Plant Membrane-On-A-Chip: A Platform for Studying Plant Membrane Proteins and Lipids.

The cell plasma membrane is a two-dimensional, fluid mosaic material composed of lipids and proteins that create a semipermeable barrier defining the cell from its environment. Compared with soluble proteins, the methodologies for the structural and functional characterization of membrane proteins are challenging. An emerging tool for studies of membrane proteins in mammalian systems is a "plasma membrane on a chip," also known as a supported lipid bilayer. Here, we create the "plant-membrane-on-a-chip,″ a supported bilayer made from the plant plasma membranes of Arabidopsis thaliana, Nicotiana benthamiana, or Zea mays. Membrane vesicles from protoplasts containing transgenic membrane proteins and their native lipids were incorporated into supported membranes in a defined orientation. Membrane vesicles fuse and orient systematically, where the cytoplasmic side of the membrane proteins faces the chip surface and constituents maintain mobility within the membrane plane. We use plant-membrane-on-a-chip to perform fluorescent imaging to examine protein-protein interactions and determine the protein subunit stoichiometry of FLOTILLINs. We report here that like the mammalian FLOTILLINs, FLOTILLINs expressed in Arabidopsis form a tetrameric complex in the plasma membrane. This plant-membrane-on-a-chip approach opens avenues to studies of membrane properties of plants, transport phenomena, biophysical processes, and protein-protein and protein-lipid interactions in a convenient, cell-free platform.

Serine proteinase inhibitors from Nicotiana benthamiana, a nonpreferred host plant, inhibit the growth of Myzus persicae (green peach aphid).

BACKGROUND: The green peach aphid (Myzus persicae) is a severe agricultural crop pest that has developed resistance to most current control methods, requiring the urgent development of novel strategies. Plant proteinase inhibitors (PINs) are small proteins that protect plants against pathogens and/or herbivores, likely by preventing efficient protein digestion. RESULTS: We identified 67 protease genes in the transcriptomes of three M. persicae lineages (USDA-Red, G002 and G006). Comparison of gene expression levels in aphid guts and whole aphids showed that several proteases, including a highly expressed serine protease, are significantly overexpressed in the guts. Furthermore, we identified three genes encoding serine protease inhibitors (SerPIN-II1, 2 and 3) in Nicotiana benthamiana, which is a nonpreferred host for M. persicae. Using virus-induced gene silencing (VIGS) with a tobacco rattle virus (TRV) vector and overexpression with a turnip mosaic virus (TuMV) vector, we demonstrated that N. benthamiana SerPIN-II1 and SerPIN-II2 cause reduced survival and growth, but do not affect aphid protein content. Likewise, SerPIN-II3 overexpression reduced survival and growth, and serpin-II3 knockout mutations, which we generated using CRISPR/Cas9, increased survival and growth. Protein content was significantly increased in aphids fed on SerPIN-II3 overexpressing plants, yet it was decreased in aphids fed on serpin-II3 mutants. CONCLUSION: Our results show that three PIN-IIs from N. benthamiana, a nonpreferred host plant, effectively inhibit M. persicae survival and growth, thereby representing a new resource for the development of aphid-resistant crop plants. © 2024 Society of Chemical Industry.

Punicalagin, a pomegranate polyphenol sensitizes the activity of antibiotics against three MDR pathogens of the Enterobacteriaceae.

