Rapid and specific detection of wheat spindle streak mosaic virus using RT-LAMP in durum wheat crude leaf extract.

To accurately determine the spread of any pathogen, including plant viruses, a quick, sensitive, cost-effective, point-of-care diagnostic assay is necessary. Wheat spindle streak mosaic virus (WSSMV) is a Bymovirus, transmitted by the plasmodiophorid Polymyxa graminis Led, which causes yellow mosaic and reduces the grain yield in wheat. Currently, detection protocols for WSSMV use ELISA or more sensitive PCR-based approaches requiring specialized laboratory and personnel. A protocol for reverse transcription loop mediated isothermal amplification (RT-LAMP) has been developed and optimized for the rapid detection of viruses using crude extracts from wheat leaves. The protocol was specific for WSSMV detection, while no reaction was observed with SBCMV or SBWMV, the non-target viruses transmitted by the same vector. The RT-LAMP assay was shown to be as sensitive as the one-step WSSMV specific RT-PCR. The RT-LAMP assay can be performed under field conditions using a portable instrument, and can help the actual spread of WSSMV, an aspect of this virus not yet well understood, to be explored.

Development and application of sugarcane streak mosaic virus vectors.

Sugarcane streak mosaic virus (SCSMV) is one of the major pathogens of sugarcane in the world. Molecular studies and disease management of SCSMV are hindered by the lack of efficient infectious clones. In this study, we successfully constructed Agrobacterium infiltration based infectious clone of SCSMV with different variants. Infectious clones of wild type SCSMV could efficiently infect Nicotiana benthamiana and sugarcane plants resulting in streak and mosaic symptoms on systemic leaves which were further confirmed with RT-PCR and serological assays. SCSMV variants of less adenylation displayed attenuated pathogenicity on N.benthamiana. SCSMV-based recombinant heterologous EGFP protein vector was also developed. The EGFP-tagged recombinant SCSMV could highly expressed in vegetative organs including roots. These infectious clones of SCSMV could be further developed for platform tools for both biotechnological studies and management of SCSMV disease.

Differential regulation of miRNAs involved in the susceptible and resistance responses of wheat cultivars to wheat streak mosaic virus and Triticum mosaic virus.

BACKGROUND: Wheat streak mosaic virus (WSMV) and Triticum mosaic virus (TriMV) are components of the wheat streak mosaic virus disease complex in the Great Plains region of the U.S.A. and elsewhere. Co-infection of wheat with WSMV and TriMV causes synergistic interaction with more severe disease symptoms compared to single infections. Plants are equipped with multiple antiviral mechanisms, of which regulation of microRNAs (miRNAs) is a potentially effective constituent. In this investigation, we have analyzed the total and relative expression of miRNA transcriptome in two wheat cultivars, Arapahoe (susceptible) and Mace (temperature-sensitive-resistant), that were mock-inoculated or inoculated with WSMV, TriMV, or both at 18 °C and 27 °C. RESULTS: Our results showed that the most abundant miRNA family among all the treatments was miRNA166, followed by 159a and 168a, although the order of the latter two changed depending on the infections. When comparing infected and control groups, twenty miRNAs showed significant upregulation, while eight miRNAs were significantly downregulated. Among them, miRNAs 9670-3p, 397-5p, and 5384-3p exhibited the most significant upregulation, whereas miRNAs 319, 9773, and 9774 were the most downregulated. The comparison of infection versus the control group for the cultivar Mace showed temperature-dependent regulation of these miRNAs. The principal component analysis confirmed that less abundant miRNAs among differentially expressed miRNAs were strongly correlated with the inoculated symptomatic wheat cultivars. Notably, miRNAs 397-5p, 398, and 9670-3p were upregulated in response to WSMV and TriMV infections, an observation not yet reported in this context. The significant upregulation of these three miRNAs was further confirmed with RT-qPCR analysis; in general, the RT-qPCR results were in agreement with our computational analysis. Target prediction analysis showed that the miRNAs standing out in our analysis targeted genes involved in defense response and regulation of transcription. CONCLUSION: Investigation into the roles of these miRNAs and their corresponding targets holds promise for advancing our understanding of the mechanisms of virus infection and possible manipulation of these factors for developing durable virus resistance in crop plants.

Mixed infection of ITPase-encoding potyvirid and secovirid in Mercurialis perennis: evidences for a convergent euphorbia-specific viral counterstrike.

