CDNA clone of transmissible umbra-like associated rnas

Abstract

An isolated infectious cDNA clone of wheat umbra-like virus or in vitro transcript thereof. An expression vector comprising a cDNA clone and / or a host cell comprising a cDNA clone or a RNA transcript thereof, or expression vector comprising the same. A method of infecting a host plant with cDNA clone of wheat umbra-like virus or in vitro transcripts (RNAs) thereof for gene editing, gene silencing, introduction of foreign nucleic acids and the like.

Description

[0001] CDNA CLONE OF TRANSMISSIBLE UMBRA-LIKE ASSOCIATED RNAS

[0002] CROSS-REFERENCE TO RELATED APPLICATIONS

[0003] The present application claims the priority benefit of U.S. Provisional Patent Application Serial No. 63 / 579,117, filed August 28, 2023, entitled CDNA CLONE OF TRANSMISSIBLE UMBRA-LIKE ASSOCIATED RNAS, incorporated by reference in its entirety herein.

[0004] SEQUENCE LISTING

[0005] The following application contains a sequence listing submitted electronically as a Standard ST.26 compliant XML file entitled “59340_SequenceListing.xml,” created on August 28, 2024, as 62,225 bytes in size, the contents of which are incorporated herein.

[0006] BACKGROUND OF THE INVENTION

[0007] Field of the Invention

[0008] The present invention relates to a cDNA infectious clone of wheat umbra-like virus, a construction method, and applications thereof.

[0009] Description of Related Art

[0010] Wheat is considered as one of the leading staple crops in the world. However, the viral pathogens of this important crop cause significant yield loss globally. Plant RNA viruses are frequently found in association with subviral RNAs. All subviral RNAs are characterized by their inability to independently complete an infection cycle, which includes transmission to the next host plant. Subviral RNAs can markedly affect disease symptoms associated with their helper virus, either attenuating or exacerbating symptoms, which can vary depending on the host. Most subviral RNAs exist in a mutualistic association with their helper virus by supporting the helper’s infection cycle.

[0011] Umbraviruses are a more recently identified type of subviral RNAs, which are coat proteindependent RNA replicons that encode their own RNA-dependent RNA polymerase (RdRp) but are dependent on a helper virus for at least encapsidation. Another group of coat protein-dependent replicons are umbravirus-like associated RNAs (ulaRNAs), which generate their RdRp similar to umbraviruses. Our recent metagenomics study of the wheat virome identified Wheat umbra-like virus (WULV), a new subviral RNA belonging to ulaRNAs, in Kansas wheat fields. Since then, similar viral sequences have been identified and reported from other parts of the Great Plains including Colorado and Texas fields. Therefore, WULV should be considered as a new emerging virus associated with wheat. Our current knowledge about the biology and molecular biology of this group of subviral RNAs, including WULV, as well as their role in disease / symptom development in their interactions with other viruses including helper viruses is very limited. Therefore, there remains a need for tools and methods to investigate genome function of WULV and interaction between these viruses, ulaRNAs, and host plants.

[0012] SUMMARY OF THE INVENTION

[0013] The present disclosure is broadly concerned with ulaRNAs, a new category of coat-protein dependent subviral RNA replicons that infect plants. The present disclosure is also concerned with a full-length complementary deoxyribonucleic acid (cDNA) infectious clone of wheat umbra-like virus (WULV). Embodiments include an isolated recombinant cDNA clone of wheat umbra-like virus comprising a nucleic acid sequence encoding amino acid sequences SEQ ID NO:4 and SEQ ID NO:6, wherein said nucleic acid sequence is operably linked to a promoter for expression of said amino acid sequences in a host cell. Embodiments also include expression vectors or constructs comprising the cDNA clone. Embodiments also include host cells comprising cDNA clones and / or in vitro transcripts thereof. Embodiments also include methods that involve infecting a host plant with cDNA clone of wheat umbra-like virus or in vitro transcripts (free RNAs) thereof. The methods generally comprise introducing a cDNA clone according to any embodiment herein, or in vitro transcripts (free RNAs) thereof to a plant tissue, seeds, roots, leaves, stems, and / or seeds using any suitable manner for introducing the nucleic acid molecules to the target.

[0014] In the present work, the clone was prepared by generating the cDNA from the full viral RNA genome and inserting the cDNA into a binary vector that carries 2X 35 S promoter (cauliflower mosaic virus (CaMV) 35S promoter). The cDNA clone efficiently replicated and systemically moved inV. benthamiana. While agroinfiltrated N. be nt ham i ana leaves demonstrated severe necrosis, upper leaves were symptomless. The present disclosure also shows that WULV is capable of autonomous replication in wheat without causing symptoms. Furthermore, the present disclosure shows that through sap inoculation WULV is transmissible in the form of free RNA. This is the first report of a mechanically transmissible ulaRNA in the absence of a helper virus. Together, these findings contribute to advancing our knowledge of the biology of this emerging wheat virus. Moreover, the construction of the WULV infectious clone provides a valuable research tool for further investigations of the biology and molecular biology of the virus as well as its contribution to disease development in interactions with other important wheat viruses.

[0015] Construction of infectious clones for plant viruses plays a central role in studies of the genome function of viruses as well as interaction between viruses and host plants. In fact, constructing WULV cDNA infectious clone provides us a powerful tool for studies on WULV biology and molecular biology using reverse genetics, for example, to investigate the function of unknown proteins encoded by WULV genome, the interaction between WULV and its host, and determine the host range of the virus. In addition, the full-length WULV cDNA clone will be useful for the development of WULV-based vectors for gene function (over gene expression), gene silencing, gene editing (viral-mediated gene editing), and expression of foreign proteins in host plants where WULV can replicate, such as wheat, oat, maize, sorghum, and the like.

[0016] The invention provides a mature system for researching the structure and the function of the virus genome and the interaction between the virus genome and host plants such as wheat.

[0017] Researchers can use infectious clones to engineer expression vectors for biotechnology applications. The infectious cDNA clone of plant viruses is a powerful tool for studying the function of viral proteins and the interaction of viruses with hosts. Researchers can use infectious clones derived from virus-infected natural hosts to study disease causality. Researchers can use infectious clones to study how viruses interact with their hosts, how viruses replicate, and how plants resist viruses. The targeted modification of plant invasive cloned genes can also be used to study the function of plant viral genes or sequences. For example, researchers can incorporate visual markers or reporter genes like green fluorescent protein (GFP) into the clones to study how viruses move through plant cells and vascular systems. Vectors comprising cDNA clones and use of infectious cloning vectors are also contemplated, such as in pathogenicity analysis of WULV and in functional studies of viral genes. Also, contemplated are recombinant bacterial strains, such as an agrobacterium strain carrying a WULV cDNA infectious clone.

[0018] In summary, the present invention relates to an infectious clone containing the full-length genome of WULV that is able to infect model plants as well as several cereal crops such as wheat, maize and sorghum. The present invention can easily induce infection by using recombinant plasmids including WULV and transforming strains. Accordingly, the present invention can be applied to various studies such as the host range of WULV and the correlation with the host, as well as investigation the interaction with other important wheat viruses and the outcome of this interaction on pathogenicity, thereby preventing economic loss due to virus infection in advance.

[0019] BRIEF DESCRIPTION OF THE DRAWINGS

[0020] The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee.

[0021] Figure (Fig.) 1A shows a schematic representation of WULV genome organization and construction of the pIL89-WULV construct cloning strategy. Full-length cDNAcopy of the WULV genome was amplified by RT-PCR using total RNA isolated from field positive wheat samples and WULV-F and WUL-R primers (Table SI) containing 20 nt of overlap with the pIL89 vector. p L89 backbone was linearized using inverse PCR and Vec F and Vec R primers (Table SI). WULV cDNA was inserted into the linearized pJL89 by In-Fusion cloning and assembly.

[0022] Fig. IB shows a schematic representation of the WULV full genome in p L89. LB and RB denote borders of the binary vector containing the T-DNAfrom Ti plasmid as well as a virus helper plasmid; 2 x 35 S: double cauliflower mosaic virus 35 S promoter; HDV-Rbz: hepatitis delta virus ribozyme; NOS: nopaline synthase terminator sequence; KanR: kanamycin resistance gene; trFA: plasmid replication initiator protein sequence. Viral predicted open reading frames (ORFs) are depicted as boxes.

[0023] Fig. 2A shows photographs demonstrating that the full-length WULV cloned cDNA replicates and systemically moves in N. benlhamiana. where symptoms of WULV in agroinfiltrated leaves of HC-Pro transgenic N. benthamiana show mild necrosis started to appear at 4 dpi and developed within 12 days. No symptoms appeared in upper non-infiltrated leaves. Agroinfiltrated plants with buffers were used as the control.

[0024] Fig. 2B shows absolute quantification of the genome of WULV in agroinfiltrated leaves over three time points (1, 3 and 10 dpi) by RT-qPCR using primers to target the WULV ORF3. The plot shows the viral copy number (logarithmic scale, y axis) over the time course along with the error bars shown as the standard variation.

[0025] Fig. 2C shows image from Northern blot analysis of the total RNA isolated from agroinfiltrated leaves using specific probe for the WULV ORF3. Sub-genomic RNA (sgRNA) was detected at 3 and 8 dpi, showing that ORF3 is expressed through sgRNA. L, RNA Millennium marker. The rRNA in the methylene blue-stained blot was used as a loading control.

[0026] Fig. 2D shows image from Western blot analysis of the total protein extracted from agroinfdtrated leaves at 6 dpi using the anti-WULV-P23 antibody. Methylene blue-stained was used as a loading control for the total protein. M, PageRuler Plus prestained protein ladder.

[0027] Fig. 2E shows WULV detection in upper non-infiltrated leaves of N. benthamiana using RT-PCR at 6 and 10 dpi. L, 1 Kb Plus DNA ladder. 1, 2, 3: plant 1, plant 2, plant 3, respectively.

[0028] Fig. 3 contains photographs from the infectivity assessment of WULV in wheat. (A) WULV detection in inoculated and upper non-inoculated leaves using RT-PCR. L, 1 kb plus DNA ladder; I, inoculated leaf; SI, first upper leaf; S2, second upper leaf; Mock, buffer inoculated upper leaf. (B) ‘Tomahawk’ wheat plants inoculated with WULV in vitro transcripts did not exhibit observable viral symptoms neither inculcated nor upper leaves. The picture of the plant was taken at 25 dpi.

[0029] Fig. 4 shows gel images demonstrating that WULV is mechanically transmissible. (A) Detection of WULV in "Tomahawk" wheat plants inoculated with the sap of infected field samples by RT-PCR. Inoculated (I) and upper non-inoculated (S) leaves were tested for WULV at 12 dpi. L, 1 kb plus DNA ladder. Inoculated plant with only the buffer was used as a negative control (mock). (B) Detection of WULV in "Tomahawk" wheat plants inoculated with the sap of agroinfiltrated N. benthamina leaves with pJL89-WULV clone by RT-PCR. WULV was detected in inoculated (I) and upper non-inoculated (S) leaves at 8 dpi and 16 dpi. L, 1 kb plus DNA ladder.

[0030] DETAILED DESCRIPTION

[0031] To address the current knowledge gaps, in this study we constructed the first full-length cDNA infectious clone of WULV and studied it in Nicotiana benthamiana. Our results showed that WULV cDNA clone efficiently replicated and systemically moved in N. benthamiana. In particular, RNA viruses including this subviral RNA replicate in cytoplasm.

[0032] Agroinfiltrated leaves showed necrosis resulting in the death of the leaves within two weeks post inoculation, while upper non-infiltrated leaves were symptomless. Moreover, since the efficiency of agroinfiltration is low in cereals including wheat, we used the WULV clone to generate RNA in vitro transcripts (i.e., using DNA to produce custom RNAs via in vitro transcription) to study the WULV in wheat. We showed unexpectedly that WULV is capable of autonomous replication in the absence of any helper viruses and systemic infection of wheat without causing observable symptoms. Furthermore, and through sap inoculation, we showed that WULV is mechanically transmissible in the form of free RNA. To the best of our knowledge, this is the first reported mechanically transmissible ulaRNA. To determine the host range of WULV, we also inoculated maize, oat, and sorghum. For maize and sorghum, we used vascular puncture inoculation (VPI) method, which is an effective technique for transmission of virus or clone in vitro transcripts where the seed of a plant, such as maize, is inoculated rather than the plant. The infectious clone is delivered by puncturing the seed with fine insect pins with the goal of delivering the virus or clone to the vascular tissue present near the embryo in the seed. For oat, we used in vitro transcripts of WULV and mechanically inoculated plants with the transcripts. Our observation here was similar to the wheat infection. WULV genome was detected in inoculated leaves as well as upper leaves; however, the plants remained otherwise asymptomatic and no visible symptoms, such as necrosis, were observed.

[0033] Together, these findings helped advancing our current knowledge about the WULV biology in wheat as its host and N benthamiana model plant. Furthermore, the developed WULV infectious clone is a valuable research tool for further investigations of the biology and molecular biology of the virus as well as its contribution to disease / symptom development in interactions with other important wheat viruses. Thus, embodiments described herein contemplate a full-length cDNA infectious clone of wheat umbra-like virus (WULV). In one or more embodiments, the cDNA infectious clone has a nucleotide sequence comprising coding sequence SEQ ID NO: 1 or SEQ ID NO:2, or a sequence having 95% sequence homology to SEQ ID NO: 1 or SEQ ID NO:2. In one or more embodiments, the cDNA infectious clone comprises a nucleic acid sequence comprising at least SEQ ID NO:3 (ORF1) and SEQ ID NO:5 (ORF2), and preferably further comprising SEQ ID NO: 7 (ORF3) and / or SEQ ID N0 9 (ORF4). In one or more embodiments, the cDNA infectious clone comprises a nucleic acid sequence encoding for one or more of SEQ ID NO:4, 6, 8, or 10. Preferably, the cDNA infectious clone comprises a nucleic acid sequence encoding for at least SEQ ID NO:4 and SEQ ID NO: 6, or a sequence having at least 95% sequence homology to SEQ ID NO:4 and SEQ ID NO:6. More preferably, the cDNA clone further comprises a nucleic acid encoding for SEQ ID NO:8 and / or SEQ ID NO: 10 or a sequence having at least 95% sequence homology to SEQ ID NO:8 and / or SEQ ID NO: 10. In one or more embodiments, the cDNA infection clone comprises a nucleic acid sequence encoding for each of SEQ ID NO:4, 6, 8, and 10, or a sequence having at least 95% sequence homology as SEQ ID NO:4, 6, 8, and 10, respectively. The sequences can be directly linked in the construct, and / or can include intervening sequences. Moreover, as illustrated in the figures, the coding sequences can overlap, such as in the case of coding sequences for ORF2 and ORF3. In any one of the disclosed embodiments, the coding sequences are located on the expression constructs adjacent at least one regulatory sequence, such as a promoter for the expression of the operably linked coding sequence to generate the transcripts and expression products.

[0034] Further, the cDNA clone of the transmissible ulaRNA can serve as a platform or vector for delivering or introducing other nucleic acids to plant systems, which could likewise introduce various advantageous expression products to the treated plant in the case, e.g., of crops, or to introduce various deleterious expression products, e.g., in the case of weeds or other undesired plants. Thus, embodiments described herein concern expression vectors comprising a cDNA infectious clone of WULV according to embodiments herein, such as for gene silencing, gene function, gene editing, and / or expression of foreign proteins in host plants, such as wheat, maize, sorghum, and other important cereal crops WULV is capable of infecting. Thus, the cDNA can deliver foreign nucleic acids for silencing of a gene, gene editing, and / or for expression of foreign proteins in the host. In general, such nucleic acids could be inserted into the cDNA construct at the C -terminal of 0RF3 (in frame with 0RF3, but between ORF3 and ORF4), or at the N- or C- terminal of ORF 4. The nucleic acid sequence could also be inserted between 0RF1 and 0RF2. The cDNA clone can be used to deliver nucleic acids with sizes up to 700 nucleotides (nts) long, and in some cases up to 500 nts, and in some cases 300 nts or less. As used herein, references to a “foreign” nucleic acid mean that the nucleic acid sequence inserted into the cDNA clone is a nucleic acid sequence that would nor normally be present at that location (e.g., in frame with ORF3, or between ORF3 and ORF4, etc.). The foreign nucleic acid may be foreign to WULV (i.e., a nucleic acid sequence not naturally found in WULV), such as a plant-derived or pest-derived nucleic acid, or a sequence for a different virus. The foreign nucleic acid may also be a sequence that is otherwise sometimes associated with WULV, but present in a different location and / or in different copy numbers or some other modified form as compared to the wild type version of the sequence.

