Ivermectin-induced gene expression changes in adult Parascaris univalens and Caenorhabditis elegans: a comparative approach to study anthelminthic metabolism and resistance in vitro.

BACKGROUND: The nematode Parascaris univalens is one of the most prevalent parasitic pathogens infecting horses but anthelmintic resistance undermines treatment approaches. The molecular mechanisms underlying drug activity and resistance remain poorly understood in this parasite since experimental in vitro models are lacking. The aim of this study was to evaluate the use of Caenorhabditis elegans as a model for P. univalens drug metabolism/resistance studies by a comparative gene expression approach after in vitro exposure to the anthelmintic drug ivermectin (IVM). METHODS: Twelve adult P. univalens worms in groups of three were exposed to ivermectin (IVM, 10-13 M, 10-11 M, 10-9 M) or left unexposed for 24 h at 37 °C, and total RNA, extracted from the anterior end of the worms, was sequenced using Illumina NovaSeq. Differentially expressed genes (DEGs) involved in metabolism, transportation, or gene expression with annotated Caernorhabditis elegans orthologues were identified as candidate genes to be involved in IVM metabolism/resistance. Similarly, groups of 300 adult C. elegans worms were exposed to IVM (10-9 M, 10-8 M and 10-7 M) or left unexposed for 4 h at 20 °C. Quantitative RT-PCR of RNA extracted from the C. elegans worm pools was used to compare against the expression of selected P. univalens candidate genes after drug treatment. RESULTS: After IVM exposure, 1085 DEGs were found in adult P. univalens worms but the relative gene expression changes were small and large variabilities were found between different worms. Fifteen of the DEGs were chosen for further characterization in C. elegans after comparative bioinformatics analyses. Candidate genes, including the putative drug target lgc-37, responded to IVM in P. univalens, but marginal to no responses were observed in C. elegans despite dose-dependent behavioral effects observed in C. elegans after IVM exposure. Thus, the overlap in IVM-induced gene expression in this small set of genes was minor in adult worms of the two nematode species. CONCLUSION: This is the first time to our knowledge that a comparative gene expression approach has evaluated C. elegans as a model to understand IVM metabolism/resistance in P. univalens. Genes in P. univalens adults that responded to IVM treatment were identified. However, identifying conserved genes in P. univalens and C. elegans involved in IVM metabolism/resistance by comparing gene expression of candidate genes proved challenging. The approach appears promising but was limited by the number of genes studied (n = 15). Future studies comparing a larger number of genes between the two species may result in identification of additional candidate genes involved in drug metabolism and/or resistance.

DORMANCY/AUXIN ASSOCIATED FAMILY PROTEIN 2 of Arabidopsis thaliana is a negative regulator of local and systemic acquired resistance.

To fine tune defense response output, plants recruit both positive and negative regulators. Here we report Arabidopsis DORMANCY/AUXIN ASSOCIATED FAMILY PROTEIN 2(DAP2) gene as a negative regulator of basal defense against virulent bacterial pathogens. Expression of DAP2 enhances upon pathogen inoculation. Our experiments show that DAP2 suppressed resistance against virulent strains of bacterial pathogens, pathogen-induced callose deposition, and ROS accumulation; however, it did not influence effector-triggered immunity. In addition, DAP2 negatively regulated systemic acquired resistance (SAR). DAP2 expression was enhanced in the pathogen-free systemic tissues of SAR-induced plants. Previously, Arabidopsis Flowering locus D (FLD) gene has been shown to be essential for SAR but not for local resistance. We show here that FLD function is necessary for SAR-induced expression of DAP2, suggesting DAP2 as a target of FLD for activation of SAR in Arabidopsis.

MEDEA-interacting protein LONG-CHAIN BASE KINASE 1 promotes pattern-triggered immunity in Arabidopsis thaliana.

