Tissue-specific mRNA profiling of the Brassica napus-Sclerotinia sclerotiorum interaction uncovers novel regulators of plant immunity.

White mold is caused by the fungal pathogen Sclerotinia sclerotiorum and leads to rapid and significant loss in plant yield. Among its many brassicaceous hosts, including Brassica napus (canola) and Arabidopsis, the response of individual tissue layers directly at the site of infection has yet to be explored. Using laser microdissection coupled with RNA sequencing, we profiled the epidermis, mesophyll, and vascular leaf tissue layers of B. napus in response to S. sclerotiorum. High-throughput tissue-specific mRNA sequencing increased the total number of detected transcripts compared with whole-leaf assessments and provided novel insight into the conserved and specific roles of ontogenetically distinct leaf tissue layers in response to infection. When subjected to pathogen infection, the epidermis, mesophyll, and vasculature activate both specific and shared gene sets. Putative defense genes identified through transcription factor network analysis were then screened for susceptibility against necrotrophic, hemi-biotrophic, and biotrophic pathogens. Arabidopsis deficient in PR5-like RECEPTOR KINASE (PR5K) mRNA levels were universally susceptible to all pathogens tested and were further characterized to identify putative interacting partners involved in the PR5K signaling pathway. Together, these data provide insight into the complexity of the plant defense response directly at the site of infection.

Identification and application of exogenous dsRNA confers plant protection against Sclerotinia sclerotiorum and Botrytis cinerea.

Sclerotinia sclerotiorum, the causal agent of white stem rot, is responsible for significant losses in crop yields around the globe. While our understanding of S. sclerotiorum infection is becoming clearer, genetic control of the pathogen has been elusive and effective control of pathogen colonization using traditional broad-spectrum agro-chemical protocols are less effective than desired. In the current study, we developed species-specific RNA interference-based control treatments capable of reducing fungal infection. Development of a target identification pipeline using global RNA sequencing data for selection and application of double stranded RNA (dsRNA) molecules identified single gene targets of the fungus. Using this approach, we demonstrate the utility of this technology through foliar applications of dsRNAs to the leaf surface that significantly decreased fungal infection and S. sclerotiorum disease symptoms. Select target gene homologs were also tested in the closely related species, Botrytis cinerea, reducing lesion size and providing compelling evidence of the adaptability and flexibility of this technology in protecting plants against devastating fungal pathogens.

RNA sequencing of Brassica napus reveals cellular redox control of Sclerotinia infection.

Brassica napus is one of the world's most valuable oilseeds and is under constant pressure by the necrotrophic fungal pathogen, Sclerotinia sclerotiorum, the causal agent of white stem rot. Despite our growing understanding of host pathogen interactions at the molecular level, we have yet to fully understand the biological processes and underlying gene regulatory networks responsible for determining disease outcomes. Using global RNA sequencing, we profiled gene activity at the first point of infection on the leaf surface 24 hours after pathogen exposure in susceptible (B. napus cv. Westar) and tolerant (B. napus cv. Zhongyou 821) plants. We identified a family of ethylene response factors that may contribute to host tolerance to S. sclerotiorum by activating genes associated with fungal recognition, subcellular organization, and redox homeostasis. Physiological investigation of redox homeostasis was further studied by quantifying cellular levels of the glutathione and ascorbate redox pathway and the cycling enzymes associated with host tolerance to S. sclerotiorum. Functional characterization of an Arabidopsis redox mutant challenged with the fungus provides compelling evidence into the role of the ascorbate-glutathione redox hub in the maintenance and enhancement of plant tolerance against fungal pathogens.

The biocontrol agent Pseudomonas chlororaphis PA23 primes Brassica napus defenses through distinct gene networks.

BACKGROUND: The biological control agent Pseudomonas chlororaphis PA23 is capable of protecting Brassica napus (canola) from the necrotrophic fungus Sclerotinia sclerotiorum via direct antagonism. While we have elucidated bacterial genes and gene products responsible biocontrol, little is known about how the host plant responds to bacterial priming on the leaf surface, including global changes in gene activity in the presence and absence of S. sclerotiorum. RESULTS: Application of PA23 to the aerial surfaces of canola plants reduced the number of S. sclerotiorum lesion-forming petals by 91.1%. RNA sequencing of the host pathogen interface showed that pretreatment with PA23 reduced the number of genes upregulated in response to S. sclerotiorum by 16-fold. By itself, PA23 activated unique defense networks indicative of defense priming. Genes encoding MAMP-triggered immunity receptors detecting flagellin and peptidoglycan were downregulated in PA23 only-treated plants, consistent with post-stimulus desensitization. Downstream, we observed reactive oxygen species (ROS) production involving low levels of H2O2 and overexpression of genes associated with glycerol-3-phosphate (G3P)-mediated systemic acquired resistance (SAR). Leaf chloroplasts exhibited increased thylakoid membrane structures and chlorophyll content, while lipid metabolic processes were upregulated. CONCLUSION: In addition to directly antagonizing S. sclerotiorum, PA23 primes the plant defense response through induction of unique local and systemic defense networks. This study provides novel insight into the effects of biocontrol agents applied to the plant phyllosphere. Understanding these interactions will aid in the development of biocontrol systems as an alternative to chemical pesticides for protection of important crop systems.

Integrating Large-Scale Data and RNA Technology to Protect Crops from Fungal Pathogens.

