Probing strigolactone perception mechanisms with rationally designed small-molecule agonists stimulating germination of root parasitic weeds.

The development of potent strigolactone (SL) agonists as suicidal germination inducers could be a useful strategy for controlling root parasitic weeds, but uncertainty about the SL perception mechanism impedes real progress. Here we describe small-molecule agonists that efficiently stimulate Phelipanchce aegyptiaca, and Striga hermonthica, germination in concentrations as low as 10-8 to 10-17 M. We show that full efficiency of synthetic SL agonists in triggering signaling through the Striga SL receptor, ShHTL7, depends on the receptor-catalyzed hydrolytic reaction of the agonists. Additionally, we reveal that the stereochemistry of synthetic SL analogs affects the hydrolytic ability of ShHTL7 by influencing the probability of the privileged conformations of ShHTL7. Importantly, an alternative ShHTL7-mediated hydrolysis mechanism, proceeding via nucleophilic attack of the NE2 atom of H246 to the 2'C of the D-ring, is reported. Together, our findings provide insight into SL hydrolysis and structure-perception mechanisms, and potent suicide germination stimulants, which would contribute to the elimination of the noxious parasitic weeds.

Cell-Trappable BODIPY-NBD Dyad for Imaging of Basal and Stress-Induced H2S in Live Biosystems.

H2S is a gaseous signaling molecule that is involved in many physiological and pathological processes. In general, the level of intracellular H2S (<1 µM) is much lower than that of GSH (∼1-10 mM), leading to the remaining challenge of selective detection and differentiation of endogenous H2S in live biosystems. To this end, we quantitatively demonstrate that the thiolysis of NBD amine has much higher selectivity for H2S over GSH than that of the reduction of aryl azide. Subsequently, we developed the first NBD-based cell-trappable probe 1 (AM-BODIPY-NBD) for highly selective and ultrasensitive imaging of intracellular H2S. Probe 1 demonstrates a 207-fold fluorescence enhancement at 520 nm after reaction with H2S/esterase to produce a bright BODIPY (quantum yield 0.42) and a detection limit of 15.7 nM. Probe 1 is water-soluble, cell-trappable, and not cytotoxic. Based on this excellent chemical tool, relative levels of basal H2S in different cell lines were measured to reveal a positive correlation between endogenous H2S and the metastatic potential of colon and breast cancer cells. In addition, H2S biogenesis in vivo was also validated by probe 1 both in tobacco leaves under viral infection and in zebrafish after tail amputation. It is anticipated that probe 1 will have widespread applications in imaging and for investigating different H2S-related biological processes and diseases.

Discovery of a Broad-Spectrum Fluorogenic Agonist for Strigolactone Receptors through a Computational Approach.

Strigolactones (SLs) are plant hormones that play various roles in plant physiology, including provoking the germination of parasitic weeds Orobanche and Striga. A family of α/ß-hydrolases have been proposed to be the SL receptor proteins. Effective assays for measuring the activity of SL receptors could promote the development of SL-related biology and chemistry. In this study, we developed a new approach called pharmacophore-linked probe virtual screening (PPVS). Its application yielded an effective "off-on" probe named Xilatone Red (XLR). This probe showed a broad spectrum and excellent sensitivity toward SL receptors, including ShD14 (Striga D14), for which the detection limit was determined to be in the micromolar range, outperforming that of the commercial fluorogenic agonist Yoshimulactone Green (YLG). Upon hydrolysis by SL receptors, XLR provided fluorogenic and colorimetric signaling responses. Furthermore, XLR could induce germination of Phelipanche aegyptiaca seeds and prevent Arabidopsis max4-1 branching defects at micromolar concentrations. Our molecular simulations revealed the essential factors in the molecular perception of XLR. We anticipate that this study can prompt the discovery of high-performance SL agonists/antagonists to combat parasitic weeds.

A H2 S-Specific Ultrasensitive Fluorogenic Probe Reveals TMV-Induced H2 S Production to Limit Virus Replication.

