Effective plant virus enrichment using carbon nanotubes and microfluidics.

Plant virus detection and identification in crops is a pillar for disease management, import of crop material, production of clean stock plants and basic plant virology studies. In this report, we present a platform for the enrichment and isolation of known or unknown viruses. This platform is based on carbon nanotube arrays inside a microfluidic device that can be a solution for the identification of low titer viruses from plants. Using our microfluidic devices, we achieved enrichment of two economically important viruses, the orthotospovirus, tomato spotted wilt orthotospovirus (TSWV) and the potyvirus, zucchini yellow mosaic virus (ZYMV). The carbon nanotube arrays integrated in these microfluidic devices are capable of trapping viruses discriminated by their size; the virus rich arrays can be then analyzed by common downstream techniques including immunoassays, PCR, HTS and electron microscopy. This procedure offers a simple to operate and portable sample preparation device capable of trapping viruses from raw plant extracts while reducing the host contamination.

Putative transcription antiterminator RfaH contributes to Erwinia amylovora virulence.

The gram-negative bacterium Erwinia amylovora causes fire blight disease of apple and pear trees. The exopolysaccharide amylovoran and lipopolysaccharides are essential E. amylovora virulence factors. Production of amylovoran and lipopolysaccharide is specified in part by genes that are members of long operons. Here, we show that full virulence of E. amylovora in apple fruitlets and tree shoots depends on the predicted transcription antiterminator RfaH. RfaH reduces pausing in the production of long transcripts having an operon polarity suppressor regulatory element within their promoter region. In E. amylovora, only the amylovoran operon and a lipopolysaccharide operon have such regulatory elements within their promoter regions and in the correct orientation. These operons showed dramatically increased polarity in the ΔrfaH mutant compared to the wild type as determined by RNA sequencing. Amylovoran and lipopolysaccharide production in vitro was reduced in rfaH mutants compared to the wild type, which probably contributes to the rfaH mutant virulence phenotype. Furthermore, type VI secretion cluster 1, which contributes to E. amylovora virulence, showed reduced expression in ΔrfaH compared to the wild type, although without an increase in polarity. The data suggest that E. amylovora RfaH directly, specifically, and exclusively suppresses operon polarity in the amylovoran operon and a lipopolysaccharide operon.

Annotation of glycolysis, gluconeogenesis, and trehaloneogenesis pathways provide insight into carbohydrate metabolism in the Asian citrus psyllid.

Citrus greening disease is caused by the pathogen Candidatus Liberibacter asiaticus and transmitted by the Asian citrus psyllid, Diaphorina citri. No curative treatment or significant prevention mechanism exists for this disease, which causes economic losses from reduced citrus production. A high-quality genome of D. citri is being manually annotated to provide accurate gene models to identify novel control targets and increase understanding of this pest. Here, we annotated 25 D. citri genes involved in glycolysis and gluconeogenesis, and seven in trehaloneogenesis. Comparative analysis showed that glycolysis genes in D. citri are highly conserved but copy numbers vary. Analysis of expression levels revealed upregulation of several enzymes in the glycolysis pathway in the thorax, consistent with the primary use of glucose by thoracic flight muscles. Manually annotating these core metabolic pathways provides accurate genomic foundation for developing gene-targeting therapeutics to control D. citri.

Annotation of putative circadian rhythm-associated genes in Diaphorina citri (Hemiptera: Liviidae).

The circadian rhythm involves multiple genes that generate an internal molecular clock, allowing organisms to anticipate environmental conditions produced by the Earth's rotation on its axis. Here, we present the results of the manual curation of 27 genes that are associated with circadian rhythm in the genome of Diaphorina citri, the Asian citrus psyllid. This insect is the vector for the bacterial pathogen Candidatus Liberibacter asiaticus (CLas), the causal agent of citrus greening disease (Huanglongbing). This disease severely affects citrus industries and has drastically decreased crop yields worldwide. Based on cry1 and cry2 identified in the psyllid genome, D. citri likely possesses a circadian model similar to the lepidopteran butterfly, Danaus plexippus. Manual annotation will improve the quality of circadian rhythm gene models, allowing the future development of molecular therapeutics, such as RNA interference or antisense technologies, to target these genes to disrupt the psyllid biology.

Annotation of yellow genes in Diaphorina citri, the vector for Huanglongbing disease.

Huanglongbing (HLB), also known as citrus greening disease, is caused by the bacterium Candidatus Liberibacter asiaticus (CLas). It is a serious threat to global citrus production. This bacterium is transmitted by the Asian citrus psyllid, Diaphorina citri (Hemiptera). There are no effective in planta treatments for CLas. Therefore, one strategy is to manage the psyllid population. Manual annotation of the D. citri genome can identify and characterize gene families that could be novel targets for psyllid control. The yellow gene family is an excellent target because yellow genes, which have roles in melanization, are linked to development and immunity. Combined analysis of the genome with RNA-seq datasets, sequence homology, and phylogenetic trees were used to identify and annotate nine yellow genes in the D. citri genome. Manual curation of genes in D. citri provided in-depth analysis of the yellow family among hemipteran insects and provides new targets for molecular control of this psyllid pest. Manual annotation was done as part of a collaborative Citrus Greening community annotation project.

Utilizing a chromosomal-length genome assembly to annotate the Wnt signaling pathway in the Asian citrus psyllid, Diaphorina citri.

The Asian citrus psyllid, Diaphorina citri, is an insect vector that transmits Candidatus Liberibacter asiaticus, the causal agent of the Huanglongbing (HLB), or citrus greening disease. This disease has devastated Florida's citrus industry, and threatens California's industry as well as other citrus producing regions around the world. To find novel solutions to the disease, a better understanding of the vector is needed. The D. citri genome has been used to identify and characterize genes involved in Wnt signaling pathways. Wnt signaling is utilized for many important biological processes in metazoans, such as patterning and tissue generation. Curation based on RNA sequencing data and sequence homology confirms 24 Wnt signaling genes within the D. citri genome, including homologs for beta-catenin, Frizzled receptors, and seven Wnt-ligands. Through phylogenetic analysis, we classify D. citri Wnt ligands as Wg/Wnt1, Wnt5, Wnt6, Wnt7, Wnt10, Wnt11, and WntA. The D. citri version 3.0 genome with chromosomal length scaffolds reveals a conserved Wnt1-Wnt6-Wnt10 gene cluster with a gene configuration like that in Drosophila melanogaster. These findings provide greater insight into the evolutionary history of D. citri and Wnt signaling in this important hemipteran vector. Manual annotation was essential for identifying high quality gene models. These gene models can be used to develop molecular systems, such as CRISPR and RNAi, which target and control psyllid populations to manage the spread of HLB. Manual annotation of Wnt signaling pathways was done as part of a collaborative community annotation project.