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."

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.

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.

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.

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.

A Sugarcane G-Protein-Coupled Receptor, ShGPCR1, Confers Tolerance to Multiple Abiotic Stresses.

Sugarcane (Saccharum spp.) is a prominent source of sugar and serves as bioenergy/biomass feedstock globally. Multiple biotic and abiotic stresses, including drought, salinity, and cold, adversely affect sugarcane yield. G-protein-coupled receptors (GPCRs) are components of G-protein-mediated signaling affecting plant growth, development, and stress responses. Here, we identified a GPCR-like protein (ShGPCR1) from sugarcane and energy cane (Saccharum spp. hybrids) and characterized its function in conferring tolerance to multiple abiotic stresses. ShGPCR1 protein sequence contained nine predicted transmembrane (TM) domains connected by four extracellular and four intracellular loops, which could interact with various ligands and heterotrimeric G proteins in the cells. ShGPCR1 sequence displayed other signature features of a GPCR, such as a putative guanidine triphosphate (GTP)-binding domain, as well as multiple myristoylation and protein phosphorylation sites, presumably important for its biochemical function. Expression of ShGPCR1 was upregulated by drought, salinity, and cold stresses. Subcellular imaging and calcium (Ca2+) measurements revealed that ShGPCR1 predominantly localized to the plasma membrane and enhanced intracellular Ca2+ levels in response to GTP, respectively. Furthermore, constitutive overexpression of ShGPCR1 in sugarcane conferred tolerance to the three stressors. The stress-tolerance phenotype of the transgenic lines corresponded with activation of multiple drought-, salinity-, and cold-stress marker genes, such as Saccharum spp. LATE EMBRYOGENESIS ABUNDANT, DEHYDRIN, DROUGHT RESPONSIVE 4, GALACTINOL SYNTHASE, ETHYLENE RESPONSIVE FACTOR 3, SALT OVERLY SENSITIVE 1, VACUOLAR Na+/H+ ANTIPORTER 1, NAM/ATAF1/2/CUC2, COLD RESPONSIVE FACTOR 2, and ALCOHOL DEHYDROGENASE 3. We suggest that ShGPCR1 plays a key role in conferring tolerance to multiple abiotic stresses, and the engineered lines may be useful to enhance sugarcane production in marginal environments with fewer resources.

Phenotypic Diversity and Association Mapping of Ascorbic Acid Content in Spinach.

Ascorbic acid (AsA), or vitamin C, is an essential nutrient for humans. In plants, AsA functions as an antioxidant during normal metabolism or in response to stress. Spinach is a highly nutritious green leafy vegetable that is consumed fresh, cooked or as a part of other dishes. One current goal in spinach breeding programs is to enhance quality and nutritional content. However, little is known about the diversity of nutritional content present in spinach germplasm, especially for AsA content. In this study, a worldwide panel of 352 accessions was screened for AsA content showing that variability in spinach germplasm is high and could be utilized for cultivar improvement. In addition, a genome-wide association study for marker-trait association was performed using three models, and associated markers were searched in the genome for functional annotation analysis. The generalized linear model (GLM), the compressed mixed linear model (CMLM) based on population parameters previously determined (P3D) and the perMarker model together identified a total of 490 significant markers distributed across all six spinach chromosomes indicating the complex inheritance of the trait. The different association models identified unique and overlapping marker sets, where 27 markers were identified by all three models. Identified high AsA content accessions can be used as parental lines for trait introgression and to create segregating populations for further genetic analysis. Bioinformatic analysis indicated that identified markers can differentiate between high and low AsA content accessions and that, upon validation, these markers should be useful for breeding programs.

Plant hairy roots enable high throughput identification of antimicrobials against Candidatus Liberibacter spp.

A major bottleneck in identifying therapies to control citrus greening and other devastating plant diseases caused by fastidious pathogens is our inability to culture the pathogens in defined media or axenic cultures. As such, conventional approaches for antimicrobial evaluation (genetic or chemical) rely on time-consuming, low-throughput and inherently variable whole-plant assays. Here, we report that plant hairy roots support the growth of fastidious pathogens like Candidatus Liberibacter spp., the presumptive causal agents of citrus greening, potato zebra chip and tomato vein greening diseases. Importantly, we leverage the microbial hairy roots for rapid, reproducible efficacy screening of multiple therapies. We identify six antimicrobial peptides, two plant immune regulators and eight chemicals which inhibit Candidatus Liberibacter spp. in plant tissues. The antimicrobials, either singly or in combination, can be used as near- and long-term therapies to control citrus greening, potato zebra chip and tomato vein greening diseases.

