Legume-type glutamate dehydrogenase: Structure, activity, and inhibition studies.

Glutamate dehydrogenases (GDHs) are key enzymes at the crossroads of N and C metabolism in plants. Legumes, whose N metabolism is particularly intricate, possess a unique type of GDH. This study presents an analysis of a legume-type GDH (isoform 2) from Medicago truncatula (MtGDH2). We measured MtGDH2 activity in both the Glu â†’ 2-oxoglutarate (2OG) and 2OG â†’ Glu reaction directions and obtained kinetic parameters for Glu, 2OG, NAD+, and NADH. Inhibition assays revealed that compounds possessing di- or tricarboxylates act as inhibitors of plant GDHs. Interestingly, 2,6-pyridinedicarboxylate (PYR) weakly inhibits MtGDH2 compared to Arabidopsis thaliana homologs. Furthermore, we explored tetrazole derivatives to discover 3-(1H-tetrazol-5-yl)benzoic acid (TBA) as an MtGDH2 inhibitor. The kinetic experiments are supported by six crystal structures, solved as: (i) unliganded enzyme, (ii) trapping the reaction intermediate 2-amino-2-hydroxyglutarate and NAD+, and also complexed with NAD+ and inhibitors such as (iii) citrate, (iv) PYR, (v) isophthalate, and (vi) TBA. The complex with TBA revealed a new mode of action that, in contrast to other inhibitors, prevents domain closure. This discovery points to TBA as a starting point for the development of novel GDH inhibitors to study the functions of GDH in plants and potentially boost biomass production.

Rational Design, Synthesis, and Computational Investigation of Dihydropyridine [2,3-d] Pyrimidines as Polyphenol Oxidase Inhibitors with Improved Potency.

Polyphenol oxidase (PPO) is an industrially important enzyme associated with browning reactions. In the present study, a set of ten new dihydropyridine [2,3-d] pyrimidines (TD-Hid-1-10) were synthesized and was found to be proven characteristically by 1H NMR, 13C NMR, IR, elemental analysis, and assessed as possible PPO inhibitors. PPO was purified from banana using three-phase partitioning, achieving an 18.65-fold purification and 136.47% activity recovery. Enzyme kinetics revealed that the compounds TD-Hid-6 and TD-Hid-7 are to be the most potent inhibitors, exhibiting mixed-type inhibition profile with IC50 values of 1.14 µM, 5.29 µM respectively against purified PPO enzyme. Electronic structure calculations at the B3LYP/PBE0 level of theories using def-2 SVP, def2-TZVP basis sets with various molecular descriptors characterized the electronic behavior of studied derivatives TD-Hid-1-10. Molecular electrostatic potential (MEP) and reduced density gradient analyses of RDG-NCI provided insights into charge distributions and weak intermolecular interactions. Docking study simulations predicted binding poses within crucial amino acid sequence in the 2y9x enzyme's active site, which is typically similar in sequence to the PPO form is not allowed. Ligands were analysed in terms of binding energies, inhibitor concentrations (mM) and various molecular interactions such as H-bonds, H-carbon, π-carbon, π-sigma, π-sigma, π-π T-shaped, π-π stacked, π-alkyl, Van der Waals and Cu interactions. The lowest binding energy (-7.83 kcal/mol) and the highest inhibitory effect (1.83 mM) were shown by the ligand Td-Hid-6, which forms H-bonds with Met280 and Asn260, exhibits π-sigma interactions with His61 and π-alkyl interactions with Val283. Other ligands also showed different interactions with various amino acids; for example, the Td-Hid-1 ligand formed H-bonds with His244 and showed π-sigma interactions with His244 and Val283.

Discovery of Novel (5-Mercapto-4-phenyl-4H-1,2,4-triazol-3-yl)methyl Phenyl Carbamate as a Potent Phytoene Desaturase Inhibitor through Scaffold Hopping and Linker Modification.

