Cabbage looper (Trichoplusia ni Hübner) labial glands contain unique bacterial flora in contrast with their alimentary canal, mandibular glands, and Malpighian tubules.

In recent years, several studies have examined the gut microbiome of lepidopteran larvae and how factors such as host plant affect it, and in turn, how gut bacteria affect host plant responses to herbivory. In addition, other studies have detailed how secretions of the labial (salivary) glands can alter host plant defense responses. We examined the gut microbiome of the cabbage looper (Trichoplusia ni) feeding on collards (Brassica oleracea) and separately analyzed the microbiomes of various organs that open directly into the alimentary canal, including the labial glands, mandibular glands, and the Malpighian tubules. In this study, the gut microbiome of T. ni was found to be generally consistent with those of other lepidopteran larvae in prior studies. The greatest diversity of bacteria appeared in the Firmicutes, Actinobacteria, Proteobacteria, and Bacteriodetes. Well-represented genera included Staphylococcus, Streptococcus, Corynebacterium, Pseudomonas, Diaphorobacter, Methylobacterium, Flavobacterium, and Cloacibacterium. Across all organs, two amplicon sequence variants (ASVs) associated with the genera Diaphorobacter and Cloacibacterium appeared to be most abundant. In terms of the most prevalent ASVs, the alimentary canal, Malpighian tubules, and mandibular glands appeared to have similar complements of bacteria, with relatively few significant differences evident. However, aside from the Diaphorobacter and Cloacibacterium ASVs common to all the organs, the labial glands appeared to possess a distinctive complement of bacteria which was absent or poorly represented in the other organs. Among these were representatives of the Pseudomonas, Flavobacterium, Caulobacterium, Anaerococcus, and Methylobacterium. These results suggest that the labial glands present bacteria with different selective pressures than those occurring in the mandibular gland, Malpighian tubules and the alimentary canal. Given the documented effects that labial gland secretions and the gut microbiome can exert on host plant defenses, the effects exerted by the bacteria inhabiting the labial glands themselves deserve further study.

Detached Leaf Assays to Simplify Gene Expression Studies in Potato During Infestation by Chewing Insect Manduca sexta.

The multitrophic nature of gene expression studies of insect herbivory demands large numbers of biological replicates, creating the need for simpler, more streamlined herbivory protocols. Perturbations of chewing insects are usually studied in whole plant systems. While this whole organism strategy is popular, it is not necessary if similar observations can be replicated in a single detached leaf. The assumption is that basic elements required for signal transduction are present within the leaf itself. In the case of early events in signal transduction, cells need only to receive the signal from the perturbation and transmit that signal to neighboring cells which are assayed for gene expression. The proposed method simply changes the timing of the detachment. In whole plant experiments, larvae are confined to a single leaf which is eventually detached from the plant and assayed for gene expression. If the order of excision is reversed, from last in whole plant studies, to first in the detached study, the feeding experiment is simplified. Solanum tuberosum var. Kennebec is propagated by nodal transfer in a simple tissue culture medium and transferred to soil for further growth if desired. Leaves are excised from the parent plant and relocated to Petri dishes where the feeding assay is conducted with the larval stages of M. sexta. Damaged leaf tissue is assayed for the expression of relatively early events in signal transduction. Gene expression analysis identified infestation-specific Cys2-His2 (C2H2) transcription factors, confirming the success of using detached leaves in early response studies. The method is easier to perform than whole plant infestations and uses less space.

Comparative analysis of the genetic variability within the Q-type C2H2 zinc-finger transcription factors in the economically important cabbage, canola and Chinese cabbage genomes.

