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Phytophthora diseases cause devastation to crops and native ecosystems worldwide. In New Zealand, Phytophthora agathidicida is threatening the survival of kauri, an endemic, culturally and ecologically important tree species. The current method for detecting P. agathidicida is a soil bating assay that is time-consuming and requires high levels of expertise to assess, thus limiting the analytical sample throughput. Here, we characterized the fatty acid methyl ester (FAME) profile of P. agathidicida. We also compared it with the FAME profile of P. cinnamomi and assessed the efficacy of FAME analysis as a diagnostic tool for detecting the pathogen in soil samples. In FAME analysis, the total fatty acid content is isolated from a sample and converted to FAMEs for analysis, a process that takes less than a day. Unique fatty acid acyl chains can serve as biomarkers for specific organisms. We detected 12 fatty acids in P. agathidicida, two of which (20:4ω6 and 20:5ω3) show promise as potential Phytophthora specific biomarkers. Collectively, these findings advance our fundamental understanding of P. agathidicida biology and provide a promising technique to increase the rate of sample processing and the speed of pathogen detection for P. agathidicida in soil., The fatty acid methyl ester (FAME) profile of P. agathidicida was characterized and assessed for its potential use in detecting P. agathidicida in soil.

Phytophthoradiseases cause devastation to crops and native ecosystems worldwide. In New Zealand,Phytophthora agathidicidais threatening the survival of kauri, an endemic, culturally and ecologically important tree species. The current method for detectingP. agathidicidais a soil bating assay that is time-consuming and requires high levels of expertise to assess, thus limiting the analytical sample throughput. Here, we characterized the fatty acid methyl ester (FAME) profile ofP. agathidicida. We also compared it with the FAME profile ofP. cinnamomiand assessed the efficacy of FAME analysis as a diagnostic tool for detecting the pathogen in soil samples. In FAME analysis, the total fatty acid content is isolated from a sample and converted to FAMEs for analysis, a process that takes less than a day. Unique fatty acid acyl chains can serve as biomarkers for specific organisms. We detected 12 fatty acids inP. agathidicida, two of which (20:4ω6 and 20:5ω3) show promise as potentialPhytophthoraspecific biomarkers. Collectively, these findings advance our fundamental understanding ofP. agathidicidabiology and provide a promising technique to increase the rate of sample processing and the speed of pathogen detection forP. agathidicidain soil.

Phytophthoraspecies cause disease and devastation of plants in ecological and horticultural settings worldwide. A recently identified species,P. agathidicida, infects and ultimately kills the treasured kauri trees that are endemic to New Zealand. Currently there are few options for controlling or treatingP. agathidicida. In this study, we sought to assess the toxicity of the oomycide oxathiapiprolin against several lifecycle stages of two geographically distinctP. agathidicidaisolates. Half maximal effective concentration (EC50) values were determined to be approximately 0.1 ng/ml for inhibiting mycelial growth, indicating thatP. agathidicidamycelia are more sensitive to oxathiapiprolin than those from most otherPhytophthoraspecies that have been studied. Oxathiapiprolin was also highly effective at inhibiting the germination of zoospores (EC50= 2-9 ng/ml for the two isolates) and oospores (complete inhibition at 100 ng/ml). In addition, oxathiapiprolin delayed the onset of detached kauri leaf infection in a dose-dependent manner. Collectively, the results presented here highlight the significant potential of oxathiapiprolin as a tool to aid in the control of kauri dieback disease.

Phytophthora species cause disease and devastation of plants in ecological and horticultural settings worldwide. A recently identified species, Phytophthoraagathidicida, infects and ultimately kills the treasured kauri trees (Agathis australis) that are endemic to New Zealand. Currently, there are few options for managing kauri dieback disease. In this study, we sought to assess the efficacy of the oomycide oxathiapiprolin against several life cycle stages of two geographically distinct P. agathidicida isolates. The effective concentration to inhibit 50% of mycelial growth (EC50) was determined to be ∼0.1 ng/ml, indicating that P. agathidicida mycelia are more sensitive to oxathiapiprolin than those from most other Phytophthora species that have been studied. Oxathiapiprolin was also highly effective at inhibiting the germination of zoospores (EC50 = 2–9 ng/ml for the two isolates) and oospores (complete inhibition at 100 ng/ml). In addition, oxathiapiprolin delayed the onset of detached kauri leaf infection in a dose-dependent manner. Collectively, the results presented here highlight the significant potential of oxathiapiprolin as a tool to aid in the control of kauri dieback disease.

