Whitefly-Plant Interactions: An Integrated Molecular Perspective.

The rapid advances in available transcriptomic and genomic data and our understanding of the physiology and biochemistry of whitefly-plant interactions have allowed us to gain new and significant insights into the biology of whiteflies and their successful adaptation to host plants. In this review, we provide a comprehensive overview of the mechanisms that whiteflies have evolved to overcome the challenges of feeding on phloem sap. We also highlight the evolution and functions of gene families involved in host perception, evaluation, and manipulation; primary metabolism; and metabolite detoxification. We discuss the emerging themes in plant immunity to whiteflies, focusing on whitefly effectors and their sites of action in plant defense-signaling pathways. We conclude with a discussion of advances in the genetic manipulation of whiteflies and the potential that they hold for exploring the interactions between whiteflies and their host plants, as well as the development of novel strategies for the genetic control of whiteflies.

Integrative transcriptomics reveals association of abscisic acid and lignin pathways with cassava whitefly resistance.

BACKGROUND: Whiteflies are a global threat to crop yields, including the African subsistence crop cassava (Manihot esculenta). Outbreaks of superabundant whitefly populations throughout Eastern and Central Africa in recent years have dramatically increased the pressures of whitefly feeding and virus transmission on cassava. Whitefly-transmitted viral diseases threaten the food security of hundreds of millions of African farmers, highlighting the need for developing and deploying whitefly-resistant cassava. However, plant resistance to whiteflies remains largely poorly characterized at the genetic and molecular levels. Knowledge of cassava-defense programs also remains incomplete, limiting characterization of whitefly-resistance mechanisms. To better understand the genetic basis of whitefly resistance in cassava, we define the defense hormone- and Aleurotrachelus socialis (whitefly)-responsive transcriptome of whitefly-susceptible (COL2246) and whitefly-resistant (ECU72) cassava using RNA-seq. For broader comparison, hormone-responsive transcriptomes of Arabidopsis thaliana were also generated. RESULTS: Whitefly infestation, salicylic acid (SA), jasmonic acid (JA), ethylene (ET), and abscisic acid (ABA) transcriptome responses of ECU72 and COL2246 were defined and analyzed. Strikingly, SA responses were largely reciprocal between the two cassava genotypes and we suggest candidate regulators. While susceptibility was associated with SA in COL2246, resistance to whitefly in ECU72 was associated with ABA, with SA-ABA antagonism observed. This was evidenced by expression of genes within the SA and ABA pathways and hormone levels during A. socialis infestation. Gene-enrichment analyses of whitefly- and hormone-responsive genes suggest the importance of fast-acting cell wall defenses (e.g., elicitor recognition, lignin biosynthesis) during early infestation stages in whitefly-resistant ECU72. A surge of ineffective immune and SA responses characterized the whitefly-susceptible COL2246's response to late-stage nymphs. Lastly, in comparison with the model plant Arabidopsis, cassava's hormone-responsive genes showed striking divergence in expression. CONCLUSIONS: This study provides the first characterization of cassava's global transcriptome responses to whitefly infestation and defense hormone treatment. Our analyses of ECU72 and COL2246 uncovered possible whitefly resistance/susceptibility mechanisms in cassava. Comparative analysis of cassava and Arabidopsis demonstrated that defense programs in Arabidopsis may not always mirror those in crop species. More broadly, our hormone-responsive transcriptomes will also provide a baseline for the cassava community to better understand global responses to other yield-limiting pests/pathogens.

Geographical survey of the mycobiome and microbiome of Southern California glassy-winged sharpshooters.

