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.

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.

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.

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.

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.