PDF1.2a , PDF1.2b , PDF1.2c , PDF1.3 , and JEDI1 .

Here the development of transgenic lines in which select JEDI genes were fused to reporter genes to develop a high-throughput chemical screening system is described. In addition, the creation of transgenic plants with an RNAi transgene silencing transcripts of the closely related PDF members within the JEDI set is reported. Furthermore, JEDI transcripts were hyper induced at 48 hpi in nahG compared to Col-0 . The Hpa-inducibility of the JEDI transcripts was confirmed via reverse transcription -PCRs at 0, 6, and 12 hpi with HpaEmoy2 in two-week-old Col-0 and nahG plants. Compared to their levels at 0 hpi, transcript levels of all JEDI genes were enhanced at 6 hpi and 12 hpi in both Arabidopsis lines. Thus, the trends observed in these RT-PCRs were similar to those in the original microarray data, suggesting that JEDI transcript accumulation in response to HpaEmoy2 recognition is at least partly SA independent. Additional time points were examined after Hpa in Col-0 and nahG.PDF genes are known to be positively controlled by the JA-dependent branch of the plant defense network. However, JA-controlled defense responses have been traditionally associated with immunity against necrotrophic or hemibiotrophic pathogens, such as the fungus B. cinerea or the bacterial pathogen P. syringae . Immunity against strict biotrophs, such as Hpa is believed to be controlled by SA dependent immune responses . However, our observation that JEDIPDF genes are upregulated during incompatible Hpa interactions suggested that JA-dependent signaling processes can also contribute to immunity to this strictly biotrophic pathogen. Thus, this set of Hpa-responsive genes provided us with an opportunity to study aspects of JA-dependent processes in immunity against Hpa. In order to design a high-throughput screening protocol for the identification of synthetic elicitors targeting the JA-dependent branch of the plant defense network, a previously characterized pPDF1.2b::GUS line was obtained and tested.

Seven-day-old seedlings were grown in liquid growth medium in 96-well plates and incubated for 24 h with 100 µM , 45 µM , or mock solution . A concentration of 45 µM MeJA induces expression of pPDF1.2b::GUS . SA should not induce this reporter . At 24 h after the respective treatments, seedlings were histochemically stained to visualize GUS expression .Even the mock-treatment resulted in GUS expression . Due to the apparent lack of specificity in expression responses a screen with this pPDF1.2b::GUS line, this screen was deemed unfeasible. Therefore, a set of new transgenic lines with fusions of JEDI promoters to reporter genes was developed and tested for their suitability for high-throughput chemical screens. Reporter lines ideal for the planned chemical screens should show no or extremely low background expression and exhibit clear pathogen or JA-inducibility. In addition, the variability of reporter gene expression levels in response to the same treatment should be minimal. A reduction in transcript levels of genes important for plant defense can affect R-gene-mediated resistance and/or basal defense responses. A reduction in basal defense may result in enhanced susceptibility to virulent pathogens . To identify effects on basal defense, the homozygous PDF-RNAi lines were infected with the Col-0 virulent Hpa isolateNoco2 . The interaction of HpaNoco2 with Col-0 is compatible, as this Hpa isolate is not recognized by any Col-0 R gene . Three-week-old plants were spray-infected with 2 ml/pot and 7 days post inoculation the extent of spore formation was quantified. Homozygous PDF-RNAi lines 38, 41, and 48 were in the wrky70-3 background.

Lines were created in the wrky70-3 background to see if knocking down gene expression in this mutant line might display different results than in the Col-0 wild type line. PDF-RNAi lines transformed into the wrky70-3 background displayed similar levels of infection as wrky70-3. In addition, wrky70- 3 and its transgenic lines consistently supported similar levels of spores as Col-0. This is contrary to previously reported data, which demonstrated that wrky70-3 supported more pathogen growth than Col-0 . Experiments performed with Arabidopsis plants at a range of ages indicated that WRKY70 transcript levels are upregulated in an age-dependent manner . In two-week-old Arabidopsis plants WRKY70 transcript levels were lower than in three-week-old plants. While basal defense up to an age of two weeks seems to be dependent on WRKY70, this transcription factor seems not to be required for this immune response in older plants. No morphological abnormalities were apparent in any of the PDF-RNAi lines. Pathogen recognition triggers a highly intricate set of defense responses which are coordinated by a complex regulatory network . Two branches of this network, which are dependent on the signaling molecules SA or JA, respectively, have been previously characterized. In Arabidopsis, SA-dependent signaling can processes induce antimicrobial proteins . Alternately, induction of PDF genes such as PDF1.2 is dependent on JA-dependent regulatory processes. While SA-dependent plant immune response have been primarily associated with resistance against biotrophic pathogens, defense reactions promoted by JA seem mainly to be effective against necrotrophs . Extensive crosstalk between JA- and SA-signaling has been reported as both types of defense signaling processes can be additive, antagonistic,flood table or synergistic dependent on the extent of their induction . Furthermore, successful defense against certain pathogens or pests often requires the coordinated induction of both SA- and JA-dependent defense reactions . High-throughput chemical screens previously identified synthetic elicitors activating SA-dependent plant immune response . A complementary screen to identify synthetic elicitors stimulating JA-dependent defense signaling processes would have great potential for the further decipher plant defense networks and provide molecular probes to uncover crosstalk mechanisms coordinating SA- and JA-dependent defense reactions. Interestingly, in nahG Arabidopsis plants JEDI transcripts appeared to be hyper induced in response to infection by Hpa suggesting they are upregulated by signaling mechanisms normally antagonized or suppressed by SA. As the JEDI set includes several PDF genes which are known to be positively regulated by JA , the hyperinduction of JEDI transcripts in the nahG background may point to a JA-dependent gene induction mechanisms that is counteracted by SA. To decipher roles that the JEDI genes may play in defense and stress responses, publically available microarray data were examined. The expression profiles of PDF1.2a, PDF1.2b, and JEDI1 were available through the Botany Array Resource website . These genes were highly upregulated in response to the oomycete Phytophthora infestans, the fungi Erysiphe orontii, Botrytis cinerea as well the abiotic stimuli wounding, osmotic stress, ultra-violet light B treatment, oxidative stress, and drought.