The glassy-winged sharpshooter, Homalodisca vitripennis Germar, is an invasive xylem-feeding leafhopper with a devastating economic impact on California agriculture through transmission of the plant pathogen, Xylella fastidiosa. While studies have focused on X. fastidiosa or known symbionts of H. vitripennis, little work has been done at the scale of the microbiome (the bacterial community) or mycobiome (the fungal community). Here, we characterize the mycobiome and the microbiome of H. vitripennis across Southern California and explore correlations with captivity and host insecticide resistance status. Using high-throughput sequencing of the ribosomal internal transcribed spacer 1 region and the 16S rRNA gene to profile the mycobiome and microbiome, respectively, we found that while the H. vitripennis mycobiome significantly varied across Southern California, the microbiome did not. We also observed a significant difference in both the mycobiome and microbiome between captive and wild H. vitripennis. Finally, we found that the mycobiome, but not the microbiome, was correlated with insecticide resistance status in wild H. vitripennis. This study serves as a foundational look at the H. vitripennis mycobiome and microbiome across Southern California. Future work should explore the putative link between microbes and insecticide resistance status and investigate whether microbial communities should be considered in H. vitripennis management practices. IMPORTANCE The glassy-winged sharpshooter is an invasive leafhopper that feeds on the xylem of plants and transmits the devastating pathogen, Xylella fastidiosa, resulting in significant economic damage to California's agricultural system. While studies have focused on this pathogen or obligate symbionts of the glassy-winged sharpshooter, there is limited knowledge of the bacterial and fungal communities that make up its microbiome and mycobiome. To address this knowledge gap, we explored the composition of the mycobiome and the microbiome of the glassy-winged sharpshooter across Southern California and identified differences associated with geography, captivity, and host insecticide resistance status. Understanding sources of variation in the microbial communities associated with the glassy-winged sharpshooter is an important consideration for developing management strategies to control this invasive insect. This study is a first step toward understanding the role microbes may play in the glassy-winged sharpshooter's resistance to insecticides.

The tomato chloroplast stromal proteome compendium elucidated by leveraging a plastid protein-localization prediction Atlas.

Tomato (Solanum lycopersicum) is a model species for studying fruit development, wounding, herbivory, and pathogen attack. Despite tomato's world-wide economic importance and the role of chloroplasts as metabolic hubs and integrators of environmental cues, little is known about the stromal proteome of tomato. Using a high-yielding protocol for chloroplast and stromal protein isolation, MudPIT nano-LC-MS/MS analyses, a robust in-house protein database (the Atlas) for predicting the plastid localization of tomato proteins, and rigorous selection criteria for inclusion/exclusion in the stromal proteome, we identified 1,278 proteins of the tomato stromal proteome. We provide one of the most robust stromal proteomes available to date with empirical evidence for 545 and 92 proteins not previously described for tomato plastids and the Arabidopsis stroma, respectively. The relative abundance of tomato stromal proteins was determined using the exponentially modified protein abundance index (emPAI). Comparison of the abundance of tomato and Arabidopsis stromal proteomes provided evidence for the species-specific nature of stromal protein homeostasis. The manual curation of the tomato stromal proteome classified proteins into ten functional categories resulting in an accessible compendium of tomato chloroplast proteins. After curation, only 91 proteins remained as unknown, uncharacterized or as enzymes with unknown functions. The curation of the tomato stromal proteins also indicated that tomato has a number of paralogous proteins, not present in Arabidopsis, which accumulated to different levels in chloroplasts. As some of these proteins function in key metabolic pathways or in perceiving or transmitting signals critical for plant adaptation to biotic and abiotic stress, these data suggest that tomato may modulate the bidirectional communication between chloroplasts and nuclei in a novel manner. The stromal proteome provides a fertile ground for future mechanistic studies in the field of tomato chloroplast-nuclear signaling and are foundational for our goal of elucidating the dynamics of the stromal proteome controlled by the solanaceous-specific, stromal, and wound-inducible leucine aminopeptidase A of tomato.

A tripartite rheostat controls self-regulated host plant resistance to insects.