BACKGROUND: Brassica oleracea, B. rapa and B. napus encompass many economically important vegetable and oil crops; such as cabbage, broccoli, canola and Chinese cabbage. The genome sequencing of these species allows for gene discovery with an eye towards discerning the natural variability available for future breeding. The Q-type C2H2 zinc-finger protein (ZFP) transcription factors contain zinc finger motifs with a conserved QALGGH as part of the motif and they may play a critical role in the plants response to stress. While they may contain from one to five ZF domains (ZFD) this work focuses on the ZFPs that contain two zinc-fingers, which bind to the promoter of genes, and negatively regulate transcription via the EAR motif. B. oleracea and rapa are diploid and evolved into distinct species about 3.7 million years ago. B. napus is polyploid and formed by fusion of the diploids about 7500 years ago. RESULTS: This work identifies a total of 146 Q-type C2H2-ZFPs with 37 in B. oleracea, 35 in B. rapa and 74 in B. napus. The level of sequence similarity and arrangement of these genes on their chromosomes have mostly remained intact in B. napus, when compared to the chromosomes inherited from either B. rapa or oleracea. In contrast, the difference between the protein sequences of the orthologs of B. rapa and oleracea is greater and their organization on the chromosomes is much more divergent. In general, the 146 proteins are highly conserved especially within the known motifs. Differences within subgroups of ZFPs were identified. Considering that B. napus has twice the number of these proteins in its genome, RNA-Seq data was mined and the expression of 68 of the 74 genes was confirmed. CONCLUSION: Alignment of these proteins gives a snapshot of the variability that may be available naturally in Brassica species. The aim is to study how different ZFPs bind different genes or how dissimilar EAR motifs alter the negative regulation of the genes bound to the ZFP. Results from such studies could be used to enhance tolerance in future Brassica breeding programs.

The remarkable plethora of infestation-responsive Q-type C2H2 transcription factors in potato.

OBJECTIVE: Q-type C2H2 transcription factors (TF) play crucial roles in the plant response to stress, often leading to regulation of downstream genes required for tolerance to these challenges. An infestation-responsive Q-type C2H2 TF (StZFP2) is induced by wounding and infestation in potato. While mining the Solanum tuberosum Group Phureja genome for additional members of this family of proteins, five StZFP2-like genes were found on a portion of chromosome 11. The objective of this work was to differentiate these genes in tissue specificity and expression upon infestation. RESULTS: Examination of different tissues showed that young roots had the highest amounts of transcripts for five of the genes. Expression of their transcripts upon excision or infestation by Manduca sexta, showed that all six genes were induced. Overall, each gene showed variations in its response to infestation and specificity for tissue expression. The six genes encode very similar proteins but most likely play unique roles in the plant response to infestation. In contrast, only two homologs have been identified in Arabidopsis and tomato. Overexpression of similar genes has led to enhanced tolerance to, for example, salinity, drought and pathogen stress. Discovery of these new StZFP2 homologs could provide additional resources for potato breeders.

Herbivory responsive C2H2 zinc finger transcription factor protein StZFP2 from potato.

While C2H2 zinc finger transcription factors (TF) are often regulated by abiotic stress, their role during insect infestation has been overlooked. This study demonstrates that the transcripts of the zinc finger transcription factors StZFP1 and StZFP2 are induced in potato (Solanum tuberosum L.) upon infestation by either the generalist tobacco hornworm (THW, Manduca sexta L.) or the specialist Colorado potato beetle (CPB, Leptinotarsa decemlineata Say). StZFP1 has been previously characterized as conferring salt tolerance to transgenic tobacco and its transcript is induced by Phytophthora infestans and several abiotic stresses. StZFP2 has not been characterized previously, but contains the hallmarks of a C2H2 zinc finger TF, with two conserved zinc finger domains and DLN motif, which encodes a transcriptional repressor domain. Expression studies demonstrate that StZFP2 transcript is also induced by tobacco hornworm and Colorado potato beetle. These observations expand the role of the C2H2 transcription factor in potato to include the response to chewing insect pests.

Herbivory of maize by southern corn rootworm induces expression of the major intrinsic protein ZmNIP1;1 and leads to the discovery of a novel aquaporin ZmPIP2;8.

