CoGRIM19 is required for invasive hyphal growth of Colletotrichum orbiculare inside epidermal cells of cucumber cotyledons.

Colletotrichum orbiculare, an anthracnose disease fungus of cucurbit plants, extends penetration hyphae inside the epidermal cells of host plants. Unlike vegetative hyphae formed on a nutrient rich medium, this pathogen initially develops biotrophic penetration hyphae, which acquire nutrient resources from living host cells and secret effector proteins to suppress host defense responses. Subsequently, the nature of penetration hyphae changes from biotrophy to necrotrophy in response to the interaction with a host plant. Hence, controlling the extension of penetration hyphae is crucial for C. orbiculare infection. Here, we identified CoGRIM19 encoding Nadh-ubiquinone oxidoreductase subunit as a pathogenicity gene. Pathogenicity assays showed that the cogrim19 mutant caused no visible symptoms on cucumber cotyledons. Microscopic observations revealed that the cogrim19 mutant developed an appressorium and penetration hyphae under artificial conditions such as on coverslips or cellulose membranes, but the penetration hyphae of the mutant were retarded in the cucumber cotyledons. Microscopic observations of biotrophy-specific expression fluorescent signals revealed that the biotrophic stage was maintained in the retarded penetration hyphae of the cogrim19 mutant as the penetration of the wild type. In addition to cytological observations, pathogenicity assays using wounded leaves showed that the cogrim19 mutant had an attenuated pathogenesis. Taking our results together, CoGRIM19 is required for invasive hyphal growth inside the epidermal cells of cucumber cotyledons in C. orbiculare.

Trade-Off Relation between Fungicide Sensitivity and Melanin Biosynthesis in Plant Pathogenic Fungi.

Circumventing the emergence of fungicide-resistant strains is a crucial issue for robust disease management in agriculture. The agricultural fungicide ferimzone has been used for the control of rice diseases including rice blast. The emergence of ferimzone-resistant strains in rice fields has not been reported. Here, we identified the copper transport CoICT1 gene as the ferimzone sensitivity gene in Colletotrichum orbiculare and the rice blast fungus Magnaporthe oryzae. Genetic and cytological analyses showed that functional defects in the copper transport pathways, consisting of CoIct1 and P-type ATPase CoCcc2, led to the low sensitivity to ferimzone and the pathogenicity defect due to attenuated melanization in the appressorium. Importantly, the presence of CuSO4 induced high sensitivity to ferimzone even in the coict1 mutant. Our study shows that there is a trade-off relation between the sensitivity to ferimzone and fungal pathogenicity.

Hijacking of host cellular components as proviral factors by plant-infecting viruses.

Plant viruses are important pathogens that cause serious crop losses worldwide. They are obligate intracellular parasites that commandeer a wide array of proteins, as well as metabolic resources, from infected host cells. In the past two decades, our knowledge of plant-virus interactions at the molecular level has exploded, which provides insights into how plant-infecting viruses co-opt host cellular machineries to accomplish their infection. Here, we review recent advances in our understanding of how plant viruses divert cellular components from their original roles to proviral functions. One emerging theme is that plant viruses have versatile strategies that integrate a host factor that is normally engaged in plant defense against invading pathogens into a viral protein complex that facilitates viral infection. We also highlight viral manipulation of cellular key regulatory systems for successful virus infection: posttranslational protein modifications for fine control of viral and cellular protein dynamics; glycolysis and fermentation pathways to usurp host resources, and ion homeostasis to create a cellular environment that is beneficial for viral genome replication. A deeper understanding of viral-infection strategies will pave the way for the development of novel antiviral strategies.

Complete genome sequence of a novel partitivirus from a wild brassicaceous plant, Arabidopsis halleri.

Two contigs with high similarity to partitivirus sequences were identified by de novo assembly of sequences obtained by RNA-Seq from a wild brassicaceous plant, Arabidopsis halleri subsp. gemmifera. Here, we report the complete genome sequence of a putative novel partitivirus. Excluding the poly-A tail, it consists of two RNA genome segments of 1912 and 1769 bp, which are predicted to encode a 585-amino-acid-long putative RNA-dependent RNA polymerase (RdRp) and a 487-amino-acid-long putative capsid protein (CP), respectively. Phylogenetically, this virus belongs to the genus Alphapartitivirus and is most closely related to Raphanus sativus partitivirus 1 from radish. We propose the name "Arabidopsis halleri partitivirus 1" (AhPV1) for this novel virus.

