Specifically targeting antimicrobial peptides for inhibition of Candidatus Liberibacter asiaticus.

AIMS: Huanglongbing (citrus greening) is a plant disease putatively caused by the unculturable Gram-negative bacterium Candidatus Liberibacter asiaticus (CLas), and it has caused severe damage to citrus plantations worldwide. There are no definitive treatments for this disease, and conventional disease control techniques have shown limited efficacy. This work presents an in silico evaluation of using specifically targeting anti-microbial peptides (STAMPs) consisting of a targeting segment and an antimicrobial segment to inhibit citrus greening by inhibiting the BamA protein of CLas, which is an outer membrane protein crucial for bacterial viability. METHODS AND RESULTS: Initially, a set of peptides with a high affinity toward BamA protein were screened and evaluated via molecular docking and molecular dynamics simulations and were verified in vitro via bio-layer interferometry (BLI). In silico studies and BLI experiments indicated that two peptides, HASP2 and HASP3, showed stable binding to BamA. Protein structures for STAMPs were created by fusing known anti-microbial peptides (AMPs) with the selected short peptides. The binding of STAMPs to BamA was assessed using molecular docking and binding energy calculations. The attachment of high-affinity short peptides significantly reduced the free energy of binding for AMPs, suggesting that it would make it easier for the STAMPs to bind to BamA. Efficacy testing in vitro using a closely related CLas surrogate bacterium showed that STAMPs had greater inhibitory activity than AMP alone. CONCLUSIONS: In silico and in vitro results indicate that the STAMPs can inhibit CLas surrogate Rhizobium grahamii more effectively compared to AMPs, suggesting that STAMPs can achieve better inhibition of CLas, potentially via enhancing the site specificity of AMPs.

Inhibition of a conserved bacterial dual-specificity phosphatase confers plant tolerance to Candidatus Liberibacter spp.

"Candidatus Liberibacter spp." are insect-vectored, fastidious, and vascular-limited phytopathogens. They are the presumptive causal agents of potato zebra chip, tomato vein clearing, and the devastating citrus greening disease worldwide. There is an urgent need to develop new strategies to control them. In this study, we characterized a dual-specificity serine/tyrosine phosphatase (STP) that is well conserved among thirty-three geographically diverse "Candidatus Liberibacter spp." and strains that infect multiple Solanaceaea and citrus spp. The STP is expressed in infected plant tissues, localized at the plant cytosol and plasma membrane, and interferes with plant cell death responses. We employed an in silico target-based molecular modeling and ligand screen to identify two small molecules with high binding affinity to STP. Efficacy studies demonstrated that the two molecules can inhibit "Candidatus Liberibacter spp." but not unrelated pathogens and confer plant disease tolerance. The inhibitors and strategies are promising means to control "Candidatus Liberibacter spp."

A Perspective on Current Therapeutic Molecule Screening Methods Against 'Candidatus Liberibacter asiaticus', the Presumed Causative Agent of Citrus Huanglongbing.

Huanglongbing (HLB), referred to as citrus greening disease, is a bacterial disease impacting citrus production worldwide and is fatal to young trees and mature trees of certain varieties. In some areas, the disease is devastating the citrus industry. A successful solution to HLB will be measured in economics: citrus growers need treatments that improve tree health, fruit production, and most importantly, economic yield. The profitability of citrus groves is the ultimate metric that truly matters when searching for solutions to HLB. Scientific approaches used in the laboratory, greenhouse, or field trials are critical to the discovery of those solutions and to estimate the likelihood of success of a treatment aimed at commercialization. Researchers and the citrus industry use a number of proxy evaluations of potential HLB solutions; understanding the strengths and limitations of each assay, as well as how best to compare different assays, is critical for decision-making to advance therapies into field trials and commercialization. This perspective aims to help the reader compare and understand the limitations of different proxy evaluation systems based on the treatment and evaluation under consideration. The researcher must determine the suitability of one or more of these metrics to identify treatments and predict the usefulness of these treatments in having an eventual impact on citrus production and HLB mitigation. As therapies advance to field trials in the next few years, a reevaluation of these metrics will be useful to guide future research efforts on strategies to mitigate HLB and vascular bacterial pathogens in other perennial crops.

