Plant-Derived, Nodule-Specific Cysteine-Rich Peptides as a Novel Source of Biopesticides for Controlling Citrus Greening Disease.

Nodule-specific cysteine-rich (NCR) peptides, encoded in the genome of the Mediterranean legume Medicago truncatula (barrelclover), are known to regulate plant-microbe interactions. A subset of computationally derived 20-mer peptide fragments from 182 NCR peptides was synthesized to identify those with activity against the unculturable vascular pathogen associated with citrus greening disease, 'Candidatus Liberibacter asiaticus' (CLas). Grounded in a design of experiments framework, we evaluated the peptides in a screening pipeline involving three distinct assays: a bacterial culture assay with Liberibacter crescens, a CLas-infected excised citrus leaf assay, and an assay to evaluate effects on bacterial acquisition by the nymphal stage of hemipteran vector Diaphorina citri. A subset of the 20-mer NCR peptide fragments inhibits both CLas growth in citrus leaves and CLas acquisition by D. citri. Two peptides induced higher levels of D. citri mortality. These findings reveal 20-mer NCR peptides as a new class of plant-derived biopesticide molecules to control citrus greening disease.

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.

Temporal and spatial detection of Candidatus Liberibacter asiaticus putative effector transcripts during interaction with Huanglongbing-susceptible, -tolerant, and -resistant citrus hosts.

BACKGROUND: Citrus Huanglongbing (HLB) is a bacterial disease with high economic significance. The associated agent Candidatus Liberibacter asiaticus is a fastidious, phloem-limited, intracellular bacterium that is transmitted by an insect vector the Asian citrus psyllid (ACP). The genome of Ca. L. asiaticus contains protein secretion machinery that suggests host cell modulation capacity of this bacterium. RESULTS: A total of 28 candidate effectors, an important class of secreted proteins, were predicted from the Ca. L. asiaticus genome. Sequence specific primers were designed for reverse transcription (RT) and quantitative PCR (qPCR), and expression was validated for 20 of the effector candidates in infected citrus with multiple genetic background. Using detached leaf inoculation, the mRNA of effectors was detected from 6 h to 7 days post ACP exposure. It was observed that higher bacterial titers were associated with a larger number of effectors showing amplification across all samples. The effectors' expression were compared in citrus hosts with various levels of HLB tolerance, including susceptible Duncan grapefruit and Washington navel orange, tolerant citron and Cleopatra mandarin, and resistant Pomeroy trifoliate and Carrizo citrange. Across all genotypes relatively high expression was observed for CLIBASIA_03695, CLIBASIA_00460, CLIBASIA_00420, CLIBASIA_04580, CLIBASIA_05320, CLIBASIA_04425, CLIBASIA_00525 and CLIBASIA_05315 in either a host-specific or -nonspecific manners. The two genotypes in each HLB-response group also show effector-expression profiles that seem to be different. In a companion study, the expression of effectors was compared between leaves and roots of own-rooted citrus that had been Ca. L. asiaticus-infected for more than a year. Results indicated relatively high expression of CLIBASIA_03875, CLIBASIA_04800 and CLIBASIA_05640 in all leaf and some root tissues of citron, Duncan and Cleopatra. CONCLUSION: This temporal and spatial expression analysis of Ca. L. asiaticus effectors identified candidates possibly critical for early bacterial colonization, host tolerance suppression and long-term survival which are all worthy of further investigation.

Diaphorina citri Nymphs Are Resistant to Morphological Changes Induced by "Candidatus Liberibacter asiaticus" in Midgut Epithelial Cells.