The anthropogenic release of toxic metals into the environment poses danger to the health of both humans and the local ecosystem. Biosensors for the detection of metals have been developed to improve our ability to monitor these environmental contaminants, yet most of these sensors use heterotrophic bacterial hosts, which require a fixed carbon source and do not typically grow in natural waterways. In this study, we constructed and characterized metal sensors for development of a photoautotrophic biosensor using Synechococcus sp. PCC 7002. We characterized gold and copper sensors based on modified MerR transcriptional activators: GolSA113T, with improved gold binding, and GolSCL, containing the metal-binding loop from CueR which binds both gold and copper. The metal-sensing constructs were first optimized and characterized in Escherichia coli MG1655. The addition of a strong ribosome binding site to the optical reporter protein increased translation of the fluorescent reporter, and expression of golSA113T from the rbc promoter of Synechococcus sp. PCC 7002 improved the response to gold in MG1655. In rich medium, the GolSA113T-based E. coli sensor detected gold at concentrations as low as 100 nM, while the GolSCL-based E. coli sensor detected gold and copper at sensitivities of 100 nM and 10 μM, respectively. Both E. coli sensors responded to gold and copper yet showed no detectable response to other metals. Abiotic factors, such as medium complexity, were found to influence the response of the E. coli sensors, with minimal medium resulting in higher sensitivities of detection. Expression of the GolSA113T- and GolSCL-based sensor constructs in the cyanobacterium Synechococcus sp. PCC 7002 resulted in photoautotrophic gold sensors, but these biosensors failed to produce a significant response to copper. Moreover, the fluorescence response of the cyanobacterial sensors to gold was significantly reduced compared to that of analogous E. coli sensors. While this effort demonstrates feasibility for the development of photoautotrophic biosensors, additional efforts to optimize sensor performance will be required.

Ethylene is well known as a plant hormone, but its role in bacteria is poorly studied. We recently showed that Synechocystis sp. Strain PCC 6803 has a functional receptor for ethylene, ethylene response 1 (Etr1), that is involved in various processes such as phototaxis in response to directional light and biofilm formation. Here, we use RNA sequencing to examine the changes in gene transcripts caused by ethylene under phototaxis conditions. Over 500 gene transcripts across many functional categories, of approximately 3700 protein‐encoding genes, were altered by application of ethylene. In general, ethylene caused both up‐ and downregulation of genes within a functional category. However, the transcript levels of amino acid metabolism genes were mainly upregulated and cell envelope genes were mostly downregulated by ethylene. The changes in cell envelope genes correlate with our prior observation that ethylene affects cell surface properties to alter cell motility. Ethylene caused a twofold or more change in 62 transcripts with the largest category of upregulated genes annotated as transporters and the largest category of downregulated genes annotated as glycosyltransferases which sometimes are involved in changing the composition of sugars on the cell surface. Consistent with changes in cell envelope, glycosyltransferase, and transporter gene transcripts, application of ethylene altered the levels of specific sugar moieties on the surface of cells. Light signaling from Etr1 involves two proteins (Slr1213 and Slr1214) and a small, noncoding RNA, carbon stress‐induced RNA1 (csiR1). Application of ethylene caused a rapid, but transient, decrease in the transcript levels of etr1, slr1213, and slr1214 and a rapid and prolonged decrease in csiR1 transcript. Deletion of Slr1214 caused a large increase in csiR1 transcript levels and ethylene lowered csiR1 transcript. These data combined with prior reports indicate that ethylene functions as a signal to affect a variety of processes altering the physiology of Synechocystis cells.