[0035] For example, the cDNA clone could be used to deliver guide RNA (i.e., DNA sequence encoding for guide RNA, gRNA or sgRNA) designed to be complementary to the target transcript for gene editing and / or silencing. This technology can be used for enhanced CRISPR / Cas systems. It has also been shown that gRNA or sgRNA can be used to induce gene silencing and reduction of target RNA in plants via the endogenous RNAi machinery, even the absence of introducing CRISPR Cas enzymes (nucleases) typically provided with a CRISPR / Cas editing approach. Thus, a Cas independent guide-induced gene silencing system holds great promise as a compact, multigene silencing editing tool, that could be even further enhanced using the cDNA clone herein to deliver the guide RNA. The guide sequences can be complementary to an endogenous target site of the host organism. Alternatively, the guide sequences can be complementary to an RNA target for an RNA virus that has infected the host plant. For example, this technology could be applied to carry out targeted genome modification in maize, which can be demonstrated as a proof of concept for example by delivering guide RNA targeting a marker gene in wheat or maize. For example, the PDS gene encoding the phytoene desaturase enzyme, which is involved in chlorophyll biosynthesis, is used as a popular marker for CRISPR / Cas9 editing. Its knockout leads to an albino phenotype. Similarly, knockout of Zmzb7 in maize results in an albino plant. Preliminary studies with such marker genes can be used to demonstrate effective delivery and expression of the guide RNA for the selected target via the cDNA clone.

[0036] The disclosure also concerns methods of manipulating a plant characteristic, said method comprising delivering to a plant cell, tissue, or plant the cDNA infectious clone according to any one of the embodiments or combinations of embodiments disclosed herein, wherein the cDNA clone contains or encodes for an RNA target associated with a gene encoding for the plant characteristic. In one or more embodiments, the plant characteristic is vigorous growth, abundant foliage, longer primary roots, yield, height, and / or shoot water potential, pest resistance, drought tolerance, heat tolerance, salt tolerance, cold resistance, herbicide resistance, fungal resistance or reduced fungal susceptibility, viral resistance, or reduced viral susceptibility. In one or more embodiments, the plant is an invasive plant or weed, wherein said plant characteristic is altered to kill said plant or render said plant susceptible to herbicides. Alternatively, the RNA target is that of a pest or pathogen that has infected or might infect the host plant.

[0037] In any one of the disclosed embodiments, the modification of the RNA target comprises reduced expression of one or more transcripts of the RNA target. In any one of the disclosed embodiments, the modification of the RNA target comprises degradation of one or more transcripts of the RNAtarget. In any one of the disclosed embodiments, modifying said RNA target comprises partially or completely silencing trans or endogenous gene expression associated with said RNA target.

[0038] In any one of the disclosed embodiments, the modification of the RNA target comprises altered transcription or translation of at least one RNA product. In any one of the disclosed embodiments, the expression of the at least one product is decreased. In any one of the disclosed embodiments, the RNA target is an RNA virus. In any one of the disclosed embodiments, the RNA virus has infected a plant cell, tissue, or seed. In any one of the disclosed embodiments, the method comprises delivering said constructs to said plant cell, tissue, or seed to treat or prevent said infection.

[0039] The cDNA clone can incorporate visual markers or reporter genes like GFP to study and visualize how viruses move throughout the plant system. For example, the sequence of eGFP could be inserted into one of three sites in the cDNA clone: N-terminal of ORF3, N-terminal of ORF4, or C- terminal of ORF4. In-fusion cloning can be used to insert the eGFP (SEQ ID NO: 13) in frame with the denoted ORF in each construct. Constructs were made as shown in SEQ ID NO:26, 27, and 28.

[0040] The cDNA clones and / or vectors comprising the same, as well as in vitro transcripts therefrom, can be used to infect host plants, such as for screening potential active agents for treating or preventing pathogen infection and / or for screening plant varieties for tolerance to pathogens.

[0041] In one or more embodiments, the present disclosure concerns a host cell comprising a cDNA infectious clone of WULV, and preferably a full length cDNA infectious clone of WULV according to embodiments herein. Embodiments of the invention also include recombinant bacterial strains, such as an agrobacterium strain, carrying a WULV cDNA infectious clone.

[0042] Methods described herein include infecting a host plant with a WULV cDNA infectious clone or WULV in vitro transcripts according to the embodiments herein. This can include the steps of introducing WULV transcripts (RNAs) in a buffer using rub-inoculation or introducing the cDNA clone / recombinant vector using agrobacterium. This can include first introducing the cDNA or vector comprising the cDNA into agrobacterium, and inoculating the agrobacterium- mediated recombinant vector or cDNA into the host plant. The method can include observing symptoms of infection and / or detecting viral genome in the plant tissues, such as in the leaves, stems, roots, etc. The method may include co-infecting the host plant with a second pathogen, such as a virus suspected as being a helper virus or a virus otherwise associated with WULV infection. The method may include observing the effect (or lack thereof) of the second virus on the plant and / or the effect or lack thereof of co-infection in the plant. The method may include treating the plant with a compound (or compounds) that are candidates as having activity to prevent or inhibit viral infection in the plant, and observing the affect (or lack thereof) of the compound on viral infection in the plant.

[0043] The methods described herein may include collecting tissue samples from the host plant for analysis. Such analysis can include extraction of proteins, detecting the presence of one or more WULV viral proteins or genome in the tissues, and the like. Any or all of the methods described herein may include comparing the results to control plants and wild type plants.

[0044] The technology could be useful in a variety of host plants, such as wheat as well as in other cereals, grasses, and the like, as well as other types of monocot or dicot plants, including without limitation rice, wheat, rye, oats, barley, millet, maize, sorghum, and the like. Advantageously, while not necessarily heritable (since most if not all plant viruses do not incorporate their genome to the genome of plant), this subviral RNA, like plant viruses, can be transmitted by seeds. Thus, in a similar mechanism to seed-borne viruses, it is hypothesized that the ulaRNA can be transmitted by planting “infected” seeds of the parent plant. Nevertheless, while the exact mechanism is not fully understood, the data and results described herein do suggest that these ulaRNAs are transmissible from parent to progeny plant (and in this way can be considered to confer somewhat “heritable” traits that can be passed or transmitted to progeny, although not necessarily through incorporation into the genome).

[0045] As used herein “regulatory elements” refers to promoters, enhancers, and other expression control signals that direct constitutive expression of a nucleotide sequence. Such elements may be host-specific or may drive expression broadly across various host cell types. Such elements may also be inducible and direct expression only under certain conditions (e.g., active or “on” only under an external stimulus, tissue-specific, or developmentally determined parameter). Advantageously, regulatory sequences can be selected to achieve targeted expression of the cDNA constructs. That is, a native promoter can be identified for the host organism that has a specific expression pattern (e.g., time or tissue specific expression). Using this promoter in the constructs ensures that the construct is thus, only expressed in the desired tissue or at the desired time. That way, you only get target mRNA silencing in that tissue (e.g., only in the roots, or root hairs, or only in the root epidermis in the elongation zone, etc.) or at the particular time of development. In this way, the constructs can be selectively activated.

[0046] The term “control” when used with respect to control plants includes wild-type (native) plants, as well as cultivars and genetically altered plants that otherwise contain a wild-type, nonmodified, or native (endogenous) gene targeted for editing (e.g., inhibition) according to the invention. Such control plants are compared to “modified” plants into which the cDNA infectious clones have been introduced according to the invention.

[0047] Additional advantages of the various embodiments of the invention will be apparent to those skilled in the art upon review of the disclosure herein and the working examples below. It will be appreciated that the various embodiments described herein are not necessarily mutually exclusive unless otherwise indicated herein. For example, a feature described or depicted in one embodiment may also be included in other embodiments, but is not necessarily included. Thus, the present invention encompasses a variety of combinations and / or integrations of the specific embodiments described herein.

[0048] As used herein, the phrase "and / or," when used in a list of two or more items, means that any one of the listed items can be employed by itself or any combination of two or more of the listed items can be employed. For example, if a composition is described as containing or excluding components A, B, and / or C, the composition can contain or exclude A alone; B alone; C alone; A and B in combination; A and C in combination; B and C in combination; or A, B, and C in combination.

[0049] The term “sequence homology” or “sequence identity” means the proportion of base matches between two nucleic acid sequences or the proportion amino acid matches between two amino acid sequences. When sequence homology is expressed as a percentage, e.g., 50%, the percentage denotes the proportion of matches over the length of a selected sequence that is compared to some other sequence. Gaps (in either of the two sequences) are permitted to maximize matching; gap lengths of 15 bases or less are usually used, or 6 bases or less or 2 bases or less for nucleotide sequences. Two amino acid sequences are homologous if there is a partial or complete identity between their sequences. For example, 85% homology means that 85% of the amino acids are identical when the two sequences are aligned for maximum matching. Gaps (in either of the two sequences being matched) are allowed in maximizing matching; gap lengths of 5 or less or 2 or less are typically permitted. Percent sequence identity between any two sequences can be calculated using publicly available sequence alignment programs and parameters described herein.

[0050] The present description also uses numerical ranges to quantify certain parameters relating to various embodiments of the invention. It should be understood that when numerical ranges are provided, such ranges are to be construed as providing literal support for claim limitations that only recite the lower value of the range as well as claim limitations that only recite the upper value of the range. For example, a disclosed numerical range of about 10 to about 100 provides literal support for a claim reciting "greater than about 10" (with no upper bounds) and a claim reciting "less than about 100" (with no lower bounds).

[0051] EXAMPLE

[0052] The following examples set forth methods in accordance with the invention. It is to be understood, however, that these examples are provided by way of illustration and nothing therein should be taken as a limitation upon the overall scope of the invention.

[0053] Construction and Biological Characterization of a cDNA Infectious Clone of Wheat umbra-like virus in Wheat (Triticum aestivuni) and Nicotiana benthamiana

[0054] 1. Introduction

[0055] Plant RNA viruses are frequently found in association with subviral RNAs in nature. Subviral RNAs are diverse, but one characteristic that they share is that they are incapable of completing their life cycle independently. In fact, they rely on other viruses as helper viruses either for replication or movement or transmission from plant to plant. Most subviral RNAs do not encode a coat protein (CP) and they fall into three classes: satellite RNAs, viroids, and two groups of coat protein-dependent RNA replicons (CdRr) with more simplified genome structure: Umbra-like associated RNAs (ulaRNAs) and tombusvirus-like associated RNAs (tlaRNAs). The impact of subviral RNAs on disease outcome (attenuating or exacerbating) can vary depending on their relationship with their helper viruses as well as their hosts.

[0056] Umbra-like associated RNAs (ulaRNAs) are a new group of subviral RNAs identified recently in association with various plant species, such as papaya, babaco, strawberry, citrus, opuntia, fig, maize, johnsongrass, grapevine, sugarcane, and evergreen trees. ulaRNAs are capsid-dependent RNA infectious agents sharing high RdRp sequence homology to the well-known plant umbraviruses belonging to the genus Umbravirus, family Tombusviridae . However, their genome size and features are different. Therefore, ulaRNAs are considered as new unclassified subviral RNAs with a different evolutionary history. Like umbraviruses, ulaRNAs encode their own RdRp which enable them to independently replicate in the host, but they are probably dependent on their helper viruses for encapsidation and transmission. The helper virus of umbraviruses is always a member of Luteoviridae . However, potential helper viruses of most ulaRNAs are currently unknown.

[0057] In the last two decades, Papaya meleira or sticky disease has been a major concern for papaya growers in Brazil and Mexico. For a long time, a dsRNA virus named Papaya meleira virus (PMeV), a virus related to mycoviruses from the Totiviridae family was recognized as the causal agent. However, in 2016, the presence of the second virus named Papaya meleira virus-2 (PMeV- 2), an ulaRNA, was reported in infected trees in Brazil. This study clearly revealed that synergistic interaction between PMeV-2 and PMeV resulted in severe symptoms. Interestingly, it was shown that PMeV-2 RNA is encapsidated in PMeV particles. Therefore, PMeV was determined as the potential helper virus of PMeV-2. In 2018, a new ulaRNA named Papaya virus Q (PpQV) was identified in Ecuador shared 70% sequence identity with PMeV-2. Unlike PMeV-2, PpQV was found most in co-infection with a potyvirus, Papaya ringspot virus (PRSV) with common symptoms of PRSV. More interestingly, PpQV was also found in single infections in asymptomatic trees, suggesting a synergistic relationship between PpQV and PRSV which needs to be investigated further. In 2021, Kwon et al. reported a new ulaRNA (Citrus yellow-vein associated virus (CYVaV) associated with Citrus yellow-vein disease (CYVD), a disease which was first reported in California in 1957 with an unknown causal agent for a long time. There is not much known about the potential helper virus(es) of CYVaV and its mode of transmission in nature like many other ulaRNAs. A newly studied strawberry-ulaRNA patho-system illustrated genetic variation among naturally infected strawberry plants with a novel ulaRNA. In this patho-system, ulaRNA was mostly found in mixed infections with viruses from Luteoviridae and Rhabdoviridcie families, but it was also detected in asymptomatic plants (certified propagation materials). Although, this limited knowledge revealed the complexity of ulaRNAs in their pathogenicity and their interactions with the plant host and other viruses (helper viruses), our understanding of the biological and molecular characteristics of ulaRNAs need to be expanded. Therefore, developing stable infectious clones of ulaRNAs and applying them as tools to study the biology of this group of RNA infectious agents is necessary.

[0058] Our recent metagenomics study of the wheat virome in Kansas fields reported a novel ulaRNA, Wheat umbra-like virus (WULV) (Redila et al., 2021b, Redila, C.D., Prakash, V., Nouri, S., 2021b. Metagenomics analysis of the wheat virome identifies novel plant and fungal-associated viral sequences. Viruses). The costing sequences from ORF1-ORF4 are in SEQ ID NO: 1. The full genome size of WULV is 3,527 nucleotides with a short 5'UTR of 10 nt and a longer 3'UTR of 237 nt, (SEQ ID NO:2), GenBank: OK573479.1, incorporated by reference herein.