Plants deploy receptor-like kinases and nucleotide-binding leucine-rich repeat receptors to confer host plant resistance (HPR) to herbivores1. These gene-for-gene interactions between insects and their hosts have been proposed for more than 50 years2. However, the molecular and cellular mechanisms that underlie HPR have been elusive, as the identity and sensing mechanisms of insect avirulence effectors have remained unknown. Here we identify an insect salivary protein perceived by a plant immune receptor. The BPH14-interacting salivary protein (BISP) from the brown planthopper (Nilaparvata lugens Stål) is secreted into rice (Oryza sativa) during feeding. In susceptible plants, BISP targets O. satvia RLCK185 (OsRLCK185; hereafter Os is used to denote O. satvia-related proteins or genes) to suppress basal defences. In resistant plants, the nucleotide-binding leucine-rich repeat receptor BPH14 directly binds BISP to activate HPR. Constitutive activation of Bph14-mediated immunity is detrimental to plant growth and productivity. The fine-tuning of Bph14-mediated HPR is achieved through direct binding of BISP and BPH14 to the selective autophagy cargo receptor OsNBR1, which delivers BISP to OsATG8 for degradation. Autophagy therefore controls BISP levels. In Bph14 plants, autophagy restores cellular homeostasis by downregulating HPR when feeding by brown planthoppers ceases. We identify an insect saliva protein sensed by a plant immune receptor and discover a three-way interaction system that offers opportunities for developing high-yield, insect-resistant crops.

Transcriptome and population structure of glassy-winged sharpshooters (Homalodisca vitripennis) with varying insecticide resistance in southern California.

BACKGROUND: Homalodisca vitripennis Germar, the glassy-winged sharpshooter, is an invasive insect in California and a critical threat to agriculture through its transmission of the plant pathogen, Xylella fastidiosa. Quarantine, broad-spectrum insecticides, and biological control have been used for population management of H. vitripennis since its invasion and subsequent proliferation throughout California. Recently wide-spread neonicotinoid resistance has been detected in populations of H. vitripennis in the southern portions of California's Central Valley. In order to better understand potential mechanisms of H. vitripennis neonicotinoid resistance, we performed RNA sequencing on wild-caught insecticide-resistant and relatively susceptible sharpshooters to profile their transcriptome and population structure. RESULTS: We identified 81 differentially expressed genes with higher expression in resistant individuals. The significant largest differentially expressed candidate gene linked to resistance status was a cytochrome P450 gene with similarity to CYP6A9. Furthermore, we observed an over-enrichment of GO terms representing functions supportive of roles in resistance mechanisms (cytochrome P450s, M13 peptidases, and cuticle structural proteins). Finally, we saw no evidence of broad-scale population structure, perhaps due to H. vitripennis' relatively recent introduction to California or due to the relatively small geographic scale investigated here. CONCLUSIONS: In this work, we characterized the transcriptome of insecticide-resistant and susceptible H. vitripennis and identified candidate genes that may be involved in resistance mechanisms for this species. Future work should seek to build on the transcriptome profiling performed here to confirm the role of the identified genes, particularly the cytochrome P450, in resistance in H. vitripennis. We hope this work helps aid future population management strategies for this and other species with growing insecticide resistance.

Gene Editing and Genetic Control of Hemipteran Pests: Progress, Challenges and Perspectives.

The origin of the order Hemiptera can be traced to the late Permian Period more than 230 MYA, well before the origin of flowering plants 100 MY later in during the Cretaceous period. Hemipteran species consume their liquid diets using a sucking proboscis; for phytophagous hemipterans their mouthparts (stylets) are elegant structures that enable voracious feeding from plant xylem or phloem. This adaptation has resulted in some hemipteran species becoming globally significant pests of agriculture resulting in significant annual crop losses. Due to the reliance on chemical insecticides for the control of insect pests in agricultural settings, many hemipteran pests have evolved resistance to insecticides resulting in an urgent need to develop new, species-specific and environmentally friendly methods of pest control. The rapid advances in CRISPR/Cas9 technologies in model insects such as Drosophila melanogaster, Tribolium castaneum, Bombyx mori, and Aedes aegypti has spurred a new round of innovative genetic control strategies in the Diptera and Lepidoptera and an increased interest in assessing genetic control technologies for the Hemiptera. Genetic control approaches in the Hemiptera have, to date, been largely overlooked due to the problems of introducing genetic material into the germline of these insects. The high frequency of CRISPR-mediated mutagenesis in model insect species suggest that, if the delivery problem for Hemiptera could be solved, then gene editing in the Hemiptera might be quickly achieved. Significant advances in CRISPR/Cas9 editing have been realized in nine species of Hemiptera over the past 4 years. Here we review progress in the Hemiptera and discuss the challenges and opportunities for extending contemporary genetic control strategies into species in this agriculturally important insect orderr.