KEY MESSAGE: Arabidopsis LONG-CHAIN BASE KINASE 1 (LCBK1) interacts with MEDEA, a component of PCR2 complex that negatively regulates immunity. LCBK1 phosphorylates phytosphingosine and thereby promotes stomatal immunity against bacterial pathogens. Arabidopsis polycomb-group repressor complex2 (PRC2) protein MEDEA (MEA) suppresses both pattern-triggered immunity (PTI) and effector-triggered immunity (ETI). MEA represses the expression of RPS2 and thereby attenuates AvrRpt2 effector-mediated ETI. However, the mechanism of MEA-mediated PTI diminution was not known. By screening the Arabidopsis cDNA library using yeast-2-hybrid interaction, we identified LONG-CHAIN BASE KINASE1 (LCBK1) as an MEA-interacting protein. We found that lcbk1 mutants are susceptible to virulent bacterial pathogens, such as Pseudomonas syringae pv maculicola (Psm) and P. syringae pv tomato (Pst) but not the avirulent strain of Pst that carries AvrRpt2 effector. Pathogen inoculation induces LCBK1 expression, especially in guard cells. We found that LCBK1 has a positive regulatory role in stomatal closure after pathogen inoculation. WT plants close stomata within an hour of Pst inoculation or flg22 (a 22 amino acid peptide from bacterial flagellin protein that activates PTI) treatment, but not lcbk1 mutants. LCBK1 phosphorylates phytosphingosine (PHS). Exogenous application of phosphorylated PHS (PHS-P) induces stomatal closure and rescues loss-of-PTI phenotype of lcbk1 mutant plants. MEA overexpressing (MEA-Oex) plants are defective, whereas loss-of-function mea-6 mutants are hyperactive in PTI-induced stomatal closure. Exogenous application of PHS-P rescues loss-of-PTI in MEA-Oex plants. Results altogether demonstrate that LCBK1 is an interactor of MEA that positively regulates PTI-induced stomatal closure in Arabidopsis.

Arabidopsis protein l-ISOASPARTYL METHYLTRANSFERASE repairs isoaspartyl damage to antioxidant enzymes and increases heat and oxidative stress tolerance.

Stressful environments accelerate the formation of isoaspartyl (isoAsp) residues in proteins, which detrimentally affect protein structure and function. The enzyme PROTEIN l-ISOASPARTYL METHYLTRANSFERASE (PIMT) repairs other proteins by reverting deleterious isoAsp residues to functional aspartyl residues. PIMT function previously has been elucidated in seeds, but its role in plant survival under stress conditions remains undefined. Herein, we used molecular, biochemical, and genetic approaches, including protein overexpression and knockdown experiments, in Arabidopsis to investigate the role of PIMTs in plant growth and survival during heat and oxidative stresses. We demonstrate that these stresses increase isoAsp accumulation in plant proteins, that PIMT activity is essential for restricting isoAsp accumulation, and that both PIMT1 and PIMT2 play an important role in this restriction and Arabidopsis growth and survival. Moreover, we show that PIMT improves stress tolerance by facilitating efficient reactive oxygen species (ROS) scavenging by protecting the functionality of antioxidant enzymes from isoAsp-mediated damage during stress. Specifically, biochemical and MS/MS analyses revealed that antioxidant enzymes acquire deleterious isoAsp residues during stress, which adversely affect their catalytic activities, and that PIMT repairs the isoAsp residues and thereby restores antioxidant enzyme function. Collectively, our results suggest that the PIMT-mediated protein repair system is an integral part of the stress-tolerance mechanism in plants, in which PIMTs protect antioxidant enzymes that maintain proper ROS homeostasis against isoAsp-mediated damage in stressful environments.

The Polycomb-Group Repressor MEDEA Attenuates Pathogen Defense.