[0059] 1 gggtaaataa atggcttggc aagcgatcag acgtttgttt acccgcaagc agcgtccacg

[0060] 61 cgctgctagc agcatctctg aggagatcta cgtgggcgac ttccccattg gagtcacccg

[0061] 121 cgaggtccca gcgccacgag tggagaccgt ggtggctgat gagctgagtc ctgtgaaccg

[0062] 181 ggagtccatg atgttggatg acgactgggt cgcagtgatc aacaccgcca gatacatgga

[0063] 241 ggctcaagct caactcaggg agcgcttgga cgaggtggtg caggaggagc tgcgccaatg

[0064] 301 ggtcctggga aacgtggcta gccccgcagc ccgtctagca gagcaacttc gcgtgcgttt

[0065] 361 cggcgcgcgc gcccccacgg ccgctaatgc ggttatggga gagcggttct tgctggacaa

[0066] 421 ggcagcggag gctggaaacg accacctgga tgccagcaac ctcgcaagct tggctgtgaa

[0067] 481 tctctggtta gtgcgcaccg cttgggacca gtccgtcggg ctgggttttc cgagtgggtg

[0068] 541 gtaactgagc tccccggggt gtcaaggagt ttcgaatgcc ctcctgacgc tcccgtaatc

[0069] 601 tggcgttcgc gggctgccac cccgatgaag tacggcaggc gaactgtgaa cttcagcaat

[0070] 661 ctgatggcta atgatattta taaggtgcac gccaactcga aggacaatct gctcagggga 721 ttttgtaccc gagtgtggcg atataagggc gccgatccga tcccgtgtct gcggtcccga 781 ctgccgcgat ttgaacgggc gttccgtggg ttcccggtac gccccgtctc gtatgattca 841 gtcgtggaga acgcattgcc ccgtcgcaga aaactgttac agacggctgt ggaaagccta 901 aaaacgaagc ctctacaact cgccgattca catgttaata cctttatcaa ggctgagaag 961 ttttgttgga gtgcgaagcc tgacgcggat cctcgtctaa tacagccccg cggtaatcgc 1021 ttccttgtct cacacggccg gtacataaag gcgattgagc cgatggtcta taaagcactg 1081 ggccgactca ccggacttcc catggtggcg aagggattca acgctcgtga aacagcggag 1141 atcctgcgca agaagtggga ccggtttaga gaccccgtgt gcatatcttt ggacgccagt 1201 cgctttgatc agcacgtcag tgtggacatg ttgaagttta ctcaccgcat ataccgacga 1261 ttcaatcgag atccggagtt taaaatgatg tgtgattggc aattacacaa cacaggcttt 1321 gcctcctgcc cagaggggag cttcaagtat aaaatcgcgg gaaggcgaat gagtggagat 1381 atggatacgt cgctgggcaa ctgcgtcatc atgtgtggtc taacatatga attatttggc

[0071] 1441 cccggcgtgg agatattcaa caacggtgac gattgtttag tgatcggcga gcgtcatgag 1501 ctagtggacg cgcccactgt ccgcgagcac tataagcagt acggtttcca tgtggtcgag 1561 gaagacagag ttgatgtgtt cgagcgcatc ctcttttgtc aaactcaacc tatctgggcc 1621 gagggatggg tcatgtgccg tcacccacgg atgatggcaa aggatttgac ctatattggg 1681 ccacacactg ctgatagggc ttggctcgat gcaattggac agtgtgggat cgctcttgct 1741 gatggagtgc ctataatgtc gcggttctac cacaatctca tcgacacaag tcaccgtaaa 1801 gcgaatatca ccaacagtat gctctactca tgcggattga cgaatttagc gaagcgcatg 1861 gaatataagg gactgcccat ctcagaccgc tctcggtatt cattttatct ggggttcgga 1921 gtcgagccgg atgttcaggt tgcgatagaa gccagtatgg gcccgctggg taatatacaa 1981 cgaattgatg gcaaactctt cgaacagacg atcacaaact acgagagcgc tctcgaagcg 2041 gcggagaagg ggtcggggca cgcgtggaag cgtcccgtct cccttcccat tccctaatcg 2101 cacacagatc gacacgcgca cctgcctcca ggacttcaag attgatgcac cagctgcgga 2161 caagtctact ggggtagcca tggtatgtat gcagctcgac cggacgctag acgccgtacg 2221 catggctgga ttctccaccc actatcgcat agtgtcagtg gaggcgcacg tgatacccgc 2281 aacaatacat gcacaggcag ttgctagtat aggcaactcc acccatcgca gcgggccggg 2341 cgcacctgga gatcttgtgg acggatccac gacgatcttc tcgtattccg aatatgtgcg 2401 atggcaatct ttgatgtcga gcacacgcaa ggctgccccc ggagcaacat tagtcgcgac 2461 gttccgacct cagaatcctg tttgggttga gaacaccgct aacgcacggg ctgatggtgc 2521 catgtcgctc ctggttggta gtattagtgc gcagaatcac acctcagcga cgtgggtgtc 2581 gttgaagtat gtgattcagt tccagatgga cgtcgccgtc tattgacggg ccggggacgc 2641 ccgtcctcca gctggctcgc gccattctga ggatatgcag ccattagtgg aagagttgca 2701 caggatggag cgatctagtg ggccgtccca gcgacctagg agggacgtct ctgagcaaat 2761 tcaactggcc ccgcctcagt ccggttctga ggataacagt aaggccgggt ggtctctgag 2821 tgccaccgct ggagcagccg ccggaactgc tgcggtggtg gcgggaggcg tggcggctgc 2881 cgaagccgtc gcaggtctcg gtgccgctgc ggcgtcggtg cccaccgata gtgtgccatt 2941 gctggacatg tcggcgagca cctcgtcact tgtggaacca tcgctgcaag tggtcgaaac 3001 cgacttggac gatgtctcct tgtgggacgc ggccgaggag gaattgaccg cagccgccga 3061 gcagacctcg ctcaccagca tacctgaaag cctgaagacc acatggcgcc agtggattga 3121 agacgaatgt cgacaaggaa tgataaagga gggggtgaag aatcggttta cagaatcact 3181 gaaccgcccc gaagcgctta acgcagcccg cacctcttgg ttcagacgct atggaaccga 3241 gcctacagac gctgaagttt tgcagcgcct tctcgaggac atcatccggt cgtaagtact

[0072] 3301 ggtcacagca ggaggtaaga cgtcccgatt ggcaactttc ggaagtaccc ggtgagggga

[0073] 3361 aacgcgccta ggacctgctc gttcgccgtg gaacgcacct gatgactact cgagcgacac

[0074] 3421 tgccgttggc tgtcacccag acagtcacct gagcttacac ttcccgaggg gggggacata

[0075] 3481 atacctctcg gtaccgaacc atctataggt agcgtgcccg cgctaccgcc cc (SEQ ID NO:2)

[0076] Four predicted open reading frames (ORF) were determined for WULV and only ORF2 (SEQ ID NO: 5) showed significant similarity to the RdRp sequence of viruses with a conserved GDD motif, while other ORFs did not demonstrate significant similarities to known proteins in databases. Sequences and their proteins are below.

[0077] Methyltransferase (ORF1): SEQ ID NO:3

[0078] ATGGCTTGGCAAGCGATCAGACGTTTGTTTACCCGCAAGCAGCgtcCACGCGCTGCTA GCAGCATCTCTGAGGAGATCTACGTGGGCGACTTCCCCATTGGAGTCACCCGCGAGG TCCCAGCGCCACGAGTGGAGACCGTGGTGGCTGATGAGCTGAGTCCTGTGAACCGG GAGTCCATGATGTTGGATGACGACTGGGTCGCAGTGATCAACACCGCCAGATACATG GAGGCTCAAGCTCAACTCAGGGAGCGCTTGGACGAGGTGGTGCAGGAGGAGCTGCG CCAATGGGTCCTGGGAAACGTGGCTAGCCCCGCaGCCCGTCTAGCAGAGCaACTTCGC GTGCGTTTCGGCGCGCGCGCCCCCACGGCCGCTAAtGCGGTTATGGGAGAGCGGTTCT TGCTGGACAAGGCAgCGGAGGCTGGAAACGACCACCTGGATGCCAGcAACCTCGCA AGCTTGGCTGTGAATCTCTGGTTAGTGCGCACCGCTTGGGACCAGTCCGTCGGGCTG GGTTTTCCGAGTGGGTGGTAA

[0079] MAWQAIRRLFTRKQRPRAASSISEEIYVGDFPIGVTREVPAPRVETVVADELSPVNRESM MLDDDWVAVINTARYMEAQAQLRERLDEVVQEELRQWVGNVASPAARLAEQLRVRFG ARAPTAANAVMGERFLLDKAAEAGNDHLDASNLASLAVNLWLVRTAWDQSVGLGFPSG W (SEQ ID NO:4)

[0080] RNA dependent RNA polymerase (ORF2):SEQ ID NO: 5 ATGAAGTACGGCAGGCGAACTGTGAACTTCAGCAATCTGATGGCTAATGATATTTAtA AGGTGCACGCCAACTCGAAGGACAATCTGCTCAGGGGATTTTGTACCCGAGTGTGGC GATATAAGGGCGCCGATCCGATCCCGTGTCTGCGGTCCCGACTGCCGCGATTTGAACG

[0081] GGCGTTCCGTGGGTTCCCGGTACGCCCCGTCTCGTATGATTCAGTCGTGGAGAACGC

[0082] ATTGCCCCGTCGCAGAAAACTGTTACAGACGGCTGTGGAAAGCCTAAAAACGAAGC

[0083] CTCTACAACTCGCCGATTCACATGTTAATACCTTTATCAAGGCTGAGAAGTTTTGTTGG

[0084] AGTGCGAAGCCTGACGCGGATCCTCGTCTAATACAGCCCCGCGGTAATCGCTTCCTTG

[0085] TCTCACACGGCCGGTACATAAAGGCGATTGAGCCGATGGTCTATAAAGCACTGGGCC

[0086] GACTCACCGGACTTCCCATGGTGGCGAAGGGATTCAACGCTCGTGAAACAGCGGAG

[0087] ATCCTGCGCAAGAAGTGGGACCGGTTTAGAGACCCCGTGTGCATATCTTTGGACGCC

[0088] AGTCGCTTTGATCAGCACGTCAGTGTGGACATGTTGAAGTTTACTCACCGCATATACC

[0089] GACGATTCAATCGAGATCCGGAGTTTAAAATGATGTGTGATTGGCAATTACACAACAC

[0090] AGGCTTTGCCTCCTGCCCAGAGGGGAGCTTCAAGTATAAAATCGCGGGAAGGCGAAT

[0091] GAGTGGAGATATGGATACGTCGCTGGGCAACTGCGTCATCATGTGTGGTCTAACATAT

[0092] GAATTATTTGGCCCCGGCGTGGAGATATTCAACAACGGTGACGATTGTTTAGTGATCG

[0093] GCGAGCGTCATGAGCTAGTGGACGCGCCCACTGTCCGCGAGCACTATAAGCAGTACG

[0094] GTTTCCATGTGGTCGAGGAAGACAGAGTTGATGTGTTCGAGCGCATCCTCTTTTGtCA

[0095] AACTCAACCTATCTGGGCCGAGGGATGGGTCATGTGCCGTCACCCACGGATGATGGC

[0096] AAAGGATTTGACCTATATTGGGCCACACACTGCTGATAGGGCTTGGCTCGATGCAATT

[0097] GGACAGTGTGGGATCGCTCTTGCTGATGGAGTGCCTATAATGTCGCGGTTCTACCACA

[0098] ATCTCATCGACACAAGTCACCGtAAAGCGAATATCACCAACAGTATGCTCTACTCATGC

[0099] GGATTGACGAATTTAGCGAAGCGCATGGAATATAAGGGACTGCCCATCTCAGACCGCT

[0100] CTCGGTATTCATTTTATCTGGGGTTCGGAGTCGAGCCGGATGTTCAGGTTGCGATAGA

[0101] AGCCAGTATGGGCCCGCTGGGTAATATACAACGAATTGATGGCAAACTCTTCGAACAG

[0102] ACGATCACAAACTACGAGAGCGCTCTCGAAGCGGCGGAGAAGGGGTCGGGGCACGC

[0103] GTGGAAGCGTCCCGTCTCCCTTCCCATTCCCTAA

[0104] MKYGRRTVNFSNLMANDIYKVHANSKDNLLRGFCTRVWRYKGADPIPCLRSRLPRFER

[0105] AFRGFPVRPVSYDSVVENALPRRRKLLQTAVESLKTKPLQLADSHVNTFIKAEKFCWSA

[0106] KPDADPRLIQPRGNRFLVSHGRYIKAIEPMVYKALGRLTGLPMVAKGFNARETAEILRKK

[0107] WDRFRDPVCISLDASRFDQHVSVDMLKFTHRIYRRFNRDPEFKMMCDWQLHNTGFASC

[0108] PEGSFKYKIAGRRMSGDMDTSLGNCVIMCGLTYELFGPGVEIFNNGDDCLVIGERHELV

[0109] DAPTVREHYKQYGFHVVEEDRVDVFERILFCQTQPIWAEGWVMCRHPRMMAKDLTYIG PHTADRAWLDAIGQCGIALADGVPIMSRFYHNLIDTSHRKANITNSMLYSCGLTNLAKR

[0110] MEYKGLPISDRSRYSFYLGFGVEPDVQVAIEASMGPLGNIQRIDGKLFEQTITNYESALEA

[0111] AEKGSGHAWKRPVSLPIP (SEQ ID NO: 6)

[0112] Putative protein 1 (ORF3):SEQ ID NO:7

[0113] ATGGCAAACTCTTCGAACAGACGATCACAAACTACGAGAGCGCTCTCGAAGCGGCG

[0114] GAGAAGGGGTCGGGGCACGCGTGGAAGCGTCCCGTCTCCCTTCCCATTCCCTAA

[0115] TCGCACACAGATCGACACGCGCACCTGCCTCCAGGACTTCAAGATTGATGCACCAGc

[0116] TGCGGACAAGTCTACTGGGGTaGCCATGGTATGTATGCAGCTCGACCGGACGCTAGAC

[0117] GCCGTACGCATGGCTGGATTCTCCACCCACTATCGCATAGTGTCAGTGGAGGCGCACG

[0118] TGATACCCgCAACAATACATGCACAGGCAGTTGCTAGTATAGGCAACTCCACCCATCG

[0119] CAGCGGGCCGGGCGCACCTGGAGATCTTGTGGACGGATCCACGACGATCTTCTCGTA

[0120] TTCCGAATATGTGCGATGGCAATCTTTGATGTCGAGCACACGCAAGGCTGCCCCCGGA

[0121] GCAACATTAGTCGCGACGTTCCGACCTCAGAATCCTGTTTGGGTTGAGAACACCGCT

[0122] AACGCACGGGCTGATGGTGCCATGTCGCTCCTGGTTGGTAGTATTAGTGCGCAGAATC

[0123] ACACCTCAGCGACGTGGGTGTCGTTGAAGTATGTGATTCAGTTCCAGATGGACGTCG CCGTCTATTGA

[0124] MANSSNRRSQTTRALSKRRRRGRGTRGSVPSPFPFPNRTQIDTRTCLQDFKIDAPAADKS

[0125] TGVAMVCMQLDRTLDAVRMAGFSTHYRIVSVEAHVIPATIHAQAVASIGNSTHRSGPGAP

[0126] GDLVDGSTTIFSYSEYVRWQSLMSSTRKAAPGATLVATFRPQNPVWVENTANARADGA

[0127] MSLLVGSISAQNHTSATWVSLKYVIQFQMDVAVY (SEQ ID NO: 8)

[0128] Putative protein 2 (ORF4): SEQ ID NO:9

[0129] ATGCAGCCATTAGTGGAAGAGTTGCACAGGATGGAGCGATCTAGTGGGCCGTCCCAG

[0130] CGACCTAGGAGGGACGTCTCTGAGCAAATTCAACTGGCCCCGCCTCAGTCCGGTTCT

[0131] GAGGATAACAGTAAGGCCGGGTGGTCTCTGAGTGCCACCGCTGGAGCAGCCGCCGG

[0132] AACTGCTGCGGTGGTGGCGGGAGGCGTGGCGGCTGCCGAAGCCGTCGCAGGTCTCG

[0133] GTGCCGCTGCGGCGTCGGTGCCCACCGATAGTGTGCCATTGCTGGACATGTCGGCGA

[0134] GCACCTCGTCACTTGTGGAACCATCGCTGCAAGTGGTcGAAACCGACtTGGACGATGT

[0135] CTCCTTGTGGGACGCGGCCGAGGAGGAATTGACCGCAGCCGCCGAGCAGACCTCGC TCACCAGCATACCTGAAAGCCTGAAGACCACATGGCGCCAGTGGATTGAAGACGAAT GTCGACAaGGAATGATAAAGGAGGGGGTGAAGAATCGGTTTACAGAATCACTGAACC GCCCCGAAGCGCTTAACGCAGCCCGCACCTCTTGGTTCAGACGCTATGGAACCGAGC CTACAGACGCTGAAGTTTTGCAGCGCCTTcTCGAGGACATCATCCGGTCGTAA

[0136] MQPLVEELHRMERSSGPSQRPRRDVSEQIQLAPPQSGSEDNSKAGWSLSATAGAAAGTA AVVAGGVAAAEAVAGLGAAAASVPTDSVPLLDMSASTSSLVEPSLQVVETDLDDVSLWD AAEEELTAAAEQTSLTSIPESLKTTWRQWIEDECRQGMIKEGVKNRFTESLNRPEALNAA RTSWFRRYGTEPTDAEVLQRLLEDIIRS (SEQ ID NO: 10)

[0137] While ORF1 probably encodes an auxiliary protein, ORF1 and ORF2 encode a larger product through stop codon readthrough that represents the replication complex. The function of the expressed proteins from ORF3 and ORF4 is unknown. Furthermore, we detected a sub-genomic RNA (sgRNA) using the total RNA from WULV positive field samples and specific probes designed for ORF3 and ORF4 (Fig. 2C).

