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In summer 2018, brown stripe symptoms were observed on leaf blades of perennial ryegrass (Lolium perenne L.) used as a turfgrass in an athletic field in Saitama Prefecture, Japan. Bacterial ooze exuded from lesions, from which a pathogenic bacterium was isolated. Inoculation of blades of perennial ryegrass with the isolates reproduced the same symptoms. Pathogenicity and bacteriological properties of the isolates were almost identical to those of Acidovorax avenae, and the identification was supported by phylogenetic analysis. This is the first report of the brown stripe on perennial ryegrass caused by A. avenae (= A. avenae subsp. avenae) in Japan. [ABSTRACT FROM AUTHOR]

Rice grassy stunt virus (RGSV) is a member of the genus Tenuivirus , which includes rice stripe virus (RSV), as the type species. A viral suppressor of RNA silencing (VSR) of RGSV has not been identified, whereas the p3 protein of RSV (RSVp3) encoded by the viral-sense (v) strand of RNA3 has been reported to act as a VSR. In this study, we examined the VSR function of the p5 protein of RGSV (RGSVp5), encoded by vRNA5. Expecting it to correspond to the vRNA3 of RSV, we compared the VSR function of RGSVp5 with that of RSVp3. In an Agrobacterium -mediated transient expression assay using a transgenic line of Nicotiana benthamiana that expressed green fluorescent protein and the wild type, RGSVp5 suppressed sense transgene-mediated post-transcriptional gene silencing (S-PTGS), inverted-repeat (IR) transgene-induced PTGS (IR-PTGS), and the systemic spread of GFP silencing, as in the case with RSVp3. By contrast, a gel mobility shift assay revealed that RGSVp5 did not have any distinct binding activity with 21-, 22-, or 24-nucleotide small interfering RNA (siRNA) duplexes, whereas RSVp3 binds to all three sizes of siRNA duplexes. Furthermore, the transiently expressed p5 protein fused with GFP was dispersed mainly in the cytoplasm, whereas the GFP-fused p3 protein of RSV was localized both in the nucleus and in the cytoplasm. Our results suggest that RGSVp5 functions as a VSR but that the suppression mechanism of RNA silencing and the subcellular localization of RGSVp5 differ from those of the analogous VSR, RSVp3, even in the same genus. [ABSTRACT FROM AUTHOR]

Since 2006, a commercial grower in Japan has noted a leaf blight symptom on potted cosmos plants grown in a field. In August 2012, a Pseudomonas-like bacterium was isolated from the symptomatic leaves and found to cause the same symptom on cosmos seedlings after inoculation. On the basis of bacteriological and phylogenetic analyses, the causative bacterium was identified as Pseudomonas cichorii. This is the first report of bacterial leaf blight on cosmos caused by P. cichorii in Japan. [ABSTRACT FROM AUTHOR]

The biological and genetic diversity of Wheat yellow mosaic virus (WYMV) isolates in Japan was characterized. On the basis of wheat cultivar reactions, 14 WYMV isolates from various places were classified into pathotypes I, II, or III. These were distributed in central, northern, and southern areas of Japan, respectively. WYMV isolates comprised three genotypes (A, A' and B) based on amino acid differences in RNA1 and two genotypes (a and b) based on amino acid differences in RNA2. A correlation was found between the WYMV RNA1-based genotype and pathotype, suggesting that factors associated with pathogenicity map to RNA1. Genotype Aa and A'a were distributed mainly in the central to southern areas of Japan, and genotype Bb was found in northern areas of Japan, as shown by reverse-transcription polymerase chain reaction restriction fragment length polymorphism analysis. Chinese isolates YA and YZ were closely related to genotypes Bb and Aa, respectively. Wheat was introduced from China to Japan in the 4th and 5th centuries, and the two genotypes of WYMV might also have been introduced with the crop from China and later adapted to local wheat cultivars in Japan. [ABSTRACT FROM AUTHOR]

Bacterial leaf spot disease of hemp was observed in Tochigi Prefecture, Japan in 1982 and characterized by necrotic lesions ca. 1-2 mm diameter on leaves with a yellow halo 2-3 mm wide. In this report, we describe the pathological, physiological and genetic properties of the causal bacterium. Our results indicated that this bacterium is identical with Xanthomonas campestris pv. cannabis reported in Romania. [ABSTRACT FROM AUTHOR]