BACKGROUND: In cellular organisms, inosine triphosphate pyrophosphatases (ITPases) prevent the incorporation of mutagenic deaminated purines into nucleic acids. These enzymes have also been detected in the genomes of several plant RNA viruses infecting two euphorbia species. In particular, two ipomoviruses produce replicase-associated ITPases to cope with high concentration of non-canonical nucleotides found in cassava tissues. METHOD: Using high-throughput RNA sequencing on the wild euphorbia species Mercurialis perennis, two new members of the families Potyviridae and Secoviridae were identified. Both viruses encode for a putative ITPase, and were found in mixed infection with a new partitivirid. Following biological and genomic characterization of these viruses, the origin and function of the phytoviral ITPases were investigated. RESULTS: While the potyvirid was shown to be pathogenic, the secovirid and partitivirid could not be transmitted. The secovirid was found belonging to a proposed new Comovirinae genus tentatively named "Mercomovirus", which also accommodates other viruses identified through transcriptome mining, and for which an asymptomatic pollen-associated lifestyle is suspected. Homology and phylogenetic analyses inferred that the ITPases encoded by the potyvirid and secovirid were likely acquired through independent horizontal gene transfer events, forming lineages distinct from the enzymes found in cassava ipomoviruses. Possible origins from cellular organisms are discussed for these proteins. In parallel, the endogenous ITPase of M. perennis was predicted to encode for a C-terminal nuclear localization signal, which appears to be conserved among the ITPases of euphorbias but absent in other plant families. This subcellular localization is in line with the idea that nucleic acids remain protected in the nucleus, while deaminated nucleotides accumulate in the cytoplasm where they act as antiviral molecules. CONCLUSION: Three new RNA viruses infecting M. perennis are described, two of which encoding for ITPases. These enzymes have distinct origins, and are likely required by viruses to circumvent high level of cytoplasmic non-canonical nucleotides. This putative plant defense mechanism has emerged early in the evolution of euphorbias, and seems to specifically target certain groups of RNA viruses infecting perennial hosts.

Distribution of Wheat-Infecting Viruses and Genetic Variability of Wheat Streak Mosaic Virus and Barley Stripe Mosaic Virus in Kazakhstan.

Wheat is an essential cereal crop for the economy and food safety of Kazakhstan. In the present work, a screening of wheat and barley from different regions of Kazakhstan was conducted using newly developed specific primers for reverse transcription PCR and loop-mediated isothermal amplification (LAMP) assays. In total, 82 and 19 of 256 samples of wheat and barley tested positive for wheat streak mosaic virus (WSMV) and barley stripe mosaic virus (BSMV), respectively. A phylogenetic analysis using two independent methods revealed that most of the analyzed isolates had a European origin. Molecular data on the distribution and diversity of cereal viruses in Kazakhstan were obtained for the first time and will help lay a foundation for the implementation of genetics and genomics in wheat phyto-epidemiology in the country.

Characterization of a New, Country Bean (Lablab purpureus) Lineage of Bean Common Mosaic Necrosis Virus.

Country bean (Lablab purpureus, family Fabaceae) is grown in subsistence agriculture in Bangladesh as a multipurpose crop for food, animal feed, and green manure. This study was undertaken to investigate the genetic diversity of bean common mosaic necrosis virus (BCMNV, genus Potyvirus, family Potyviridae) in country beans. Leaf samples from country beans showing yellowing, vein banding, and mosaic symptoms were collected during field surveys between 2015 and 2019 cropping seasons from farmers' fields in different geographic regions. These samples were tested by serological and molecular diagnostic assays for the presence of BCMNV. Virus-positive samples were subjected to high-throughput Illumina sequencing to generate near-complete genomes of BCMNV isolates. In pairwise comparisons, the polyprotein sequences of BCMNV isolates from Bangladesh showed greater than 98% identities among themselves and shared less than 84% sequence identity at the nucleotide level with virus isolates reported from other countries. In the phylogenetic analysis, BCMNV isolates from Bangladeshi country beans formed a separate clade from virus isolates reported from common beans in other countries in the Americas, Africa, Europe, and from East Timor. Grow-out studies showed seed-to-seedling transmission of BCMNV, implying a possible seedborne nature of the virus in country beans.

The HC-Pro cistron of Triticum mosaic virus is dispensable for systemic infection in wheat but is required for symptom phenotype and efficient genome amplification.