Plants recruit positive and negative regulators for fine tuning the balance between growth and development. Negative regulators of pathogen defense generally modulate defense hormone biosynthesis and signaling. Here, we report a mechanism for attenuation of the defense response in Arabidopsis (Arabidopsis thaliana), which is mediated by the polycomb-group repressor MEDEA (MEA). Our results showed that pathogen inoculation or exogenous application of salicylic acid, methyl jasmonate, or the bacterial 22-amino acid domain of flagellin peptide induces the expression of MEAMEA expression was higher when plants were inoculated with the avirulent strain of Pseudomonas syringae pv. tomato (Pst) carrying the AvrRpt2 effector (Pst-AvrRpt2) compared to the virulent Pst strain. MEA remains suppressed during the vegetative phase via DNA and histone (H3K27) methylation, and only the maternal copy is expressed in the female gametophyte and endosperm via histone and DNA demethylation. In contrast, Pst-AvrRpt2 induces high levels of MEA expression via hyper-accumulation of H3K4me3 at the MEA locus. MEA-overexpressing transgenic plants are susceptible to the fungal pathogen Botrytis cinerea and bacterial pathogens Pst and Pst-AvrRpt2, whereas mea mutant plants are more resistant to bacterial pathogens. AvrRpt2-mediated immunity requires the function of RESISTANCE TO P. SYRINGAE2 (RPS2) in Arabidopsis. Using transcriptional analysis and chromatin immunoprecipitation, we established that MEA directly targets RPS2 and suppresses its transcription. We screened an Arabidopsis cDNA library using MEA as the bait in a yeast two-hybrid assay and identified DROUGHT-INDUCED19, a transcription factor that interacts with MEA and recruits it at the RPS2 promoter. The results identified a previously unknown mechanism of defense response attenuation in plants.

Arabidopsis thaliana methionine sulfoxide reductase B8 influences stress-induced cell death and effector-triggered immunity.

KEY MESSAGE: Reactive oxygen species (ROS) oxidize methionine to methionine sulfoxide (MetSO) and thereby inactivate proteins. Methionine sulfoxide reductase (MSR) enzyme converts MetSO back to the reduced form and thereby detoxifies the effect of ROS. Our results show that Arabidopsis thaliana MSR enzyme coding gene MSRB8 is required for effector-triggered immunity and containment of stress-induced cell death in Arabidopsis. Plants activate pattern-triggered immunity (PTI), a basal defense, upon recognition of evolutionary conserved molecular patterns present in the pathogens. Pathogens release effector molecules to suppress PTI. Recognition of certain effector molecules activates a strong defense, known as effector-triggered immunity (ETI). ETI induces high-level accumulation of reactive oxygen species (ROS) and hypersensitive response (HR), a rapid programmed death of infected cells. ROS oxidize methionine to methionine sulfoxide (MetSO), rendering several proteins nonfunctional. The methionine sulfoxide reductase (MSR) enzyme converts MetSO back to the reduced form and thereby detoxifies the effect of ROS. Though a few plant MSR genes are known to provide tolerance against oxidative stress, their role in plant-pathogen interaction is not known. We report here that activation of cell death by avirulent pathogen or UV treatment induces expression of MSRB7 and MSRB8 genes. The T-DNA insertion mutant of MSRB8 exaggerates HR-associated and UV-induced cell death and accumulates a higher level of ROS than wild-type plants. The negative regulatory role of MSRB8 in HR is further supported by amiRNA and overexpression lines. Mutants and overexpression lines of MSRB8 are susceptible and resistant respectively, compared to the wild-type plants, against avirulent strains of Pseudomonas syringae pv. tomato DC3000 (Pst) carrying AvrRpt2, AvrB, or AvrPphB genes. However, the MSRB8 gene does not influence resistance against virulent Pst or P. syringae pv. maculicola (Psm) pathogens. Our results altogether suggest that MSRB8 function is required for ETI and containment of stress-induced cell death in Arabidopsis.

Design, development, and acceleration trials of radio-frequency quadrupole.

A deuteron radio frequency quadrupole (RFQ) accelerator has been designed, fabricated, and tested at BARC, which will be used for neutron generation. The RFQ operates at a frequency of 350 MHz and needs an inter-vane voltage of 44 kV to accelerate the deuteron beam to 400 keV within a length of 1.03 m. The error analysis shows that the offset of two opposite vanes in the same direction by 100 µm leads to a change in resonant frequency by 1.3 MHz and a significant change of fields in the quadrants (∼±40% with respect to average field). From the 3D analysis, we have observed that the unwanted dipole mode frequencies are very near to the quadrupole mode frequency which will make structure sensitive to the perturbations. In order to move the dipole modes away from the quadrupole modes, we have used the dipole stabilizer rods. The 5 wire transmission line theory was used to study the perturbative analysis of the RFQ and based on this a computer program has been written to tune the cavity to get required field distribution. Based on these studies, a 1.03 m long RFQ made of OFE copper has been fabricated and tested. Even though the RFQ was designed for deuteron (D(+)) beam, we tested it by accelerating both the proton (H(+)) and D(+) beams. The RFQ was operated in pulsed mode and accelerated both H(+) and D(+) beams to designed values of 200 and 400 keV, respectively. The measured parameters are in good agreement with the designed values validating our simulations and fabrication processes. In this paper, simulations, RF measurements, and beam commissioning results are presented.

