Laura L. Hladky

Co-Infection by Two Criniviruses Alters Accumulation of Each Virus in a Host-Specific Manner and Influences Efficiency of Virus Transmission

Tomato chlorosis virus (ToCV), and Tomato infectious chlorosis virus (TICV), family Closteroviridae, genus Crinivirus, cause interveinal chlorosis, leaf brittleness, and limited necrotic flecking or bronzing on tomato leaves. Both viruses cause a decline in plant vigor and reduce fruit yield, and are emerging as serious production problems for field and greenhouse tomato growers in many parts of the world. The viruses have been found together in tomato, indicating that infection by one Crinivirus sp. does not prevent infection by a second. Transmission efficiency and virus persistence in the vector varies significantly among the four different whitefly vectors of ToCV; Bemisia tabaci biotypes A and B, Trialeurodes abutilonea, and T. vaporariorum. Only T. vaporariorum can transmit TICV. In order to elucidate the effects of co-infection on Crinivirus sp. accumulation and transmission efficiency, we established Physalis wrightii and Nicotiana benthamiana source plants, containing either TICV or ToCV alone or both viruses together. Vectors were allowed to feed separately on all virus sources, as well as virus-free plants, then were transferred to young plants of both host species. Plants were tested by quantitative reverse-transcription polymerase chain reaction, and results indicated host-specific differences in accumulation by TICV and ToCV and alteration of accumulation patterns during co-infection compared with single infection. In N. benthamiana, TICV titers increased during co-infection compared with levels in single infection, while ToCV titers decreased. However, in P. wrightii, titers of both TICV and ToCV decreased during mixed infection compared with single infection, although to different degrees. Vector transmission efficiency of both viruses corresponded with virus concentration in the host in both single and mixed infections. This illustrates that Crinivirus epidemiology is impacted not only by vector transmission specificity and incidence of hosts but also by interactions between viruses and efficiency of accumulation in host plants.

A New Expanded Host Range of Cucurbit yellow stunting disorder virus Includes Three Agricultural Crops

Cucurbit yellow stunting disorder virus (CYSDV) was identified in the fall of 2006 affecting cucurbit production in the southwestern United States (California, Arizona), as well as in nearby Sonora, Mexico, resulting in nearly universal infection of fall melon crops in 2006 and 2007, and late infection of 2007 spring melons. Survival of CYSDV through the largely cucurbit-free winter months suggested the presence of weed or alternate crop hosts, although previous studies indicated a limited host range restricted to members of the Cucurbitaceae. To determine potential reservoir hosts for CYSDV in desert production, weed and crop hosts were collected from throughout the region over a period of 26 months, and were tested for the presence of CYSDV by reverse transcription-polymerase chain reaction (RT-PCR) using CYSDV HSP70h- and coat protein gene-specific primers. Many noncucurbits collected from infected melon fields and nearby areas were symptomless and virus free; however, CYSDV was detected in alfalfa (Medicago sativa), lettuce (Lactuca sativa), and snap bean (Phaseolus vulgaris), as well as in several weed species widely prevalent in the region. Typical crinivirus symptoms of interveinal yellowing and leaf brittleness were observed on CYSDV-infected snap bean, alkali mallow (Sida hederacea) and Wrights groundcherry (Physalis wrightii), while other infected crop and weed hosts were symptomless. Transmission tests demonstrated that lettuce, snap bean, alkali mallow, Wrights groundcherry, and buffalo gourd (Cucurbita foetidissima) could serve as virus reservoir hosts for transmission of CYSDV to melon and other cucurbits. These results expand the previously known host range of CYSDV, demonstrating that the virus is capable of infecting not only members of the Cucurbitaceae, but also plants in seven additional taxonomic families.

Methods for detection and differentiation of existing and new crinivirus species through multiplex and degenerate primer RT-PCR.

A method was developed for rapid identification and differentiation of both known and novel crinivirus species involving both multiplex and degenerate reverse transcription-polymerase chain reaction (RT-PCR). The multiplex method can discriminate among known criniviruses infecting vegetable and small fruit crops, and rapidly identify viruses associated with disease symptoms, as well as identification of mixed crinivirus infections. Four host groups for multiplex detection of criniviruses were selected based on the types of crops where specific criniviruses would be expected to occur. Each detection group contained three to four crop-specific primers designed to the same region of the gene encoding the highly conserved RNA-dependent RNA polymerase gene (RdRp) of criniviruses for rapid, single-reaction determination of which crinivirus(es) may be infecting a plant. Degenerate reverse primers used for RT and in PCR were designed to amplify all members of each host group, and were coupled with species-specific forward primers resulting in four separate single-reaction cocktails for detection of most criniviruses sequenced to date, whether present in single or mixed virus infections. Additional viruses can be added to multiplex detection by adjustment of primer concentration for balanced detection of target viruses. In order to identify unknown putative criniviruses or those for which sequence information is not yet available, a genus-wide, universal degenerate primer set was developed. These primers also targeted the crinivirus RdRp gene, and amplify a wide range of crinivirus sequences. Both detection systems can be used with most RNA extraction methods, and with RT-PCR reagents common in most laboratories.

The complete nucleotide sequence and genome organization of tomato infectious chlorosis virus: a distinct crinivirus most closely related to lettuce infectious yellows virus.