Efficient CRISPR/Cas9-mediated genome modification of the glassy-winged sharpshooter Homalodisca vitripennis (Germar).

CRISPR/Cas9 technology enables the extension of genetic techniques into insect pests previously refractory to genetic analysis. We report the establishment of genetic analysis in the glassy-winged sharpshooter (GWSS), Homalodisca vitripennis, which is a significant leafhopper pest of agriculture in California. We use a novel and simple approach of embryo microinjection in situ on the host plant and obtain high frequency mutagenesis, in excess of 55%, of the cinnabar and white eye pigmentation loci. Through pair matings, we obtained 100% transmission of w and cn alleles to the G3 generation and also established that both genes are located on autosomes. Our analysis of wing phenotype revealed an unexpected discovery of the participation of pteridine pigments in wing and wing-vein coloration, indicating a role for these pigments beyond eye color. We used amplicon sequencing to examine the extent of off-target mutagenesis in adults arising from injected eggs, which was found to be negligible or non-existent. Our data show that GWSS can be easily developed as a genetic model system for the Hemiptera, enabling the study of traits that contribute to the success of invasive pests and vectors of plant pathogens. This will facilitate novel genetic control strategies.

Improved draft reference genome for the Glassy-winged Sharpshooter (Homalodisca vitripennis), a vector for Pierce's disease.

Homalodisca vitripennis (Hemiptera: Cicadellidae), known as the glassy-winged sharpshooter, is a xylem feeding leafhopper and an important agricultural pest as a vector of Xylella fastidiosa, which causes Pierce's disease in grapes and a variety of other scorch diseases. The current H. vitripennis reference genome from the Baylor College of Medicine's i5k pilot project is a 1.4-Gb assembly with 110,000 scaffolds, which still has significant gaps making identification of genes difficult. To improve on this effort, we used a combination of Oxford Nanopore long-read sequencing technology combined with Illumina sequencing reads to generate a better assembly and first-pass annotation of the whole genome sequence of a wild-caught Californian (Tulare County) individual of H. vitripennis. The improved reference genome assembly for H. vitripennis is 1.93-Gb in length (21,254 scaffolds, N50 = 650 Mb, BUSCO completeness = 94.3%), with 33.06% of the genome masked as repetitive. In total, 108,762 gene models were predicted including 98,296 protein-coding genes and 10,466 tRNA genes. As an additional community resource, we identified 27 orthologous candidate genes of interest for future experimental work including phenotypic marker genes like white. Furthermore, as part of the assembly process, we generated four endosymbiont metagenome-assembled genomes, including a high-quality near complete 1.7-Mb Wolbachia sp. genome (1 scaffold, CheckM completeness = 99.4%). The improved genome assembly and annotation for H. vitripennis, curated set of candidate genes, and endosymbiont MAGs will be invaluable resources for future research of H. vitripennis.

Bph30 confers resistance to brown planthopper by fortifying sclerenchyma in rice leaf sheaths.

