Asymmetry in Synergistic Interaction Between Wheat streak mosaic virus and Triticum mosaic virus in Wheat.

Wheat streak mosaic virus (WSMV) and Triticum mosaic virus (TriMV), distinct members in the family Potyviridae, are economically important wheat-infecting viruses in the Great Plains region. Previously, we reported that coinfection of wheat by WSMV and TriMV caused disease synergism with increased concentration of both viruses. The mechanisms of synergistic interaction between WSMV and TriMV and the effects of prior infection of wheat by either of these "synergistically interacting partner" (SIP) viruses on the establishment of local and systemic infection by the other SIP virus are not known. In this study, using fluorescent protein-tagged viruses, we found that prior infection of wheat by WSMV or TriMV negatively affected the onset and size of local foci elicited by subsequent SIP virus infection compared with those in buffer-inoculated wheat. These data revealed that prior infection of wheat by an SIP virus has no measurable advantage for another SIP virus on the initiation of infection and cell-to-cell movement. In TriMV-infected wheat, WSMV exhibited accelerated long-distance movement and increased accumulation of genomic RNAs compared with those in buffer-inoculated wheat, indicating that TriMV-encoded proteins complemented WSMV for efficient systemic infection. In contrast, TriMV displayed delayed systemic infection in WSMV-infected wheat, with fewer genomic RNA copies in early stages of infection compared with those in buffer-inoculated wheat. However, during late stages of infection, TriMV accumulation in WSMV-infected wheat increased rapidly with accelerated long-distance movement compared with those in buffer-inoculated wheat. Taken together, these data suggest that interactions between synergistically interacting WSMV and TriMV are asymmetrical; thus, successful establishment of synergistic interaction between unrelated viruses will depend on the order of infection of plants by SIP viruses.

Differences in Fusarium Species in brown midrib Sorghum and in Air Populations in Production Fields.

Several Fusarium spp. cause sorghum (Sorghum bicolor) grain mold, resulting in deterioration and mycotoxin production in the field and during storage. Fungal isolates from the air (2005 to 2006) and from leaves and grain from wild-type and brown midrib (bmr)-6 and bmr12 plants (2002 to 2003) were collected from two locations. Compared with the wild type, bmr plants have reduced lignin content, altered cell wall composition, and different levels of phenolic intermediates. Multilocus maximum-likelihood analysis identified two Fusarium thapsinum operational taxonomic units (OTU). One was identified at greater frequency in grain and leaves of bmr and wild-type plants but was infrequently detected in air. Nine F. graminearum OTU were identified: one was detected at low levels in grain and leaves while the rest were only detected in air. Wright's F statistic (FST) indicated that Fusarium air populations differentiated between locations during crop anthesis but did not differ during vegetative growth, grain development, and maturity. FST also indicated that Fusarium populations from wild-type grain were differentiated from those in bmr6 or bmr12 grain at one location but, at the second location, populations from wild-type and bmr6 grain were more similar. Thus, impairing monolignol biosynthesis substantially effected Fusarium populations but environment had a strong influence.

The Coat Protein and NIa Protease of Two Potyviridae Family Members Independently Confer Superinfection Exclusion.

