Susan D. Lawrence

Q-type C2H2 zinc finger proteins (ZFPs) play a role in plant stress response. Overexpression of individual ZFPs enhances tolerance to abiotic stresses such as drought or salt. Overexpression of ZFP, Zat18 enhances tolerance to drought, whereas plants with lower amounts are less resilient. The effect of Zat18 loss on gene expression has not been characterized. Overexpression of an ortholog of Zat18 in potato, StZFP2 enhances tolerance to infestation by Manduca sexta. This work focuses on two Zat18 mutant lines, a knockout and knockdown. The two lines differ in response to infestation. Typically, a chewing insect induces the jasmonic acid (JA) pathway. While infestation induces JA pathway genes, Zat18 mutant lines downregulate abscisic acid signaling and induce ethylene signaling genes ERF1 and EBF2 as well as salicylic acid response gene NPR3. Knockdown Zat18, cause far more changes in the infestation response compared with Col-0 or the Zat18 knockout line.

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.

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.

Late blight in potato caused by Phytophthora infestans remains a major impediment to potato growers worldwide. Utilization of regulators that affect defence may augment control measures to achieve a basal defence against late blight. This work examines the effect of over-expressing the transcription factor StZFP2 in potato on P. infestans infection. StZFP2 is induced upon infestation by chewing insects such as Manduca sexta and the pathogen P. infestans. StZFP2 was transformed into potato under the control of the CaMV 35S promoter. Three over-expressing lines (OE-StZFP2) resulted in significantly lower lesion size five days after infection (DAI). The increased expression of StZFP2 slowed the spread of infection at 5DAI. Levels of StZFP2 and classic pathogen response marker genes, StPR-1b and StPR-2 were significantly lower in more resistant lines, suggesting that OE-StZFP2 affects this pathogen response.

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. 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. 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.

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.

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. 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.

The diamondback moth, Plutella xylostella (L.), is a pest of the Brassicaceae, including the model plant Arabidopsis thaliana (L.) Heynhold. Ecotypes of Arabidopsis have previously been reported to vary in the amounts of leaf area consumed when fed on by diamondback moth larvae. In the present study, food consumption varied among plant lines previously identified as either susceptible or resistant, but larval growth did not differ. Digestive efficiency varied inversely with food consumption. Thus, larvae compensated for reduced digestive efficiency by increasing consumption such that they achieved similar growth rates on all the Arabidopsis lines tested. We conclude that leaf area consumed alone may not be an ideal parameter for determining insect resistance.