Phloem-feeding insects cause massive losses in agriculture and horticulture. Host plant resistance to phloem-feeding insects is often mediated by changes in phloem composition, which deter insect settling and feeding and decrease viability. Here, we report that rice plant resistance to the phloem-feeding brown planthopper (BPH) is associated with fortification of the sclerenchyma tissue, which is located just beneath the epidermis and a cell layer or two away from the vascular bundle in the rice leaf sheath. We found that BPHs prefer to feed on the smooth and soft region on the surface of rice leaf sheaths called the long-cell block. We identified Bph30 as a rice BPH resistance gene that prevents BPH stylets from reaching the phloem due to the fortified sclerenchyma. Bph30 is strongly expressed in sclerenchyma cells and enhances cellulose and hemicellulose synthesis, making the cell walls stiffer and sclerenchyma thicker. The structurally fortified sclerenchyma is a formidable barrier preventing BPH stylets from penetrating the leaf sheath tissues and arriving at the phloem to feed. Bph30 belongs to a novel gene family, encoding a protein with two leucine-rich domains. Another member of the family, Bph40, also conferred resistance to BPH. Collectively, the fortified sclerenchyma-mediated resistance mechanism revealed in this study expands our understanding of plant-insect interactions and opens a new path for controlling planthoppers in rice.

Virus-induced phytohormone dynamics and their effects on plant-insect interactions.

Attacks on plants by both viruses and their vectors is common in nature. Yet the dynamics of the plant-virus-vector tripartite system, in particular the effects of viral infection on plant-insect interactions, have only begun to emerge in the last decade. Viruses can modulate the interactions between insect vectors and plants via the jasmonate, salicylic acid and ethylene phytohormone pathways, resulting in changes in fitness and viral transmission capacity of their insect vectors. Virus infection of plants may also modulate other phytohormones, such as auxin, gibberellins, cytokinins, brassinosteroids and abscisic acid, with yet undefined consequences on plant-insect interactions. Moreover, virus infection in plants may incur changes to other plant traits, such as nutrition and secondary metabolites, that potentially contribute to virus-associated, phytohormone-mediated manipulation of plant-insect interactions. In this article, we review the research progress, discuss issues related to the complexity and variability of the viral modulation of plant interactions with insect vectors, and suggest future directions of research in this field.

Can CRISPR gene drive work in pest and beneficial haplodiploid species?

Gene drives based on CRISPR/Cas9 have the potential to reduce the enormous harm inflicted by crop pests and insect vectors of human disease, as well as to bolster valued species. In contrast with extensive empirical and theoretical studies in diploid organisms, little is known about CRISPR gene drive in haplodiploids, despite their immense global impacts as pollinators, pests, natural enemies of pests, and invasive species in native habitats. Here, we analyze mathematical models demonstrating that, in principle, CRISPR homing gene drive can work in haplodiploids, as well as at sex-linked loci in diploids. However, relative to diploids, conditions favoring the spread of alleles deleterious to haplodiploid pests by CRISPR gene drive are narrower, the spread is slower, and resistance to the drive evolves faster. By contrast, the spread of alleles that impose little fitness cost or boost fitness was not greatly hindered in haplodiploids relative to diploids. Therefore, altering traits to minimize damage caused by harmful haplodiploids, such as interfering with transmission of plant pathogens, may be more likely to succeed than control efforts based on introducing traits that reduce pest fitness. Enhancing fitness of beneficial haplodiploids with CRISPR gene drive is also promising.

Methodology: an optimized, high-yield tomato leaf chloroplast isolation and stroma extraction protocol for proteomics analyses and identification of chloroplast co-localizing proteins.