Aquaporins channel water and other neutral molecules through cell membranes. Aquaporin gene expression is subject to transcriptional control and can be modulated by factors affecting water balance such as salt, abscisic acid and drought. During infestation of maize by southern corn rootworm (SCR), an insect that chews into and significantly damages maize roots, three maize aquaporins were differentially expressed upon prolonged infestation. Using a brief infestation of maize roots ZmNIP1;1 transcript abundance again increased under infestation while expression of a new aquaporin, ZmPIP2;8 and ZmTIP2;2 expression did not change. Since ZmPIP2;8 has not been described previously, the deduced protein sequence was analyzed in silico and found to contain the hallmarks of plant aquaporins, with a predicted protein structure similar to other functionally characterized PIP2s. NIPs characterized to date have been implicated in facilitating the movement of a variety of small molecules, while TIPs and PIPs often have the capacity to facilitate trans-membrane movement of water. Functional assays (using heterologous expression in Xenopus laevis oocytes) of ZmTIP2;2 and ZmPIP2;8 confirmed that these aquaporins demonstrate water channel capacity.

Expression of biologically active human interferon alpha 2 in Aloe vera.

Methods necessary for the successful transformation and regeneration of Aloe vera were developed and used to express the human protein, interferon alpha 2 (IFNα2). IFNα2 is a secreted cytokine that plays a vital role in regulating the cellular response to viral infection. Transgenic plants were regenerated from callus cultures initiated from zygotic embryos. Expression of the IFNA2 transgene in transformed plants was confirmed by RT-PCR and IFNα2 protein was detected by immunoblot analysis. Human A549 cells treated with transgenic aloe extracts for 6 h induced expression of the interferon stimulated gene 54, indicating activation of the IFN signaling pathway. The biological activity of the aloe produced IFNα2 was assessed using an antiviral assay with A549 cells treated with extracts from both the rind and pulp fractions of the shoot and subsequently infected with the lytic encephalomyocarditis virus. The highest level of activity attributable to recombinant IFNα2 was determined to be 625 IU/mg of total soluble protein (TSP) in the rind and 2,108 IU/mg TSP in the pulp. Two daughter plants that vegetatively budded during the course of this study were also confirmed to express IFNα2. These results confirm that Aloe vera is capable of expressing a human protein with biological activity, and that a secreted protein targeting the apoplast can be detected in the pulp fraction of the plant.

Root herbivory: molecular analysis of the maize transcriptome upon infestation by Southern corn rootworm, Diabrotica undecimpunctata howardi.

While many studies have characterized changes to the transcriptome of plants attacked by shoot-eating insect pests, few have examined transcriptome-level effects of root pests. Maize (Zea mays) seedlings were subjected to infestation for approximately 2 weeks by the root herbivore southern corn rootworm (SCR) Diabrotica undecimpunctata howardi, and changes in transcript abundance within both roots and shoots were analyzed using a 57K element microarray. A total of 541 genes showed statistically significant changes in transcript abundance in infested roots, including genes encoding many pathogenesis-related proteins such as chitinases, proteinase inhibitors, peroxidases and ß-1,3-glucanases. Several WRKY transcription factors--often associated with biotic responses--exhibited increased transcript abundance upon SCR feeding. Differentially expressed (DE) genes were also detected in shoots of infested vs control plants. Quantitative Reverse Transcriptase Polymerase Chain Reaction (RT-PCR) was used to confirm patterns of transcript abundance for several significant DE genes using an independent experiment with a 2-6 day period of SCR infestation. Because of the well-documented roles that jasmonic acid (JA) or salicylic acid (SA) play in herbivory responses, the effect of exogenous JA or SA application on transcript abundance corresponding to the same subset of SCR-responsive genes was assessed. The response of these genes at the level of transcript abundance to SA and JA differed between roots and shoots and also differed among the genes that were examined. These data suggested that SA- and JA-dependent and independent signals contributed to the transcriptome-level changes in maize roots and shoots in response to SCR infestation.