Inoculation of Tomato With Plant Growth Promoting Rhizobacteria Affects the Tomato-Potato Psyllid-Candidatus Liberibacter Solanacearum Interactions.

The Rio Grande Valley (RGV) in southern Texas is well-suited for vegetable production due to its relatively mild/warm weather conditions in the fall and winter. Consequently, insects inflict year-round, persistent damage to crops in the RGV and regions with similar climate. Bactericera cockerelli (Sulc) (Hemiptera: Triozidae), commonly known as the potato psyllid, is a known vector of Candidatus Liberibacter solanacearum (CLso) (Hyphomicrobiales: Rhizobiaceae), a fastidious phloem-limited bacterium associated to vein-greening in tomatoes and Zebra Chip in potatoes. Vector control is the primary approach of integrated pest management (IPM) strategies that aim to prevent plant diseases in commercial agricultural systems. However, resistance-selective pressures that decrease the effectiveness of chemical control (insecticide) applications over time are of increasing concern. Therefore, we explore an ecological approach to devising alternative IPM methodologies to manage the psyllid-transmitted CLso pathogen to supplement existing chemical products and application schedules without increasing resistance. In this study, our objective was to examine the effects of plant-growth promoting rhizobacteria (PGPR) on host-vector-pathogen interactions. Soil-drench applications of PGPRs to Solanum lycopersicum (Solanales: Solanaceae) seedlings revealed structural and possible physiological changes to the plant host and indirect changes on psyllid behavior: host plants had increased length and biomass of roots and exhibited delayed colonization by CLso, while psyllids displayed changes in parental (F0) psyllid behavior (orientation and oviposition) in response to treated hosts and in the sex ratio of their progeny (F1). Based on our results, we suggest that PGPR may have practical use in commercial tomato production.

Evaluation of Candidatus Liberibacter Asiaticus Efflux Pump Inhibition by Antimicrobial Peptides.

Citrus greening, also known as Huanglongbing (HLB), is caused by the unculturable bacterium Candidatus Liberibacter spp. (e.g., CLas), and has caused a devastating decline in citrus production in many areas of the world. As of yet, there are no definitive treatments for controlling the disease. Antimicrobial peptides (AMPs) that have the potential to block secretion-dependent effector proteins at the outer-membrane domains were screened in silico. Predictions of drug-receptor interactions were built using multiple in silico techniques, including molecular docking analysis, molecular dynamics, molecular mechanics generalized Born surface area analysis, and principal component analysis. The efflux pump TolC of the Type 1 secretion system interacted with natural bacteriocin plantaricin JLA-9, blocking the ß barrel. The trajectory-based principal component analysis revealed the possible binding mechanism of the peptides. Furthermore, in vitro assays using two closely related culturable surrogates of CLas (Liberibacter crescens and Rhizobium spp.) showed that Plantaricin JLA-9 and two other screened AMPs inhibited bacterial growth and caused mortality. The findings contribute to designing effective therapies to manage plant diseases associated with Candidatus Liberibacter spp.

Plant-based expression platforms to produce high-value metabolites and proteins.

Plant-based heterologous expression systems can be leveraged to produce high-value therapeutics, industrially important proteins, metabolites, and bioproducts. The production can be scaled up, free from pathogen contamination, and offer post-translational modifications to synthesize complex proteins. With advancements in molecular techniques, transgenics, CRISPR/Cas9 system, plant cell, tissue, and organ culture, significant progress has been made to increase the expression of recombinant proteins and important metabolites in plants. Methods are also available to stabilize RNA transcripts, optimize protein translation, engineer proteins for their stability, and target proteins to subcellular locations best suited for their accumulation. This mini-review focuses on recent advancements to enhance the production of high-value metabolites and proteins necessary for therapeutic applications using plants as bio-factories.