Threonine synthase CoTHR4 is involved in infection-related morphogenesis during the pre-penetration stage in Colletotrichum orbiculare.

Upon recognition of host plants, Colletotrichum orbiculare, an anthracnose disease fungus of cucurbitaceous plants, initiates morphological differentiation, including conidial germination and appressorium formation on the cuticle layer. The series of infection processes of C. orbiculare requires enormous nutrient and energy, but the surface of the cucurbitaceous hosts is hardly nutrient-rich. Hence, C. orbiculare must exert tight management of its intracellular nutrients in order to properly induce infection-related morphogenesis. Here, we carried out a large-scale insertional mutagenesis screen using Agrobacterium tumefaciens-mediated transformation to identify novel genes involved in the pathogenicity of C. orbiculare and found that CoTHR4-encoded threonine synthase, a homolog of Saccharomyces cerevisiae THR4, is required for pathogenicity and conidiation in C. orbiculare. Threonine supplementation allowed the cothr4 mutant to produce conidia to a level equivalent to that of the wild-type. The conidia produced from the threonine-treated cothr4 mutant failed to germinate in the absence of threonine, but retained the ability to germinate and to form appressoria in the presence of threonine. However, the conidia produced from the threonine-treated cothr4 mutant remained attenuated in pathogenicity on cucumber cotyledons even in the presence of threonine. Cytorrhysis assays revealed that appressoria of the cothr4 mutant induced by exogenous threonine treatment showed low turgor generation. Taken together, these results showed that threonine synthase CoThr4 plays a pivotal role in infection-related morphogenesis during the pre-penetration stage of C. orbiculare.

5'-Terminal stem-loop of carnation ringspot virus RNA1 is required for the efficient amplification of viral RNAs.

Carnation ringspot virus (CRSV) is the prototype virus of the genus Dianthovirus. Full-length cDNAs of CRSV strainsPV-0097 and PV-21 were constructed and the infectivity of in vitro transcripts was analyzed. Infectivity of PV-0097 and PV-21 to several plants was markedly higher than that of 1.30, a previously reported infectious CRSV clone. Overall RNA sequences of these viruses were similar, but PV-0097 and PV-21 contained additional nucleotides at the 5' end of RNA1. Stem-loop structures were predicted in the 5'-terminal region of PV-0097 and PV-21 RNA1 but not in 1.30 RNA1. Mutant CRSV 1.30 RNA1 that contains the terminal 4 nucleotides of PV-0097, predicted to fold a 5'-terminal stem-loop structure, recovered higher level accumulation of viral RNAs in the inoculated protoplasts and leaves of Nicotiana benthamiana. These results suggest that the 5'-terminal stem-loop structure of CRSV RNA1 plays an important role in efficient amplification of the virus.

Hijacking a host scaffold protein, RACK1, for replication of a plant RNA virus.

Receptor for activated C kinase 1 (RACK1) is strictly conserved across eukaryotes and acts as a versatile scaffold protein involved in various signaling pathways. Plant RACK1 is known to exert important functions in innate immunity against fungal and bacterial pathogens. However, the role of the RACK1 in plant-virus interactions remains unknown. Here, we addressed the role of RACK1 of Nicotiana benthamiana during infection by red clover necrotic mosaic virus (RCNMV), a plant positive-stranded RNA virus. NbRACK1 was shown to be recruited by the p27 viral replication protein into endoplasmic reticulum-derived aggregated structures (possible replication sites). Downregulation of NbRACK1 by virus-induced gene silencing inhibited viral cap-independent translation and p27-mediated reactive oxygen species (ROS) accumulation, which are prerequisite for RCNMV replication. We also found that NbRACK1 interacted with a host calcium-dependent protein kinase (NbCDPKiso2) that activated a ROS-generating enzyme. Interestingly, NbRACK1 was required for the interaction of p27 with NbCDPKiso2, suggesting that NbRACK1 acts as a bridge between the p27 viral replication protein and NbCDPKiso2. Collectively, our findings provide an example of a viral strategy in which a host multifaceted scaffold protein RACK1 is highjacked for promoting viral protein-triggered ROS production necessary for robust viral replication.