BACKGROUND: Multidrug resistance (MDR) in the family Enterobacteriaceae is a perniciously increasing threat to global health security. The discovery of new antimicrobials having the reversing drug resistance potential may contribute to augment and revive the antibiotic arsenal in hand. This study aimed to explore the anti-Enterobacteriaceae capability of bioactive polyphenols from Punica granatum (P. granatum) and their co-action with antibiotics against clinical isolates of Enterobacteriaceae predominantly prevalent in South Asian countries. METHODS: The Kandhari P. granatum (Pakistani origin) extracts were tested for anti-Enterobacteriaceae activity by agar well diffusion assay against MDR Salmonella enterica serovar Typhi, serovar Typhimurium and Escherichia coli. Predominant compounds of active extract were determined by mass spectrometry and screened for bioactivity by agar well diffusion and minimum inhibitory concentration (MIC) assay. The active punicalagin was further evaluated at sub-inhibitory concentrations (SICs) for coactivity with nine conventional antimicrobials using a disc diffusion assay followed by time-kill experiments that proceeded with SICs of punicalagin and antimicrobials. RESULTS: Among all P. granatum crude extracts, pomegranate peel methanol extract showed the largest inhibition zones of 25, 22 and 19 mm, and the MICs as 3.9, 7.8 and 7.8 mg/mL for S. typhi, S. typhimurium and E. coli, respectively. Punicalagin and ellagic acid were determined as predominant compounds by mass spectrometry. In plate assay, punicalagin (10 mg/mL) was active with hazy inhibition zones of 17, 14, and 13 mm against S. typhi, S. typhimurium and E. coli, respectively. However, in broth dilution assay punicalagin showed no MIC up to 10 mg/mL. The SICs 30 µg, 100 µg, and 500 µg of punicalagin combined with antimicrobials i.e., aminoglycoside, ß-lactam, and fluoroquinolone act in synergy against MDR strains with % increase in inhibition zone values varying from 3.4 ± 2.7% to 73.8 ± 8.4%. In time-kill curves, a significant decrease in cell density was observed with the SICs of antimicrobials/punicalagin (0.03-60 µg/mL/30, 100, 500 µg/mL of punicalagin) combinations. CONCLUSIONS: The P. granatum peel methanol extract exhibited antimicrobial activity against MDR Enterobacteriaceae pathogens. Punicalagin, the bacteriostatic flavonoid act as a concentration-dependent sensitizing agent for antimicrobials against Enterobacteriaceae. Our findings for the therapeutic punicalagin-antimicrobial combination prompt further evaluation of punicalagin as a potent activator for drugs, which otherwise remain less or inactive against MDR strains.

Fatty alcohols, a minor component of the tree tobacco surface wax, are associated with defence against caterpillar herbivory.

Despite decades of research resulting in a comprehensive understanding of epicuticular wax metabolism, the function of these almost ubiquitous metabolites in plant-herbivore interactions remains unresolved. In this study, we examined the effects of CRISPR-induced knockout mutations in four Nicotiana glauca (tree tobacco) wax metabolism genes. These mutations cause a wide range of changes in epicuticular wax composition, leading to altered interactions with insects and snails. Three interaction classes were examined: chewing herbivory by seven caterpillars and one snail species, phloem feeding by Myzus persicae (green peach aphid) and oviposition by Bemisia tabaci (whitefly). Although total wax load and alkane abundance did not affect caterpillar growth, a correlation across species, showed that fatty alcohols, a minor component of N. glauca surface waxes, negatively affected the growth of both a generalist caterpillar (Spodoptera littoralis) and a tobacco-feeding specialist (Manduca sexta). This negative correlation was overshadowed by the stronger effect of anabasine, a nicotine isomer, and was apparent when fatty alcohols were added to an artificial lepidopteran diet. By contrast, snails fed more on waxy leaves. Aphid reproduction and feeding activity were unaffected by wax composition but were potentially affected by altered cutin composition. Wax crystal morphology could explain the preference of B. tabaci to lay eggs on waxy wild-type plants relative to both alkane and fatty alcohol-deficient mutants. Together, our results suggest that the varied responses among herbivore classes and species are likely to be a consequence of the co-evolution that shaped the specific effects of different surface wax components in plant-herbivore interactions.

Quantification of plant cardenolides by HPLC, measurement of Na+/K+-ATPase inhibition activity, and characterization of target enzymes.