BACKGROUND: Chloroplasts are critical organelles that perceive and convey metabolic and stress signals to different cellular components, while remaining the seat of photosynthesis and a metabolic factory. The proteomes of intact leaves, chloroplasts, and suborganellar fractions of plastids have been evaluated in the model plant Arabidopsis, however fewer studies have characterized the proteomes of plastids in crops. Tomato (Solanum lycopersicum) is an important world-wide crop and a model system for the study of wounding, herbivory and fruit ripening. While significant advances have been made in understanding proteome and metabolome changes in fruit ripening, far less is known about the tomato chloroplast proteome or its subcompartments. RESULTS: With the long-term goal of understanding chloroplast proteome dynamics in response to stress, we describe a high-yielding method to isolate intact tomato chloroplasts and stromal proteins for proteomic studies. The parameters that limit tomato chloroplast yields were identified and revised to increase yields. Compared to published data, our optimized method increased chloroplast yields by 6.7- and 4.3-fold relative to published spinach and Arabidopsis leaf protocols, respectively; furthermore, tomato stromal protein yields were up to 79-fold higher than Arabidopsis stromal proteins yields. We provide immunoblot evidence for the purity of the stromal proteome isolated using our enhanced methods. In addition, we leverage our nanoliquid chromatography tandem mass spectrometry (nanoLC-MS/MS) data to assess the quality of our stromal proteome. Using strict criteria, proteins detected by 1 peptide spectral match, by one peptide, or were sporadically detected were designated as low-level contaminating proteins. A set of 254 proteins that reproducibly co-isolated with the tomato chloroplast stroma were identified. The subcellular localization, frequency of detection, normalized spectral abundance, and functions of the co-isolating proteins are discussed. CONCLUSIONS: Our optimized method for chloroplast isolation increased the yields of tomato chloroplasts eightfold enabling the proteomics analysis of the chloroplast stromal proteome. The set of 254 proteins that co-isolate with the chloroplast stroma provides opportunities for developing a better understanding of the extensive and dynamic interactions of chloroplasts with other organelles. These co-isolating proteins also have the potential for expanding our knowledge of proteins that are co-localized in multiple subcellular organelles.

Intracellular symbionts drive sex ratio in the whitefly by facilitating fertilization and provisioning of B vitamins.

Symbionts can regulate animal reproduction in multiple ways, but the underlying physiological and biochemical mechanisms remain largely unknown. The presence of multiple lineages of maternally inherited, intracellular symbionts (the primary and secondary symbionts) in terrestrial arthropods is widespread in nature. However, the biological, metabolic, and evolutionary role of co-resident secondary symbionts for hosts is poorly understood. The bacterial symbionts Hamiltonella and Arsenophonus have very high prevalence in two globally important pests, the whiteflies Bemisia tabaci and Trialeurodes vaporariorum, respectively. Both symbionts coexist with the primary symbiont Portiera in the same host cell (bacteriocyte) and are maternally transmitted. We found that elimination of both Hamiltonella and Arsenophonous by antibiotic treatment reduced the percentage of female offspring in whiteflies. Microsatellite genotyping and cytogenetic analysis revealed that symbiont deficiency inhibited fertilization in whiteflies, leading to more haploid males with one maternal allele, which is consistent with distorted sex ratio in whiteflies. Quantification of essential amino acids and B vitamins in whiteflies indicated that symbiont deficiency reduced B vitamin levels, and dietary B vitamin supplementation rescued fitness of whiteflies. This study, for the first time, conclusively demonstrates that these two intracellular symbionts affect sex ratios in their whitefly hosts by regulating fertilization and supplying B vitamins. Our results reveal that both symbionts have the convergent function of regulating reproduction in phylogenetically-distant whitefly species. The 100% frequency, the inability of whiteflies to develop normally without their symbiont, and rescue with B vitamins suggests that both symbionts may be better considered co-primary symbionts.

Genome-wide analyses of cassava Pathogenesis-related (PR) gene families reveal core transcriptome responses to whitefly infestation, salicylic acid and jasmonic acid.