Triticum mosaic virus (TriMV), the type species of the genus Poacevirus in the family Potyviridae, is an economically important wheat curl mite-transmitted wheat-infecting virus in the Great Plains region of the USA. In this study, the functional genomics of helper component-proteinase (HC-Pro) encoded by TriMV was examined using a reverse genetics approach. TriMV with complete deletion of HC-Pro cistron elicited systemic infection in wheat, indicating that HC-Pro cistron is dispensable for TriMV systemic infection. However, TriMV lacking HC-Pro caused delayed systemic infection with mild symptoms that resulted in little or no stunting of plants with a significant reduction in the accumulation of genomic RNA copies and coat protein (CP). Sequential deletion mutagenesis from the 5' end of HC-Pro cistron in the TriMV genome revealed that deletions within amino acids 3 to 25, except for amino acids 3 and 4, elicited mild symptoms with reduced accumulation of genomic RNA and CP. Surprisingly, TriMV with deletion of amino acids 3 to 50 or 3 to 125 in HC-Pro elicited severe symptoms with a substantial increase in genomic RNA copies but a drastic reduction in CP accumulation. Additionally, TriMV with heterologous HC-Pro from other potyvirids produced symptom phenotype and genomic RNA accumulation similar to that of TriMV without HC-Pro, suggesting that HC-Pros of other potyvirids were not effective in complementing TriMV in wheat. Our data indicate that HC-Pro is expendable for replication of TriMV but is required for efficient viral genomic RNA amplification and symptom development. The availability of TriMV with various deletions in the HC-Pro cistron will facilitate the examination of the requirement of HC-Pro for wheat curl mite transmission.

Alfalfa vein mottling virus, a novel potyvirid infecting Medicago sativa L.

BACKGROUND: We have recently identified a novel virus detected in alfalfa seed material. The virus was tentatively named alfalfa-associated potyvirus 1, as its genomic fragments bore similarities with potyvirids. In this study, we continued investigating this novel species, expanding information on its genomic features and biological characteristics. METHODS: This research used a wide range of methodology to achieve end results: high throughput sequencing, bioinformatics tools, reverse transcription-polymerase chain reactions, differential diagnostics using indicator plants, virus purification, transmission electron microscopy, and others. RESULTS: In this study, we obtained a complete genome sequence of the virus and classified it as a tentative species in the new genus, most closely related to the members of the genus Ipomovirus in the family Potyviridae. This assumption is based on the genome sequence and structure, phylogenetic relationships, and transmission electron microscopy investigations. We also demonstrated its mechanical transmission to the indicator plant Nicotiana benthamiana and to the natural host Medicago sativa, both of which developed characteristic symptoms therefore suggesting a pathogenic nature of the disease. CONCLUSIONS: Consistent with symptomatology, the virus was renamed to alfalfa vein mottling virus. A name Alvemovirus was proposed for the new genus in the family Potyviridae, of which alfalfa vein mottling virus is a tentative member.

Whitefly-Transmitted Viruses of Cucurbits in the Southern United States.

Cucurbits are economically important crops that are widely cultivated in many parts of the world, including the southern US. In recent years, higher temperatures have favored the rapid build-up of whiteflies in the fall-grown cucurbits in this region. As a result, whitefly-transmitted viruses (WTVs) have severely impacted the marketable yield of cucurbits. In this review, we discuss three major groups of WTVs negatively impacting cucurbit cultivation in the southern US, including begomoviruses, criniviruses, and ipomoviruses. Here, we discuss the available information on the biology, epidemiology and advances made toward detecting and managing these viruses, including sources of resistance and cultural practices.

A papain-like cysteine protease-released small signal peptide confers wheat resistance to wheat yellow mosaic virus.

Wheat yellow mosaic virus (WYMV), a soil-borne pathogen, poses a serious threat to global wheat production. Here, we identify a WYMV resistance gene, TaRD21A, that belongs to the papain-like cysteine protease family. Through genetic manipulation of TaRD21A expression, we establish its positive role in the regulation of wheat to WYMV resistance. Furthermore, our investigation shows that the TaRD21A-mediated plant antiviral response relies on the release of a small peptide catalyzed by TaRD21A protease activity. To counteract wheat resistance, WYMV-encoded nuclear inclusion protease-a (NIa) suppress TaRD21A activity to promote virus infection. In resistant cultivars, a natural variant of TaRD21A features a glycine-to-threonine substitution and this substitution enables the phosphorylation of threonine, thereby weakening the interaction between NIa and TaRD21A, reinforcing wheat resistance against WYMV. Our study not only unveils a WYMV resistance gene but also offers insights into the intricate mechanisms underpinning resistance against WYMV.