The biosynthesis of cardiac glycosides, broadly classified as cardenolides and bufadienolides, has evolved repeatedly among flowering plants. Individual species can produce dozens or even hundreds of structurally distinct cardiac glycosides. Although all cardiac glycosides exhibit biological activity by inhibiting the function of the essential Na+/K+-ATPase in animal cells, they differ in their level of inhibitory activity. For within- and between-species comparisons of cardiac glycosides to address ecological and evolutionary questions, it is necessary to not only quantify their relative abundance, but also their effectiveness in inhibiting the activity of different animal Na+/K+-ATPases. Here we describe protocols for characterizing the amount and toxicity of cardenolides from plant samples and the degree of insect Na+/K+-ATPase tolerance to inhibition: (1) an HPLC-based assay to quantify the abundance of individual cardenolides in plant extracts, (2) an assay to quantify inhibition of Na+/K+-ATPase activity by plant extracts, and (3) extraction of insect Na+/K+-ATPases for inhibition assays.

Tree tobacco (Nicotiana glauca) cuticular wax composition is essential for leaf retention during drought, facilitating a speedy recovery following rewatering.

Despite decades of extensive study, the role of cuticular lipids in sustaining plant fitness is far from being understood. We utilized genome-edited tree tobacco (Nicotiana glauca) to investigate the significance of different classes of epicuticular wax in abiotic stress such as cuticular water loss, drought, and light response. We generated mutants displaying a range of wax compositions. Four wax mutants and one cutin mutant were extensively investigated for alterations in their response to abiotic factors. Although the mutations led to elevated cuticular water loss, the wax mutants did not display elevated transpiration or reduced growth under nonstressed conditions. However, under drought, plants lacking alkanes were unable to reduce their transpiration, leading to leaf death, impaired recovery, and stem cracking. By contrast, plants deficient in fatty alcohols exhibited elevated drought tolerance, which was part of a larger trend of plant phenotypes not clustering by a glossy/glaucous appearance in the parameters examined in this study. We conclude that although alkanes have little effect on whole N. glauca transpiration and biomass gain under normal, nonstressed conditions, they are essential during drought responses, since they enable plants to seal their cuticle upon stomatal closure, thereby reducing leaf death and facilitating a speedy recovery.

Horizontally transferred genes as RNA interference targets for aphid and whitefly control.

RNA interference (RNAi)-based technologies are starting to be commercialized as a new approach for agricultural pest control. Horizontally transferred genes (HTGs), which have been transferred into insect genomes from viruses, bacteria, fungi or plants, are attractive targets for RNAi-mediated pest control. HTGs are often unique to a specific insect family or even genus, making it unlikely that RNAi constructs targeting such genes will have negative effects on ladybugs, lacewings and other beneficial predatory insect species. In this study, we sequenced the genome of a red, tobacco-adapted isolate of Myzus persicae (green peach aphid) and bioinformatically identified 30 HTGs. We then used plant-mediated virus-induced gene silencing (VIGS) to show that several HTGs of bacterial and plant origin are important for aphid growth and/or survival. Silencing the expression of fungal-origin HTGs did not affect aphid survivorship but decreased aphid reproduction. Importantly, although there was uptake of plant-expressed RNA by Coccinella septempunctata (seven-spotted ladybugs) via the aphids that they consumed, we did not observe negative effects on ladybugs from aphid-targeted VIGS constructs. To demonstrate that this approach is more broadly applicable, we also targeted five Bemisia tabaci (whitefly) HTGs using VIGS and demonstrated that knockdown of some of these genes affected whitefly survival. As functional HTGs have been identified in the genomes of numerous pest species, we propose that these HTGs should be explored further as efficient and safe targets for control of insect pests using plant-mediated RNA interference.

Characterizing serotonin biosynthesis in Setaria viridis leaves and its effect on aphids.