"Candidatus Liberibacter asiaticus" is the causative bacterium associated with citrus greening disease. "Ca Liberibacter asiaticus" is transmitted by Diaphorina citri more efficiently when it is acquired by nymphs rather than adults. Why this occurs is not known. We compared midguts of D. citri insects reared on healthy or "Ca Liberibacter asiaticus"-infected citrus trees using quantitative PCR, confocal microscopy, and mitochondrial superoxide staining for evidence of oxidative stress. Consistent with its classification as propagative, "Ca Liberibacter asiaticus" titers were higher in adults than in nymphs. Our previous work showed that adult D. citri insects have basal levels of karyorrhexis (fragmentation of the nucleus) in midgut epithelial cells, which is increased in severity and frequency in response to "Ca Liberibacter asiaticus." Here, we show that nymphs exhibit lower levels of early-stage karyorrhexis than adults and are refractory to the induction of advanced karyorrhexis by "Ca Liberibacter asiaticus" in the midgut epithelium. MitoSox Red staining showed that guts of infected adults, particularly males, experienced oxidative stress in response to "Ca Liberibacter asiaticus." A positive correlation between the titers of "Ca Liberibacter asiaticus" and the Wolbachia endosymbiont was observed in adult and nymph midguts, suggesting an interplay between these bacteria during development. We hypothesize that the resistance of the nymph midgut to late-stage karyorrhexis through as yet unknown molecular mechanisms benefits "Ca Liberibacter asiaticus" for efficient invasion of midgut epithelial cells, which may be a factor explaining the developmental dependency of "Ca Liberibacter asiaticus" acquisition by the vector.

Juvenile hormone affects the splicing of Culex quinquefasciatus early trypsin messenger RNA.

The full length of Culex quiquefasciatus early trypsin has been cloned and sequenced and a three-dimensional (3D) model of the enzyme was built showing that the enzyme has the canonical trypsin's active pocket containing H78, D123, S129, and D128. The biosynthesis of juvenile hormone (JH) III by the corpora allata (CA) in female Cx. quiquefasciatus is sugar-dependent. Females that were maintained on water after emergence synthesize very little JH III, JH III bisepoxide, and methyl farnesoate (MF) (3.8, 1.1, and 0.8 fmol/4 hr/CA, respectively). One hour after sugar feeding, the synthesis of JH III and JH III bisepoxide reached a maximum (11.3 and 5.9 fmol/4 hr/CA, respectively) whereas MF biosynthesis reached a maximum at 24 hr (5.2 fmol/4 hr/CA). The early trypsin is transcribed with a short intron (51 nt) is spliced when JH III biosynthesis is high in sugar fed and at 1 hr after the blood meal (22 and 15 fmol/4 hr/CA, respectively). We investigated the transcriptional and posttranscriptional regulation of the early trypsin gene showing that JH III concentrations influence splicing. In the absence JH III the unspliced transcript is linked by a phosphoamide bond at the 5'-end to RNA ribonuleoprotein (RNP). The biosynthesis of the early trypsin was followed in ligated abdomens (without CA) of newly emerged females that fed blood by enema. Our results show that the early trypsin biosynthesis depends on sugar and blood feeding, whereas the late trypsin biosynthesis does not depend on sugar feeding, or JH III biosynthesis. Downregulating the early trypsin transcript does not affect the late trypsin.

Expression of Bacillus thuringiensis cytolytic toxin (Cyt2Ca1) in citrus roots to control Diaprepes abbreviatus larvae.

Diaprepes abbreviatus (L.) is an important pest of citrus in the USA. Currently, no effective management strategies of D. abbreviatus exist in citriculture, and new methods of control are desperately sought. To protect citrus against D. abbreviatus a transgenic citrus rootstock expressing Bacillus thuringiensis Cyt2Ca1, an insect toxin protein, was developed using Agrobacterium-mediated transformation of 'Carrizo' citrange [Citrus sinensis (L) Osbeck Poncirus trifoliate (L) Raf]. The transgenic citrus root stock expressed the cytolytic toxin Cyt2Ca1 constitutively under the control of a 35S promoter in the transgenic Carrizo citrange trifoliate hybrid including the roots that are the food source of larval D. abbreviatus. The engineered citrus was screened by Western blot and RT-qPCR analyses for cyt2Ca1 and positive citrus identified. Citrus trees expressing different levels of cyt2Ca1 transcripts were identified (Groups A-C). High expression of the toxin in the leaves (109 transcripts/ng RNA), however, retarded plant growth. The transgenic plants were grown in pots and the roots exposed to 3week old D. abbreviatus larvae using no-choice plant bioassays. Three cyt2Ca1 transgenic plants were identified that sustained less root damage belonging to Group B and C. One plant caused death to 43% of the larvae that fed on its roots expressed 8×106cyt2Ca1 transcripts/ng RNA. These results show, for the first time, that Cyt2Ca1 expressed in moderate amounts by the roots of citrus does not retard citrus growth and can protect it from larval D. abbreviatus.

Unexpected High Intragenomic Variation in Two of Three Major Pest Thrips Species Does Not Affect Ribosomal Internal Transcribed Spacer 2 (ITS2) Utility for Thrips Identification.

