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.

Longitudinal Transcriptomic, Proteomic, and Metabolomic Response of Citrus sinensis to Diaphorina citri Inoculation of Candidatus Liberibacter asiaticus.

Huanglongbing (HLB) is a fatal citrus disease that is currently threatening citrus varieties worldwide. One putative causative agent, Candidatus Liberibacter asiaticus (CLas), is vectored by Diaphorina citri, known as the Asian citrus psyllid (ACP). Understanding the details of CLas infection in HLB disease has been hindered by its Candidatus nature and the inability to confidently detect it in diseased trees during the asymptomatic stage. To identify early changes in citrus metabolism in response to inoculation of CLas using its natural psyllid vector, leaves from Madam Vinous sweet orange (Citrus sinensis (L.) Osbeck) trees were exposed to CLas-positive ACP or CLas-negative ACP and longitudinally analyzed using transcriptomics (RNA sequencing), proteomics (liquid chromatography-tandem mass spectrometry; data available in Dryad: 10.25338/B83H1Z), and metabolomics (proton nuclear magnetic resonance). At 4 weeks postexposure (wpe) to psyllids, the initial HLB plant response was primarily to the ACP and, to a lesser extent, the presence or absence of CLas. Additionally, analysis of 4, 8, 12, and 16 wpe identified 17 genes and one protein as consistently differentially expressed between leaves exposed to CLas-positive ACP versus CLas-negative ACP. This study informs identification of early detection molecular targets and contributes to a broader understanding of vector-transmitted plant pathogen interactions.

Laser capture of tomato pericarp tissues for microscale carotenoid analysis by supercritical fluid chromatography.

Plant organs and tissues are comprised of an array of cell types often superimposed on a gradient of developmental stages. As a result, the ability to analyze and understand the synthesis, metabolism, and accumulation of plant biomolecules requires improved methods for cell- and tissue-specific analysis. Tomato (Solanum lycopersicum) is the world's most valuable fruit crop and is an important source of health-promoting dietary compounds, including carotenoids. Furthermore, tomato possesses unique genetic activities at the cell and tissue levels, making it an ideal system for tissue- and cell-type analysis of important biochemicals. A sample preparation workflow was developed for cell-type-specific carotenoid analysis in tomato fruit samples. Protocols for hyperspectral imaging of tomato fruit samples, cryoembedding and sectioning of pericarp tissue, laser microdissection of specific cell types, metabolite extraction using cell wall digestion enzymes and pressure cycling, and carotenoid quantification by supercritical fluid chromatography were optimized and integrated into a working protocol. The workflow was applied to quantify carotenoids in the cuticle and noncuticle component of the tomato pericarp during fruit development from the initial ripening to full ripe stages. Carotenoids were extracted and quantified from cell volumes less than 10nL. This workflow for cell-type-specific metabolite extraction and quantification can be adapted for the analysis of diverse metabolites, cell types, and organisms.

Host Plant Adaptation Drives Changes in Diaphorina citri Proteome Regulation, Proteoform Expression, and Transmission of 'Candidatus Liberibacter asiaticus', the Citrus Greening Pathogen.

The Asian citrus psyllid (Diaphorina citri) is a pest of citrus and the primary insect vector of the bacterial pathogen, 'Candidatus Liberibacter asiaticus' (CLas), which is associated with citrus greening disease. The citrus relative Murraya paniculata (orange jasmine) is a host plant of D. citri but is more resistant to CLas compared with all tested Citrus genotypes. The effect of host switching of D. citri between Citrus medica (citron) and M. paniculata plants on the acquisition and transmission of CLas was investigated. The psyllid CLas titer and the proportion of CLas-infected psyllids decreased in the generations after transfer from CLas-infected citron to healthy M. paniculata plants. Furthermore, after several generations of feeding on M. paniculata, pathogen acquisition (20 to 40% reduction) and transmission rates (15 to 20% reduction) in psyllids transferred to CLas-infected citron were reduced compared with psyllids continually maintained on infected citron. Top-down (difference gel electrophoresis) and bottom-up (shotgun MS/MS) proteomics methods were used to identify changes in D. citri protein expression resulting from host plant switching between Citrus macrophylla and M. paniculata. Changes in expression of insect metabolism, immunity, and cytoskeleton proteins were associated with host plant switching. Both transient and sustained feeding on M. paniculata induced distinct patterns of protein expression in D. citri compared with psyllids reared on C. macrophylla. The results point to complex interactions that affect vector competence and may lead to strategies to control the spread of citrus greening disease.

