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.

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.

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.

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.

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.

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.

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.

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.

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.

Wheat cultivar-specific disease synergism and alteration of virus accumulation during co-infection with Wheat streak mosaic virus and Triticum mosaic virus.

Triticum mosaic virus (TriMV), the type member of the newly proposed Poacevirus genus, and Wheat streak mosaic virus (WSMV), the type member of Tritimovirus genus of the family Potyviridae, infect wheat naturally in the Great Plains and are transmitted by wheat curl mites. In this study, we examined the ability of these viruses to infect selected cereal hosts, and found several differential hosts between TriMV and WSMV. Additionally, we examined the interaction between WSMV and TriMV in three wheat cultivars at two temperature regimens (19 and 20 to 26 degrees C), and quantified the virus concentration in single and double infections by real-time reverse-transcription polymerase chain reaction. Double infections in wheat cvs. Arapahoe and Tomahawk at both temperature regimens induced disease synergism with severe leaf deformation, bleaching, and stunting, with a 2.2- to 7.4-fold increase in accumulation of both viruses over single infections at 14 days postinoculation (dpi). However, at 28 dpi, in double infections at 20 to 26 degrees C, TriMV concentration was increased by 1.4- to 1.8-fold in Arapahoe and Tomahawk but WSMV concentration was decreased to 0.5-fold. WSMV or TriMV replicated poorly in Mace at 19 degrees C with no synergistic interaction whereas both viruses accumulated at moderate levels at 20 to 26 degrees C and induced mild to moderate disease synergism in doubly infected Mace compared with Arapahoe and Tomahawk. Co-infections in Mace at 20 to 26 degrees C caused increased TriMV accumulation at 14 and 28 dpi by 2.6- and 1.4-fold and WSMV accumulated at 0.5- and 1.6-fold over single infections, respectively. Our data suggest that WSMV and TriMV induced cultivar-specific disease synergism in Arapahoe, Tomahawk, and Mace, and these findings could have several implications for management of wheat viruses in the Great Plains.

Wheat streak mosaic virus genotypes introduced to Argentina are closely related to isolates from the American Pacific Northwest and Australia.

Wheat streak mosaic virus (WSMV) was first detected in Argentina in 2002. Comparison of 78 WSMV coat protein sequences revealed that three Argentine isolates were closely related to isolates from the American Pacific Northwest (APNW) and Australia. Complete sequences were determined for one Argentine isolate, four APNW isolates, and three additional isolates from other regions of the USA. Comparison of these eight new sequences with five previously sequenced isolates of WSMV confirmed close affinity of WSMV from Argentina with APNW isolates. Collectively, these results indicate concurrent establishment of the same WSMV lineage in both Argentina and Australia.

Triticum mosaic virus: a distinct member of the family potyviridae with an unusually long leader sequence.

The complete genome sequence of Triticum mosaic virus (TriMV), a member in the family Potyviridae, has been determined to be 10,266 nucleotides (nt) excluding the 3' polyadenylated tail. The genome encodes a large polyprotein of 3,112 amino acids with the "hall-mark proteins" of potyviruses, including a small overlapping gene, PIPO, in the P3 cistron. The genome of TriMV has an unusually long 5' nontranslated region of 739 nt with 12 translation initiation codons and three small open reading frames, which resemble those of the internal ribosome entry site containing 5' leader sequences of the members of Picornaviridae. Pairwise comparison of 10 putative mature proteins of TriMV with those of representative members of genera in the family Potyviridae revealed 33 to 44% amino acid identity within the highly conserved NIb protein sequence and 15 to 29% amino acid identity within the least conserved P1 protein, suggesting that TriMV is a distinct member in the family Potyviridae. In contrast, TriMV displayed 47 to 65% amino acid sequence identity with available sequences of mature proteins of Sugarcane streak mosaic virus (SCSMV), an unassigned member of the Potyviridae. Phylogenetic analyses of the complete polyprotein, NIa-Pro, NIb, and coat protein sequences of representative species of six genera and unassigned members of the family Potyviridae suggested that TriMV and SCSMV are sister taxa and share a most recent common ancestor with tritimoviruses or ipomoviruses. These results suggest that TriMV and SCSMV should be classified in a new genus, and we propose the genus Poacevirus in the family Potyviridae, with TriMV as the type member.

