In silico screening leads to novel scaffolds with both antifungal and anti-NLRP3 inflammasome activity

Due to structural similarities that exist between established inhibitors of the NLRP3-inflammasome, sulfonylureas Glyburide and MCC-950, and herbicidal-sulfonylureas, that specifically target fungal acetohydroxyacid synthase (AHAS), we sought to determine the potential for compounds to block both inflammation and inhibit fungal growth. In silico screening of ∼250,000 compounds was used to identify a prioritized list of chemical structures capable of inhibiting both targets. Prioritization of the top 1% of scores identified ∼70 compounds with a diverse set of scaffolds for testing in vitro. Selected hits were used to assess anti-inflammatory function in a THP-1 challenge model with LPS+ATP and resulting IC50 values were obtained. MIC and hyphal-growth assays were conducted to determine potential antifungal activity using media depleted of branched chain amino acids isoleucine and valine, to confirm on target AHAS inhibition. Identification of hits that exhibited low micromolar activity for NLRP3 and AHAS inhibition were selected for SAR study. In vitro testing of the analogs along with molecular docking led to increased knowledge for lead optimization of the potential hits. In silico screening has resulted in IC50 (IL-1β release) and MIC50 (fungal growth) values with low μM potency against several Candida species. In vivo validation will further confirm the potential of the scaffolds for further synthetic-modification for the rationale design of novel dual-purpose drugs

Novel Mutation in the Acetohydroxyacid Synthase (AHAS), Gene Confers Imidazolinone Resistance in Chickpea Cicer arietinum L. Plants

Chickpea (Cicer arietinum L.) is an important crop in crop-rotation management in Israel. Imidazolinone herbicides have a wide spectrum of weed control, but chickpea plants are sensitive to acetohydroxyacid synthase (AHAS; also known as acetolactate synthase [ALS]) inhibitors. Using the chemical mutagen ethyl methanesulfonate (EMS), we developed a chickpea line (M2033) that is resistant to imidazolinone herbicides. A point mutation was detected in one of the two genes encoding the AHAS catalytic subunit of M2033. The transition of threonine to isoleucine at position 192 (203 according to Arabidopsis) conferred resistance of M2033 to imidazolinones, but not to other groups of AHAS inhibitors. The role of this substitution in the resistance of line M2033 was proven by genetic transformation of tobacco plants. This resistance showed a single-gene semidominant inheritance pattern. Conclusion: A novel mutation, T192I (T203I according to Arabidopsis), providing resistance to IMI herbicides but not to other groups of AHAS inhibitors, is described in the AHAS1 protein of EMS-mutagenized chickpea line M2033.

Evidence for the Application of Emerging Technologies to Accelerate Crop Improvement – A Collaborative Pipeline to Introgress Herbicide Tolerance Into Chickpea

Accelerating genetic gain in crop improvement is required to ensure improved yield and yield stability under increasingly challenging climatic conditions. This case study demonstrates the effective confluence of innovative breeding technologies within a collaborative breeding framework to develop and rapidly introgress imidazolinone Group 2 herbicide tolerance into an adapted Australian chickpea genetic background. A well-adapted, high-yielding desi cultivar PBA HatTrick was treated with ethyl methanesulfonate to generate mutations in the ACETOHYDROXYACID SYNTHASE 1 (CaAHAS1) gene. After 2 years of field screening with imidazolinone herbicide across >20 ha and controlled environment progeny screening, two selections were identified which exhibited putative herbicide tolerance. Both selections contained the same single amino acid substitution, from alanine to valine at position 205 (A205V) in the AHAS1 protein, and KASP™ markers were developed to discriminate between tolerant and intolerant genotypes. A pipeline combining conventional crossing and F2 production with accelerated single seed descent from F2:4 and marker-assisted selection at F2 rapidly introgressed the herbicide tolerance trait from one of the mutant selections, D15PAHI002, into PBA Seamer, a desi cultivar adapted to Australian cropping areas. Field evaluation of the derivatives of the D15PAHI002 × PBA Seamer cross was analyzed using a factor analytic mixed model statistical approach designed to accommodate low seed numbers resulting from accelerated single seed descent. To further accelerate trait introgression, field evaluation trials were undertaken concurrent with crop safety testing trials. In 2020, 4 years after the initial cross, an advanced line selection CBA2061, bearing acetohydroxyacid synthase (AHAS) inhibitor tolerance and agronomic and disease resistance traits comparable to parent PBA Seamer, was entered into Australian National Variety Trials as a precursor to cultivar registration. The combination of cross-institutional collaboration and the application of novel pre-breeding platforms and statistical technologies facilitated a 3-year saving compared to a traditional breeding approach. This breeding pipeline can be used as a model to accelerate genetic gain in other self-pollinating species, particularly food legumes.

