A pathogenicity determinant maps to the N‐terminal coat protein region of the Pepino mosaic virus genome

Pepino mosaic virus (PepMV) poses a worldwide threat to the tomato industry. Considerable differences at the genetic level allow for the distinction of four main genotypic clusters; however, the basis of the phenotypic outcome is difficult to elucidate. This work reports the generation of wild‐type PepMV infectious clones of both EU (mild) and CH2 (aggressive) genotypes, from which chimeric infectious clones were created. Phenotypic analysis in three solanaceous hosts, Nicotiana benthamiana, Datura stramonium and Solanum lycopersicum, indicated that a PepMV pathogenicity determinant mapped to the 3′‐terminal region of the genome. Increased aggression was only observed in N. benthamiana, showing that this factor is host specific. The determinant was localized to amino acids 11–26 of the N‐terminal coat protein (CP) region; this is the first report of this region functioning as a virulence factor in PepMV.     

Gain of virulence by Soybean mosaic virus on Rsv4‐genotype soybeans is associated with a relative fitness loss in a susceptible host

‘Gene‐for‐gene’ theory predicts that gain of virulence by an avirulent pathogen on plants expressing resistance (R) genes is associated with fitness loss in susceptible hosts. However, the validity of this prediction has been studied in only a few plant viral pathosystems. In this study, the Soybean mosaic virus (SMV)–Rsv4 pathosystem was exploited to test this prediction. In Rsv4‐genotype soybeans, P3 of avirulent SMV strains provokes an as yet uncharacterized resistance mechanism that restricts the invading virus to the inoculated leaves. A single amino acid substitution in P3 functionally converts an avirulent to a virulent strain, suggesting that the genetic composition of P3 plays a crucial role in virulence on Rsv4‐genotype soybeans. In this study, we examined the impact of gain of virulence mutation(s) on the fitness of virulent variants derived from three avirulent SMV strains in a soybean genotype lacking the Rsv4 gene. Our data demonstrate that gain of virulence mutation(s) by all avirulent viruses on Rsv4‐genotype soybean is associated with a relative fitness loss in a susceptible host. The implications of this finding on the durable deployment of the Rsv4 gene in soybean are discussed.     

Four sequence positions of the movement protein of Cucumber mosaic virus determine the virulence against cmv1‐mediated resistance in melon

The resistance to a set of strains of Cucumber mosaic virus (CMV) in the melon accession PI 161375, cultivar ‘Songwhan Charmi’, is dependent on one recessive gene, cmv1, which confers total resistance, whereas a second set of strains is able to overcome it. We tested 11 strains of CMV subgroups I and II in the melon line SC12‐1‐99, which carries the gene cmv1, and showed that this gene confers resistance to strains of subgroup II only and that restriction is not related to either viral replication or cell‐to‐cell movement. This is the first time that a resistant trait has been correlated with CMV subgroups. Using infectious clones of the CMV strains LS (subgroup II) and FNY (subgroup I), we generated rearrangements and viral chimaeras between both strains and established that the determinant of virulence against the gene cmv1 resides in the first 209 amino acids of the movement protein, as this region from FNY is sufficient to confer virulence to the LS clone in the line SC12‐1‐99. A comparison of the sequences of the strains of both subgroups in this region shows that there are five main positions shared by all strains of subgroup II, which are different from those of subgroup I. Site‐directed mutagenesis of the CMV‐LS clone to substitute these residues for those of CMV‐FNY revealed that a combination of four of these changes [the group 64–68 (SNNLL to HGRIA), and the point mutations R81C, G171T and A195I] was required for a complete gain of function of the LS MP in the resistant melon plant.     

The P2 of Wheat yellow mosaic virus rearranges the endoplasmic reticulum and recruits other viral proteins into replication‐associated inclusion bodies

Viruses commonly modify host endomembranes to facilitate biological processes in the viral life cycle. Infection by viruses belonging to the genus Bymovirus (family Potyviridae) has long been known to induce the formation of large membranous inclusion bodies in host cells, but their assembly and biological roles are still unclear. Immunoelectron microscopy of cells infected with the bymovirus Wheat yellow mosaic virus (WYMV) showed that P1, P2 and P3 are the major viral protein constituents of the membranous inclusions, whereas NIa‐Pro (nuclear inclusion‐a protease) and VPg (viral protein genome‐linked) are probable minor components. P1, P2 and P3 associated with the endoplasmic reticulum (ER), but only P2 was able to rearrange ER and form large aggregate structures. Bioinformatic analyses and chemical experiments showed that P2 is an integral membrane protein and depends on the active secretory pathway to form aggregates of ER membranes. In planta and in vitro assays demonstrated that P2 interacts with P1, P3, NIa‐Pro or VPg and recruits these proteins into the aggregates. In vivo RNA labelling using WYMV‐infected wheat protoplasts showed that the synthesis of viral RNAs occurs in the P2‐associated inclusions. Our results suggest that P2 plays a major role in the formation of membranous compartments that house the genomic replication of WYMV.     