A versatile Agrobacterium-based plant transformation system for genetic engineering of diverse citrus cultivars.

Developing an efficient transformation system is vital in genetically engineering recalcitrant crops, particularly trees. Here, we outline an Agrobacterium tumefaciens-based stable plant transformation methodology for citrus genetic engineering. The process was optimized to suit the requirements of fourteen citrus varieties by establishing appropriate infection, co-cultivation, selection, and culture media conditions. The procedure includes transforming seedling-derived epicotyl segments with an A. tumefaciens strain, then selecting and regenerating transformed tissues. Transgenic shoots were further identified by a visual reporter (e.g., ß-glucuronidase) and confirmed by Northern and Southern blot analysis. Transgene integrations among the transgenic lines ranged between one to four. The methodology can yield transformation efficiencies of up to 11%, and transgenic plants can be recovered as early as six months, depending on the variety. In addition, we show that incorporating A. tumefaciens helper virulence genes (virG and virE), spermidine, and lipoic acid in the resuspension buffer before transformation improved the transformation efficiency of specific recalcitrant cultivars, presumably by enhancing T-DNA integration and alleviating oxidative stress on the explant tissues. In conclusion, the optimized methodology can be utilized to engineer diverse recalcitrant citrus varieties towards trait improvement or functional genetics applications.

Applications of CRISPR/Cas13-Based RNA Editing in Plants.

The Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/CRISPR-associated (Cas) system is widely used as a genome-editing tool in various organisms, including plants, to elucidate the fundamental understanding of gene function, disease diagnostics, and crop improvement. Among the CRISPR/Cas systems, Cas9 is one of the widely used nucleases for DNA modifications, but manipulation of RNA at the post-transcriptional level is limited. The recently identified type VI CRISPR/Cas systems provide a platform for precise RNA manipulation without permanent changes to the genome. Several studies reported efficient application of Cas13 in RNA studies, such as viral interference, RNA knockdown, and RNA detection in various organisms. Cas13 was also used to produce virus resistance in plants, as most plant viruses are RNA viruses. However, the application of CRISPR/Cas13 to studies of plant RNA biology is still in its infancy. This review discusses the current and prospective applications of CRISPR/Cas13-based RNA editing technologies in plants.

Identification and Characterization of Potato Zebra Chip Resistance Among Wild Solanum Species.

Potato zebra chip (ZC) disease, associated with the uncultured phloem-limited bacterium, Candidatus Liberibacter solanacearum (CLso), is transmitted by the potato psyllid Bactericera cockerelli. Potato ZC disease poses a significant threat to potato production worldwide. Current management practices mainly rely on the control of the psyllid to limit the spread of CLso. The present study investigated new sources of ZC resistance among wild Solanum species. A taxonomically diverse collection of tuber-bearing Solanum species was screened; one ZC-resistant accession and three ZC-tolerant accessions were identified among the 52 screened accessions. Further characterization of the resistant accession showed that the resistance was primarily associated with antibiosis effects due to differences in leaf trichome density and morphology of the wild accession, which could limit the psyllid feeding and oviposition. This germplasm offers a good resource for further understanding ZC and psyllid resistance mechanisms, contributing to potato breeding efforts to develop ZC resistance cultivars. Alternatively, it could be used as a potential trap crop to manage psyllid and control ZC disease.

Role of tyrosine autophosphorylation and methionine residues in BRI1 function in Arabidopsis thaliana.