The nonstructural protein pC6 encoded by rice grassy stunt virus is thought to correspond functionally to the nonstructural protein pC4 of rice stripe virus, which can support viral cell-to-cell movement. In a trans-complementation experiment with a movement-defective tomato mosaic virus, pC6 and pC4 facilitated intercellular transport of the virus. Transient expression of pC6, fused with green fluorescent protein, in epidermal cells was predominantly observed close to the cell wall as well as in a few punctate structures, presumably associated with plasmodesmata. These results suggest that pC6 has a role similar to that of pC4 in viral cell-to-cell movement. [ABSTRACT FROM AUTHOR]

Abstract: A reverse-transcription loop-mediated isothermal amplification (RT-LAMP) assay was established for the detection of nine viruses from infected rice plants, including rice black-streaked dwarf virus (RBSDV), rice dwarf virus (RDV), rice gall dwarf virus (RGDV), rice ragged stunt virus (RRSV), rice transitory yellowing virus (RTYV), rice stripe virus (RSV), rice grassy stunt virus (RGSV), rice tungro spherical virus (RTSV), and rice tungro bacilliform virus (RTBV). Virus-specific primer sets were designed from the genome sequences of these viruses. By the combination of RNA rapid extraction and RT-LAMP, these nine viruses could be detected within 2h from infected rice plants. The sensitivities of the assays were either higher than (for RSV, RTBV, and RTYV) or similar (for RDV) to those of one-step RT-PCR. Furthermore, RTBV and RTSV were detected not only in infected rice plants but also in viruliferous insect vectors. The RT-LAMP assays may facilitate studies on rice disease epidemiology, outbreak surveillance, and molecular pathology. [Copyright &y& Elsevier]

In July 2013, black spots were observed on leaves of calanthe in a flowerbed of a park in Tokyo, Japan. The spots were circular to oval and often accompanied by a yellow halo. A bacterium was isolated from the lesions and was found to cause the same symptoms on calanthe leaves after inoculation. Based on phylogenetic and bacteriological analyses, the causal bacterium was identified as Burkholderia andropogonis. This is the first report of bacterial black spot on calanthe caused by B. andropogonis in Japan. [ABSTRACT FROM AUTHOR]

Fig mosaic virus (FMV), a negative-strand RNA virus, is a causal agent of fig mosaic disease, which occurs in almost all countries where figs are grown and severely affects worldwide fig production. Reverse transcription-polymerase chain reaction (RT-PCR) using FMV-specific primers has typically been used to detect FMV, but faster and easier detection methods for FMV are required because RT-PCR requires multiple steps and special equipment. In this study, we established a reverse transcription loop-mediated isothermal amplification (RT-LAMP) method to detect FMV. We designed LAMP primer sets based on sequence conservation among FMV isolates. The RT-LAMP reaction using a primer set targeting RNA3 in total RNA extracted from FMV-infected fig leaves resulted in rapid detection within 15 min. In addition, we established a fast and simple method of direct sampling from fig leaves using a wooden toothpick. The RT-LAMP reaction specificity and reactivity when using the direct sampling method were almost comparable to those obtained using isolated total RNA. Moreover, geographically and phylogenetically distinct FMV isolates were detectable using the FMV RT-LAMP assay. The assay presented here provides a practical method to detect FMV. [ABSTRACT FROM AUTHOR]