[0138] WULV has unique genome features which differentiate it from most ulaRNAs. While either 2 or 3 predicated ORFs have been identified for most ulaRNAs, WULV genome contains 4 predicted ORFs. Recently, a new ulaRNA has been identified and reported from grapevine showing similar genome organization to WULV with 4 predicated ORFs. Others conducted a comprehensive structural analysis and genome mapping of ulaRNAs and classified this group of the coat proteindependent RNAs into 3 classes. Based on this classification, WULV is placed in class 3 along with the strawberry associated virus A (SbaVA). Interestingly, this classification placed ulaRNAs associated with maize, johnsongrass and sugarcane together in a separate well-supported clad than WULV, suggesting that WULV is phylogenetically different from ulaRNAs infecting other cereal and grasses, and it has been probably evolved from a different ancestor. Recent metagenomic studies of wheat virome in Colorado and Texas fields identified viral sequences illustrating similarities to WULV, suggesting WULV as a newly emerging virus associated with wheat which needs to be characterized.

[0139] In this work, we constructed a full-length cDNA infectious clone for WULV (pJL89-WULV, SEQ ID NO: 12) to study its biological activity in Nicotiana benthamiana and wheat. We demonstrated that our WULV cDNA clone efficiently replicates and systemically moves in N. benthamiana, causing necrosis in infiltrated leaves, while upper non-infiltrated leaves were symptomless. In addition, we showed that WULV in vitro transcripts are infectious in wheat and the virus systemically moves to upper leaves without causing observable disease symptoms. Furthermore, our study showed that WULV RNAis able to be mechanically transmitted from plant to plant by crude sap inoculation. To the best of our knowledge, this is the first reported mechanically transmissible ulaRNA. This study is the first to generate a stable infectious WULV clone as a valuable research tool to study the biology and molecular biology of an ulaRNAs in association with one of the most important crops.

[0140] 2. Materials and Methods

[0141] 2.1. Virus Source

[0142] WULV positive wheat leaves collected from KS fields in 2019 (Redila et al., 2021b) kept at -80C freezer were used as the source of WULV in this study. It is worth mentioning that our WULV positive field samples were co-infected with wheat poty viruses including WSMV and TriMV and we did not find any single WULV infection in our two years survey. Total RNAs were isolated from 100 mg of plant tissues using TRI reagent (Zymo Research, CA, USA) following the manufacturer’s instruction. The extracted RNAs were treated with DNase I (Zymo Research, CA, USA).

[0143] 2.2. Construction of a full-length cDNA clone of WULV

[0144] The full-length cDNA clone of WULV was constmcted using the In-Fusion cloning system (Takara Bio, CA, USA) (Fig. 1A). A set of primers (Table SI) was designed using the SnapGene software version 6.1.1 to amplify the full-length WULV and linearize the pJL89 vector (Lindbo 2007), Addgene plasmid #98558, with 35S promoter (SEQ ID NO: 11) and HDV Ribozyme sequences in T-DNA region of Agrobacterium vector. WULV fragment was inserted into the pJL89 between the double 35S promoter (2 x 35S) and the hepatitis delta virus ribozyme (HD-Rbz) (Fig. IB).

[0145] Table SI. Primers and Probes To amplify the WULV genome, the first strand cDNA was synthesized using the total RNA isolated from infected wheat field samples and SuperScript III Reverse Transcriptase (Invitrogen, CA, USA). 1 pL of the cDNA was used in 25 pL PCR reaction containing IX CloneAmp HiFi PCR Premix (Takara Bio, CA, USA), 0.3 pM primers WUUV-F and WUUV-R (Table SI) and nuclease free water. 10 ng of pJL89 was linearized (Fig. 1A) by PCR using Vec-F and Vec-R primers (Table SI) and IX CloneAmp HiFi PCR Premix. Both amplicons were gel purified using the Zymoclean Gel DNA Recovery Kit (Zymo Research, CA, USA) according to the manufacturer’s instruction. The In-Fusion reaction was conducted using an insert to vector ratio of 2: 1, 5X In-Fusion HD Enzyme Premix (Takara Bio, CA, USA) and nuclease free water to 10 pL of total volume. The reaction was incubated at 50°C for 15 min and then transformed into Escherichia coli Stellar competent cells (Takara Bio, CA, USA). The transformants were screened by colony PCR using specific WUEV-F2 and WUEV-R6 primers (Table SI). The plasmids were purified from the selected PCR-positive colonies using the ZymoPURE II Plasmid Maxiprep Kit (Zymo Research, CA, USA) according to the manufacturer’s instructions. The presence of the full-length cDNA clones of WULV was confirmed by enzyme digestion and Sanger sequencing (GeneWiz, NJ, USA). pJL89-WULV Clone Sequence (SEQ ID NO: 12)

[0146] GGGTCGGCATGGCATCTCCACCTCCTCGCGGTCCGACCTGGGCATCCGAAGGAGGAC GTCGTCCACTCGGATGGCTAAGGGAGAGCTCGAATTTCCCCGATCGTTCAAACATTTG GCAATAAAGTTTCTTAAGATTGAAtcctgttgccggtcttgcgactagagtccgcaaatcaccagtctctctctacaaat ctatctctctctattttctccagaataatgtgtgagtagttcccagataagggaattagggttcttatagggtttcgctcatgtgttgagcatataag aaacccttagtatgtatttgtatttgtaaaatacttctatcaataaaatttctaattcctaaaaccaaaatccagtgacctgcagcCCGGccggg ggatccactagcAGATTGTCGTTTCCCGCCTTCAGTTTAAACTATCAGTGTTTGACAGGATAT ATTGGCGGGTAAACCTAAGAGAAAAGAGCGTTTATTAGAATAATCGGATATTTAAAAG GGCGTGAAAAGGTTTATCCGTTCGTCCATTTGTATGTGCATGCCAACCACAGGAGATC TCAgtaaagcgctggctgaacccccagccggaactgaccccacaaggccctagcgtttgcaatgcaccaggtcatcattgacccaggc gtgttccaccaggccgctgcctcgcaactcttcgcaggcttcgccgacctgctcgcgccacttcttcacgcgggtggaatccgatccgcaca tgaggcggaaggtttccagcttgagcgggtacggctcccggtgcgagctgaaatagtcgaacatccgtcgggccgtcggcgacagcttgc ggtacttctcccatatgaatttcgtgtagtggtcgccagcaaacagcacgacgatttcctcgtcgatcaggacctggcaacgggacgttttctt gccacggtccaggacgcggaagcggtgcagcagcgacaccgattccaggtgcccaacgcggtcggacgtgaagcccatcgccgtcgc ctgtaggcgcgacaggcattcctcggccttcgtgtaataccggccattgatcgaccagcccaggtcctggcaaagctcgtagaacgtgaag gtgatcggctcgccgataggggtgcgcttcgcgtactccaacacctgctgccacaccagttcgtcatcgtcggcccgcagctcgacgccg gtgtaggtgatcttcacgtccttgttgacgtggaaaatgaccttgttttgcagcgcctcgcgcgggattttcttgttgcgcgtggtgaacagggc agagcgggccgtgtcgtttggcatcgctcgcatcgtgtccggccacggcgcaatatcgaacaaggaaagctgcatttccttgatctgctgctt cgtgtgtttcagcaacgcggcctgcttggcctcgctgacctgttttgccaggtcctcgccggcggtttttcgcttcttggtcgtcatagttcctcg cgtgtcgatggtcatcgacttcgccaaacctgccgcctcctgttcgagacgacgcgaacgctccacggcggccgatggcgcgggcaggg cagggggagccagttgcacgctgtcgcgctcgatcttggccgtagcttgctggaccatcgagccgacggactggaaggtttcgcggggc gcacgcatgacggtgcggcttgcgatggtttcggcatcctcggcggaaaaccccgcgtcgatcagttcttgcctgtatgccttccggtcaaa cgtccgattcattcaccctccttgcgggattgccccgactcacgccggggcaatgtgcccttattcctgatttgacccgcctggtgccttggtg tccagataatccaccttatcggcaatgaagtcggtcccgtagaccgtctggccgtccttctcgtacttggtattccgaatcttgccctgcacgaa taccagcgaccccttgcccaaatacttgccgtgggcctcggcctgagagccaaaacacttgatgcggaagaagtcggtgcgctcctgcttg tcgccggcatcgttgcgccacatctaggtactaaaacaattcatccagtaaaatataatattttattttctcccaatcaggcttgatccccagtaa gtcaaaaaatagctcgacatactgttcttccccgatatcctccctgatcgaccggacgcagaaggcaatgtcataccacttgtccgccctgcc gcttctcccaagatcaataaagccacttactttgccatctttcacaaagatgttgctgtctcccaggtcgccgtgggaaaagacaagttcctctt cgggctttccgtctttaaaaaatcatacagctcgcgcggatctttaaatggagtgtcttcttcccagttttcgcaatccacatcggccagatcgtt attcagtaagtaatccaattcggctaagcggctgtctaagctattcgtatagggacaatccgatatgtcgatggagtgaaagagcctgatgca ctccgcatacagctcgataatcttttcagggctttgttcatcttcatactcttccgagcaaaggacgccatcggcctcactcatgagcagattgct ccagccatcatgccgttcaaagtgcaggacctttggaacaggcagctttccttccagccatagcatcatgtccttttcccgttccacatcatag gtggtccctttataccggctgtccgtcatttttaaatataggttttcattttctcccaccagcttatataccttagcaggagacattccttccgtatctt ttacgcagcggtatttttcgatcagttttttcaattccggtgatattctcattttagccatttattatttccttcctcttttctacagtatttaaagataccc caagaagctaattataacaagacgaactccaattcactgttccttgcattctaaaaccttaaataccagaaaacagctttttcaaagttgttttcaa agttggcgtataacatagtatcgacggagccgattttgaaaccacaattatgggtgatgctgccaactcgagagcgggccgggagggttcg agaagggggggcaccccccttcggcgtgcgcggtcacgcgcacagggcgcagccctggttaaaaacaaggtttataaatattggtttaaa agcaggttaaaagacaggttagcggtggccgaaaaacgggcggaaacccttgcaaatgctggattttctgcctgtggacagcccctcaaat gtcaataggtgcgcccctcatctgtcagcactctgcccctcaagtgtcaaggatcgcgcccctcatctgtcagtagtcgcgcccctcaagtgt caataccgcagggcacttatccccaggcttgtccacatcatctgtgggaaactcgcgtaaaatcaggcgttttcgccgatttgcgaggctggc cagctccacgtcgccggccgaaatcgagcctgcccctcatctgtcaacgccgcgccgggtgagtcggcccctcaagtgtcaacgtccgcc cctcatctgtcagtgagggccaagtttccgcgaggtatccacaacgccggcggccggccgcggtgtctcgcacacggctcgacggcgt ttctggcgcgtttgcagggccatagacggccgccagcccagcggcgagggcaaccagcccggtgagctCTAGTGGACTGAT GGGCTGCCTGTATCGAGTGGTGATTTTGTGCCGAGCTGCCGGTCGGGGAGCTGTTGG

[0147] CTGGCTGGTGGCAGGATATATTGTGGTGTAAACAAATTGACGCTTAGACAACTTAATA ACACATTGCGGACGTTTTTAATGTACTGgggtggttttggtaccgggccccccctcgaggtcgacggtatcgataa gcttgatatcgaattcctgcaggtcaacatggtggagcacGACACTCTCGTCTACTCCAAGAATATCAAAGAT

[0148] ACAGTCTCAGAAGACCAAAGGGCTATTGAGACTTTTCAACAAAGGGTAATATCGGGA

[0149] AACCTCCTCGGATTCCATTGCCCAGCTATCTGTCACTTCATCAAAAGGACAGTAGAAA

[0150] AGGAAGGTGGCACCTACAAATGCCATCATTGCGATAAAGGAAAGGCTATCGTTCAAG

[0151] ATGCCTCTGCCGACAGTGGTCCCAAAGATGGACCCCCACCCACGAGGAGCATCGTGG

[0152] AAAAAGAAGACGTTCCAACCACGTCTTCAAAGCAAGTGGATTGATGTGATAACATGG

[0153] TGGAGCACGACACTCTCGTCTACTCCAAGAATATCAAAGATACAGTCTCAGAAGACC

[0154] AAAGGGCTATTGAGACTTTTCAACAAAGGGTAATATCGGGAAACCTCCTCGGATTCCA

[0155] TTGCCCAGCTATCTGTCACTTCATCAAAAGGACAGTAGAAAAGGAAGGTGGCACCTA

[0156] CAAATGCCATCATTGCGATAAAGGAAAGGCTATCGTTCAAGATGCCTCTGCCGACAGT

[0157] GGTCCCAAAGATGGACCCCCACCCACGAGGAGCATCGTGGAAAAAGAAGACGTTCC

[0158] AACCACGTCTTCAAAGCAAGTGGATTGATGTGATATCTCCACTGACGTAAGGGATGAC

[0159] GCACAATCCCACTATCCTTCGCAAGACCTTCCTCTATATAAGGAAGTTCATTTCATTTG

[0160] GAGAGGGGGTAAATAAATGGCTTGGCAAGCGATCAGACGTTTGTTTACCCGCAAGCA

[0161] GCgtcCACGCGCTGCTAGCAGCATCTCTGAGGAGATCTACGTGGGCGACTTCCCCATTG

[0162] GAGTCACCCGCGAGGTCCCAGCGCCACGAGTGGAGACCGTGGTGGCTGATGAGCTG

[0163] AGTCCTGTGAACCGGGAGTCCATGATGTTGGATGACGACTGGGTCGCAGTGATCAAC

[0164] ACCGCCAGATACATGGAGGCTCAAGCTCAACTCAGGGAGCGCTTGGACGAGGTGGT

[0165] GCAGGAGGAGCTGCGCCAATGGGTCCTGGGAAACGTGGCTAGCCCCGCaGCCCGTCT

[0166] AGCAGAGCaACTTCGCGTGCGTTTCGGCGCGCGCGCCCCCACGGCCGCTAAtGCGGTT

[0167] ATGGGAGAGCGGTTCTTGCTGGACAAGGCAgCGGAGGCTGGAAACGACCACCTGGA

[0168] TGCCAGcAACCTCGCAAGCTTGGCTGTGAATCTCTGGTTAGTGCGCACCGCTTGGGAC

[0169] CAGTCCGTCGGGCTGGGTTTTCCGAGTGGGTGGTAACTGAGCTCCCCGGGGTGTCAA

[0170] GGAGTTTCGAATGCCCTCCTGACGCTCCCGTAATCTGGCGTTCGCGGGCTGCCACCCC

[0171] GATGAAGTACGGCAGGCGAACTGTGAACTTCAGCAATCTGATGGCTAATGATATTTAt

[0172] AAGGTGCACGCCAACTCGAAGGACAATCTGCTCAGGGGATTTTGTACCCGAGTGTGG

[0173] CGATATAAGGGCGCCGATCCGATCCCGTGTCTGCGGTCCCGACTGCCGCGATTTGAAC

[0174] GGGCGTTCCGTGGGTTCCCGGTACGCCCCGTCTCGTATGATTCAGTCGTGGAGAACG

[0175] CATTGCCCCGTCGCAGAAAACTGTTACAGACGGCTGTGGAAAGCCTAAAAACGAAG

[0176] CCTCTACAACTCGCCGATTCACATGTTAATACCTTTATCAAGGCTGAGAAGTTTTGTTG

[0177] GAGTGCGAAGCCTGACGCGGATCCTCGTCTAATACAGCCCCGCGGTAATCGCTTCCTT GTCTCACACGGCCGGTACATAAAGGCGATTGAGCCGATGGTCTATAAAGCACTGGGC