BACKGROUND: Brassinosteroids (BRs), a group of plant growth hormones, control biomass accumulation and biotic and abiotic stress tolerance, and therefore are highly relevant to agriculture. BRs bind to the BR receptor protein, brassinosteroid insensitive 1 (BRI1), which is classified as a serine/threonine (Ser/Thr) protein kinase. Recently, we reported that BRI1 acts as a dual-specificity kinase both in vitro and in vivo by undergoing autophosphorylation at tyrosine (Tyr) residues. OBJECTIVE: In this study, we characterized the increased leaf growth and early flowering phenotypes of transgenic lines expressing the mutated recombinant protein, BRI1(Y831F)-Flag, compared with those expressing BRI1-Flag. BRI1(Y831F)-Flag transgenic plants showed a reduction in hypocotyl and petiole length compared with BRI1-Flag seedlings. Transcriptome analysis revealed differential expression of flowering time-associated genes (AP1, AP2, AG, FLC, and SMZ) between BRI1(Y831F)-Flag and BRI1-Flag transgenic seedlings. We also performed site-directed mutagenesis of the BRI1 gene, and investigated the effect of methionine (Met) substitution in the extracellular domain (ECD) of BRI1 on plant growth and BR sensitivity by evaluating hypocotyl elongation and root growth inhibition. METHODS: The pBIB-Hyg+-pBR-BRI1-Flag construct(Li et al. 2002) was used as the template for SDM with QuickChange XL Site Directed Mutagenesis Kit (Stratagene, La Jolla, CA, USA) to make the SDM mutants. After PCR with SDM kit, add 1 µl of Dpn1 to PCR reaction. Incubate at 37 °C for 2 h to digest parental DNA and then transformed into XL10-gold competent cells. Transcriptome analysis was carried out at the University of Illinois (Urbana-Champaign, Illinois, USA). RNA was prepared and hybridized to the Affymetrix GeneChip Arabidopsis ATH1 Genome Array using the Gene Chip Express Kit (Ambion, Austin, TX, USA). RESULTS: Tyrosine 831 autophosphorylation of BRI1 regulates Arabidopsis flowering time, and mutation of methionine residues in the extracellular domain of BRI1 affects hypocotyl and root length. BRI1(M656Q)-Flag, BRI1(M657Q)-Flag, and BRI1(M661Q)-Flag seedlings were insensitive to the BL treatment and showed no inhibition of root elongation. However, BRI1(M665Q)-Flag and BRI1(M671Q)-Flag seedlings were sensitive to the BL treatment, and exhibited root elongation inhibition. the early flowering phenotype of BRI1(Y831F)-Flag transgenic plants is consistent with the expression levels of key flowering-related genes, including those promoting flowering (AP1, AP2, and AG) and repressing flowering (FLC and SMZ).

A BTB-TAZ protein is required for gene activation by Cauliflower mosaic virus 35S multimerized enhancers.

The Arabidopsis (Arabidopsis thaliana) BTB-TAZ DOMAIN PROTEIN 2 (BT2) contains an N-terminal BTB domain, a central TAZ zinc-finger protein-protein interaction domain, and a C-terminal calmodulin-binding domain. We previously demonstrated that BT2 regulates telomerase activity and mediates multiple responses to nutrients, hormones, and abiotic stresses in Arabidopsis. Here, we describe the essential role of BT2 in activation of genes by multimerized Cauliflower mosaic virus 35S (35S) enhancers. Loss of BT2 function in several well-characterized 35S enhancer activation-tagged lines resulted in suppression of the activation phenotypes. Suppression of the phenotypes was associated with decreased transcript abundance of the tagged genes. Nuclear run-on assays, mRNA decay studies, and bisulfite sequencing revealed that BT2 is required to maintain the transcriptionally active state of the multimerized 35S enhancers, and lack of BT2 leads to hypermethylation of the 35S enhancers. The TAZ domain and the Ca++/calmodulin-binding domain of BT2 are critical for its function and 35S enhancer activity. We further demonstrate that BT2 requires CULLIN3 and two bromodomain-containing Global Transcription factor group E proteins (GTE9 and GTE11), to regulate 35S enhancer activity. We propose that the BT2-CULLIN3 ubiquitin ligase, through interactions with GTE9 and GTE11, regulates 35S enhancer activity in Arabidopsis.