Amino acid substitution in P3 of Soybean mosaic virus to convert avirulence to virulence on Rsv4‐genotype soybean is influenced by the genetic composition of P3

The modification of avirulence factors of plant viruses by one or more amino acid substitutions converts avirulence to virulence on hosts containing resistance genes. Limited experimental studies have been conducted on avirulence/virulence factors of plant viruses, in particular those of potyviruses, to determine whether avirulence/virulence sites are conserved among strains. In this study, the Soybean mosaic virus (SMV)–Rsv4 pathosystem was exploited to determine whether: (i) avirulence/virulence determinants of SMV reside exclusively on P3 regardless of virus strain; and (ii) the sites residing on P3 and crucial for avirulence/virulence of isolates belonging to strain G2 are also involved in virulence of avirulent isolates belonging to strain G7. The results confirm that avirulence/virulence determinants of SMV on Rsv4‐genotype soybean reside exclusively on P3. Furthermore, the data show that sites involved in the virulence of SMV on Rsv4‐genotype soybean vary among strains, with the genetic composition of P3 playing a crucial role.     

The interaction of soybean reticulon homology domain protein (GmRHP) with Soybean mosaic virus encoded P3 contributes to the viral infection

Soybean mosaic virus (SMV), a member of the Potyvirus genus, is a prevalent and devastating viral pathogen in soybean-growing regions worldwide. Potyvirus replication occurs in the 6K2-induced viral replication complex at endoplasmic reticulum exit sites. Potyvirus-encoded P3 is also associated with the endoplasmic reticulum and is as an essential component of the viral replication complex, playing a key role in viral replication. This study provides evidence that the soybean (Glycine max) reticulon homology domain protein (designated as GmRHP) interacts with SMV-P3 by using a two-hybrid yeast system to screen a soybean cDNA library. A bimolecular fluorescence complementation assay further confirmed the interaction, which occurred on the cytomembrane, endoplasmic reticulum and cytoskeleton in Nicotiana benthamiana cells. The transient expression of GmRHP can promote the coupling of Turnip mosaic virus replication and cell-to-cell movement in N. benthamiana. The interaction between the membrane protein SMV-P3 and GmRHP may contribute to the potyvirus infection, and GmRHP may be an essential host factor for P3's involvement in potyvirus replication.     

Hijacking of the nucleolar protein fibrillarin by TGB1 is required for cell‐to‐cell movement of Barley stripe mosaic virus

Barley stripe mosaic virus (BSMV) Triple Gene Block1 (TGB1) is a multifunctional movement protein with RNA‐binding, ATPase and helicase activities which mainly localizes to the plasmodesmata (PD) in infected cells. Here, we show that TGB1 localizes to the nucleus and the nucleolus, as well as the cytoplasm, and that TGB1 nuclear‐cytoplasmic trafficking is required for BSMV cell‐to‐cell movement. Prediction analyses and laser scanning confocal microscopy (LSCM) experiments verified that TGB1 possesses a nucleolar localization signal (NoLS) (amino acids 95–104) and a nuclear localization signal (NLS) (amino acids 227–238). NoLS mutations reduced BSMV cell‐to‐cell movement significantly, whereas NLS mutations almost completely abolished movement. Furthermore, neither the NoLS nor NLS mutant viruses could infect Nicotiana benthamiana systemically, although the NoLS mutant virus was able to establish systemic infections of barley. Protein interaction experiments demonstrated that TGB1 interacts directly with the glycine–arginine‐rich (GAR) domain of the nucleolar protein fibrillarin (Fib2). Moreover, in BSMV‐infected cells, Fib2 accumulation increased by about 60%–70% and co‐localized with TGB1 in the plasmodesmata. In addition, BSMV cell‐to‐cell movement in fib2 knockdown transgenic plants was reduced to less than one‐third of that of non‐transgenic plants. Fib2 also co‐localized with both TGB1 and BSMV RNA, which are the main components of the ribonucleoprotein (RNP) movement complex. Collectively, these results show that TGB1–Fib2 interactions play a direct role in cell‐to‐cell movement, and we propose that Fib2 is hijacked by BSMV TGB1 to form a BSMV RNP which functions in cell‐to‐cell movement.     

