The Characterization of SaPIN2b,a Plant Trichome-Localized Proteinase Inhibitor from Solanum americanum

Proteinase inhibitors play an important role in plant resistance of insects and pathogens. In this study, we characterized the serine proteinase inhibitor SaPIN2b, which is constitutively expressed in Solanum americanum trichomes and contains two conserved motifs of the proteinase inhibitor II (PIN2) family. The recombinant SaPIN2b (rSaPIN2b), which was expressed in Escherichia coli, was demonstrated to be a potent proteinase inhibitor against a panel of serine proteinases, including subtilisin A, chymotrypsin and trypsin. Moreover, rSaPIN2b also effectively inhibited the proteinase activities of midgut trypsin-like proteinases that were extracted from the devastating pest Helicoverpa armigera. Furthermore, the overexpression of SaPIN2b in transgenic tobacco plants resulted in enhanced resistance against H. armigera. Taken together, our results demonstrated that SaPIN2b is a potent serine proteinase inhibitor that may act as a protective protein in plant defense against insect attacks.     

Overexpression of Glutamate Decarboxylase in Transgenic Tobacco Plants Deters Feeding by Phytophagous Insect Larvae

Gamma-aminobutyrate (GABA) is a ubiquitous four-carbon, non-protein amino acid synthesized by glutamate decarboxylase. Previous research suggests that the endogenous synthesis of GABA, a naturally occurring inhibitory neurotransmitter at neuromuscular junctions, serves as a plant resistance mechanism against invertebrate pests. In this study, two homozygous transgenic tobacco lines constitutively overexpressing a single copy of a full-length chimeric glutamate decarboxylase cDNA and possessing enhanced capacity for GABA accumulation (GAD plants), a homozygous transgenic line lacking the gene insert, and wild-type tobacco were employed. Tobacco budworm larvae were presented with plantattached wild type and transgenic leaves for 4 hr in a feeding preference study. Larvae consumed six to twelve times more leaf tissue from wild-type plants than from GAD plants. These results suggest that leaf GABA accumulation, which is known to occur in response to insect larval walking and feeding, represents a rapidly deployed localresistance mechanism.     

Constitutive and Jasmonate-Inducible Traits of Datura wrightii

Plants in the family Solanaceae possess numerous traits that are induced from damage from herbivores. Many of these also can be induced by exposing plants to the plant hormone jasmonic acid or its volatile ester methyl jasmonate. Datura wrightii (Solanaceae) is dimorphic for leaf trichome morphology in most southern California populations. Trichome phenotype is governed by a single gene, and the glandular trichome condition is dominant and under developmental control. This study addressed two major objectives. The first was to determine if mature plants with glandular or nonglandular trichomes responded differentially to methyl jasmonate. The second objective was to determine if exposure of seedlings to methyl jasmonate during the period of trichome differentiation altered either the phenotype or the density of trichomes that mature plants expressed. Methyl jasmonate induced from 200 to 800 μg/ml of proteinase inhibitor activity and increased the activity of polyphenol oxidase by more than threefold depending on the experiment. These increases did not differ significantly between plants expressing glandular or nonglandular trichomes. Methyl jasmonate exposure did not increase the activity of peroxidase or the concentration of scopolamine or hyoscyamine, the two major alkaloids of Datura. Exposure to methyl jasmonate during trichome differentiation did not affect either the final trichome phenotype or the density of either type of trichome, but did increase the production of acylsugars in glandular trichomes by 44%. Because trichome phenotype was not inducible, and because both trichome phenotypes showed similar increases in proteinase inhibitors and polyphenol oxidase activity, the methyl-jasmonate-inducible responses of D. wrightii are independent of trichome phenotype in D. wrightii.     

Tryptamine-induced resistance in tryptophan decarboxylase transgenic poplar and tobacco plants against their specific herbivores

The presence of amines and their derivatives in plant tissues is known to influence insect feeding and reproduction. The enzyme tryptophan decarboxylase (TDC) catalyzes the decarboxylation of tryptophan to tryptamine, which is both a bioactive amine and a precursor of other indole derivatives. Transgenic poplar and tobacco plants ectopically expressing TDC1 accumulated elevated levels of tryptamine without affecting plant growth and development. This accumulation was consistently associated with adverse effects on feeding behavior and physiology of Malacosoma disstria Hub. (forest tent caterpillar, FTC) and Manduca sexta L. (tobacco hornworm, THW). Behavior studies with FTC and THW larvae showed that acceptability of the leaf tissue to larvae was inversely related to foliar tryptamine levels. Physiological studies with FTC and THW larvae showed that consumption of leaf tissue from the transgenic lines is deleterious to larvae growth, apparently due to a postingestive mechanism. Thus, ectopic expression of TDC1 can allow sufficient tryptamine to accumulate in poplar and tobacco leaf tissue to suppress significantly the growth of insect pests that normally feed on these plants.     