Understanding the role of H2 S in host defense mechanisms against RNA viruses may provide opportunities for the development of antivirals to combat viral infections. Here, we have developed a green-emitting fluorogenic probe, which exhibits a large fluorescence response at 520 nm (>560-fold) when treated with 100 µM H2 S for 1 h. It is highly selective for H2 S over biothiols (>400-fold F/F0 ) and has a detection limit of 12.9 nM. We demonstrate the application of the probe for endogenous H2 S detection in vivo for the understanding of its roles in antiviral host defense. Such virus-induced H2 S inhibits viral replication by reducing gene expression of RNA-dependent RNA polymerase (RdRp) and coat protein (CP). Additionally, a H2 S donor GYY4137 showed significantly antiviral activity as ribavirin, a broad-spectrum drug against RNA viruses. Furtherly, we propose a possible molecular mechanism for the TMV-induced H2 S biogenesis. This work provides a proof-of-principle in support of further studies identifying endogenous H2 S and its donors as potential antivirals toward RNA viruses.

Genetically encoding ε-N-benzoyllysine in proteins.

Lysine ε-N-benzoylation is a recently identified PTM occurring on histone proteins, and herein we genetically encoded ε-N-benzoyl-lysine (BzK) into recombinant proteins in E. coli and mammalian cells, and applied it for the modification of histone proteins and the analysis of sirtuin debenzoylase activity.

Comparative Studies of Potential Binding Pocket Residues Reveal the Molecular Basis of ShHTL Receptors in the Perception of GR24 in Striga.

Root parasitic weeds such as Striga spp. have caused significant losses in agriculture production worldwide. The seed germination of the weeds depends on strigolactones (SLs) that target a series of HYPOSENSITIVE TO LIGHT/KARRIKIN INSENSITIVE2 in Striga hermonthica (ShHTL) proteins. In the present study, 60 ShHTL7 mutants were constructed, and the equilibrium dissociation constants for GR24 (a synthetic SL analogue, commonly used as a standard in SL germination studies) against these mutants were measured by surface plasmon resonance. Based on these data, the SL binding pocket residues were distinguished. Of them, some specific residues for ShHTL7 were found, such as T142, T190, and M219. A model showing quite well internal and external predictive abilities was established by the mutation-dependent biomacromolecular quantitative structure-activity relationship method. It provided an expanded understanding for GR24 binding to a series of ShHTL receptors and should help design broad-spectrum agrochemicals with cross interactions with several members of SL receptors.

Strigolactone Analogues Derived from Dihydroflavonoids as Potent Seed Germinators for the Broomrapes.

The broomrapes (Orobanche and Phelipanche spp.) and witchweeds (Striga spp.) are a class of parasitic weeds, which are distributed widely in the tropical, subtropical, and temperate areas of the globe. Since they have completely consistent lifecycles with the host plants, it is difficult to control them selectively through using the conventional herbicides. Inducing suicidal germination of these weed seeds by small molecular signaling agents proved to be a promising strategy for the management of parasitic weeds. As a class of naturally occurring terpenoid metabolites, strigolactones (SLs) show significant biological activities including stimulation germination of weed seeds, inhibition of shoot-branching, and so on. However, the widespread application of these natural SLs is greatly limited by their extremely low natural abundance and complex molecular structures. Design and synthesis of the simplified analogues as natural SLs alternatives provide a viable avenue for the efficient control of these parasitic weeds. We herein disclose the development of a novel class of SLs analogues derived from dihydroflavonoids as potent seed germinators of parasitic weeds. It was shown that one of them displayed a higher potential toward the seed germination of the broomrapes than the positive control GR24. The structure-activity relationship of these SLs analogues was further validated on the basis of the binding affinity experiment to strigolactone receptor protein HTL7 by using a YLG fluorescent probe method.

Quantitative proteomics links metabolic pathways to specific developmental stages of the plant-pathogenic oomycete Phytophthora capsici.

The oomycete Phytophthora capsici is a plant pathogen responsible for important losses to vegetable production worldwide. Its asexual reproduction plays an important role in the rapid propagation and spread of the disease in the field. A global proteomics study was conducted to compare two key asexual life stages of P. capsici, i.e. the mycelium and cysts, to identify stage-specific biochemical processes. A total of 1200 proteins was identified using qualitative and quantitative proteomics. The transcript abundance of some of the enriched proteins was also analysed by quantitative real-time polymerase chain reaction. Seventy-three proteins exhibited different levels of abundance between the mycelium and cysts. The proteins enriched in the mycelium are mainly associated with glycolysis, the tricarboxylic acid (or citric acid) cycle and the pentose phosphate pathway, providing the energy required for the biosynthesis of cellular building blocks and hyphal growth. In contrast, the proteins that are predominant in cysts are essentially involved in fatty acid degradation, suggesting that the early infection stage of the pathogen relies primarily on fatty acid degradation for energy production. The data provide a better understanding of P. capsici biology and suggest potential metabolic targets at the two different developmental stages for disease control.

