Transcriptome profile of pecan scab resistant and susceptible trees from a pecan provenance collection.

Pecan scab is a devastating disease that causes damage to pecan (Carya illinoinensis (Wangenh.) K. Koch) fruit and leaves. The disease is caused by the fungus Venturia effusa (G. Winter) and the main management practice for controlling the disease is by application of fungicides at 2-to-3-week intervals throughout the growing season. Besides disease-related yield loss, application of fungicides can result in considerable cost and increases the likelihood of fungicide resistance developing in the pathogen. Resistant cultivars are available for pecan growers; although, in several cases resistance has been overcome as the pathogen adapts to infect resistant hosts. Despite the importance of host resistance in scab management, there is little information regarding the molecular basis of genetic resistance to pecan scab.The purpose of this study was to elucidate mechanisms of natural pecan scab resistance by analyzing transcripts that are differentially expressed in pecan leaf samples from scab resistant and susceptible trees. The leaf samples were collected from trees in a provenance collection orchard that represents the natural range of pecan in the US and Mexico. Trees in the orchard have been exposed to natural scab infections since planting in 1989, and scab ratings were collected over three seasons. Based on this data, ten susceptible trees and ten resistant trees were selected for analysis. RNA-seq data was collected and analyzed for diseased and non-diseased parts of susceptible trees as well as for resistant trees. A total of 313 genes were found to be differentially expressed when comparing resistant and susceptible trees without disease. For susceptible samples showing scab symptoms, 1,454 genes were identified as differentially expressed compared to non-diseased susceptible samples. Many genes involved in pathogen recognition, defense responses, and signal transduction were up-regulated in diseased samples of susceptible trees, whereas differentially expressed genes in pecan scab resistant samples were generally down-regulated compared to non-diseased susceptible samples.Our results provide the first account of candidate genes involved in resistance/susceptibility to pecan scab under natural conditions in a pecan orchard. This information can be used to aid pecan breeding programs and development of biotechnology-based approaches for generating pecan cultivars with more durable scab resistance.

An updated de novo transcriptome for green ash (Fraxinus pennsylvanica).

De novo transcriptome assembly of next-generation sequencing information has become a powerful tool for the study of non-model species. Transcriptomes generated by this method can have high variability due to endless combinations of user-defined variables and programs available for assembly. Many methods have been developed for evaluating the quality of these assemblies. Here, raw sequencing information for Green ash (Fraxinus pennsylvanica Marshall) that was previously published has been re-evaluated. An updated assembly has been developed by including additional sequencing information not used for the currently accepted transcriptome in combination with more stringent trimming parameters. Input reads were assembled with Trinity and Abyss assembly programs. The resulting Trinity assembly has a 7.3-fold increase in genomic breadth of coverage, a 2.4-fold increase in predicted complete open reading frames, an increased L50 value, and increased Benchmarking Universal Single-Copy Ortholog completeness compared to the earlier published transcriptome. This updated transcriptome can be leveraged to help fight the rapid decline of green ash due to pathogens.

A simplified method for producing laboratory grade recombinant TEV protease from E. coli.

The tobacco etch virus (TEV) protease has become a popular choice for cleaving fusion proteins because of its high stringency in sequence recognition. Procedures for isolating recombinant protein from the cytoplasm of E. coli require rupturing of the cell wall via enzymatic treatment combined with sonication or French press. Here we present an expedited method for producing laboratory-grade TEV protease in E. coli using a freeze-thaw method, followed by purification with immobilized metal affinity chromatography. Protease is obtained by expression from the pDZ2087 plasmid in BL21 (DE3) cells. Proteolysis resulting from this product, cleaves a maltose-binding protein fusion to completion at a fusion-to-protease molar ratio of 50:1.

Biolistic Transformation of Wheat.

Biolistic transformation of wheat is one of the most commonly used methods for gene function study and trait discovery. It has been widely adapted as a fundamental platform to generate wheat plants with new traits and has become a powerful tool for facilitating the crop improvement. In this chapter, we present a complete and straightforward protocol for wheat transformation via biolistic bombardment system. Although wheat is still one of the hardest plant species to transform, this protocol offers an optimized and efficient system to produce transgenic plants. To demonstrate the application of this protocol, in this chapter we describe an example of obtaining transgenic wheat by the co-bombardment of two plasmids, containing a green fluorescent protein gene and a glufosinate herbicide selection gene, respectively. In addition, procedures for the screening and testing of putative transgenic plants are described. This protocol has been successfully applied to generate stable transgenic bread wheat (Triticum aestivum) in both spring and winter varieties.