An Automated Method for Rapid Identification of Putative Gene Family Members in Plants

BACKGROUND: Gene duplication events have played a significant role in genome evolution, particularly in plants. Exhaustive searches for all members of a known gene family as well as the identification of new gene families has become increasingly important. Subfunctionalization via changes in regulatory sequences following duplication (adaptive selection) appears to be a common mechanism of evolution in plants and can be accompanied by purifying selection on the coding region. Such negative selection can be detected by a bias toward synonymous over nonsynonymous substitutions. However, the process of identifying this bias requires many steps usually employing several different software programs. We have simplified the process and significantly shortened the time required by condensing many steps into a few scripts or programs to rapidly identify putative gene family members beginning with a single query sequence. RESULTS: In this report we 1) describe the software tools (SimESTs, PCAT, and SCAT) developed to automate the gene family identification, 2) demonstrate the validity of the method by correctly identifying 3 of 4 PAL gene family members from Arabidopsis using EST data alone, 3) identify 2 to 6 CAD gene family members from Glycine max (previously unidentified), and 4) identify 2 members of a putative Glycine max gene family previously unidentified in any plant species. CONCLUSION: Gene families in plants, particularly that subset where purifying selection has occurred in the coding region, can be identified quickly and easily by integrating our software tools and commonly available contig assembly and ORF identification programs

Quantitative and functional post-translational modification proteomics reveals that TREPH1 plays a role in plant thigmomorphogenesis

Plants can sense both intracellular and extracellular mechanical forces and can respond through morphological changes. The signaling components responsible for mechanotransduction of the touch response are largely unknown. Here, we performed a high-throughput SILIA (stable isotope labeling in Arabidopsis)-based quantitative phosphoproteomics analysis to profile changes in protein phosphorylation resulting from 40 seconds of force stimulation in Arabidopsis thaliana. Of the 24 touch-responsive phosphopeptides identified, many were derived from kinases, phosphatases, cytoskeleton proteins, membrane proteins and ion transporters. TOUCH-REGULATED PHOSPHOPROTEIN1 (TREPH1) and MAP KINASE KINASE 2 (MKK2) and/or MKK1 became rapidly phosphorylated in touch-stimulated plants. Both TREPH1 and MKK2 are required for touch-induced delayed flowering, a major component of thigmomorphogenesis. The treph1-1 and mkk2 mutants also exhibited defects in touch-inducible gene expression. A non-phosphorylatable site-specific isoform of TREPH1 (S625A) failed to restore touch-induced flowering delay of treph1-1, indicating the necessity of S625 for TREPH1 function and providing evidence consistent with the possible functional relevance of the touch-regulated TREPH1 phosphorylation. Bioinformatic analysis and biochemical subcellular fractionation of TREPH1 protein indicate that it is a soluble protein. Altogether, these findings identify new protein players in Arabidopsis thigmomorphogenesis regulation, suggesting that protein phosphorylation may play a critical role in plant force responses

Computational methods for the discovery and analysis of genes and other functional DNA sequences

The need for automating genome analysis is a result of the tremendous amount of genomic data. As of today, a high-throughput DNA sequencing machine can run millions of sequencing reactions in parallel, and it is becoming faster and cheaper to sequence the entire genome of an organism. Public databases containing genomic data are growing exponentially, and hence the rise in demand for intuitive automated methods of DNA analysis and subsequent gene identification. However, the complexity of gene organization makes automation a challenging task, and smart algorithm design and parallelization are necessary to perform accurate analyses in reasonable amounts of time. This work describes two such automated methods for the identification of novel genes within given DNA sequences. The first method utilizes negative selection patterns as an evolutionary rationale for the identification of additional members of a gene family. As input it requires a known protein coding gene in that family. The second method is a massively parallel data mining algorithm that searches a whole genome for inverted repeats (palindromic sequences) and identifies potential precursors of non-coding RNA genes. Both methods were validated successfully on the fully sequenced and well studied plant species, Arabidopsis thaliana --Abstract, page iv

Resistance gene enrichment sequencing (RenSeq) enables reannotation of the NB-LRR gene family from sequenced plant genomes and rapid mapping of resistance loci in segregating populations

RenSeq is a NB-LRR (nucleotide binding-site leucine-rich repeat) gene-targeted, Resistance gene enrichment and sequencing method that enables discovery and annotation of pathogen resistance gene family members in plant genome sequences. We successfully applied RenSeq to the sequenced potato Solanum tuberosum clone DM, and increased the number of identified NB-LRRs from 438 to 755. The majority of these identified R gene loci reside in poorly or previously unannotated regions of the genome. Sequence and positional details on the 12 chromosomes have been established for 704 NB-LRRs and can be accessed through a genome browser that we provide. We compared these NB-LRR genes and the corresponding oligonucleotide baits with the highest sequence similarity and demonstrated that ~80% sequence identity is sufficient for enrichment. Analysis of the sequenced tomato S. lycopersicum ‘Heinz 1706’ extended the NB-LRR complement to 394 loci. We further describe a methodology that applies RenSeq to rapidly identify molecular markers that co-segregate with a pathogen resistance trait of interest. In two independent segregating populations involving the wild Solanum species S. berthaultii (Rpi-ber2) and S. ruiz-ceballosii (Rpi-rzc1), we were able to apply RenSeq successfully to identify markers that co-segregate with resistance towards the late blight pathogen Phytophthora infestans. These SNP identification workflows were designed as easy-to-adapt Galaxy pipelines

Identification and analysis of seven effector protein families with different adaptive and evolutionary histories in plant-associated members of the Xanthomonadaceae.

