Microarray analysis of tomato plants exposed to the nonviruliferous or viruliferous whitefly vector harboring Pepper golden mosaic virus.

Plants are routinely exposed to biotic and abiotic stresses to which they have evolved by synthesizing constitutive and induced defense compounds. Induced defense compounds are usually made, initially, at low levels; however, following further stimulation by specific kinds of biotic and abiotic stresses, they can be synthesized in relatively large amounts to abate the particular stress. cDNA microarray hybridization was used to identify an array of genes that were differentially expressed in tomato plants 15 d after they were exposed to feeding by nonviruliferous whiteflies or by viruliferous whiteflies carrying Pepper golden mosaic virus (PepGMV) (Begomovirus, Geminiviridae). Tomato plants inoculated by viruliferous whiteflies developed symptoms characteristic of PepGMV, whereas plants exposed to nonviruliferous whitefly feeding or nonwounded (negative) control plants exhibited no disease symptoms. The microarray analysis yielded over 290 spotted probes, with significantly altered expression of 161 putative annotated gene targets, and 129 spotted probes of unknown identities. The majority of the differentially regulated "known" genes were associated with the plants exposed to viruliferous compared with nonviruliferous whitefly feeding. Overall, significant differences in gene expression were represented by major physiological functions including defense-, pathogen-, photosynthesis-, and signaling-related responses and were similar to genes identified for other insect-plant systems. Viruliferous whitefly-stimulated gene expression was validated by real-time quantitative polymerase chain reaction of selected, representative candidate genes (messenger RNA): arginase, dehydrin, pathogenesis-related proteins 1 and -4, polyphenol oxidase, and several protease inhibitors. This is the first comparative profiling of the expression of tomato plants portraying different responses to biotic stress induced by viruliferous whitefly feeding (with resultant virus infection) compared with whitefly feeding only and negative control nonwounded plants exposed to neither. These results may be applicable to many other plant-insect-pathogen system interactions.

Larval Helicoverpa zea Transcriptional, Growth and Behavioral Responses to Nicotine and Nicotiana tabacum.

The polyphagous feeding habits of the corn earworm, Helicoverpa zea (Boddie), underscore its status as a major agricultural pest with a wide geographic distribution and host plant repertoire. To study the transcriptomic response to toxins in diet, we conducted a microarray analysis of H. zea caterpillars feeding on artificial diet, diet laced with nicotine and Nicotiana tabacum (L.) plants. We supplemented our analysis with growth and aversion bioassays. The transcriptome reflects an abundant expression of proteases, chitin, cytochrome P450 and immune-related genes, many of which are shared between the two experimental treatments. However, the tobacco treatment tended to elicit stronger transcriptional responses than nicotine-laced diet. The salivary factor glucose oxidase, known to suppress nicotine induction in the plant, was upregulated by H. zea in response to tobacco but not to nicotine-laced diet. Reduced caterpillar growth rates accompanied the broad regulation of genes associated with growth, such as juvenile hormone epoxide hydrolase. The differential expression of chemosensory proteins, such as odorant binding-protein-2 precursor, as well as the neurotransmitter nicotinic-acetylcholine-receptor subunit 9, highlights candidate genes regulating aversive behavior towards nicotine. We suggest that an observed coincidental rise in cannibalistic behavior and regulation of proteases and protease inhibitors in H. zea larvae signify a compensatory response to induced plant defenses.

Gut Transcription in Helicoverpa zea is Dynamically Altered in Response to Baculovirus Infection.

The Helicoverpa zea transcriptome was analyzed 24 h after H. zea larvae fed on artificial diet laced with Helicoverpa zea single nucleopolyhedrovirus (HzSNPV). Significant differential regulation of 1,139 putative genes (p < 0.05 T-test with Benjamini and Hochberg False Discovery Rate) was detected in the gut epithelial tissue; where 63% of these genes were down-regulated and 37% of genes were up-regulated compared to the mock-infected control. Genes that play important roles in digestive physiology were noted as being generally down-regulated. Among these were aminopeptidases, trypsin-like serine proteases, lipases, esterases and serine proteases. Genes related to the immune response reacted in a complex nature having peptidoglycan binding and viral antigen recognition proteins and antiviral pathway systems down-regulated, whereas antimicrobial peptides and prophenoloxidase were up-regulated. In general, detoxification genes, specifically cytochrome P450 and glutathione S-transferase were down-regulated as a result of infection. This report offers the first comparative transcriptomic study of H. zea compared to HzSNPV infected H. zea and provides further groundwork that will lead to a larger understanding of transcriptional perturbations associated with viral infection and the host response to the viral insult in what is likely the most heavily infected tissue in the insect.

