W. Patrick Wechter

Comparative Genomics of Plant-Associated Pseudomonas spp.: Insights into Diversity and Inheritance of Traits Involved in Multitrophic Interactions

We provide here a comparative genome analysis of ten strains within the Pseudomonas fluorescens group including seven new genomic sequences. These strains exhibit a diverse spectrum of traits involved in biological control and other multitrophic interactions with plants, microbes, and insects. Multilocus sequence analysis placed the strains in three sub-clades, which was reinforced by high levels of synteny, size of core genomes, and relatedness of orthologous genes between strains within a sub-clade. The heterogeneity of the P. fluorescens group was reflected in the large size of its pan-genome, which makes up approximately 54% of the pan-genome of the genus as a whole, and a core genome representing only 45–52% of the genome of any individual strain. We discovered genes for traits that were not known previously in the strains, including genes for the biosynthesis of the siderophores achromobactin and pseudomonine and the antibiotic 2-hexyl-5-propyl-alkylresorcinol; novel bacteriocins; type II, III, and VI secretion systems; and insect toxins. Certain gene clusters, such as those for two type III secretion systems, are present only in specific sub-clades, suggesting vertical inheritance. Almost all of the genes associated with multitrophic interactions map to genomic regions present in only a subset of the strains or unique to a specific strain. To explore the evolutionary origin of these genes, we mapped their distributions relative to the locations of mobile genetic elements and repetitive extragenic palindromic (REP) elements in each genome. The mobile genetic elements and many strain-specific genes fall into regions devoid of REP elements (i.e., REP deserts) and regions displaying atypical tri-nucleotide composition, possibly indicating relatively recent acquisition of these loci. Collectively, the results of this study highlight the enormous heterogeneity of the P. fluorescens group and the importance of the variable genome in tailoring individual strains to their specific lifestyles and functional repertoire.

Deep Sequencing of Small RNAs in Tomato for Virus and Viroid Identification and Strain Differentiation

Small RNAs (sRNA), including microRNAs (miRNA) and small interfering RNAs (siRNA), are produced abundantly in plants and animals and function in regulating gene expression or in defense against virus or viroid infection. Analysis of siRNA profiles upon virus infection in plant may allow for virus identification, strain differentiation, and de novo assembly of virus genomes. In the present study, four suspected virus-infected tomato samples collected in the U.S. and Mexico were used for sRNA library construction and deep sequencing. Each library generated between 5–7 million sRNA reads, of which more than 90% were from the tomato genome. Upon in-silico subtraction of the tomato sRNAs, the remaining highly enriched, virus-like siRNA pools were assembled with or without reference virus or viroid genomes. A complete genome was assembled for Potato spindle tuber viroid (PSTVd) using siRNA alone. In addition, a near complete virus genome (98%) also was assembled for Pepino mosaic virus (PepMV). A common mixed infection of two strains of PepMV (EU and US1), which shared 82% of genome nucleotide sequence identity, also could be differentially assembled into their respective genomes. Using de novo assembly, a novel potyvirus with less than 60% overall genome nucleotide sequence identity to other known viruses was discovered and its full genome sequence obtained. Taken together, these data suggest that the sRNA deep sequencing technology will likely become an efficient and powerful generic tool for virus identification in plants and animals.