KEY MESSAGE: A combined transcriptomic and metabolic analysis of Setaria viridis leaves responding to aphid infestation was used to identify genes related to serotonin biosynthesis. Setaria viridis (green foxtail), a short life-cycle C4 plant in the Poaceae family, is the wild ancestor of Setaria italica (foxtail millet), a resilient crop that provides good yields in dry and marginal land. Although S. viridis has been studied extensively in the last decade, the molecular mechanisms of insect resistance in this species remain under-investigated. To address this issue, we performed a metabolic analysis of S. viridis and discovered that these plants accumulate the tryptophan-derived compounds tryptamine and serotonin. To elucidate the defensive functions of serotonin, Rhophalosiphum padi (bird cherry-oat aphids) were exposed to this compound, either by exogenous application to the plant medium or with artificial diet bioassays. In both cases, exposure to serotonin increased aphid mortality. To identify genes that are involved in serotonin biosynthesis, we conducted a transcriptome analysis and identified several predicted S. viridis tryptophan decarboxylase (TDC) and tryptamine 5-hydroxylase (T5H) genes. Two candidate genes were ectopically expressed in Nicotiana tabacum, where SvTDC1 (Sevir.6G066200) had tryptophan decarboxylase activity, and SvT5H1 (Sevir.8G219600) had tryptamine hydroxylase activity. Moreover, the function of the SvTDC1 gene was validated using virus-induced gene silencing in S. italica, which caused a reduction in serotonin levels. This study provides the first evidence of serotonin biosynthesis in Setaria leaves. The biosynthesis of serotonin may play an important role in defense responses and could prove to be useful for developing more pest-tolerant Setaria italica cultivars.

Acylsugars protect Nicotiana benthamiana against insect herbivory and desiccation.

KEY MESSAGE: Nicotiana benthamiana acylsugar acyltransferase (ASAT) is required for protection against desiccation and insect herbivory. Knockout mutations provide a new resource for investigation of plant-aphid and plant-whitefly interactions. Nicotiana benthamiana is used extensively as a transient expression platform for functional analysis of genes from other species. Acylsugars, which are produced in the trichomes, are a hypothesized cause of the relatively high insect resistance that is observed in N. benthamiana. We characterized the N. benthamiana acylsugar profile, bioinformatically identified two acylsugar acyltransferase genes, ASAT1 and ASAT2, and used CRISPR/Cas9 mutagenesis to produce acylsugar-deficient plants for investigation of insect resistance and foliar water loss. Whereas asat1 mutations reduced accumulation, asat2 mutations caused almost complete depletion of foliar acylsucroses. Three hemipteran and three lepidopteran herbivores survived, gained weight, and/or reproduced significantly better on asat2 mutants than on wildtype N. benthamiana. Both asat1 and asat2 mutations reduced the water content and increased leaf temperature. Our results demonstrate the specific function of two ASAT proteins in N. benthamiana acylsugar biosynthesis, insect resistance, and desiccation tolerance. The improved growth of aphids and whiteflies on asat2 mutants will facilitate the use of N. benthamiana as a transient expression platform for the functional analysis of insect effectors and resistance genes from other plant species. Similarly, the absence of acylsugars in asat2 mutants will enable analysis of acylsugar biosynthesis genes from other Solanaceae by transient expression.

Rapid Screening of Myzus persicae (Green Peach Aphid) RNAi Targets Using Tobacco Rattle Virus.

Plant-mediated RNA interference (RNAi) can be used to reduce the growth of insect pests, including Myzus persicae (green peach aphid), a prolific pest of numerous dicot crop species. In one approach, viruses that have been engineered to carry an aphid gene fragment are used to infect plants and thereby silence target gene expression in the aphids feeding on these plants, a process called virus-induced gene silencing, or VIGS. Tobacco Rattle Virus (TRV) in the model plant, Nicotiana benthamiana, was the first of many VIGS systems that have been developed for different plant species. In this chapter, we describe a method for silencing M. persicae gene expression using an established TRV-VIGS vector that infects and spreads in N. benthamiana. The two parts of the TRV genome, RNA1 and RNA2, have been cloned into Agrobacterium T-DNA vectors for initiation of plant infections. The RNA2 construct is modified with a Gateway-compatible cloning site to allow insertion of aphid genes. When feeding on TRV-infected N. benthamiana plants, aphids ingest dsRNAs that silence specific target genes. TRV-VIGS of aphid genes allows rapid identification of essential gene targets that can be used for the control of M. persicae by this and other RNAi methods.

