Rubisco small subunit (RbCS) is co-opted by potyvirids as the scaffold protein in assembling a complex for viral intercellular movement.

Plant viruses must move through plasmodesmata (PD) to complete their life cycles. For viruses in the Potyviridae family (potyvirids), three viral factors (P3N-PIPO, CI, and CP) and few host proteins are known to participate in this event. Nevertheless, not all the proteins engaging in the cell-to-cell movement of potyvirids have been discovered. Here, we found that HCPro2 encoded by areca palm necrotic ring spot virus (ANRSV) assists viral intercellular movement, which could be functionally complemented by its counterpart HCPro from a potyvirus. Affinity purification and mass spectrometry identified several viral factors (including CI and CP) and host proteins that are physically associated with HCPro2. We demonstrated that HCPro2 interacts with both CI and CP in planta in forming PD-localized complexes during viral infection. Further, we screened HCPro2-associating host proteins, and identified a common host protein in Nicotiana benthamiana-Rubisco small subunit (NbRbCS) that mediates the interactions of HCPro2 with CI or CP, and CI with CP. Knockdown of NbRbCS impairs these interactions, and significantly attenuates the intercellular and systemic movement of ANRSV and three other potyvirids (turnip mosaic virus, pepper veinal mottle virus, and telosma mosaic virus). This study indicates that a nucleus-encoded chloroplast-targeted protein is hijacked by potyvirids as the scaffold protein to assemble a complex to facilitate viral movement across cells.

MdASMT9-mediated melatonin biosynthesis enhances basal thermotolerance in apple plants.

High temperatures negatively impact the yield and quality of fruit crops. Exogenous melatonin (MT) application has been shown to enhance heat tolerance, but the response of endogenous MT to heat stress, particularly in perennial fruit trees, remains unclear. The present study investigated the effects of high temperatures on transgenic apple plants overexpressing the MT biosynthesis gene N-acetylserotonin methyltransferase 9 (MdASMT9). Endogenous MT protected transgenic plants from heat stress by increasing antioxidant enzyme activity and scavenging reactive oxygen species (ROS), and protecting the chloroplasts from damage. Application of MT and overexpression of MdASMT9 also reduced abscisic acid accumulation through promoting MdWRKY33-mediated transcriptional inhibition of MdNCED1 and MdNCED3, thus inducing stomatal opening for better heat dissipation. Furthermore, MT-enhanced autophagic activity through promoting MdWRKY33-mediated transcriptional enhancement of MdATG18a under heat stress. These findings provide new insights into the regulation of endogenous MT and its role in improving basal thermotolerance in perennial fruit trees.

Development of cassava common mosaic virus-based vector for protein expression and gene editing in cassava.

BACKGROUND: Plant virus vectors designed for virus-mediated protein overexpression (VOX), virus-induced gene silencing (VIGS), and genome editing (VIGE) provide rapid and cost-effective tools for functional genomics studies, biotechnology applications and genome modification in plants. We previously reported that a cassava common mosaic virus (CsCMV, genus Potexvirus)-based VIGS vector was used for rapid gene function analysis in cassava. However, there are no VOX and VIGE vectors available in cassava. RESULTS: In this study, we developed an efficient VOX vector (CsCMV2-NC) for cassava by modifying the CsCMV-based VIGS vector. Specifically, the length of the duplicated putative subgenomic promoter (SGP1) of the CsCMV CP gene was increased to improve heterologous protein expression in cassava plants. The modified CsCMV2-NC-based VOX vector was engineered to express genes encoding green fluorescent protein (GFP), bacterial phytoene synthase (crtB), and Xanthomonas axonopodis pv. manihotis (Xam) type III effector XopAO1 for viral infection tracking, carotenoid biofortification and Xam virulence effector identification in cassava. In addition, we used CsCMV2-NC to deliver single guide RNAs (gMePDS1/2) targeting two loci of the cassava phytoene desaturase gene (MePDS) in Cas9-overexpressing transgenic cassava lines. The CsCMV-gMePDS1/2 efficiently induced deletion mutations of the targeted MePDS with the albino phenotypes in systemically infected cassava leaves. CONCLUSIONS: Our results provide a useful tool for rapid and efficient heterologous protein expression and guide RNA delivery in cassava. This expands the potential applications of CsCMV-based vector in gene function studies, biotechnology research, and precision breeding for cassava.