Resistance Assessment for Oxathiapiprolin in Phytophthora capsici and the Detection of a Point Mutation (G769W) in PcORP1 that Confers Resistance.

The potential for oxathiapiprolin resistance in Phytophthora capsici was evaluated. The baseline sensitivities of 175 isolates to oxathiapiprolin were initially determinated and found to conform to a unimodal curve with a mean EC50 value of 5.61 × 10(-4) µg/ml. Twelve stable oxathiapiprolin-resistant mutants were generated by fungicide adaptation in two sensitive isolates, LP3 and HNJZ10. The fitness of the LP3-mutants was found to be similar to or better than that of the parental isolate LP3, while the HNJZ10-mutants were found to have lost the capacity to produce zoospores. Taken together these results suggest that the risk of P. capsici developing resistance to oxathiapiprolin is moderate. Comparison of the PcORP1 genes in the LP3-mutants and wild-type parental isolate, which encode the target protein of oxathiapiprolin, revealed that a heterozygous mutation caused the amino acid substitution G769W. Transformation and expression of the mutated PcORP1-769W allele in the sensitive wild-type isolate BYA5 confirmed that the mutation in PcORP1 was responsible for the observed oxathiapiprolin resistance. Finally diagnostic tests including As-PCR and CAPs were developed to detect the oxathiapiprolin resistance resulting from the G769W point mutation in field populations of P. capsici.

Insights into the adaptive response of the plant-pathogenic oomycete Phytophthora capsici to the fungicide flumorph.

Phytophthora capsici is an important oomycete plant pathogen that causes significant losses worldwide. The carboxylic acid amide fungicide flumorph has shown excellent activity against oomycete plant pathogens. Despite its potential, there remains concern that the sexual reproduction of oomycete pathogens, which results in genetic recombination, could result in the rapid development of resistance to flumorph. The current study utilized an iTRAQ (isobaric tags for relative and absolute quantitation) based method to compare differences between the proteome of the parental P. capsici isolate PCAS1 and its sexual progeny S2-838, which exhibits significant resistance to flumorph. A total of 2396 individual proteins were identified, of these, 181 were considered to be associated with the adaptive response of P. capsici to flumorph. The subsequent bioinformatic analysis revealed that the adaptive response of P. capsici to flumorph was complex and regulated by multiple mechanisms, including utilising carbohydrate from the host environment to compensate for the cell wall stress induced by flumorph, a shift in energy generation, decreased amino acids biosynthesis, and elevated levels of proteins associated with the pathogen's response to stimulus and transmembrane transport. Moreover, the results of the study provided crucial data that could provide the basis for early monitoring of flumorph resistance in field populations of P. capsici.

Proteomic profile of the plant-pathogenic oomycete Phytophthora capsici in response to the fungicide pyrimorph.

Pyrimorph is a novel fungicide from the carboxylic acid amide (CAA) family used to control plant-pathogenic oomycetes such as Phytophthora capsici. The proteomic response of P. capsici to pyrimorph was investigated using the iTRAQ technology to determine the target site of the fungicide and potential biomarker candidates of drug efficacy. A total of 1336 unique proteins were identified from the mycelium of wild-type P. capsici isolate (Hd3) and two pyrimorph-resistant mutants (R3-1 and R3-2) grown in the presence or absence of pyrimorph. Comparative analysis revealed that the three P. capsici isolates Hd3, R3-1, and R3-2 produced 163, 77, and 13 unique proteins, respectively, which exhibited altered levels of abundance in response to the pyrimorph treatment. Further investigations, using Cluster of Orthologous Groups of Proteins (COG) analysis and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis identified 35 proteins related to the mode of action of pyrimorph against P. capsici and 62 proteins involved in the stress response of P. capsici to pyrimorph. Many of the proteins with altered expression were associated with glucose and energy metabolism. Biochemical analysis using d-[U-(14) C]glucose verified the proteomics data, suggesting that the major mode of action of pyrimorph in P. capsici is the inhibition of cell wall biosynthesis. These results also illustrate that proteomics approaches are useful tools for determining the pathways targeted by novel fungicides as well as for evaluating the tolerance of plant pathogens to environmental challenges, such as the presence of fungicides.