With a rapidly growing human population it is expected that plant science researchers and the agricultural community will need to increase food productivity using less arable land. This challenge is complicated by fungal pathogens and diseases, many of which can severely impact crop yield. Current measures to control fungal pathogens are either ineffective or have adverse effects on the agricultural enterprise. Thus, developing new strategies through research innovation to protect plants from pathogenic fungi is necessary to overcome these hurdles. RNA sequencing technologies are increasing our understanding of the underlying genes and gene regulatory networks mediating disease outcomes. The application of invigorating next generation sequencing strategies to study plant-pathogen interactions has and will provide unprecedented insight into the complex patterns of gene activity responsible for crop protection. However, questions remain about how biological processes in both the pathogen and the host are specified in space directly at the site of infection and over the infection period. The integration of cutting edge molecular and computational tools will provide plant scientists with the arsenal required to identify genes and molecules that play a role in plant protection. Large scale RNA sequence data can then be used to protect plants by targeting genes essential for pathogen viability in the production of stably transformed lines expressing RNA interference molecules, or through foliar applications of double stranded RNA.

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.

Transcriptome atlas of the Arabidopsis funiculus--a study of maternal seed subregions.

The funiculus anchors the structurally complex seed to the maternal plant, and is the only direct route of transport for nutrients and maternal signals to the seed. While our understanding of seed development is becoming clearer, current understanding of the genetics and cellular mechanisms that contribute to funiculus development is limited. Using laser microdissection combined with global RNA-profiling experiments we compared the genetic profiles of all maternal and zygotic regions and subregions during seed development. We found that the funiculus is a dynamic region of the seed that is enriched for mRNAs associated with hormone metabolism, molecular transport, and metabolic activities corresponding to biological processes that have yet to be described in this maternal seed structure. We complemented our genetic data with a complete histological analysis of the funiculus from the earliest stages of development through to seed maturation at the light and electron microscopy levels. The anatomy revealed signs of photosynthesis, the endomembrane system, cellular respiration, and transport within the funiculus, all of which supported data from the transcriptional analysis. Finally, we studied the transcriptional programming of the funiculus compared to other seed subregions throughout seed development. Using newly designed in silico algorithms, we identified a number of transcriptional networks hypothesized to be responsible for biological processes like auxin response and glucosinolate biosynthesis found specifically within the funiculus. Taken together, patterns of gene activity and histological observations reveal putative functions of the understudied funiculus region and identify predictive transcriptional circuits underlying these biological processes in space and time.

Vitamin C deficiency improves somatic embryo development through distinct gene regulatory networks in Arabidopsis.

Changes in the endogenous ascorbate redox status through genetic manipulation of cellular ascorbate levels were shown to accelerate cell proliferation during the induction phase and improve maturation of somatic embryos in Arabidopsis. Mutants defective in ascorbate biosynthesis such as vtc2-5 contained ~70 % less cellular ascorbate compared with their wild-type (WT; Columbia-0) counterparts. Depletion of cellular ascorbate accelerated cell division processes and cellular reorganization and improved the number and quality of mature somatic embryos grown in culture by 6-fold compared with WT tissues. To gain insight into the molecular mechanisms underlying somatic embryogenesis (SE), we profiled dynamic changes in the transcriptome and analysed dominant patterns of gene activity in the WT and vtc2-5 lines across the somatic embryo culturing process. Our results provide insight into the gene regulatory networks controlling SE in Arabidopsis based on the association of transcription factors with DNA sequence motifs enriched in biological processes of large co-expressed gene sets. These data provide the first detailed account of temporal changes in the somatic embryo transcriptome starting with the zygotic embryo, through tissue dedifferentiation, and ending with the mature somatic embryo, and impart insight into possible mechanisms for the improved culture of somatic embryos in the vtc2-5 mutant line.

Predicting transcriptional circuitry underlying seed coat development.

Filling, protection, and dispersal of angiosperm seeds are largely dependent on the development of the maternally derived seed coat. The development of the seed coat in plants such as Arabidopsis thaliana and Glycine max (soybean) is regulated by a complex network of genes and gene products responsible for the establishment and identity of this multicellular structure. Recent studies support the hypothesis that the structure, development, and function of the seed coat are under the control of transcriptional regulators that are specified in space and time. Furthermore, these transcriptional regulators can act in combination to orchestrate the expression of large gene sets. We discuss the underlying transcriptional circuits of the seed coat sub-regions through the interrogation of large-scale datasets, and also provide some ideas on how the identification and analysis of these datasets can be further improved in these two model oilseed systems.

Transcriptional circuitry underlying seed coat development in Arabidopsis.

We analyzed two sub-regions of the maternal seed coat, chalazal (CZSC) and distal (SC), using transcriptomic and histological analyses in the model plant Arabidopsis thaliana. Hierarchical clustering analysis showed that the CZSC and SC are transcriptionally distinct, though the two sub-regions are more similar during early stages of seed development. Robust statistical and network analysis revealed novel roles for both sub-regions during the course of the seed lifecycle and provides insight into the regulatory circuitry underlying these poorly studied sub-regions of the seed. Data show many of the processes that characterize the SC including starch deposition during the morphogenesis phase, and mucilage deposition and cell wall thickening during the maturation phase, are either absent or expressed to a much lesser extent in the CZSC. We further analyzed the CZSC in detail and show that this sub-region is likely involved in the control of information into the seed from the maternal plant and that some of these processes are predicted to operate through the activity of bZIP transcription factors through the G-box DNA sequence motif.