High-Level Production of Recombinant Snowdrop Lectin in Sugarcane and Energy Cane.

Sugarcane and energy cane (Saccharum spp. hybrids) are ideal for plant-based production of recombinant proteins because their high resource-use efficiency, rapid growth and efficient photosynthesis enable extensive biomass production and protein accumulation at a cost-effective scale. Here, we aimed to develop these species as efficient platforms to produce recombinant Galanthus nivalis L. (snowdrop) agglutinin (GNA), a monocot-bulb mannose-specific lectin with potent antiviral, antifungal and antitumor activities. Initially, GNA levels of 0.04% and 0.3% total soluble protein (TSP) (0.3 and 3.8 mg kg-1 tissue) were recovered from the culms and leaves, respectively, of sugarcane lines expressing recombinant GNA under the control of the constitutive maize ubiquitin 1 (Ubi) promoter. Co-expression of recombinant GNA from stacked multiple promoters (pUbi and culm-regulated promoters from sugarcane dirigent5-1 and Sugarcane bacilliform virus) on separate expression vectors increased GNA yields up to 42.3-fold (1.8% TSP or 12.7 mg kg-1 tissue) and 7.7-fold (2.3% TSP or 29.3 mg kg-1 tissue) in sugarcane and energy cane lines, respectively. Moreover, inducing promoter activity in the leaves of GNA transgenic lines with stress-regulated hormones increased GNA accumulation to 2.7% TSP (37.2 mg kg-1 tissue). Purification by mannose-agarose affinity chromatography yielded a functional sugarcane recombinant GNA with binding substrate specificity similar to that of native snowdrop-bulb GNA, as shown by enzyme-linked lectin and mannose-binding inhibition assays. The size and molecular weight of recombinant GNA were identical to those of native GNA, as determined by size-exclusion chromatography and MALDI-TOF mass spectrometry. This work demonstrates the feasibility of producing recombinant GNA at high levels in Saccharum species, with the long-term goal of using it as a broad-spectrum antiviral carrier molecule for hemopurifiers and in related therapeutic applications.

Unprecedented enhancement of recombinant protein production in sugarcane culms using a combinatorial promoter stacking system.

Plants represent a safe and cost-effective platform for producing high-value proteins with pharmaceutical properties; however, the ability to accumulate these in commercially viable quantities is challenging. Ideal crops to serve as biofactories would include low-input, fast-growing, high-biomass species such as sugarcane. The objective of this study was to develop an efficient expression system to enable large-scale production of high-value recombinant proteins in sugarcane culms. Bovine lysozyme (BvLz) is a potent broad-spectrum antimicrobial enzyme used in the food, cosmetics and agricultural industries. Here, we report a novel strategy to achieve high-level expression of recombinant proteins using a combinatorial stacked promoter system. We demonstrate this by co-expressing BvLz under the control of multiple constitutive and culm-regulated promoters on separate expression vectors and combinatorial plant transformation. BvLz accumulation reached 1.4% of total soluble protein (TSP) (10.0 mg BvLz/kg culm mass) in stacked multiple promoter:BvLz lines, compared to 0.07% of TSP (0.56 mg/kg) in single promoter:BvLz lines. BvLz accumulation was further boosted to 11.5% of TSP (82.5 mg/kg) through event stacking by re-transforming the stacked promoter:BvLz lines with additional BvLz expression vectors. The protein accumulation achieved with the combinatorial promoter stacking expression system was stable in multiple vegetative propagations, demonstrating the feasibility of using sugarcane as a biofactory for producing high-value proteins and bioproducts.

Linking metabolic phenotypes to pathogenic traits among "Candidatus Liberibacter asiaticus" and its hosts.

