Long-term passages of Plantago asiatica mosaic virus alter virulence and multiplication in Nicotiana benthamiana plants.

We demonstrated the infectivity and host adaptation of a viola isolate of Plantago asiatica mosaic virus (PlAMV-Vi) in an asymptomatic host, Nicotiana benthamiana, through long-term serial passages. Serial passaging of a green fluorescent protein-tagged full-length cDNA clone of PlAMV-Vi (PlAMV-ViGFP) in N. benthamiana plants resulted in the appearance of a new virus line inducing leaf-crinkle symptoms, the Leaf Crinkle (LC) line. Virus titers were higher for both in the LC and the 14th passage line(s) of PlAMV-ViGFP compared with the original line. The LC line was found to have seven unique nucleotide mutations that may have contributed to its higher virulence and multiplication rate in N. benthamiana.

Quantitative trait locus (QTL) analysis and fine-mapping for Fusarium oxysporum disease resistance in Raphanus sativus using GRAS-Di technology.

Fusarium wilt is a significant disease in radish, but the genetic mechanisms controlling yellows resistance (YR) are not well understood. This study aimed to identify YR-QTLs and to fine-map one of them using F2:3 populations developed from resistant and susceptible radish parents. In this study, two high-density genetic maps each containing shared co-dominant markers and either female or male dominant markers that spanned 988.6 and 1127.5 cM with average marker densities of 1.40 and 1.53 cM, respectively, were generated using Genotyping by Random Amplicon Sequencing-Direct (GRAS-Di) technology. We identified two YR-QTLs on chromosome R2 and R7, and designated the latter as ForRs1 as the major QTL. Fine mapping narrowed down the ForRs1 locus to a 195 kb region. Among the 16 predicted genes in the delimited region, 4 genes including two receptor-like protein and -kinase genes (RLP/RLK) were identified as prime candidates for ForRs1 based on the nucleotide sequence comparisons between the parents and their predicted functions. This study is the first to use a GRAS-Di for genetic map construction of cruciferous crops and fine map the YR-QTL on the R7 chromosome of radish. These findings will provide groundbreaking insights into radish YR breeding and understanding the genetics of YR mechanism.

In Planta Recognition of a Double-Stranded RNA Synthesis Protein Complex by a Potexviral RNA Silencing Suppressor.

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.

Recognition of floral homeotic MADS domain transcription factors by a phytoplasmal effector, phyllogen, induces phyllody.

Plant pathogens alter the course of plant developmental processes, resulting in abnormal morphology in infected host plants. Phytoplasmas are unique plant-pathogenic bacteria that transform plant floral organs into leaf-like structures and cause the emergence of secondary flowers. These distinctive symptoms have attracted considerable interest for many years. Here, we revealed the molecular mechanisms of the floral symptoms by focusing on a phytoplasma-secreted protein, PHYL1, which induces morphological changes in flowers that are similar to those seen in phytoplasma-infected plants. PHYL1 is a homolog of the phytoplasmal effector SAP54 that also alters floral development. Using yeast two-hybrid and in planta transient co-expression assays, we found that PHYL1 interacts with and degrades the floral homeotic MADS domain proteins SEPALLATA3 (SEP3), APETALA1 (AP1) and CAULIFLOWER (CAL). This degradation of MADS domain proteins was dependent on the ubiquitin-proteasome pathway. The expression of floral development genes downstream of SEP3 and AP1 was disrupted in 35S::PHYL1 transgenic plants. PHYL1 was genetically and functionally conserved among other phytoplasma strains and species. We designate PHYL1, SAP54 and their homologs as members of the phyllody-inducing gene family of 'phyllogens'.

Lectin-mediated resistance impairs plant virus infection at the cellular level.