Interaction of eukaryotic proliferating cell nuclear antigen (PCNA) with the replication-associated protein (Rep) of cotton leaf curl Multan virus and pedilanthus leaf curl virus.

The replication-associated (Rep) proteins of pathogenic begomoviruses, including cotton leaf curl Multan virus (CLCuMuV) and pedilanthus leaf curl virus (PeLCV), interact with the DNA replication machinery of their eukaryotic hosts. The analysis of Rep protein sequences showed that there is 13-28% sequence variation among CLCuMuV and PeLCV isolates, with phylogenetic clusters that can separated at least in part based on the country of origin of the respective viruses. To identify specific host factors involved in the virus replication cycle, we conducted yeast two-hybrid assays to detect possible interactions between the CLCuMuV and PeLCV Rep proteins and 30 protein components of the Saccharomyces cerevisiae DNA replication machinery. This showed that the proliferating cell nuclear antigen (PCNA) protein of S. cerevisiae interacts with Rep proteins from both CLCuMuV and PeLCV. We used the yeast PCNA sequence in BLAST comparisons to identify two PCNA orthologs each in Gossypium hirsutum (cotton), Arabidopsis thaliana (Arabidopsis), and Nicotiana benthamiana (tobacco). Sequence comparisons showed 38-40% identity between the yeast and plant PCNA proteins, and > 91% identity among the plant PCNA proteins, which clustered together in one phylogenetic group. The expression of the six plant PCNA proteins in the yeast two-hybrid system confirmed interactions with the CLCuMuV and PeLCV Rep proteins. Our results demonstrate that the interaction of begomovirus Rep proteins with eukaryotic PCNA proteins is strongly conserved, despite significant evolutionary variation in the protein sequences of both of the interacting partners.

Less Is More: a Mutation in the Chemical Defense Pathway of Erysimum cheiranthoides (Brassicaceae) Reduces Total Cardenolide Abundance but Increases Resistance to Insect Herbivores.

Erysimum cheiranthoides L (Brassicaceae; wormseed wallflower) accumulates not only glucosinolates, which are characteristic of the Brassicaceae, but also abundant and diverse cardenolides. These steroid toxins, primarily glycosylated forms of digitoxigenin, cannogenol, and strophanthidin, inhibit the function of essential Na+/K+-ATPases in animal cells. We screened a population of 659 ethylmethanesulfonate-mutagenized E. cheiranthoides plants to identify isolates with altered cardenolide profiles. One mutant line exhibited 66% lower cardenolide content, resulting from greatly decreased cannogenol and strophanthidin glycosides, partially compensated for by increases in digitoxigenin glycosides. This phenotype was likely caused by a single-locus recessive mutation, as evidenced by a wildtype phenotype of F1 plants from a backcross, a 3:1 wildtype:mutant segregation in the F2 generation, and genetic mapping of the altered cardenolide phenotype to one position in the genome. The mutation created a more even cardenolide distribution, decreased the average cardenolide polarity, but did not impact most glucosinolates. Growth of generalist herbivores from two feeding guilds, Myzus persicae Sulzer (Hemiptera: Aphididae; green peach aphid) and Trichoplusia ni Hübner (Lepidoptera: Noctuidae; cabbage looper), was decreased on the mutant line compared to wildtype. Both herbivores accumulated cardenolides in proportion to the plant content, with T. ni accumulating higher total concentrations than M. persicae. Helveticoside, a relatively abundant cardenolide in E. cheiranthoides, was not detected in M. persicae feeding on these plants. Our results support the hypothesis that increased digitoxigenin glycosides provide improved protection against M. persicae and T. ni, despite an overall decrease in cardenolide content of the mutant line.

Comparison of in Vitro and in Planta Toxicity of Vip3A for Lepidopteran Herbivores.