HTSQualC is a flexible and one-step quality control software for high-throughput sequencing data analysis.

Use of high-throughput sequencing (HTS) has become indispensable in life science research. Raw HTS data contains several sequencing artifacts, and as a first step it is imperative to remove the artifacts for reliable downstream bioinformatics analysis. Although there are multiple stand-alone tools available that can perform the various quality control steps separately, availability of an integrated tool that can allow one-step, automated quality control analysis of HTS datasets will significantly enhance handling large number of samples parallelly. Here, we developed HTSQualC, a stand-alone, flexible, and easy-to-use software for one-step quality control analysis of raw HTS data. HTSQualC can evaluate HTS data quality and perform filtering and trimming analysis in a single run. We evaluated the performance of HTSQualC for conducting batch analysis of HTS datasets with 322 samples with an average ~ 1 M (paired end) sequence reads per sample. HTSQualC accomplished the QC analysis in ~ 3 h in distributed mode and ~ 31 h in shared mode, thus underscoring its utility and robust performance. In addition to command-line execution, we integrated HTSQualC into the free, open-source, CyVerse cyberinfrastructure resource as a GUI interface, for wider access to experimental biologists who have limited computational resources and/or programming abilities.

Biochemical Origin of Raman-Based Diagnostics of Huanglongbing in Grapefruit Trees.

Biotic and abiotic stresses cause substantial changes in plant biochemistry. These changes are typically revealed by high-performance liquid chromatography (HPLC) and mass spectroscopy-coupled HPLC (HPLC-MS). This information can be used to determine underlying molecular mechanisms of biotic and abiotic stresses in plants. A growing body of evidence suggests that changes in plant biochemistry can be probed by Raman spectroscopy, an emerging analytical technique that is based on inelastic light scattering. Non-invasive and non-destructive detection and identification of these changes allow for the use of Raman spectroscopy for confirmatory diagnostics of plant biotic and abiotic stresses. In this study, we couple HPLC and HPLC-MS findings on biochemical changes caused by Candidatus Liberibacter spp. (Ca. L. asiaticus) in citrus trees to the spectroscopic signatures of plant leaves derived by Raman spectroscopy. Our results show that Ca. L. asiaticus cause an increase in hydroxycinnamates, the precursors of lignins, and flavones, as well as a decrease in the concentration of lutein that are detected by Raman spectroscopy. These findings suggest that Ca. L. asiaticus induce a strong plant defense response that aims to exterminate bacteria present in the plant phloem. This work also suggests that Raman spectroscopy can be used to resolve stress-induced changes in plant biochemistry on the molecular level.

Development of an in vitro Microtuberization and Temporary Immersion Bioreactor System to Evaluate Heat Stress Tolerance in Potatoes (Solanum tuberosum L.).

High temperature (heat) stress reduces tuber yield and quality of potatoes. Screening potatoes for heat tolerance is increasingly important, considering the climate change scenario and expansion of potatoes to countries where heat stress is an issue. In vitro screening for tolerance to abiotic stresses offers several advantages, including quick evaluation of numerous genotypes (clones) in reduced space, controlled environmental conditions (temperature and photoperiod), and free from confounding variables inherent to greenhouse and field conditions. In this study, we explored the feasibility of using a temporary immersion bioreactor system for heat tolerance screening of potatoes. We determined the best hormone-free microtuberizing media for this system (MSG with 8% sucrose) to enhance microtuber number and size. Comparisons of microtubers produced at 30°C as heat treatment, with 16°C as normal condition, allowed to identify heat tolerant and susceptible potato clones. The use of bioreactors allowed distinguishing well-formed (non-deformed) from deformed microtubers. Heat stress increased the total biomass of plant tissues in all the clones. However, the effect of heat stress on microtuber number and weight varied among the clones. Incubation at 30°C decreased the weight and number of non-deformed microtubers in all the clones except for Reveille Russet in which the weight of non-deformed microtubers was significantly increased and the count of non-deformed microtubers was not affected. The potato variety Reveille Russet, which was selected under high-temperature field conditions in Texas, had many non-deformed microtubers per explant and the highest microtuber weight among four clones evaluated under heat stress. We described a faster and reliable in vitro microtuberization system for abiotic stress tolerance screening, identified Reveille Russet as a promising heat-tolerant potato variety, and confirmed Russet Burbank and Atlantic as susceptible heat-tolerant checks.