The Xanthomonadaceae family consists of species of non-pathogenic and pathogenic γ-proteobacteria that infect different hosts, including humans and plants. In this study, we performed a comparative analysis using 69 fully sequenced genomes belonging to this family, with a focus on identifying proteins enriched in phytopathogens that could explain the lifestyle and the ability to infect plants. Using a computational approach, we identified seven phytopathogen-enriched protein families putatively secreted by type II secretory system: PheA (CM-sec), LipA/LesA, VirK, and four families involved in N-glycan degradation, NixE, NixF, NixL, and FucA1. In silico and phylogenetic analyses of these protein families revealed they all have orthologs in other phytopathogenic or symbiotic bacteria, and are involved in the modulation and evasion of the immune system. As a proof of concept, we performed a biochemical characterization of LipA from Xac306 and verified that the mutant strain lost most of its lipase and esterase activities and displayed reduced virulence in citrus. Since this study includes closely related organisms with distinct lifestyles and highlights proteins directly related to adaptation inside plant tissues, novel approaches might use these proteins as biotechnological targets for disease control, and contribute to our understanding of the coevolution of plant-associated bacteria

Characterisation of dairy strains of Geobacillus stearothermophilus and a genomics insight into its growth and survival during dairy manufacture : a thesis presented in partial fulfilment of the requirements for the degree of Doctor of Philosophy in Microbiology at Massey University, Palmerston North, New Zealand

The thermophilic bacilli, such as G. stearothermophilus, are an important group of contaminants in the dairy industry. Although these bacilli are generally not pathogenic, their presence in dairy products is an indicator of poor hygiene and high numbers are unacceptable to customers. In addition, their growth may result in milk product defects caused by the production of acids or enzymes, potentially leding to off-flavours. These bacteria are able to grow in sections of dairy manufacturing plants where temperatures reach 40 – 65 °C. Furthermore, because they are spore formers, they are difficult to eliminate. In addition, they exhibit a fast growth rate and tend to readily form biofilms. Many strategies have been tested to prevent the formation of thermophilic bacilli biofilms in dairy manufacture, but with limited success. This is, in part, because little is known about the diversity of strains found in dairy manufacture, the structure of thermophilic bacilli biofilms and how these bacteria have adapted to grow in a dairy environment. In Chapters 2 and 3, phenotypic approaches were taken to understand the diversity of strains within a manufacturing plant. Specifically in Chapter 2, strains of the most dominant thermphilic bacilli, G. stearothermophilus, were isolated from the surface of various locations within the evaporator section and ten strains were evaluated for different phenotypic characteristics. Biochemical profiling, matrix-assisted laser desorption/ionization time-of-flight mass spectrometry and fatty profiling demonstrated that the population was diverse. In Chapter 3, it was shown that the same ten strains varied in their ability to form biofilms and produce spores. Three strains of G. stearothermophilus, A1, P3 and D1, were selected for further analysis. SEM demonstrated that there were differences in biofilm morphologies between the three strains, particularly D1 versus the other two strains, A1 and P3. In Chapters 4, 5 and 6 a comparative genomics approach was taken to determine how these bacteria are able to grow and survive within a dairy manufacturing environment, as well as how they differ from other strains of Geobacillus. In Chapter 4 draft genome sequences were generated for three strains of G.stearothermophilus. Identification of a putative lactose operon in the three dairy strains provided evidence of dairy adaptation. In Chapter 5 a phylogenomics approach was taken to resolve relationships within the Geobacillus genus and to identify differences within the G. stearothermophilus group itself. Finally in Chapter 6 comparison with the model organism B. subtilis, gave a genomics insight into the potential mechanisms of sporulation for Geobacillus spp

Expressed sequence tags from the oomycete fish pathogen Saprolegnia parasitica reveal putative virulence factors

Peer reviewedPublisher PD

Molecular phenotyping of the pal1 and pal2 mutants of Arabidopsis thaliana reveals far-reaching consequences on phenylpropanoid, amino acid, and carbohydrate metabolism

The first enzyme of the phenylpropanoid pathway, Phe ammonia-lyase (PAL), is encoded by four genes in Arabidopsis thaliana. Whereas PAL function is well established in various plants, an insight into the functional significance of individual gene family members is lacking. We show that in the absence of clear phenotypic alterations in the Arabidopsis pall and pal2 single mutants and with limited phenotypic alterations in the pall pal2 double mutant, significant modifications occur in the transcriptome and metabolome of the pal mutants. The disruption of PAL led to transcriptomic adaptation of components of the phenylpropanoid biosynthesis, carbohydrate metabolism, and amino acid metabolism, revealing complex interactions at the level of gene expression between these pathways. Corresponding biochemical changes included a decrease in the three major flavonol glycosides, glycosylated vanillic acid, scopolin, and two novel feruloyl malates coupled to coniferyl alcohol. Moreover, Phe overaccumulated in the double mutant, and the levels of many other amino acids were significantly imbalanced. The lignin content was significantly reduced, and the syringyl/guaiacyl ratio of lignin monomers had increased. Together, from the molecular phenotype, common and specific functions of PAL1 and PAL2 are delineated, and PAL1 is qualified as being more important for the generation of phenylpropanoids