Superinfection exclusion (SIE) is an antagonistic virus-virus interaction whereby initial infection by one virus prevents subsequent infection by closely related viruses. Although SIE has been described in diverse viruses infecting plants, humans, and animals, its mechanisms, including involvement of specific viral determinants, are just beginning to be elucidated. In this study, SIE determinants encoded by two economically important wheat viruses, Wheat streak mosaic virus (WSMV; genus Tritimovirus, family Potyviridae) and Triticum mosaic virus (TriMV; genus Poacevirus, family Potyviridae), were identified in gain-of-function experiments that used heterologous viruses to express individual virus-encoded proteins in wheat. Wheat plants infected with TriMV expressing WSMV P1, HC-Pro, P3, 6K1, CI, 6K2, NIa-VPg, or NIb cistrons permitted efficient superinfection by WSMV expressing green fluorescent protein (WSMV-GFP). In contrast, wheat infected with TriMV expressing WSMV NIa-Pro or coat protein (CP) substantially excluded superinfection by WSMV-GFP, suggesting that both of these cistrons are SIE effectors encoded by WSMV. Importantly, SIE is due to functional WSMV NIa-Pro or CP rather than their encoding RNAs, as altering the coded protein products by minimally changing RNA sequences led to abolishment of SIE. Deletion mutagenesis further revealed that elicitation of SIE by NIa-Pro requires the entire protein while CP requires only a 200-amino-acid (aa) middle fragment (aa 101 to 300) of the 349 aa. Strikingly, reciprocal experiments with WSMV-mediated expression of TriMV proteins showed that TriMV CP, and TriMV NIa-Pro to a lesser extent, likewise excluded superinfection by TriMV-GFP. Collectively, these data demonstrate that WSMV- and TriMV-encoded CP and NIa-Pro proteins are effectors of SIE and that these two proteins trigger SIE independently of each other. IMPORTANCE: Superinfection exclusion (SIE) is an antagonistic virus-virus interaction that prevents secondary invasions by identical or closely related viruses in the same host cells. Although known to occur in diverse viruses, SIE remains an enigma in terms of key molecular determinants and action mechanisms. In this study, we found that Wheat streak mosaic virus (WSMV) and Triticum mosaic virus (TriMV) encode two independently functioning cistrons that serve as effectors of SIE at the protein but not the RNA level. The coat protein and NIa-Pro encoded by these two viruses, when expressed from a heterologous virus, exerted SIE to the cognate viruses. The identification of virus-encoded effectors of SIE and their transgenic expression could potentially facilitate the development of virus-resistant crop plants. Additionally, functional conservation of SIE in diverse virus groups suggests that a better understanding of the underlying mechanisms of SIE could facilitate the development of novel antiviral therapies against viral diseases.

Triticum mosaic virus exhibits limited population variation yet shows evidence of parallel evolution after replicated serial passage in wheat.

An infectious cDNA clone of Triticum mosaic virus (TriMV) (genus Poacevirus; family Potyviridae) was used to establish three independent lineages in wheat to examine intra-host population diversity levels within protein 1 (P1) and coat protein (CP) cistrons over time. Genetic variation was assessed at passages 9, 18 and 24 by single-strand conformation polymorphism, followed by nucleotide sequencing. The founding P1 region genotype was retained at high frequencies in most lineage/passage populations, while the founding CP genotype disappeared after passage 18 in two lineages. We found that rare TriMV genotypes were present only transiently and lineages followed independent evolutionary trajectories, suggesting that genetic drift dominates TriMV evolution. These results further suggest that experimental populations of TriMV exhibit lower mutant frequencies than that of Wheat streak mosaic virus (genus Tritimovirus; family Potyviridae) in wheat. Nevertheless, there was evidence for parallel evolution at a synonymous site in the TriMV CP cistron.

Dynamics of small RNA profiles of virus and host origin in wheat cultivars synergistically infected by Wheat streak mosaic virus and Triticum mosaic virus: virus infection caused a drastic shift in the endogenous small RNA profile.

Co-infection of wheat (Triticum aestivum L.) by Wheat streak mosaic virus (WSMV, a Tritimovirus) and Triticum mosaic virus (TriMV, a Poacevirus) of the family Potyviridae causes synergistic interaction. In this study, the effects of the synergistic interaction between WSMV and TriMV on endogenous and virus-derived small interfering RNAs (vsiRNAs) were examined in susceptible ('Arapahoe') and temperature-sensitive resistant ('Mace') wheat cultivars at 18°C and 27°C. Single and double infections in wheat caused a shift in the profile of endogenous small RNAs from 24 nt being the most predominant in healthy plants to 21 nt in infected wheat. Massive amounts of 21 and 22 nt vsiRNAs accumulated in singly and doubly infected Arapahoe at both temperatures and in Mace at 27°C but not 18°C. The plus- and minus-sense vsiRNAs were distributed throughout the genomic RNAs in Arapahoe at both temperature regimens and in Mace at 27°C, although some regions served as hot-spots, spawning an excessive number of vsiRNAs. The vsiRNA peaks were conserved among cultivars, suggesting that the Dicer-like enzymes in susceptible and resistant cultivars similarly accessed the genomic RNAs of WSMV or TriMV. Accumulation of large amounts of vsiRNAs in doubly infected plants suggests that the silencing suppressor proteins encoded by TriMV and WSMV do not prevent the formation of vsiRNAs; thus, the synergistic effect observed is independent from RNA-silencing mediated vsiRNA biogenesis. The high-resolution map of endogenous and vsiRNAs from WSMV- and/or TriMV-infected wheat cultivars may form a foundation for understanding the virus-host interactions, the effect of synergistic interactions on host defense, and virus resistance mechanisms in wheat.