Potato Zebra Chip: An Overview of the Disease, Control Strategies, and Prospects.

Potato (Solanum tuberosum L.) is an important food crop worldwide. As the demand for fresh and processed potato products is increasing globally, there is a need to manage and control devastating diseases such as zebra chip (ZC). ZC disease causes major yield losses in many potato-growing regions and is associated with the fastidious, phloem-limited bacterium Candidatus Liberibacter solanacearum (CLso) that is vectored by the potato-tomato psyllid (Bactericera cockerelli Sulc). Current management measures for ZC disease mainly focus on chemical control and integrated pest management strategies of the psyllid vector to limit the spread of CLso, however, they add to the costs of potato production. Identification and deployment of CLso and/or the psyllid resistant cultivars, in combination with integrated pest management, may provide a sustainable long-term strategy to control ZC. In this review, we provide a brief overview of the ZC disease, epidemiology, current management strategies, and potential new approaches to manage ZC disease in the future.

Brachypodium Phenylalanine Ammonia Lyase (PAL) Promotes Antiviral Defenses against Panicum mosaic virus and Its Satellites.

Brachypodium distachyon has recently emerged as a premier model plant for monocot biology, akin to Arabidopsis thaliana We previously reported genome-wide transcriptomic and alternative splicing changes occurring in Brachypodium during compatible infections with Panicum mosaic virus (PMV) and its satellite virus (SPMV). Here, we dissected the role of Brachypodium phenylalanine ammonia lyase 1 (PAL1), a key enzyme for phenylpropanoid and salicylic acid (SA) biosynthesis and the induction of plant defenses. Targeted metabolomics profiling of PMV-infected and PMV- plus SPMV-infected (PMV/SPMV) Brachypodium plants revealed enhanced levels of multiple defense-related hormones and metabolites such as cinnamic acid, SA, and fatty acids and lignin precursors during disease progression. The virus-induced accumulation of SA and lignin was significantly suppressed upon knockdown of B. distachyonPAL1 (BdPAL1) using RNA interference (RNAi). The compromised SA accumulation in PMV/SPMV-infected BdPAL1 RNAi plants correlated with weaker induction of multiple SA-related defense gene markers (pathogenesis related 1 [PR-1], PR-3, PR-5, and WRKY75) and enhanced susceptibility to PMV/SPMV compared to that of wild-type (WT) plants. Furthermore, exogenous application of SA alleviated the PMV/SPMV necrotic disease phenotypes and delayed plant death caused by single and mixed infections. Together, our results support an antiviral role for BdPAL1 during compatible host-virus interaction, perhaps as a last resort attempt to rescue the infected plant.IMPORTANCE Although the role of plant defense mechanisms against viruses are relatively well studied in dicots and in incompatible plant-microbe interactions, studies of their roles in compatible interactions and in grasses are lagging behind. In this study, we leveraged the emerging grass model Brachypodium and genetic resources to dissect Panicum mosaic virus (PMV)- and its satellite virus (SPMV)-compatible grass-virus interactions. We found a significant role for PAL1 in the production of salicylic acid (SA) in response to PMV/SPMV infections and that SA is an essential component of the defense response preventing the plant from succumbing to viral infection. Our results suggest a convergent role for the SA defense pathway in both compatible and incompatible plant-virus interactions and underscore the utility of Brachypodium for grass-virus biology.