Sugarcane streak mosaic virus P1 protein inhibits unfolded protein response through direct suppression of bZIP60U splicing.

The unfolded protein response (UPR) is a cell-designated strategy that maintains the balance of protein folding in the endoplasmic reticulum (ER). UPR features a network of signal transduction pathways that reprogram the transcription, mRNA translation, and protein post-translational modification to relieve the ER stresses from unfolded/misfolded proteins. Infection with plant viruses can induce the UPR, and activated UPR often promotes plant viral infections in turn. However, the mechanism used by plant viruses to balance UPR and achieve robust infection remain largely unknown. In this study, P1SCSMV was identified as a virus-encoded RNA silencing suppressor (VSR). Heterologous overexpression of P1SCSMV via potato virus X (PVX) was found lead to programmed cell death (PCD) in Nicotiana benthamiana. Furthermore, P1SCSMV was also found to inhibit the PVX infection-triggered UPR by downregulating UPR-related genes and directly induced the distortion and collapse of the ER polygonal meshes on PVX-P1SCSMV infected N. benthamiana. Moreover, self-interaction, VSR activity, UPR inhibition, and cell death phenotype of P1SCSMV were also found to be dependent on its bipartite nuclear localization signal (NLS) (251RKRKLFPRIPLK262). P1SCSMV was found to directly bind to the stem-loop region of NbbZIP60U via its NLS and inhibit the UPR pathways, ultimately resulting in a PCD phenotype in PVX-P1SCSMV infected N. benthamiana leaves. This study also revealed the balancing role of potyviruses encoded P1SCSMV in the UPR pathway to achieve robust viral infection. This may represent a novel virulence strategy for plant viruses.

Complete genome sequence of a divergent strain of cardamom mosaic virus.

Cardamom mosaic virus (CdMV; genus Macluravirus), which causes mosaic (katte) disease in cardamom, is a highly variable member of the family Potyviridae. So far, the complete genome sequence of one isolate from Karnataka (KS) has been reported. In the present study, we determined the complete genome sequence of a CdMV isolate from Kerala (KI) and the complete CP gene sequences of nine isolates of CdMV from Kerala, Karnataka, and Tamil Nadu, India. The complete genome of CdMV (KI) consists of 8255 nucleotides (nt) with two open reading frames (ORFs). The large ORF, potentially coding for a polyprotein of 2638 amino acids (aa), is further processed into nine mature proteins at eight cleavage sites. The second ORF, PIPO (pretty interesting Potyviridae ORF) starting with a C(A)6 motif, encodes a small protein of 56 aa. The viral genome contains an additional 13 nt in the 5' untranslated region (UTR) and 6 nt in the CP gene, as well as a deletion of 13 nt at the 3' UTR in comparison to the KS isolate of CdMV. The complete viral genome and polyprotein share 76% and 85% sequence identity with the KS isolate of CdMV, indicating that the present isolate is highly divergent from the KS isolate. Sequencing and analysis of the CP sequences of 16 CdMV isolates from different regions revealed high heterogeneity among them, suggesting that they should be considered members of more than one species.

Molecular characterization of a divergent genetic variant of wheat Eqlid mosaic virus from a Texas wheat field.

Investigations conducted during the spring 2020 season to diagnose the associated viral agent of a severe mosaic disease of wheat in a Texas Panhandle field revealed the presence of wheat Eqlid mosaic virus (WEqMV; genus Tritimovirus, family Potyviridae) in the analyzed samples. The complete genome sequences of two WEqMV isolates were determined, and each was found to be 9,634 nucleotides (nt) in length (excluding the polyA tail) and to contain 5' and 3' untranslated regions of 135 nt and 169 nt, respectively, based on rapid amplification of cDNA ends (RACE) assays. Both sequences contained an open reading frame (ORF) of 9,330 nt encoding a polyprotein of 3,109 amino acids (aa). The ORF sequences of the two isolates were 100% identical to each other, but only 74.7% identical to that of the exemplar WEqMV-Iran isolate, with 85.7% aa sequence identity in the encoded polyprotein. The Texas WEqMV isolates also diverged significantly from WEqMV-Iran in the individual proteins at the nt and aa levels. This is the first report of WEqMV in the United States and the first report of this virus outside of Iran, indicating an expansion of its geographical range.

