Anthony J. McMechan

Efficient and stable expression of GFP through Wheat streak mosaic virus-based vectors in cereal hosts using a range of cleavage sites: Formation of dense fluorescent aggregates for sensitive virus tracking

A series of Wheat streak mosaic virus (WSMV)-based expression vectors were developed by engineering a cycle 3 GFP (GFP) cistron between P1 and HC-Pro cistrons with several catalytic/cleavage peptides at the C-terminus of GFP. WSMV-GFP vectors with the Foot-and-mouth disease virus 1D/2A or 2A catalytic peptides cleaved GFP from HC-Pro but expressed GFP inefficiently. WSMV-GFP vectors with homologous NIa-Pro heptapeptide cleavage sites did not release GFP from HC-Pro, but efficiently expressed GFP as dense fluorescent aggregates. However, insertion of one or two spacer amino acids on either side of NIb/CP heptapeptide cleavage site or deletion in HC-Pro cistron improved processing by NIa-Pro. WSMV-GFP vectors were remarkably stable in wheat for seven serial passages and for 120 days postinoculation. Mite transmission efficiencies of WSMV-GFP vectors correlated with the amount of free GFP produced. WSMV-GFP vectors infected the same range of cereal hosts as wild-type virus, and GFP fluorescence was detected in most wheat tissues.

An Eriophyid Mite-Transmitted Plant Virus Contains Eight Genomic RNA Segments with Unusual Heterogeneity in the Nucleocapsid Protein

ABSTRACT Eriophyid mite-transmitted, multipartite, negative-sense RNA plant viruses with membrane-bound spherical virions are classified in the genus Emaravirus. We report here that the eriophyid mite-transmitted Wheat mosaic virus (WMoV), an Emaravirus, contains eight genomic RNA segments, the most in a known negative-sense RNA plant virus. Remarkably, two RNA 3 consensus sequences, encoding the nucleocapsid protein, were found with 12.5% sequence divergence, while no heterogeneity was observed in the consensus sequences of additional genomic RNA segments. The RNA-dependent RNA polymerase, glycoprotein precursor, nucleocapsid, and P4 proteins of WMoV exhibited limited sequence homology with the orthologous proteins of other emaraviruses, while proteins encoded by additional genomic RNA segments displayed no significant homology with proteins reported in GenBank, suggesting that the genus Emaravirus evolved further with a divergent octapartite genome. Phylogenetic analyses revealed that WMoV formed an evolutionary link between members of the Emaravirus genus and the family Bunyaviridae. Furthermore, genomic-length virus- and virus-complementary (vc)-sense strands of all WMoV genomic RNAs accumulated asymmetrically in infected wheat, with 10- to 20-fold more virus-sense genomic RNAs than vc-sense RNAs. These data further confirm the octapartite negative-sense polarity of the WMoV genome. In WMoV-infected wheat, subgenomic-length mRNAs of vc sense were detected for genomic RNAs 3, 4, 7, and 8 but not for other RNA species, suggesting that the open reading frames present in the complementary sense of genomic RNAs are expressed through subgenomic- or near-genomic-length vc-sense mRNAs. IMPORTANCE Wheat mosaic virus (WMoV), an Emaravirus, is the causal agent of High Plains disease of wheat and maize. In this study, we demonstrated that the genome of WMoV comprises eight negative-sense RNA segments with an unusual sequence polymorphism in an RNA encoding the nucleocapsid protein but not in the additional genomic RNA segments. WMoV proteins displayed weak or no homology with reported emaraviruses, suggesting that the genus Emaravirus further evolved with a divergent octapartite genome. The current study also examined the profile of WMoV RNA accumulation in wheat and provided evidence for the synthesis of subgenomic-length mRNAs of virus complementary sense. This is the first report to demonstrate that emaraviruses produce subgenomic-length mRNAs that are most likely utilized for genome expression. Importantly, this study facilitates the examination of gene functions and virus diversity and the development of effective diagnostic methods and management strategies for an economically important but poorly understood virus.

