Discovery of antiplasmodial pyridine carboxamides and thiocarboxamides.

Malaria continues to be a significant burden, particularly in Africa, which accounts for 95% of malaria deaths worldwide. Despite advances in malaria treatments, malaria eradication is hampered by insecticide and antimalarial drug resistance. Consequently, the need to discover new antimalarial lead compounds remains urgent. To help address this need, we evaluated the antiplasmodial activity of twenty-two amides and thioamides with pyridine cores and their non-pyridine analogues. Twelve of these compounds showed in vitro anti-proliferative activity against the intraerythrocytic stage of Plasmodium falciparum, the most virulent species of Plasmodium infecting humans. Thiopicolinamide 13i was found to possess submicromolar activity (IC50 = 142 nM) and was >88-fold less active against a human cell line. The compound was equally effective against chloroquine-sensitive and -resistant parasites and did not inhibit ß-hematin formation, pH regulation or PfATP4. Compound 13i may therefore possess a novel mechanism of action.

Optimization of 2,3-Dihydroquinazolinone-3-carboxamides as Antimalarials Targeting PfATP4.

There is an urgent need to populate the antimalarial clinical portfolio with new candidates because of resistance against frontline antimalarials. To discover new antimalarial chemotypes, we performed a high-throughput screen of the Janssen Jumpstarter library against the Plasmodium falciparum asexual blood-stage parasite and identified the 2,3-dihydroquinazolinone-3-carboxamide scaffold. We defined the SAR and found that 8-substitution on the tricyclic ring system and 3-substitution of the exocyclic arene produced analogues with potent activity against asexual parasites equivalent to clinically used antimalarials. Resistance selection and profiling against drug-resistant parasite strains revealed that this antimalarial chemotype targets PfATP4. Dihydroquinazolinone analogues were shown to disrupt parasite Na+ homeostasis and affect parasite pH, exhibited a fast-to-moderate rate of asexual kill, and blocked gametogenesis, consistent with the phenotype of clinically used PfATP4 inhibitors. Finally, we observed that optimized frontrunner analogue WJM-921 demonstrates oral efficacy in a mouse model of malaria.

A G358S mutation in the Plasmodium falciparum Na+ pump PfATP4 confers clinically-relevant resistance to cipargamin.

Diverse compounds target the Plasmodium falciparum Na+ pump PfATP4, with cipargamin and (+)-SJ733 the most clinically-advanced. In a recent clinical trial for cipargamin, recrudescent parasites emerged, with most having a G358S mutation in PfATP4. Here, we show that PfATP4G358S parasites can withstand micromolar concentrations of cipargamin and (+)-SJ733, while remaining susceptible to antimalarials that do not target PfATP4. The G358S mutation in PfATP4, and the equivalent mutation in Toxoplasma gondii ATP4, decrease the sensitivity of ATP4 to inhibition by cipargamin and (+)-SJ733, thereby protecting parasites from disruption of Na+ regulation. The G358S mutation reduces the affinity of PfATP4 for Na+ and is associated with an increase in the parasite's resting cytosolic [Na+]. However, no defect in parasite growth or transmissibility is observed. Our findings suggest that PfATP4 inhibitors in clinical development should be tested against PfATP4G358S parasites, and that their combination with unrelated antimalarials may mitigate against resistance development.

Over-expression of a γ-tocopherol methyltransferase gene in vitamin E pathway confers PEG-simulated drought tolerance in alfalfa.

