Genomic Sequencing , Discovery , and Characterization of Viral Pathogens in the Glassy - Winged Sharpshooter

A new viral pathogen of the glassy-winged sharpshooter, GWSS, (Homalodisca vitripennis, aka H. coagulata) was discovered and characterized. Few pathogens of leafhoppers have been discovered which have potential for use as a biological control agent. To identify new pathogens of GWSS we used a genomic approach to isolate, sequence, and identify expressed sequence tags, ESTs, produced from field collected GWSS populations. Viral sequences were identified out of the initial 9,620 ESTs generated from single-pass 5’ end sequencing of the GWSS expression library. Processing produced 8,795 ESTs which had lengths greater than 100 nucleotides post quality and vector trimming. The ESTs had an average read length of 689 bp, and an average inset size of 899 bp. Sequences shorter than 200 contiguous bases were removed from analyses. After assembly there were 3,008 sequences, 799 contigs with an average length of 1,113 bp, and 2,209 singlets. Using these sequences to get a start, it was possible to completely sequence the full virus genome, and the virus was labeled, HoCV-1. Further analyses and characterization of HoCV-1 demonstrated that it infected and crossed the midgut barrier of GWSS. The virus was classified as a member of the family Dicistroviridae, which are single-stranded RNA viruses which do not have a DNA stage. Two other viruses were also identified which are currently being characterized and which were taxonomically unrelated to HoCV-1. These leafhopper viral pathogens appear to induce increased mortality, 40% or more, during the nymphal stages of leafhopper development and may have further applications in the management of leafhopper pests to reduce the spread of Pierce’s disease of grapes. INTRODUCTION Leafhoppers are the second most serious agricultural pest, after aphids, both of which transmit plant diseases. Few leafhopper pathogens are known and efforts to discover pathogens can be costly. Where it occurs the glassy-winged sharpshooter, GWSS, Homalodisca vitripennis Germar 1821 (Hemiptera: Cicadellidae) (Takiya et al., 2006) is the primary vector in the spread of Pierce’s disease, PD, of grapes. Pierce’s disease is caused by strains of the plant-infecting, Xylella bacteria, which cause severe economic losses to viticulture and other tree crops in the USA. The GWSS readily flies long distances, thus spreading PD throughout and across grapes growing regions. To maximize our efforts we chose an approach which would advance our understanding of the genetic basis of leafhopper biology, while optimizing efforts to discover leafhopper pathogens. Therefore, we chose to create a large-scale 5′ end sequencing project of cDNA clones produced from adult GWSS. The use of expressed sequence tags (EST) have proven to be a rapid method to generate important genomic information which permits researchers to address difficult questions concerning insect biology, pathology and disease transmission. OBJECTIVES Search for viral pathogens of sharpshooters using the molecular approach of cDNA libraries which provide a rapid, cost effective method that advances our understanding of an organism, plus identifies the invisible, unknown, internal/external organisms associated with the target species, the GWSS, or other leafhopper. Viral pathogens are tools for leafhopper management and open new avenues to reduce PD. RESULTS & DISCUSSION GWSS Genomics: Adult GWSS were collected from a citrus grove near Riverside, CA. Of the initial 9,620 ESTs generated from single-pass 5’ end sequencing of the GWSS expression library, 8,795 ESTs had lengths greater than 100 nucleotides post quality and vector trimming. The ESTs had an average read length of 689 bp, and an average inset size of 899 bp. Approximately 500 of these sequences were identified as having significant homology to a virus. After assembly of the dataset there were 3,008 sequences, 799 contigs with an average length of 1,113 bp, and 2,209 singlets, average length of 681 bp. Of the total assembled 3,008 sequences, 1,574 or 52.3% corresponded with putative matches in GenBank at an E-value of <-10, while 1,434 sequences, or 47.7%, had ‘no significant homology’ to sequences currently listed in GenBank,nr database by in silico analyses (BLASTX, TBLASTX, BLASTN). The sequence data described in this paper have been submitted GenBank Accession Numbers CF194966 through CF195393. National Center for Biotechnology Information, NCBI. The capsid protein sequence of HoCV-1 was submitted into GenBank (accession number: DQ308403). The genomic architecture was determined (Figure 1) and phylogenetic analyses performed as in Hunnicutt et al., 2006, (Figure 2), (Table 1).