Identification of a novel viral factor inducing tumorous symptoms by disturbing vascular development in planta.

Plant viruses induce various disease symptoms that substantially impact agriculture, but the underlying mechanisms of viral disease in plants are poorly understood. Kobu-sho is a disease in gentian that shows gall formation with ectopic development of lignified cells and vascular tissues such as xylem. Here, we show that a gene fragment of gentian Kobu-sho-associated virus, which is designated as Kobu-sho-inducing factor (KOBU), induces gall formation accompanied by ectopic development of lignified cells and xylem-like tissue in Nicotiana benthamiana. Transgenic gentian expressing KOBU exhibited tumorous symptoms, confirming the gall-forming activity of KOBU. Surprisingly, KOBU expression can also induce differentiation of an additional leaf-like tissue on the abaxial side of veins in normal N. benthamiana and gentian leaves. Transcriptome analysis with Arabidopsis thaliana expressing KOBU revealed that KOBU activates signaling pathways that regulate xylem development. KOBU protein forms granules and plate-like structures and co-localizes with mRNA splicing factors within the nucleus. Our findings suggest that KOBU is a novel pleiotropic virulence factor that stimulates vascular and leaf development. IMPORTANCE While various mechanisms determine disease symptoms in plants depending on virus-host combinations, the details of how plant viruses induce symptoms remain largely unknown in most plant species. Kobu-sho is a disease in gentian that shows gall formation with ectopic development of lignified cells and vascular tissues such as xylem. Our findings demonstrate that a gene fragment of gentian Kobu-sho-associated virus (GKaV), which is designated as Kobu-sho-inducing factor, induces the gall formation accompanied by the ectopic development of lignified cells and xylem-like tissue in Nicotiana benthamiana. The molecular mechanism by which gentian Kobu-sho-associated virus induces the Kobu-sho symptoms will provide new insight into not only plant-virus interactions but also the regulatory mechanisms underlying vascular and leaf development.

Microbial Diversity in the Phyllosphere and Rhizosphere of an Apple Orchard Managed under Prolonged "Natural Farming" Practices.

Microbial diversity in an apple orchard cultivated with natural farming practices for over 30 years was compared with conventionally farmed orchards to analyze differences in disease suppression. In this long-term naturally farmed orchard, major apple diseases were more severe than in conventional orchards but milder than in a short-term natural farming orchard. Among major fungal species in the phyllosphere, we found that Aureobasidium pullulans and Cryptococcus victoriae were significantly less abundant in long-term natural farming, while Cladosporium tenuissimum predominated. However, diversity of fungal species in the phyllosphere was not necessarily the main determinant in the disease suppression observed in natural farming; instead, the maintenance of a balanced, constant selection of fungal species under a suitable predominant species such as C. tenuissimum seemed to be the important factors. Analysis of bacteria in the phyllosphere revealed Pseudomonas graminis, a potential inducer of plant defenses, predominated in long-term natural farming in August. Rhizosphere metagenome analysis showed that Cordyceps and Arthrobotrys, fungal genera are known to include insect- or nematode-infecting species, were found only in long-term natural farming. Among soil bacteria, the genus Nitrospira was most abundant, and its level in long-term natural farming was more than double that in the conventionally farmed orchard.

Chemical Synthesis of Sialyl N-Glycans and Analysis of Their Recognition by Neuraminidase.

The chemical synthesis of a fully sialylated tetraantennary N-glycan has been achieved for the first time by using the diacetyl strategy, in which NHAc is protected as NAc2 to improve reactivity by preventing intermolecular hydrogen bonds. Another key was the glycosylation to the branched mannose in an ether solvent, which promoted the desired glycosylation by stabilizing the oxocarbenium ion intermediate. Furthermore, high α-selectivity of these glycosylation reactions was realized by utilizing remote participation. Two asymmetrically deuterium labeled sialyl N-glycans were also synthesized by the same strategy. The synthesized N-glycans were used to probe the molecular basis of H1N1 neuraminidase recognition. The asymmetrically deuterated N-glycans revealed a difference in the recognition of sialic acid on each branch. Meanwhile, the tetraantennary N-glycan was used to evaluate the effects of multivalency and steric hinderance by forming branching structures.

Recent Advances in the Chemical Biology of N-Glycans.

Asparagine-linked N-glycans on proteins have diverse structures, and their functions vary according to their structures. In recent years, it has become possible to obtain high quantities of N-glycans via isolation and chemical/enzymatic/chemoenzymatic synthesis. This has allowed for progress in the elucidation of N-glycan functions at the molecular level. Interaction analyses with lectins by glycan arrays or nuclear magnetic resonance (NMR) using various N-glycans have revealed the molecular basis for the recognition of complex structures of N-glycans. Preparation of proteins modified with homogeneous N-glycans revealed the influence of N-glycan modifications on protein functions. Furthermore, N-glycans have potential applications in drug development. This review discusses recent advances in the chemical biology of N-glycans.

Identification and characterization of a tombusvirus isolated from Japanese gentian.

The DECS (dsRNA isolation, exhaustive amplification, cloning and sequencing) analysis technique for viral diagnosis detected a tombusvirus in Japanese gentian not displaying severe symptoms. We tentatively named this virus "gentian virus A" (GeVA). GeVA systemically but inefficiently infected Japanese gentian without causing visible symptoms, while it led to severe symptoms in some other plants. The complete genome sequence of GeVA indicated a typical tombusvirus-like structure. Phylogenetic analysis of the deduced amino acid sequences of four tombusvirus-encoded proteins did not reveal other known tombusviruses that were closely-related to GeVA, suggesting that it is a novel tombusvirus.

Horizontal pollen transmission of Gentian ovary ring-spot virus is initiated during penetration of the stigma and style by infected pollen tubes.

Gentian ovary ring-spot virus (GORV) infected gentian plants by pollination with GORV-infected gentian pollen grains, but the virus was not horizontally transmitted to gentian plants by transfer of pollen from GORV-infected Nicotiana benthamiana plants. However, N. benthamiana plants were infected with the virus by pollination with infected gentian pollen as well as by pollination with infected N. benthamiana pollen. When infected gentian pollen grains were placed on N. benthamiana stigmas, germinating pollen tubes penetrated into the stigmas and the styles (stigma-style). Virus infection occurred during penetration of the stigma-style, and the virus subsequently spread systemically to the mother plant. On the other hand, most infected N. benthamiana pollen grains failed to germinate on gentian stigmas, and virus infections were not detected in the stigma-style.