Targeting Vancomycin-Resistant Enterococci (VRE) Infections and Van Operon-Mediated Drug Resistance Using Dimeric Cholic Acid-Peptide Conjugates.

Emergence of vancomycin resistance in Gram-positive bacteria and the prevalence of vancomycin-resistant Enterococci (VRE) infections are highly alarming as very limited antibiotic options are available against VRE infections. Here, we present the synthesis of cholic acid-derived dimeric amphiphiles where two cholic acid moieties are tethered through carboxyl terminals using different alkylene spacers. Our investigations revealed that dimer 5 possessing a propylene spacer and glycine-valine peptides tethered on hydroxyl groups is the most effective antimicrobial against VRE. Dimer 5 can permeabilize bacterial membranes, generate reactive oxygen species, and clear preformed biofilms. We further demonstrate that dimer 5 downregulates vancomycin-mediated transcriptional activation of the vanHAX gene cluster and does not allow VSE to develop vancomycin resistance until 100 generations. Therefore, this study, for the first time, presents a bacterial membrane-targeting amphiphile that can mitigate VRE infections and inhibit the emergence of vancomycin resistance.

Foliar Application or Seed Priming of Cholic Acid-Glycine Conjugates can Mitigate/Prevent the Rice Bacterial Leaf Blight Disease via Activating Plant Defense Genes.

Xanthomonas Oryzae pv. oryzae (Xoo) causes bacterial blight and Rhizoctonia solani (R. solani) causes sheath blight in rice accounting for >75% of crop losses. Therefore, there is an urgent need to develop strategies for the mitigation of these pathogen infections. In this study, we report the antimicrobial efficacy of Cholic Acid-Glycine Conjugates (CAGCs) against Xoo and R. solani. We show that CAGC C6 is a broad-spectrum antimicrobial and is also able to degrade biofilms. The application of C6 did not hamper plant growth and showed minimal effect on the plant cell membranes. Exogenous application of C6 on pre-infection or post-infection of Xoo on rice susceptible genotype Taichung native (TN1) can mitigate the bacterial load and improve resistance through upregulation of plant defense genes. We further demonstrate that C6 can induce plant defense responses when seeds were primed with C6 CAGC. Therefore, this study demonstrates the potential of CAGCs as effective antimicrobials for crop protection that can be further explored for field applications.

Proteasomal Degradation of JAZ9 by Salt- and Drought-Induced Ring Finger 1 During Pathogen Infection.

E3 ubiquitin ligase salt- and drought-induced ring finger 1 (SDIR1) plays a novel role in modulating plant immunity against pathogens. The molecular interactors of SDIR1 during pathogen infection are not known. SDIR1-interacting jasmonate zinc-finger inflorescence meristem domain (JAZ) proteins were identified through a yeast two-hybrid (Y2H) screen. Full-length JAZ9 interacts with SDIR1 only in the presence of coronatine (a bacteria-secreted toxin) or jasmonic acid (JA) in a Y2H assay. The bimolecular fluorescence complementation and pull-down assays confirm the in planta interaction of these proteins. JAZ9 proteins, negative regulators of JA-mediated plant defense, were degraded during the pathogen infection by SDIR1 through a proteasomal pathway causing disease susceptibility against hemibiotrophic pathogens.[Formula: see text] The author(s) have dedicated the work to the public domain under the Creative Commons CC0 "No Rights Reserved" license by waiving all of his or her rights to the work worldwide under copyright law, including all related and neighboring rights, to the extent allowed by law, 2021.

Comparative Transcriptome Analysis of Rice Resistant and Susceptible Genotypes to Xanthomonas oryzae pv. oryzae Identifies Novel Genes to Control Bacterial Leaf Blight.