Overexpression of an Osa-miR162a Derivative in Rice Confers Cross-Kingdom RNA Interference-Mediated Brown Planthopper Resistance without Perturbing Host Development

Rice is a main food crop for more than half of the global population. The brown planthopper (BPH, Nilaparvata lugens) is one of the most destructive insect pests of rice. Currently, repeated overuse of chemical insecticides represents a common practice in agriculture for BPH control, which can induce insect tolerance and provoke environmental concerns. This situation calls for innovative and widely applicable strategies for rice protection against BPH. Here we report that the rice osa-miR162a can mediate cross-kingdom RNA interference (RNAi) by targeting the NlTOR (Target of rapamycin) gene of BPH that regulates the reproduction process. Through artificial diet or injection, osa-miR162a mimics repressed the NlTOR expression and impaired the oviposition of BPH adults. Consistently, overproduced osa-miR162a in transgenic rice plants compromised the fecundity of BPH adults fed with these plants, but meanwhile perturbed root and grain development. To circumvent this issue, we generated osa-miR162a-m1, a sequence-optimized osa-miR162a, by decreasing base complementarity to rice endogenous target genes while increasing base complementarity to NlTOR. Transgenic overexpression of osa-miR162a-m1 conferred rice resistance to BPH without detectable developmental penalty. This work reveals the first cross-kingdom RNAi mechanism in rice-BPH interactions and inspires a potentially useful approach for improving rice resistance to BPH. We also introduce an effective strategy to uncouple unwanted host developmental perturbation from desirable cross-kingdom RNAi benefits for overexpressed plant miRNAs.     

Water Deficiency and Induced Defense Against a Generalist Insect Herbivore in Desert and Mediterranean Populations of Eruca sativa

In natural and agricultural ecosystems, plants are often simultaneously or sequentially exposed to combinations of stressors. Here we tested whether limited water availability (LWA) affects plant response to insect herbivory using two populations of Eruca sativa from desert and Mediterranean habitats that differ in their induced defenses. Considering that such differences evolved as responses to biotic and possibly abiotic stress factors, the two populations offered an opportunity to study ecological aspects in plant response to combined stresses. Analysis of chemical defense mechanisms showed that LWA significantly induced total glucosinolate concentrations in the Mediterranean plants, but their concentrations were reduced in the desert plants. However, LWA, with and without subsequent jasmonate elicitation, significantly induced the expression of proteinase inhibitor in the desert plants. Results of a no-choice feeding experiment showed that LWA significantly increased desert plant resistance to Spodoptera littoralis larvae, whereas it did not affect the relatively strong basal resistance of the Mediterranean plants. LWA and subsequent jasmonate elicitation increased resistance against the generalist insect in Mediterranean plants, possibly due to both increased proteinase inhibitor expression and glucosinolate accumulation. The effect of LWA on the expression of genes involved in phytohormone signaling, abscisic acid (ABA-1) and jasmonic acid (AOC1), and the jasmonate responsive PDF1.2, suggested the involvement of abscisic acid in the regulation of defense mechanisms in the two populations. Our results indicate that specific genotypic responses should be considered when estimating general patterns in plant response to herbivory under water deficiency conditions.

     

Chloroplast-Expressed MSI-99 in Tobacco Improves Disease Resistance and Displays Inhibitory Effect against Rice Blast Fungus

Rice blast is a major destructive fungal disease that poses a serious threat to rice production and the improvement of blast resistance is critical to rice breeding. The antimicrobial peptide MSI-99 has been suggested as an antimicrobial peptide conferring resistance to bacterial and fungal diseases. Here, a vector harboring the MSI-99 gene was constructed and introduced into the tobacco chloroplast genome via particle bombardment. Transformed plants were obtained and verified to be homoplastomic by PCR and Southern hybridization. In planta assays demonstrated that the transgenic tobacco plants displayed an enhanced resistance to the fungal disease. The evaluation of the antimicrobial activity revealed that the crude protein extracts from the transgenic plants manifested an antimicrobial activity against E. coli, even after incubation at 120 °C for 20 min, indicating significant heat stability of MSI-99. More importantly, the MSI-99-containing protein extracts were firstly proved in vitro and in vivo to display significant suppressive effects on two rice blast isolates. These findings provide a strong basis for the development of new biopesticides to combat rice blast.     