[0178] CGACTCACCGGACTTCCCATGGTGGCGAAGGGATTCAACGCTCGTGAAACAGCGGA

[0179] GATCCTGCGCAAGAAGTGGGACCGGTTTAGAGACCCCGTGTGCATATCTTTGGACGC

[0180] CAGTCGCTTTGATCAGCACGTCAGTGTGGACATGTTGAAGTTTACTCACCGCATATAC

[0181] CGACGATTCAATCGAGATCCGGAGTTTAAAATGATGTGTGATTGGCAATTACACAACA

[0182] CAGGCTTTGCCTCCTGCCCAGAGGGGAGCTTCAAGTATAAAATCGCGGGAAGGCGAA

[0183] TGAGTGGAGATATGGATACGTCGCTGGGCAACTGCGTCATCATGTGTGGTCTAACATA

[0184] TGAATTATTTGGCCCCGGCGTGGAGATATTCAACAACGGTGACGATTGTTTAGTGATC

[0185] GGCGAGCGTCATGAGCTAGTGGACGCGCCCACTGTCCGCGAGCACTATAAGCAGTAC

[0186] GGTTTCCATGTGGTCGAGGAAGACAGAGTTGATGTGTTCGAGCGCATCCTCTTTTGtC

[0187] AAACTCAACCTATCTGGGCCGAGGGATGGGTCATGTGCCGTCACCCACGGATGATGG

[0188] CAAAGGATTTGACCTATATTGGGCCACACACTGCTGATAGGGCTTGGCTCGATGCAAT

[0189] TGGACAGTGTGGGATCGCTCTTGCTGATGGAGTGCCTATAATGTCGCGGTTCTACCAC

[0190] AATCTCATCGACACAAGTCACCGtAAAGCGAATATCACCAACAGTATGCTCTACTCATG

[0191] CGGATTGACGAATTTAGCGAAGCGCATGGAATATAAGGGACTGCCCATCTCAGACCGC

[0192] TCTCGGTATTCATTTTATCTGGGGTTCGGAGTCGAGCCGGATGTTCAGGTTGCGATAG

[0193] AAGCCAGTATGGGCCCGCTGGGTAATATACAACGAATTGATGGCAAACTCTTCGAAC

[0194] AGACGATCACAAACTACGAGAGCGCTCTCGAAGCGGCGGAGAAGGGGTCGGG

[0195] GCACGCGTGGAAGCGTCCCGTCTCCCTTCCCATTCCCTAATCGCACACAGATCGA

[0196] CACGCGCACCTGCCTCCAGGACTTCAAGATTGATGCACCAGcTGCGGACAAGTCTAC

[0197] TGGGGTaGCCATGGTATGTATGCAGCTCGACCGGACGCTAGACGCCGTACGCATGGCT

[0198] GGATTCTCCACCCACTATCGCATAGTGTCAGTGGAGGCGCACGTGATACCCgCAACAA

[0199] TACATGCACAGGCAGTTGCTAGTATAGGCAACTCCACCCATCGCAGCGGGCCGGGCG

[0200] CACCTGGAGATCTTGTGGACGGATCCACGACGATCTTCTCGTATTCCGAATATGTGCG

[0201] ATGGCAATCTTTGATGTCGAGCACACGCAAGGCTGCCCCCGGAGCAACATTAGTCGC

[0202] GACGTTCCGACCTCAGAATCCTGTTTGGGTTGAGAACACCGCTAACGCACGGGCTGA

[0203] TGGTGCCATGTCGCTCCTGGTTGGTAGTATTAGTGCGCAGAATCACACCTCAGCGACG

[0204] TGGGTGTCGTTGAAGTATGTGATTCAGTTCCAGATGGACGTCGCCGTCTATTGACGGG

[0205] CCGGGGACGCCCGTCCTCCAGCTGGCTCGCGCCATTCTGAGGATATGCAGCCATTAGT

[0206] GGAAGAGTTGCACAGGATGGAGCGATCTAGTGGGCCGTCCCAGCGACCTAGGAGGG

[0207] ACGTCTCTGAGCAAATTCAACTGGCCCCGCCTCAGTCCGGTTCTGAGGATAACAGTA AGGCCGGGTGGTCTCTGAGTGCCACCGCTGGAGCAGCCGCCGGAACTGCTGCGGTG GTGGCGGGAGGCGTGGCGGCTGCCGAAGCCGTCGCAGGTCTCGGTGCCGCTGCGGC GTCGGTGCCCACCGATAGTGTGCCATTGCTGGACATGTCGGCGAGCACCTCGTCACTT GTGGAACCATCGCTGCAAGTGGTcGAAACCGACtTGGACGATGTCTCCTTGTGGGACG CGGCCGAGGAGGAATTGACCGCAGCCGCCGAGCAGACCTCGCTCACCAGCATACCT GAAAGCCTGAAGACCACATGGCGCCAGTGGATTGAAGACGAATGTCGACAaGGAATG ATAAAGGAGGGGGTGAAGAATCGGTTTACAGAATCACTGAACCGCCCCGAAGCGCTT AACGCAGCCCGCACCTCTTGGTTCAGACGCTATGGAACCGAGCCTACAGACGCTGAA GTTTTGCAGCGCCTTcTCGAGGACATCATCCGGTCGTAAGTACTGGTCACAGCAGGAG GTAAGACGTCCCGATTGGCAACTTTCGGAAGTACCCGGTGAGGGGAAACGCGCCTAG GACCTGCTCGTTCGCCGTGGAACGCACCTGATGACTACTCGAGCGACACTGCCGTTG GCTGTCACCCAGACAGTCACCTGAGCTTACACTTCCCGAGGGGGGGGACATAATACC TCTCGGTACCGAACCATCTATAGGTAGCGTGCCCGCGCTACCGCCCC (SEQ ID NO:1)

[0208] In the sequence above, the bolded portion represents the coding sequence that overlaps for 0RF2 and ORF 3.

[0209] 2.3. Agroinfiltration of N. benthamiana with WULV cDNA Clone

[0210] The selected WULV cDNA clones (pJL89-WULV) were transformed into GV3101 strain of Agrobacterium tumefaciens on Luria-Bertani (LB) agar plates containing 50 pM kanamycin, 30 pM gentamycin and 10 pM rifampicin. Positive colonies were inoculated in to 3 ml of LB media containing the same antibiotics and incubated for 24-40 hours at 28°C with shaking at 220 rpm. 200pl of the fully grown culture was added to fresh 100 ml LB antibiotics media and incubated at 28°C with shaking at 220 rpm. After 24 hours, culture was pelleted at 5000 rpm in Sorvall RC6 (Thermo Fisher Scientific, MA, USA) centrifuge and resuspended into infiltration buffer containing 10 mM of 2-(N-morpholino) ethanesulfonic acid (MES), 10 mM magnesium sulfate and 150 pM acetosyringone to 1.0 as a final optical density at 600 nm (OD600). Using a needleless syringe, the viral suspension was infiltrated in the abaxial side of fully expanded leaves of four weeks old N. benthamiana plants expressing the potyviral silencing suppressor HC-Pro (Wang et al., 2009). Non-infiltrated plants were used as negative controls. 2.4. Validation of WULV cDNA clone Infection in N. benthamiana

[0211] The infectious ability and stability of pJL89-WULV cDNA clone in N. benthamiana was examined by three methods:

[0212] 2.4.1. RT-(q)PCR

[0213] Total RNAs were extracted using TRI reagent (Zymo Research, CA, USA) from HC-Pro N. benthamina infiltrated at three time points (1, 3 and 10 dpi). cDNA was generated using SuperScript III Reverse Transcriptase (Invitrogen, CA, USA) and gene specific WULV-R3 (Table SI). qPCR reaction was performed using SsoAdvanced Universal SYBR Green Supermix (BioRad, CA, USA) and qPCR-F and qPCR-R primers (Table SI). Each sample was analyzed in three technical replicates. The qPCR cycling conditions were as follows: pre-denaturation of 95 °C for 2 min, 40 cycles of denaturation of 95 °C for 10 s and an annealing / extension step at 55 °C for 30 s. Detection of WULV ORF3 replication was carried out by the CFX96 Touch™ Real-Time PCR detection system (Bio-Rad, CA, USA).

[0214] To calculate the viral copy number, serial dilutions of plasmid pJL89-WULV were used to generate the standard curve and virus copy number was calculated as described by Plumet and Gerlier (2005) using CFX96 Touch™ Real-Time PCR software.

[0215] To study the WULV movement in N. benthamiana, total RNAs were isolated from upper noninfiltrated leaves at 6 and 10 dpi using TRI reagent. The first strand cDNA was produced as mentioned above. Then, PCR was carried out using 1 pL of cDNA in 25 pL reaction containing IX PrimeSTAR Max Premix (2X) (Takara Bio, CA, USA), 0.3 pM gene specific primers WULV- F2 and WULV-R6 (Table SI), and nuclease free water. PCR products were run on a 0.8% agarose gel and imaged by Odyssey XF Imaging System (LLCOR, NE, USA).

[0216] 2.4.2. Northern Blot

[0217] Total RNAs were extracted from N. benthamina leaves using TRI reagent (Zymo Research, CA, USA) following the manufacturer’s instruction. RNAs were denatured with glyoxal at 55°C for 30 min followed by electrophoresis on 1% agarose gels and transferring to Nylon Membrane, positively charged (Roche, Basel, Switzerland). RNAs were fixed to the membrane by crosslinking under UV light and the blots were stained with methylene blue. After removing glyoxal by soaking the blots in 100 mM TAE IX and boiling for 30 s, the membranes were hybridized with DIG-labeled RNA probes to detect the positive strand RNA. For making the probe, DNA fragment covering nucleotides 2221-2590 of the ORF3 was amplified using WULV-Probe-F3 and WULV- Probe-R3 primers (Table SI). The amplicons were used as templates for in vitro transcription reactions with Digoxigenin- 11 -UTP using DIG labeling mix (Roche, Basel, Switzerland). The transcripts were cleaned up using the RNA Clean & Concentrator kit (Zymo Research, CA, USA). The membranes were hybridized in the NorthernMax Prehybridization / Hybridization Buffer (Invitrogen, CA, USA) overnight and washed twice in 2X SSC (IX SSC is 0.15 M sodium chloride and 0.015 M sodium citrate) and 0.1% SDS for 5 min, twice in 0.5X SSC and 0.1% SDS for 5 min, and twice in 0.1X SSC and 0.1% SDS for 5 min. The first two washings were performed at room temperature, while the last washing was performed at 65°C. The membranes were incubated in the blocking buffer (Roche, Basel, Switzerland) for one hour followed by incubation in the alkaline phosphatase conjugated antibody against DIG (Roche, Basel, Switzerland) for 30 min. After washing twice at room temperature, the membranes were incubated in the detection buffer (0.1 M Tris-HCl, 0.1 M NaCl, pH 9.5) for 5 min and imaged with chemiluminescence on Odyssey XF Imaging System (LI-COR, NE, USA).

[0218] 2.4.3. Western Blot

[0219] Total proteins were extracted from 100 mg of agroinfiltrated N. benthamiana in 300 pL of protein extraction buffer (100 mM Tris-HCl pH 7.5, 100 mM EDTApH 8.0, 5 mM DDT, 150 mM NaCl and 0.1% v / v Triton X-100). 12 pL of the extracts were separated by SDS-PAGE using precast 10% polyacrylamide gels (Bio-Rad, CA, USA) and transferred to nitrocellulose membranes using the iBlot Dry Blotting System (Invitrogen, CA, USA). Immunodetection of the WUEV-ORF3 protein using an anti-WUEV-P23 antibody against the synthesized C- RPQNPVWVENTANARADG (SEQ ID NO: 29) peptide was performed using the iBind Western System (Invitrogen, CA, USA). The goat anti -rabbit IgG-HRP conjugate (Bio-Rad, CA, USA) was used as secondary antibody and the protein was detected by using the SuperSignal West Pico Chemiluminescent Substrate (Thermo Fisher Scientific, MA, USA) and Odyssey XF Imaging System (LI-COR, NE, USA).

[0220] 2.5. In vitro Transcription and Infectivity Assay in Wheat

[0221] The full genome sequence of WULV (OK573479) described in our previous work (Redila et al., 2021b) was used for designing primers targeting the extreme 5' and 3' ends of the WULV genome. The full genome of WULV was amplified using pJL89-WULV clone as template in 25 pL PCR reaction containing IX PrimeSTAR Max Premix (2X) (Takara Bio, CA, USA), 0.3 pM gene specific primers WULV-F7+T7 and WULV-R3 (Table SI), and nuclease free water. PCR products were gel purified using the Zymoclean Gel DNA Recovery Kit (Zymo Research, CA, USA) according to the manufacturer’s instruction. In vitro transcripts were synthesized using the MEGAscript™ T7 Kit (Invitrogen, CA, USA) and 250 ng purified DNAs as template followed by cleaning up of RNA transcripts using the RNA Clean & Concentrator kit (Zymo Research, CA, USA). “Tomahawk” wheat plants at the single-leaf stage were inoculated with purified RNA transcripts or FES (1% Sodium Pyrophosphate, 1% Bentonite, 1% Celite, 0.1M glycine, 0.06M dibasic potassium phosphate) buffer. Inoculated plants were kept in a growth chamber on a 22°C, 16 h light / 8 h dark cycle and monitored for any symptoms. Inoculated and upper non-inoculated leaves were collected at 12 dpi. Collected leaves were kept at -80°C for RNA isolation.

[0222] 2.5.1. RT-PCR

[0223] Total RNAs were isolated from 100 mg of collected leaves using TRI reagent (Zymo Research, CA, USA). 1 pL of the first strand cDNA synthesized as described above was used in 25 pL PCR reaction containing IX PrimeSTAR Max Premix (2X) (Takara Bio, CA, USA) and 0.3 pM gene specific primers WULV-F2 and WULV-R6 (Table SI). PCR products were run on a 0.8% agarose gel and imaged by Odyssey XF Imaging System (LI-COR, NE, USA).

[0224] 2.6. Mechanical Transmission of WULV in Wheat

[0225] To examine if WULV is mechanically transmitted, sap inoculations were carried out in wheat using two inoculum sources: WULV positive field wheat leaves and agroinfiltrated N. benthamiana leaves. Briefly, 100 mg of either field wheat sample or agroinfiltrated N. benthamiana leaves were grounded in 0.2 M phosphate buffer (pH 7.0). The prepared sap was used to inoculate onto carborundum-dusted leaves of two-leaf stage wheat “Tomahawk” variety. Buffer-inoculated plants were used as negative controls. Total RNAs isolation and cDNA synthesis were performed as described above. Subsequently, the PCR was performed using WULV-F2 and WULV-R6 primer pair (Table SI) and GoTaq® Flexi DNA Polymerase (Promega, WI, USA) in a final volume of 20 pl. The reaction conditions were as follows: 95°C for 2 min followed by 35 cycles of 95°C for 30 s, 55°C for 30 s, 72°C for 1 min and a final elongation at 72°C for 5 min. The PCR products were verified by electrophoresis in 0.8% agarose gel. 3. Results

[0226] 3.1. Full-length cDNA clone of WULV is infectious in N. benthamiana

[0227] The infectivity and pathogenicity of WULV full-length cDNA clone (pJL89-WULV) was examined by agroinfdtration of four-week-old HC-Pro transgenic N benthamiana. At 4 dpi, mild necrosis appeared on agroinfiltrated leaves which was developed rapidly over the period of 12 dpi (Fig. 2A) and leaves eventually died at 2 weeks post inoculation (wpi). No notable symptoms except mild yellowing were observed on upper non-infiltrated leaves (Fig. 2A). We monitored upper leaves for developing any symptoms within 35 dpi but they stayed symptomless (Fig. 2A). To monitor WULV infection, time-course RT-qPCR was conducted using the total RNAs from infiltrated leaves at 1, 3 and 10 dpi. The viral copy number over the course of infection showed a gradual increase in infiltrated leaves (Fig. 2B). We observed a slight decline in the titer after 5 dpi which is because of the necrotic cell death response of the infiltrated leaves. The replication of WULV cDNA clone in N. benthamiana was further confirmed by northern blot analysis using the ORF3 probe which detected both gRNA and sgRNA (Fig. 2C). In addition, total proteins were isolated from infiltrated leaves at 6 dpi and analyzed by western blotting. A strong expressed protein with a molecular weight of ~23 KDa (p23) corresponding to WULV-0RF3 was detected (Fig. 2D), validating the infectivity of pJL89-WULV clone in N. benthamiana. In addition, and to examine if WULV can systemically infect A benthamiana, upper leaves were collected and tested at 6 and 10 dpi. RT-PCR detected WULV in upper non-infiltrated leaves (Fig. 2E), suggesting the systemic movement of WULV in N. benthamiana.