Phytoene desaturase (PDS) is a key rate-limiting enzyme in the carotenoid biosynthesis pathway. Although commercial PDS inhibitors have been developed for decades, it remains necessary to develop novel PDS inhibitors with higher bioactivity. In this work, we used the scaffold hopping and linker modification approaches to design and synthesize a series of compounds (7a-7o, 8a-8l, and 14a-14d). The postemergence application assay demonstrated that 8e and 7e separately showed the best herbicidal activity at 750 g a.i./ha and lower doses (187.5 g, 375g a.i./ha) without no significant toxicity to maize and wheat. The surface plasmon resonance revealed strong binding affinity between 7e and Synechococcus PDS (SynPDS). The HPLC analysis confirmed that 8e at 750 g a.i./ha caused significant phytoene accumulation in Arabidopsis seedlings. This work demonstrates the efficacy of structure-guided optimization through scaffold hopping and linker modification to design potent PDS inhibitors with enhanced bioactivity and crop safety.

Design, Synthesis and Proteomics-Based Analysis of Novel Triazinone Derivatives Containing Amide Structures as Safer Protoporphyrinogen IX Oxidase Inhibitors.

Resistant weeds severely threaten crop yields as they compete with crops for resources required for survival. Trifludimoxazin, a protoporphyrinogen IX oxidase (PPO) inhibitor, can effectively control resistant weeds. However, its crop safety record is unsatisfactory. Consequently, a scaffold-hopping strategy is employed in this study to develop a series of new triazinone derivatives featuring an amide structure. Most compounds depicted excellent herbicidal activity across a broad spectrum at 37.5-150 g ai/ha, among which (R)-I-5 was equivalent to flumioxazin. (R)-I-5 demonstrated significant crop tolerance to rice and wheat, even at 150 g ai/ha. (R)-I-5 exhibited superior pharmacokinetic features compared to flumioxazin and trifludimoxazin. This was depicted by the absorption, distribution, metabolism, excretion, and toxicity predictions. Notably, proteomics-based analysis was applied for the first time to investigate variations among plant proteins before and after herbicide application, shedding light on the conservative and divergent roles of PPO.

A Novel AHAS-Inhibiting Herbicide Candidate for Controlling Leptochloa chinensis: A Devastating Weedy Grass in Rice Fields.

Given the prevalence of the malignant weed Chinese Sprangletop (Leptochloa chinensis (L.) Nees) in rice fields, the development of novel herbicides against this weed has aroused wide interest. Here, we report a novel diphenyl ether-pyrimidine hybrid, DEP-5, serving as a systematic pre/postemergence herbicide candidate for broad-spectrum weed control in rice fields, specifically for L. chinensis. Notably, DEP-5 exhibits over 80% herbicidal activity against the resistant biotypes even at 37.5 g a.i./ha under greenhouse conditions and has complete control of L. chinensis at 150 g a.i./ha in the rice fields. We uncover that DEP-5 acts as a noncompetitive inhibitor of acetohydroxyacid synthase (AHAS) with an inhibition constant (Ki) of 39.4 µM. We propose that DEP-5 binds to AHAS in two hydrophobic-driven binding modes that differ from commercial AHAS inhibitors. Overall, these findings demonstrate that DEP-5 has great potential to be developed into a herbicide for L. chinensis control and inspire fresh concepts for novel AHAS-inhibiting herbicide design.

Design, Synthesis, and Herbicidal Activity of Novel Tetrahydrophthalimide Derivatives Containing Oxadiazole/Thiadiazole Moieties.

Protoporphyrinogen oxidase (PPO, EC 1.3.3.4) has a high status in the development of new inhibitors. To develop novel and highly effective PPO inhibitors, active substructure linking and bioisosterism replacement strategies were used to design and synthesize novel tetrahydrophthalimide derivatives containing oxadiazole/thiadiazole moieties, and their inhibitory effects on Nicotiana tobacco PPO (NtPPO) and herbicidal activity were evaluated. Among them, compounds B11 (Ki = 9.05 nM) and B20 (Ki = 10.23 nM) showed significantly better inhibitory activity against NtPPO than that against flumiclorac-pentyl (Ki = 46.02 nM). Meanwhile, compounds A20 and B20 were 100% effective against three weeds (Abutilon theophrasti, Amaranthus retroflexus, and Portulaca oleracea) at 37.5 g a.i./ha. It was worth observing that compound B11 was more than 90% effective against three weeds (Abutilon theophrasti, Amaranthus retroflexus, and Portulaca oleracea) at 18.75 and 9.375 g a.i./ha. It was also safer to rice, maize, and wheat than flumiclorac-pentyl at 150 g a.i./ha. In addition, the molecular docking results showed that compound B11 could stably bind to NtPPO and it had a stronger hydrogen bond with Arg98 (2.9 Å) than that of flumiclorac-pentyl (3.2 Å). This research suggests that compound B11 could be used as a new PPO inhibitor, and it could help control weeds in agricultural production.