The bacterial leaf blight in rice caused by Xanthomonas oryzae pv. oryzae (Xoo) affects crop losses worldwide. In spite of developing resistant varieties by introgressing different Xa genes, the occurrence of diseases is evident. Here we report identification of several genes that are associated with improved plant immunity against Xoo in a resistant genotype BPT-5204 in comparison with susceptible genotype TN-1. The RNA sequencing information was developed to identify the genes that could provide durable resistance in rice. Xoo-resistant rice genotype BPT-5204 with Xa 5, 13 and 21 genes is compared with sensitive Taichung Native 1 (TN-1) to identify the genetic pathways and gene networks involved in resistance mechanisms. The higher levels of salicylic acid resulted in upregulation of many pathogenesis-related (PR) and redox protein encoding transcripts which resulted in higher hypersensitive response in BPT-5204. Many Serine/threonine protein kinase, leucine-rich repeat (LRR) transmembrane protein kinase, protein kinase family genes, Wall-associated kinase (WAK) were upregulated that resulted in activation of bZIP, WRKY, MYB, DOF and HSFs transcription factors that are associated with improved plant immunity. The study provided roles of many genes and their associated plant immunity pathways that can be used for developing resistant rice cultivars.

Disruption in the DNA Mismatch Repair Gene MSH2 by CRISPR-Cas9 in Indica Rice Can Create Genetic Variability.

Genetic variation is crucial for crop improvement. We adopted a gene editing approach to create variations in the rice genome by targeting the mutator locus homolog 2 (MSH2), a DNA mismatch repair gene. The hypothesis is that disruption of the MSH2 gene leads to a reduced DNA mismatch repair that creates INDELs, resulting in altered phenotypes. The Indica rice (IR-64) genotype was transformed with a guide RNA targeted to the MSH2 gene using an Agrobacterium-mediated in planta method. Many plants showed integration of Cas9 and gRNA constructs in rice plants. One of the msh2 mutants showed a superior phenotype due to editing and possible INDELs in the whole genome. The stable integration of the transgene and its flanking sequence analysis confirms no disruption of any gene, and the observed phenotype is due to the mutations in the MSH2 gene. Few transgenic plants showed disruption of genes due to T-DNA integration that led to altered phenotypes. The plants with altered phenotypes having more tiller number, early flowering, and robust growth with a high biomass were identified. These genetically reprogrammed rice plants could be a potential resource to create more segregating population or act as donor lines to stabilize the important agronomic traits that may help in a speed breeding process.

Stress-Induced Detoxification Enzymes in Rice Have Broad Substrate Affinity.

Reactive carbonyl compounds (RCCs) such as hydroxynonenol, malondialdehyde, acrolein, crotonaldehyde, methylglyoxal, and glyoxal accumulate at higher levels under stress in plants and damage the cell metabolic activities. Plants have evolved several detoxifying enzymes such as aldo-keto reductases (AKRs), aldehyde/alcohol dehydrogenases (ALDH/ADH), and glyoxalases. We report the phylogenetic relationship of these proteins and in silico analysis of rice-detoxifying protein structures and their substrate affinity with cofactors using docking and molecular simulation studies. Molecular simulations with nicotinamide adenine dinucleotide phosphate or glutathione cofactor docking with commonly known reactive substrates suggests that the AKRs, ALDH, and ADH proteins attain maximum conformational changes, whereas glyoxalase has fewer conformational changes with cofactor binding. Several AKRs showed a significant binding affinity with many RCCs. The rice microarray studies showed enhanced expression of many AKRs in resistant genotypes, which also showed higher affinity to RCCs, signifying their importance in managing carbonyl stress. The higher expression of AKRs is regulated by stress-responsive transcription factors (TFs) as we identified stress-specific cis-elements in their promoters. The study reports the stress-responsive nature of AKRs, their regulatory TFs, and their best RCC targets, which may be used for crop improvement programs.

A Novel Role of Salt- and Drought-Induced RING 1 Protein in Modulating Plant Defense Against Hemibiotrophic and Necrotrophic Pathogens.