Dual function of a cis-acting RNA element that acts as a replication enhancer and a translation repressor in a plant positive-stranded RNA virus.

The genome of red clover necrotic mosaic virus is divided into two positive-stranded RNA molecules of RNA1 and RNA2, which have no 5' cap structure and no 3' poly(A) tail. Previously, we showed that any mutations in the cis-acting RNA replication elements of RNA2 abolished its cap-independent translational activity, suggesting a strong link between RNA replication and translation. Here, we investigated the functions of the 5' untranslated region (UTR) of RNA2 and revealed that the basal stem-structure (5'BS) predicted in the 5' UTR is essential for robust RNA replication. Interestingly, RNA2 mutants with substitution or deletion in the right side of the 5'BS showed strong translational activity, despite their impaired replication competency. Furthermore, nucleotide sequences other than the 5'BS of the 5' UTR were essential to facilitate the replication-associated translation. Overall, these cis-acting RNA elements seem to coordinately regulate the balance between RNA replication and replication-associated translation.

Roles of superoxide anion and hydrogen peroxide during replication of two unrelated plant RNA viruses in Nicotiana benthamiana.

Reactive oxygen species (ROS), including superoxide anion (O2-), hydrogen peroxide (H2O2), and hydroxyl radical, act as signaling molecules to transduce biotic and abiotic stimuli into stress adaptations in plants. A respiratory burst oxidase homolog B of Nicotiana benthamiana (NbRBOHB) is responsible for O2- production to inhibit pathogen infection during plant innate immunity. RBOH-derived O2- can be immediately converted into H2O2 by the action of superoxide dismutase. Interestingly, we recently showed that red clover necrotic mosaic virus (RCNMV), a plant positive-strand RNA [(+)RNA] virus, hijacks the host's ROS-generating machinery during infection. An RCNMV replication protein associates with NbRBOHB and triggers intracellular ROS bursts. These bursts are required for robust viral RNA replication. However, what types of ROS are required for viral replication is currently unknown. Here, we found that RCNMV replication was sensitive to an O2- scavenger but insensitive to an H2O2 scavenger. Interestingly, replication of another plant (+)RNA virus, brome mosaic virus, was sensitive to both types of scavengers. These results indicate a virus-specific pattern requirement of O2- and H2O2 for (+)RNA virus replication and suggest a conserved nature of the roles of ROS in (+)RNA virus replication.

Requirement for eukaryotic translation initiation factors in cap-independent translation differs between bipartite genomic RNAs of red clover necrotic mosaic virus.

The bipartite genomic RNAs of red clover necrotic mosaic virus (RCNMV) lack a 5' cap and a 3' poly(A) tail. RNA1 encodes viral replication proteins, and RNA2 encodes a movement protein (MP). These proteins are translated in a cap-independent manner. We previously identified two cis-acting RNA elements that cooperatively recruit eukaryotic translation initiation factor (eIF) complex eIF4F or eIFiso4F to RNA1. Such cis-acting RNA elements and host factors have not been identified in RNA2. Here we found that translation of RNA1 was significantly compromised in Arabidopsis thaliana carrying eif4f mutation. RNA1 replicated efficiently in eifiso4f1 mutants, suggesting vigorous translation of the replication proteins from RNA1 in the plants. In contrast, MP accumulation was decreased in eifiso4f1 mutants but not in eif4f mutants. Collectively, these results suggest that RCNMV uses different eIF complexes for translation of its bipartite genomic RNAs, which may contribute to fine-tuning viral gene expression during infection.

Harnessing host ROS-generating machinery for the robust genome replication of a plant RNA virus.