Cotyledon peeling method for passion fruit protoplasts: a versatile cell system for transient gene expression in passion fruit (Passiflora edulis).

Passion fruit (Passiflora edulis) is a perennial evergreen vine that grows mainly in tropical and subtropical regions due to its nutritional, medicinal and ornamental values. However, the molecular biology study of passion fruit is extremely hindered by the lack of an easy and efficient method for transformation. The protoplast transformation system plays a vital role in plant regeneration, gene function analysis and genome editing. Here, we present a new method ('Cotyledon Peeling Method') for simple and efficient passion fruit protoplast isolation using cotyledon as the source tissue. A high yield (2.3 × 107 protoplasts per gram of fresh tissues) and viability (76%) of protoplasts were obtained upon incubation in the enzyme solution [1% (w/v) cellulase R10, 0.25% (w/v) macerozyme R10, 0.4 M mannitol, 10 mM CaCl2, 20 mM KCl, 20 mM MES and 0.1% (w/v) BSA, pH 5.7] for 2 hours. In addition, we achieved high transfection efficiency of 83% via the polyethylene glycol (PEG)-mediated transformation with a green fluorescent protein (GFP)-tagged plasmid upon optimization. The crucial factors affecting transformation efficiency were optimized as follows: 3 µg of plasmid DNA, 5 min transfection time, PEG concentration at 40% and protoplast density of 100 × 104 cells/ml. Furthermore, the established protoplast system was successfully applied for subcellular localization analysis of multiple fluorescent organelle markers and protein-protein interaction study. Taken together, we report a simple and efficient passion fruit protoplast isolation and transformation system, and demonstrate its usage in transient gene expression for the first time in passion fruit. The protoplast system would provide essential support for various passion fruit biology studies, including genome editing, gene function analysis and whole plant regeneration.

The nucleoprotein of influenza A virus inhibits the innate immune response by inducing mitophagy.

Mitophagy is a form of autophagy that plays a key role in maintaining the homeostasis of functional mitochondria in the cell. Viruses have evolved various strategies to manipulate mitophagy to escape host immune responses and promote virus replication. In this study, the nucleoprotein (NP) of H1N1 virus (PR8 strain) was identified as a regulator of mitophagy. We revealed that NP-mediated mitophagy leads to the degradation of the mitochondria-anchored protein MAVS, thereby blocking MAVS-mediated antiviral signaling and promoting virus replication. The NP-mediated mitophagy is dependent on the interaction of NP with MAVS and the cargo receptor TOLLIP. Moreover, Y313 of NP is a key residue for the MAVS-NP interaction and NP-mediated mitophagy. The NPY313F mutation significantly attenuates the virus-induced mitophagy and the virus replication in vitro and in vivo. Taken together, our findings uncover a novel mechanism by which the NP of influenza virus induces mitophagy to attenuate innate immunity.Abbreviations: ACTB: actin beta; ATG7: autophagy related 7; ATG12: autophagy related 12; CCCP: carbonyl cyanide 3-chlorophenyl hydrazone; co-IP: co-immunoprecipitation; COX4/COXIV: cytochrome c oxidase subunit 4; DAPI: 4',6-diamidino-2-phenylindole, dihydrochloride; EID50: 50% egg infective dose; GAPDH: glyceraldehyde-3-phosphate dehydrogenase; GFP: green fluorescent protein; HEK: human embryonic kidney; hpi: hours post-infection; IAV: influenza A virus; IFN: interferon; IP: immunoprecipitation; LAMP1: lysosomal associated membrane protein 1; MAP1LC3/LC3: microtubule associated protein 1 light chain 3; MAVS: mitochondrial antiviral signaling protein; Mdivi-1: mitochondrial division inhibitor 1; MLD50: 50% mouse lethal dose; MOI: multiplicity of infection; NBR1: NBR1 autophagy cargo receptor; NP: nucleoprotein; PB1: basic polymerase 1; RFP: red fluorescent protein; RIGI: RNA sensor RIG-I; RIGI-N: RIGI-CARD; SeV: Sendai virus; SQSTM1/p62: sequestosome 1; TIMM23: translocase of inner mitochondrial membrane 23; TOLLIP: toll interacting protein; TOMM20: translocase of outer mitochondrial membrane 20; TUBA: tubulin alpha; Vec: empty vector; vRNP: viral ribonucleoprotein.