Seed Transmission of Wheat Streak Mosaic Virus and Triticum Mosaic Virus in Differentially Resistant Wheat Cultivars.

Wheat streak mosaic virus (WSMV) and Triticum mosaic virus (TriMV) are important viral pathogens of wheat in the Great Plains. These viruses individually or in mixed infections with High Plains wheat mosaic virus cause a devastating wheat streak mosaic (WSM) disease. Although seed transmission of WSMV has been studied, no information is currently available on that of TriMV. Furthermore, no study has explored the implications of mixed infections of WSMV and TriMV on seed transmission of one or both viruses. To study both aspects, seeds from differentially resistant field-grown wheat plants (cv. TAM 304 (susceptible), Joe (WSMV resistant, Wsm2 gene), and Breakthrough (BT) (WSMV and TriMV resistant, Wsm1 gene)) showing characteristic WSM symptoms were collected and analyzed to quantify both viruses using qRT-PCR. The percentage of seeds tested positive for WSMV or TriMV individually and in mixed infection varied with cultivar and virus combinations; 13% of TAM 304 seeds tested positive for WSMV, followed by 8% of BT and 4% of Joe seeds. Similarly, TriMV was detected in 12% of BT seeds, followed by 11% of TAM 304 and 8% of Joe seeds. Lastly, mixed infection was detected in 7% of TAM 304 seeds, followed by 4% in BT, and 2% in Joe. Dissection of field-collected seeds into three parts, embryo, endosperm, and seed coat, revealed both WSMV and TriMV accumulated only in the seed coat. Consistent with seeds, percent infection of WSMV or TriMV in the plants that emerged from infected seeds in each treatment varied with cultivar and virus combinations (WSMV: BT 3%; Joe 2%; TAM 304 9%; TriMV: BT 7%; Joe 8%; and TAM 304 10%). Plants infected with mixed viruses showed more pronounced WSM symptoms compared to individual infections. However, both viruses were present only in a few plants (BT: 2%, Joe: 1%, and TAM 304: 4%). Taken together, this study showed that TriMV was transmitted vertically at a higher frequency than WSMV in resistant cultivars, and the seed transmission of TriMV with WSMV increased the virulence of both pathogens (measured via WSM symptom severity) in the emerged plants. Furthermore, Wsm1 and Wsm2 genes considerably reduced WSMV transmission via infected seeds. However, no such effects were observed on TriMV, especially in progeny plants. These results reiterated the importance of planting clean seeds and highlighted the immediate need to identify/develop new sources of TriMV resistance to effectively manage the recurring WSM epidemic.

P3 and NIa-Pro of Turnip Mosaic Virus Are Independent Elicitors of Superinfection Exclusion.

Superinfection exclusion (SIE) is an antagonistic interaction between identical or closely related viruses in host cells. Previous studies by us and others led to the hypothesis that SIE was elicited by one or more proteins encoded in the genomes of primary viruses. Here, we tested this hypothesis using Turnip mosaic virus (TuMV), a member of the genus Potyvirus of the family Potyviridae, with significant economic consequences. To this end, individual TuMV-encoded proteins were transiently expressed in the cells of Nicotiana benthamiana leaves, followed by challenging them with a modified TuMV expressing the green fluorescent protein (TuMV-GFP). Three days after TuMV-GFP delivery, these cells were examined for the replication-dependent expression of GFP. Cells expressing TuMV P1, HC-Pro, 6K1, CI, 6K2, NIa-VPg, NIb, or CP proteins permitted an efficient expression of GFP, suggesting that these proteins failed to block the replication of a superinfecting TuMV-GFP. By contrast, N. benthamiana cells expressing TuMV P3 or NIa-Pro did not express visible GFP fluorescence, suggesting that both of them could elicit potent SIE against TuMV-GFP. The SIE elicitor activity of P3 and NIa-Pro was further confirmed by their heterologous expression from a different potyvirus, potato virus A (PVA). Plants systemically infected with PVA variants expressing TuMV P3 or NIa-Pro blocked subsequent infection by TuMV-GFP. A +1-frameshift mutation in P3 and NIa-Pro cistrons facilitated superinfection by TuMV-GFP, suggesting that the P3 and NIa-Pro proteins, but not the RNA, are involved in SIE activity. Additionally, deletion mutagenesis identified P3 amino acids 3 to 200 of 352 and NIa-Pro amino acids 3 to 40 and 181 to 242 of 242 as essential for SIE elicitation. Collectively, our study demonstrates that TuMV encodes two spatially separated proteins that act independently to exert SIE on superinfecting TuMV. These results lay the foundation for further mechanistic interrogations of SIE in this virus.