Plants possess a multilayered defense response, known as plant innate immunity, to infection by a wide variety of pathogens. Lectins, sugar binding proteins, play essential roles in the innate immunity of animal cells, but the role of lectins in plant defense is not clear. This study analyzed the resistance of certain Arabidopsis thaliana ecotypes to a potexvirus, plantago asiatica mosaic virus (PlAMV). Map-based positional cloning revealed that the lectin gene JACALIN-TYPE LECTIN REQUIRED FOR POTEXVIRUS RESISTANCE1 (JAX1) is responsible for the resistance. JAX1-mediated resistance did not show the properties of conventional resistance (R) protein-mediated resistance and was independent of plant defense hormone signaling. Heterologous expression of JAX1 in Nicotiana benthamiana showed that JAX1 interferes with infection by other tested potexviruses but not with plant viruses from different genera, indicating the broad but specific resistance to potexviruses conferred by JAX1. In contrast with the lectin gene RESTRICTED TEV MOVEMENT1, which inhibits the systemic movement of potyviruses, which are distantly related to potexviruses, JAX1 impairs the accumulation of PlAMV RNA at the cellular level. The existence of lectin genes that show a variety of levels of virus resistance, their targets, and their properties, which are distinct from those of known R genes, suggests the generality of lectin-mediated resistance in plant innate immunity.

Efficient foreign gene expression in planta using a plantago asiatica mosaic virus-based vector achieved by the strong RNA-silencing suppressor activity of TGBp1.

Plant virus expression vectors provide a powerful tool for basic research as well as for practical applications. Here, we report the construction of an expression vector based on plantago asiatica mosaic virus (PlAMV), a member of the genus Potexvirus. Modification of a vector to enhance the expression of a foreign gene, combined with the use of the foot-and-mouth disease virus 2A peptide, allowed efficient expression of the foreign gene in two model plant species, Arabidopsis thaliana and Nicotiana benthamiana. Comparison with the widely used potato virus X (PVX) vector demonstrated that the PlAMV vector retains an inserted foreign gene for a longer period than PVX. Moreover, our results showed that the GFP expression construct PlAMV-GFP exhibits stronger RNA silencing suppression activity than PVX-GFP, which is likely to contribute to the stability of the PlAMV vector.

Dynamin-related protein 2 interacts with the membrane-associated methyltransferase domain of plantago asiatica mosaic virus replicase and promotes viral replication.

Positive-strand RNA viruses replicate their RNA in the viral replication complex, a spherical structure formed by remodeling of host intracellular membranes. This process also requires the interaction between viral membrane-associated replication proteins and host factors. We previously identified the membrane-associated determinant of the replicase of plantago asiatica mosaic virus (PlAMV), a positive-strand RNA virus of the genus Potexvirus, in its methyltransferase (MET) domain, and suggested that its interaction with host factors is required to establish viral replication. Here we identified Nicotiana benthamiana dynamin-related protein 2 (NbDRP2) as an interactor of the MET domain of the PlAMV replicase by co-immunoprecipitation (Co-IP) and mass spectrometry analysis. NbDRP2 is closely related to the DRP2 subfamily proteins in Arabidopsis thaliana, AtDRP2A and AtDRP2B. Confocal microscopy observation and Co-IP confirmed the interaction between the MET domain and NbDRP2. Also, the expression of NbDRP2 was induced by PlAMV infection. PlAMV accumulation was reduced when the expression of NbDRP2 gene was suppressed by virus-induced gene silencing. In addition, PlAMV accumulation was reduced in protoplasts treated with dynamin inhibitor. These results indicate a proviral role of the interaction of NbDRP2 with the MET domain in PlAMV replication.

Unique morphological changes in plant pathogenic phytoplasma-infected petunia flowers are related to transcriptional regulation of floral homeotic genes in an organ-specific manner.

Abnormal flowers are often induced by infection of certain plant pathogens, e.g. phytoplasma, but the molecular mechanisms underlying these malformations have remained poorly understood. Here, we show that infection with OY-W phytoplasma (Candidatus Phytoplasma asteris, onion yellows phytoplasma strain, line OY-W) affects the expression of the floral homeotic genes of petunia plants in an organ-specific manner. Upon infection with OY-W phytoplasma, floral morphological changes, including conversion to leaf-like structures, were observed in sepals, petals and pistils, but not in stamens. As the expression levels of homeotic genes differ greatly between floral organs, we examined the expression levels of homeotic genes in each floral organ infected by OY-W phytoplasma, compared with healthy plants. The expression levels of several homeotic genes required for organ development, such as PFG, PhGLO1 and FBP7, were significantly downregulated by the phytoplasma infection in floral organs, except the stamens, suggesting that the unique morphological changes caused by the phytoplasma infection might result from the significant decrease in expression of some crucial homeotic genes. Moreover, the expression levels of TER, ALF and DOT genes, which are known to participate in floral meristem identity, were significantly downregulated in the phytoplasma-infected petunia meristems, implying that phytoplasma would affect an upstream signaling pathway of floral meristem identity. Our results suggest that phytoplasma infection may have complex effects on floral development, resulting in the unique phenotypes that were clearly distinct from the mutant flower phenotypes produced by the knock-out or the overexpression of certain homeotic genes.

