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.