Many plant-encoded E3 ligases are known to be involved in plant defense. Here, we report a novel role of E3 ligase SALT- AND DROUGHT-INDUCED RING FINGER1 (SDIR1) in plant immunity. Even though SDIR1 is reasonably well-characterized, its role in biotic stress response is not known. The silencing of SDIR1 in Nicotiana benthamiana reduced the multiplication of the virulent bacterial pathogen Pseudomonas syringae pv. tabaci. The Arabidopsis sdir1 mutant is resistant to virulent pathogens, whereas SDIR1 overexpression lines are susceptible to both host and nonhost hemibiotrophic bacterial pathogens. However, sdir1 mutant and SDIR1 overexpression lines showed hypersusceptibility and resistance, respectively, against the necrotrophic pathogen Erwinia carotovora. The mutant of SDIR1 target protein, i.e., SDIR-interacting protein 1 (SDIR1P1), also showed resistance to host and nonhost pathogens. In SDIR1 overexpression plants, transcripts of NAC transcription factors were less accumulated and the levels of jasmonic acid (JA) and abscisic acid were increased. In the sdir1 mutant, JA signaling genes JAZ7 and JAZ8 were downregulated. These data suggest that SDIR1 is a susceptibility factor and its activation or overexpression enhances disease caused by P. syringae pv. tomato DC3000 in Arabidopsis. Our results show a novel role of SDIR1 in modulating plant defense gene expression and plant immunity.[Formula: see text] Copyright © 2021 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

Ribosomal protein QM/RPL10 positively regulates defence and protein translation mechanisms during nonhost disease resistance.

Ribosomes play an integral part in plant growth, development, and defence responses. We report here the role of ribosomal protein large (RPL) subunit QM/RPL10 in nonhost disease resistance. The RPL10-silenced Nicotiana benthamiana plants showed compromised disease resistance against nonhost pathogen Pseudomonas syringae pv. tomato T1. The RNA-sequencing analysis revealed that many genes involved in defence and protein translation mechanisms were differentially affected due to silencing of NbRPL10. Arabidopsis AtRPL10 RNAi and rpl10 mutant lines showed compromised nonhost disease resistance to P. syringae pv. tomato T1 and P. syringae pv. tabaci. Overexpression of AtRPL10A in Arabidopsis resulted in reduced susceptibility against host pathogen P. syringae pv. tomato DC3000. RPL10 interacts with the RNA recognition motif protein and ribosomal proteins RPL30, RPL23, and RPS30 in the yeast two-hybrid assay. Silencing or mutants of genes encoding these RPL10-interacting proteins in N. benthamiana or Arabidopsis, respectively, also showed compromised disease resistance to nonhost pathogens. These results suggest that QM/RPL10 positively regulates the defence and translation-associated genes during nonhost pathogen infection.

pssRNAit: A Web Server for Designing Effective and Specific Plant siRNAs with Genome-Wide Off-Target Assessment.

We report an advanced web server, the plant-specific small noncoding RNA interference tool pssRNAit, which can be used to design a pool of small interfering RNAs (siRNAs) for highly effective, specific, and nontoxic gene silencing in plants. In developing this tool, we integrated the transcript dataset of plants, several rules governing gene silencing, and a series of computational models of the biological mechanism of the RNA interference (RNAi) pathway. The designed pool of siRNAs can be used to construct a long double-strand RNA and expressed through virus-induced gene silencing (VIGS) or synthetic transacting siRNA vectors for gene silencing. We demonstrated the performance of pssRNAit by designing and expressing the VIGS constructs to silence Phytoene desaturase (PDS) or a ribosomal protein-encoding gene, RPL10 (QM), in Nicotiana benthamiana We analyzed the expression levels of predicted intended-target and off-target genes using reverse transcription quantitative PCR. We further conducted an RNA-sequencing-based transcriptome analysis to assess genome-wide off-target gene silencing triggered by the fragments that were designed by pssRNAit, targeting different homologous regions of the PDS gene. Our analyses confirmed the high accuracy of siRNA constructs designed using pssRNAit The pssRNAit server, freely available at https://plantgrn.noble.org/pssRNAit/, supports the design of highly effective and specific RNAi, VIGS, or synthetic transacting siRNA constructs for high-throughput functional genomics and trait improvement in >160 plant species.