A dual strategy for the suppression of host antiviral silencing: two distinct suppressors for viral replication and viral movement encoded by potato virus M.

Viruses encode RNA silencing suppressors to counteract host antiviral silencing. In this study, we analyzed the suppressors encoded by potato virus M (PVM), a member of the genus Carlavirus. In the conventional green fluorescent protein transient coexpression assay, the cysteine-rich protein (CRP) of PVM inhibited both local and systemic silencing, whereas the triple gene block protein 1 (TGBp1) showed suppressor activity only on systemic silencing. Furthermore, to elucidate the roles of these two suppressors during an active viral infection, we performed PVX vector-based assays and viral movement complementation assays. CRP increased the accumulation of viral RNA at the single-cell level and also enhanced viral cell-to-cell movement by inhibiting RNA silencing. However, TGBp1 facilitated viral movement but did not affect viral accumulation in protoplasts. These data suggest that CRP inhibits RNA silencing primarily at the viral replication step, whereas TGBp1 is a suppressor that acts at the viral movement step. Thus, our findings demonstrate a sophisticated viral infection strategy that suppresses host antiviral silencing at two different steps via two mechanistically distinct suppressors. This study is also the first report of the RNA silencing suppressor in the genus Carlavirus.

Manipulation of Insect Vectors' Host Selection Behavior by Barley Yellow Dwarf Virus Is Dependent on the Host Plant Species and Viral Co-Infection.

Previous studies have shown that vector-borne viruses can manipulate the host selection behavior of insect vectors, yet the tripartite interactions of pathogens, host plants and insect vectors have been documented only in a limited number of pathosystems. Here, we report that the host selection behavior of the insect vector of barley yellow dwarf virus-PAV (BYDV-PAV) and cereal yellow dwarf virus-RPS (CYDV-RPS) is dependent on the host plant species and viral co-infection. This study shows that a model cereal plant, Brachypodium distachyon, is a suitable host plant for examining tripartite interactions with BYDV-PAV and CYDV-RPS. We reveal that BYDV-PAV has a different effect on the host selection behavior of its insect vector depending on the host plant species. Viruliferous aphids significantly prefer non-infected plants to virus-infected wheat plants, whereas viral infection on a novel host plant, B. distachyon, is not implicated in the attraction of either viruliferous or nonviruliferous aphids. Furthermore, our findings show that multiple virus infections of wheat with BYDV-PAV and CYDV-RPS alter the preference of their vector aphid. This result indicates that BYDV-PAV acquisition alters the insect vector's host selection, thereby varying the spread of multiple viruses.

A necrosis-inducing elicitor domain encoded by both symptomatic and asymptomatic Plantago asiatica mosaic virus isolates, whose expression is modulated by virus replication.

Systemic necrosis is the most destructive symptom induced by plant pathogens. We previously identified amino acid 1154, in the polymerase domain (POL) of RNA-dependent RNA polymerase (RdRp) of Plantago asiatica mosaic virus (PlAMV), which affects PlAMV-induced systemic necrosis in Nicotiana benthamiana. By point-mutation analysis, we show that amino acid 1,154 alone is not sufficient for induction of necrotic symptoms. However, PlAMV replicons that can express only RdRp, derived from a necrosis-inducing PlAMV isolate, retain their ability to induce necrosis, and transient expression of PlAMV-encoded proteins indicated that the necrosis-eliciting activity resides in RdRp. Moreover, inducible-overexpression analysis demonstrated that the necrosis was induced in an RdRp dose-dependent manner. In addition, during PlAMV infection, necrotic symptoms are associated with high levels of RdRp accumulation. Surprisingly, necrosis-eliciting activity resides in the helicase domain (HEL), not in the amino acid 1,154-containing POL, of RdRp, and this activity was observed even in HELs of PlAMV isolates of which infection does not cause necrosis. Moreover, HEL-induced necrosis had characteristics similar to those induced by PlAMV infection. Overall, our data suggest that necrotic symptoms induced by PlAMV infection depend on the accumulation of a non-isolate specific elicitor HEL (even from nonnecrosis isolates), whose expression is indirectly regulated by amino acid 1,154 that controls replication.