BACKGROUND: α-Tocopherol is one of the most important vitamin E components present in plant. α-Tocopherol is a potent antioxidant, which can deactivate photoproduced reactive oxygen species (ROS) and prevent lipids from oxidation when plants suffer drought stress. γ-Tocopherol methyltransferase (γ-TMT) catalyzes the formation of α-tocopherol in the tocopherol biosynthetic pathway. Our previous studies showed that over-expression of γ-TMT gene can increase the accumulation of α-tocopherol in alfalfa (Medicago sativa). However, whether these transgenic plants confer increased drought tolerance and the underlying mechanism are still unknown. RESULTS: In the present study, we further evaluate transgenic alfalfa lines, and found that over-expression of MsTMT led to an increase in α-tocopherol and total tocopherol level in the transgenic lines compared with the control plant. It was revealed that drought tolerance of the transgenic alfalfa was remarkably increased, with alleviated oxidative damage and accumulation of more osmolytic substances. The stomatal development in transgenic plants was significantly inhibited on both sides of leaves, which may be resulted from the repression of MsSPCHLESS (MsSPCH) gene. The reduced stomatal density of transgenic plants contributes to a lower stomatal conductance and higher water use efficiency (WUE). Moreover, both RNA-seq and qRT-PCR analyses indicate that regulatory mechanism of MsTMT in drought involved in both ABA-dependent and ABA-independent pathways. CONCLUSION: Our results suggest that MsTMT gene plays a positive role in regulating alfalfa response to PEG-simulated drought stress, which might involve complex mechanisms, including ROS scavenging system, stomatal development and multiple phytohormone signaling pathways. This study will broaden our view on the function of γ-TMT gene and provide new strategy for genetic engineering in alfalfa breeding.

NMT1 and NMT3 N-Methyltransferase Activity Is Critical to Lipid Homeostasis, Morphogenesis, and Reproduction.

Phosphatidylcholine (PC) is a major membrane phospholipid and a precursor for major signaling molecules. Understanding its synthesis is important for improving plant growth, nutritional value, and resistance to stress. PC synthesis is complex, involving several interconnected pathways, one of which proceeds from serine-derived phosphoethanolamine to form phosphocholine through three sequential phospho-base methylations catalyzed by phosphoethanolamine N-methyltransferases (PEAMTs). The contribution of this pathway to the production of PC and plant growth has been a matter of some debate. Although a handful of individual PEAMTs have been described, there has not been any in planta investigation of a PEAMT family. Here, we provide a comparative functional analysis of two Arabidopsis (Arabidopsis thaliana) PEAMTs, NMT1 and the little known NMT3. Analysis of loss-of-function mutants demonstrates that NMT1 and NMT3 synergistically regulate PC homeostasis, phase transition at the shoot apex, coordinated organ development, and fertility through overlapping but also specific functions. The nmt1 nmt3 double mutant shows extensive sterility, drastically reduced PC concentrations, and altered lipid profiles. These findings demonstrate that the phospho-base methylation pathway makes a major contribution to PC synthesis in Arabidopsis and that NMT1 and NMT3 play major roles in its catalysis and the regulation of PC homeostasis as well as in plant growth and reproduction.

A pair of allelic WRKY genes play opposite roles in rice-bacteria interactions.

Although allelic diversity of genes has been reported to play important roles in different physiological processes, information on allelic diversity of defense-responsive genes in host-pathogen interactions is limited. Here, we report that a pair of allelic genes, OsWRKY45-1 and OsWRKY45-2, which encode proteins with a 10-amino acid difference, play opposite roles in rice (Oryza sativa) resistance against bacterial pathogens. Bacterial blight caused by Xanthomonas oryzae pv oryzae (Xoo), bacterial streak caused by Xanthomonas oryzae pv oryzicola (Xoc), and fungal blast caused by Magnaporthe grisea are devastating diseases of rice worldwide. OsWRKY45-1-overexpressing plants showed increased susceptibility and OsWRKY45-1-knockout plants showed enhanced resistance to Xoo and Xoc. In contrast, OsWRKY45-2-overexpressing plants showed enhanced resistance and OsWRKY45-2-suppressing plants showed increased susceptibility to Xoo and Xoc. Interestingly, both OsWRKY45-1- and OsWRKY45-2-overexpressing plants showed enhanced resistance to M. grisea. OsWRKY45-1-regulated Xoo resistance was accompanied by increased accumulation of salicylic acid and jasmonic acid and induced expression of a subset of defense-responsive genes, while OsWRKY45-2-regulated Xoo resistance was accompanied by increased accumulation of jasmonic acid but not salicylic acid and induced expression of another subset of defense-responsive genes. These results suggest that both OsWRKY45-1 and OsWRKY45-2 are positive regulators in rice resistance against M. grisea, but the former is a negative regulator and the latter is a positive regulator in rice resistance against Xoo and Xoc. The opposite roles of the two allelic genes in rice-Xoo interaction appear to be due to their mediation of different defense signaling pathways.