[0228] 3.2. In vitro transcripts of WULV are infectious in wheat

[0229] The infectivity of wheat by WULV in vitro transcripts was confirmed by amplifying a 1.3 kb fragment of WULV genome in inoculated and non-inoculated (newly emerged) leaves at 12 dpi (Fig. 3 A), suggesting the solo replication without a helper virus and systemic movement of WULV in wheat. Interestingly, none of the WULV positive wheat plants expressed observable symptoms during the period of the symptom monitoring time which was till 30 dpi (Fig. 3B).

[0230] 3.3. WULV is mechanically transmissible in the form of free RNA

[0231] The crude sap inoculation of wheat “Tomahawk” with two inoculum sources revealed that WULV is able to transmit mechanically from plant to plant. At 12 dpi, inoculated and non- inoculated (newly emerged) leaves of “Tomahawk” plants with the sap of field positive samples were tested for WULV by RT-PCR, and ~1.3 kb DNA fragments were detected (Fig. 4A); while no PCR products were detected in plants inoculated with the buffer. WULV was also detected in “Tomahawk” plants inoculated with the sap of agroinflitrated N. benthamiana (Fig. 4B). While inoculated “Tomahawk” with WULV positive field samples demonstrated common WSM symptoms because of the co-infection with WSMV, no obvious symptoms were observed in the positive PCR wheat plants inoculated with the sap of infected N benthamiana. This was consistent with the result that we obtained from infecting wheat with WULV in vitro transcript (Fig. 3B), revealed that WULV solo infection in wheat is symptomless.

[0232] 4. Discussion

[0233] Our recent metagenomics study of the wheat virome identified WULV as a new ulaRNA associated with field collected wheat in KS (Redila et al., 2021b). Since then, similar viral sequences to WULV have been identified and reported from Colorado and Texas fields. Although not much yet known about the distribution of WULV in the Great Plains fields, it seems that WULV should be considered as a new emerging virus. Hence, molecular and biological characterization as well as the role of WULV in disease development in single and mixed infections with other wheat viruses and its routes of transmission are necessary. In this study, we constructed a full- length cDNA clone of WULV which was infectious in N. benthamiana. Detection of a strong sgRNA (~ 1.5 kb) in agroinfiltrated leaves indicated the highly efficient replication of the WULV clone. The size of the detected sgRNA was approximately the sum of the sizes of two predicted ORFs of WULV, ORF3 and ORF4, suggesting that these two ORFs are probably transcribed as a single mRNA from the sgRNA. However, the sgRNA may encode for two viral proteins of 23 (ORF3) and 22 KDa (ORF4), respectively. This was confirmed by the western blot analysis using an antibody against the ORF3 clearly produced a single strong small protein of the size of -23 KDa (p23). Our attempts to detect p22 were not successful probably because of the low level of the protein accumulation at that specific stage of WULV life cycle. Hence, these results suggest that WULV sgRNA may function as a bicistronic mRNA with high expression of ORF3 and low translational efficiency for synthesis of ORF4. Moreover, WULV was detected in upper leaves of N. benthamiana, suggesting the capability of systemic movement of WULV in solo infections. Agroinfiltrated N. benthamiana leaves showed severe necrosis, appeared at 4 dpi and developed over the period of two weeks (Fig. 2A). Affected leaves eventually died, while no obvious symptoms except mild yellowing were observed in systemic leaves. This observation was different from another report that studied the biology of CYVaV infectious clone in N. benthamiana in that infiltrated leaves did not develop any symptoms, but systemic leaves showed unusual symptoms including heart-shaped apical leaf, abnormal developed flowers along with interveinal leaf necrosis at the late stage of infection (31 and 54 dpi), suggesting that the biology of ulaRNAs is different and the specific interaction between the specific RNA and the plant determines the outcome of the infection. We should mention again that we monitored symptoms in upper leaves for 35 days post inoculation, therefore, we cannot rule out the possibility of the appearance of further symptoms in upper leaves at the later stage of the infection, however the probability is low. Although the functions of the ORF3 and ORF4 are not yet determined, we observed the high accumulation of the p23, encoded protein by ORF3 in agroinfiltrated leaves in this study. Therefore, our hypothesis was that this protein is probably the key player in inducing necrosis. The study with mutant transcripts derived from pJL89-WULV infectious clone was conducted to test this hypothesis. We used site-directed mutagenesis to disrupt producing p23 and inoculated N. benthamiana plants with both wild type (WT) ORF3 mutant clones. Our results showed that WULV still can replicate and move in plants inoculated with ORF3 mutant clone. Therefore, p23 encoded by ORF3 is not a movement protein. Our new hypothesis is that p23 protects RNA by forming virus-like particles (VLPs). We have used infected plants to purify potential VLPs and we will use electron microscopy to observe any formed VLPs.

[0234] This study also provided evidence that WULV is capable of autonomous replication and movement in wheat without causing detectable disease phenotypes. Although, this result was consistent with the previous reports of the presence of ulaRNAs in asymptomatic plants in solo infections, it was different from the results observed in infected citrus plants with CYVaV in which the leaves of infected plants showed yellow-vein symptoms. This again indicates that not all ulaRNAs behave the same.

[0235] The co-infections of ulaRNAs with plant viruses from different families have been reported in natural patho-systems. However, not yet enough studies have been conducted to investigate the role of ulaRNAs in disease / symptom severity in mixed infections. To date, only one study has revealed the clear role of an ulaRNA, PMeV-2, in exacerbation of symptom severity of Papaya sticky disease in mixed infections with its helper virus. Our WULV positive field collected samples were co-infected with wheat potyviruses including Wheat streak mosaic virus (WSMV) and Triticum mosaic virus (TriMV) exhibiting common wheat streak mosaic (WSM) symptoms (Redila et al., 2021a). This co-infection raises this question whether the presence of WULV in mixed infections increases the titer of WSMV and / or TriMV and severity of WSM symptoms, the knowledge gaps that need yet to be addressed.

[0236] Furthermore, this study demonstrated that WULV is mechanically transmissible from plant to plant through sap inoculation. Although the transmission using the sap of WULV positive field samples can bring up this assumption that the co-infection with potyviruses helps WULV mechanical transmission, the result obtained by infecting wheat using the sap of infected N. benthamiana clearly showed that WULV is capable to transmit in the form of free RNA in the absence of any helper virus. To the best of our knowledge, this is the first reported mechanically transmissible ulaRNA. Although, our study confirmed this route of transmission by at least two serial passages in wheat (data has not been shown) under experimental conditions, further studies are needed to determine the natural transmission mode(s) of WULV.

[0237] 5. Conclusion

[0238] WULV, a new subviral RNA associated with wheat, has been recently identified and reported from important wheat growing regions of the U.S. As an emergent virus, there are large gaps in our knowledge about WULV molecular and biological characterizations, its distribution in fields, and its transmission. To address these knowledge gaps, this study provides details of the construction of the first infectious clone of WULV as a valuable research tool for further investigations of the biology and molecular biology of the virus as well as its contribution to disease development in interactions with other important wheat viruses. For example, we have since generated GFP constructs. The sequence of eGFP was inserted in three sites: N-terminal of ORF3, N-terminal of ORF4, and C- terminal of ORF4 (the below shows the sequence of all three constructs and the inserted sites), using in-Fusion cloning to insert eGFP in frame (this is important that eGFP be in frame with the ORF) with each ORF in each construct.

[0239] Fusing eGFP into the C-terminal of ORF3 (ORF3 and eGFP are in frame): SEQ ID NO:26 aggtcaacatggtggagcacGACACTCTCGTCTACTCCAAGAATATCAAAGATACAGTCTCAGAA GACCAAAGGGCTATTGAGACTTTTCAACAAAGGGTAATATCGGGAAACCTCCTCGGA TTCCATTGCCCAGCTATCTGTCACTTCATCAAAAGGACAGTAGAAAAGGAAGGTGGC

[0240] ACCTACAAATGCCATCATTGCGATAAAGGAAAGGCTATCGTTCAAGATGCCTCTGCCG

[0241] ACAGTGGTCCCAAAGATGGACCCCCACCCACGAGGAGCATCGTGGAAAAAGAAGAC

[0242] GTTCCAACCACGTCTTCAAAGCAAGTGGATTGATGTGATAACATGGTGGAGCACGAC

[0243] ACTCTCGTCTACTCCAAGAATATCAAAGATACAGTCTCAGAAGACCAAAGGGCTATTG

[0244] AGACTTTTCAACAAAGGGTAATATCGGGAAACCTCCTCGGATTCCATTGCCCAGCTAT

[0245] CTGTCACTTCATCAAAAGGACAGTAGAAAAGGAAGGTGGCACCTACAAATGCCATCA

[0246] TTGCGATAAAGGAAAGGCTATCGTTCAAGATGCCTCTGCCGACAGTGGTCCCAAAGA

[0247] TGGACCCCCACCCACGAGGAGCATCGTGGAAAAAGAAGACGTTCCAACCACGTCTT

[0248] CAAAGCAAGTGGATTGATGTGATATCTCCACTGACGTAAGGGATGACGCACAATCCC

[0249] ACTATCCTTCGCAAGACCTTCCTCTATATAAGGAAGTTCATTTCATTTGGAGAGGGGGT

[0250] AAATAAATGGCTTGGCAAGCGATCAGACGTTTGTTTACCCGCAAGCAGCgtcCACGCG

[0251] CTGCTAGCAGCATCTCTGAGGAGATCTACGTGGGCGACTTCCCCATTGGAGTCACCCG

[0252] CGAGGTCCCAGCGCCACGAGTGGAGACCGTGGTGGCTGATGAGCTGAGTCCTGTGA

[0253] ACCGGGAGTCCATGATGTTGGATGACGACTGGGTCGCAGTGATCAACACCGCCAGAT

[0254] ACATGGAGGCTCAAGCTCAACTCAGGGAGCGCTTGGACGAGGTGGTGCAGGAGGAG

[0255] CTGCGCCAATGGGTCCTGGGAAACGTGGCTAGCCCCGCaGCCCGTCTAGCAGAGCaA

[0256] CTTCGCGTGCGTTTCGGCGCGCGCGCCCCCACGGCCGCTAAtGCGGTTATGGGAGAGC

[0257] GGTTCTTGCTGGACAAGGCAgCGGAGGCTGGAAACGACCACCTGGATGCCAGcAACC

[0258] TCGCAAGCTTGGCTGTGAATCTCTGGTTAGTGCGCACCGCTTGGGACCAGTCCGTCG

[0259] GGCTGGGTTTTCCGAGTGGGTGGTAACTGAGCTCCCCGGGGTGTCAAGGAGTTTCGA

[0260] ATGCCCTCCTGACGCTCCCGTAATCTGGCGTTCGCGGGCTGCCACCCCGATGAAGTAC

[0261] GGCAGGCGAACTGTGAACTTCAGCAATCTGATGGCTAATGATATTTAtAAGGTGCACG

[0262] CCAACTCGAAGGACAATCTGCTCAGGGGATTTTGTACCCGAGTGTGGCGATATAAGG

[0263] GCGCCGATCCGATCCCGTGTCTGCGGTCCCGACTGCCGCGATTTGAACGGGCGTTCC

[0264] GTGGGTTCCCGGTACGCCCCGTCTCGTATGATTCAGTCGTGGAGAACGCATTGCCCCG

[0265] TCGCAGAAAACTGTTACAGACGGCTGTGGAAAGCCTAAAAACGAAGCCTCTACAAC

[0266] TCGCCGATTCACATGTTAATACCTTTATCAAGGCTGAGAAGTTTTGTTGGAGTGCGAA

[0267] GCCTGACGCGGATCCTCGTCTAATACAGCCCCGCGGTAATCGCTTCCTTGTCTCACAC

[0268] GGCCGGTACATAAAGGCGATTGAGCCGATGGTCTATAAAGCACTGGGCCGACTCACC

[0269] GGACTTCCCATGGTGGCGAAGGGATTCAACGCTCGTGAAACAGCGGAGATCCTGCGC AAGAAGTGGGACCGGTTTAGAGACCCCGTGTGCATATCTTTGGACGCCAGTCGCTTT

[0270] GATCAGCACGTCAGTGTGGACATGTTGAAGTTTACTCACCGCATATACCGACGATTCA

[0271] ATCGAGATCCGGAGTTTAAAATGATGTGTGATTGGCAATTACACAACACAGGCTTTGC

[0272] CTCCTGCCCAGAGGGGAGCTTCAAGTATAAAATCGCGGGAAGGCGAATGAGTGGAG

[0273] ATATGGATACGTCGCTGGGCAACTGCGTCATCATGTGTGGTCTAACATATGAATTATTT

[0274] GGCCCCGGCGTGGAGATATTCAACAACGGTGACGATTGTTTAGTGATCGGCGAGCGT

[0275] CATGAGCTAGTGGACGCGCCCACTGTCCGCGAGCACTATAAGCAGTACGGTTTCCATG

[0276] TGGTCGAGGAAGACAGAGTTGATGTGTTCGAGCGCATCCTCTTTTGtCAAACTCAACC

[0277] TATCTGGGCCGAGGGATGGGTCATGTGCCGTCACCCACGGATGATGGCAAAGGATTT

[0278] GACCTATATTGGGCCACACACTGCTGATAGGGCTTGGCTCGATGCAATTGGACAGTGT

[0279] GGGATCGCTCTTGCTGATGGAGTGCCTATAATGTCGCGGTTCTACCACAATCTCATCGA

[0280] CACAAGTCACCGtAAAGCGAATATCACCAACAGTATGCTCTACTCATGCGGATTGACG

[0281] AATTTAGCGAAGCGCATGGAATATAAGGGACTGCCCATCTCAGACCGCTCTCGGTATT

[0282] CATTTTATCTGGGGTTCGGAGTCGAGCCGGATGTTCAGGTTGCGATAGAAGCCAGTAT

[0283] GGGCCCGCTGGGTAATATACAACGAATTGATGGCAAACTCTTCGAACAGACGATCAC

[0284] AAACTACGAGAGCGCTCTCGAAGCGGCGGAGAAGGGGTCGGGGCACGCGTGGAAG

[0285] CGTCCCGTCTCCCTTCCCATTCCCTAATCGCACACAGATCGACACGCGCACCTGCCTC

[0286] CAGGACTTCAAGATTGATGCACCAGcTGCGGACAAGTCTACTGGGGTaGCCATGGTAT

[0287] GTATGCAGCTCGACCGGACGCTAGACGCCGTACGCATGGCTGGATTCTCCACCCACTA

[0288] TCGCATAGTGTCAGTGGAGGCGCACGTGATACCCgCAACAATACATGCACAGGCAGTT

[0289] GCTAGTATAGGCAACTCCACCCATCGCAGCGGGCCGGGCGCACCTGGAGATCTTGTG

[0290] GACGGATCCACGACGATCTTCTCGTATTCCGAATATGTGCGATGGCAATCTTTGATGTC

[0291] GAGCACACGCAAGGCTGCCCCCGGAGCAACATTAGTCGCGACGTTCCGACCTCAGA

[0292] ATCCTGTTTGGGTTGAGAACACCGCTAACGCACGGGCTGATGGTGCCATGTCGCTCCT

[0293] GGTTGGTAGTATTAGTGCGCAGAATCACACCTCAGCGACGTGGGTGTCGTTGAAGTAT

[0294] GTGATTCAGTTCCAGATGGACGTCGCCGTCTATGTGAGCAAGGGCGAGGAGCTGTTC

[0295] ACCGGGGTGGTGCCCATCCTGGTCGAGCTGGACGGCGACGTAAACGGCCACAAGTT

[0296] CAGCGTGTCCGGCGAGGGCGAGGGCGATGCCACCTACGGCAAGCTGACCCTGAAGT

[0297] TCATCTGCACCACCGGCAAGCTGCCCGTGCCCTGGCCCACCCTCGTGACCACCCTGA

[0298] CCTACGGCGTGCAGTGCTTCAGCCGCTACCCCGACCACATGAAGCAGCACGACTTCT

[0299] TCAAGTCCGCCATGCCCGAAGGCTACGTCCAGGAGCGCACCATCTTCTTCAAGGACG ACGGCAACTACAAGACCCGCGCCGAGGTGAAGTTCGAGGGCGACACCCTGGTGAAC