4-Hydroxyphenylpyruvate Dioxygenase Inhibitors: From Molecular Design to Synthesis.

Weed resistance is a critical issue in crop production. Among the known herbicides, 4-hydroxyphenylpyruvate dioxygenase (HPPD) inhibitors are crucial for addressing weed resistance. HPPD inhibitors constitute a pivotal aspect of contemporary crop protection strategies. The advantages of these herbicides are their broad weed spectrum, flexible application, and excellent compatibility with other herbicides. They also exhibit satisfactory crop selectivity and low toxicity and are environmentally friendly. An increasing number of new HPPD inhibitors have been designed by combining computer-aided drug design with conventional design approaches. Herein, the molecular design and structural features of innovative HPPD inhibitors are reviewed to guide the development of new HPPD inhibitors possessing an enhanced biological efficacy.

Oligomerization-mediated autoinhibition and cofactor binding of a plant NLR.

Nucleotide-binding leucine-rich repeat (NLR) proteins play a pivotal role in plant immunity by recognizing pathogen effectors1,2. Maintaining a balanced immune response is crucial, as excessive NLR expression can lead to unintended autoimmunity3,4. Unlike most NLRs, the plant NLR required for cell death 2 (NRC2) belongs to a small NLR group characterized by constitutively high expression without self-activation5. The mechanisms underlying NRC2 autoinhibition and activation are not yet understood. Here we show that Solanum lycopersicum (tomato) NRC2 (SlNRC2) forms dimers and tetramers and higher-order oligomers at elevated concentrations. Cryo-electron microscopy shows an inactive conformation of SlNRC2 in these oligomers. Dimerization and oligomerization not only stabilize the inactive state but also sequester SlNRC2 from assembling into an active form. Mutations at the dimeric or interdimeric interfaces enhance pathogen-induced cell death and immunity in Nicotiana benthamiana. The cryo-electron microscopy structures unexpectedly show inositol hexakisphosphate (IP6) or pentakisphosphate (IP5) bound to the inner surface of the C-terminal leucine-rich repeat domain of SlNRC2, as confirmed by mass spectrometry. Mutations at the inositol phosphate-binding site impair inositol phosphate binding of SlNRC2 and pathogen-induced SlNRC2-mediated cell death in N. benthamiana. Our study indicates a negative regulatory mechanism of NLR activation and suggests inositol phosphates as cofactors of NRCs.

Discovery of Novel Pyridazine Herbicides Targeting Phytoene Desaturase with Scaffold Hopping.

Phytoene desaturase (PDS) is a critical functional enzyme in blocking ζ-carotene biosynthesis and is one of the bleaching herbicide targets. At present, norflurazon (NRF) is the only commercial pyridazine herbicide targeting PDS. Therefore, developing new and diverse pyridazine herbicides targeting PDS is urgently required. In this study, diflufenican (BF) was used as the lead compound, and a scaffold-hopping strategy was employed to design and synthesize some pyridazine derivatives based on the action mode of BF and PDS. The preemergence herbicidal activity tests revealed that compound 6-chloro-N-(2,4-difluorophenyl)-3-(3-(trifluoromethyl)phenoxy)pyridazine-4-carboxamide (B1) with 2,4-diF substitution in the benzeneamino ring showed 100% inhibition rates against the roots and stems of Echinochloa crus-galli and Portulaca oleracea at 100 µg/mL, superior to the inhibition rates of BF. Meanwhile, compound B1 demonstrated excellent postemergence herbicidal activity against broadleaf weeds, which was similar to that of BF (inhibition rate of 100%) but superior to that of NRF. This indicated that 6-Cl in the pyridazine ring is the key group for postemergence herbicidal activity. In addition, compound B1 could induce downregulation of PDS gene expression, 15-cis-phytoene accumulation, and Y(II) deficiency and prevent photosynthesis. Therefore, B1 can be considered as a promising candidate for developing high-efficiency PDS inhibitors.

Release of a ubiquitin brake activates OsCERK1-triggered immunity in rice.