Direct leaf-peeling method for areca protoplasts: a simple and efficient system for protoplast isolation and transformation in areca palm (Areca catechu).

BACKGROUND: Areca palm (Areca catechu) is a woody perennial plant of both economical and medicinal importance grown in tropical and subtropical climates. Yet, the molecular biology study of areca palm is extremely impeded by its unavailability of a transformation method. An efficient protoplast isolation and transformation system could be highly desirable to overcome this barrier. RESULTS: Here, we described a simple and efficient method for protoplast isolation and transformation from the perennial plant areca palm. A high yield of protoplasts (2.5 × 107 protoplasts per gram of fresh leaf tissues) was obtained from the fresh light green leaflet from the newly-emerged leaf digested overnight in the enzyme solution [2% (w/v) cellulase R10, 0.5% (w/v) macerozyme R10, 0.7 M mannitol, 10 mM CaCl2, 20 mM KCl, 20 mM MES and 0.1% (w/v) BSA, pH 5.7] by the direct leaf-peeling method. The isolated areca protoplasts maintain viability of 86.6% and have been successfully transformed with a green fluorescent protein (GFP)-tagged plasmid (pGreen0029-GFP, 6.0 kb) via the polyethylene glycol (PEG)-mediated transformation. Moreover, the mannitol concentration (optimal: 0.7 M) was determined as a key factor affecting areca protoplast isolation. We also demonstrated that the optimal density of areca protoplast for efficient transformation was at 1.0-1.5 × 106 cells/ml. With the optimization of transformation parameters, we have achieved a relatively high transformation efficiency of nearly 50%. CONCLUSION: We have established the first efficient protocol for the high-yield isolation and transformation of areca palm protoplasts. This method shall be applied in various biological studies of areca palm, such as gene function analysis, genome editing, protein trafficking and localization and protein-protein interaction. In addition, the protoplast system offers a great genetic transformation approach for the woody perennial plant-areca palm. Moreover, the established platform may be applied in protoplast isolation and transformation for other important species in the palm family, including oil palm and coconut.

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.

The Single Distinct Leader Protease Encoded by Alpinia oxyphylla Mosaic Virus (Genus Macluravirus) Suppresses RNA Silencing Through Interfering with Double-Stranded RNA Synthesis.

The genomic 5'-terminal regions of viruses in the family Potyviridae (potyvirids) encode two types of leader proteases: serine-protease (P1) and cysteine-protease (HCPro), which differ greatly in the arrangement and sequence composition among inter-genus viruses. Most potyvirids have the same tandemly arranged P1 and HCPro, whereas viruses in the genus Macluravirus encode a single distinct leader protease, a truncated version of HCPro with yet-unknown functions. We investigated the RNA silencing suppression (RSS) activity and its underpinning mechanism of the distinct HCPro from alpinia oxyphylla mosaic macluravirus (aHCPro). Sequence analysis revealed that macluraviral HCPros have obvious truncations in the N-terminal and middle regions when aligned to their counterparts in potyviruses (well-characterized viral suppressors of RNA silencing). Nearly all defined elements essential for the RSS activity of potyviral counterparts are not distinguished in macluraviral HCPros. Here, we demonstrated that aHCPro exhibits a similar anti-silencing activity with the potyviral counterpart. However, aHCPro fails to block both the local and systemic spreading of RNA silencing. In line, aHCPro interferes with the dsRNA synthesis, an upstream step in the RNA silencing pathway. Affinity-purification and NanoLC-MS/MS analysis revealed that aHCPro has no association with core components or their potential interactors involving in dsRNA synthesis from the protein layer. Instead, the ectopic expression of aHCPro significantly reduces the transcript abundance of RDR2, RDR6, SGS3, and SDE5. This study represents the first report on the anti-silencing function of Macluravirus-encoded HCPro and the underlying molecular mechanism.