Ethylene influences the growth and development of Arabidopsis (Arabidopsis thaliana) via five receptor isoforms. However, the ETHYLENE RESPONSE1 (ETR1) ethylene receptor has unique, and sometimes contrasting, roles from the other receptor isoforms. Prior research indicates that the receiver domain of ETR1 is important for some of these noncanonical roles. We determined that the ETR1 receiver domain is not needed for ETR1’s predominant role in mediating responses to the ethylene antagonist, silver. To understand the structure-function relationship underlying the unique roles of the ETR1 receiver domain in the control of specific traits, we performed alanine-scanning mutagenesis. We chose amino acids that are poorly conserved and are in regions predicted to have altered tertiary structure compared with the receiver domains of the other two receptors that contain a receiver domain, ETR2 and ETHYLENE INSENSITIVE4. The effects of these mutants on various phenotypes were examined in transgenic, receptor-deficient Arabidopsis plants. Some traits, such as growth in air and growth recovery after the removal of ethylene, were unaffected by these mutations. By contrast, three mutations on one surface of the receiver domain rendered the transgene unable to rescue ethylene-stimulated nutations. Additionally, several mutations on another surface altered germination on salt. Some of these mutations conferred hyperfunctionality to ETR1 in the context of seed germination on salt, but not for other traits, that correlated with increased responsiveness to abscisic acid. Thus, the ETR1 receiver domain has multiple functions where different surfaces are involved in the control of different traits. Models are discussed for these observations.

Prior work resulted in identification of an Arabidopsis mutant, eer5-1, with extreme ethylene response in conjunction with failure to induce a subset of ethylene-responsive genes, including AtEBP. EER5, which is a TREX-2 homolog that is part of a nucleoporin complex, functions as part of a cryptic aspect of the ethylene signaling pathway that is required for regulating the magnitude of ethylene response. A suppressor mutagenesis screen was carried out to identify second site mutations that could restore the growth of ethylene-treated eer5-1 to wild-type levels. A dominant gain-of-function mutation in the ethylene receptor ETHYLENE RESPONSE SENSOR 1 (ERS1) was identified, with the ers1-4 mutation being located in transmembrane domain III at a point nearly equivalent to the previously described etr1-2 mutation in the other Arabidopsis subfamily I ethylene receptor, ETHYLENE RESPONSE 1 (ETR1). Although both ers1-4 and etr1-2 partially suppress the ethylene hypersensitivity of eer5-1 and are at least in part REVERSION TO ETHYLENE SENSITIVITY 1 (RTE1)-dependent, ers1-4 was additionally found to restore the expression of AtEBP in ers1-4;eer5-1 etiolated seedlings after ethylene treatment in an EIN3-dependent manner. Our work indicates that ERS1-regulated expression of a subset of ethylene-responsive genes is related to controlling the magnitude of ethylene response, with hyperinduction of these genes correlated with reduced ethylene-dependent growth inhibition.

Copper ions play an important role in ethylene receptor biogenesis and proper function. The copper transporter RESPONSIVE-TO-ANTAGONIST1 (RAN1) is essential for copper ion transport in Arabidopsis thaliana. However it is still unclear how copper ions are delivered to RAN1 and how copper ions affect ethylene receptors. There is not a specific copper chelator which could be used to explore these questions. Here, by chemical genetics, we identified a novel small molecule, triplin, which could cause a triple response phenotype on dark-grown Arabidopsis seedlings through ethylene signaling pathway. ran1-1 and ran1-2 are hypersensitive to triplin. Adding copper ions in growth medium could partially restore the phenotype on plant caused by triplin. Mass spectrometry analysis showed that triplin could bind copper ion. Compared to the known chelators, triplin acts more specifically to copper ion and it suppresses the toxic effects of excess copper ions on plant root growth. We further showed that mutants of ANTIOXIDANT PROTEIN1 (ATX1) are hypersensitive to tiplin, but with less sensitivity comparing with the ones of ran1-1 and ran1-2. Our study provided genetic evidence for the first time that, copper ions necessary for ethylene receptor biogenesis and signaling are transported from ATX1 to RAN1. Considering that triplin could chelate copper ions in Arabidopsis, and copper ions are essential for plant and animal, we believe that, triplin not only could be useful for studying copper ion transport of plants, but also could be useful for copper metabolism study in animal and human., Author summary Copper ions are cofactors of protein functions, and their disorder is closely related to many human diseases which drive to develop new copper chelator related drugs. In plants, copper ions are essential for ethylene receptors, but many details are unclear. Researchers need novel specific copper chelators to study these questions. Here, by using plant chemical genetics, we identified a novel chemical triplin, which could activate ethylene signaling pathway by chelating copper ions essential for ethylene receptors. Ethylene resistance mutant etr1-1 and ein2 were resistant to triplin, and copper transporter mutants ran1-1 and ran1-2 were hypersensitive to triplin. Using triplin as a molecular genetics tool, we showed copper chaperone ATX1 acts the upstream of RAN1 which transports copper ions to ethylene receptors. We provide a sample that metabolism could be studied by combining chemical genetics and known signaling pathway. Moreover, triplin could chelate copper ions effectively and specifically in Arabidopsis, and could be a useful tool to study copper ion transport in plant, and valuable for designing copper chelator related drug.