Analysis of the coat protein gene and untranslated region of RNA3 of Cucumber Mosaic Virus isolates infecting various Lilium species and hybrids: association of the isolate infecting asiatic hybrid lily with subgroup II

The variability in the coat protein gene of Cucumber mosaic virus (CMV) isolates from various Lilium species and hybrids namely L. longiflorum, L. tigrinum, Asiatic hybrid and Oriental hybrid lilies was studied by sequence comparison of ～900 bp regions spanning the entire coat protein, intercistronic regions and 3′-UTR. CMV isolate characterised from Asiatic hybrid lily showed the highest homology with subgroup II isolates (94 – 97%), whereas 73 – 76% homology was observed with those belonging to subgroup I. Similarly, another three isolates showed 91 – 98% amino acid sequence homology with subgroup I and 74 – 76% sequence homology with subgroup II. Based on the criteria for classification of CMV isolates all the Indian isolates fall in subgroup I, except the one characterized from Asiatic Hybrid lily which falls into subgroup II. Other lily isolates from world were placed in subgroup II. This is the first case of Asiatic hybrid lily CMV isolate belonging to subgroup II.     

N‐terminal region of cysteine‐rich protein (CRP) in carlaviruses is involved in the determination of symptom types

Plant viruses in the genus Carlavirus include more than 65 members. Plants infected with carlaviruses exhibit various symptoms, including leaf malformation and plant stunting. Cysteine‐rich protein (CRP) encoded by carlaviruses has been reported to be a pathogenicity determinant. Carlavirus CRPs contain two motifs in their central part: a nuclear localization signal (NLS) and a zinc finger motif (ZF). In addition to these two conserved motifs, carlavirus CRPs possess highly divergent, N‐terminal, 34 amino acid residues with unknown function. In this study, to analyse the role of these distinct domains, we tested six carlavirus CRPs for their RNA silencing suppressor activity, ability to enhance the pathogenicity of a heterologous virus and effects on virus accumulation levels. Although all six tested carlavirus CRPs showed RNA silencing suppressor activity at similar levels, symptoms induced by the Potato virus X (PVX) heterogeneous system exhibited two different patterns: leaf malformation and whole‐plant stunting. The expression of each carlavirus CRP enhanced PVX accumulation levels, which were not correlated with symptom patterns. PVX‐expressing CRP with mutations in either NLS or ZF did not induce symptoms, suggesting that both motifs play critical roles in symptom expression. Further analysis using chimeric CRPs, in which the N‐terminal region was replaced with the corresponding region of another CRP, suggested that the N‐terminal region of carlavirus CRPs determined the exhibited symptom types. The up‐regulation of a plant gene upp‐L, which has been reported in a previous study, was also observed in this study; however, the expression level was not responsible for symptom types.     

Mitogen‐activated protein kinase phosphatase 1 reduces the replication efficiency of Bamboo mosaic virus in Nicotiana benthamiana

In plants, the mitogen‐activated protein kinase (MAPK) cascades are the central signaling pathways of the complicated defense network triggered by the perception of pathogen‐associated molecular patterns to repel pathogens. The Arabidopsis thaliana MAPK phosphatase 1 (AtMKP1) negatively regulates the activation of MAPKs. Recently, the AtMKP1 homolog of Nicotiana benthamiana (NbMKP1) was found in association with the Bamboo mosaic virus (BaMV) replication complex. This study aimed to investigate the role of NbMKP1 in BaMV multiplication in N. benthamiana. Silencing of NbMKP1 increased accumulations of the BaMV‐encoded proteins and the viral genomic RNA, although the same condition reduced the infectivity of Pseudomonas syringae pv. tomato DC3000 in N. benthamiana. On the other hand, overexpression of NbMKP1 decreased the BaMV coat protein accumulation in a phosphatase activity‐dependent manner in protoplasts. NbMKP1 also negatively affected the in vitro RNA polymerase activity of the BaMV replication complex. Collectively, the activity of NbMKP1 seems to reduce BaMV multiplication, inconsistent with the negatively regulatory role of MKP1 in MAPK cascades in terms of warding off fungal and bacterial invasion. In addition, silencing of NbMKP1 increased the accumulation of Foxtail mosaic virus but decreased Potato virus X. The discrepant effects exerted by NbMKP1 on different pathogens foresee the difficulty to develop plants with broad‐spectrum resistance through genetically manipulating a single player in MAPK cascades.     