Effects of elevated peroxidase levels and corn earworm feeding on gene expression in tomato.

Microarray analysis was used to measure the impact of herbivory by Helicoverpa zea, (corn earworm caterpillar) on wild-type and transgenic tomato, Solanum lycopersicum, plants that over-express peroxidase. Caterpillar herbivory had by far the greatest affect on gene expression, but the peroxidase transgene also altered the expression of a substantial number of tomato genes. Particularly high peroxidase activity resulted in the up-regulation of genes encoding proteinase inhibitors, pathogenesis-related (PR) proteins, as well as proteins associated with iron and calcium transport, and flowering. In a separate experiment conducted under similar conditions, real-time quantitative polymerase chain reaction (qPCR) analysis confirmed our microarray results for many genes. There was some indication that multiple regulatory interactions occurred due to the interaction of the different treatments. While herbivory had the greatest impact on tomato gene expression, our results suggest that levels of expression of a multifunctional gene, such as peroxidase and its products, can influence other gene expression systems distinct from conventional signaling pathways, further indicating the complexity of plant defensive responses to insects.

Caterpillar labial saliva alters tomato plant gene expression.

We examined the effects of Helicoverpa zea caterpillar labial saliva on tomato plant gene expression. Caterpillars with labial salivary glands (mock-ablated) and without (ablated) were fed on tomato plants for 24 hr; then, the leaf mRNA was analyzed with tomato microarrays. Analysis of the transcript profiles revealed 384 expressed sequence tags (ESTs) that were significantly altered due to herbivory compared to the non-wounded plants. The majority of the ESTs were quantitatively altered more so by mock-ablated caterpillars with labial salivary glands than ablated caterpillars. Particularly notable, ESTs encoding acid phosphatase, arginase, acidic endochitinase, dehydrin, polyphenol oxidase, protease inhibitors, and threonine deaminase were more highly stimulated by mock-ablated caterpillars than ablated caterpillars. In addition, tomato leaves were mechanically wounded with scissors and painted with labial salivary gland extract, autoclaved salivary gland extract, or water, and compared to non-wounded tomato plants. After 4 hr, these leaves were collected and a tomato microarray analysis of the mRNA revealed correlation of the gene expression of these leaves altered by mechanical wounding and painted with salivary gland extract to the gene expression of leaves fed on by mock-ablated caterpillars. We show that caterpillar labial saliva is an important component of herbivory that can alter plant gene expression.

Molecular, biochemical, and organismal analyses of tomato plants simultaneously attacked by herbivores from two feeding guilds.

Previous work identified aphids and caterpillars as having distinct effects on plant responses to herbivory. We sought to decipher these interactions across different levels of biological organization, i.e., molecular, biochemical, and organismal, with tomato plants either damaged by one 3rd-instar beet armyworm caterpillar (Spodoptera exigua), damaged by 40 adult potato aphids (Macrosiphum euphorbiae), simultaneous damaged by both herbivores, or left undamaged (controls). After placing insects on plants, plants were transferred to a growth chamber for 5 d to induce a systemic response. Subsequently, individual leaflets from non-damaged parts of plants were excised and used for gene expression analysis (microarrays and quantitative real-time PCR), C/N analysis, total protein analysis, proteinase inhibitor (PI) analysis, and for performance assays. At the molecular level, caterpillars up-regulated 56 and down-regulated 29 genes systemically, while aphids up-regulated 93 and down-regulated 146 genes, compared to controls. Although aphids induced more genes than caterpillars, the magnitude of caterpillar-induced gene accumulation, particularly for those associated with plant defenses, was often greater. In dual-damaged plants, aphids suppressed 27% of the genes regulated by caterpillars, while caterpillars suppressed 66% of the genes regulated by aphids. At the biochemical level, caterpillars induced three-fold higher PI activity compared to controls, while aphids had no effects on PIs either alone or when paired with caterpillars. Aphid feeding alone reduced the foliar C/N ratio, but not when caterpillars also fed on the plants. Aphid and caterpillar feeding alone had no effect on the amount of protein in systemic leaves; however, both herbivores feeding on the plant reduced the amount of protein compared to aphid-damaged plants. At the organismal level, S. exigua neonate performance was negatively affected by prior caterpillar feeding, regardless of whether aphids were present or absent. This study highlights areas of concordance and disjunction between molecular, biochemical, and organismal measures of induced plant resistance when plants are attacked by multiple herbivores. In general, our data produced consistent results when considering each herbivore separately but not when considering them together.