Exploring transcriptional signalling mediated by OsWRKY13, a potential regulator of multiple physiological processes in rice.

BACKGROUND: Rice transcription regulator OsWRKY13 influences the functioning of more than 500 genes in multiple signalling pathways, with roles in disease resistance, redox homeostasis, abiotic stress responses, and development. RESULTS: To determine the putative transcriptional regulation mechanism of OsWRKY13, the putative cis-acting elements of OsWRKY13-influenced genes were analyzed using the whole genome expression profiling of OsWRKY13-activated plants generated with the Affymetrix GeneChip Rice Genome Array. At least 39 transcription factor genes were influenced by OsWRKY13, and 30 of them were downregulated. The promoters of OsWRKY13-upregulated genes were overrepresented with W-boxes for WRKY protein binding, whereas the promoters of OsWRKY13-downregulated genes were enriched with cis-elements putatively for binding of MYB and AP2/EREBP types of transcription factors. Consistent with the distinctive distribution of these cis-elements in up- and downregulated genes, nine WRKY genes were influenced by OsWRKY13 and the promoters of five of them were bound by OsWRKY13 in vitro; all seven differentially expressed AP2/EREBP genes and six of the seven differentially expressed MYB genes were suppressed by in OsWRKY13-activated plants. A subset of OsWRKY13-influenced WRKY genes were involved in host-pathogen interactions. CONCLUSION: These results suggest that OsWRKY13-mediated signalling pathways are partitioned by different transcription factors. WRKY proteins may play important roles in the monitoring of OsWRKY13-upregulated genes and genes involved in pathogen-induced defence responses, whereas MYB and AP2/EREBP proteins may contribute most to the control of OsWRKY13-downregulated genes.

Molecular analyses of the rice tubby-like protein gene family and their response to bacterial infection.

Tubby-like protein family has been identified in various multicellular organisms, indicating its fundamental functions in the organisms. However, the roles of plant tubby-like proteins are unknown. In this study, we have defined the tubby-like protein gene (OsTLP) family with 14 members in rice. Most of the OsTLPs harbor a tubby domain in their carboxyl terminus and an F-box domain in the amino terminus. The expression of all the OsTLPs was induced on infection of Xanthomonas oryzae pv. oryzae, which causes bacterial blight, one of the most devastating diseases of rice worldwide. The maximal expression levels were observed at 2-8 h after infection for all the genes. Eight of the 14 OsTLPs were also responsive to wounding. All the OsTLPs showed differential expression in different tissues at different developmental stages. However, four pairs of the 14 OsTLPs, with each pair having high sequence similarity and distributing on the similar position of different chromosomes, showed similar expression pattern in different tissues, indicating their direct relationship in evolution. These results suggest that the OsTLP family is involved in host-pathogen interaction and it may be also associated with other physiological and developmental activities.

Rice gene network inferred from expression profiling of plants overexpressing OsWRKY13, a positive regulator of disease resistance.