Agricultural pest infestation is as old as domestication of food crops and contributes a major share to the cost of crop production. In a transgenic pest control approach, plant production of Vip3A, an insecticidal protein from Bacillus thuringiensis, is effective against lepidopteran pests. A synthetic Vip3A gene was evaluated for efficacy against Spodoptera litura Fabricius (Lepidoptera: Noctuidae; cotton leafworm), Spodoptera exigua Hübner (Lepidoptera: Noctuidae; beet armyworm), Spodoptera frugiperda Smith (Lepidoptera: Noctuidae; fall armyworm), Helicoverpa armigera Hübner (Lepidoptera: Noctuidae; cotton bollworm), Helicoverpa zea Boddie (Lepidoptera: Noctuidae; corn earworm), Heliothis virescens Fabricius (Lepidoptera: Noctuidae; tobacco budworm), and Manduca sexta L. (Lepidoptera: Sphingidae; tobacco hornworm) in tobacco. In artificial diet assays, the concentration required to achieve 50% mortality was highest for H. zea followed by H. virescens > S. exigua > H. armigera > M. sexta > S. frugiperda > S. litura. By contrast, in bioassays with detached leaves from Vip3A transgenic tobacco, the time until 50% lethality was M. sexta > H. virescens > S. litura > H. zea > H. armigera > S. exigua. There was no significant correlation between the artificial diet and transgenic plant bioassay results. Notably, the two insect species that are best-adapted for growth on tobacco, M. sexta and H. virescens, showed the greatest time to 50% mortality on Vip3A-transgenic tobacco. Together, our results suggest that artificial diet assays may be a poor predictor of Vip3A efficacy in transgenic plants, lepidopteran species vary in their sensitivity to Vip3A in diet-dependent manner, and host plant adaptation of the targeted herbivores should be considered when designing transgenic plants for pest control.

Expression of Pinellia ternata leaf agglutinin under rolC promoter confers resistance against a phytophagous sap sucking aphid, Myzus persicae

BACKGROUND: Piercing/sucking insect pests in the order Hemiptera causes substantial crop losses by removing photoassimilates and transmitting viruses to their host plants. Cloning and heterologous expression of plantderived insect resistance genes is a promising approach to control aphids and other sap-sucking insect pests. While expression from the constitutive 35S promoter provides broad protection, the phloem-specific rolC promoter provides better defense against sap sucking insects. The selection of plant-derived insect resistance genes for expression in crop species will minimize bio-safety concerns. RESULTS: Pinellia ternata leaf agglutinin gene (pta), encodes an insecticidal lectin, was isolated and cloned under the 35S and rolC promoters in the pGA482 plant transformation vector for Agrobacterium-mediated tobacco transformation. Integration and expression of the transgene was validated by Southern blotting and qRT-PCR, respectively. Insect bioassays data of transgenic tobacco plants showed that expression of pta under rolC promoter caused 100% aphid mortality and reduced aphid fecundity up to 70% in transgenic tobacco line LRP9. These results highlight the better effectivity of pta under rolC promoter to control phloem feeders, aphids. CONCLUSIONS: These findings suggested the potential of PTA against aphids and other sap sucking insect pests. Evaluation of gene in tobacco under two different promoters; 35S constitutive promoter and rolC phloemspecific promoter could be successfully use for other crop plants particularly in cotton. Development of transgenic cotton plants using plant-derived insecticidal, PTA, would be key step towards commercialization of environmentally safe insect-resistant crops.

In-planta expression of insecticidal proteins provides protection against lepidopteran insects.

The development of advanced biotechnological control strategies opens a new era of environment friendly pest management. The current study is part of such an effort, in which we developed a control strategy based on gene pyramiding that confers broad-spectrum resistance against lepidopteran (Helicoverpa armigera and Spodoptera litura) and hemipteran (Myzus persicae, Phenacoccus solenopsis, and Bemisia tabaci) insect pests. Previously, we reported a double gene construct expressing Hvt and lectin in tobacco (Nicotiana tabacum) plants under phloem specific promoters which confers resistance against hemipteran insects. Here we extended our studies by evaluating the advanced generation of these tobacco plants expressing hvt-lectin against lepidopteran insects. Tobacco plants expressing both toxins were tested against H. armigera and S. litura. Insect bioassay results showed 100% mortality of H. armigera within 48-72 hours and 100% mortality of S. litura within 72-96 hours. Our results suggest that the use of both toxins as a gene pyramiding strategy to control both lepidopteran and hemipterans insects on commercial basis to reduce the use of chemical pesticides.