As sessile organisms, plants have to accommodate to rapid changes in their surrounding environment. Reactive oxygen species (ROS) act as signaling molecules to transduce biotic and abiotic stimuli into plant stress adaptations. It is established that a respiratory burst oxidase homolog B of Nicotiana benthamiana (NbRBOHB) produces ROS in response to microbe-associated molecular patterns to inhibit pathogen infection. Plant viruses are also known as causative agents of ROS induction in infected plants; however, the function of ROS in plant-virus interactions remains obscure. Here, we show that the replication of red clover necrotic mosaic virus (RCNMV), a plant positive-strand RNA [(+)RNA] virus, requires NbRBOHB-mediated ROS production. The RCNMV replication protein p27 plays a pivotal role in this process, redirecting the subcellular localization of NbRBOHB and a subgroup II calcium-dependent protein kinase of N. benthamiana (NbCDPKiso2) from the plasma membrane to the p27-containing intracellular aggregate structures. p27 also induces an intracellular ROS burst in an RBOH-dependent manner. NbCDPKiso2 was shown to be an activator of the p27-triggered ROS accumulations and to be required for RCNMV replication. Importantly, this RBOH-derived ROS is essential for robust viral RNA replication. The need for RBOH-derived ROS was demonstrated for the replication of another (+)RNA virus, brome mosaic virus, suggesting that this characteristic is true for plant (+)RNA viruses. Collectively, our findings revealed a hitherto unknown viral strategy whereby the host ROS-generating machinery is diverted for robust viral RNA replication.

Pathogenesis mediated by proviral host factors involved in translation and replication of plant positive-strand RNA viruses.

Viral pathogenesis comes from complex interactions between viruses and hosts. All the processes of viral infection, including translation of viral factors and replication of viral genomes, define viral pathogenesis; therefore, molecular insights into the mechanisms underlying viral replication strategies unambiguously pave the way for our comprehensive understanding of viral pathogenesis and disease outcome, as well as for developing new antiviral strategies against plant virus disease. Recent studies of plant positive-strand RNA [(+)RNA] viruses have advanced our understanding of co-opted host factors and their roles in viral translation and replication. It is becoming clear that plant (+)RNA viruses harness host factors involved in membrane trafficking and lipid metabolism to establish the viral replication complex (VRC). In this review, we aim to discuss the contribution of co-opted host factors in translation and genome replication of plant (+)RNA viruses mainly focusing on those involved in the biogenesis of the VRC, which may act as a central hub in almost all the processes of viral infection as well as viral pathogenesis.

Phosphatidic acid produced by phospholipase D promotes RNA replication of a plant RNA virus.

Eukaryotic positive-strand RNA [(+)RNA] viruses are intracellular obligate parasites replicate using the membrane-bound replicase complexes that contain multiple viral and host components. To replicate, (+)RNA viruses exploit host resources and modify host metabolism and membrane organization. Phospholipase D (PLD) is a phosphatidylcholine- and phosphatidylethanolamine-hydrolyzing enzyme that catalyzes the production of phosphatidic acid (PA), a lipid second messenger that modulates diverse intracellular signaling in various organisms. PA is normally present in small amounts (less than 1% of total phospholipids), but rapidly and transiently accumulates in lipid bilayers in response to different environmental cues such as biotic and abiotic stresses in plants. However, the precise functions of PLD and PA remain unknown. Here, we report the roles of PLD and PA in genomic RNA replication of a plant (+)RNA virus, Red clover necrotic mosaic virus (RCNMV). We found that RCNMV RNA replication complexes formed in Nicotiana benthamiana contained PLDα and PLDß. Gene-silencing and pharmacological inhibition approaches showed that PLDs and PLDs-derived PA are required for viral RNA replication. Consistent with this, exogenous application of PA enhanced viral RNA replication in plant cells and plant-derived cell-free extracts. We also found that a viral auxiliary replication protein bound to PA in vitro, and that the amount of PA increased in RCNMV-infected plant leaves. Together, our findings suggest that RCNMV hijacks host PA-producing enzymes to replicate.

GAPDH--a recruits a plant virus movement protein to cortical virus replication complexes to facilitate viral cell-to-cell movement.