[0300] CGCATCGAGCTGAAGGGCATCGACTTCAAGGAGGACGGCAACATCCTGGGGCACAA

[0301] GCTGGAGTACAACTACAACAGCCACAACGTCTATATCATGGCCGACAAGCAGAAGAA

[0302] CGGCATCAAGGTGAACTTCAAGATCCGCCACAACATCGAGGACGGCAGCGTGCAGC

[0303] TCGCCGACCACTACCAGCAGAACACCCCCATCGGCGACGGCCCCGTGCTGCTGCCCG

[0304] ACAACCACTACCTGAGCACCCAGTCCGCCCTGAGCAAAGACCCCAACGAGAAGCGC

[0305] GATCACATGGTCCTGCTGGAGTTCGTGACCGCCGCCGGGATCACTCTCGGCATGGAC

[0306] GAGCTGTACAAGTGACGGGCCGGGGACGCCCGTCCTCCAGCTGGCTCGCGCCATTCT

[0307] GAGGATATGCAGCCATTAGTGGAAGAGTTGCACAGGATGGAGCGATCTAGTGGGCCG

[0308] TCCCAGCGACCTAGGAGGGACGTCTCTGAGCAAATTCAACTGGCCCCGCCTCAGTCC

[0309] GGTTCTGAGGATAACAGTAAGGCCGGGTGGTCTCTGAGTGCCACCGCTGGAGCAGCC

[0310] GCCGGAACTGCTGCGGTGGTGGCGGGAGGCGTGGCGGCTGCCGAAGCCGTCGCAGG

[0311] TCTCGGTGCCGCTGCGGCGTCGGTGCCCACCGATAGTGTGCCATTGCTGGACATGTCG

[0312] GCGAGCACCTCGTCACTTGTGGAACCATCGCTGCAAGTGGTcGAAACCGACtTGGACG

[0313] ATGTCTCCTTGTGGGACGCGGCCGAGGAGGAATTGACCGCAGCCGCCGAGCAGACC

[0314] TCGCTCACCAGCATACCTGAAAGCCTGAAGACCACATGGCGCCAGTGGATTGAAGAC

[0315] GAATGTCGACAaGGAATGATAAAGGAGGGGGTGAAGAATCGGTTTACAGAATCACTG

[0316] AACCGCCCCGAAGCGCTTAACGCAGCCCGCACCTCTTGGTTCAGACGCTATGGAACC

[0317] GAGCCTACAGACGCTGAAGTTTTGCAGCGCCTTcTCGAGGACATCATCCGGTCGTAAG

[0318] TACTGGTCACAGCAGGAGGTAAGACGTCCCGATTGGCAACTTTCGGAAGTACCCGGT

[0319] GAGGGGAAACGCGCCTAGGACCTGCTCGTTCGCCGTGGAACGCACCTGATGACTACT

[0320] CGAGCGACACTGCCGTTGGCTGTCACCCAGACAGTCACCTGAGCTTACACTTCCCGA

[0321] GGGGGGGGACATAATACCTCTCGGTACCGAACCATCTATAGGTAGCGTGCCCGCGCTA CCGCCCC

[0322] Fusing eGFP into the N-terminal of ORF4 (ORF4 and eGFP are in frame): SEQ ID

[0323] NO:27 aggtcaacatggtggagcacGACACTCTCGTCTACTCCAAGAATATCAAAGATACAGTCTCAGAA

[0324] GACCAAAGGGCTATTGAGACTTTTCAACAAAGGGTAATATCGGGAAACCTCCTCGGA

[0325] TTCCATTGCCCAGCTATCTGTCACTTCATCAAAAGGACAGTAGAAAAGGAAGGTGGC

[0326] ACCTACAAATGCCATCATTGCGATAAAGGAAAGGCTATCGTTCAAGATGCCTCTGCCG ACAGTGGTCCCAAAGATGGACCCCCACCCACGAGGAGCATCGTGGAAAAAGAAGAC

[0327] GTTCCAACCACGTCTTCAAAGCAAGTGGATTGATGTGATAACATGGTGGAGCACGAC

[0328] ACTCTCGTCTACTCCAAGAATATCAAAGATACAGTCTCAGAAGACCAAAGGGCTATTG

[0329] AGACTTTTCAACAAAGGGTAATATCGGGAAACCTCCTCGGATTCCATTGCCCAGCTAT

[0330] CTGTCACTTCATCAAAAGGACAGTAGAAAAGGAAGGTGGCACCTACAAATGCCATCA

[0331] TTGCGATAAAGGAAAGGCTATCGTTCAAGATGCCTCTGCCGACAGTGGTCCCAAAGA

[0332] TGGACCCCCACCCACGAGGAGCATCGTGGAAAAAGAAGACGTTCCAACCACGTCTT

[0333] CAAAGCAAGTGGATTGATGTGATATCTCCACTGACGTAAGGGATGACGCACAATCCC

[0334] ACTATCCTTCGCAAGACCTTCCTCTATATAAGGAAGTTCATTTCATTTGGAGAGGGGGT

[0335] AAATAAATGGCTTGGCAAGCGATCAGACGTTTGTTTACCCGCAAGCAGCgtcCACGCG

[0336] CTGCTAGCAGCATCTCTGAGGAGATCTACGTGGGCGACTTCCCCATTGGAGTCACCCG

[0337] CGAGGTCCCAGCGCCACGAGTGGAGACCGTGGTGGCTGATGAGCTGAGTCCTGTGA

[0338] ACCGGGAGTCCATGATGTTGGATGACGACTGGGTCGCAGTGATCAACACCGCCAGAT

[0339] ACATGGAGGCTCAAGCTCAACTCAGGGAGCGCTTGGACGAGGTGGTGCAGGAGGAG

[0340] CTGCGCCAATGGGTCCTGGGAAACGTGGCTAGCCCCGCaGCCCGTCTAGCAGAGCaA

[0341] CTTCGCGTGCGTTTCGGCGCGCGCGCCCCCACGGCCGCTAAtGCGGTTATGGGAGAGC

[0342] GGTTCTTGCTGGACAAGGCAgCGGAGGCTGGAAACGACCACCTGGATGCCAGcAACC

[0343] TCGCAAGCTTGGCTGTGAATCTCTGGTTAGTGCGCACCGCTTGGGACCAGTCCGTCG

[0344] GGCTGGGTTTTCCGAGTGGGTGGTAACTGAGCTCCCCGGGGTGTCAAGGAGTTTCGA

[0345] ATGCCCTCCTGACGCTCCCGTAATCTGGCGTTCGCGGGCTGCCACCCCGATGAAGTAC

[0346] GGCAGGCGAACTGTGAACTTCAGCAATCTGATGGCTAATGATATTTAtAAGGTGCACG

[0347] CCAACTCGAAGGACAATCTGCTCAGGGGATTTTGTACCCGAGTGTGGCGATATAAGG

[0348] GCGCCGATCCGATCCCGTGTCTGCGGTCCCGACTGCCGCGATTTGAACGGGCGTTCC

[0349] GTGGGTTCCCGGTACGCCCCGTCTCGTATGATTCAGTCGTGGAGAACGCATTGCCCCG

[0350] TCGCAGAAAACTGTTACAGACGGCTGTGGAAAGCCTAAAAACGAAGCCTCTACAAC

[0351] TCGCCGATTCACATGTTAATACCTTTATCAAGGCTGAGAAGTTTTGTTGGAGTGCGAA

[0352] GCCTGACGCGGATCCTCGTCTAATACAGCCCCGCGGTAATCGCTTCCTTGTCTCACAC

[0353] GGCCGGTACATAAAGGCGATTGAGCCGATGGTCTATAAAGCACTGGGCCGACTCACC

[0354] GGACTTCCCATGGTGGCGAAGGGATTCAACGCTCGTGAAACAGCGGAGATCCTGCGC

[0355] AAGAAGTGGGACCGGTTTAGAGACCCCGTGTGCATATCTTTGGACGCCAGTCGCTTT

[0356] GATCAGCACGTCAGTGTGGACATGTTGAAGTTTACTCACCGCATATACCGACGATTCA ATCGAGATCCGGAGTTTAAAATGATGTGTGATTGGCAATTACACAACACAGGCTTTGC

[0357] CTCCTGCCCAGAGGGGAGCTTCAAGTATAAAATCGCGGGAAGGCGAATGAGTGGAG

[0358] ATATGGATACGTCGCTGGGCAACTGCGTCATCATGTGTGGTCTAACATATGAATTATTT

[0359] GGCCCCGGCGTGGAGATATTCAACAACGGTGACGATTGTTTAGTGATCGGCGAGCGT

[0360] CATGAGCTAGTGGACGCGCCCACTGTCCGCGAGCACTATAAGCAGTACGGTTTCCATG

[0361] TGGTCGAGGAAGACAGAGTTGATGTGTTCGAGCGCATCCTCTTTTGtCAAACTCAACC

[0362] TATCTGGGCCGAGGGATGGGTCATGTGCCGTCACCCACGGATGATGGCAAAGGATTT

[0363] GACCTATATTGGGCCACACACTGCTGATAGGGCTTGGCTCGATGCAATTGGACAGTGT

[0364] GGGATCGCTCTTGCTGATGGAGTGCCTATAATGTCGCGGTTCTACCACAATCTCATCGA

[0365] CACAAGTCACCGtAAAGCGAATATCACCAACAGTATGCTCTACTCATGCGGATTGACG

[0366] AATTTAGCGAAGCGCATGGAATATAAGGGACTGCCCATCTCAGACCGCTCTCGGTATT

[0367] CATTTTATCTGGGGTTCGGAGTCGAGCCGGATGTTCAGGTTGCGATAGAAGCCAGTAT

[0368] GGGCCCGCTGGGTAATATACAACGAATTGATGGCAAACTCTTCGAACAGACGATCAC

[0369] AAACTACGAGAGCGCTCTCGAAGCGGCGGAGAAGGGGTCGGGGCACGCGTGGAAG

[0370] CGTCCCGTCTCCCTTCCCATTCCCTAATCGCACACAGATCGACACGCGCACCTGCCTC

[0371] CAGGACTTCAAGATTGATGCACCAGcTGCGGACAAGTCTACTGGGGTaGCCATGGTAT

[0372] GTATGCAGCTCGACCGGACGCTAGACGCCGTACGCATGGCTGGATTCTCCACCCACTA

[0373] TCGCATAGTGTCAGTGGAGGCGCACGTGATACCCgCAACAATACATGCACAGGCAGTT

[0374] GCTAGTATAGGCAACTCCACCCATCGCAGCGGGCCGGGCGCACCTGGAGATCTTGTG

[0375] GACGGATCCACGACGATCTTCTCGTATTCCGAATATGTGCGATGGCAATCTTTGATGTC

[0376] GAGCACACGCAAGGCTGCCCCCGGAGCAACATTAGTCGCGACGTTCCGACCTCAGA

[0377] ATCCTGTTTGGGTTGAGAACACCGCTAACGCACGGGCTGATGGTGCCATGTCGCTCCT

[0378] GGTTGGTAGTATTAGTGCGCAGAATCACACCTCAGCGACGTGGGTGTCGTTGAAGTAT

[0379] GTGATTCAGTTCCAGATGGACGTCGCCGTCTATTGACGGGCCGGGGACGCCCGTCCT

[0380] CCAGCTGGCTCGCGCCATTCTGAGGATATGGTGAGCAAGGGCGAGGAGCTGTTCACC

[0381] GGGGTGGTGCCCATCCTGGTCGAGCTGGACGGCGACGTAAACGGCCACAAGTTCAG

[0382] CGTGTCCGGCGAGGGCGAGGGCGATGCCACCTACGGCAAGCTGACCCTGAAGTTCAT

[0383] CTGCACCACCGGCAAGCTGCCCGTGCCCTGGCCCACCCTCGTGACCACCCTGACCTA

[0384] CGGCGTGCAGTGCTTCAGCCGCTACCCCGACCACATGAAGCAGCACGACTTCTTCAA

[0385] GTCCGCCATGCCCGAAGGCTACGTCCAGGAGCGCACCATCTTCTTCAAGGACGACGG

[0386] CAACTACAAGACCCGCGCCGAGGTGAAGTTCGAGGGCGACACCCTGGTGAACCGCA TCGAGCTGAAGGGCATCGACTTCAAGGAGGACGGCAACATCCTGGGGCACAAGCTG

[0387] GAGTACAACTACAACAGCCACAACGTCTATATCATGGCCGACAAGCAGAAGAACGGC

[0388] ATCAAGGTGAACTTCAAGATCCGCCACAACATCGAGGACGGCAGCGTGCAGCTCGCC

[0389] GACCACTACCAGCAGAACACCCCCATCGGCGACGGCCCCGTGCTGCTGCCCGACAAC

[0390] CACTACCTGAGCACCCAGTCCGCCCTGAGCAAAGACCCCAACGAGAAGCGCGATCA

[0391] CATGGTCCTGCTGGAGTTCGTGACCGCCGCCGGGATCACTCTCGGCATGGACGAGCT

[0392] GTACAAGCAGCCATTAGTGGAAGAGTTGCACAGGATGGAGCGATCTAGTGGGCCGTC

[0393] CCAGCGACCTAGGAGGGACGTCTCTGAGCAAATTCAACTGGCCCCGCCTCAGTCCGG

[0394] TTCTGAGGATAACAGTAAGGCCGGGTGGTCTCTGAGTGCCACCGCTGGAGCAGCCGC

[0395] CGGAACTGCTGCGGTGGTGGCGGGAGGCGTGGCGGCTGCCGAAGCCGTCGCAGGTC

[0396] TCGGTGCCGCTGCGGCGTCGGTGCCCACCGATAGTGTGCCATTGCTGGACATGTCGG

[0397] CGAGCACCTCGTCACTTGTGGAACCATCGCTGCAAGTGGTcGAAACCGACtTGGACGA

[0398] TGTCTCCTTGTGGGACGCGGCCGAGGAGGAATTGACCGCAGCCGCCGAGCAGACCT

[0399] CGCTCACCAGCATACCTGAAAGCCTGAAGACCACATGGCGCCAGTGGATTGAAGACG

[0400] AATGTCGACAaGGAATGATAAAGGAGGGGGTGAAGAATCGGTTTACAGAATCACTGA

[0401] ACCGCCCCGAAGCGCTTAACGCAGCCCGCACCTCTTGGTTCAGACGCTATGGAACCG

[0402] AGCCTACAGACGCTGAAGTTTTGCAGCGCCTTcTCGAGGACATCATCCGGTCGTAAGT

[0403] ACTGGTCACAGCAGGAGGTAAGACGTCCCGATTGGCAACTTTCGGAAGTACCCGGTG

[0404] AGGGGAAACGCGCCTAGGACCTGCTCGTTCGCCGTGGAACGCACCTGATGACTACTC

[0405] GAGCGACACTGCCGTTGGCTGTCACCCAGACAGTCACCTGAGCTTACACTTCCCGAG

[0406] GGGGGGGACATAATACCTCTCGGTACCGAACCATCTATAGGTAGCGTGCCCGCGCTAC

[0407] CGCCCC

[0408] Fusing eGFP into the C-terminal of ORF4 (ORF4 and eGFP are in frame): SEQ ID