The Effect of Temperature, Relative Humidity, and Virus Infection Status on Off-Host Survival of the Wheat Curl Mite (Acari: Eriophyidae)

ABSTRACT The wheat curl mite, Aceria tosichella Keifer, is an eriophyid pest of wheat, although its primary economic impact on wheat is due to the transmission of Wheat streak mosaic (WSMV), Wheat mosaic (also known as High Plains virus), and Triticum mosaic (TriMV) viruses. These viruses cause significant annual losses in winter wheat production throughout the western Great Plains. Temperature and humidity are factors that often influence arthropod survival, especially during dispersal from their hosts, yet the impact of these two factors on off-host survival has not been documented for wheat curl mite. Pathogen-infected host plants often influence the biology and behavior of vectors, yet it is not known if virus-infected wheat affects off-host survival of wheat curl mite. The objectives of this study were to 1) determine if temperature, relative humidity, and mite genotype impact off-host survival of wheat curl mite and 2) determine the effect of WSMV- and TriMV-infected host plants on off-host survival of wheat curl mite. Temperature and relative humidity significantly affected off-host survival of wheat curl mite. Length of survival decreased with increasing temperature (106.2 h at 10°Cand 17.0 h at 30°C) and decreasing relative humidity (78.1 h at 95 and 21.3 h at 2%). Mites from TriMV-infected host plants had ∼20% reduction in survival at 20°Ccompared with those from WSMV-infected plants. The duration of off-host survival of wheat curl mite is influenced by environmental conditions. Management strategies that target a break in host presence will greatly reduce mite densities and virus spread and need to account for these limits.

Planting Date and Variety Selection for Management of Viruses Transmitted by the Wheat Curl Mite (Acari: Eriophyidae).

Abstract Wheat is an important food grain worldwide, and it is the primary dryland crop in the western Great Plains. A complex of three viruses (Wheat streak mosaic, Wheat mosaic, and Triticum mosaic viruses) is a common cause of loss in winter wheat production in the Great Plains. All these viruses are transmitted by the wheat curl mite (Aceria tosichella Keifer). Once these viruses are established, there are no curative actions; therefore, prevention is the key to successful management. A study was designed to evaluate preventative management tactics (planting date, resistant varieties) for reducing the impact from this virus complex. The main plot treatments were three planting dates, and split-plot treatments were three wheat varieties. Varieties were planted at three different times during the fall to simulate early, recommended, and late planting dates. The varieties evaluated in this study were Mace (virus resistant), Millennium (mild tolerance), and Tomahawk (susceptible). Measurements of virus symptomology and yield were used to determine virus impact. Results consistently showed that the resistant Mace yielded more than Millennium or Tomahawk under virus pressure. In some years, delayed planting improved the yields for all varieties, regardless of their background; however, under the most severe virus pressure the combination of both management strategies was not sufficient to provide practical control of this complex. These results illustrate the importance of using a combination of management tactics for this complex, but also reinforce the importance for producers to use additional management strategies (e.g., control preharvest volunteer wheat) to manage this complex.

In Vitro Transcripts of Wild-Type and Fluorescent Protein-Tagged Triticum mosaic virus (Family Potyviridae ) are Biologically Active in Wheat

Triticum mosaic virus (TriMV) (genus Poacevirus, family Potyviridae) is a recently described eriophyid mite-transmitted wheat virus. In vitro RNA transcripts generated from full-length cDNA clones of TriMV proved infectious on wheat. Wheat seedlings inoculated with in vitro transcripts elicited mosaic and mottling symptoms similar to the wild-type virus, and the progeny virus was efficiently transmitted by wheat curl mites, indicating that the cloned virus retained pathogenicity, movement, and wheat curl mite transmission characteristics. A series of TriMV-based expression vectors was constructed by engineering a green fluorescent protein (GFP) or red fluorescent protein (RFP) open reading frame with homologous NIa-Pro cleavage peptides between the P1 and HC-Pro cistrons. We found that GFP-tagged TriMV with seven or nine amino acid cleavage peptides efficiently processed GFP from HC-Pro. TriMV-GFP vectors were stable in wheat for more than 120 days and for six serial passages at 14-day intervals by mechanical inoculation and were transmitted by wheat curl mites similarly to the wild-type virus. Fluorescent protein-tagged TriMV was observed in wheat leaves, stems, and crowns. The availability of fluorescent protein-tagged TriMV will facilitate the examination of virus movement and distribution in cereal hosts and the mechanisms of cross protection and synergistic interactions between TriMV and Wheat streak mosaic virus.