The mitochondrial cytochrome oxidase I gene (mtCO1) and the ribosomal internal transcribed spacer 2 region (ITS2) are among the most widely used molecular markers for insect taxonomic characterization. Three economically important species of thrips, Scirtothripsdorsalis, Thripspalmi, and Frankliniellaoccidentalis were selected to examine the extent of intragenomic variation within these two marker regions in the family Thripidae, and determine if this variation would affect the utility of markers in thrips molecular diagnostics. For each species, intragenomic (within individual) variation and intergenomic (among individuals) variation was assessed by cloning and sequencing PCR-amplified copies. Intergenomic variation was generally higher than intragenomic variation except in cases where intergenomic variation was very low, as in mtCO1 from S.dorsalis and F.occidentalis. Intragenomic variation was detected in both markers in all three of the thrips species, however, 2-3 times more intragenomic variation was observed for ITS2 than mtCO1 in both S.dorsalis and T.palmi. Furthermore, levels of intragenomic variation were low for both of the genes in F.occidentalis. In all of the three thrips species, no sex-based clustering of haplotypes was observed in either marker. Unexpected high intragenomic variation in ITS2 for two of three thrips species did not interfere with thrips diagnostics. However, caution should be taken in applying ITS2 to certain studies of S.dorsalis and T.palmi when high levels of intragenomic variation could be problematic or confounding. In such studies, mtCO1 may be a preferable marker. Possible reasons for discrepancies in intragenomic variation among genomic regions are discussed.

A novel mitochondrial genome architecture in thrips (Insecta: Thysanoptera): extreme size asymmetry among chromosomes and possible recent control region duplication.

BACKGROUND: Multipartite mitochondrial genomes are very rare in animals but have been found previously in two insect orders with highly rearranged genomes, the Phthiraptera (parasitic lice), and the Psocoptera (booklice/barklice). RESULTS: We provide the first report of a multipartite mitochondrial genome architecture in a third order with highly rearranged genomes: Thysanoptera (thrips). We sequenced the complete mitochondrial genomes of two divergent members of the Scirtothrips dorsalis cryptic species complex. The East Asia 1 species has the single circular chromosome common to animals while the South Asia 1 species has a genome consisting of two circular chromosomes. The fragmented South Asia 1 genome exhibits extreme chromosome size asymmetry with the majority of genes on the large, 14.28 kb, chromosome and only nad6 and trnC on the 0.92 kb mini-circle chromosome. This genome also features paralogous control regions with high similarity suggesting a very recent origin of the nad6 mini-circle chromosome in the South Asia 1 cryptic species. CONCLUSIONS: Thysanoptera, along with the other minor paraenopteran insect orders should be considered models for rapid mitochondrial genome evolution, including fragmentation. Continued use of these models will facilitate a greater understanding of recombination and other mitochondrial genome evolutionary processes across eukaryotes.

Direct Evidence of Egestion and Salivation of Xylella fastidiosa Suggests Sharpshooters Can Be "Flying Syringes".

Xylella fastidiosa is unique among insect-transmitted plant pathogens because it is propagative but noncirculative, adhering to and multiplying on the cuticular lining of the anterior foregut. Any inoculation mechanism for X. fastidiosa must explain how bacterial cells exit the vector's stylets via the food canal and directly enter the plant. A combined egestion-salivation mechanism has been proposed to explain these unique features. Egestion is the putative outward flow of fluid from the foregut via hypothesized bidirectional pumping of the cibarium. The present study traced green fluorescent protein-expressing X. fastidiosa or fluorescent nanoparticles acquired from artificial diets by glassy-winged sharpshooters, Homalodisca vitripennis, as they were egested into simultaneously secreted saliva. X. fastidiosa or nanoparticles were shown to mix with gelling saliva to form fluorescent deposits and salivary sheaths on artificial diets, providing the first direct, conclusive evidence of egestion by any hemipteran insect. Therefore, the present results strongly support an egestion-salivation mechanism of X. fastidiosa inoculation. Results also support that a column of fluid is transiently held in the foregut without being swallowed. Evidence also supports (but does not definitively prove) that bacteria were suspended in the column of fluid during the vector's transit from diet to diet, and were egested with the held fluid. Thus, we hypothesize that sharpshooters could be true "flying syringes," especially when inoculation occurs very soon after uptake of bacteria, suggesting the new paradigm of a nonpersistent X. fastidiosa transmission mechanism.