A Nimble Cloning-compatible vector system for high-throughput gene functional analysis in plants.

Plant expression vectors are essential tools for gene functional analysis and molecular plant breeding. The gene of interest is transferred to the vector by molecular cloning technology. Nimble Cloning is a newly developed molecular cloning method with the advantages of simplicity, efficiency, and standardization. In this study, we developed a "pNC" vector system that contains 55 Nimble Cloning-compatible vectors for functional analysis of genes in plants. These vectors contain the NC frame flanked by unique adapters for one-step and standardized Nimble Cloning. We demonstrate that the pNC vectors are convenient and effective for the functional analysis of plant genes, including the study of gene ectopic expression, protein subcellular localization, protein-protein interaction, gene silencing (RNAi), virus-induced gene silencing, promoter activity, and CRISPR-Cas9-mediated genome editing. The "pNC" vector system represents a high-throughput toolkit that can facilitate the large-scale analysis of plant functional genomics.

Internal electric field induced photocarriers separation of nickel-doped pyrite/pyrite homojunction with rich sulfur vacancies for superior Cr(VI) reduction.

Improving the separation efficiency and transfer ability of photoinduced electrons/holes in pyrite (FeS2)-based photocatalytic materials is significant for the photoreduction of hexavalent chromium (Cr(VI)) but still remains a challenge. Herein, a novel homojunction was prepared through in-situ growth of nickel (Ni) doped FeS2 nanoparticles on FeS2 nanobelts (denoted as Ni-FeS2/FeS2). Systematical characterizations revealed that Ni doped FeS2 nanoparticles have been successfully in situ grown along the lattice of FeS2 nanobelts. Photoreduction experiments demonstrated that the Ni-FeS2/FeS2 homojunction with 2 mmol Ni doping contents (denoted as 2Ni-FeS2/FeS2) exhibited the optimum Cr(VI) reduction efficiency among the studied catalysts. Density Functional Theory (DFT) calculated results verified that Ni doping could not only be advantageous for the formation of sulfur vacancies but also modify the band gap and band structure of FeS2 nanoparticles. Moreover, several doping energy levels caused by Ni doping have also appeared near the Fermi level of FeS2 nanoparticles. The migration paths of electrons and the existence of internal electric field (IEF) in homojunction were further verified by the calculation of work function. To sum up, the doping energy levels and IEF that produced by homojunction played important roles in accelerating the separation efficiency of its photogenerated carriers.

Specific Monoclonal Antibodies Targeting Unique HA Epitopes Block H7N9 Influenza A Viral Replication.

The H7N9 subtype influenza A viruses pose a serious threat to public health, and there is still a lack of vaccines or drugs for humans against H7N9 influenza viruses. In this study, we screened two monoclonal antibodies (MAbs), 4H1E8 and 7H9A6, that specifically recognize the hemagglutinin (HA) protein of H7N9 influenza virus and display highly neutralizing activity against H7N9 virus. The epitopes recognized by two MAbs are nearly all conserved within all known H7 subtypes. Characteristic identification showed that two MAbs have high avidity for the HA protein but no hemagglutinin inhibition activity or antibody-dependent cellular cytotoxicity. Mechanistically, the 4H1E8 and 7H9A6 antibodies inhibit the pH-dependent conformational change of HA and block the HA-mediated membrane fusion. More importantly, 4H1E8 and 7H9A6 exhibit promising prophylactic and therapeutic effects against lethal challenge with H7N9 virus. Moreover, 4H1E8- and 7H9A6-treated mice displayed inhibition of pulmonary viral replication and reduced lung lesions after viral challenge. Together, these findings indicate that antibodies 4H1E8 and 7H9A6 recognize unique epitopes in the HA protein and possess the neutralizing activity and protective efficacy against the H7N9 influenza A viruses. IMPORTANCE In 2013, H7N9 influenza viruses appeared in China and other countries resulting in more than 1,500 individual infections or death. There are still limited studies on vaccines or drugs for humans against H7N9 influenza viruses. Alternative approaches against H7N9 virus infection need to be developed. Here, we identified two monoclonal antibodies (4H1E8 and 7H9A6) that possess neutralizing activity by blocking the pH-dependent HA-mediated membrane fusion. Additionally, the two monoclonal antibodies protect mice against the H7N9 virus challenge prophylactically or therapeutically. Therefore, our study demonstrates that 4H1E8 and 7H9A6 could be used for the prevention and treatment of the H7N9 influenza virus, and the conserved epitopes we identified may contribute to the development of a broad H7N9 vaccine and provide insights into unique antiviral approaches.