The formation of virus movement protein (MP)-containing punctate structures on the cortical endoplasmic reticulum is required for efficient intercellular movement of Red clover necrotic mosaic virus (RCNMV), a bipartite positive-strand RNA plant virus. We found that these cortical punctate structures constitute a viral replication complex (VRC) in addition to the previously reported aggregate structures that formed adjacent to the nucleus. We identified host proteins that interacted with RCNMV MP in virus-infected Nicotiana benthamiana leaves using a tandem affinity purification method followed by mass spectrometry. One of these host proteins was glyceraldehyde 3-phosphate dehydrogenase-A (NbGAPDH-A), which is a component of the Calvin-Benson cycle in chloroplasts. Virus-induced gene silencing of NbGAPDH-A reduced RCNMV multiplication in the inoculated leaves, but not in the single cells, thereby suggesting that GAPDH-A plays a positive role in cell-to-cell movement of RCNMV. The fusion protein of NbGAPDH-A and green fluorescent protein localized exclusively to the chloroplasts. In the presence of RCNMV RNA1, however, the protein localized to the cortical VRC as well as the chloroplasts. Bimolecular fluorescence complementation assay and GST pulldown assay confirmed in vivo and in vitro interactions, respectively, between the MP and NbGAPDH-A. Furthermore, gene silencing of NbGAPDH-A inhibited MP localization to the cortical VRC. We discuss the possible roles of NbGAPDH-A in the RCNMV movement process.

Host and viral RNA-binding proteins involved in membrane targeting, replication and intercellular movement of plant RNA virus genomes.

Many plant viruses have positive-strand RNA [(+)RNA] as their genome. Therefore, it is not surprising that RNA-binding proteins (RBPs) play important roles during (+)RNA virus infection in host plants. Increasing evidence demonstrates that viral and host RBPs play critical roles in multiple steps of the viral life cycle, including translation and replication of viral genomic RNAs, and their intra- and intercellular movement. Although studies focusing on the RNA-binding activities of viral and host proteins, and their associations with membrane targeting, and intercellular movement of viral genomes have been limited to a few viruses, these studies have provided important insights into the molecular mechanisms underlying the replication and movement of viral genomic RNAs. In this review, we briefly overview the currently defined roles of viral and host RBPs whose RNA-binding activity have been confirmed experimentally in association with their membrane targeting, and intercellular movement of plant RNA virus genomes.

Base-paired structure in the 5' untranslated region is required for the efficient amplification of negative-strand RNA3 in the bromovirus melandrium yellow fleck virus.

Melandrium yellow fleck virus belongs to the genus Bromovirus, which is a group of tripartite plant RNA viruses. This virus has an approximately 200-nucleotide direct repeat sequence in the 5' untranslated region (UTR) of RNA3 that encodes the 3a movement protein. In the present study, protoplast assays suggested that the duplicated region contains amplification-enhancing elements. Deletion analyses of the 5' UTR of RNA3 showed that mutations in the short base-paired region, which is located dozens of bases upstream of the initiation codon of the 3a gene, greatly reduced the accumulation of RNA3. Disruption and restoration of the base-paired structure caused the accumulation of RNA3 to be decreased and restored, respectively. In vitro translation/replication assays demonstrated that the base-paired structure is important for the efficient amplification of negative-stand RNA3. A similar base-paired structure in RNA3 of another bromovirus, brome mosaic virus (BMV), also facilitated the efficient amplification of BMV RNA3, but only in combination with melandrium yellow fleck virus (MYFV) replicase and not with BMV replicase, thereby suggesting specific interactions between base-paired structures and MYFV replicase.

Traffic jam on the cellular secretory pathway generated by a replication protein from a plant RNA virus.

Although positive-strand RNA [(+)RNA] viruses have a limited coding capacity, they can replicate efficiently in host cells because of their ability to use host-derived proteins, membranes, lipids, and metabolites, and to rewire cellular trafficking pathways. Previously, we showed that a plant RNA virus, the Red clover necrotic mosaic virus (RCNMV), hijacked Arf1 and Sar1, which are small GTPases that regulate the biogenesis of COPI and COPII vesicles, respectively, for viral RNA replication. These small GTPases are relocated from appropriate subcellular compartments to the viral RNA replication sites by p27 replication protein, which raises the possibility that RCNMV interferes with the cellular secretory pathway. Here, we examined this possibility by using green fluorescent protein-fused rice SCAMP1 and Arabidopsis LRR84A as secretory pathway marker proteins and showed that p27 inhibited the trafficking of these proteins. RCNMV-mediated inhibition of the host secretion pathway and its possible impact on plant-virus interaction are discussed.