Wheat streak mosaic virus coat protein is a determinant for vector transmission by the wheat curl mite

Wheat streak mosaic virus (WSMV; genus Tritimovirus; family Potyviridae), is transmitted by the wheat curl mite (Aceria tosichella Keifer). The requirement of coat protein (CP) for WSMV transmission by the wheat curl mite was examined using a series of viable deletion and point mutations. Mite transmission of WSMV was completely abolished with deletions comprising CP amino acids 58-100. In contrast, the amino-proximal (amino acids 6-27 and 36-57) and carboxy-terminal (14 amino acids) regions of CP were expendable for mite transmission. Mutation of aspartic acid residues at amino acid positions 289 or 326 (D289A or D326A) at the carboxy-proximal region of CP significantly reduced mite transmission. Remarkably, every wheat plant infected by mutants D289A or D326A through mite transmission but not with in vitro transcripts contained a second-site mutation of R131C and N275H, respectively. Collectively, these data demonstrate for the first time that CP is a determinant for an eriophyid-transmitted plant virus.

Impact of timing and method of virus inoculation on the severity of wheat streak mosaic disease

Wheat streak mosaic virus (WSMV), transmitted by the wheat curl mite Aceria tosichella, frequently causes significant yield loss in winter wheat throughout the Great Plains of the United States. A field study was conducted in the 2013-14 and 2014-15 growing seasons to compare the impact of timing of WSMV inoculation (early fall, late fall, or early spring) and method of inoculation (mite or mechanical) on susceptibility of winter wheat cultivars Mace (resistant) and Overland (susceptible). Relative chlorophyll content, WSMV incidence, and yield components were determined. The greatest WSMV infection occurred for Overland, with the early fall inoculations resulting in the highest WSMV infection rate (up to 97%) and the greatest yield reductions relative to the control (up to 94%). In contrast, inoculation of Mace resulted in low WSMV incidence (1 to 28.3%). The findings from this study indicate that both method of inoculation and wheat cultivar influenced severity of wheat streak mosaic; however, timing of inoculation also had a dramatic influence on disease. In addition, mite inoculation provided much more consistent infection rates and is considered a more realistic method of inoculation to measure disease impact on wheat cultivars.

Correction for Tatineni et al., An Eriophyid Mite-Transmitted Plant Virus Contains Eight Genomic RNA Segments with Unusual Heterogeneity in the Nucleocapsid Protein

Satyanarayana Tatineni, Anthony J. McMechan, Everlyne N. Wosula, Stephen N. Wegulo, Robert A. Graybosch, Roy French, Gary L. Hein USDA Agricultural Research Service and Department of Plant Pathology, University of Nebraska—Lincoln, Lincoln, Nebraska, USA; Department of Entomology and Department of Plant Pathology, University of Nebraska—Lincoln, Lincoln, Nebraska, USA; USDA Agricultural Research Service and Department of Agronomy and Horticulture, University of Nebraska—Lincoln, Lincoln, Nebraska, USA

New Zealand Stresses that It Is High Plains Virus Free, and the Virus Struggles with an Identity Crisis