Rapid detection of sweepoviruses through lateral flow dipstick-based recombinase polymerase amplification.

Sweepoviruses represent a phylogenetic group of begomoviruses that cause significant sweet potato (Ipomoea batatas) production losses in various countries across the world. For rapid identification of sweepoviruses, we developed a technique based on isothermal recombinase polymerase amplification in conjunction with lateral flow dipsticks (RPA-LFD). The optimum reaction conditions for the RPA were 20 min incubation at 37°C. The RPA-LFD specifically detected distinct sweepovirus species, with no other viruses infecting sweet potato causing a cross-reaction. The detection limit of the RPA-LFD was 1.0×104 copies of the target DNA molecule per reaction, and it exhibited a 10-fold greater sensitivity than the conventional PCR. Furthermore, when coupled with an alkaline polyethylene glycol-based crude genomic DNA extraction, the entire procedure was completed in 30 min without the use of any special instruments other than a water bath. Therefore, the RPA-LFD technique is a potential sweepovirus diagnostic tool that can be used in the field with fewer available resources. Keywords: detection; sweepoviruses; recombinase polymerase amplification; lateral flow dipstick.

Biological and Molecular Characterization of Two Closely Related Arepaviruses and Their Antagonistic Interaction in Nicotiana benthamiana.

Previously, our group characterized two closely related viruses from Areca catechu, areca palm necrotic ringspot virus (ANRSV) and areca palm necrotic spindle-spot virus (ANSSV). These two viruses share a distinct genomic organization of leader proteases and represent the only two species of the newly established genus Arepavirus of the family Potyviridae. The biological features of the two viruses are largely unknown. In this study, we investigated the pathological properties, functional compatibility of viral elements, and interspecies interactions in the model plant, Nicotiana benthamiana. Using a newly obtained infectious clone of ANRSV, we showed that this virus induces more severe symptoms compared with ANSSV and that this is related to a rapid virus multiplication in planta. A series of hybrid viruses were constructed via the substitution of multiple elements in the ANRSV infectious clone with the counterparts of ANSSV. The replacement of either 5'-UTR-HCPro1-HCPro2 or CI effectively supported replication and systemic infection of ANRSV, whereas individual substitution of P3-7K, 9K-NIa, and NIb-CP-3'-UTR abolished viral infectivity. Finally, we demonstrated that ANRSV confers effective exclusion of ANSSV both in coinfection and super-infection assays. These results advance our understanding of fundamental aspects of these two distinct but closely related arepaviruses.

An efficient papaya leaf distortion mosaic potyvirus vector for virus-induced gene silencing in papaya.

Papaya (Carica papaya L.) is regarded as an excellent model for genomic studies of tropical trees because of its short generation time and its small genome that has been sequenced. However, functional genomic studies in papaya depend on laborious genetic transformations because no rapid tools exist for this species. Here, we developed a highly efficient virus-induced gene silencing (VIGS) vector for use in papaya by modifying an artificially attenuated infectious clone of papaya leaf distortion mosaic virus (PLDMV; genus: Potyvirus), PLDMV-E, into a stable Nimble Cloning (NC)-based PLDMV vector, pPLDMV-NC, in Escherichia coli. The target fragments for gene silencing can easily be cloned into pPLDMV-NC without multiple digestion and ligation steps. Using this PLDMV VIGS system, we silenced and characterized five endogenous genes in papaya, including two common VIGS marker genes, namely, phytoene desaturase, Mg-chelatase H subunit, putative GIBBERELLIN (GA)-INSENSITIVE DWARF1A and 1B encoding GA receptors; and the cytochrome P450 gene CYP83B1, which encodes a key enzyme involved in benzylglucosinolate biosynthesis. The results demonstrate that our newly developed PLDMV VIGS vector is a rapid and convenient tool for functional genomic studies in papaya.