Development on Citrus medica infected with 'Candidatus Liberibacter asiaticus' has sex-specific and -nonspecific impacts on adult Diaphorina citri and its endosymbionts.

Huanglongbing (HLB) is a deadly, incurable citrus disease putatively caused by the unculturable bacterium, 'Candidatus Liberibacter asiaticus' (CLas), and transmitted by Diaphorina citri. Prior studies suggest D. citri transmits CLas in a circulative and propagative manner; however, the precise interactions necessary for CLas transmission remain unknown, and the impact of insect sex on D. citri-CLas interactions is poorly understood despite reports of sex-dependent susceptibilities to CLas. We analyzed the transcriptome, proteome, metabolome, and microbiome of male and female adult D. citri reared on healthy or CLas-infected Citrus medica to determine shared and sex-specific responses of D. citri and its endosymbionts to CLas exposure. More sex-specific than shared D. citri responses to CLas were observed, despite there being no difference between males and females in CLas density or relative abundance. CLas exposure altered the abundance of proteins involved in immunity and cellular and oxidative stress in a sex-dependent manner. CLas exposure impacted cuticular proteins and enzymes involved in chitin degradation, as well as energy metabolism and abundance of the endosymbiont 'Candidatus Profftella armatura' in both sexes similarly. Notably, diaphorin, a toxic Profftella-derived metabolite, was more abundant in both sexes with CLas exposure. The responses reported here resulted from a combination of CLas colonization of D. citri as well as the effect of CLas infection on C. medica. Elucidating these impacts on D. citri and their endosymbionts contributes to our understanding of the HLB pathosystem and identifies the responses potentially critical to limiting or promoting CLas acquisition and propagation in both sexes.

Longitudinal Transcriptomic, Proteomic, and Metabolomic Analysis of Citrus limon Response to Graft Inoculation by Candidatus Liberibacter asiaticus.

Presymptomatic detection of citrus trees infected with Candidatus Liberibacter asiaticus (CLas), the bacterial pathogen associated with Huanglongbing (HLB; citrus greening disease), is critical to controlling the spread of the disease. To test whether infected citrus trees produce systemic signals that may be used for indirect disease detection, lemon (Citrus limon) plants were graft-inoculated with either CLas-infected or control (CLas-) budwood, and leaf samples were longitudinally collected over 46 weeks and analyzed for plant changes associated with CLas infection. RNA, protein, and metabolite samples extracted from leaves were analyzed using RNA-Seq, mass spectrometry, and 1H NMR spectroscopy, respectively. Significant differences in specific transcripts, proteins, and metabolites were observed between CLas-infected and control plants as early as 2 weeks post graft (wpg). The most dramatic differences between the transcriptome and proteome of CLas-infected and control plants were observed at 10 wpg, including coordinated increases in transcripts and proteins of citrus orthologs of known plant defense genes. This integrated approach to quantifying plant molecular changes in leaves of CLas-infected plants supports the development of diagnostic technology for presymptomatic or early disease detection as part of efforts to control the spread of HLB into uninfected citrus groves.

Longitudinal Transcriptomic, Proteomic, and Metabolomic Analyses of Citrus sinensis (L.) Osbeck Graft-Inoculated with "Candidatus Liberibacter asiaticus".

"Candidatus Liberibacter asiaticus" (CLas) is the bacterium associated with the citrus disease Huanglongbing (HLB). Current CLas detection methods are unreliable during presymptomatic infection, and understanding CLas pathogenicity to help develop new detection techniques is challenging because CLas has yet to be isolated in pure culture. To understand how CLas affects citrus metabolism and whether infected plants produce systemic signals that can be used to develop improved detection techniques, leaves from Washington Navel orange (Citrus sinensis (L.) Osbeck) plants were graft-inoculated with CLas and longitudinally studied using transcriptomics (RNA sequencing), proteomics (liquid chromatography-tandem mass spectrometry), and metabolomics (proton nuclear magnetic resonance). Photosynthesis gene expression and protein levels were lower in infected plants compared to controls during late infection, and lower levels of photosynthesis proteins were identified as early as 8 weeks post-grafting. These changes coordinated with higher sugar concentrations, which have been shown to accumulate during HLB. Cell wall modification and degradation gene expression and proteins were higher in infected plants during late infection. Changes in gene expression and proteins related to plant defense were observed in infected plants as early as 8 weeks post-grafting. These results reveal coordinated changes in greenhouse navel leaves during CLas infection at the transcript, protein, and metabolite levels, which can inform of biomarkers of early infection.