A soluble form of P74 can act as a per os infectivity factor to the Autographa californica multiple nucleopolyhedrovirus.

The baculovirus occlusion-derived virion (ODV) is required to spread virus infection among insect hosts via the per os route. The Autographa californica multicapsid nucleopolyhedrovirus P74 protein is an ODV envelope protein that is essential for ODVs to be infectious. P74 is anchored in the ODV envelope by a C-terminal transmembrane anchor domain and is N-terminally exposed on the ODV surface. In the present study, a series of N-terminal and C-terminal truncation mutants of P74 were evaluated for their ability to rescue per os infectivity of the P74-null virus, AcLP4. It was discovered that a P74 truncation mutant lacking the C-terminal transmembrane anchor domain of P74 was able to rescue per os infection. This result shows that a soluble form of P74 retains per os infectivity factor function and suggests that P74 may be complexed with other proteins in the ODV envelope.

Trypsin cleavage of the baculovirus occlusion-derived virus attachment protein P74 is prerequisite in per os infection.

Baculovirus occlusion-derived virions (ODVs) contain a number of infectivity factors essential for the initiation of infection in larval midgut cells. Deletion of any of these factors neutralizes infectivity by the per os route. We have observed that P74 of the group I alphabaculovirus Autographa californica multiple nucleopolyhedrovirus (AcMNPV) is N-terminally cleaved when a soluble form of the protein was incubated with insect midgut tissues under alkaline conditions and that cleavage was prevented by soybean trypsin inhibitor (SBTI). Presently, biological assays were carried out that suggest SBTI inhibits and trypsin enhances baculovirus per os infectivity. We developed a method to rescue per os infectivity of a P74 null virus involving co-transfection of viral DNA with a plasmid that transiently expresses p74. We used this plasmid rescue method to functionally characterize P74. A series of site-directed mutants were generated at the N terminus to evaluate if trypsin cleavage sites were necessary for function. Mutagenesis of R195, R196 and R199 compromised per os infectivity and rendered P74 resistant to midgut trypsin.

Potato, Solanum tuberosum, defense against Colorado potato beetle, Leptinotarsa decemlineata (Say): microarray gene expression profiling of potato by Colorado potato beetle regurgitant treatment of wounded leaves.

Colorado potato beetle (CPB) is a leading pest of solanaceous plants. Despite the economic importance of this pest, surprisingly few studies have been carried out to characterize its molecular interaction with the potato plant. In particular, little is known about the effect of CPB elicitors on gene expression associated with the plant's defense response. In order to discover putative CPB elicitor-responsive genes, the TIGR 11,421 EST Solanaceae microarray was used to identify genes that are differentially expressed in response to the addition of CPB regurgitant to wounded potato leaves. By applying a cutoff corresponding to an adjusted P-value of <0.01 and a fold change of >1.5 or <0.67, we found that 73 of these genes are induced by regurgitant treatment of wounded leaves when compared to wounding alone, whereas 54 genes are repressed by this treatment. This gene set likely includes regurgitant-responsive genes as well as wounding-responsive genes whose expression patterns are further enhanced by the presence of regurgitant. Real-time polymerase chain reaction was used to validate differential expression by regurgitant treatment for five of these genes. In general, genes that encoded proteins involved in secondary metabolism and stress were induced by regurgitant; genes associated with photosynthesis were repressed. One induced gene that encodes aromatic amino acid decarboxylase is responsible for synthesis of the precursor of 2-phenylethanol. This is significant because 2-phenylethanol is recognized by the CPB predator Perillus bioculatis. In addition, three of the 16 type 1 and type 2 proteinase inhibitor clones present on the potato microarray were repressed by application of CPB regurgitant to wounded leaves. Given that proteinase inhibitors are known to interfere with digestion of proteins in the insect midgut, repression of these proteinase inhibitors by CPB may inhibit this component of the plant's defense arsenal. These data suggest that beyond the wound response, CPB elicitors play a role in mediating the plant/insect interaction.