A cassava common mosaic virus vector for virus-induced gene silencing in cassava.

BACKGROUND: Cassava is an important crop for food security and industry in the least-developed and developing countries. The completion of the cassava genome sequence and identification of large numbers of candidate genes by next-generation sequencing provide extensive resources for cassava molecular breeding and increase the need for rapid and efficient gene function analysis systems in cassava. Several plant virus-induced gene silencing (VIGS) systems have been developed as reverse genetic tools for rapid gene function analysis in cassava. However, these VIGS vectors could cause severe viral symptoms or inefficient gene silencing. RESULTS: In this study, we constructed agroinfection-compatible infectious cDNA clones of cassava common mosaic virus isolate CM (CsCMV-CM, genus Potexvirus, family Alphaflexiviridae) that causes systemic infection with mild symptoms in cassava. CsCMV-CM was then modified to a viral vector carrying the Nimble cloning frame, which facilitates the rapid and high-throughput cloning of silencing fragments into the viral genome. The CsCMV-based vector successfully silenced phytoene desaturase (PDS) and magnesium chelatase subunit I (ChlI) in different cassava varieties and Nicotiana benthamiana. The silencing of the ChlI gene could persist for more than two months. CONCLUSIONS: This CsCMV-based VIGS system provides a new tool for rapid and efficient gene function studies in cassava.

A Newly Identified Virus in the Family Potyviridae Encodes Two Leader Cysteine Proteases in Tandem That Evolved Contrasting RNA Silencing Suppression Functions.

Potyviridae is the largest family of plant-infecting RNA viruses and includes many agriculturally and economically important viral pathogens. The viruses in the family, known as potyvirids, possess single-stranded, positive-sense RNA genomes with polyprotein processing as a gene expression strategy. The N-terminal regions of potyvirid polyproteins vary greatly in sequence. Previously, we identified a novel virus species within the family, Areca palm necrotic spindle-spot virus (ANSSV), which was predicted to encode two cysteine proteases, HCPro1 and HCPro2, in tandem at the N-terminal region. Here, we present evidence showing self-cleavage activity of these two proteins and define their cis-cleavage sites. We demonstrate that HCPro2 is a viral suppressor of RNA silencing (VSR), and both the variable N-terminal and conserved C-terminal (protease domain) moieties have antisilencing activity. Intriguingly, the N-terminal region of HCPro1 also has RNA silencing suppression activity, which is, however, suppressed by its C-terminal protease domain, leading to the functional divergence of HCPro1 and HCPro2 in RNA silencing suppression. Moreover, the deletion of HCPro1 or HCPro2 in a newly created infectious clone abolishes viral infection, and the deletion mutants cannot be rescued by addition of corresponding counterparts of a potyvirus. Altogether, these data suggest that the two closely related leader proteases of ANSSV have evolved differential and essential functions to concertedly maintain viral viability.IMPORTANCE The Potyviridae represent the largest group of known plant RNA viruses and account for more than half of the viral crop damage worldwide. The leader proteases of viruses within the family vary greatly in size and arrangement and play key roles during the infection. Here, we experimentally demonstrate the presence of a distinct pattern of leader proteases, HCPro1 and HCPro2 in tandem, in a newly identified member within the family. Moreover, HCPro1 and HCPro2, which are closely related and typically characterized with a short size, have evolved contrasting RNA silencing suppression activity and seem to function in a coordinated manner to maintain viral infectivity. Altogether, the new knowledge fills a missing piece in the evolutionary relationship history of potyvirids and improves our understanding of the diversification of potyvirid genomes.

The RNA-Dependent RNA Polymerase NIb of Potyviruses Plays Multifunctional, Contrasting Roles during Viral Infection.