Gene expression in developing watermelon fruit

BackgroundCultivated watermelon form large fruits that are highly variable in size, shape, color, and content, yet have extremely narrow genetic diversity. Whereas a plethora of genes involved in cell wall metabolism, ethylene biosynthesis, fruit softening, and secondary metabolism during fruit development and ripening have been identified in other plant species, little is known of the genes involved in these processes in watermelon. A microarray and quantitative Real-Time PCR-based study was conducted in watermelon [Citrullus lanatus (Thunb.) Matsum. & Nakai var. lanatus] in order to elucidate the flow of events associated with fruit development and ripening in this species. RNA from three different maturation stages of watermelon fruits, as well as leaf, were collected from field grown plants during three consecutive years, and analyzed for gene expression using high-density photolithography microarrays and quantitative PCR.ResultsHigh-density photolithography arrays, composed of probes of 832 EST-unigenes from a subtracted, fruit development, cDNA library of watermelon were utilized to examine gene expression at three distinct time-points in watermelon fruit development. Analysis was performed with field-grown fruits over three consecutive growing seasons. Microarray analysis identified three hundred and thirty-five unique ESTs that are differentially regulated by at least two-fold in watermelon fruits during the early, ripening, or mature stage when compared to leaf. Of the 335 ESTs identified, 211 share significant homology with known gene products and 96 had no significant matches with any database accession. Of the modulated watermelon ESTs related to annotated genes, a significant number were found to be associated with or involved in the vascular system, carotenoid biosynthesis, transcriptional regulation, pathogen and stress response, and ethylene biosynthesis. Ethylene bioassays, performed with a closely related watermelon genotype with a similar phenotype, i.e. seeded, bright red flesh, dark green rind, etc., determined that ethylene levels were highest during the green fruit stage followed by a decrease during the white and pink fruit stages. Additionally, quantitative Real-Time PCR was used to validate modulation of 127 ESTs that were differentially expressed in developing and ripening fruits based on array analysis.ConclusionThis study identified numerous ESTs with putative involvement in the watermelon fruit developmental and ripening process, in particular the involvement of the vascular system and ethylene. The production of ethylene during fruit development in watermelon gives further support to the role of ethylene in fruit development in non-climacteric fruits.

High frequency oligonucleotides: targeting active gene (HFO-TAG) markers revealed wide genetic diversity among Citrullus spp. accessions useful for enhancing disease or pest resistance in watermelon cultivars

There is a continuous need to enhance watermelon cultivars for disease and pest resistance. Different U.S. Plant Introductions (PIs) of Citrullus lanatus subsp. lanatus var. lanatus [also known as C. lanatus (Thunb.) Matsum. et Nakai subsp. lanatus var. citroides (Bailey) Mansf. ex Greb.] (CLC) collected in southern Africa are a useful source for enhancing disease or pest resistance in watermelon cultivars. They are also valuable as rootstocks for grafted watermelon, particularly in fields infested with root-knot nematodes or Fusarium wilt. However, there is little information about genetic relationships among these PIs. In this study, genetic diversity was examined among 74 CLC PIs collected from their center of origin in southern Africa. Also, 15 Citrullus lanatus subsp. lanatus (CLL) PIs and the American heirloom cultivars Charleston Gray and Black Diamond (Citrullus lanatus subsp. vulgaris (Schrader ex Eckl. et Zeyh.) Fursa) (CLV) and five Citrullus colocynthis (L.) Schrader (CC) PIs collected in different locations throughout the world were used as out-groups in the phylogenetic analysis for the CLC PIs. Twenty-three high frequency oligonucleotides—targeting active gene (HFO-TAG) primers were used in polymerase chain reaction (PCR) experiments to produce a total of 562 polymorphic markers among the Citrullus PIs and cultivars. Cluster and multidimensional scaling plot analysis produced distinct groups of CLC, CLL, and CC PIs. Several PIs that were designated as CLC or CLL were in transitional positions, indicating that they are the result of gene flow between the major Citrullus groups or subgroups. Population structure analysis indicated that CLC comprises two subgroups; each containing a set of unique alleles. Also, unique alleles exist in the CLL and the CC genotypes. Overall, broad genetic diversity exists among the Citrullus PIs. The data in this study should be useful for identifying PIs with a wide genetic distance between them that could be used in breeding programs aiming to develop heterotic F1 hybrid rootstock lines for grafted watermelon.

Development Of An Engineered Bioluminescent Reporter Phage For Detection Of Bacterial Blight Of Crucifers

ABSTRACT Bacterial blight, caused by the phytopathogen Pseudomonas cannabina pv. alisalensis, is an emerging disease afflicting important members of the Brassicaceae family. The disease is often misdiagnosed as pepper spot, a much less severe disease caused by the related pathogen Pseudomonas syringae pv. maculicola. We have developed a phage-based diagnostic that can both identify and detect the causative agent of bacterial blight and differentiate the two pathogens. A recombinant “light”-tagged reporter phage was generated by integrating bacterial luxAB genes encoding luciferase into the genome of P. cannabina pv. alisalensis phage PBSPCA1. The PBSPCA1::luxAB reporter phage is viable and stable and retains properties similar to those of the wild-type phage. PBSPCA1::luxAB rapidly and sensitively detects P. cannabina pv. alisalensis by conferring a bioluminescent signal response to cultured cells. Detection is dependent on cell viability. Other bacterial pathogens of Brassica species such as P. syringae pv. maculicola, Pseudomonas marginalis, Pectobacterium carotovorum, Xanthomonas campestris pv. campestris, and X. campestris pv. raphani either do not produce a response or produce significantly attenuated signals with the reporter phage. Importantly, the reporter phage detects P. cannabina pv. alisalensis on diseased plant specimens, indicating its potential for disease diagnosis.