High Plains virus (HPV), a tentative member of the genus Emaravirus, causes a potentially serious economic disease in cereals. Recently, in this journal, Tatineni et al. (1) mistakenly reported HPV as being present in New Zealand, citing the paper by Lebas et al. from 2005 (2). The 2005 report clearly states that New Zealand is HPV free in both the abstract and the introduction (2). To date, HPV is not known to occur in New Zealand. The Ministry for Primary Industries of New Zealand has very strict regulations in place to prevent the importation of unwanted organisms such as HPV. For example, the importation of Zea mays seeds must follow the requirements stated in Import Health Standard 155.02.05 (for seed for sowing) (3), which includes testing of HPV by enzyme-linked immunosorbent assay (ELISA) or PCR. The Tatineni et al. statement (1) will mislead regulatory officials of New Zealand’s trading partners who regularly monitor world microbe dynamics in the scientific literature. In fact, there are plant biosecurity actions in place (4) that directly affect New Zealand’s international trade when a regulated plant virus like HPV is reported as present. The situation above is aggravated by the lack of a definitive virus name yet to be ruled on by the Emaravirus Study Group, Plant Viruses Subcommittee, International Committee on Taxonomy of Viruses (ICTV). Historically, many names have been used to refer to this virus. The name Wheat spot mosaic virus (WSpM virus) was given to a virus associated with High Plains disease studied in the 1950s (5). WSpM virus-diseased samples were not preserved, and therefore it is not possible to do comparative studies (6, 7). The name High Plains virus, which causes red striping symptoms on maize, was first coined by Jensen et al. in 1996 (8). Subsequently, a virus associated with High Plains disease also eliciting red striping symptoms was partially characterized and sequenced and the name maize red stripe virus (MRStV) was introduced as a replacement for HPV (9). A note added in proof for this paper suggested the name of Wheat mosaic virus (WMoV) based on the fact that the virus is more prevalent in wheat than in maize (9). WMoV was subsequently referred to into recent publications (10, 11). All proposed names reflect diverse symptoms observed among infected wheat and sweet corn varieties and the fact that the virus is commonly detected in mixed infections. HPV is reported to coinfect with Wheat streak mosaic virus (WSMV), Triticum mosaic virus (TriMV), Barley yellow dwarf virus PAV (BYDVPAV), and Cereal yellow dwarf virus RPV (CYDV-RPV) in the Great Plains region of the United States (10, 12). In general, all proposed names do not conflict with ICTV definitions of species, construction of names, and/or rules for orthography. The fact that the HPV name does not refer to a specific host name, but to a geographical location, and its elusiveness for years may have sparked this populated name list. However, other plant viruses have been named based only on geographical origin; examples are the tombusviruses Havel River virus, Lato River virus, and Neckar River virus, which are all ICTV-approved names (13). MacDiarmid et al. in 2013 (4) had proposed and justified new recommendations for plant virus characterization and classification in the context of new virus discovery. Both the ICTV and MacDiarmid et al. (4) are seeking (i) fluent communication among scientists, (ii) stability, (iii) to not use names that might cause error or confusion, and (iv) to avoid the unnecessary creation of names as stated by the ICTV principles of nomenclature. Certainly, HPV is not known to be present in New Zealand, and acknowledgment of the HPV-free status of New Zealand is urgently required. This also implies the requirement of a more meticulous review of manuscripts by referees and editors of the journal. Regarding HPV naming, less confusion in the long term will be expected if HPV is kept as a name, which is justified by the existence of precedent plant virus naming using geographical location markers and HPV’s broader usage in literature and sequence repositories.

Population Dynamics of the Wheat Curl Mite (Acari: Eriophyidae) During the Heading Stages of Winter Wheat

Abstract The wheat curl mite (Aceria tosichella Keifer) is the only known vector of three viruses in wheat—Wheat streak mosaic virus, Wheat mosaic virus, and Triticum mosaic virus. The economic impact of this disease complex is linked to the presence of suitable hosts prior to winter wheat maturing in early summer and the movement of wheat curl mite from wheat to oversummering hosts prior to wheat harvest. Previous research has documented the prevalence and density of mite populations on maturing wheat heads; however, these studies were limited to a few late stages of wheat. A study was conducted to evaluate mite population densities across all stages of head development to determine when wheat curl mites are most abundant and the relative increase in abundance over time. In addition, a study was conducted to evaluate the impact of rainfall on mite populations during wheat heading. A final study was conducted to determine the potential for direct infestation of seedlings germinating from wheat curl mite-infested wheat heads. Results showed a rapid buildup in mite populations from low densities in early heading and peaking at the hard dough stage, with nearly all wheat heads having some mite presence. In addition, high mite populations resulted in direct infestation of germinated seedlings from the early through hard dough stages. Rainfall applications had no observable impact on mite population densities in wheat heads. These results demonstrate the increased potential for mites to infest hosts prior to winter wheat maturing and illustrate the increased risk for these hosts to serve as oversummering hosts.