Potyviruses represent the largest group of known plant RNA viruses and include many agriculturally important viruses, such as Plum pox virus, Soybean mosaic virus, Turnip mosaic virus, and Potato virus Y. Potyviruses adopt polyprotein processing as their genome expression strategy. Among the 11 known viral proteins, the nuclear inclusion protein b (NIb) is the RNA-dependent RNA polymerase responsible for viral genome replication. Beyond its principal role as an RNA replicase, NIb has been shown to play key roles in diverse virus-host interactions. NIb recruits several host proteins into the viral replication complexes (VRCs), which are essential for the formation of functional VRCs for virus multiplication, and interacts with the sumoylation pathway proteins to suppress NPR1-mediated immunity response. On the other hand, NIb serves as a target of selective autophagy as well as an elicitor of effector-triggered immunity, resulting in attenuated virus infection. These contrasting roles of NIb provide an excellent example of the complex co-evolutionary arms race between plant hosts and potyviruses. This review highlights the current knowledge about the multifunctional roles of NIb in potyvirus infection, and discusses future research directions.

A Double Mutation in the Conserved Motifs of the Helper Component Protease of Papaya Leaf Distortion Mosaic Virus for the Generation of a Cross-Protective Attenuated Strain.

Potyviral helper component protease (HC-Pro), as a major determinant of symptom expression in susceptible plants, is a likely target candidate in the production of attenuated strains for cross-protection. In this study, single or double mutations of Lys (K) to Glu (E) in the Lys-Ile-Thr-Cys motif and Arg (R) to Ile (I) in the Phe-Arg-Asn-Lys motif of the HC-Pro from the severe papaya leaf distortion mosaic virus strain DF (PLDMV-DF) reduced symptom expression and virus accumulation in infected papaya (Carica papaya) plants. The papaya plants infected with the attenuated double mutant of PLDMV-EI presented as symptomless. PLDMV-EI provided effective protection against PLDMV-DF infection in three papaya cultivars and had no effect on plant growth and development. Our result showed that PLDMV-EI is a promising mild strain for the practical use of cross-protection in the field.

Rapid detection of two emerging viruses associated with necrotic symptoms in Areca catechu L. by reverse transcription loop-mediated isothermal amplification (RT-LAMP).

Two reverse-transcription loop-mediated isothermal amplification (RT-LAMP) assays were developed for the detection of areca palm necrotic ringspot virus (ANRSV) and areca palm necrotic spindle-spot virus (ANSSV), respectively. These two emerging viruses both induce necrotic symptoms in areca palms. The coat protein (CP) gene of ANRSV and the 9 K gene of ANSSV were used to design the respective RT-LAMP primers for the assays. Each set of four primers designed for each of these viruses was found to be highly specific in the detection of the respective targeted virus. The optimal incubation conditions for the RT-LAMP assays were 63 °C for 40 min for ANRSV and at 61 °C for 40 min for ANSSV. The sensitivity of the RT-LAMP method for each of these viruses was 10-fold greater than that of the corresponding conventional reverse-transcription polymerase chain reaction (RT-PCR). The RT-LAMP assays may be useful for the rapid early detection of ANSSV and ANRSV in commercial areca palm production.

Areca Palm Necrotic Ringspot Virus, Classified Within a Recently Proposed Genus Arepavirus of the Family Potyviridae, Is Associated With Necrotic Ringspot Disease in Areca Palm.

Areca palm (Areca catechu), one of the two most important commercial crops in Hainan, China, has been severely damaged by a variety of pathogens and insects. Here, we report a new disease, tentatively referred to as areca palm necrotic ringspot disease (ANRSD), which is highly epidemic in the main growing regions in Hainan. Transmission electron microscopy observation and small RNA deep sequencing revealed the existence of a viral agent of the family Potyviridae in a diseased areca palm plant (XC1). The virus was tentatively named areca palm necrotic ringspot virus (ANRSV). Subsequently, the positive-sense single-stranded genome of ANRSV isolate XC1 was completely determined. The genome annotation revealed the existence of two cysteine proteinases in tandem (HC-Pro1 and HC-Pro2) in the genomic 5' terminus of ANRSV. Sequence comparison and phylogenetic analysis suggested the taxonomic classification of ANRSV into the recently proposed genus Arepavirus in the family Potyviridae. Given the close relationship of ANRSV with another newly reported arepavirus (areca palm necrotic spindle-spot virus), the exact taxonomic status of ANRSV needs to be further investigated. In this study, a reverse transcription polymerase chain reaction assay for ANRSV-specific detection was developed and a close association between ANRSV and ANRSD was found.