“Light-tagged” bacteriophage as a diagnostic tool for the detection of phytopathogens

Detection of the phytopathogen Pseudomonas cannabina pv alisalensis, the causal agent of bacterial blight of crucifers is essential for managing this disease. A phage-based diagnostic assay was developed that detects and identifies P. cannabina pv alisalensis from cultures and diseased plant specimens. A recombinant “light-tagged” reporter phage was generated by integrating the luxAB genes into the P. cannabina pv alisalensis phage PBSPCA1 genome. PBSPCA1::luxAB is viable, stable and detects P. cannabina pv alisalensis within minutes and with high sensitivity by conferring a bioluminescent signal. Detection is dependent on cell viability since cells treated with a bactericidal disinfectant are unable to elicit a signal. Importantly, the reporter phage detects P. cannabina pv alisalensis from diseased plant specimens indicating the potential of the diagnostic for disease identification. The reporter phage displays promise for the rapid and specific diagnostic detection of cultivated isolates, and infected plant specimens.

Genetic Resources of Watermelon

As a result of many years of domestication and selection for desirable fruit quality, most of the modern dessert watermelon cultivars share a narrow genetic base. Africa is the center of origin and diversity of the genus Citrullus and is thus the focus of efforts to collect and conserve germplasm for enhancing dessert watermelons with resistance to diseases and pests. In addition to C. lanatus, accessions of several other species of Citrullus have been used as sources of disease and pest resistance. These are C. amarus (citron watermelon), which is native to southern Africa, C. mucosospermus (egusi watermelon) of sub-Saharan/western Africa origin, and C. colocynthis (colocynth) native to the deserts of northern Africa, the Middle East and Asia. Citrullus amarus, C. lanatus, and C. mucosospermus are readily intercrossed with one another and thus C. amarus and C. mucosospermus have at times been classified as subspecies or botanical varieties within C. lanatus. Genetic resources within Citrullus contain genes conferring resistance to a broad range of fungal diseases such as Fusarium wilt, anthracnose, gummy stem blight; oomycete diseases including Phytophthora capsici, powdery mildew, downy mildew; viruses such as the watermelon strain of Papaya ringspot virus, Zucchini yellow mosaic virus, and Squash vein yellowing virus (SqVYV); and insect pests such as root-knot nematodes, whiteflies, and mites. Watermelon germplasm collections are maintained in China, South Africa and Zimbabwe. The United States Department of Agriculture (USDA), Agricultural Research Service (ARS), National Plant Germplasm System (NPGS), maintains a large collection of watermelon and related Citrullus spp. germplasm. The USDA/ARS/NPGS, Germplasm Resources Information Network (GRIN) http://www.ars-grin.gov/npgs contains general information on accessions held within the USDA/NPGS collection.

Cucurbit Genomics Database (CuGenDB): a central portal for comparative and functional genomics of cucurbit crops

Abstract The Cucurbitaceae family (cucurbit) includes several economically important crops, such as melon, cucumber, watermelon, pumpkin, squash and gourds. During the past several years, genomic and genetic data have been rapidly accumulated for cucurbits. To store, mine, analyze, integrate and disseminate these large-scale datasets and to provide a central portal for the cucurbit research and breeding community, we have developed the Cucurbit Genomics Database (CuGenDB; http://cucurbitgenomics.org) using the Tripal toolkit. The database currently contains all available genome and expressed sequence tag (EST) sequences, genetic maps, and transcriptome profiles for cucurbit species, as well as sequence annotations, biochemical pathways and comparative genomic analysis results such as synteny blocks and homologous gene pairs between different cucurbit species. A set of analysis and visualization tools and user-friendly query interfaces have been implemented in the database to facilitate the usage of these large-scale data by the community. In particular, two new tools have been developed in the database, a ‘SyntenyViewer’ to view genome synteny between different cucurbit species and an ‘RNA-Seq’ module to analyze and visualize gene expression profiles. Both tools have been packed as Tripal extension modules that can be adopted in other genomics databases developed using the Tripal system.