Five Newly Collected Turnip Mosaic Virus (TuMV) Isolates from Jeju Island, Korea are Closely Related to Previously Reported Korean TuMV Isolates but Show Distinctive Symptom Development.

For several years, temperatures in the Korean peninsula have gradually increased due to climate change, resulting in a changing environment for growth of crops and vegetables. An associated consequence is that emerging species of insect vector have caused increased viral transmission. In Jeju Island, Korea, occurrences of viral disease have increased. Here, we report characterization of five newly collected turnip mosaic virus (TuMV) isolates named KBJ1, KBJ2, KBJ3, KBJ4 and KBJ5 from a survey on Jeju Island in 2017. Full-length cDNAs of each isolate were cloned into the pJY vector downstream of cauliflower mosaic virus 35S and bacteriophage T7 RNA polymerase promoters. Their fulllength sequences share 98.9-99.9% nucleotide sequence identity and were most closely related to previously reported Korean TuMV isolates. All isolates belonged to the BR group and infected both Chinese cabbage and radish. Four isolates induced very mild symptoms in Nicotiana benthamiana but KBJ5 induced a hypersensitive response. Symptom differences may result from three amino acid differences uniquely present in KBJ5; Gly(382)Asp, Ile(891)Val, and Lys(2522)Glu in P1, P3, and NIb, respectively.

A Turnip Mosaic Virus Determinant of Systemic Necrosis in Nicotiana benthamiana and a Novel Resistance-Breaking Determinant in Chinese Cabbage Identified from Chimeric Infectious Clones.

Infectious clones of Korean turnip mosaic virus (TuMV) isolates KIH1 and HJY1 share 88.1% genomic nucleotides and 96.4% polyprotein amino acid identity, and they induce systemic necrosis or mild mosaic, respectively, in Nicotiana benthamiana. Chimeric constructs between these isolates exchanged the 5', central, and 3' domains of KIH1 (K) and HJY1 (H), where the order of the letters indicates the origin of these domains. KIH1 and chimeras KHH and KKH induced systemic necrosis, whereas HJY1 and chimeras HHK, HKK, and HKH induced mild symptoms, indicating the determinant of necrosis to be within the 5' 3.9 kb of KIH1; amino acid identities of the included P1, Helper component protease, P3, 6K1, and cylindrical inclusion N-terminal domain were 90.06, 98.91, 93.80, 100, and 100%, respectively. Expression of P1 or P3 from a potato virus X vector yielded symptom differences only between P3 of KIH1 and HJY1, implicating a role for P3 in necrosis in N. benthamiana. Chimera KKH infected Brassica rapa var. pekinensis 'Norang', which was resistant to both KIH1 and HJY1, indicating that two separate TuMV determinants are required to overcome the resistance. Ability of diverse TuMV isolates, chimeras, and recombinants to overcome resistance in breeding lines may allow identification of novel resistance genes.

A single nucleotide change in the overlapping MP and CP reading frames results in differences in symptoms caused by two isolates of Youcai mosaic virus.

Two isolates of Youcai mosaic virus (YoMV) were obtained, and their full-length genomic sequences were determined. Full-length infectious cDNA clones of each isolate were generated in which the viral sequence was under the control of dual T7 and 35S promoters for both in vitro transcript production and agro-infiltration. Comparison of the predicted amino acid sequences of the encoded proteins revealed only four differences between the isolates: three in the RNA-dependent RNA polymerase (RdRp) (V383I and M492I in the 125-kDa protein and T1245M in the 182-kDa protein); and one in the overlapping region of the movement protein (MP) and coat protein (CP) genes, affecting only the N-terminal domain of CP (CP M17T). One of the isolates caused severe symptoms in Nicotiana benthamiana plants, while the other caused only mild symptoms. In order to identify the amino acid residues associated with symptom severity, chimeric constructs were generated by combining parts of the two infectious YoMV clones, and the symptoms in infected plants were compared to those induced by the parental isolates. This allowed us to conclude that the M17T substitution in the N-terminal domain of CP was responsible for the difference in symptom severity. The M17T variation was found to be unique among characterized YoMV isolates. A difference in potential post-translational modification resulting from the presence of a predicted casein kinase II phosphorylation site only in the CP of isolate HK2 may be responsible for the symptom differences.

Sequence Variations Among 17 New Radish Isolates of Turnip mosaic virus Showing Differential Pathogenicity and Infectivity in Nicotiana benthamiana, Brassica rapa, and Raphanus sativus.