BACKGROUND: Whiteflies are a threat to cassava (Manihot esculenta), an important staple food in many tropical/subtropical regions. Understanding the molecular mechanisms regulating cassava's responses against this pest is crucial for developing control strategies. Pathogenesis-related (PR) protein families are an integral part of plant immunity. With the availability of whole genome sequences, the annotation and expression programs of the full complement of PR genes in an organism can now be achieved. An understanding of the responses of the entire complement of PR genes during biotic stress and to the defense hormones, salicylic acid (SA) and jasmonic acid (JA), is lacking. Here, we analyze the responses of cassava PR genes to whiteflies, SA, JA, and other biotic aggressors. RESULTS: The cassava genome possesses 14 of the 17 plant PR families, with a total of 447 PR genes. A cassava PR gene nomenclature is proposed. Phylogenetic relatedness of cassava PR proteins to each other and to homologs in poplar, rice and Arabidopsis identified cassava-specific PR gene family expansions. The temporal programs of PR gene expression in response to the whitefly (Aleurotrachelus socialis) in four whitefly-susceptible cassava genotypes showed that 167 of the 447 PR genes were regulated after whitefly infestation. While the timing of PR gene expression varied, over 37% of whitefly-regulated PR genes were downregulated in all four genotypes. Notably, whitefly-responsive PR genes were largely coordinately regulated by SA and JA. The analysis of cassava PR gene expression in response to five other biotic stresses revealed a strong positive correlation between whitefly and Xanthomonas axonopodis and Cassava Brown Streak Virus responses and negative correlations between whitefly and Cassava Mosaic Virus responses. Finally, certain associations between PR genes in cassava expansions and response to biotic stresses were observed among PR families. CONCLUSIONS: This study represents the first genome-wide characterization of PR genes in cassava. PR gene responses to six biotic stresses and to SA and JA are demonstrably different to other angiosperms. We propose that our approach could be applied in other species to fully understand PR gene regulation by pathogens, pests and the canonical defense hormones SA and JA.

A metabolomics characterisation of natural variation in the resistance of cassava to whitefly.

BACKGROUND: Cassava whitefly outbreaks were initially reported in East and Central Africa cassava (Manihot esculenta Crantz) growing regions in the 1990's and have now spread to other geographical locations, becoming a global pest severely affecting farmers and smallholder income. Whiteflies impact plant yield via feeding and vectoring cassava mosaic and brown streak viruses, making roots unsuitable for food or trading. Deployment of virus resistant varieties has had little impact on whitefly populations and therefore development of whitefly resistant varieties is also necessary as part of integrated pest management strategies. Suitable sources of whitefly resistance exist in germplasm collections that require further characterization to facilitate and assist breeding programs. RESULTS: In the present work, a hierarchical metabolomics approach has been employed to investigate the underlying biochemical mechanisms associated with whitefly resistance by comparing two naturally occurring accessions of cassava, one susceptible and one resistant to whitefly. Quantitative differences between genotypes detected at pre-infestation stages were consistently observed at each time point throughout the course of the whitefly infestation. This prevalent differential feature suggests that inherent genotypic differences override the response induced by the presence of whitefly and that they are directly linked with the phenotype observed. The most significant quantitative changes relating to whitefly susceptibility were linked to the phenylpropanoid super-pathway and its linked sub-pathways: monolignol, flavonoid and lignan biosynthesis. These findings suggest that the lignification process in the susceptible variety is less active, as the susceptible accession deposits less lignin and accumulates monolignol intermediates and derivatives thereof, differences that are maintained during the time-course of the infestation. CONCLUSIONS: Resistance mechanism associated to the cassava whitefly-resistant accession ECU72 is an antixenosis strategy based on reinforcement of cell walls. Both resistant and susceptible accessions respond differently to whitefly attack at biochemical level, but the inherent metabolic differences are directly linked to the resistance phenotype rather than an induced response in the plant.

Extreme resistance: The GLK-Rx1 alliance.

Plants immune surveillance systems depend on nucleotide-binding leucine-rich repeat receptors (NLRs). A subset of NLRs are nuclear-localized, including Rx1, which confers an extreme immunity against potato virus X (PVX). As with many NLRs, the downstream signaling partners of Rx1 are unknown. Townsend et al. identify a Golden-like transcription factor that interacts with Rx1 and mediates antiviral immunity, providing the first insights into the specificity factors that enable the nonspecific DNA-binding Rx1 to confer extreme resistance to PVX.

Chlorophyte aspartyl aminopeptidases: Ancient origins, expanded families, new locations, and secondary functions.