12-Oxo-Phytodienoic Acid Acts as a Regulator of Maize Defense against Corn Leaf Aphid.

The corn leaf aphid (CLA; Rhopalosiphum maidis) is a phloem sap-sucking insect that attacks many cereal crops, including maize (Zea mays). We previously showed that the maize inbred line Mp708, which was developed by classical plant breeding, provides enhanced resistance to CLA. Here, using electrophysiological monitoring of aphid feeding behavior, we demonstrate that Mp708 provides phloem-mediated resistance to CLA. Furthermore, feeding by CLA on Mp708 plants enhanced callose deposition, a potential defense mechanism utilized by plants to limit aphid feeding and subsequent colonization. In maize, benzoxazinoids (BX) or BX-derived metabolites contribute to enhanced callose deposition by providing heightened resistance to CLA. However, BX and BX-derived metabolites were not significantly altered in CLA-infested Mp708 plants, indicating BX-independent defense against CLA. Evidence presented here suggests that the constitutively higher levels of 12-oxo-phytodienoic acid (OPDA) in Mp708 plants contributed to enhanced callose accumulation and heightened CLA resistance. OPDA enhanced the expression of ethylene biosynthesis and receptor genes, and the synergistic interactions of OPDA and CLA feeding significantly induced the expression of the transcripts encoding Maize insect resistance1-Cysteine Protease, a key defensive protein against insect pests, in Mp708 plants. Furthermore, exogenous application of OPDA on maize jasmonic acid-deficient plants caused enhanced callose accumulation and heightened resistance to CLA, suggesting that the OPDA-mediated resistance to CLA is independent of the jasmonic acid pathway. We further demonstrate that the signaling function of OPDA, rather than a direct toxic effect, contributes to enhanced CLA resistance in Mp708.

Ethylene signaling regulates natural variation in the abundance of antifungal acetylated diferuloylsucroses and Fusarium graminearum resistance in maize seedling roots.

The production and regulation of defensive specialized metabolites play a central role in pathogen resistance in maize (Zea mays) and other plants. Therefore, identification of genes involved in plant specialized metabolism can contribute to improved disease resistance. We used comparative metabolomics to identify previously unknown antifungal metabolites in maize seedling roots, and investigated the genetic and physiological mechanisms underlying their natural variation using quantitative trait locus mapping and comparative transcriptomics approaches. Two maize metabolites, smilaside A (3,6-diferuloyl-3',6'-diacetylsucrose) and smiglaside C (3,6-diferuloyl-2',3',6'-triacetylsucrose), were identified that could contribute to maize resistance against Fusarium graminearum and other fungal pathogens. Elevated expression of an ethylene signaling gene, ETHYLENE INSENSITIVE 2 (ZmEIN2), co-segregated with a decreased smilaside A : smiglaside C ratio. Pharmacological and genetic manipulation of ethylene availability and sensitivity in vivo indicated that, whereas ethylene was required for the production of both metabolites, the smilaside A : smiglaside C ratio was negatively regulated by ethylene sensitivity. This ratio, rather than the absolute abundance of these two metabolites, was important for maize seedling root defense against F. graminearum. Ethylene signaling regulates the relative abundance of the two F. graminearum-resistance-related metabolites and affects resistance against F. graminearum in maize seedling roots.

Computational and biological characterization of fusion proteins of two insecticidal proteins for control of insect pests.