ESTIMATING BACTERIAL DIVERSITY IN SCIRTOTHRIPS DORSALIS (THYSANOPTERA: THRIPIDAE) VIA NEXT GENERATION SEQUENCING.

The last 2 decades have produced a better understanding of insect-microbial associations and yielded some important opportunities for insect control. However, most of our knowledge comes from model systems. Thrips (Thysanoptera: Thripidae) have been understudied despite their global importance as invasive species, plant pests and disease vectors. Using a culture and primer independent next-generation sequencing and metagenomics pipeline, we surveyed the bacteria of the globally important pest, Scirtothrips dorsalis Hood. The most abundant bacterial phyla identified were Actinobacteria and Proteobacteria and the most abundant genera were Propionibacterium, Stenotrophomonas, and Pseudomonas. A total of 189 genera of bacteria were identified. The absence of any vertically transferred symbiont taxa commonly found in insects is consistent with other studies suggesting that thrips primarilly acquire resident microbes from their environment. This does not preclude a possible beneficial/intimate association between S. dorsalis and the dominant taxa identified and future work should determine the nature of these associations.

Population genetics of invasive Bemisia tabaci (Hemiptera: Aleyrodidae) cryptic species in the United States based on microsatellite markers.

The Bemisia tabaci (Gennadius) (Hemiptera: Aleyrodidae) cryptic species complex of whiteflies contains two species, MEAM1 and MED, that are highly invasive in supportive climates the world over. In the United States, MEAM1 occurs both in the field and in the greenhouse, but MED is only found in the greenhouse. To make inferences about the population structure of both species, and the origin and recent spread of MED within the United States, 987 MEAM1 whiteflies and 340 MED whiteflies were genotyped at six and seven microsatellite loci, respectively, for population genetic analyses. Major results of the study are 1) MED exhibits more population structure and genetic differentiation than MEAM1, 2) nuclear microsatellite markers exhibit a high degree of concordance with mitochondrial markers recovering a major east-west phylogeographic break within MED, 3) both eastern and western MED are found throughout the continental United States and eastern MED is present in Hawaii, and 4) MEAM1 contains two greenhouse U.S. populations significantly differentiated from other U.S. MEAM1. The results suggest that MED was introduced into the United States on at least three occasions and rapidly spread throughout the United States, showing no discernible differentiation across 7,000 km. The results further suggest that there is an enhanced role of the protected agricultural environment in promoting genetic differentiation in both invasive B. tabaci cryptic species.

Direct Infusion Device for Molecule Delivery in Plants.

Testing the function of therapeutic compounds in plants is an important component of agricultural research. Foliar and soil-drench methods are routine but have drawbacks, including variable uptake and the environmental breakdown of tested molecules. Trunk injection of trees is well-established, but most methods for this require expensive, proprietary equipment. To screen various treatments for Huanglongbing, a simple, low-cost method to deliver these compounds to the vascular tissue of small greenhouse-grown citrus trees infected with the phloem-limited bacterium Candidatus Liberibacter asiaticus (CLas) or infested with the phloem-feeding CLas insect vector Diaphorina citri Kuwayama (D. citri) is needed. To meet these screening requirements, a direct plant infusion (DPI) device was designed that connects to the plant's trunk. The device is made using a nylon-based 3D-printing system and easily obtainable auxiliary components. The compound uptake efficacy of this device was tested in citrus plants using the fluorescent marker 5,6-carboxyfluorescein-diacetate. Uniform compound distribution of the marker throughout the plants was routinely observed. Furthermore, this device was used to deliver antimicrobial and insecticidal molecules to determine their effects on CLas and D. citri respectively. The aminoglycoside antibiotic streptomycin was delivered into CLas-infected citrus plants using the device, which resulted in a reduction in the CLas titer from 2 weeks to 4 weeks post treatment. Delivering the neonicotinoid insecticide imidacloprid into D. citri-infested citrus plants resulted in a significant increase in psyllid mortality after 7 days. These results suggest that this DPI device represents a useful system for delivering molecules into plants for testing and facilitate research and screening purposes.

Impacts of Oak Mulch Amendments on Rhizosphere Microbiome of Citrus Trees Grown in Florida Flatwood Soils.