Accumulating information indicates that plant disease resistance signaling pathways frequently interact with other pathways regulating developmental processes or abiotic stress responses. However, the molecular mechanisms of these types of crosstalk remain poorly understood in most cases. Here we report that OsWRKY13, an activator of rice resistance to both bacterial and fungal pathogens, appears to function as a convergent point for crosstalk among the pathogen-induced salicylate-dependent defense pathway and five other physiologic pathways. Genome-wide analysis of the expression profiles of OsWRKY13-overexpressing lines suggests that OsWRKY13 directly or indirectly regulates the expression of more than 500 genes that are potentially involved in different physiologic processes according to the classification of the Gene Ontology database. By comparing the expression patterns of genes functioning in known pathways or cellular processes of pathogen infection and the phenotypes between OsWRKY13-overexpressing and wild-type plants, our data suggest that OsWRKY13 is also a regulator of other physiologic processes during pathogen infection. The OsWRKY13-associated disease resistance pathway synergistically interacts via OsWRKY13 with the glutathione/glutaredoxin system and flavonoid biosynthesis pathway to monitor redox homeostasis and to putatively enhance the biosynthesis of antimicrobial flavonoid phytoalexins, respectively, in OsWRKY13-overexpressing lines. Meanwhile, the OsWRKY13-associated disease resistance pathway appears to interact antagonistically with the SNAC1-mediated abiotic stress defense pathway, jasmonic acid signaling pathway, and terpenoid metabolism pathway via OsWRKY13 to suppress salt and cold defense responses as well as to putatively retard rice growth and development.

Isolation and manipulation of quantitative trait loci for disease resistance in rice using a candidate gene approach.

Bacterial blight caused by Xanthomonas oryzae pv. oryzae and fungal blast caused by Magnaporthe grisea result in heavy production losses in rice, a main staple food for approximately 50% of the world's population. Application of host resistance to these pathogens is the most economical and environment-friendly approach to solve this problem. Quantitative trait loci (QTLs) controlling quantitative resistance are valuable sources for broad-spectrum and durable disease resistance. Although large numbers of QTLs for bacterial blight and blast resistance have been identified, these sources have not been used effectively in rice improvement because of the complex genetic control of quantitative resistance and because the genes underlying resistance QTLs are unknown. To isolate disease resistance QTLs, we established a candidate gene strategy that integrates linkage map, expression profile, and functional complementation analyses. This strategy has proven to be applicable for identifying the genes underlying minor resistance QTLs in rice-Xoo and rice-M. grisea systems and it may also help to shed light on disease resistance QTLs of other cereals. Our results also suggest that a single minor QTL can be used in rice improvement by modulating the expression of the gene underlying the QTL. Pyramiding two or three minor QTL genes, whose expression can be managed and that function in different defense signal transduction pathways, may allow the breeding of rice cultivars that are highly resistant to bacterial blight and blast.

Identification of novel pathogen-responsive cis-elements and their binding proteins in the promoter of OsWRKY13, a gene regulating rice disease resistance.

The WRKY transcription factor superfamily controls diverse developmental and physiological processes in plants. However, little is known about the factors that directly regulate the function of WRKY genes. In this study, we identified cis-acting elements and their binding proteins of rice OsWRKY13, a gene that plays a pivotal role in disease resistance against bacterial and fungal pathogens. Two novel pathogen-responsive cis-elements, PRE2 and PRE4, were characterized from the promoter region of OsWRKY13. The two cis-elements negatively regulate gene expression without pathogen challenge, and positively regulate gene expression after pathogen-induced protein binding. OsWRKY13 binds to PRE4, which harbours a novel W-like box. Another five proteins (Rad51-like; tubby-like; SWIM zinc finger and nucleotide-binding adaptor shared by APAF-1, certain R proteins and CED-4 (NB-ARC) domain containing proteins; and an unknown protein) also bind to one of the two cis-elements. Different proteins interacting with the same cis-element appear to have different DNA-binding core sequences. These proteins localize in the nucleus and show differential expression upon pathogen challenge. These results suggest that OsWRKY13 expression is regulated by multiple factors to achieve disease resistance.

OsWRKY13 mediates rice disease resistance by regulating defense-related genes in salicylate- and jasmonate-dependent signaling.