Cytokinin Confers Brown Planthopper Resistance by Elevating Jasmonic Acid Pathway in Rice

Plants have evolved a sophisticated defense system that employs various hormone pathways to defend against attacks by insect pests. Cytokinin (CK) plays an important role in plant growth and stress tolerance, but the role of CKs in plant–insect interaction remains largely unclear. Here, we report that CKs act as a positive regulator in rice resistance against brown planthopper (BPH), a devastating insect pest of rice. We found that BPH feeding promotes CK biosynthesis and signaling in rice. Exogenous application of CKs significantly increased the rice resistance to BPH. Increasing endogenous CKs by knocking out cytokinin oxidase/dehydrogenase (OsCKXs) led to enhanced resistance to BPH. Moreover, the levels of the plant hormone jasmonic acid (JA) and the expression of JA-responsive genes were elevated by CK treatment and in OsCKXs knockout plants. Furthermore, JA-deficient mutant og1 was more susceptible to BPH, and CK-induced BPH resistance was suppressed in og1. These results indicate that CK-mediated BPH resistance is JA-dependent. Our findings provide the direct evidence for the novel role of CK in promoting insect resistance, and demonstrate that CK-induced insect resistance is JA-dependent. These results provide important guidance for effective pest management strategies in the future.     

Overexpression of Rice BSR2 Confers Disease Resistance and Induces Enlarged Flowers in Torenia fournieri Lind

Plant pathogens evade basal defense systems and attack different organs and tissues of plants. Genetic engineering of plants with genes that confer resistance against pathogens is very effective in pathogen control. Conventional breeding for disease resistance in ornamental crops is difficult and lagging relative to that in non-ornamental crops due to an inadequate number of disease-resistant genes. Therefore, genetic engineering of these plants with defense-conferring genes is a practical approach. We used rice BSR2 encoding CYP78A15 for developing transgenic Torenia fournieri Lind. lines. The overexpression of BSR2 conferred resistance against two devastating fungal pathogens, Rhizoctonia solani and Botrytis cinerea. In addition, BSR2 overexpression resulted in enlarged flowers with enlarged floral organs. Histological observation of the petal cells suggested that the enlargement in the floral organs could be due to the elongation and expansion of the cells. Therefore, the overexpression of BSR2 confers broad-spectrum disease resistance and induces the production of enlarged flowers simultaneously. Therefore, this could be an effective strategy for developing ornamental crops that are disease-resistant and economically more valuable.     

Genetic and Environmental Variation in Acyl Glucose Ester Production and Glandular and Nonglandular Trichome Densities in Datura wrightii

Natural populations of Datura wrightii in southern California are dimorphic for trichome type. Some plants within populations produce greater than 85% glandular trichomes, whereas other produce mainly nonglandular trichomes. Glandular trichome exudates in D. wrightii consist of glucose esterified with straight chain C₆–C₉ acids. These exudates, and similar exudates in other species, confer resistance to several insect herbivore species. We tested the hypothesis that water was limiting sugar ester production and examined the extent to which trichome density was determined by environmental factors by measuring the concentrations of sugar esters and the densities of trichomes on leaves of plants grown under different irrigation treatments. Water did not limit sugar ester production, as unwatered plants produced 36% more millimoles of glucose esters per square centimeter of leaf surface than did watered plants. Although the addition of water increased leaf size, densities of both nonglandular and glandular trichomes did not change with leaf length or area, suggesting that plants having larger leaves initiated more trichomes in order to maintain nearly constant densities. Millimoles of sugar esters produced did not correlate with densities of glandular trichomes, suggesting that other factors in addition to glandular trichome number govern the production of sugar esters for plant defense.     