An eriophyid mite-transmitted plant virus contains eight genomic RNA segments with unusual heterogeneity in the nucleocapsid protein.

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 C-terminus of Wheat streak mosaic virus coat protein is involved in differential infection of wheat and maize through host-specific long-distance transport.

Viral determinants and mechanisms involved in extension of host range of monocot-infecting viruses are poorly understood. Viral coat proteins (CP) serve many functions in almost every aspect of the virus life cycle. The role of the C-terminal region of Wheat streak mosaic virus (WSMV) CP in virus biology was examined by mutating six negatively charged aspartic acid residues at positions 216, 289, 290, 326, 333, and 334. All of these amino acid residues are dispensable for virion assembly, and aspartic acid residues at positions 216, 333, and 334 are expendable for normal infection of wheat and maize. However, mutants D289N, D289A, D290A, DD289/290NA, and D326A exhibited slow cell-to-cell movement in wheat, which resulted in delayed onset of systemic infection, followed by a rapid recovery of genomic RNA accumulation and symptom development. Mutants D289N, D289A, and D326A inefficiently infected maize, eliciting milder symptoms, while D290A and DD289/290NA failed to infect systemically, suggesting that the C-terminus of CP is involved in differential infection of wheat and maize. Mutation of aspartic acid residues at amino acid positions 289, 290, and 326 severely debilitated virus ingress into the vascular system of maize but not wheat, suggesting that these amino acids facilitate expansion of WSMV host range through host-specific long-distance transport.

Wheat streak mosaic virus infects systemically despite extensive coat protein deletions: identification of virion assembly and cell-to-cell movement determinants.

Viral coat proteins function in virion assembly and virus biology in a tightly coordinated manner with a role for virtually every amino acid. In this study, we demonstrated that the coat protein (CP) of Wheat streak mosaic virus (WSMV; genus Tritimovirus, family Potyviridae) is unusually tolerant of extensive deletions, with continued virion assembly and/or systemic infection found after extensive deletions are made. A series of deletion and point mutations was created in the CP cistron of wild-type and/or green fluorescent protein-tagged WSMV, and the effects of these mutations on cell-to-cell and systemic transport and virion assembly of WSMV were examined. Mutants with overlapping deletions comprising N-terminal amino acids 6 to 27, 36 to 84, 85 to 100, 48 to 100, and 36 to 100 or the C-terminal 14 or 17 amino acids systemically infected wheat with different efficiencies. However, mutation of conserved amino acids in the core domain, which may be involved in a salt bridge, abolished virion assembly and cell-to-cell movement. N-terminal amino acids 6 to 27 and 85 to 100 are required for efficient virion assembly and cell-to-cell movement, while the C-terminal 65 amino acids are dispensable for virion assembly but are required for cell-to-cell movement, suggesting that the C terminus of CP functions as a dedicated cell-to-cell movement determinant. In contrast, amino acids 36 to 84 are expendable, with their deletion causing no obvious effects on systemic infection or virion assembly. In total, 152 amino acids (amino acids 6 to 27 and 36 to 100 and the 65 amino acids at the C-terminal end) of 349 amino acids of CP are dispensable for systemic infection and/or virion assembly, which is rare for multifunctional viral CPs.