Wheat streak mosaic virus Lacking Helper Component-Proteinase Is Competent to Produce Disease Synergism in Double Infections with Maize chlorotic mottle virus.

ABSTRACT The tritimovirus Wheat streak mosaic virus (WSMV) and the machlomovirus Maize chlorotic mottle virus (MCMV) each cause systemic chlorosis in infected maize plants. Infection of maize with both viruses produces corn lethal necrosis disease (CLND). Here, we report that complete deletion of the WSMV helper component-proteinase (HC-Pro) coding region had no effect on induction of CLND symptoms following coinoculation of maize with WSMV and MCMV. We further demonstrated that elevation of virus titers in double infections, relative to single infections, also was independent of WSMV HC-Pro. Thus, unlike potyvirus HC-Pro, WSMV HC-Pro was dispensable for disease synergism. Because disease synergism involving potyviruses requires HC-Pro-mediated suppression of posttranscriptional gene silencing (PTGS), we hypothesized that WSMV HC-Pro may not be a suppressor of PTGS. Indeed, WSMV HC-Pro did not suppress PTGS of a green fluorescent protein (GFP) transgene in an Agrobacterium-mediated coinfiltration assay in which potyvirus HC-Pro acted as a strong suppressor. Furthermore, coinfiltration with potyvirus HC-Pro, but not WSMV HC-Pro, resulted in elevated levels of the GFP target mRNA under conditions which trigger PTGS. Collectively, these results revealed significant differences in HC-Pro function among divergent genera of the family Potyviridae and suggest that the tritimovirus WSMV utilizes a gene other than HC-Pro to suppress PTGS and mediate synergistic interactions with unrelated viruses.

Evolution of Wheat streak mosaic virus: dynamics of population growth within plants may explain limited variation.

Like many other plant RNA viruses, Wheat streak mosaic virus (WSMV) sequence diversity within and among infected plants is low given the large number of virions produced. This may be explained by considering aspects of plant virus life history. Intracellular replication of RNA viruses is predominately linear, not exponential, which means that the rate at which mutations accumulate also is linear. Bottlenecks during systemic movement further limit diversity. Analysis of mixed infections with two WSMV isolates suggests that about four viral genomes participate in systemic invasion of each tiller. Low effective population size increases the role of stochastic processes on dynamics of plant virus population genetics and evolution. Despite low pair-wise diversity among isolates, the number of polymorphic sites within the U.S. population is about the same as between divergent strains or a sister species. Characteristics of polymorphism in the WSMV coat protein gene suggest that most variation appears neutral.

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.

Spinach curly top virus: A Newly Described Curtovirus Species from Southwest Texas with Incongruent Gene Phylogenies.

ABSTRACT A curtovirus associated with a disease of spinach was isolated in southwest Texas during 1996. Disease symptoms included severe stunting and chlorosis, with younger leaves curled, distorted, and dwarfed. Viral DNA was purified and an infectious clone obtained. Agroinoculation using a construct bearing full-length tandem repeats of the cloned viral genome resulted in systemic infection of species in six of seven plant families tested, indicating that the virus has a wide host range. Symptoms produced in spinach agroinoculated with cloned viral DNA were similar to those observed in the field. Viral single-stranded and double-stranded DNA forms typical of curtovirus infection were detected in host plants by Southern blot hybridization. The complete sequence of the infectious clone comprised 2,925 nucleotides, with seven open reading frames encoding proteins homologous to those of other curtoviruses. Complete genome comparisons revealed that the spinach curtovirus shared 64.2 to 83.9% nucleotide sequence identity relative to four previously characterized curtovirus species: Beet curly top virus, Beet severe curly top virus, Beet mild curly top virus, and Horseradish curly top virus. Phylogenetic analysis of individual open reading frames indicated that the evolutionary history of the three virion-sense genes was different from that of the four complementary-sense genes, suggesting that recombination among curtoviruses may have occurred. Collectively, these results indicate that the spinach curtovirus characterized here represents a newly described species of the genus Curtovirus, for which we propose the name Spinach curly top virus.

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.