Carbonyl Cytotoxicity Affects Plant Cellular Processes and Detoxifying Enzymes Scavenge These Compounds to Improve Stress Tolerance.

Oxidative stress is ubiquitous in environmental stresses and prevails over the cellular metabolic and phenotypic responses in plants. Reactive oxygen species (ROS) generated under stress affect macromolecules to form another group of toxic compounds called reactive carbonyl compounds (RCCs). These molecules have a longer half-life than ROS and cause carbonyl stress that affects cellular metabolism, cellular homeostasis, and crop productivity. The later effect of oxidative stress in terms of the generation of RCCs and glycation products and their effects on plant processes have not been explored well in plant biology. Therefore, how these molecules are produced and a few important effects of RCCs on plants have been discussed in this review article. Further, the plant adaptive detoxification mechanisms of RCCs have been discussed. The enzymes that were identified in plants to detoxify these cytotoxic compounds have broad substrate specificity and the potential for use in breeding programs. The review should provide a comprehensive understanding of the cytotoxic compounds beyond ROS and subsequently their mitigation strategies for crop improvement programs.

Induction of Acquired Tolerance Through Gradual Progression of Drought Is the Key for Maintenance of Spikelet Fertility and Yield in Rice Under Semi-irrigated Aerobic Conditions.

Plants have evolved several adaptive mechanisms to cope with water-limited conditions. While most of them are through constitutive traits, certain "acquired tolerance" traits also provide significant improvement in drought adaptation. Most abiotic stresses, especially drought, show a gradual progression of stress and hence provide an opportunity to upregulate specific protective mechanisms collectively referred to as "acquired tolerance" traits. Here, we demonstrate a significant genetic variability in acquired tolerance traits among rice germplasm accessions after standardizing a novel gradual stress progress protocol. Two contrasting genotypes, BPT 5204 (drought susceptible) and AC 39000 (tolerant), were used to standardize methodology for capturing acquired tolerance traits at seedling phase. Seedlings exposed to gradual progression of stress showed higher recovery with low free radical accumulation in both the genotypes compared to rapid stress. Further, the gradual stress progression protocol was used to examine the role of acquired tolerance at flowering phase using a set of 17 diverse rice genotypes. Significant diversity in free radical production and scavenging was observed among these genotypes. Association of these parameters with yield attributes showed that genotypes that managed free radical levels in cells were able to maintain high spikelet fertility and hence yield under stress. This study, besides emphasizing the importance of acquired tolerance, explains a high throughput phenotyping approach that significantly overcomes methodological constraints in assessing genetic variability in this important drought adaptive mechanism.

The ATPase Activity of Escherichia coli Expressed AAA+-ATPase Protein.

ATPases are the enzymes that breakdown ATP to ADP and release inorganic phosphate (Pi). Here we provide a detailed protocol to determine the ATPase activity of a recombinant AAA+-ATPase protein (GENERAL CONTROL NON-REPRESSIBLE-4 [GCN4]) by spectrophotometric absorption at 360 nm to measure the accumulated inorganic phosphate. In general, the substrate 2-amino-6-mercapto-7-methylpurine riboside (methylthioguanosine, a guanosine analog: MESG) is enzymatically converted in the presence of Pi by purine nucleoside phosphorylase (PNP) to ribose 1-phosphate and 2-amino-6-mercapto-7-methylpurine. The spectrophotometric shift in maximum absorbance at 330 nm for the MESG substrate and subsequent conversion product at 360 nm due to enzymatic conversion was measured. The GCN4-His-tagged recombinant protein was expressed in Escherichia coli BL21 cells and purified using Ni-NTA column. This purified protein was then used for the quantitation of Pi in solution or the continuous determination of Pi released due to the ATPase activity of GCN4, an AAA+-ATPase protein conserved in many eukaryotes, which in plants regulates stomatal aperture during biotic and abiotic stress in plants.