Differential Transmission of Triticum mosaic virus by Wheat Curl Mite Populations Collected in the Great Plains.

Wheat is an important food grain worldwide and the primary dryland crop in the western Great Plains. A complex of three wheat curl mite (WCM)-transmitted viruses (Wheat streak mosaic virus, High plains virus, and Triticum mosaic virus [TriMV]) is a cause of serious loss in winter wheat production in the Great Plains. TriMV was first reported in Kansas in 2006 and later found in most other Great Plains states. Currently, three populations of WCM have been identified by genetic characterization and differential responses to mite resistance genes in wheat. In this study, we examined TriMV transmission by these three WCM populations: 'Nebraska' (NE), 'Montana' (MT), and 'South Dakota' (SD). Mite transmission using single-mite transfers revealed that the NE WCM population transmitted TriMV at 41%, while the MT and SD WCM populations failed to transmit TriMV. In multi-mite transfers, the NE WCM population transmitted TriMV at 100% level compared with 2.5% transmission by MT and SD WCM populations. Interestingly, NE mites transferred during the quiescent stages following the first and second instar transmitted TriMV at a 39 to 40% rate, suggesting that immature mites were able to acquire the virus and maintain it through molting. In addition, mite survival for single-mite transfers was significantly lower for NE mites when transferred from TriMV-inoculated source plants (60%) compared with mock-inoculated source plants (84%). This demonstrates potentially negative effects on WCM survival from TriMV. TriMV transmission differences demonstrated in this study underscore the importance of identification of mite genotypes for future studies with TriMV.

Occurrence, sequence polymorphism and population structure of Circulifer tenellus virus 1 in a field population of the beet leafhopper.

Circulifer tenellus virus 1 (CiTV1) is the prototypical example of an unusual group of dsRNA viruses associated with insects and for which ecological data are lacking. A San Joaquin Valley (SJV), California population of the beet leafhopper (BLH; Circulifer tenellus [Baker]) was sampled for CiTV1 in 2010. Among 365 BLH sampled, 119 (32.6%) were positive for CiTV1, with at least one CiTV1-positive BLH collected from each of 35 locations. Chi-square tests indicated that observed CiTV1 incidence differed from expected values based on collection season but not geography within the SJV. Sequence comparisons identified three CiTV1 strains, designated A, B, and C. Strain A predominated (82.4%), strain B was less common (16.8%), and only one (0.8%) strain C isolate was encountered. Chi-square tests demonstrated that observed frequencies of strains A and B did not differ from expected values in space or time, indicating that the SJV population of CiTV1 was unstructured.

Immunodetection of Triticum mosaic virus by DAS- and DAC-ELISA using antibodies produced against coat protein expressed in Escherichia coli: potential for high-throughput diagnostic methods.

Triticum mosaic virus (TriMV), an economically important virus infecting wheat in the Great Plains region of the USA, is the type species of the Poacevirus genus in the family Potyviridae. Sensitive and high-throughput serology-based detection methods are crucial for the management of TriMV and germplasm screening in wheat breeding programs. In this study, TriMV coat protein (CP) was expressed in Escherichia coli, and polyclonal antibodies were generated against purified soluble native form recombinant CP (rCP) in rabbits. Specificity and sensitivity of resulting antibodies were tested in Western immuno-blot and enzyme-linked immunosorbent assays (ELISA). In direct antigen coating (DAC)-ELISA, antibodies reacted specifically, beyond 1:20,000 dilution with TriMV in crude sap, but not with healthy extracts, and antiserum at a 1:10,000 dilution detected TriMV in crude sap up to 1:4860 dilution. Notably, rabbit anti-TriMV IgG and anti-TriMV IgG-alkaline phosphatase conjugate reacted positively with native virions in crude sap in a double antibody sandwich-ELISA, suggesting that these antibodies can be used as coating antibodies which is crucial for any 'sandwich' type of assays. Finally, the recombinant antibodies reacted positively in ELISA with representative TriMV isolates collected from fields, suggesting that antibodies generated against rCP can be used for sensitive, large-scale, and broad-spectrum detection of TriMV.