Triazolopyrimidine herbicides are potent inhibitors of Aspergillus fumigatus acetohydroxyacid synthase and potential antifungal drug leads

Aspergillus fumigatus is a fungal pathogen whose effects can be debilitating and potentially fatal in immunocompromised patients. Current drug treatment options for this infectious disease are limited to just a few choices (e.g. voriconazole and amphotericin B) and these themselves have limitations due to potentially adverse side effects. Furthermore, the likelihood of the development of resistance to these current drugs is ever present. Thus, new treatment options are needed for this infection. A new potential antifungal drug target is acetohydroxyacid synthase (AHAS; EC 2.2.1.6), the first enzyme in the branched chain amino acid biosynthesis pathway, and a target for many commercial herbicides. In this study, we have expressed, purified and characterised the catalytic subunit of AHAS from A. fumigatus and determined the inhibition constants for several known herbicides. The most potent of these, penoxsulam and metosulam, have Ki values of 1.8 ± 0.9 nM and 1.4 ± 0.2 nM, respectively. Molecular modelling shows that these compounds are likely to bind into the herbicide binding pocket in a mode similar to Candida albicans AHAS. We have also shown that these two compounds inhibit A. fumigatus growth at a concentration of 25 µg/mL. Thus, AHAS inhibitors are promising leads for the development of new anti-aspergillosis therapeutics.

Pro197Thr Substitution in Ahas Gene Causing Resistance to Pyroxsulam Herbicide in Rigid Ryegrass (Lolium Rigidum Gaud.)

Lolium rigidum Gaud. is a cross-pollinated species characterized by high genetic diversity and it was detected as one of the most herbicide resistance-prone weeds, globally. Acetohydroxyacid synthase (AHAS) resistant populations cause significant problems in cereal production; therefore, monitoring the development of AHAS resistance is widely recommended. Using next-generation sequencing (NGS), a de novo transcriptome sequencing dataset was presented to identify the complete open reading frame (ORF) of AHAS enzyme in L. rigidum and design markers to amplify fragments consisting of all of the eight resistance-conferring amino acid mutation sites. Pro197Thr, Pro197Ala, Pro197Ser, Pro197Gln, and Trp574Leu amino acid substitutions have been observed in samples. Although the Pro197Thr amino acid substitution was already described in SU and IMI resistant populations, this is the first report to reveal that the Pro197Thr in AHAS enzyme confers a high level of resistance (ED50 3.569) to pyroxsulam herbicide (Triazolopyrimidine).

A Cisgenic Approach in the Transformation of Bread Wheat cv. Saratovskaya 29 with Class I Chitinase Gene

Background: Bread wheat is one of the major crops grown worldwide, showing high demand for new varieties with traits such as pathogen resistance. As the public acceptance of transgenic plants remains low, a novel approach – cisgenesis – is being developed to introduce the genes from the same or closely related species. Objective: This study presents a cisgenic approach used for the transformation of wheat with class I chitinase gene derived from T. aestivum cv. Stepnaya 15, co-transformed with acetohydroxyacid synthase gene that provides tolerance to imidazolinone herbicides. Methods: Calli from immature embryos of spring bread wheat Triticum aestivum cv. Saratovskaya 29 were used for co-transformation with two independent Minimal Expression Units (MEUs): class I chitinase and Acetohydroxyacid Synthase (AHAS) gene. For identification of cisgenic plants, genomic DNA was extracted from the leaves of imazethapyr-resistant regenerant plants at the plantlets stage and screened by polymerase chain reaction. The efficiency of transformation was calculated as the relation of regenerated plants with chitinase gene insert to the total number of calli in the experiment. Results: The average transformation efficiency in four series of experiments (total number of calli - 2299) was found to be 1.84% (ranging from 0.3% to 3.4%), while total co-transformation efficiency reached 87.93%. Conclusion: The high efficiency of co-transformation in the experiment promotes it as a very useful technique for the production of wheat lines, free of the selectable marker gene. To our knowledge, this is the first report of cisgenic bread wheat, where both target and selectable genes are derived from wheat.