Surveillance for Sri Lankan cassava mosaic virus (SLCMV) in Cambodia and Vietnam one year after its initial detection in a single plantation in 2015.

Cassava mosaic disease, one of the ten most economically important crop viral diseases in the world, was first reported in Southeast Asia from a single plantation in Cambodia in 2015. To determine the presence and incidence of Sri Lankan cassava mosaic virus (SLCMV) one year after first detection, a total of 6,480 samples from 419 fields were systematically collected from cassava production areas across Cambodia (3,840 samples; 240 fields) and Vietnam (2,640samples; 179 fields) in the 2016 cropping season. Using PCR-based diagnostics, we identified 49 SLCMV-infected plants from nine fields, representing 2% of the total number of fields sampled. Infected fields were geographically restricted to two provinces of Eastern Cambodia, while no infection was detected from any of the other sampled sites in either country. Symptom expression patterns in infected plants suggested that SLCMV may have been transmitted both through infected planting materials, and by Bemisia tabaci, the known whitefly vector of SLCMV. In addition, 14% of virus infected plants did not express typical symptoms of cassava mosaic disease on their leaves, highlighting that molecular-based validation is needed to confirm the presence of SLCMV in the field. None of the owners of the SLCMV-infected fields indicated acquired planting materials from the plantation in Ratanakiri where SLCMV was first reported. The surveillance baseline data generated for both countries is discussed in light of future options to control and manage cassava mosaic disease.

Emerging pests and diseases of South-east Asian cassava: a comprehensive evaluation of geographic priorities, management options and research needs.

Cassava is a major staple, bio-energy and industrial crop in many parts of the developing world. In Southeast Asia, cassava is grown on >4 million ha by nearly 8 million (small-scale) farming households, under (climatic, biophysical) conditions that often prove unsuitable for many other crops. While SE Asian cassava has been virtually free of phytosanitary constraints for most of its history, a complex of invasive arthropod pests and plant diseases has recently come to affect local crops. We describe results from a region-wide monitoring effort in the 2014 dry season, covering 429 fields across five countries. We present geographic distribution and field-level incidence of the most prominent pest and disease invaders, introduce readily-available management options and research needs. Monitoring work reveals that several exotic mealybug and (red) mite species have effectively colonised SE Asia's main cassava-growing areas, occurring in respectively 70% and 54% of fields, at average field-level incidence of 27 ± 2% and 16 ± 2%. Cassava witches broom (CWB), a systemic phytoplasma disease, was reported from 64% of plots, at incidence levels of 32 ± 2%. Although all main pests and diseases are non-natives, we hypothesise that accelerating intensification of cropping systems, increased climate change and variability, and deficient crop husbandry are aggravating both organism activity and crop susceptibility. Future efforts need to consolidate local capacity to tackle current (and future) pest invaders, boost detection capacity, devise locally-appropriate integrated pest management (IPM) tactics, and transfer key concepts and technologies to SE Asia's cassava growers. Urgent action is needed to mobilise regional as well as international scientific support, to effectively tackle this phytosanitary emergency and thus safeguard the sustainability and profitability of one of Asia's key agricultural commodities. © 2016 Society of Chemical Industry.

The phytoplasmal virulence factor TENGU causes plant sterility by downregulating of the jasmonic acid and auxin pathways.

Despite plants infected by pathogens are often unable to produce offspring, it remains unclear how sterility is induced in host plants. In this study, we demonstrate that TENGU, a phytoplasmal virulence peptide known as a dwarfism inducer, acts as an inducer of sterility. Transgenic expression of TENGU induced both male and female sterility in Arabidopsis thaliana flowers similar to those observed in double knockout mutants of auxin response factor 6 (ARF6) and ARF8, which are known to regulate floral development in a jasmonic acid (JA)-dependent manner. Transcripts of ARF6 and ARF8 were significantly decreased in both tengu-transgenic and phytoplasma-infected plants. Furthermore, JA and auxin levels were actually decreased in tengu-transgenic buds, suggesting that TENGU reduces the endogenous levels of phytohormones by repressing ARF6 and ARF8, resulting in impaired flower maturation. TENGU is the first virulence factor with the effects on plant reproduction by perturbation of phytohormone signaling.