Ethylene is a plant hormone that plays a crucial role in the growth and development of plants. The ethylene receptors in plants are well studied, and it is generally assumed that they are found only in plants. In a search of sequenced genomes, we found that many bacterial species contain putative ethylene receptors. Plants acquired many proteins from cyanobacteria as a result of the endosymbiotic event that led to chloroplasts. We provide data that the cyanobacterium Synechocystis (Synechocystis sp. PCC 6803) has a functional receptor for ethylene, Synechocystis Ethylene Response1 (SynEtr1). We first show that SynEtr1 directly binds ethylene. Second, we demonstrate that application of ethylene to Synechocystis cells or disruption of the SynEtr1 gene affects several processes, including phototaxis, type IV pilus biosynthesis, photosystem II levels, biofilm formation, and spontaneous cell sedimentation. Our data suggest a model where SynEtr1 inhibits downstream signaling and ethylene inhibits SynEtr1. This is similar to the inverse-agonist model of ethylene receptor signaling proposed for plants and suggests a conservation of structure and function that possibly originated over 1 billion years ago. Prior research showed that SynEtr1 also contains a light-responsive phytochrome-like domain. Thus, SynEtr1 is a bifunctional receptor that mediates responses to both light and ethylene. To our knowledge, this is the first demonstration of a functional ethylene receptor in a nonplant species and suggests that that the perception of ethylene is more widespread than previously thought.

Prior work resulted in identification of an Arabidopsis mutant, eer5-1, with extreme ethylene response in conjunction with failure to induce a subset of ethylene-responsive genes, including AtEBP. EER5, which is a TREX-2 homolog that is part of a nucleoporin complex, functions as part of a cryptic aspect of the ethylene signaling pathway that is required for regulating the magnitude of ethylene response. A suppressor mutagenesis screen was carried out to identify second site mutations that could restore the growth of ethylene-treated eer5-1 to wild-type levels. A dominant gain-of-function mutation in the ethylene receptor ETHYLENE RESPONSE SENSOR 1 (ERS1) was identified, with the ers1-4 mutation being located in transmembrane domain III at a point nearly equivalent to the previously described etr1-2 mutation in the other Arabidopsis subfamily I ethylene receptor, ETHYLENE RESPONSE 1 (ETR1). Although both ers1-4 and etr1-2 partially suppress the ethylene hypersensitivity of eer5-1 and are at least in part REVERSION TO ETHYLENE SENSITIVITY 1 (RTE1)-dependent, ers1-4 was additionally found to restore the expression of AtEBP in ers1-4;eer5-1 etiolated seedlings after ethylene treatment in an EIN3-dependent manner. Our work indicates that ERS1-regulated expression of a subset of ethylene-responsive genes is related to controlling the magnitude of ethylene response, with hyperinduction of these genes correlated with reduced ethylene-dependent growth inhibition.

The first step in ethylene perception occurs when the ethylene molecule binds to a receptor. A large number of studies have increased our understanding about how ethylene binds to the receptors and a signal is transduced. These studies have shown that a copper ion is a required cofactor that is delivered by the RAN1 (RESPONSIVE TO ANTAGONIST1) copper transporter. Additionally, biochemical studies have determined that the receptors are functional protein kinases. However, receptor protein kinase activity is not required for responses to ethylene. Rather, this activity seems to modulate responses to ethylene. Even though the exact nature of receptor output is unknown, it is clear that the receptors affect the activity of the CTR1 (CONSITUTIVE TRIPLE RESPONSE1) protein kinase. A model for how ethylene affects receptor signaling is presented.

Ethylene is a hormone that affects many processes important for plant growth, development, and responses to stresses. The first step in ethylene signal transduction is when ethylene binds to its receptors. Numerous studies have examined how these receptors function. In this review we summarize many of these studies and present our current understanding about how ethylene binds to the receptors. The biochemical output of the receptors is not known but current models predict that when ethylene binds to the receptors, the activity of the associated protein kinase, CTR1 (constitutive triple response1), is reduced. This results in downstream transcriptional changes leading to ethylene responses. We present a model where a copper cofactor is required and the binding of ethylene causes the receptor to pass through a transition state to become non-signaling leading to lower CTR1 activity.