Plant pattern-recognition receptors perceive microorganism-associated molecular patterns to activate immune signalling1,2. Activation of the pattern-recognition receptor kinase CERK1 is essential for immunity, but tight inhibition of receptor kinases in the absence of pathogen is crucial to prevent autoimmunity3,4. Here we find that the U-box ubiquitin E3 ligase OsCIE1 acts as a molecular brake to inhibit OsCERK1 in rice. During homeostasis, OsCIE1 ubiquitinates OsCERK1, reducing its kinase activity. In the presence of the microorganism-associated molecular pattern chitin, active OsCERK1 phosphorylates OsCIE1 and blocks its E3 ligase activity, thus releasing the brake and promoting immunity. Phosphorylation of a serine within the U-box of OsCIE1 prevents its interaction with E2 ubiquitin-conjugating enzymes and serves as a phosphorylation switch. This phosphorylation site is conserved in E3 ligases from plants to animals. Our work identifies a ligand-released brake that enables dynamic immune regulation.

Discovery of Novel N-Phenyltriazinone Derivatives Containing Oxime Ether or Oxime Ester Moieties as Promising Protoporphyrinogen IX Oxidase Inhibitors.

Protoporphyrinogen IX oxidase (PPO, EC 1.3.3.4) is one of the most important targets for the discovery of green herbicides. In order to find novel PPO inhibitors with a higher herbicidal activity, a series of novel N-phenyltriazinone derivatives containing oxime ether and oxime ester groups were designed and synthesized based on the strategy of pharmacophore and scaffold hopping. Bioassay results revealed that some compounds showed herbicidal activities; especially, compound B16 exhibited broad-spectrum and excellent 100% herbicidal effects to Echinochloa crusgalli, Digitaria sanguinalis, Setaria faberii, Abutilon juncea, Amaranthus retroflexus, and Portulaca oleracea at a concentration of 37.5 g a.i./ha, which were comparable to trifludimoxazin. Nicotiana tabacum PPO (NtPPO) enzyme inhibitory assay indicated that B16 showed an excellent enzyme inhibitory activity with a value of 32.14 nM, which was similar to that of trifludimoxazin (31.33 nM). Meanwhile, compound B16 revealed more safety for crops (rice, maize, wheat, peanut, soybean, and cotton) than trifludimoxazin at a dose of 150 g a.i./ha. Moreover, molecular docking and molecular dynamics simulation further showed that B16 has a very strong and stable binding to NtPPO. It indicated that B16 can be used as a potential PPO inhibitor and herbicide candidate for application in the field.

Design, Synthesis, and Biological Evaluation of Pyridazinone-Containing Derivatives As Novel Protoporphyrinogen IX Oxidase Inhibitor.

Protoporphyrinogen IX oxidase (PPO, E.C. 1.3.3.4) plays a pivotal role in chlorophyll biosynthesis in plants, making it a prime target for herbicide development. In this study, we conducted an investigation aimed at discovering PPO-inhibiting herbicides. Through this endeavor, we successfully identified a series of novel compounds based on the pyridazinone scaffold. Following structural optimization and biological assessment, compound 10ae, known as ethyl 3-((6-fluoro-5-(6-oxo-4-(trifluoromethyl)pyridazin-1(6H)-yl)benzo[d]thiazol-2-yl)thio)propanoate, emerged as a standout performer. It exhibited robust activity against Nicotiana tabacum PPO (NtPPO) with an inhibition constant (Ki) value of 0.0338 µM. Concurrently, we employed molecular simulations to obtain further insight into the binding mechanism with NtPPO. Additionally, another compound, namely, ethyl 2-((6-fluoro-5-(5-methyl-6-oxo-4-(trifluoromethyl)pyridazin-1(6H)-yl)benzo[d]thiazol-2-yl)thio)propanoate (10bh), demonstrated broad-spectrum and highly effective herbicidal properties against all six tested weeds (Leaf mustard, Chickweed, Chenopodium serotinum, Alopecurus aequalis, Poa annua, and Polypogon fugax) at the dosage of 150 g a.i./ha through postemergence application in a greenhouse. This work identified a novel lead compound (10bh) that showed good activity in vitro and excellent herbicidal activity in vivo and had promising prospects as a new PPO-inhibiting herbicide lead.

Differential Resistance to Acetyl-CoA Carboxylase Inhibitors in Rice: Insights from Two Distinct Target-Site Mutations.