Variants of Triticum mosaic virus Isolated From Wheat in Colorado Show Divergent Biological Behavior.

Triticum mosaic virus (TriMV) is a recently discovered virus infecting wheat. We compared Colorado isolates C10-492 and C11-775 with the 06-123 isolate. Comparisons were made using enzyme-linked immunosorbent assay (ELISA), infectivity assay, host range, dry weight (DW), inoculation of 'Mace' wheat with temperature-sensitive resistance to Wheat streak mosaic virus, and the deduced amino acid sequence of the coat proteins (CP) and P1 proteins. Both C10-492 and C11-775 infected 'Gallatin' barley and, when compared with 06-123, had significantly reduced ELISA values and virus titers in wheat. Both Colorado isolates caused symptomless infections in Mace, whereas 06-123 caused mosaic symptoms. The amino acid sequences of the CP differed by two and one amino acids for C10-492 and C11-775, respectively, compared with 06-123. The sequence of C10-492 differed from C11-775 by one amino acid. The P1 amino acid sequence of C10-492 and C11-775 differed from 06-123 by three and one amino acids, respectively. The C10-492 and C11-775 isolates reduced DW significantly in 'Karl 92' but significantly less than 06-123. In '2317' wheat, the Colorado isolates did not consistently cause significant reduction in DW, while 06-123 did. The data collectively indicate that C10-492 and C11-775 are isolates of TriMV showing biological behavior diverse from that of 06-123.

Genetic characterization of North American populations of the wheat curl mite and dry bulb mite.

The wheat curl mite, Aceria tosichella Keifer, transmits at least three harmful viruses, wheat streak mosaic virus (WSMV), high plains virus (HPV), and Triticum mosaic virus (TriMV) to wheat (Triticum aestivum L.) throughout the Great Plains. This virus complex is considered to be the most serious disease of winter wheat in the western Great Plains. One component of managing this disease has been developing mite resistance in wheat; however, identification of mite biotypes has complicated deployment and stability of resistance. This biotypic variability in mites and differential virus transmission by different mite populations underscores the need to better understand mite identity. However, A. tosichella has a history of serious taxonomic confusion, especially as it relates to A. tulipae Keifer, the dry bulb mite. Molecular techniques were used to genetically characterize multiple A. tosichella populations and compare them to populations of A. tulipae. DNA from these populations was polymerase chain reaction amplified and the ribosomal ITS2 region sequenced and compared. These results indicated limited variability between these two species, but two distinct types within A. tosichella were found that corresponded to previous work with Australian mite populations. Further work using sequencing of several mitochondrial DNA genes also demonstrated two distinct types of A. tosichella populations. Furthermore, the separation between these two A. tosichella types is comparable to their separation with A. tulipae, suggesting that species scale differences exist between these two types ofA. tosichella. These genetic differences correspond to important biological differences between the types (e.g., biotypic and virus transmission differences). In light of these differences, it is important that future studies on biological response differences account for these mite differences.

Triticum mosaic poacevirus enlists P1 rather than HC-Pro to suppress RNA silencing-mediated host defense.

Triticum mosaic virus (TriMV) is the type species of the newly established Poacevirus genus in the family Potyviridae. In this study, we demonstrate that in contrast to the helper component-proteinase (HC-Pro) of Potyvirus species, the P1 proteins of TriMV and Sugarcane streak mosaic poacevirus function in suppression of RNA silencing (SRS). TriMV P1 effectively suppressed silencing induced by single- or double-stranded RNAs (ss/ds RNAs), and disrupted the systemic spread of silencing signals at a step after silencing signal production. Interestingly, contrary to enhanced SRS activity of potyviral HC-Pro by co-expression with P1, the presence of TriMV HC-Pro reduced SRS activity of TriMV P1. Furthermore, TriMV P1 suppressed systemic silencing triggered by dsRNA more efficiently than the HC-Pro of Turnip mosaic potyvirus. Furthermore, TriMV P1 enhanced the pathogenicity of a heterologous virus. Our results established poaceviral P1 as a potent RNA silencing suppressor that probably employs a novel mechanism to suppress RNA silencing-based antiviral defense.

