Tissue-specific localization of polyketide synthase and other associated genes in the lichen, Cladonia rangiferina, using laser microdissection.

The biosynthesis of two polyketides, atranorin and fumarprotocetraric acid, produced from a lichen-forming fungus, Cladonia rangiferina (L.) F. H. Wigg. was correlated with the expression of eight fungal genes (CrPKS1, CrPKS3, CrPKS16, Catalase (CAT), Sugar Transporter (MFsug), Dioxygenase (YQE1), C2H2 Transcription factor (C2H2), Transcription Factor PacC (PacC), which are thought to be involved in polyketide biosynthesis, and one algal gene, NAD-dependent deacetylase sirtuin 2 (AsNAD)), using laser microdissection (LMD). The differential gene expression levels within the thallus tissue layers demonstrate that the most active region for potential polyketide biosynthesis within the lichen is the outer apical region proximal to the photobiont but some expression also occurs in reproductive tissue. This is the first study using laser microdissection to explore gene expression of these nine genes and their location of expression; it provides a proof-of-concept for future experiments exploring tissue-specific gene expression within lichens; and it highlights the utility of LMD for use in lichen systems.

Transcriptome analysis of the Brassica napus-Leptosphaeria maculans pathosystem identifies receptor, signaling and structural genes underlying plant resistance.

The hemibiotrophic fungal pathogen Leptosphaeria maculans is the causal agent of blackleg disease in Brassica napus (canola, oilseed rape) and causes significant loss of yield worldwide. While genetic resistance has been used to mitigate the disease by means of traditional breeding strategies, there is little knowledge about the genes that contribute to blackleg resistance. RNA sequencing and a streamlined bioinformatics pipeline identified unique genes and plant defense pathways specific to plant resistance in the B. napus-L. maculans LepR1-AvrLepR1 interaction over time. We complemented our temporal analyses by monitoring gene activity directly at the infection site using laser microdissection coupled to quantitative PCR. Finally, we characterized genes involved in plant resistance to blackleg in the Arabidopsis-L. maculans model pathosystem. Data reveal an accelerated activation of the plant transcriptome in resistant host cotyledons associated with transcripts coding for extracellular receptors and phytohormone signaling molecules. Functional characterization provides direct support for transcriptome data and positively identifies resistance regulators in the Brassicaceae. Spatial gradients of gene activity were identified in response to L. maculans proximal to the site of infection. This dataset provides unprecedented spatial and temporal resolution of the genes required for blackleg resistance and serves as a valuable resource for those interested in host-pathogen interactions.

Tissue-specific laser microdissection of the Brassica napus funiculus improves gene discovery and spatial identification of biological processes.

The three primary tissue systems of the funiculus each undergo unique developmental programs to support the growth and development of the filial seed. To understand the underlying transcriptional mechanisms that orchestrate development of the funiculus at the globular embryonic stage of seed development, we used laser microdissection coupled with RNA-sequencing to produce a high-resolution dataset of the mRNAs present in the epidermis, cortex, and vasculature of the Brassica napus (canola) funiculus. We identified 7761 additional genes in these tissues compared with the whole funiculus organ alone using this technology. Differential expression and enrichment analyses were used to identify several biological processes associated with each tissue system. Our data show that cell wall modification and lipid metabolism are prominent in the epidermis, cell growth and modification occur in the cortex, and vascular tissue proliferation and differentiation occur in the central vascular strand. We provide further evidence that each of the three tissue systems of the globular stage funiculus are involved in specific biological processes that all co-ordinate to support seed development. The identification of genes and gene regulators responsible for tissue-specific developmental processes of the canola funiculus now serves as a valuable resource for seed improvement research.