Two non-target recessive genes confer resistance to the anti-oomycete microtubule inhibitor zoxamide in Phytophthora capsici.

This study characterized isolates of P. capsici that had developed a novel mechanism of resistance to zoxamide, which altered the minimum inhibition concentration (MIC) but not the EC50. Molecular analysis revealed that the ß-tubulin gene of the resistant isolates contained no mutations and was expressed at the same level as in zoxamide-sensitive isolates. This suggested that P. capsici had developed a novel non-target-site-based resistance to zoxamide. Analysis of the segregation ratio of zoxamide-resistance in the sexual progeny of the sensitive isolates PCAS1 and PCAS2 indicated that the resistance to zoxamide was controlled by one or more recessive nuclear genes. Furthermore, the segregation of resistance in the F1, F2, and BC1 progeny was in accordance with the theoretical ratios of the χ(2) test (P>0.05), which suggested that the resistance to zoxamide was controlled by two recessive genes, and that resistance to zoxamide occurred when at least one pair of these alleles was homozygous. This implies that the risk of zoxamide-resistance in P. capsici is low to moderate. Nevertheless this potential for resistance should be monitored closely, especially if two compatible mating types co-exist in the same field.

Competition between pyrimorph-sensitive and pyrimorph-resistant isolates of Phytophthora capsici.

Phytophthora capsici causes significant losses to vegetable production worldwide. Pyrimorph, a new carboxylic acid amide fungicide, has been registered to control P. capsici in China. A mutation (Q1077K) in cellulose synthase 3 has been reported to confer resistance to pyrimorph. In this study, we measured the competition between pyrimorph-resistant and pyrimorph-sensitive isolates of P. capsici. Mixed zoospore suspensions of resistant (R) and sensitive (S) isolates at five ratios (1R:9S, 3R:7S, 5R:5S, 7R:3S, and 9R:1S) were applied to carrot agar in vitro test (with five successive transfers) and to the soil surface around pepper plants in planta test (with 10 successive disease cycles). The proportion of resistant isolates was measured by a conventional assay in which single zoospore isolates recovered after transfers or disease cycles were grown on agar medium with a discriminatory concentration of pyrimorph. The results were then compared with those of a real-time polymerase chain reaction (PCR)-based method developed here, the results were similar. Both assays showed that the competitive ability of the resistant isolates was similar to or less than that of the sensitive isolates. The real-time PCR assay developed will be useful for high-throughput analysis and monitoring the development of pyrimorph resistance in field populations of P. capsici.

Loss of heterozygosity drives clonal diversity of Phytophthora capsici in China.

Phytophthora capsici causes significant loss to pepper (Capsicum annum) in China and our goal was to develop single nucleotide polymorphism (SNP) markers for P. capsici and characterize genetic diversity nationwide. Eighteen isolates of P. capsici from locations worldwide were re-sequenced and candidate nuclear and mitochondrial SNPs identified. From 2006 to 2012, 276 isolates of P. capsici were recovered from 136 locations in 27 provinces and genotyped using 45 nuclear and 2 mitochondrial SNPs. There were two main mitochondrial haplotypes and 95 multi-locus genotypes (MLGs) identified. Genetic diversity was geographically structured with a high level of genotypic diversity in the north and on Hainan Island in the south, suggesting outcrossing contributes to diversity in these areas. The remaining areas of China are dominated by four clonal lineages that share mitochondrial haplotypes, are almost exclusively the A1 or A2 mating type and appear to exhibit extensive diversity based on loss of heterozygosity (LOH). Analysis of SNPs directly from infected peppers confirmed LOH in field populations. One clonal lineage is dominant throughout much of the country. The overall implications for long-lived genetically diverse clonal lineages amidst a widely dispersed sexual population are discussed.

Genetically diverse long-lived clonal lineages of Phytophthora capsici from pepper in Gansu, China.