Weeds present a significant challenge to agricultural productivity, and acetyl-CoA carboxylase (ACCase)-inhibiting herbicides have proven to be effective in managing weed populations in rice fields. To develop ACCase-inhibiting herbicide-resistant rice, we generated mutants of rice ACCase (OsACC) featuring Ile-1792-Leu or Gly-2107-Ser substitutions through ethyl methyl sulfonate (EMS) mutagenesis. The Ile-1792-Leu mutant displayed cross-resistance to aryloxyphenoxypropionate (APP) and phenylpyrazoline (DEN) herbicides, whereas the Gly-2107-Ser mutants primarily exhibited cross-resistance to APP herbicides with diminished resistance to the DEN herbicide. In vitro assays of the OsACC activity revealed an increase in resistance to haloxyfop and quizalofop, ranging from 4.84- to 29-fold in the mutants compared to that in wild-type. Structural modeling revealed that both mutations likely reduce the binding affinity between OsACC and ACCase inhibitors, thereby imparting resistance. This study offers insights into two target-site mutations, contributing to the breeding of herbicide-resistant rice and presenting alternative weed management strategies in rice cultivation.

Design, Synthesis, and Herbicidal Evaluation of Pyrrolidinone-Containing 2-Phenylpyridine Derivatives as Novel Protoporphyrinogen Oxidase Inhibitors.

In this work, a series of pyrrolidinone-containing 2-phenylpyridine derivatives were synthesized and evaluated as novel protoporphyrinogen IX oxidase (PPO, EC 1.3.3.4) inhibitors for herbicide development. At 150 g ai/ha, compounds 4d, 4f, and 4l can inhibit the grassy weeds of Echinochloa crus-galli (EC), Digitaria sanguinalis (DS), and Lolium perenne (LP) with a range of 60 to 90%. Remarkably, at 9.375 g ai/ha, these compounds showed 100% inhibition effects against broadleaf weeds of Amaranthus retroflexus (AR) and Abutilon theophrasti (AT), which were comparable to the performance of the commercial herbicides flumioxazin (FLU) and saflufenacil (SAF) and better than that of acifluorfen (ACI). Molecular docking analyses revealed significant hydrogen bonding and π-π stacking interactions between compounds 4d and 4l with Arg98, Asn67, and Phe392, respectively. Additionally, representative compounds were chosen for in vivo assessment of PPO inhibitory activity, with compounds 4d, 4f, and 4l demonstrating excellent inhibitory effects. Notably, compounds 4d and 4l induced the accumulation of reactive oxygen species (ROS) and a reduction in the chlorophyll (Chl) content. Consequently, compounds 4d, 4f, and 4l are promising lead candidates for the development of novel PPO herbicides.

Confirmation and characterization of the first case of acetolactate synthase (ALS)-inhibitor resistance in Japanese brome (Bromus japonicus) in the US.

BACKGROUND: Japanese brome (Bromus japonicus Thumb.) is one of the problematic annual weeds in winter wheat (Triticum aestivum L.) and is generally controlled by acetolactate synthase (ALS) inhibitors. Repeated use of the ALS inhibitor propoxycarbazone-Na resulted in the evolution of resistance to this herbicide in three B. japonicus populations, i.e., R1, R2, and R3 in Kansas (KS). However, the level of resistance and mechanism conferring resistance in these populations is unknown. The objectives of this research were to (i) evaluate the level of resistance to propoxycarbazone-Na in R1, R2, and R3 in comparison with a known susceptible population (S1), (ii) investigate the mechanism of resistance involved in conferring ALS-inhibitor resistance, and (iii) investigate the cross-resistance to other ALS inhibitors. RESULTS: Dose-response (0 to 16x; x = 44 g ai ha-1 of propoxycarbazone-Na) assay indicated 167, 125, and 667-fold resistance in R1, R2 and R3 populations, respectively, compared to S1 population. ALS gene sequencing confirmed the mutations resulting in amino acid substitutions, i.e., Pro-197-Thr (R3, R1)/Ser (R2, R1) bestowing resistance to these ALS inhibitors. Such amino acid substitutions also showed differential cross-resistance to sulfosulfuron, mesosulfuron-methyl, pyroxsulam, and imazamox among resistant populations. Pretreatment with malathion (a cytochrome P450 enzyme-inhibitor) followed by imazamox treatment suggested cross-resistance to this herbicide possibly via metabolism only in R3 population. CONCLUSION: Overall, these results confirm the first case of target-site based resistance to ALS inhibitors in B. japonicus in the US, highlighting the need for exploring herbicides with alternative modes of action to enhance weed control in winter wheat. © 2024 Society of Chemical Industry.