Candidatus Liberibacter asiaticus Minimally Alters Expression of Immunity and Metabolism Proteins in Hemolymph of Diaphorina citri, the Insect Vector of Huanglongbing.

Huanglongbing (HLB), also known as citrus greening disease, is the most serious disease of citrus plants. It is associated with the Gram-negative bacterium ' Candidatus Liberibacter asiaticus' ( CLas), which is transmitted between host plants by the hemipteran insect vector Diaphorina citri in a circulative, propagative manner involving specific interactions with various insect tissues including the hemolymph, fluid that occupies the body cavity akin to insect blood. High resolution quantitative mass spectrometry was performed to investigate the effect of CLas exposure on D. citri hemolymph at the proteome level. In contrast to the broad proteome effects on hundreds of proteins and a diverse array of metabolic pathways previously reported in gut and whole insect proteome analyses, the effect of CLas on the hemolymph was observed to be highly specific, restricted to key immunity and metabolism pathways, and lower in magnitude than that previously observed in the whole insect body and gut. Vitellogenins were abundantly expressed and CLas-responsive. Gene-specific RNA expression analysis suggests that these proteins are expressed in both male and female insects and may have roles outside of reproductive vitellogenesis. Proteins for fatty acid synthesis were found to be up-regulated, along with metabolic proteins associated with energy production, supported at the organismal level by the previously published observation that D. citri individuals experience a higher level of hunger when reared on CLas-infected plants. Prediction of post-translational modifications identified hemolymph proteins with phosphorylation and acetylation upon CLas exposure. Proteins derived from the three most prominent bacterial endosymbionts of the psyllid were also detected in the hemolymph, and several of these have predicted secretion signals. A DNAK protein, the bacterial HSP70, detected in the hemolymph expressed from Wolbachia pipientis was predicted to encode a eukaryotic nuclear localization signal. Taken together, these data show specific changes to immunity and metabolism in D. citri hemolymph involving host and endosymbiont proteins. These data provide a novel context for proteomic changes seen in other D. citri tissues in response to CLas and align with organismal data on the effects of CLas on D. citri metabolism and reproduction.

Crystal structure of diaphorin methanol monosolvate isolated from Diaphorina citri Kuwayama, the insect vector of citrus greening disease.

The title compound C22H39NO9·CH3OH [systematic name: (S)-N-((S)-{(2S,4R,6R)-6-[(S)-2,3-di-hydroxy-prop-yl]-4-hy-droxy-5,5-di-methyl-tetra-hydro-2H-pyran-2-yl}(hy-droxy)meth-yl)-2-hy-droxy-2-[(2R,5R,6R)-2-meth-oxy-5,6-dimeth-yl-4-methyl-ene-tetra-hydro-2H-pyran-2-yl]acetamide methanol monosolvate], was isolated from the Asian citrus psyllid, Diaphorina citri Kuwayama, and crystallizes in the space group P21. 'Candidatus Profftella armatura' a bacterial endosymbiont of D. citri, biosynthesizes diaphorin, which is a hybrid polyketide-nonribosomal peptide comprising two highly substituted tetra-hydro-pyran rings joined by an N-acyl aminal bridge [Nakabachi et al. (2013 ▸). Curr. Biol.23, 1478-1484]. The crystal structure of the title compound establishes the complete relative configuration of diaphorin, which agrees at all nine chiral centers with the structure of the methanol monosolvate of the di-p-bromo-benzoate derivative of pederin, a biogenically related compound whose crystal structure was reported previously [Furusaki et al. (1968 ▸). Tetra-hedron Lett.9, 6301-6304]. Thus, the absolute configuration of diaphorin is proposed by analogy to that of pederin.

Metabolic Interplay between the Asian Citrus Psyllid and Its Profftella Symbiont: An Achilles' Heel of the Citrus Greening Insect Vector.

