Nicole G. Novak

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.

Cytokinin Primes Plant Responses to Wounding and Reduces Insect Performance

We report a potential role of endogenous cytokinin supply in priming plant defense against herbivory. Cytokinin priming significantly reduced weight gain by insect larvae. Unlike previously described priming by volatile compounds, priming by cytokinin did not overcome vascular restrictions on systemic wound signaling. However, similar to priming by volatile compounds, cytokinin priming occurred upstream of accumulation of jasmonic acid and its precursor linolenic acid in mechanically wounded source leaves. Cytokinin priming significantly altered wound-induced accumulation of transcripts encoding homologs of allene oxide synthase, trypsin inhibitor, and chitinase. Cytokinin priming may reflect coordination between aboveground inducible defense against herbivory and belowground processes such as nutrient availability. These findings should encourage investigations of how genetic alterations in cytokinin signaling and response pathways may affect plant–herbivore interactions.

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.

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.

Remarkable susceptibility of the diamondback moth (Plutella xylostella) to ingestion of Pir toxins from Photorhabdus luminescens

Genes encoding Pir toxins were cloned and sequenced from Photorhabdus luminescens (Enterobacteriaceae) strain Hm. Cultures of Escherichia coli expressing the Pir A and B proteins were highly toxic when fed to larvae of Plutella xylostella L. (Lepidoptera: Plutellidae), as had been reported previously. Histological examination of P. xylostella larvae fed with recombinant E. coli revealed gross abnormalities of the midgut epithelium, with profound swelling and shedding of the apical membranes. However, the recombinant E. coli had no effect on the growth or mortality of larval Heliothis virescens F. (Lepidoptera: Noctuidae), Manduca sexta L. (Lepidoptera: Sphingidae), Lymantria dispar L. (Lepidoptera: Lymantriidae), or Leptinotarsa decemlineata Say (Coleoptera: Chrysomelidae). Based on these results, P. xylostella is at least 300‐fold more susceptible to Pir toxins than other insect species tested, suggesting that they may not be broadly useful as insecticidal proteins. Because Pir B has sequence similarities with N‐terminal portions of Cry proteins from Bacillus thuringiensis, we also tested the recombinant E. coli against a strain of P. xylostella that is resistant to the Cry 1A toxin, but found no difference in mortality between resistant and susceptible strains.

Examining the molecular interaction between potato (Solanum tuberosum) and Colorado potato beetle Leptinotarsa decemlineata

Colorado potato beetle (CPB) is a devastating herbivorous pest of solanaceous plants. Despite the economic im- pact, little is known about the molecular interaction of CPB with these plants. Using an 11 421 expressed sequence tag (EST) potato microarray, we identified 320 genes differentially expressed in potato leaves in response to CPB herbivory. Amongst these were genes putatively encoding proteinase inhibitors along with enzymes of terpenoid, alkaloid, and phe- nylpropanoid biosynthetic pathways, suggesting the defensive chemistries that constitute potatos defense against CPB her- bivory. Several genes, such as those encoding proteinase inhibitors, represent mechanisms implicated in other plant- herbivory interactions, and could correspond with general defensive chemistry strategies. In other cases, products of the differentially expressed genes may represent taxa-specific defensive chemistry. For example, the presumed alkaloid prod- ucts of a putative tropinone reductase I are specific to a subset of the Solanaceae. Two herbivory-induced genes, not spe- cific to potato, are implicated in the synthesis of volatiles known to attract CPB predators. Comparison of continuous herbivore attack versus recovery from CPB attack indicates that fewer genes involved in defensive chemistry are induced after continuous feeding than after feeding and recovery, suggesting the plants ability to mount a full defense response is enhanced under light versus heavy attack.

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.

Mining the Brassica oleracea Genome for Q-type C2H2 Zinc Finger Transcription Factor Genes

Q-type zinc finger proteins have been studied in several plant species and have been associated with response to stress. A whole genome analysis of Arabidopsis identified 176 putative C2H2 transcription factors (TF). Q-type C2H2 TFs containing the QALGGH motif and are a subset of these. In Arabidopsis, 18 have been described that contain two zinc finger motifs per protein, and several of these have been associated with a response to abiotic stress. The zinc fingers bind to the promoters of genes and negatively regulate transcription via the ethylene-responsive element-binding factor-associated amphiphilic repression (EAR) motif. Previously, we have identified StZFP2 from potato as induced upon infestation by insect pests. Interest in finding similar proteins in the newly annotated cabbage genome (Brassica oleracea var. capitata) led to the discovery of 25 proteins with 2-Q-type zinc finger domains. Examination of several transcriptome studies of B. oleracea submitted to the National Center for Biotechnology Information (NCBI) Sequence Read Archive (SRA) confirms the expression of all of these genes in at least one tissue type. Identification of the nearest homologs of these proteins in Arabidopsis and comparison of the map positions of both Arabidopsis and B. oleracea genes supports both the synteny between the genetic maps of these related species and the triplication event in Brassica species hypothesized to occur prior to the split from the ancestral progenitor of Arabidopsis and B. oleracea.

Over-expression of StZFP2 in Solanum tuberosum L. var. Kennebec (potato) inhibits growth of Tobacco Hornworm larvae (THW, Manduca sexta L.)

ABSTRACT Tobacco hornworm (Manduca sexta, THW) is a voracious pest of tomato and potato. StZFP2 is a Q-type C2H2 zinc finger transcription factor (TF) that is induced upon wounding and infestation. Previous work has shown that Q-type C2H2 TFs are involved in stress responses and when over expressed, can enhance protection against drought, salinity or pathogen infection. Twelve transgenic lines (S1-S12) were tested that over-express StZFP2. Feeding S6 or S8 to THW significantly lowered larval weight (21–37%) as well as increased expression of StPIN2 in comparison to untransformed Kennebec. The increase in StPIN2, a classic marker for insect defense in potato, is consistent with the decreases in larval weight gain.