Viruses inhibit TIR gcADPR signalling to overcome bacterial defence.

The Toll/interleukin-1 receptor (TIR) domain is a key component of immune receptors that identify pathogen invasion in bacteria, plants and animals1-3. In the bacterial antiphage system Thoeris, as well as in plants, recognition of infection stimulates TIR domains to produce an immune signalling molecule whose molecular structure remains elusive. This molecule binds and activates the Thoeris immune effector, which then executes the immune function1. We identified a large family of phage-encoded proteins, denoted here as Thoeris anti-defence 1 (Tad1), that inhibit Thoeris immunity. We found that Tad1 proteins are 'sponges' that bind and sequester the immune signalling molecule produced by TIR-domain proteins, thus decoupling phage sensing from immune effector activation and rendering Thoeris inactive. Tad1 can also efficiently sequester molecules derived from a plant TIR-domain protein, and a high-resolution crystal structure of Tad1 bound to a plant-derived molecule showed a unique chemical structure of 1 ''-2' glycocyclic ADPR (gcADPR). Our data furthermore suggest that Thoeris TIR proteins produce a closely related molecule, 1''-3' gcADPR, which activates ThsA an order of magnitude more efficiently than the plant-derived 1''-2' gcADPR. Our results define the chemical structure of a central immune signalling molecule and show a new mode of action by which pathogens can suppress host immunity.

Silencing an E3 Ubiquitin Ligase Gene OsJMJ715 Enhances the Resistance of Rice to a Piercing-Sucking Herbivore by Activating ABA and JA Signaling Pathways.

The RING-type E3 ubiquitin ligases play an important role in plant growth, development, and defense responses to abiotic stresses and pathogens. However, their roles in the resistance of plants to herbivorous insects remain largely unknown. In this study, we isolated the rice gene OsJMJ715, which encodes a RING-domain containing protein, and investigated its role in rice resistance to brown planthopper (BPH, Nilaparvata lugens). OsJMJ715 is a nucleus-localized E3 ligase whose mRNA levels were upregulated by the infestation of gravid BPH females, mechanical wounding, and treatment with JA or ABA. Silencing OsJMJ715 enhanced BPH-elicited levels of ABA, JA, and JA-Ile as well as the amount of callose deposition in plants, which in turn increased the resistance of rice to BPH by reducing the feeding of BPH and the hatching rate of BPH eggs. These findings suggest that OsJMJ715 negative regulates the BPH-induced biosynthesis of ABA, JA, and JA-Ile and that BPH benefits by enhancing the expression of OsJMJ715.

Resistance to post-emergent herbicides is becoming common for grass weeds on New Zealand wheat and barley farms.

To estimate the prevalence of herbicide-resistant weeds, 87 wheat and barley farms were randomly surveyed in the Canterbury region of New Zealand. Over 600 weed seed samples from up to 10 mother plants per taxon depending on abundance, were collected immediately prior to harvest (two fields per farm). Some samples provided by agronomists were tested on an ad-hoc basis. Over 40,000 seedlings were grown to the 2-4 leaf stage in glasshouse conditions and sprayed with high priority herbicides for grasses from the three modes-of-action acetyl-CoA carboxylase (ACCase)-inhibitors haloxyfop, fenoxaprop, clodinafop, pinoxaden, clethodim, acetolactate synthase (ALS)-inhibitors iodosulfuron, pyroxsulam, nicosulfuron, and the 5-enolpyruvyl shikimate 3-phosphate synthase (EPSPS)-inhibitor glyphosate. The highest manufacturer recommended label rates were applied for the products registered for use in New Zealand, often higher than the discriminatory rates used in studies elsewhere. Published studies of resistance were rare in New Zealand but we found weeds survived herbicide applications on 42 of the 87 (48%) randomly surveyed farms, while susceptible reference populations died. Resistance was found for ALS-inhibitors on 35 farms (40%) and to ACCase-inhibitors on 20 (23%) farms. The number of farms with resistant weeds (denominator is 87 farms) are reported for ACCase-inhibitors, ALS-inhibitors, and glyphosate respectively as: Avena fatua (9%, 1%, 0% of farms), Bromus catharticus (0%, 2%, 0%), Lolium spp. (17%, 28%, 0%), Phalaris minor (1%, 6%, 0%), and Vulpia bromoides (0%, not tested, 0%). Not all farms had the weeds present, five had no obvious weeds prior to harvest. This survey revealed New Zealand's first documented cases of resistance in P. minor (fenoxaprop, clodinafop, iodosulfuron) and B. catharticus (pyroxsulam). Twelve of the 87 randomly sampled farms (14%) had ALS-inhibitor chlorsulfuron-resistant sow thistles, mostly Sonchus asper but also S. oleraceus. Resistance was confirmed in industry-supplied samples of the grasses Digitaria sanguinalis (nicosulfuron, two maize farms), P. minor (iodosulfuron, one farm), and Lolium spp. (cases included glyphosate, haloxyfop, pinoxaden, iodosulfuron, and pyroxsulam, 9 farms). Industry also supplied Stellaria media samples that were resistant to chlorsulfuron and flumetsulam (ALS-inhibitors) sourced from clover and ryegrass fields from the North and South Island.