Infectious clones were generated from 17 new Korean radish isolates of Turnip mosaic virus (TuMV). Phylogenetic analysis indicated that all new isolates, and three previously characterized Korean radish isolates, belong to the basal-BR group (indicating that the pathotype can infect both Brassica and Raphanus spp.). Pairwise analysis revealed genomic nucleotide and polyprotein amino acid identities of >87.9 and >95.7%, respectively. Five clones (HJY1, HJY2, KIH2, BE, and prior isolate R007) had lower sequence identities than other isolates and produced mild symptoms in Nicotiana benthamiana. These isolates formed three distinct sequence classes (HJY1/HJY2/R007, KIH2, and BE), and several differential amino acid residues (in P1, P3, 6K2, and VPg) were present only in mild isolates HJY1, HJY2, and R007. The remaining isolates all induced systemic necrosis in N. benthamiana. Four mild isolates formed a phylogenetic subclade separate from another subclade including all of the necrosis-inducing isolates plus mild isolate KIH2. Symptom severity in radish and Chinese cabbage genotypes was not correlated with pathogenicity in N. benthamiana; indeed, Chinese cabbage cultivar Norang was not infected by any isolate, whereas Chinese cabbage cultivar Chusarang was uniformly susceptible. Four isolates were unable to infect radish cultivar Iljin, but no specific amino acid residues were correlated with avirulence. These results may lead to the identification of new resistance genes against TuMV.

Generation of an Infectious Clone of a New Korean Isolate of Apple chlorotic leaf spot virus Driven by Dual 35S and T7 Promoters in a Versatile Binary Vector.

The full-length sequence of a new isolate of Apple chlorotic leaf spot virus (ACLSV) from Korea was divergent, but most closely related to the Japanese isolate A4, at 84% nucleotide identity. The full-length cDNA of the Korean isolate of ACLSV was cloned into a binary vector downstream of the bacteriophage T7 RNA promoter and the Cauliflower mosaic virus 35S promoter. Chenopodium quinoa was successfully infected using in vitro transcripts synthesized using the T7 promoter, detected at 20 days post inoculation (dpi), but did not produce obvious symptoms. Nicotiana occidentalis and C. quinoa were inoculated through agroinfiltration. At 32 dpi the infection rate was evaluated; no C. quinoa plants were infected by agroinfiltration, but infection of N. occidentalis was obtained.

Complete nucleotide sequences and construction of full-length infectious cDNA clones of cucumber green mottle mosaic virus (CGMMV) in a versatile newly developed binary vector including both 35S and T7 promoters.

Seed-transmitted viruses have caused significant damage to watermelon crops in Korea in recent years, with cucumber green mottle mosaic virus (CGMMV) infection widespread as a result of infected seed lots. To determine the likely origin of CGMMV infection, we collected CGMMV isolates from watermelon and melon fields and generated full-length infectious cDNA clones. The full-length cDNAs were cloned into newly constructed binary vector pJY, which includes both the 35S and T7 promoters for versatile usage (agroinfiltration and in vitro RNA transcription) and a modified hepatitis delta virus ribozyme sequence to precisely cleave RNA transcripts at the 3' end of the tobamovirus genome. Three CGMMV isolates (OMpj, Wpj, and Mpj) were separately evaluated for infectivity in Nicotiana benthamiana, demonstrated by either Agroinfiltration or inoculation with in vitro RNA transcripts. CGMMV nucleotide identities to other tobamoviruses were calculated from pairwise alignments using DNAMAN. CGMMV identities were 49.89% to tobacco mosaic virus; 49.85% to pepper mild mottle virus; 50.47% to tomato mosaic virus; 60.9% to zucchini green mottle mosaic virus; and 60.96% to kyuri green mottle mosaic virus, confirming that CGMMV is a distinct species most similar to other cucurbit-infecting tobamoviruses. We further performed phylogenetic analysis to determine relationships of our new Korean CGMMV isolates to previously characterized isolates from Canada, China, India, Israel, Japan, Korea, Russia, Spain, and Taiwan available from NCBI. Analysis of CGMMV amino acid sequences showed three major clades, broadly typified as 'Russian,' 'Israeli,' and 'Asian' groups. All of our new Korean isolates fell within the 'Asian' clade. Neither the 128 nor 186 kDa RdRps of the three new isolates showed any detectable gene silencing suppressor function.

Photopatterning of cell-adhesive-modified poly(ethyleneimine) for guided neuronal growth.

We describe photopatterning technique that employs the photodegradation of cell-adhesive-modified poly(ethyleneimine) (m-PEI) to fabricate precise micropatterns on the indium tin oxide (ITO) substrate for guided neuronal growth. The photodegradation of m-PEI coated on hydroxyl group-terminated ITO substrate created micropatterns over a large area through deep UV irradiation. The photopatterned m-PEI layer can effectively guide neurite outgrowth and control neurite extensions from individual neurons.