Evidence that the caterpillar salivary enzyme glucose oxidase provides herbivore offense in solanaceous plants.

The insect salivary enzyme glucose oxidase (GOX) can inhibit wound-inducible nicotine production in tobacco, Nicotiana tabacum. We examined whether salivary gland extracts of Helicoverpa zea lacking active GOX could still suppress nicotine in tobacco, Nicotiana tabacum, and whether GOX could suppress wound-inducible defenses of another Solanaceous plant, tomato Lycopersicon esculentum. Tobacco leaves were wounded with a cork borer and treated with water, salivary gland extracts with active GOX (SxG), or salivary gland extracts with inactive GOX (SxI). After three days, leaves treated with SxG had significantly less nicotine than all other wounded treatments. Neonates that fed on the terminal leaves of tobacco plants treated with SxG had significantly higher survival than neonates that fed on leaves treated with either SxI or water. This evidence supports the assertion that GOX is the salivary factor responsible for the suppression of tobacco plant nicotine production by H. zea saliva. Results for the NahG tobacco plants, which lack salicylic acid (SA) due to a transgene for bacterial SA hydroxylase, indicate that suppression of nicotine by GOX does not require SA. However, tobacco leaves that were wounded and treated with SxG had significantly higher levels of the SA-mediated PR-1a protein than leaves treated with SxI or water. Leaves of tomato plants wounded with scissors and then treated with SxG had trypsin inhibitor levels that were moderately lower than plants wounded and treated with purified GOX, water, or SxI. However, all the wounded tomato leaves irrespective of treatment resulted in lower caterpillar growth rates than the non-wounded tomato leaves. Glucose oxidase is the first insect salivary enzyme shown to suppress wound-inducible herbivore defenses of plants.

Evidence that ribonuclease activity present in beetle regurgitant is found to stimulate virus resistance in plants.

Phaseolus vulgaris L. cv. 'Pinto' bean is a local lesion host for the plant pathogen Southern bean mosaic virus (SBMV) and its vector is the Mexican bean beetle, Epilachna varivestis Mulsant. The objective of this study was to determine if prior feeding by the beetle would affect 'Pinto' bean's resistance to SBMV and determine if ribonuclease (RNase), a major constituent of beetle regurgitant, mediated the plant's response to the virus. 'Pinto' bean plants fed upon by beetles had increased resistance to plant viruses compared to non-wounded or mechanically wounded and buffer-treated plants. Plants that were mechanically wounded and treated with RNase had increased resistance to plant viruses that was equal to plants fed upon by adult beetles. The induction of plant pathogen defenses could be a good adaptation for the plant in the presence of a beetle and pathogen threat. This evidence suggests that RNase activity in the beetle regurgitant could function as an insect-derived elicitor of plant resistance to viruses.

Herbivory: caterpillar saliva beats plant defences.

Blood-feeding arthropods secrete special salivary proteins that suppress the defensive reaction they induce in their hosts. This is in contrast to herbivores, which are thought to be helpless victims of plant defences elicited by their oral secretions. On the basis of the finding that caterpillar regurgitant can reduce the amount of toxic nicotine released by the tobacco plant Nicotiana tabacum, we investigate here whether specific salivary components from the caterpillar Helicoverpa zea might be responsible for this suppression. We find that the enzyme glucose oxidase counteracts the production of nicotine induced by the caterpillar feeding on the plant.