CryoEM and stability analysis of virus-like particles of potyvirus and ipomovirus infecting a common host.

Sweet potato feathery mottle virus (SPFMV) and Sweet potato mild mottle virus (SPMMV) are members of the genera Potyvirus and Ipomovirus, family Potyviridae, sharing Ipomoea batatas as common host, but transmitted, respectively, by aphids and whiteflies. Virions of family members consist of flexuous rods with multiple copies of a single coat protein (CP) surrounding the RNA genome. Here we report the generation of virus-like particles (VLPs) by transient expression of the CPs of SPFMV and SPMMV in the presence of a replicating RNA in Nicotiana benthamiana. Analysis of the purified VLPs by cryo-electron microscopy, gave structures with resolutions of 2.6 and 3.0 Å, respectively, showing a similar left-handed helical arrangement of 8.8 CP subunits per turn with the C-terminus at the inner surface and a binding pocket for the encapsidated ssRNA. Despite their similar architecture, thermal stability studies reveal that SPMMV VLPs are more stable than those of SPFMV.

Transmission, localization, and infectivity of seedborne maize chlorotic mottle virus.

Maize lethal necrosis is a destructive virus disease of maize caused by maize chlorotic mottle virus (MCMV) in combination with a virus in the family Potyviridae. Emergence of MLN is typically associated with the introduction of MCMV or its vectors and understanding its spread through seed is critical for disease management. Previous studies suggest that although MCMV is detected on seed, the seed transmission rate of this virus is low. However, mechanisms influencing its transmission are poorly understood. Elucidating these mechanisms is crucial for informing strategies to prevent spread on contaminated seed. In this study, we evaluated the rate of MCMV seed transmission using seed collected from plants that were artificially inoculated with MCMV isolates from Hawaii and Kenya. Grow-out tests indicated that MCMV transmission through seed was rare, with a rate of 0.004% among the more than 85,000 seed evaluated, despite detection of MCMV at high levels in the seed lots. To understand factors that limit transmission from seed, MCMV distribution in seed tissues was examined using serology and immunolocalization. The virus was present at high levels in maternal tissues, the pericarp and pedicel, but absent from filial endosperm and embryo seed tissues. The ability to transmit MCMV from seed to uninfected plants was tested to evaluate virus viability. Transmission was negatively associated with both seed maturity and moisture content. Transmission of MCMV from infested seed dried to less than 15% moisture was not detected, suggesting proper handling could be important for minimizing spread of MCMV through seed.

Differential Seasonal Prevalence of Yellowing Viruses Infecting Melon Crops in Southern California and Arizona Determined by Multiplex RT-PCR and RT-qPCR.

Viruses transmitted by the whitefly (Bemisia tabaci) are an increasing threat to cucurbit production in the southwestern United States and many other cucurbit production regions of the world. The crinivirus cucurbit yellow stunting disorder virus (CYSDV) has severely impacted melon production in California and Arizona since its 2006 introduction to the region. Within the past few years, another crinivirus, cucurbit chlorotic yellows virus (CCYV), and the whitefly-transmitted ipomovirus squash vein yellowing virus (SqVYV) were found infecting melon plants in California's Imperial Valley. CYSDV, CCYV, and an aphid-transmitted polerovirus, cucurbit aphid-borne yellows virus (CABYV), occur together in the region and produce identical yellowing symptoms on cucurbit plants. Mixed infections of these four viruses in the Sonoran Desert and other regions pose challenges for disease management and efforts to develop resistant varieties. A multiplex single-step RT-PCR method was developed that differentiates among these viruses, and this was used to determine the prevalence and distribution of the viruses in melon samples from fields in the Sonoran Desert melon production region of California and Arizona during the spring and fall melon seasons from 2019 through 2021. TaqMan probes were developed, optimized, and applied in a single-step multiplex RT-qPCR to quantify titers of these four viruses in plant samples, which frequently carry mixed infections. Results of the multiplex RT-PCR analysis demonstrated that CYSDV is the predominant virus during the fall, whereas CCYV was by far the most prevalent virus during the spring each year. Multiplex RT-qPCR was used to evaluate differential accumulation and spatiotemporal distribution of viruses within plants and suggested differences in competitive accumulation of CCYV and CYSDV within melon. This study provides the first official report of SqVYV in Arizona and offers an efficient method for virus detection and quantification for breeding and disease management in areas impacted by cucurbit yellowing viruses.