Using Multiplexed CRISPR/Cas9 for Suppression of Cotton Leaf Curl Virus

In recent decades, Pakistan has suffered a decline in cotton production due to several factors, including insect pests, cotton leaf curl disease (CLCuD), and multiple abiotic stresses. CLCuD is a highly damaging plant disease that seriously limits cotton production in Pakistan. Recently, genome editing through CRISPR/Cas9 has revolutionized plant biology, especially to develop immunity in plants against viral diseases. Here we demonstrate multiplex CRISPR/Cas-mediated genome editing against CLCuD using transient transformation in N. benthamiana plants and cotton seedlings. The genomic sequences of cotton leaf curl viruses (CLCuVs) were obtained from NCBI and the guide RNA (gRNA) were designed to target three regions in the viral genome using CRISPR MultiTargeter. The gRNAs were cloned in pHSE401/pKSE401 containing Cas9 and confirmed through colony PCR, restriction analysis, and sequencing. Confirmed constructs were moved into Agrobacterium and subsequently used for transformation. Agroinfilteration in N. benthamiana revealed delayed symptoms (3–5 days) with improved resistance against CLCuD. In addition, viral titer was also low (20–40%) in infected plants co-infiltrated with Cas9-gRNA, compared to control plants (infected with virus only). Similar results were obtained in cotton seedlings. The results of transient expression in N. benthamiana and cotton seedlings demonstrate the potential of multiplex CRISPR/Cas to develop resistance against CLCuD. Five transgenic plants developed from three experiments showed resistance (60−70%) to CLCuV, out of which two were selected best during evaluation and screening. The technology will help breeding CLCuD-resistant cotton varieties for sustainable cotton production.     

Biological Activity of Acyl Glucose Esters from <Emphasis Type="Italic">Datura wrightii</Emphasis> Glandular Trichomes against Three Native Insect Herbivores

Datura wrightii is dimorphic for leaf trichome type in southern California. “Sticky” plants produce glandular trichomes that secrete acylsugars, whereas velvety plants produce nonglandular trichomes. Glandular trichomes confer resistance to some potential insect herbivores and are associated with reduced feeding in the field by two native coleopteran herbivores: the tobacco flea beetle, Epitrix hirtipennis, and a weevil, Trichobaris compacta. In contrast, another native beetle, Lema daturaphila, damages sticky and velvety plants similarly in the field. A series of choice and no-choice “ester removal” and “ester addition” feeding experiments were performed in the laboratory to evaluate the role of acylsugars in feeding by all three insect species. Consumption of sticky leaves after their esters were removed by washing was compared to consumption of unwashed sticky leaves and velvety leaves in ester removal experiments. Consumption of velvety leaves was measured after acylsugars were applied to those leaves in controlled amounts in the ester addition experiments. Consumption by E. hirtipennis was reduced by acylsugars in all experiments. Consumption by T. compacta was reduced by acylsugars in the ester removal experiments, but not in the ester addition experiments. The location of the acylsugars at the tip of a long trichome, rather than simply on the leaf surface, may be an important component of the biological activity of acylsugars against T. compacta in nature. Consumption by L. daturaphila was not significantly reduced by acylsugars in any experiment. The acylsugars caused no significant mortality of any of the three insect species.     

Expression of Cytochrome c3 from Desulfovibrio vulgaris in Plant Leaves Enhances Uranium Uptake and Tolerance of Tobacco

Cytochrome c3 (uranyl reductase) from Desulfovibrio vulgaris can reduce uranium in bacterial cells and in cell-free systems. This gene was introduced in tobacco under control of the RbcS promoter, and the resulting transgenic plants accumulated uranium when grown on a uranyl ion containing medium. The uptaken uranium was detected by EM in chloroplasts. In the presence of uranyl ions in sublethal concentration, the transgenic plants grew phenotypically normal while the control plants’ development was impaired. The data on uranium oxidation state in the transgenic plants and the possible uses of uranium hyperaccumulation by plants for environmental cleanup are discussed.     

Microbial Toxins in Insect and Nematode Pest Biocontrol

Invertebrate pests, such as insects and nematodes, not only cause or transmit human and livestock diseases but also impose serious crop losses by direct injury as well as vectoring pathogenic microbes. The damage is global but greater in developing countries, where human health and food security are more at risk. Although synthetic pesticides have been in use, biological control measures offer advantages via their biodegradability, environmental safety and precise targeting. This is amply demonstrated by the successful and widespread use of Bacillus thuringiensis to control mosquitos and many plant pests, the latter by the transgenic expression of insecticidal proteins from B. thuringiensis in crop plants. Here, I discuss the prospects of using bacterial and fungal toxins for pest control, including the molecular basis of their biocidal activity.