Functional characterization of the mutations in Pepper mild mottle virus overcoming tomato tm-1-mediated resistance.

In tomato plants, Pepper mild mottle virus (PMMoV) cannot replicate because the tm-1 protein inhibits RNA replication. The resistance of tomato plants to PMMoV remains durable both in the field and under laboratory conditions. In this study, we constructed several mutant PMMoVs and analysed their abilities to replicate in tomato protoplasts and plants. We found that two mutants, PMMoV-899R,F976Y and PMMoV-899R,F976Y,D1098N, were able to replicate in tomato protoplasts, but only PMMoV-899R,F976Y,D1098N was able to multiply in tomato plants. Further analysis showed that the D1098N mutation of the replication proteins weakened the inhibitory effect of the tm-1 protein and enhanced the replication efficiency of PMMoV-899R,F976Y,D1098N. We also observed that the infectivity of the viruses decreased in the order wild-type PMMoV > PMMoV-899R,F976Y > PMMoV-899R,F976Y,D1098N in original host plants, pepper and tobacco plants. On the contrary, the single mutation D1098N abolished PMMoV replication in tobacco protoplasts. On the basis of these observations, it is likely that the deleterious side-effects of mutations in replication proteins prevent the emergence of PMMoV mutants that can overcome tm-1-mediated resistance.

Molecular biology and epidemiology of dianthoviruses.

The genus Dianthovirus is one of eight genera in the family Tombusviridae. All the genera have monopartite positive-stranded RNA genomes, except the dianthoviruses which have bipartite genomes. The dianthoviruses are distributed worldwide. Although they share common structural features with the other Tombusviridae viruses in their virions and the terminal structure of the genomic RNAs, the bipartite nature of the dianthovirus genome offers an ideal experimental system with which to study basic issues of virology. The two genomic RNAs seem to use distinct strategies to regulate their translation, transcription, genome replication, genome packaging, and cell-to-cell movement during infection. This review summarizes the current state of our knowledge of the dianthoviruses, with its main emphasis on the molecular biology of the virus, including the viral and host factors required for its infection of host plants. The epidemiology of the virus and the possible viral impacts on agriculture and the environment are also discussed.

ADP ribosylation factor 1 plays an essential role in the replication of a plant RNA virus.

Eukaryotic positive-strand RNA viruses replicate using the membrane-bound replicase complexes, which contain multiple viral and host components. Virus infection induces the remodeling of intracellular membranes. Virus-induced membrane structures are thought to increase the local concentration of the components that are required for replication and provide a scaffold for tethering the replicase complexes. However, the mechanisms underlying virus-induced membrane remodeling are poorly understood. RNA replication of red clover necrotic mosaic virus (RCNMV), a positive-strand RNA plant virus, is associated with the endoplasmic reticulum (ER) membranes, and ER morphology is perturbed in RCNMV-infected cells. Here, we identified ADP ribosylation factor 1 (Arf1) in the affinity-purified RCNMV RNA-dependent RNA polymerase fraction. Arf1 is a highly conserved, ubiquitous, small GTPase that is implicated in the formation of the coat protein complex I (COPI) vesicles on Golgi membranes. Using in vitro pulldown and bimolecular fluorescence complementation analyses, we showed that Arf1 interacted with the viral p27 replication protein within the virus-induced large punctate structures of the ER membrane. We found that inhibition of the nucleotide exchange activity of Arf1 using the inhibitor brefeldin A (BFA) disrupted the assembly of the viral replicase complex and p27-mediated ER remodeling. We also showed that BFA treatment and the expression of dominant negative Arf1 mutants compromised RCNMV RNA replication in protoplasts. Interestingly, the expression of a dominant negative mutant of Sar1, a key regulator of the biogenesis of COPII vesicles at ER exit sites, also compromised RCNMV RNA replication. These results suggest that the replication of RCNMV depends on the host membrane traffic machinery.