The complete nucleotide sequence of tomato infectious chlorosis virus (TICV) was determined and compared with those of other members of the genus Crinivirus. RNA 1 is 8,271 nucleotides long with three open reading frames and encodes proteins involved in replication. RNA 2 is 7,913 nucleotides long and encodes eight proteins common within the genus Crinivirus that are involved in genome protection, movement and other functions yet to be identified. Similarity between TICV and other criniviruses varies throughout the genome but TICV is related more closely to lettuce infectious yellows virus than to any other crinivirus, thus identifying a third group within the genus.

Complete Genome Sequence and Biological Characterization of Moroccan pepper virus (MPV) and Reclassification of Lettuce necrotic stunt virus as MPV

Moroccan pepper virus (MPV) and Lettuce necrotic stunt virus (LNSV) have been steadily increasing in prevalence in central Asia and western North America, respectively, over the past decade. Recent sequence analysis of LNSV demonstrated a close relationship between the coat proteins of LNSV and MPV. To determine the full extent of the relationship between LNSV and MPV, the genomes of three MPV isolates were sequenced and compared with that of LNSV. Sequence analysis demonstrated that genomic nucleotide sequences as well as virus-encoded proteins of the three MPV isolates and LNSV shared 97% or greater identity. A full-length clone of a California LNSV isolate was developed and virus derived from infectious transcripts was used to evaluate host plant reactions under controlled conditions. Symptoms of LNSV matched those described previously for MPV on most of a select series of host plants, although some differences were observed. Collectively, these molecular and biological results demonstrate that LNSV should be classified as MPV within the family Tombusviridae, genus Tombusvirus, and confirm the presence of MPV in North America.

DIFFERENTIATING Rz-1 AND Rz-2 RESISTANCE REACTIONS TO BEET NECROTIC YELLOW VEIN VIRUS THROUGH PROTEOME ANALYSIS IN SUGARBEET

Rhizomania, caused by Beet necrotic yellow vein virus (BNYVV), is one of the most economically important diseases affecting sugarbeet, and is widely distributed in most sugarbeet growing areas of the world. Control is achieved almost exclusively through planting of resistant varieties. Following the introduction of Rz1 varieties in the 1990s, new pathotypes that break resistance have appeared. Previous studies demonstrated that a relatively small number of differences in sugarbeet protein expression were associated with BNYVV infection as well as for resistance. Current studies examine protein differences among resistant (Rz1 and Rz2) and susceptible (rz1 and rz2) sugarbeet, when infected independently with the traditional (pathotype A) and Rz1 resistance-breaking BNYVV (pathotype IV, from California’s Imperial Valley). Near isogenic lines differing only for Rhizomania resistance were provided by KWS, and raised in virus-specific soils under standardized growth chamber conditions. Three independent growth chamber experiments were completed sequentially using the same growth chambers and growth parameters for all plants in all experiments to eliminate variability to the greatest extent possible. Protein was extracted from sugarbeet seedlings three weeks after planting to represent a time point early in the infection cycle. Peptides were purified and concentrated using an on-line enrichment column followed by chromatographic separation on a reverse phase nanospray column using a 90 minute linear gradient. Peptides were eluted directly into the mass spectrometer (Thermo Scientific LTQ linear ion trap). Compound lists of the resulting spectra were generated using Xcalibur 2.2 software (Thermo Scientific). Spectra obtained through mass spectrometry (MS/MS) were searched against the mouse Uniprot Amaranthaceae database (version 11/02/12) concatenated with reverse sequences for determination of the peptide FDR 1 (6,832 sequence entries) using both the Mascot database search engine (version 2.3) and SorcererTM-SEQUEST®. Search results for each independently analyzed sample were imported and combined using Scaffold software 3 (Version 3.6.4, Proteome Software, Portland, OR). Proteins containing shared peptides were grouped by Scaffold (Proteome Software, Portland, OR) to satisfy the laws of parsimony. Manual validation of MS/MS spectra was performed for all protein identifications above the probability thresholds that were based on only two unique peptides. Results identified nearly 500 proteins exhibiting variability; however, our analysis focused on only those proteins exhibiting significantly different expression between treatments. Most of the statistically significant protein differences are associated with photosynthetic pathways, supporting the effect of BNYVV infection on foliar yellowing in the field. Others are involved in pathogen defense. Of particular interest is a Beta-1,3-Glucanase identified in BNYVV-susceptible interactions, which is a protein that has been shown to be a pathogen response protein in corn with antifungal activity. Additionally, this protein has been correlated with the ability of viruses to induce symptoms and move cell-to-cell in infected plants through degradation of callose deposits between cell walls and membranes, including in plasmodesmata. Peroxidases and a chitinase were also identified as significantly different during the susceptible interactions. Peroxidases are proteins involved in active oxygen-based stress responses in plants including responses to virus infection and systemic acquired resistance. Chitinases are involved in plant defense against fungi and could be associated with a host response to P. betae. Another interesting protein is glutamine synthetase, a protein exhibiting a four fold increase in BNYVV-resistant reactions compared with levels in susceptible interactions. This protein is involved in nitrogen regulation in plants. We suspect glutamine synthetase may be involved in general BNYVV infection or P. betae pathogenesis. Other proteins of interest have been identified, and are being evaluated. The project is providing new information on physiological changes illustrated by protein expression variation among sugarbeet plants infected with a resistance-breaking BNYVV pathotype (BNYVV-IV), the resistance-breaking form of the virus from California’s Imperial Valley, and the traditional A-pathotype (common throughout the US and world) and how this is influenced by the presence or absence of either the Rz1 or Rz2 resistance genes.