Sharka disease, caused by plum pox virus (PPV), is the most serious viral disease of stone fruit trees. Among the eight known strains of the virus, PPV-D is the most important due to its recent global spread. Although enzyme-linked immunosorbent assay (ELISA) is the most common approach for diagnosing sharka, it involves time-consuming steps and requires expensive equipment and trained technicians. In this study, an on-site PPV detection kit based on immunochromatography was developed using polyclonal antibodies against the coat protein (CP) of a PPV-D isolate. The immunochromatographic (IC) assay kit was as sensitive as a commercial ELISA system for detecting Japanese PPV-D isolates. Moreover, it was easy to use (a one-step procedure), and results could be obtained on-site within 15 min without special laboratory equipment. The IC assay kit detected the virus from every aerial part of symptomatic Japanese apricot trees. In a detailed study of viral localization in leaves, the most suitable plant parts for use in the IC assay were symptomatic mesophyll tissues and the region from the petiole to the main vein. A positive reaction was also observed using the CP of other major (PPV-M and PPV-Rec) and minor (PPV-EA, PPV-W, and PPV-T) strains. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Plant virus movement proteins (MPs) localize to plasmodesmata (PD) to facilitate virus cell-to-cell movement. Numerous studies have suggested that MPs use a pathway either through the ER or through the plasma membrane (PM). Furthermore, recent studies reported that ER-PM contact sites and PM microdomains, which are subdomains found in the ER and PM, are involved in virus cell-to-cell movement. However, functional relationship of these subdomains in MP traffic to PD has not been described previously. We demonstrate here the intracellular trafficking of fig mosaic virus MP (MPFMV) using live cell imaging, focusing on its ER-directing signal peptide (SPFMV). Transiently expressed MPFMV was distributed predominantly in PD and patchy microdomains of the PM. Investigation of ER translocation efficiency revealed that SPFMV has quite low efficiency compared with SPs of well-characterized plant proteins, calreticulin and CLAVATA3. An MPFMV mutant lacking SPFMV localized exclusively to the PM microdomains, whereas SP chimeras, in which the SP of MPFMV was replaced by an SP of calreticulin or CLAVATA3, localized exclusively to the nodes of the ER, which was labeled with Arabidopsis synaptotagmin 1, a major component of ER-PM contact sites. From these results, we speculated that the low translocation efficiency of SPFMV contributes to the generation of ER-translocated and the microdomain-localized populations, both of which are necessary for PD localization. Consistent with this hypothesis, SP-deficient MPFMV became localized to PD when co-expressed with an SP chimera. Here we propose a new model for the intracellular trafficking of a viral MP. A substantial portion of MPFMV that fails to be translocated is transferred to the microdomains, whereas the remainder of MPFMV that is successfully translocated into the ER subsequently localizes to ER-PM contact sites and plays an important role in the entry of the microdomain-localized MPFMV into PD. [ABSTRACT FROM AUTHOR]

A reverse-transcription loop-mediated isothermal amplification (RT-LAMP) assay was developed to detect plantago asiatica mosaic virus (PlAMV), one of the most damaging lily-infecting viruses and a member of the genus Potexvirus in the family Alphaflexiviridae. A set of six primers was designed based on the central core region of the coat protein gene of the Li1 isolate of PlAMV, which detected the isolate most efficiently at 65 °C. The RT-LAMP assay specifically detected several PlAMV isolates with a high level of genetic and biological variation, but not potato virus X (another virus species in the same Potexvirus genus). The sensitivity of the RT-LAMP was tenfold higher than that of conventional RT-PCR. Moreover, with a simple method using a toothpick, PlAMV was directly detected from infected lily leaves using the RT-LAMP assay without RNA extraction. This simple and highly sensitive method can be used for rapid surveys for PlAMV. [ABSTRACT FROM AUTHOR]

Adhesins are microbial surface proteins that mediate the adherence of microbial pathogens to host cell surfaces. In Mollicutes, several adhesins have been reported in mycoplasmas and spiroplasmas. Adhesins P40 of Mycoplasma agalactiae and P89 of Spiroplasma citri contain a conserved amino acid sequence known as the Mollicutes adhesin motif ( MAM), whose function in the host cell adhesion remains unclear. Here, we show that phytoplasmas, which are plant-pathogenic mollicutes transmitted by insect vectors, possess an adhesion-containing MAM that was identified in a putative membrane protein, PAM289 ( P38), of the ' Candidatus Phytoplasma asteris,' OY strain. P38 homologs and their MAMs were highly conserved in related phytoplasma strains. While P38 protein was expressed in OY-infected insect and plant hosts, binding assays showed that P38 interacts with insect extract, and weakly with plant extract. Interestingly, the interaction of P38 with the insect extract depended on MAM. These results suggest that P38 is a phytoplasma adhesin that interacts with the hosts. In addition, the MAM of adhesins is important for the interaction between P38 protein and hosts. [ABSTRACT FROM AUTHOR]