Two Chloroplast-Localized Proteins: AtNHR2A and AtNHR2B, Contribute to Callose Deposition During Nonhost Disease Resistance in Arabidopsis.

Plants are naturally resistant to most pathogens through a broad and durable defense response called nonhost disease resistance. Nonhost disease resistance is a complex process that includes preformed physical and chemical barriers and induced responses. In spite of its importance, many components of nonhost disease resistance remain to be identified and characterized. Using virus-induced gene silencing in Nicotiana benthamiana, we discovered a novel gene that we named NbNHR2 (N. benthamiana nonhost resistance 2). NbNHR2-silenced plants were susceptible to the nonadapted pathogen Pseudomonas syringae pv. tomato T1, which does not cause disease in wild-type or nonsilenced N. benthamiana plants. We found two orthologous genes in Arabidopsis thaliana: AtNHR2A and AtNHR2B. Similar to the results obtained in N. benthamiana, Atnhr2a and Atnhr2b mutants were susceptible to the nonadapted bacterial pathogen of A. thaliana, P. syringae pv. tabaci. We further found that these mutants were also defective in callose deposition. AtNHR2A and AtNHR2B fluorescent protein fusions transiently expressed in N. benthamiana localized predominantly to chloroplasts and a few unidentified dynamic puncta. RFP-AtNHR2A and AtNHR2B-GFP displayed overlapping signals in chloroplasts, indicating that the two proteins could interact, an idea supported by coimmunoprecipitation studies. We propose that AtNHR2A and AtNHR2B are new components of a chloroplast-signaling pathway that activates callose deposition to the cell wall in response to bacterial pathogens.

GENERAL CONTROL NONREPRESSIBLE4 Degrades 14-3-3 and the RIN4 Complex to Regulate Stomatal Aperture with Implications on Nonhost Disease Resistance and Drought Tolerance.

Plants have complex and adaptive innate immune responses against pathogen infections. Stomata are key entry points for many plant pathogens. Both pathogens and plants regulate stomatal aperture for pathogen entry and defense, respectively. Not all plant proteins involved in stomatal aperture regulation have been identified. Here, we report GENERAL CONTROL NONREPRESSIBLE4 (GCN4), an AAA+-ATPase family protein, as one of the key proteins regulating stomatal aperture during biotic and abiotic stress. Silencing of GCN4 in Nicotiana benthamiana and Arabidopsis thaliana compromises host and nonhost disease resistance due to open stomata during pathogen infection. AtGCN4 overexpression plants have reduced H+-ATPase activity, stomata that are less responsive to pathogen virulence factors such as coronatine (phytotoxin produced by the bacterium Pseudomonas syringae) or fusicoccin (a fungal toxin produced by the fungus Fusicoccum amygdali), reduced pathogen entry, and enhanced drought tolerance. This study also demonstrates that AtGCN4 interacts with RIN4 and 14-3-3 proteins and suggests that GCN4 degrades RIN4 and 14-3-3 proteins via a proteasome-mediated pathway and thereby reduces the activity of the plasma membrane H+-ATPase complex, thus reducing proton pump activity to close stomata.

Transcriptome Analysis of Sunflower Genotypes with Contrasting Oxidative Stress Tolerance Reveals Individual- and Combined- Biotic and Abiotic Stress Tolerance Mechanisms.