M18 aspartyl aminopeptidases (DAPs) are well characterized in microbes and animals with likely functions in peptide processing and vesicle trafficking. In contrast, there is a dearth of knowledge on plant aminopeptidases with a preference for proteins and peptides with N-terminal acidic residues. During evolution of the Plantae, there was an expansion and diversification of the M18 DAPs. After divergence of the ancestral green algae from red and glaucophyte algae, a duplication yielded the DAP1 and DAP2 lineages. Subsequently DAP1 genes were lost in chlorophyte algae. A duplication of DAP2-related genes occurred early in green plant evolution. DAP2 genes were retained in land plants and picoeukaryotic algae and lost in green algae. In contrast, DAP2-like genes persisted in picoeukaryotic and green algae, while this lineage was lost in land plants. Consistent with this evolutionary path, Arabidopsis thaliana has two DAP gene lineages (AtDAP1 and AtDAP2). Similar to animal and yeast DAPs, AtDAP1 is localized to the cytosol or vacuole; while AtDAP2 harbors an N-terminal transit peptide and is chloroplast localized. His6-DAP1 and His6-DAP2 expressed in Escherichia coli were enzymatically active and dodecameric with masses exceeding 600 kDa. His6-DAP1 and His6-DAP2 preferentially hydrolyzed Asp-p-nitroanilide and Glu-p-nitroanilide. AtDAPs are highly conserved metallopeptidases activated by MnCl2 and inhibited by ZnCl2 and divalent ion chelators. The protease inhibitor PMSF inhibited and DTT stimulated both His6-DAP1 and His6-DAP2 activities suggesting a role for thiols in the AtDAP catalytic mechanism. The enzymes had distinct pH and temperature optima, as well as distinct kinetic parameters. Both enzymes had high catalytic efficiencies (kcat/Km) exceeding 1.0 x 107 M-1 sec-1. Using established molecular chaperone assays, AtDAP1 and AtDAP2 prevented thermal denaturation. AtDAP1 also prevented protein aggregation and promoted protein refolding. Collectively, these data indicate that plant DAPs have a complex evolutionary history and have evolved new biochemical features that may enable their role in vivo.

Structural insights into chaperone-activity enhancement by a K354E mutation in tomato acidic leucine aminopeptidase.

Tomato plants express acidic leucine aminopeptidase (LAP-A) in response to various environmental stressors. LAP-A not only functions as a peptidase for diverse peptide substrates, but also displays chaperone activity. A K354E mutation has been shown to abolish the peptidase activity but to enhance the chaperone activity of LAP-A. To better understand this moonlighting function of LAP-A, the crystal structure of the K354E mutant was determined at 2.15 Šresolution. The structure reveals that the K354E mutation destabilizes an active-site loop and causes significant rearrangement of active-site residues, leading to loss of the catalytic metal-ion coordination required for the peptidase activity. Although the mutant was crystallized in the same hexameric form as wild-type LAP-A, gel-filtration chromatography revealed an apparent shift from the hexamer to lower-order oligomers for the K354E mutant, showing a mixture of monomers to trimers in solution. In addition, surface-probing assays indicated that the K354E mutant has more accessible hydrophobic areas than wild-type LAP-A. Consistently, computational thermodynamic estimations of the interfaces between LAP-A monomers suggest that increased exposure of hydrophobic surfaces occurs upon hexamer breakdown. These results suggest that the K354E mutation disrupts the active-site loop, which also contributes to the hexameric assembly, and destabilizes the hexamers, resulting in much greater hydrophobic areas accessible for efficient chaperone activity than in the wild-type LAP-A.

Hemipteran and dipteran pests: Effectors and plant host immune regulators.

Hemipteran and dipteran insects have behavioral, cellular and chemical strategies for evading or coping with the host plant defenses making these insects particularly destructive pests worldwide. A critical component of a host plant's defense to herbivory is innate immunity. Here we review the status of our understanding of the receptors that contribute to perception of hemipteran and dipteran pests and highlight the gaps in our knowledge in these early events in immune signaling. We also highlight recent advances in identification of the effectors that activate pattern-triggered immunity and those involved in effector-triggered immunity.