Investigating the Viral Suppressor HC-Pro Inhibiting Small RNA Methylation through Functional Comparison of HEN1 in Angiosperm and Bryophyte.

In plants, HEN1-facilitated methylation at 3' end ribose is a critical step of small-RNA (sRNA) biogenesis. A mutant of well-studied Arabidopsis HEN1 (AtHEN1), hen1-1, showed a defective developmental phenotype, indicating the importance of sRNA methylation. Moreover, Marchantia polymorpha has been identified to have a HEN1 ortholog gene (MpHEN1); however, its function remained unfathomed. Our in vivo and in vitro data have shown MpHEN1 activity being comparable with AtHEN1, and their substrate specificity towards duplex microRNA (miRNA) remained consistent. Furthermore, the phylogenetic tree and multiple alignment highlighted the conserved molecular evolution of the HEN1 family in plants. The P1/HC-Pro of the turnip mosaic virus (TuMV) is a known RNA silencing suppressor and inhibits HEN1 methylation of sRNAs. Here, we report that the HC-Pro physically binds with AtHEN1 through FRNK motif, inhibiting HEN1's methylation activity. Moreover, the in vitro EMSA data indicates GST-HC-Pro of TuMV lacks sRNA duplex-binding ability. Surprisingly, the HC-Pro also inhibits MpHEN1 activity in a dosage-dependent manner, suggesting the possibility of interaction between HC-Pro and MpHEN1 as well. Further investigations on understanding interaction mechanisms of HEN1 and various HC-Pros can advance the knowledge of viral suppressors.

Improved bacterial leaf blight disease resistance in the major elite Vietnamese rice cultivar TBR225 via editing of the OsSWEET14 promoter.

TBR225 is one of the most popular commercial rice varieties in Northern Vietnam. However, this variety is highly susceptible to bacterial leaf blight (BLB), a disease caused by Xanthomonas oryzae pv. oryzae (Xoo) which can lead to important yield losses. OsSWEET14 belongs to the SWEET gene family that encodes sugar transporters. Together with other Clade III members, it behaves as a susceptibility (S) gene whose induction by Asian Xoo Transcription-Activator-Like Effectors (TALEs) is absolutely necessary for disease. In this study, we sought to introduce BLB resistance in the TBR225 elite variety. First, two Vietnamese Xoo strains were shown to up-regulate OsSWEET14 upon TBR225 infection. To investigate if this induction is connected with disease susceptibility, nine TBR225 mutant lines with mutations in the AvrXa7, PthXo3 or TalF TALEs DNA target sequences of the OsSWEET14 promoter were obtained using the CRISPR/Cas9 editing system. Genotyping analysis of T0 and T1 individuals showed that mutations were stably inherited. None of the examined agronomic traits of three transgene-free T2 edited lines were significantly different from those of wild-type TBR225. Importantly, one of these T2 lines, harboring the largest homozygous 6-bp deletion, displayed decreased OsSWEET14 expression as well as a significantly reduced susceptibility to a Vietnamese Xoo strains and complete resistance to another one. Our findings indicate that CRISPR/Cas9 editing conferred an improved BLB resistance to a Vietnamese commercial elite rice variety.