Tritimovirus P1 functions as a suppressor of RNA silencing and an enhancer of disease symptoms.

Wheat streak mosaic virus (WSMV) is an eriophyid mite-transmitted virus of the genus Tritimovirus, family Potyviridae. Complete deletion of helper component-proteinase (HC-Pro) has no effect on WSMV virulence or disease synergism, suggesting that a different viral protein suppresses RNA silencing. RNA silencing suppression assays using Nicotiana benthamiana 16C plants expressing GFP were conducted with each WSMV protein; only P1 suppressed RNA silencing. Accumulation of GFP siRNAs was markedly reduced in leaves infiltrated with WSMV P1 at both 3 and 6 days post infiltration relative to WSMV HC-Pro and the empty vector control. On the other hand, helper component-proteinase (HC-Pro) of two species in the mite-transmitted genus Rymovirus, family Potyviridae was demonstrated to be a suppressor of RNA silencing. Symptom enhancement assays were conducted by inoculating Potato virus X (PVX) onto transgenic N. benthamiana. Symptoms produced by PVX were more severe on transgenic plants expressing WSMV P1 or potyvirus HC-Pro compared to transgenic plants expressing GFP or WSMV HC-Pro.

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.

The N-terminal region of wheat streak mosaic virus coat protein is a host- and strain-specific long-distance transport factor.

Understanding the genetics underlying host range differences among plant virus strains can provide valuable insights into viral gene functions and virus-host interactions. In this study, we examined viral determinants and mechanisms of differential infection of Zea mays inbred line SDp2 by Wheat streak mosaic virus (WSMV) isolates. WSMV isolates Sidney 81 (WSMV-S81) and Type (WSMV-T) share 98.7% polyprotein sequence identity but differentially infect SDp2: WSMV-S81 induces a systemic infection, but WSMV-T does not. Coinoculation and sequential inoculation of SDp2 with WSMV-T and/or WSMV-S81 did not affect systemic infection by WSMV-S81, suggesting that WSMV-T does not induce a restrictive defense response but that virus-encoded proteins may be involved in differential infection of SDp2. The viral determinant responsible for strain-specific host range was mapped to the N terminus of coat protein (CP) by systematic exchanges of WSMV-S81 sequences with those of WSMV-T and by reciprocal exchanges of CP or CP codons 1 to 74. Green fluorescent protein (GFP)-tagged WSMV-S81 with CP or CP residues 1 to 74 from WSMV-T produced similar numbers of infection foci and genomic RNAs and formed virions in inoculated leaves as those produced with WSMV-S81, indicating that failure to infect SDp2 systemically is not due to defects in replication, cell-to-cell movement, or virion assembly. However, these GFP-tagged hybrids showed profound defects in long-distance transport of virus through the phloem. Furthermore, we found that four of the five differing amino acids in the N terminus of CP between the WSMV-S81 and WSMV-T isolates were collectively involved in systemic infection of SDp2. Taken together, these results demonstrate that the N-terminal region of tritimoviral CP functions in host- and strain-specific long-distance movement.

Population genetics of Homalodisca vitripennis reovirus validates timing and limited introduction to California of its invasive insect host, the glassy-winged sharpshooter.

As RNA viruses evolve rapidly, we hypothesized that a virus could serve as a surrogate to discriminate recently separated populations of an invasive insect species. Homalodisca vitripennis reovirus (HoVRV) was used as a surrogate to assess population structure of glassy-winged sharpshooter (GWSS), an invasive species detected in California ~20 years ago. HoVRV nucleotide sequence polymorphism revealed a bottleneck in the introduced population, yielded population age estimates consistent with timing of GWSS discovery in California, suggested gene flow within the native range but not among native and introduced populations, and could potentially pinpoint source of the introduced population. Collectively, the data support use of a virus surrogate to define critical attributes of invasive species populations, with the caveat that life history of the surrogate must be closely linked to that of the host.