Additional treatments reported on in BAR, such as: SA, the ET precursor 1-aminocyclopropane-1-carboxylic-acid, the cytokinin zeatin, methyl jasmonate, indole-3-acetic acid , abscisic acid , gibberellic acid , and heat caused no significant changes in the expression profiles of PDF1.2a, PDF1.2b, and JEDI1. Microarray data in the BAR database therefore indicated that a wide range of pathogens and stressors may induce JEDI expression. Wounding triggered the accumulation of JEDI transcripts . However, JEDI transcripts did not accumulate in response to treatment with MeJA . A likely explanation for this unexpected observation is that responses to MeJA were measured at early times after MeJA treatment. , while previous studies showed PDF transcripts to accumulate around 48 h after treatment with jasmonates . Microarray data sets in BAR include responses of Arabidopsis to different types of pathogens, such as the hemibiotrophic oomycete P. infestans, the biotrophic fungus E. orontii, and the necrotrophic fungus B. cinerea . Thus, transcriptome responses of Arabidopsis to a complete set of pathogenic lifestyles are represented in BAR. Of these three different types of pathogens, the hemibiotroph P. infestans induced the strongest accumulation of JEDI transcripts while the other two pathogens induced expression to a lesser degree.Besides canonical JA-response elements, additional putative cis-elements are commonly present in all PDF promoters. These include binding sites of the AP2-domain transcription factor RAV1, which binds to bipartite recognition sequence, one of which is a AP2 domain . GCC boxes are known to mediate responses to ET or JA . In addition, W-box motifs are present in JEDI promoters. W boxes have been mainly associated with SA-dependent regulatory mechanisms, but may have wider roles in defense gene regulation . Furthermore, GATA promoter, and I-box motifs are present JEDI1 and PDF1.2a suggesting that these genes have particularly diverse roles. GATA and I-boxes are light-responsive promoter elements. The JEDI genes also respond to light and many non-defense-related stimuli, which again suggest a diversity of roles for these genes. It has been previously reported that the GCC box is important, but not essential, for the defense-related up-regulation of PDF1.2b . Thus, additional promoter elements must also be involved in the defense-related up-regulation of PDF1.2b . While the created transgenic lines with pPDF::LUC or pPDF::GUS fusions seemed not suitable for new synthetic elicitor screens, as they exhibit high background expression levels, these lines may still be useful for other types of experiments. They can be used to study spatial and temporal patterns of pathogen-induced JEDI gene expression. They may also allow the rough localization of promoter elements mediating responses to various defense related stimuli, such as infection with virulent or avirulent Hpa isolates. The high degree of structural relatedness and similarities of their defense related expression among PDF genes suggested functional redundancy. Studies using single mutants for individual PDF genes have not been reported on in the literature for the defense related functions of these genes. Thus, only a quadruple mutant with combined mutations in each of the four PDF genes is likely to exhibit clear defense-related phenotypes. The construction if such a quadruple mutant, however, is not feasible due to the extremely close linkage of PDF1.3 and PDF1.2b as well as PDF1.2c and PDF1.2a . As the nucleotide sequences of these four PDF-JEDI genes share a high degree of identity, RNAi-based silencing using the pANDA-RNAi vector seemed a feasible strategy. Several Arabidopsis lines containing the PDF-RNAi vector exhibited reduced basal defense against Hpa. However, in these lines transcript levels of only PDF1.2c and PDF1.3 appeared to be reduced. Thus, co-silencing of these two PDF members may be sufficient to overcome functional redundancy within this group of defense genes. Unfortunately, the RT-PCR analyses measuring PDF transcript levels were not consistent. Measurements of the respective transcripts need to be repeated by a more reliable and robust method, such as real-time quantitative RT-PCR. Assays to examine the effect of RNAi-transgene on resistance to other pathogens should be also performed. In any case, this study, which demonstrates a potential role of some PDF genes in basal defense against Hpa indicates that members of this gene family can contribute to immunity against a strict biotroph. While this finding is novel, it is not unexpected, as PDF transcripts were found to accumulate during infections of Arabidopsis with pathogens of different lifestyles, including biotrophs .