[0409] NO:28 aggtcaacatggtggagcacGACACTCTCGTCTACTCCAAGAATATCAAAGATACAGTCTCAGAA

[0410] GACCAAAGGGCTATTGAGACTTTTCAACAAAGGGTAATATCGGGAAACCTCCTCGGA

[0411] TTCCATTGCCCAGCTATCTGTCACTTCATCAAAAGGACAGTAGAAAAGGAAGGTGGC

[0412] ACCTACAAATGCCATCATTGCGATAAAGGAAAGGCTATCGTTCAAGATGCCTCTGCCG

[0413] ACAGTGGTCCCAAAGATGGACCCCCACCCACGAGGAGCATCGTGGAAAAAGAAGAC

[0414] GTTCCAACCACGTCTTCAAAGCAAGTGGATTGATGTGATAACATGGTGGAGCACGAC

[0415] ACTCTCGTCTACTCCAAGAATATCAAAGATACAGTCTCAGAAGACCAAAGGGCTATTG AGACTTTTCAACAAAGGGTAATATCGGGAAACCTCCTCGGATTCCATTGCCCAGCTAT

[0416] CTGTCACTTCATCAAAAGGACAGTAGAAAAGGAAGGTGGCACCTACAAATGCCATCA

[0417] TTGCGATAAAGGAAAGGCTATCGTTCAAGATGCCTCTGCCGACAGTGGTCCCAAAGA

[0418] TGGACCCCCACCCACGAGGAGCATCGTGGAAAAAGAAGACGTTCCAACCACGTCTT

[0419] CAAAGCAAGTGGATTGATGTGATATCTCCACTGACGTAAGGGATGACGCACAATCCC

[0420] ACTATCCTTCGCAAGACCTTCCTCTATATAAGGAAGTTCATTTCATTTGGAGAGGGGGT

[0421] AAATAAATGGCTTGGCAAGCGATCAGACGTTTGTTTACCCGCAAGCAGCgtcCACGCG

[0422] CTGCTAGCAGCATCTCTGAGGAGATCTACGTGGGCGACTTCCCCATTGGAGTCACCCG

[0423] CGAGGTCCCAGCGCCACGAGTGGAGACCGTGGTGGCTGATGAGCTGAGTCCTGTGA

[0424] ACCGGGAGTCCATGATGTTGGATGACGACTGGGTCGCAGTGATCAACACCGCCAGAT

[0425] ACATGGAGGCTCAAGCTCAACTCAGGGAGCGCTTGGACGAGGTGGTGCAGGAGGAG

[0426] CTGCGCCAATGGGTCCTGGGAAACGTGGCTAGCCCCGCaGCCCGTCTAGCAGAGCaA

[0427] CTTCGCGTGCGTTTCGGCGCGCGCGCCCCCACGGCCGCTAAtGCGGTTATGGGAGAGC

[0428] GGTTCTTGCTGGACAAGGCAgCGGAGGCTGGAAACGACCACCTGGATGCCAGcAACC

[0429] TCGCAAGCTTGGCTGTGAATCTCTGGTTAGTGCGCACCGCTTGGGACCAGTCCGTCG

[0430] GGCTGGGTTTTCCGAGTGGGTGGTAACTGAGCTCCCCGGGGTGTCAAGGAGTTTCGA

[0431] ATGCCCTCCTGACGCTCCCGTAATCTGGCGTTCGCGGGCTGCCACCCCGATGAAGTAC

[0432] GGCAGGCGAACTGTGAACTTCAGCAATCTGATGGCTAATGATATTTAtAAGGTGCACG

[0433] CCAACTCGAAGGACAATCTGCTCAGGGGATTTTGTACCCGAGTGTGGCGATATAAGG

[0434] GCGCCGATCCGATCCCGTGTCTGCGGTCCCGACTGCCGCGATTTGAACGGGCGTTCC

[0435] GTGGGTTCCCGGTACGCCCCGTCTCGTATGATTCAGTCGTGGAGAACGCATTGCCCCG

[0436] TCGCAGAAAACTGTTACAGACGGCTGTGGAAAGCCTAAAAACGAAGCCTCTACAAC

[0437] TCGCCGATTCACATGTTAATACCTTTATCAAGGCTGAGAAGTTTTGTTGGAGTGCGAA

[0438] GCCTGACGCGGATCCTCGTCTAATACAGCCCCGCGGTAATCGCTTCCTTGTCTCACAC

[0439] GGCCGGTACATAAAGGCGATTGAGCCGATGGTCTATAAAGCACTGGGCCGACTCACC

[0440] GGACTTCCCATGGTGGCGAAGGGATTCAACGCTCGTGAAACAGCGGAGATCCTGCGC

[0441] AAGAAGTGGGACCGGTTTAGAGACCCCGTGTGCATATCTTTGGACGCCAGTCGCTTT

[0442] GATCAGCACGTCAGTGTGGACATGTTGAAGTTTACTCACCGCATATACCGACGATTCA

[0443] ATCGAGATCCGGAGTTTAAAATGATGTGTGATTGGCAATTACACAACACAGGCTTTGC

[0444] CTCCTGCCCAGAGGGGAGCTTCAAGTATAAAATCGCGGGAAGGCGAATGAGTGGAG

[0445] ATATGGATACGTCGCTGGGCAACTGCGTCATCATGTGTGGTCTAACATATGAATTATTT GGCCCCGGCGTGGAGATATTCAACAACGGTGACGATTGTTTAGTGATCGGCGAGCGT

[0446] CATGAGCTAGTGGACGCGCCCACTGTCCGCGAGCACTATAAGCAGTACGGTTTCCATG

[0447] TGGTCGAGGAAGACAGAGTTGATGTGTTCGAGCGCATCCTCTTTTGtCAAACTCAACC

[0448] TATCTGGGCCGAGGGATGGGTCATGTGCCGTCACCCACGGATGATGGCAAAGGATTT

[0449] GACCTATATTGGGCCACACACTGCTGATAGGGCTTGGCTCGATGCAATTGGACAGTGT

[0450] GGGATCGCTCTTGCTGATGGAGTGCCTATAATGTCGCGGTTCTACCACAATCTCATCGA

[0451] CACAAGTCACCGtAAAGCGAATATCACCAACAGTATGCTCTACTCATGCGGATTGACG

[0452] AATTTAGCGAAGCGCATGGAATATAAGGGACTGCCCATCTCAGACCGCTCTCGGTATT

[0453] CATTTTATCTGGGGTTCGGAGTCGAGCCGGATGTTCAGGTTGCGATAGAAGCCAGTAT

[0454] GGGCCCGCTGGGTAATATACAACGAATTGATGGCAAACTCTTCGAACAGACGATCAC

[0455] AAACTACGAGAGCGCTCTCGAAGCGGCGGAGAAGGGGTCGGGGCACGCGTGGAAG

[0456] CGTCCCGTCTCCCTTCCCATTCCCTAATCGCACACAGATCGACACGCGCACCTGCCTC

[0457] CAGGACTTCAAGATTGATGCACCAGcTGCGGACAAGTCTACTGGGGTaGCCATGGTAT

[0458] GTATGCAGCTCGACCGGACGCTAGACGCCGTACGCATGGCTGGATTCTCCACCCACTA

[0459] TCGCATAGTGTCAGTGGAGGCGCACGTGATACCCgCAACAATACATGCACAGGCAGTT

[0460] GCTAGTATAGGCAACTCCACCCATCGCAGCGGGCCGGGCGCACCTGGAGATCTTGTG

[0461] GACGGATCCACGACGATCTTCTCGTATTCCGAATATGTGCGATGGCAATCTTTGATGTC

[0462] GAGCACACGCAAGGCTGCCCCCGGAGCAACATTAGTCGCGACGTTCCGACCTCAGA

[0463] ATCCTGTTTGGGTTGAGAACACCGCTAACGCACGGGCTGATGGTGCCATGTCGCTCCT

[0464] GGTTGGTAGTATTAGTGCGCAGAATCACACCTCAGCGACGTGGGTGTCGTTGAAGTAT

[0465] GTGATTCAGTTCCAGATGGACGTCGCCGTCTATTGACGGGCCGGGGACGCCCGTCCT

[0466] CCAGCTGGCTCGCGCCATTCTGAGGATATGCAGCCATTAGTGGAAGAGTTGCACAGG

[0467] ATGGAGCGATCTAGTGGGCCGTCCCAGCGACCTAGGAGGGACGTCTCTGAGCAAATT

[0468] CAACTGGCCCCGCCTCAGTCCGGTTCTGAGGATAACAGTAAGGCCGGGTGGTCTCTG

[0469] AGTGCCACCGCTGGAGCAGCCGCCGGAACTGCTGCGGTGGTGGCGGGAGGCGTGGC

[0470] GGCTGCCGAAGCCGTCGCAGGTCTCGGTGCCGCTGCGGCGTCGGTGCCCACCGATAG

[0471] TGTGCCATTGCTGGACATGTCGGCGAGCACCTCGTCACTTGTGGAACCATCGCTGCA

[0472] AGTGGTcGAAACCGACtTGGACGATGTCTCCTTGTGGGACGCGGCCGAGGAGGAATT

[0473] GACCGCAGCCGCCGAGCAGACCTCGCTCACCAGCATACCTGAAAGCCTGAAGACCA

[0474] CATGGCGCCAGTGGATTGAAGACGAATGTCGACAaGGAATGATAAAGGAGGGGGTGA

[0475] AGAATCGGTTTACAGAATCACTGAACCGCCCCGAAGCGCTTAACGCAGCCCGCACCT CTTGGTTCAGACGCTATGGAACCGAGCCTACAGACGCTGAAGTTTTGCAGCGCCTTcT CGAGGACATCATCCGGTCGGTGAGCAAGGGCGAGGAGCTGTTCACCGGGGTGGTGC CCATCCTGGTCGAGCTGGACGGCGACGTAAACGGCCACAAGTTCAGCGTGTCCGGCG AGGGCGAGGGCGATGCCACCTACGGCAAGCTGACCCTGAAGTTCATCTGCACCACCG GCAAGCTGCCCGTGCCCTGGCCCACCCTCGTGACCACCCTGACCTACGGCGTGCAGT GCTTCAGCCGCTACCCCGACCACATGAAGCAGCACGACTTCTTCAAGTCCGCCATGC CCGAAGGCTACGTCCAGGAGCGCACCATCTTCTTCAAGGACGACGGCAACTACAAG ACCCGCGCCGAGGTGAAGTTCGAGGGCGACACCCTGGTGAACCGCATCGAGCTGAA GGGCATCGACTTCAAGGAGGACGGCAACATCCTGGGGCACAAGCTGGAGTACAACT ACAACAGCCACAACGTCTATATCATGGCCGACAAGCAGAAGAACGGCATCAAGGTGA ACTTCAAGATCCGCCACAACATCGAGGACGGCAGCGTGCAGCTCGCCGACCACTACC AGCAGAACACCCCCATCGGCGACGGCCCCGTGCTGCTGCCCGACAACCACTACCTGA GCACCCAGTCCGCCCTGAGCAAAGACCCCAACGAGAAGCGCGATCACATGGTCCTG CTGGAGTTCGTGACCGCCGCCGGGATCACTCTCGGCATGGACGAGCTGTACAAGTAA GTACTGGTCACAGCAGGAGGTAAGACGTCCCGATTGGCAACTTTCGGAAGTACCCGG TGAGGGGAAACGCGCCTAGGACCTGCTCGTTCGCCGTGGAACGCACCTGATGACTAC TCGAGCGACACTGCCGTTGGCTGTCACCCAGACAGTCACCTGAGCTTACACTTCCCG AGGGGGGGGACATAATACCTCTCGGTACCGAACCATCTATAGGTAGCGTGCCCGCGCT ACCGCCCC

[0476] The foregoing provides a proof of concept for how the cDNA constructs can be used to deliver a wide range of foreign (recombinant) nucleic acids into a plant.

[0477] By employing WULV infectious clone, this study uncovered that WULV can efficiently replicate and infect systemically wheat as its primary host and N. benlhamiana model plant in the absence of a helper virus. Moreover, WULV RNA seems very stable and can be freely mechanically transmitted.

Claims

CLAIMS:

1. An isolated cDNA clone of wheat umbra-like virus comprising a nucleic acid sequence encoding amino acid sequences SEQ ID NO:4 and SEQ ID NO:6, wherein said nucleic acid sequence is operably linked to a promoter for expression of said amino acid sequences in a host cell.

2. The cDNA clone of claim 1, further wherein said nucleic acid sequence comprises a first coding sequence encoding SEQ ID NO:4 and a second coding sequence encoding SEQ ID NO:6.

3. The cDNA clone of claim 2, wherein said nucleic acid sequence comprising a noncoding sequence between said first and second coding sequences.

4. The cDNA clone of claim 1, wherein said nucleic acid sequence comprises SEQ ID NO: 3 and SEQ ID NO 5.

6. The cDNA clone of claim 1, wherein said nucleic acid sequences further encodes amino acid sequences SEQ ID NO: 8 and / or 10.

7. The cDNA clone of claim 6, wherein said nucleic acid sequence comprises SEQ ID NO:7 and / or SEQ ID NO:9.

8. The cDNA clone of claim 1, wherein said nucleic acid sequence comprises SEQ ID NO: 1.

9. The cDNA clone of claim 1, wherein said nucleic acid sequence comprises SEQ ID NO:2.

10. The cDNA clone of claim 1, wherein said promoter comprises two promoter sequences.

11. The cDNA clone of claim 10, wherein said promoter comprises SEQ ID NO: 11.

12. The cDNA clone of claim 1, wherein said nucleic acid sequence comprising open reading frames (ORFs) 1, 2, 3, and 4.

13. The cDNA clone of claim 12, further comprising a foreign nucleic acid sequence inserted at a location selected from the group consisting of: between ORFs 1 and 2, in frame at the C- terminal of ORF3, between ORF3 and ORF4, and at the N- or C-terminal of ORF4.

14. The cDNA clone of claim 13, wherein said foreign nucleic acid is a guide RNA complementary to a target transcript for gene editing and / or silencing, or wherein said foreign nucleic acid is a fluorescent marker.

15. The cDNA clone of claim 13, wherein said foreign nucleic acid has a size of 700 nts or less.

16. An expression vector comprising a cDNA clone according to any one of claims 1-15.

17. An expression vector comprising a cDNA clone comprising SEQ ID NO: 1 or SEQ ID NO:2.

18. The expression vector of claim 17, wherein said vector comprises SEQ ID NO: 12.

19. A host cell comprising a cDNA clone according to any one of claims 1-15, or an RNA transcript thereof, or expression vector comprising the same.

20. A method of infecting a host plant with cDNA clone of wheat umbra-like virus or in vitro transcripts (RNAs) thereof, the method comprising introducing a cDNA clone according to any one of claims 1-15 or in vitro transcript thereof to a plant tissue, seeds, roots, leaves, stems, and / or seeds.21 . The method of claim 20, wherein said introducing step comprises agrobacterium-mediated transmission via a recombinant agrobacterium strain carrying said cDNA clone introduced to said plant.

22. The method of claim 20, wherein said introducing step comprising introducing the in vitro transcripts of said cDNA clone as free RNA to said plant.

23. The method of claim 20, wherein said cDNA clone comprises a foreign nucleic acid encoding for guide RNA complementary to a target transcript for gene editing and / or silencing in the plant or pest or pathogen of the plant.

24. The method of claim 20, wherein said cDNA clone comprises a foreign nucleic acid encoding an RNA target associated with a gene encoding for a plant characteristic, wherein said plant characteristic is vigorous growth, abundant foliage, longer primary roots, yield, height, and / or shoot water potential, pest resistance, drought tolerance, heat tolerance, salt tolerance, cold resistance, herbicide resistance, fungal resistance or reduced fungal susceptibility, viral resistance, and / or reduced viral susceptibility.

25. The method of claim 20, further comprising co-infecting the plant with a second plant virus.

26. The method of claim 25, further comprising collecting plant tissue samples and detecting symptoms of said second plant virus.

27. The method of claim 26, further comprising treating said plant with a candidate compound and detecting changes in said symptoms after treating said plant.

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