In nature plants are often simultaneously challenged by different biotic and abiotic stresses. Although the mechanisms underlying plant responses against single stress have been studied considerably, plant tolerance mechanisms under combined stress is not understood. Also, the mechanism used to combat independently and sequentially occurring many number of biotic and abiotic stresses has also not systematically studied. From this context, in this study, we attempted to explore the shared response of sunflower plants to many independent stresses by using meta-analysis of publically available transcriptome data and transcript profiling by quantitative PCR. Further, we have also analyzed the possible role of the genes so identified in contributing to combined stress tolerance. Meta-analysis of transcriptomic data from many abiotic and biotic stresses indicated the common representation of oxidative stress responsive genes. Further, menadione-mediated oxidative stress in sunflower seedlings showed similar pattern of changes in the oxidative stress related genes. Based on this a large scale screening of 55 sunflower genotypes was performed under menadione stress and those contrasting in oxidative stress tolerance were identified. Further to confirm the role of genes identified in individual and combined stress tolerance the contrasting genotypes were individually and simultaneously challenged with few abiotic and biotic stresses. The tolerant hybrid showed reduced levels of stress damage both under combined stress and few independent stresses. Transcript profiling of the genes identified from meta-analysis in the tolerant hybrid also indicated that the selected genes were up-regulated under individual and combined stresses. Our results indicate that menadione-based screening can identify genotypes not only tolerant to multiple number of individual biotic and abiotic stresses, but also the combined stresses.

Simultaneous expression of regulatory genes associated with specific drought-adaptive traits improves drought adaptation in peanut.

Adaptation of crops to drought-prone rain-fed conditions can be achieved by improving plant traits such as efficient water mining (by superior root characters) and cellular-level tolerance mechanisms. Pyramiding these drought-adaptive traits by simultaneous expression of genes regulating drought-adaptive mechanisms has phenomenal relevance in improving stress tolerance. In this study, we provide evidence that peanut transgenic plants expressing Alfalfa zinc finger 1 (Alfin1), a root growth-associated transcription factor gene, Pennisetum glaucum heat-shock factor (PgHSF4) and Pea DNA helicase (PDH45) involved in protein turnover and protection showed improved tolerance, higher growth and productivity under drought stress conditions. Stable integration of all the transgenes was noticed in transgenic lines. The transgenic lines showed higher root growth, cooler crop canopy air temperature difference (less CCATD) and higher relative water content (RWC) under drought stress. Low proline levels in transgenic lines substantiate the maintenance of higher water status. The survival and recovery of transgenic lines was significantly higher under gradual moisture stress conditions with higher biomass. Transgenic lines also showed significant tolerance to ethrel-induced senescence and methyl viologen-induced oxidative stress. Several stress-responsive genes such as heat-shock proteins (HSPs), RING box protein-1 (RBX1), Aldose reductase, late embryogenesis abundant-5 (LEA5) and proline-rich protein-2 (PRP2), a gene involved in root growth, showed enhanced expression under stress in transgenic lines. Thus, the simultaneous expression of regulatory genes contributing for drought-adaptive traits can improve crop adaptation and productivity under water-limited conditions.

Plant Ribosomal Proteins, RPL12 and RPL19, Play a Role in Nonhost Disease Resistance against Bacterial Pathogens.

Characterizing the molecular mechanism involved in nonhost disease resistance is important to understand the adaptations of plant-pathogen interactions. In this study, virus-induced gene silencing (VIGS)-based forward genetics screen was utilized to identify genes involved in nonhost resistance in Nicotiana benthamiana. Genes encoding ribosomal proteins, RPL12 and RPL19, were identified in the screening. These genes when silenced in N. benthamiana caused a delay in nonhost bacteria induced hypersensitive response (HR) with concurrent increase in nonhost bacterial multiplication. Arabidopsis mutants of AtRPL12 and AtRPL19 also compromised nonhost resistance. The studies on NbRPL12 and NbRPL19 double silenced plants suggested that both RPL12 and RPL19 act in the same pathway to confer nonhost resistance. Our work suggests a role for RPL12 and RPL19 in nonhost disease resistance in N. benthamiana and Arabidopsis. In addition, we show that these genes also play a minor role in basal resistance against virulent pathogens.