Arabidopsis flowering locus D influences systemic-acquired-resistance- induced expression and histone modifications of WRKY genes.

A plant that is in part infected by a pathogen is more resistant throughout its whole body to subsequent infections--a phenomenon known as systemic acquired resistance (SAR). Mobile signals are synthesized at the site of infection and distributed throughout the plant through vascular tissues. Mechanism of SAR development subsequent to reaching the mobile signal in the distal tissue is largely unknown. Recently we showed that flowering locus D (FLD) gene of Arabidopsis thaliana is required in the distal tissue to activate SAR. FLD codes for a homologue of human-lysine-specific histone demethylase. Here we show that FLD function is required for priming (SAR induced elevated expression during challenge inoculation) of WRKY29 and WRKY6 genes. FLD also differentially influences basal and SAR-induced expression of WRKY38, WRKY65 and WRKY53 genes. In addition, we also show that FLD partly localizes in nucleus and influences histone modifications at the promoters of WRKY29 and WRKY6 genes. The results altogether indicate to the possibility of FLD's involvement in epigenetic regulation of SAR.

Arabidopsis thaliana FLOWERING LOCUS D is required for systemic acquired resistance.

Localized infection in plants often induces systemic acquired resistance (SAR), which provides long-term protection against subsequent infections. A signal originating in the SAR-inducing organ is transported to the distal organs, where it stimulates salicylic acid (SA) accumulation and priming, a mechanism that results in more robust activation of defenses in response to subsequent pathogen infection. In recent years, several metabolites that promote long-distance SAR signaling have been identified. However, the mechanism or mechanisms by which plants perceive and respond to the SAR signals are largely obscure. Here, we show that, in Arabidopsis thaliana, the FLOWERING LOCUS D (FLD) is required for responding to the SAR signals leading to the systemic accumulation of SA and enhancement of disease resistance. Although the fld mutant was competent in accumulating the SAR-inducing signal, it was unable to respond to the SAR signal that accumulates in petiole exudates of wild-type leaves inoculated with a SAR-inducing pathogen. Supporting FLD's role in systemic SAR signaling, we observed that dehydroabietinal and azelaic acid, two metabolites that, in wild-type plants, promote SAR-associated systemic accumulation of SA and priming, respectively, were unable to promote SAR in the fld mutant. FLD also participates in flowering, where it functions to repress expression of the flowering repressor FLOWERING LOCUS C (FLC). However, epistasis analysis indicates that FLD's function in SAR is independent of FLC.

Arabidopsis thaliana cdd1 mutant uncouples the constitutive activation of salicylic acid signalling from growth defects.

Arabidopsis genotypes with a hyperactive salicylic acid-mediated signalling pathway exhibit enhanced disease resistance, which is often coupled with growth and developmental defects, such as dwarfing and spontaneous necrotic lesions on the leaves, resulting in reduced biomass yield. In this article, we report a novel recessive mutant of Arabidopsis, cdd1 (constitutive defence without defect in growth and development1), that exhibits enhanced disease resistance associated with constitutive salicylic acid signalling, but without any observable pleiotropic phenotype. Both NPR1 (NON-EXPRESSOR OF PATHOGENESIS-RELATED GENES1)-dependent and NPR1-independent salicylic acid-regulated defence pathways are hyperactivated in cdd1 mutant plants, conferring enhanced resistance against bacterial pathogens. However, a functional NPR1 allele is required for the cdd1-conferred heightened resistance against the oomycete pathogen Hyaloperonospora arabidopsidis. Salicylic acid accumulates at elevated levels in cdd1 and cdd1 npr1 mutant plants and is necessary for cdd1-mediated PR1 expression and disease resistance phenotypes. In addition, we provide data which indicate that the cdd1 mutation negatively regulates the npr1 mutation-induced hyperactivation of ethylene/jasmonic acid signalling.