Functional characterization and gene expression profiling of superoxide dismutase from plant pathogenic phytoplasma.

The rapid production of huge amounts of reactive oxygen species (ROS) is one of the responses of animal and plant cells induced under stress conditions, such as pathogenic bacterial infection. To protect against the cytotoxic ROS, it is important for pathogenic bacteria to inactivate ROS by employing their antioxidant enzymes like superoxide dismutase (SOD). Here, we cloned and characterized the sodA gene from the plant pathogenic bacterium, 'Candidatus Phytoplasma asteris' OY-W strain. This is the first description of gene expression and antioxidant enzymatic activity of SOD from a phytoplasma. We also demonstrated the sodA gene product (OY-SOD) functions as Mn-type SOD. Since other Mollicutes bacteria such as mycoplasmas do not possess sodA probably due to reductive evolution, it is intriguing that phytoplasmas possess sodA despite their lack of many metabolic genes, suggesting that OY-SOD may play an important role in the phytoplasma colonization of plants and insects. Moreover, Western blot analysis and real-time PCR revealed that OY-SOD is expressed when the phytoplasma is grown in both plant and insect hosts, suggesting it is functioning in both hosts. Possible role of SOD in protection against damage by host-derived ROS is discussed.

Dramatic transcriptional changes in an intracellular parasite enable host switching between plant and insect.

Phytoplasmas are bacterial plant pathogens that have devastating effects on the yields of crops and plants worldwide. They are intracellular parasites of both plants and insects, and are spread among plants by insects. How phytoplasmas can adapt to two diverse environments is of considerable interest; however, the mechanisms enabling the "host switching" between plant and insect hosts are poorly understood. Here, we report that phytoplasmas dramatically alter their gene expression in response to "host switching" between plant and insect. We performed a detailed characterization of the dramatic change that occurs in the gene expression profile of Candidatus Phytoplasma asteris OY-M strain (approximately 33% of the genes change) upon host switching between plant and insect. The phytoplasma may use transporters, secreted proteins, and metabolic enzymes in a host-specific manner. As phytoplasmas reside within the host cell, the proteins secreted from phytoplasmas are thought to play crucial roles in the interplay between phytoplasmas and host cells. Our microarray analysis revealed that the expression of the gene encoding the secreted protein PAM486 was highly upregulated in the plant host, which is also observed by immunohistochemical analysis, suggesting that this protein functions mainly when the phytoplasma grows in the plant host. Additionally, phytoplasma growth in planta was partially suppressed by an inhibitor of the MscL osmotic channel that is highly expressed in the plant host, suggesting that the osmotic channel might play an important role in survival in the plant host. These results also suggest that the elucidation of "host switching" mechanism may contribute to the development of novel pest controls.

Cloning, expression analysis, and sequence diversity of genes encoding two different immunodominant membrane proteins in poinsettia branch-inducing phytoplasma (PoiBI).

Poinsettia branch-inducing phytoplasma (PoiBI) is a phytopathogenic bacterium that infects poinsettia, and is associated with the free-branching morphotype (characterized by many axillary shoots and flowers) of many commercially grown poinsettias. The major membrane proteins of phytoplasmas are classified into three general types, that is, immunodominant membrane protein (Imp), immunodominant membrane protein A (IdpA), and antigenic membrane protein (Amp). These membrane proteins are often used as targets for the production of antibodies used in phytoplasma detection. Herein, we cloned and sequenced the imp and idpA genes of PoiBI strains from 26 commercial poinsettia cultivars. Although the amino acid sequences of the encoded IdpA proteins were invariant, those of the encoded Imp varied among the PoiBI isolates, with no synonymous nucleotide substitution. Western blotting and immunohistochemical analyses revealed that the amount of Imp expressed exceeded that of IdpA, in contrast to the case of a related phytoplasma-disease, western X-disease, for which the major membrane protein appears to be IdpA, not Imp. These results suggest that even phylogenetically close phytoplasmas express different types of major membrane proteins.