RNA silencing plays an important antiviral role in plants and invertebrates. To counteract antiviral RNA silencing, most plant viruses have evolved viral suppressors of RNA silencing (VSRs). TRIPLE GENE BLOCK PROTEIN1 (TGBp1) of potexviruses is a well-characterized VSR , but the detailed mechanism by which it suppresses RNA silencing remains unclear. We demonstrate that transgenic expression of TGBp1 of plantago asiatica mosaic virus (PlAMV) induced developmental abnormalities in Arabidopsis thaliana similar to those observed in mutants of SUPPRESSOR OF GENE SILENCING3 (SGS3) and RNA-DEPENDENT RNA POLYMERASE6 (RDR6) required for the trans -acting small interfering RNA synthesis pathway. PlAMV-TGBp1 inhibits SGS3/RDR6-dependent double-stranded RNA synthesis in the trans -acting small interfering RNA pathway. TGBp1 interacts with SGS3 and RDR6 and coaggregates with SGS3/RDR6 bodies, which are normally dispersed in the cytoplasm. In addition, TGBp1 forms homooligomers, whose formation coincides with TGBp1 aggregation with SGS3/RDR6 bodies. These results reveal the detailed molecular function of TGBp1 as a VSR and shed new light on the SGS3/RDR6-dependent double-stranded RNA synthesis pathway as another general target of VSRs. [ABSTRACT FROM AUTHOR]

Abstract: Examination of cultured insect vector cells that had been infected with Rice gall dwarf virus (RGDV), using transmission electron microscopy and confocal microscopy, revealed the presence of clusters of virus-coated mitochondria around viroplasms in which replication and assembly of RGDV occurred, suggesting a role for mitochondria in supplying the energy required for viral morphogenetic processes. Electron tomography revealed that RGDV particles on the surface of mitochondria are arrayed in an orderly but loose manner, unlike tightly packaged particles in vesicular compartments, suggesting the presence of counterpart molecules on the surface of mitochondria. The viral particles in close proximity to mitochondria were aligned along intermediate filaments, which might serve as scaffolds for the anchorage of these particles. RGDV has a putative mitochondrion-targeting sequence on the outer surface of the outer-capsid protein P8. The arrangement of RGDV particles around mitochondria suggests that the region of the P8 protein containing the mitochondrion-targeting sequence might attach to a molecule like a receptor on the outer mitochondrial membrane. Our analysis demonstrates the three-dimensional arrangement and molecular basis for the mitochondrial proximity of RGDV particles during viral replication. [Copyright &y& Elsevier]

Rice dwarf virus (RDV), with 12 double-stranded RNA (dsRNA) genome segments (S1 to S12), replicates in and is transmitted by vector insects. The RDV-plant host-vector insect system allows us to examine the evolution, adaptation, and population genetics of a plant virus. We compared the effects of long-term maintenance of RDV on population structures in its two hosts. The maintenance of RDV in rice plants for several years resulted in gradual accumulation of nonsense mutations in S2 and S10, absence of expression of the encoded proteins, and complete loss of transmissibility. RDV maintained in cultured insect cells for 6 years retained an intact protein-encoding genome. Thus, the structural P2 protein encoded by S2 and the nonstructural Pns10 protein encoded by S10 of RDV are subject to different selective pressures in the two hosts, and mutations accumulating in the host plant are detrimental in vector insects. However, one round of propagation in insect cells or individuals purged the populations of RDV that had accumulated deleterious mutations in host plants, with exclusive survival of fully competent RDV. Our results suggest that during the course of evolution, an ancestral form of RDV, of insect virus origin, might have acquired the ability to replicate in a host plant, given its reproducible mutations in the host plant that abolish vector transmissibility and viability in nature. [ABSTRACT FROM AUTHOR]

Since the propagation of plant viruses depends on various host susceptibility factors, deficiency in them can prevent viral infection in cultivated and model plants. Recently, we identified the susceptibility factor Essential for poteXvirus Accumulation 1 (EXA1) in Arabidopsis thaliana, and revealed that EXA1-mediated resistance was effective against three potexviruses. Although EXA1 homolog genes are found in tomato and rice, little is known about which viruses depend on EXA1 for their infection capability and whether the function of EXA1 homologs in viral infection is conserved across multiple plant species, including crops. To address these questions, we generated knockdown mutants using virus-induced gene silencing in two Solanaceae species, Nicotiana benthamiana and tomato. In N. benthamiana, silencing of an EXA1 homolog significantly compromised the accumulation of potexviruses and a lolavirus, a close relative of potexviruses, whereas transient expression of EXA1 homologs from tomato and rice complemented viral infection. EXA1 dependency for potexviral infection was also conserved in tomato. These results indicate that EXA1 is necessary for effective accumulation of potexviruses and a lolavirus, and that the function of EXA1 in viral infection is conserved among diverse plant species. [ABSTRACT FROM AUTHOR]