A Zinc Finger Motif in the P1 N Terminus, Highly Conserved in a Subset of Potyviruses, Is Associated with the Host Range and Fitness of Telosma Mosaic Virus.

P1 is the first protein translated from the genomes of most viruses in the family Potyviridae, and it contains a C-terminal serine-protease domain that cis-cleaves the junction between P1 and HCPro in most cases. Intriguingly, P1 is the most divergent among all mature viral factors, and its roles during viral infection are still far from understood. In this study, we found that telosma mosaic virus (TelMV, genus Potyvirus) in passion fruit, unlike TelMV isolates present in other hosts, has two stretches at the P1 N terminus, named N1 and N2, with N1 harboring a Zn finger motif. Further analysis revealed that at least 14 different potyviruses, mostly belonging to the bean common mosaic virus subgroup, encode a domain equivalent to N1. Using the newly developed TelMV infectious cDNA clones from passion fruit, we demonstrated that N1, but not N2, is crucial for viral infection in both Nicotiana benthamiana and passion fruit. The regulatory effects of N1 domain on P1 cis cleavage, as well as the accumulation and RNA silencing suppression (RSS) activity of its cognate HCPro, were comprehensively investigated. We found that N1 deletion decreases HCPro abundance at the posttranslational level, likely by impairing P1 cis cleavage, thus reducing HCPro-mediated RSS activity. Remarkably, disruption of the Zn finger motif in N1 did not impair P1 cis cleavage and HCPro accumulation but severely debilitated TelMV fitness. Therefore, our results suggest that the Zn finger motif in P1s plays a critical role in viral infection that is independent of P1 protease activity and self-release, as well as HCPro accumulation and silencing suppression. IMPORTANCE Viruses belonging to the family Potyviridae represent the largest group of plant-infecting RNA viruses, including a variety of agriculturally and economically important viral pathogens. Like all picorna-like viruses, potyvirids employ polyprotein processing as the gene expression strategy. P1, the first protein translated from most potyvirid genomes, is the most variable viral factor and has attracted great scientific interest. Here, we defined a Zn finger motif-encompassing domain (N1) at the N terminus of P1 among diverse potyviruses phylogenetically related to bean common mosaic virus. Using TelMV as a model virus, we demonstrated that the N1 domain is key for viral infection, as it is involved both in regulating the abundance of its cognate HCPro and in an as-yet-undefined key function unrelated to protease processing and RNA silencing suppression. These results advance our knowledge of the hypervariable potyvirid P1s and highlight the importance for infection of a previously unstudied Zn finger domain at the P1 N terminus.

All eggs in one basket: How potyvirus infection is controlled at a single cap-independent translation event.

Regulation of protein synthesis is a strong determinant of potyviral pathogenicity. The Potyviridae family is the largest family of plant-infecting positive sense RNA viruses. Similar to the animal-infecting Picornaviridae family, the potyviral RNA genome lacks a 5' cap, and instead has a viral protein (VPg) linked to its 5' end. Potyviral genomes are mainly translated into one large polyprotein relying on a single translation event to express all their protein repertoire. In the absence of the 5' cap, the Potyviridae family depends on cis-acting elements in their 5' untranslated regions (UTR) to recruit the translation machinery. In this review, we summarize the diverse 5'UTR-driven, cap-independent translation mechanisms employed by the Potyviridae family including scanning-dependent mechanism, internal initiation, and the stimulatory role of the VPg. These mechanisms have direct implications on potyviral pathogenicity, including host range specificity and resistance. Finally, we discuss how these viral strategies could not only inform new avenues for engineering and/or breeding for crop resistance but would also provide opportunities for the development of biotechnological tools for large-scale protein production in plant systems.