Transgenic resistance to <Emphasis Type="Italic">Cymbidium mosaic virus</Emphasis> in <Emphasis Type="Italic">Dendrobium</Emphasis> expressing the viral capsid protein gene

A Taiwan isolate of Cymbidium mosaic virus (CymMV-CS) was isolated from infected Cymbidium sinesis Willd. The cDNA of the capsid protein (CP) gene was synthesized and sequenced. Alignment of this CP gene with other reported CPs revealed homologies of 92–98% at the nucleotide level and 98–99% at the amino acid level. To generate virus-resistant varieties, the CymMV-CS CP gene was transformed into Dendrobium protocorms through particle bombardment. Transformants were selected on medium supplemented with 20 mg/L hygromycin and the presence of the transgene was confirmed by polymerase chain reaction, Southern, Northern and Western blot analyses. Transgenic Dendrobium harboring the CymMV CP gene expressed a very low level of virus accumulation four months post-inoculation with CymMV, as detected by ELISA. The transgenic plants exhibited much milder symptoms than the non-transgenic plants upon challenge with CymMV virionsSequence data reported from this article have been deposited at the GenBank Data Libraries under Accession No. AY429021.     

Dissecting the multifunctional role of the N‐terminal domain of the Melon necrotic spot virus coat protein in RNA packaging,viral movement and interference with antiviral plant defence

The coat protein (CP) of Melon necrotic spot virus (MNSV) is structurally composed of three major domains. The middle S‐domain builds a robust protein shell around the viral genome, whereas the C‐terminal protruding domain, or P‐domain, is involved in the attachment of virions to the transmission vector. Here, we have shown that the N‐terminal domain, or R‐domain, and the arm region, which connects the R‐domain and S‐domain, are involved in different key steps of the viral cycle, such as cell‐to‐cell movement and the suppression of RNA silencing and pathogenesis through their RNA‐binding capabilities. Deletion mutants revealed that the CP RNA‐binding ability was abolished only after complete, but not partial, deletion of the R‐domain and the arm region. However, a comparison of the apparent dissociation constants for the CP RNA‐binding reaction of several partial deletion mutants showed that the arm region played a more relevant role than the R‐domain in in vitro RNA binding. Similar results were obtained in in vivo assays, although, in this case, full‐length CPs were required to encapsidate full‐length genomes. We also found that the R‐domain carboxyl portion and the arm region were essential for efficient cell‐to‐cell movement, for enhancement of Potato virus X pathogenicity, for suppression of systemic RNA silencing and for binding of small RNAs. Therefore, unlike other carmovirus CPs, the R‐domain and the arm region of MNSV CP have acquired, in addition to other essential functions such as genome binding and encapsidation functions, the ability to suppress RNA silencing by preventing systemic small RNA transport.     

Identification of an amino acid residue required for differential recognition of a viral movement protein by the Tomato mosaic virus resistance gene Tm-2(2)

The Tm-2 gene of tomato and its allelic gene, Tm-2², confer resistance to Tomato mosaic virus (ToMV) and encode a member of the coiled-coil/nucleotide binding-ARC/leucine-rich repeat (LRR) protein class of plant resistance (R) genes. Despite exhibiting only four amino acid differences between the products of Tm-2 and Tm-2², Tm-2² confers resistance to ToMV mutant B7, whereas Tm-2 is broken by ToMV-B7. An Agrobacterium-mediated transient expression system was used to study the mechanism of differential recognition of the movement proteins (MPs), an avirulence factor for ToMV resistance, of ToMV-B7 by Tm-2 and Tm-2². Although resistance induced by Tm-2 and Tm-2² is not usually accompanied by hypersensitive response (HR), Tm-2 and Tm-2² induced HR-like cell death by co-expression with MP of a wild-type ToMV, a strain that causes resistance for these R genes, and Tm-2² but not Tm-2 induced cell death with B7-MP in this system. Site-directed amino acid mutagenesis revealed that Tyr-767 in the LRR of Tm-2² is required for the specific recognition of the B7-MP. These results suggest that the Tyr residue in LRR contributes to the recognition of B7-MP, and that Tm-2 and Tm-2² are involved in HR cell death.