Phytophthora capsici causes significant loss to pepper production in China, and our objective was to investigate the population structure in Gansu province. Between 2007 and 2011, 279 isolates were collected from pepper at 24 locations. Isolates (or subsets) were assessed for simple sequence repeat (SSR) genotype, metalaxyl resistance, mating type, and physiological race using cultivars from the World Vegetable Center (AVRDC) and New Mexico recombinant inbred lines (NMRILs). The A1 and A2 mating types were recovered from nine locations and metalaxyl-resistant isolates from three locations. A total of 104 isolates tested on the AVRDC panel resolved five physiological races. None of 42 isolates tested on the NMRIL panel caused visible infection. SSR genotyping of 127 isolates revealed 59 unique genotypes, with 42 present as singletons and 17 having 2 to 13 isolates. Isolates with identical genotypes were recovered from multiple sites across multiple years and, in many cases, had different race types or metalaxyl sensitivities. Isolates clustered into three groups with each group having almost exclusively the A1 or A2 mating type. Overall it appears long-lived genetically diverse clonal lineages are dispersed across Gansu, outcrossing is rare, and functionally important variation exists within a clonal framework.

Resistance to the novel fungicide pyrimorph in Phytophthora capsici: risk assessment and detection of point mutations in CesA3 that confer resistance.

Pyrimorph is a novel fungicide with high activity against the plant pathogen Phytophthora capsici. We investigated the risk that P. capsici can develop resistance to pyrimorph. The baseline sensitivities of 226 P. capsici isolates, tested by mycelial growth inhibition, showed a unimodal distribution with a mean EC(50) value of 1.4261 (± 0.4002) µg/ml. Twelve pyrimorph-resistant mutants were obtained by repeated exposure to pyrimorph in vitro with a frequency of approximately 1 × 10(-4). The resistance factors of the mutants ranged from 10.67 to 56.02. Pyrimorph resistance of the mutants was stable after 10 transfers on pyrimorph-free medium. Fitness in sporulation, cystospore germination, and pathogenicity in the pyrimorph-resistant mutants was similar to or less than that in the parental wild-type isolates. On detached pepper leaves and pepper plants treated with the recommended maximum dose of pyrimorph, however, virulence was greater for mutants with a high level of pyrimorph resistance than for the wild type. The results suggest that the risk of P. capsici developing resistance to pyrimorph is low to moderate. Among mutants with a high level of pyrimorph resistance, EC(50) values for pyrimorph and CAA fungicides flumorph, dimethomorph, and mandipropamid were positively correlated. This indicated that point mutations in cellulose synthase 3 (CesA3) may confer resistance to pyrimorph. Comparison of CesA3 in isolates with a high level of pyrimorph resistance and parental isolates showed that an amino acid change from glutamine to lysine at position 1077 resulted in stable, high resistance in the mutants. Based on the point mutations, an allele-specific PCR method was developed to detect pyrimorph resistance in P. capsici populations.

Assessing the risk that Phytophthora melonis can develop a point mutation (V1109L) in CesA3 conferring resistance to carboxylic acid amide fungicides.

The risk that the plant pathogen Phytophthora melonis develops resistance to carboxylic acid amide (CAA) fungicides was determined by measuring baseline sensitivities of field isolates, generating resistant mutants, and measuring the fitness of the resistant mutants. The baseline sensitivities of 80 isolates to flumorph, dimethomorph and iprovalicarb were described by unimodal curves, with mean EC(50) values of 0.986 (±0.245), 0.284 (±0.060) and 0.327 (±0.068) µg/ml, respectively. Seven isolates with different genetic background (as indicated by RAPD markers) were selected to generate CAA-resistance. Fifty-five resistant mutants were obtained from three out of seven isolates by spontaneous selection and UV-mutagenesis with frequencies of 1×10(-7) and 1×10(-6), respectively. CAA-resistance was stable for all mutants. The resistance factors of these mutants ranged from 7 to 601. The compound fitness index (CFI  =  mycelial growth × zoospore production × pathogenicity) was often lower for the CAA-resistant isolates than for wild-type isolates, suggesting that the risk of P. melonis developing resistance to CAA fungicides is low to moderate. Among the CAA-resistant isolates, a negative correlation between EC(50) values was found for iprovalicarb vs. flumorph and for iprovalicarb vs. dimethomorph. Comparison of the full-length cellulose synthase 3 (CesA3) between wild-type and CAA-resistant isolates revealed only one point mutation at codon position 1109: a valine residue (codon GTG in wild-type isolates) was converted to leucine (codon CTG in resistant mutants). This represents a novel point mutation with respect to mutations in CesA3 conferring resistance to CAA fungicides. Based on this mutation, an efficient allelic-specific PCR (AS-PCR) method was developed for rapid detection of CAA-resistance in P. melonis populations.