Inhibition of proteinase inhibitor transcripts by Leptinotarsa decemlineata regurgitant in Solanum lycopersicum.

One mechanism by which plants defend themselves against insect herbivores is the production of plant proteinase inhibitors, which can inhibit digestion in the midgut, thus affecting growth and survival. In this work, the effect of Colorado potato beetle (CPB) [Leptinotarsa decemlineata (Say)] regurgitant on Solanum lycopersicum defenses was investigated. When regurgitant from fourth-instar CPB was applied to wounded S. lycopersicum leaves, the wound-induced transcripts for the proteinase inhibitors pin1 and pin2 were reduced. Boiling the regurgitant abolished its ability to reduce the pin transcripts. Ultrafiltration of the regurgitant demonstrated that it contained a component between 10 and 30 kDa molecular weight that inhibited wound-induced pin1 and pin2 expression, suggesting that it may be a protein. This may represent a mechanism that the CPB has evolved to elude the plant's induced response to infestation.

Wound and insect herbivory responsive genes in poplar.

Insect herbivory leads to induced resistance to subsequent infestations in plants. This is due in part to feeding-induced expression of genes that can lead to reduced palatability and/or digestibility of the plant material. We identified 57 distinct differentially expressed genes from poplars that were either infested by gypsy moth (Lymantria dispar) or mechanically wounded. Eleven highly insect-inducible genes were also found to be wound-inducible. Time course analysis revealed diverse timing of peak transcript accumulation. Sequence analysis of promoters suggested that the wound responsive elements, W and DRE, and the jasmonic acid responsive H motif, are over-represented in wound-induced poplar promoters and should be investigated further.

Expression of poplar chitinase in tomato leads to inhibition of development in colorado potato beetle.

The previously described poplar chitinase, WIN6, is induced during infestation by gypsy moth (Lymantria dispar L.) larvae, thus suggesting a role in defense against insect pests. To test this hypothesis, we produced tomato seedlings infected with a recombinant potato virus X (PVX), which produces WIN6, and tested its insecticidal properties on Colorado potato beetle [CPB; Leptinotarsa decemlineata (Say)], which is a serious pest of tomatoes and other crops. The advantage of PVX is that plant material is ready for insect bioassay within 3-4 weeks of constructing the recombinant virus. Considering that production of transgenic tomato seedlings using Agrobacterium takes at least 6 months, this hastens the rate at which genes can be examined. Upon insect bioassay, only 47% CPB neonates feeding on leaves containing >0.3% w/w WIN6 developed to 2nd instar while 93% of controls reached 2nd instar. To our knowledge this is the first plant chitinase that retards development of an insect pest.

Evidence for proteolytic cleavage of the baculovirus occlusion-derived virion envelope protein P74.

Baculovirus occlusion-derived virions (ODVs) are released from occlusion bodies by the alkaline environment of the insect midgut. The ODV envelope protein P74 is required for oral infectivity. A soluble form of the Autographa californica multiple nucleopolyhedrovirus P74 protein, P74sol, was engineered as part of a chimeric protein with jellyfish green fluorescent protein (GFP). P74sol-GFP was overproduced by the baculovirus expression system and purified away from the wild-type P74. Brush border membrane vesicles (BBMVs) were prepared from the midguts of third-instar Helicoverpa zea larvae. When P74sol-GFP was incubated under alkaline conditions with BBMVs, a P74sol-GFP product with a smaller molecular mass was produced. Immunoblots indicated that the smaller product was generated by N-terminal cleavage of P74. This cleavage was prevented by soybean trypsin inhibitor. Analysis of the peptide sequences of P74 homologues identified a conserved trypsin cleavage site that could generate the observed P74sol-GFP BBMV-specific cleavage product.