Rhizosphere interactions are an understudied component of citrus production. This is even more important in Florida flatwood soils, which pose significant challenges in achieving sustainable and effective fruit production due to low natural fertility and organic matter. Citrus growers apply soil amendments, including oak mulch, to ameliorate their soil conditions. Thus, the aim of this research was to evaluate the effects of oak mulch on citrus nutrient uptake, soil characteristics, and rhizosphere composition. The plant material consisted of 'Valencia' sweet orange (Citrus × sinensis) trees grafted on 'US-812' (C. reticulata × C. trifoliata) rootstock. The experiment consisted of two treatments, which included trees treated with oak mulch (300 kg of mulch per plot) and a control. The soil and leaf nutrient contents, soil pH, cation exchange capacity, moisture, temperature, and rhizosphere bacterial compositions were examined over the course of one year (spring and fall 2021). During the spring samplings, the citrus trees treated with oak mulch resulted in significantly greater soil Zn and Mn contents, greater soil moisture, and greater rhizosphere bacterial diversity compared to the control, while during the fall samplings, only a greater soil moisture content was observed in the treated trees. The soil Zn and Mn content detected during the spring samplings correlated with the significant increases in the diversity of the rhizosphere bacterial community composition. Similarly, the reduced rates of leaching and evaporation (at the soil surface) of oak mulch applied to Florida sandy soils likely played a large role in the significant increase in moisture and nutrient retention.

Improved real-time PCR detection of 'Candidatus Liberibacter asiaticus' from citrus and psyllid hosts by targeting the intragenic tandem-repeats of its prophage genes.

'Candidatus Liberibacter asiaticus' (CLas) is a Gram-negative α-proteobacterium, and the prominent species of Liberibacter associated with a devastating worldwide citrus disease known as huanglongbing (HLB). This fastidious bacterium resides in phloem sieve cells of host plants and is vectored by the Asian citrus psyllid (Diaphorina citri). Due to its uneven distribution in planta and highly variable bacterial titers, detection of HLB bacteria can be challenging. Here we demonstrated a new utility of nearly identical tandem-repeats of two CLas prophage genes for real-time PCR by SYBR Green 1 (LJ900fr) and TaqMan(®) (LJ900fpr). When compared with conventional 16S rDNA-based real-time PCR, targeting the repeat sequence reduced the relative detectable threshold by approximately 9 and 3 real-time PCR cycles for LJ900fr and LJ900fpr, respectively. Additionally, both LJ900 methods detected CLas from otherwise non-detectable samples by other methods. CLas was also detected from globally derived samples including psyllids, various citrus varieties, periwinkle, dodder, and orange jasmine, suggesting the new detection method can be applicable worldwide. Additionally, we demonstrated the presence of the hyv(I)/hyv(II) repeat sequence within the 'Ca. Liberibacter americanus' strain. The method thereby provides sensitive HLB detection with broad application for scientific, regulatory, and citrus grower communities.

Distribution of Bemisia tabaci (Hemiptera: Aleyrodidae) biotypes in North America after the Q invasion.

After the 2004 discovery of the Bemisia tabaci (Gennadius) (Hemiptera Aleyrodidae) Q biotype in the United States, there was a vital need to determine the geographical and host distribution as well as its interaction with the resident B biotype because of its innate ability to rapidly develop high-level insecticide resistance that persists in the absence of exposure. As part of a coordinated country-wide effort, an extensive survey of B. tabaci biotypes was conducted in North America, with the cooperation of growers, industry, local, state, and federal agencies, to monitor the introduction and distribution of the Q biotype. The biotype status of submitted B. tabaci samples was determined either by polymerase chain reaction amplification and sequencing of a mitochondrial cytochrome oxidase I small subunit gene fragment and characterization of two biotype discriminating nuclear microsatellite markers or esterase zymogram analysis. Two hundred and eighty collections were sampled from the United States, Bermuda, Canada, and Mexico during January 2005 through December 2011. Host plants were split between ornamental plant and culinary herb (67%) and vegetable and field crop (33%) commodities. The New World biotype was detected on field-grown tomatoes (Solanum lycopersicum L.) in Mexico (two) and in commercial greenhouses in Texas (three) and represented 100% of these five collections. To our knowledge, the latter identification represents the first report of the New World biotype in the United States since its rapid displacement in the late 1980s after the introduction of biotype B. Seventy-one percent of all collections contained at least one biotype B individual, and 53% of all collections contained only biotype B whiteflies. Biotype Q was detected in 23 states in the United States, Canada (British Columbia and Ontario territories), Bermuda, and Mexico. Forty-five percent of all collections were found to contain biotype Q in samples from ornamentals, herbs and a single collection from tomato transplants located in protected commercial horticultural greenhouses, but there were no Q detections in outdoor agriculture (vegetable or field crops). Ten of the 15 collections (67%) from Canada and a single collection from Bermuda contained biotype Q, representing the first reports of biotype Q for both countries. Three distinct mitochondrial haplotypes of B. tabaci biotype Q whiteflies were detected in North America Our data are consistent with the inference of independent invasions from at least three different locations. Of the 4,641 individuals analyzed from 517 collections that include data from our previous work, only 16 individuals contained genetic or zymogram evidence of possible hybridization of the Q and B biotypes, and there was no evidence that rare hybrid B-Q marker co-occurrences persisted in any populations.