'Candidatus Liberibacter asiaticus' (CLas), the bacterial pathogen associated with citrus greening disease, is transmitted by Diaphorina citri, the Asian citrus psyllid. Interactions among D. citri and its microbial endosymbionts, including 'Candidatus Profftella armatura', are likely to impact transmission of CLas. We used quantitative mass spectrometry to compare the proteomes of CLas(+) and CLas(-) populations of D. citri, and found that proteins involved in polyketide biosynthesis by the endosymbiont Profftella were up-regulated in CLas(+) insects. Mass spectrometry analysis of the Profftella polyketide diaphorin in D. citri metabolite extracts revealed the presence of a novel diaphorin-related polyketide and the ratio of these two polyketides was changed in CLas(+) insects. Insect proteins differentially expressed between CLas(+) and CLas(-) D. citri included defense and immunity proteins, proteins involved in energy storage and utilization, and proteins involved in endocytosis, cellular adhesion, and cytoskeletal remodeling which are associated with microbial invasion of host cells. Insight into the metabolic interdependence between the insect vector, its endosymbionts, and the citrus greening pathogen reveals novel opportunities for control of this disease, which is currently having a devastating impact on citrus production worldwide.

Adaptation to nicotine feeding in Myzus persicae.

Lineages of the generalist hemipteran herbivore Myzus persicae (green peach aphid) that have expanded their host range to include tobacco often have elevated nicotine tolerance. The tobacco-adapted M. persicae lineage used in this study was able to reproduce on nicotine-containing artificial diets at concentrations that were 15-fold higher than those that were lethal to a non-adapted M. persicae lineage. Fecundity of the nicotine-tolerant M. persicae lineage was increased by 100 µM nicotine in artificial diet, suggesting that this otherwise toxic alkaloid can serve as a feeding stimulant at low concentrations. This lineage also was pre-adapted to growth on tobacco, exhibiting no drop in fecundity when it was moved onto tobacco from a different host plant. Although growth of the non-tobacco-adapted M. persicae lineage improved after three generations on tobacco, this higher reproductive rate was not associated with increased nicotine tolerance. Myzus persicae gene expression microarrays were used to identify transcripts that are up-regulated in response to nicotine in the tobacco-adapted lineage. Induced expression was found for CYP6CY3, which detoxifies nicotine in M. persicae, other genes encoding known classes of detoxifying enzymes, and genes encoding secreted M. persicae salivary proteins.

Adaptation to nicotine in the facultative tobacco-feeding hemipteran Bemisia tabaci.

BACKGROUND: Plant defensive metabolites such as nicotine can provide barriers to host-range expansion by generalist herbivores. Nicotine is one of the most abundant and toxic plant secondary metabolites in nature and is defined by high toxicity to plant-feeding insects. There is significant variation in nicotine tolerance among Bemisia tabaci (tobacco whitefly) isolates. Some nicotine-tolerant B. tabaci strains can consume 40-fold higher nicotine levels than susceptible strains, and also show cross-resistance to neonicotinoid insecticides. In this study, biological and molecular assays were used to investigate the responses of B. tabaci strains that differ in their ability to tolerate dietary nicotine. RESULTS: Egg laying and honeydew secretion bioassays as well as gene expression microarrays were used to measure B. tabaci biological parameters and gene transcripts misregulated in response to nicotine in resistant and susceptible strains. The resistant B. tabaci strain laid significantly fewer eggs and excreted more honeydew on a tobacco strain with high levels of nicotine, suggesting a fitness cost effect. The molecular response was drastic in the susceptible strain, while the resistant strain exhibited moderate response. Higher expression of the previously identified CYP6CM1 P450 monooxygenase gene related to the resistance to neonicotinoids, as well as other P450s and metabolic genes, was identified in the resistant and susceptible strains after exposure to nicotine. CONCLUSIONS: Nicotine is a very toxic plant natural compound, and its mode of action resembles that of synthetic neonicotinoids. The biological and molecular responses observed in this study suggest that nicotine may play an important role in providing barriers for host-plant expansion by generalists, and may act as a natural factor that contributes to the development of insect populations resistant to synthetic pesticides.

Insecticide resistance mechanisms in the green peach aphid Myzus persicae (Hemiptera: Aphididae) I: A transcriptomic survey.