Although 109 WRKY genes have been identified in the rice genome, the functions of most are unknown. Here, we show that OsWRKY13 plays a pivotal role in rice disease resistance. Overexpression of OsWRKY13 can enhance rice resistance to bacterial blight and fungal blast, two of the most devastating diseases of rice worldwide, at both the seedling and adult stages, and shows no influence on the fertility. This overexpression was accompanied by the activation of salicylic acid (SA) synthesis-related genes and SA-responsive genes and the suppression of jasmonic acid (JA) synthesis-related genes and JA-responsive genes. OsWRKY13 bound to the promoters of its own and at least three other genes in SA- and JA-dependent signaling pathways. Its DNA-binding activity was influenced by pathogen infection. These results suggest that OsWRKY13, as an activator of the SA-dependent pathway and a suppressor of JA-dependent pathways, mediates rice resistance by directly or indirectly regulating the expression of a subset of genes acting both upstream and downstream of SA and JA. Furthermore, OsWRKY13 will provide a transgenic tool for engineering wider-spectrum and whole-growth-stage resistance rice in breeding programs.

Dual function of rice OsDR8 gene in disease resistance and thiamine accumulation.

The function of OsDR8, a rice disease resistance-responsive gene, was studied. Silencing of OsDR8 using an RNA interference approach resulted in phenotypic alteration of the plants. The transgenic plants with repressed expression of OsDR8 showed reduced resistance or susceptibility to Xanthomonas oryzae pv. oryzae and Magnaporthe grisea causing bacterial blight and blast, which are two of the most devastating diseases in rice worldwide, respectively. The putative product of OsDR8 was highly homologous to an enzyme involved in the biosynthesis of the thiazole precursor of thiamine. Transgenic plants showing repressed expression of OsDR8 and reduced resistance had significantly lower levels of thiamine than the control plants. Exogenous application of thiamine could complement the compromised defense of the OsDR8-silenced plants. The expression level of several defense-responsive genes including the earlier functional genes of defense transduction pathway, OsPOX and OsPAL, and the downstream genes of the pathway, OsPR1a, OsPR1b, OsPR4, OsPR5 and OsPR10, was also decreased in the OsDR8-silenced plants. These results suggest that the impact of OsDR8 on disease resistance in rice may be through the regulation of expression of other defense-responsive genes and the site of OsDR8 function is on the upstream of the signal transduction pathway. In addition, the accumulation of thiamine may be essential for bacterial blight resistance and blast resistance.

Features of the expressed sequences revealed by a large-scale analysis of ESTs from a normalized cDNA library of the elite indica rice cultivar Minghui 63.

The indica subspecies of cultivated rice occupies the largest area of rice production in the world. However, a systematic analysis of cDNA sequences from the indica subspecies has not been performed. The aim of the present study was to collect and analyze the expressed sequence tags (ESTs) of indica rice on a large scale. A total of 39 208 raw sequences were generated from a normalized cDNA library prepared by use of 15 different tissues of the indica cultivar Minghui 63. After trimming, processing and analysis, 17 835 unique sequences were obtained, each of which presumably represents a unique gene. Of these sequences, 2663 were novel, and at least 70 were indica specific. Comparison of the Minghui 63 sequences with the ESTs/full-length cDNAs in GenBank revealed a large number of deletion/insertion/substitution (DIS) at both the inter- and intra-subspecific levels. The overall number of polymorphisms in the expressed sequences was higher in the inter-subspecific comparisons than in the intra-subspecific comparisons. However, the extent of DIS-based polymorphism was highly variable among different rice varieties. In total, 15 726 unique sequences, including 697 novel sequences, were assigned to regions where large numbers of quantitative trait loci (QTLs) for agronomic traits had been detected previously. These results may be useful for developing new molecular markers for genetic mapping, detecting allelic polymorphisms associated with phenotypic variations between rice varieties, and facilitating QTL cloning by providing the starting points for candidate-gene identification.