The Ribosome Is the Ultimate Receptor for Trypsin Modulating Oostatic Factor (TMOF).

Aedes aegypti Trypsin Modulating Oostatic Factor (AeaTMOF). a mosquito decapeptide that controls trypsin biosynthesis in female and larval mosquitoes. enters the gut epithelial cells of female mosquitoes using ABC-tmfA receptor/importer. To study the ultimate targeted receptor after AeaTMOF enters the cell, AeaTMOF was incubated in vitro with either Escherichia coli or Spodoptera frugiperda protein-expressing extracts containing 70S and 80S ribosomes, respectively. The effect of AeaTMOF on luciferase biosynthesis in vitro using 70S ribosomes was compared with that of oncocin112 (1-13), a ribosome-binding antibacterial peptide. The IC50 of 1 µM and 2 µM, respectively, for both peptides was determined. Incubation with a protein-expressing system and S. frugiperda 80S ribosomes determined an IC50 of 1.8 µM for Aedes aegypti larval late trypsin biosynthesis. Incubation of purified E. coli ribosome with increasing concentration of AeaTMOF shows that the binding of AeaTMOF to the bacterial ribosome exhibits a high affinity (KD = 23 ± 3.4 nM, Bmax = 0.553 ± 0.023 pmol/µg ribosome and Kassoc = 4.3 × 107 M-1). Molecular modeling and docking experiments show that AeaTMOF binds bacterial and Drosophila ribosome (50S and 60S, respectively) at the entrance of the ribosome exit tunnel, blocking the tRNA entrance and preventing protein biosynthesis. Recombinant E. coli cells that express only ABC-tmfA importer are inhibited by AeaTMOF but not by oncocin112 (1-13). These results suggest that the ribosome is the ultimate targeted receptor of AeaTMOF.

Bactericidal Properties of Proline-Rich Aedes aegypti Trypsin Modulating Oostatic Factor (AeaTMOF).

The antimicrobial properties of proline-rich Aedes aegypti decapeptide TMOF (AeaTMOF) and oncocin112 (1-13) were compared. Incubations with multidrug-resistant Escherichia coli cells showed that AeaTMOF (5 mM) was able to completely inhibit bacterial cell growth, whereas oncocin112 (1-13) (20 mM) partially inhibited bacterial growth as compared with bacterial cells that were not multidrug-resistant cells. AeaTMOF (5 mM) was very effective against Acinetobacter baumannii and Pseudomonas aeruginosa, completely inhibiting cell growth during 15 h incubations. AeaTMOF (5 mM) completely inhibited the Gram-positive bacteria Staphylococcus aureus and Bacillus thurengiensis sups. Israelensis cell growth, whereas oncocin112 (1-13) (10 and 20 mM) failed to affect bacterial cell growth. E. coli cells that lack the SbmA transporter were inhibited by AeaTMOF (5 mM) and not by oncocin112 (1-13) (10 to 20 mM), indicating that AeaTMOF can use other bacterial transporters than SbmA that is mainly used by proline-rich antimicrobial peptides. Incubation of E. coli cells with NaAzide showed that AeaTMOF does not use ABC-like transporters that use ATP hydrolysis to import molecules into bacterial cells. Three-dimensional modeling and docking of AeaTMOF to SbmA and MdtM transporters showed that AeaTMOF can bind these proteins, and the binding location of AeaTMOF inside these protein transporters allows AeaTMOF to be transported into the bacterial cytosol. These results show that AeaTMOF can be used as a future antibacterial agent against both multidrug-resistant Gram-positive and -negative bacteria.