BACKGROUND: Insecticide resistance is one of the best examples of rapid micro-evolution found in nature. Since the development of the first synthetic insecticide in 1939, humans have invested considerable effort to stay ahead of resistance phenotypes that repeatedly develop in insects. Aphids are a group of insects that have become global pests in agriculture and frequently exhibit insecticide resistance. The green peach aphid, Myzus persicae, has developed resistance to at least seventy different synthetic compounds, and different insecticide resistance mechanisms have been reported worldwide. METHODOLOGY/PRINCIPAL FINDINGS: To further characterize this resistance, we analyzed genome-wide transcriptional responses in three genotypes of M. persicae, each exhibiting different resistance mechanisms, in response to an anti-cholinesterase insecticide. The sensitive genotype (exhibiting no resistance mechanism) responded to the insecticide by up-regulating 183 genes primarily ones related to energy metabolism, detoxifying enzymes, proteins of extracellular transport, peptidases and cuticular proteins. The second genotype (resistant through a kdr sodium channel mutation), up-regulated 17 genes coding for detoxifying enzymes, peptidase and cuticular proteins. Finally, a multiply resistant genotype (carrying kdr and a modified acetylcholinesterase), up-regulated only 7 genes, appears not to require induced insecticide detoxification, and instead down-regulated many genes. CONCLUSIONS/SIGNIFICANCE: This study suggests strongly that insecticide resistance in M. persicae is more complex that has been described, with the participation of a broad array of resistance mechanisms. The sensitive genotype exhibited the highest transcriptional plasticity, accounting for the wide range of potential adaptations to insecticides that this species can evolve. In contrast, the multiply resistant genotype exhibited a low transcriptional plasticity, even for the expression of genes encoding enzymes involved in insecticide detoxification. Our results emphasize the value of microarray studies to search for regulated genes in insects, but also highlights the many ways those different genotypes can assemble resistant phenotypes depending on the environmental pressure.

Immunity and other defenses in pea aphids, Acyrthosiphon pisum.

BACKGROUND: Recent genomic analyses of arthropod defense mechanisms suggest conservation of key elements underlying responses to pathogens, parasites and stresses. At the center of pathogen-induced immune responses are signaling pathways triggered by the recognition of fungal, bacterial and viral signatures. These pathways result in the production of response molecules, such as antimicrobial peptides and lysozymes, which degrade or destroy invaders. Using the recently sequenced genome of the pea aphid (Acyrthosiphon pisum), we conducted the first extensive annotation of the immune and stress gene repertoire of a hemipterous insect, which is phylogenetically distantly related to previously characterized insects models. RESULTS: Strikingly, pea aphids appear to be missing genes present in insect genomes characterized to date and thought critical for recognition, signaling and killing of microbes. In line with results of gene annotation, experimental analyses designed to characterize immune response through the isolation of RNA transcripts and proteins from immune-challenged pea aphids uncovered few immune-related products. Gene expression studies, however, indicated some expression of immune and stress-related genes. CONCLUSIONS: The absence of genes suspected to be essential for the insect immune response suggests that the traditional view of insect immunity may not be as broadly applicable as once thought. The limitations of the aphid immune system may be representative of a broad range of insects, or may be aphid specific. We suggest that several aspects of the aphid life style, such as their association with microbial symbionts, could facilitate survival without strong immune protection.

Genomic resources for Myzus persicae: EST sequencing, SNP identification, and microarray design.

BACKGROUND: The green peach aphid, Myzus persicae (Sulzer), is a world-wide insect pest capable of infesting more than 40 plant families, including many crop species. However, despite the significant damage inflicted by M. persicae in agricultural systems through direct feeding damage and by its ability to transmit plant viruses, limited genomic information is available for this species. RESULTS: Sequencing of 16 M. persicae cDNA libraries generated 26,669 expressed sequence tags (ESTs). Aphids for library construction were raised on Arabidopsis thaliana, Nicotiana benthamiana, Brassica oleracea, B. napus, and Physalis floridana (with and without Potato leafroll virus infection). The M. persicae cDNA libraries include ones made from sexual and asexual whole aphids, guts, heads, and salivary glands. In silico comparison of cDNA libraries identified aphid genes with tissue-specific expression patterns, and gene expression that is induced by feeding on Nicotiana benthamiana. Furthermore, 2423 genes that are novel to science and potentially aphid-specific were identified. Comparison of cDNA data from three aphid lineages identified single nucleotide polymorphisms that can be used as genetic markers and, in some cases, may represent functional differences in the protein products. In particular, non-conservative amino acid substitutions in a highly expressed gut protease may be of adaptive significance for M. persicae feeding on different host plants. The Agilent eArray platform was used to design an M. persicae oligonucleotide microarray representing over 10,000 unique genes. CONCLUSION: New genomic resources have been developed for M. persicae, an agriculturally important insect pest. These include previously unknown sequence data, a collection of expressed genes, molecular markers, and a DNA microarray that can be used to study aphid gene expression. These resources will help elucidate the adaptations that allow M. persicae to develop compatible interactions with its host plants, complementing ongoing work illuminating plant molecular responses to phloem-feeding insects.