Candidatus Liberibacter asiaticus (CLas) has been associated with Huanglongbing, a lethal vector-borne disease affecting citrus crops worldwide. While comparative genomics has provided preliminary insights into the metabolic capabilities of this uncultured microorganism, a comprehensive functional characterization is currently lacking. Here, we reconstructed and manually curated genome-scale metabolic models for the six CLas strains A4, FL17, gxpsy, Ishi-1, psy62, and YCPsy, in addition to a model of the closest related culturable microorganism, L. crescens BT-1. Predictions about nutrient requirements and changes in growth phenotypes of CLas were confirmed using in vitro hairy root-based assays, while the L. crescens BT-1 model was validated using cultivation assays. Host-dependent metabolic phenotypes were revealed using expression data obtained from CLas-infected citrus trees and from the CLas-harboring psyllid Diaphorina citri Kuwayama. These results identified conserved and unique metabolic traits, as well as strain-specific interactions between CLas and its hosts, laying the foundation for the development of model-driven Huanglongbing management strategies.

Minor alleles are associated with white rust (Albugo occidentalis) susceptibility in spinach (Spinacia oleracea).

Minor alleles (MA) have been associated with disease incidence in human studies, enabling the identification of diagnostic risk factors for various diseases. However, allelic mapping has rarely been performed in plant systems. The goal of this study was to determine whether a difference in MA prevalence is a strong enough risk factor to indicate a likely significant difference in disease resistance against white rust (WR; Albugo occidentalis) in spinach (Spinacia oleracea). We used WR disease severity ratings (WR-DSRs) in a diversity panel of 267 spinach accessions to define resistant- and susceptibility-associated groups within the distribution scores and then tested the single-nucleotide polymorphism (SNP) variants to interrogate the MA prevalence in the most susceptible (MS) vs. most resistant (MR) individuals using permutation-based allelic association tests. A total of 448 minor alleles associated with WR severity were identified in the comparison between the 25% MS and the 25% MR accessions, while the MA were generally similar between the two halves of the interquartile range. The minor alleles in the MS group were distributed across all six chromosomes and made up ~71% of the markers that were also strongly associated with WR in parallel performed genome-wide association study. These results indicate that susceptibility may be highly determined by the disproportionate overrepresentation of minor alleles, which could be used to select for resistant plants. Furthermore, by focusing on the distribution tails, allelic mapping could be used to identify plant markers associated with quantitative traits on the most informative segments of the phenotypic distribution.

Panicum Mosaic Virus and Its Satellites Acquire RNA Modifications Associated with Host-Mediated Antiviral Degradation.

Positive-sense RNA viruses in the Tombusviridae family have genomes lacking a 5' cap structure and prototypical 3' polyadenylation sequence. Instead, these viruses utilize an extensive network of intramolecular RNA-RNA interactions to direct viral replication and gene expression. Here we demonstrate that the genomic RNAs of Panicum mosaic virus (PMV) and its satellites undergo sequence modifications at their 3' ends upon infection of host cells. Changes to the viral and subviral genomes arise de novo within Brachypodium distachyon (herein called Brachypodium) and proso millet, two alternative hosts of PMV, and exist in the infections of a native host, St. Augustinegrass. These modifications are defined by polyadenylation [poly(A)] events and significant truncations of the helper virus 3' untranslated region-a region containing satellite RNA recombination motifs and conserved viral translational enhancer elements. The genomes of PMV and its satellite virus (SPMV) were reconstructed from multiple poly(A)-selected Brachypodium transcriptome data sets. Moreover, the polyadenylated forms of PMV and SPMV RNAs copurify with their respective mature icosahedral virions. The changes to viral and subviral genomes upon infection are discussed in the context of a previously understudied poly(A)-mediated antiviral RNA degradation pathway and the potential impact on virus evolution.IMPORTANCE The genomes of positive-sense RNA viruses have an intrinsic capacity to serve directly as mRNAs upon viral entry into a host cell. These RNAs often lack a 5' cap structure and 3' polyadenylation sequence, requiring unconventional strategies for cap-independent translation and subversion of the cellular RNA degradation machinery. For tombusviruses, critical translational regulatory elements are encoded within the 3' untranslated region of the viral genomes. Here we describe RNA modifications occurring within the genomes of Panicum mosaic virus (PMV), a prototypical tombusvirus, and its satellite agents (i.e., satellite virus and noncoding satellite RNAs), all of which depend on the PMV-encoded RNA polymerase for replication. The atypical RNAs are defined by terminal polyadenylation and truncation events within the 3' untranslated region of the PMV genome. These modifications are reminiscent of host-mediated RNA degradation strategies and likely represent a previously underappreciated defense mechanism against invasive nucleic acids.