Sucking pests pose a serious agricultural challenge, as available transgenic technologies such as Bacillus thuringiensis crystal toxins (Bt) are not effective against them. One approach is to produce fusion protein toxins for the control of these pests. Two protein toxins, Hvt (ω-atracotoxin from Hadronyche versuta) and onion leaf lectin, were translationally fused to evaluate the negative effects of fusion proteins on Phenacoccus solenopsis (mealybug), a phloem-feeding insect pest. Hvt was cloned both N-terminally (HL) and then C-terminally (LH) in the fusion protein constructs, which were expressed transiently in Nicotiana tabacum using a Potato Virus X (PVX) vector. The HL fusion protein was found to be more effective against P. solenopsis, with an 83% mortality rate, as compared to the LH protein, which caused 65% mortality. Hvt and lectin alone caused 42% and 45%, respectively, under the same conditions. Computational studies of both fusion proteins showed that the HL protein is more stable than the LH protein. Together, these results demonstrate that translational fusion of two insecticidal proteins improved the insecticidal activity relative to each protein individually and could be expressed in transgenic plants for effective control of sucking pests.

Concurrent Overexpression of Arabidopsis thaliana Cystathionine γ-Synthase and Silencing of Endogenous Methionine γ-Lyase Enhance Tuber Methionine Content in Solanum tuberosum.

Potatoes (Solanum tuberosum) are deficient in methionine, an essential amino acid in human and animal diets. Higher methionine levels increase the nutritional quality and promote the typically pleasant aroma associated with baked and fried potatoes. Several attempts have been made to elevate tuber methionine levels by genetic engineering of methionine biosynthesis and catabolism. Overexpressing Arabidopsis thaliana cystathionine γ-synthase (AtCGS) in S. tuberosum up-regulates a rate-limiting step of methionine biosynthesis and increases tuber methionine levels. Alternatively, silencing S. tuberosum methionine γ-lyase (StMGL), which causes decreased degradation of methionine into 2-ketobutyrate, also increases methionine levels. Concurrently enhancing biosynthesis and reducing degradation were predicted to provide further increases in tuber methionine content. Here we report that S. tuberosum cv. Désirée plants with AtCGS overexpression and StMGL silenced by RNA interference are morphologically normal and accumulate higher free methionine levels than either single-transgenic line.

The draft genome of whitefly Bemisia tabaci MEAM1, a global crop pest, provides novel insights into virus transmission, host adaptation, and insecticide resistance.

BACKGROUND: The whitefly Bemisia tabaci (Hemiptera: Aleyrodidae) is among the 100 worst invasive species in the world. As one of the most important crop pests and virus vectors, B. tabaci causes substantial crop losses and poses a serious threat to global food security. RESULTS: We report the 615-Mb high-quality genome sequence of B. tabaci Middle East-Asia Minor 1 (MEAM1), the first genome sequence in the Aleyrodidae family, which contains 15,664 protein-coding genes. The B. tabaci genome is highly divergent from other sequenced hemipteran genomes, sharing no detectable synteny. A number of known detoxification gene families, including cytochrome P450s and UDP-glucuronosyltransferases, are significantly expanded in B. tabaci. Other expanded gene families, including cathepsins, large clusters of tandemly duplicated B. tabaci-specific genes, and phosphatidylethanolamine-binding proteins (PEBPs), were found to be associated with virus acquisition and transmission and/or insecticide resistance, likely contributing to the global invasiveness and efficient virus transmission capacity of B. tabaci. The presence of 142 horizontally transferred genes from bacteria or fungi in the B. tabaci genome, including genes encoding hopanoid/sterol synthesis and xenobiotic detoxification enzymes that are not present in other insects, offers novel insights into the unique biological adaptations of this insect such as polyphagy and insecticide resistance. Interestingly, two adjacent bacterial pantothenate biosynthesis genes, panB and panC, have been co-transferred into B. tabaci and fused into a single gene that has acquired introns during its evolution. CONCLUSIONS: The B. tabaci genome contains numerous genetic novelties, including expansions in gene families associated with insecticide resistance, detoxification and virus transmission, as well as numerous horizontally transferred genes from bacteria and fungi. We believe these novelties likely have shaped B. tabaci as a highly invasive polyphagous crop pest and efficient vector of plant viruses. The genome serves as a reference for resolving the B. tabaci cryptic species complex, understanding fundamental biological novelties, and providing valuable genetic information to assist the development of novel strategies for controlling whiteflies and the viruses they transmit.