Plant production of high affinity nanobodies that block SARS-CoV-2 spike protein binding with its receptor, human angiotensin converting enzyme.

Nanobodies® (VHH antibodies), are small peptides that represent the antigen binding domain, VHH of unique single domain antibodies (heavy chain only antibodies, HcAb) derived from camelids. Here, we demonstrate production of VHH nanobodies against the SARS-CoV-2 spike proteins in the solanaceous plant Nicotiana benthamiana through transient expression and their subsequent detection verified through western blot. We demonstrate that these nanobodies competitively inhibit binding between the SARS-CoV-2 spike protein receptor binding domain and its human receptor protein, angiotensin converting enzyme 2. There has been significant interest and a number of publications on the use of plants as biofactories and even some reports of producing nanobodies in plants. Our data demonstrate that functional nanobodies blocking a process necessary to initiate SARS-CoV-2 infection into mammalian cells can be produced in plants. This opens the alternative of using plants in a scheme to rapidly respond to therapeutic needs for emerging pathogens in human medicine and agriculture.

Cloning and Characterization of Aedes aegypti Trypsin Modulating Oostatic Factor (TMOF) Gut Receptor.

Trypsin Modulating Oostatic Factor (TMOF) receptor was solubilized from the guts of female Ae. Aegypti and cross linked to His6-TMOF and purified by Ni affinity chromatography. SDS PAGE identified two protein bands (45 and 61 kDa). The bands were cut digested and analyzed using MS/MS identifying a protein sequence (1306 amino acids) in the genome of Ae. aegypti. The mRNA of the receptor was extracted, the cDNA sequenced and cloned into pTAC-MAT-2. E. coli SbmA- was transformed with the recombinant plasmid and the receptor was expressed in the inner membrane of the bacterial cell. The binding kinetics of TMOF-FITC was then followed showing that the cloned receptor exhibits high affinity to TMOF (KD = 113.7 ± 18 nM ± SEM and Bmax = 28.7 ± 1.8 pmol ± SEM). Incubation of TMOF-FITC with E. coli cells that express the receptor show that the receptor binds TMOF and imports it into the bacterial cells, indicating that in mosquitoes the receptor imports TMOF into the gut epithelial cells. A 3D modeling of the receptor indicates that the receptor has ATP binding sites and TMOF transport into recombinant E. coli cells is inhibited with ATPase inhibitors Na Arsenate and Na Azide.

Crowdsourced Identification of Potential Target Genes for CTV Induced Gene Silencing for Controlling the Citrus Greening Vector Diaphorina citri.

Citrus Greening or Huanglongbing (HLB) is a disease of citrus, causing high reduction in citrus production and is transmitted by the Asian citrus psyllid Diaphorina citri Kuwayama vectoring a phloem-limited bacterium Candidatus Liberibacter sp. We report research results using crowdsourcing challenge strategy identifying potential gene targets in D. citri to control the insect using RNA interference (RNAi). From 63 submitted sequences, 43 were selected and tested by feeding them to D. citri using artificial diet assays. After feeding on artificial diet, the three most effective dsRNAs causing 30% mortality above control silenced genes expressing iron-sulfur cluster subunit of the mitochondrial electron transport chain complex (Rieske), heme iron-binding terminal oxidase enzyme (Cytochrome P450) and tetrahydrobiopterin (BH4) pathway enzyme (Pterin 4α-Carbinolamine Dehydratase). These sequences were cloned into a citrus phloem-limited virus (Citrus tristeza virus, CTV T36) expressing dsRNA against these target genes in citrus. The use of a viral mediated "para-transgenic" citrus plant system caused higher mortality to adult D. citri than what was observed using artificial diet, reaching 100% when detached citrus leaves with the engineered CTV expressing dsRNA were fed to adult D. citri. Using this approach, a virus-induced gene silencing (VIGS) can be used to test future transgenic cultivars before genetically engineering citrus. RNA Seq analysis after feeding D. citri CTV-RIE on infected leaves identified transcriptionally modified genes located upstream and downstream of the targeted RIE gene. These genes were annotated showing that many are associated with the primary function of the Rieske gene that was targeted by VIGS.