Maize genes induced by herbivory and volicitin.

In crop plants, both mechanical damage and insect attack trigger rapid changes in gene transcription. We investigated whether insect herbivory differs from a general wound response, and if so, is the induction specific to the pest/host plant interaction? Herbivory by beet armyworm (BAW; Spodoptera exigua) caterpillars on maize results in a unique pattern of volatile compounds not triggered by wounding alone that attracts the generalist parasitoid Cotesia marginiventris. Caterpillar-induced volatile emission can be mimicked when a component of the BAW oral secretions (N-(17-hydroxylinolenoyl)-L-glutamine) termed volicitin, is applied to wounded leaves. We identified genes that are affected by BAW feeding by comparing volicitin treatment with wounding alone. We compared cDNAs from these two populations by isolating genes from a subtractive library and using reverse northerns. Virtual northern blots confirmed these results and further showed that BAW infestation affected the expression of these genes. In some cases, BAW feeding inhibited the expression of volicitin-induced genes, suggesting the role of additional bioactive components in caterpillar regurgitate. Transcripts involved in volatile production are increased by volicitin and BAW infestation treatments, and are also detectable at low levels in mechanically wounded leaves. Finally, we identified three new sesquiterpene cyclase genes that are induced by volicitin.

Epitope tagging: a monoclonal antibody specific for recombinant fusion proteins in plants.

The easy identification of a recombinant protein in plant material becomes increasingly relevant as more transgenic plants are used for research and commercial applications. Tagging recombinant proteins with a small peptide (epitope) can perform such a task. However, available epitope antibodies will also cross-react with endogenous plant proteins at a level that may be unacceptable. Here we describe the new epitope antibody AcV5. Whether it is attached to the carbooxyl terminal end of enhanced green fluorescent protein (EGFP) or the Bacillus thuringiensis endotoxin Cry3A, these proteins remain functional. In addition, using less than 250 pg AcV5-tagged EGFP produces a strong signal on Western blots with no cross-reactivity of proteins from a broad range of plants of agronomic importance.

Purification of DNA for the transfection of a Spodoptera frugiperda cell line.

Spodoptera frugiperda (Sf-9) cells have been widely used in baculovirus expression systems, transient gene expression studies and transgenic cell lines. These applications commonly require the transfection of bacterial plasmid DNA. One of the most reliable methods of preparing transfection-quality plasmid DNA is cesium chloride (CsCl) density gradient centrifugation. However, the traditional CsCl DNA purification is a long and laborious process. We have made a series of modifications to the traditional method that makes it faster, safer and easier. In the current study we demonstrate that DNA prepared by our modified CsCl method was also better for the transfection of Sf-9 cells than DNA prepared by the traditional CsCl method.

A study of the Autographa californica multiple nucleopolyhedrovirus ODV envelope protein p74 using a GFP tag.

The Autographa californica multiple nucleopolyhedrovirus (AcMNPV) protein p74 is associated with the occlusion-derived virus (ODV) envelope. p74 is essential for oral infectivity of ODV and has been proposed to play a role in midgut attachment and/or fusion. In this study, p74 protein was expressed in-frame with green fluorescent protein (GFP) to create a p74-GFP chimera. The C-terminal GFP portion of the chimera facilitated visualization of the trafficking of p74 in baculovirus-infected Spodoptera frugiperda (Sf-9) cells. p74-GFP chimeric proteins localized in the intranuclear ring zone of the nucleus and were found to co-precipitate with the microvesicle fraction of cell lysates. A series of truncations of p74 was expressed as p74-GFP chimeras in recombinant baculoviruses. When C-terminal region S580-F645 was deleted from p74, p74-GFP chimera localization became non-specific and chimeras became soluble. p74 region S580-F645 directed GFP to the intranuclear ring zone in a similar pattern to full-length p74. The hydrophobic C terminus of p74 plays a role in protein localization and possibly in transmembrane anchoring and insertion.