Hong Gi Kim

Efficiency of VIGS and gene expression in a novel bipartite potexvirus vector delivery system as a function of strength of TGB1 silencing suppression.

We have developed plant virus-based vectors for virus-induced gene silencing (VIGS) and protein expression, based on Alternanthera mosaic virus (AltMV), for infection of a wide range of host plants including Nicotiana benthamiana and Arabidopsis thaliana by either mechanical inoculation of in vitro transcripts or via agroinfiltration. In vivo transcripts produced by co-agroinfiltration of bacteriophage T7 RNA polymerase resulted in T7-driven AltMV infection from a binary vector in the absence of the Cauliflower mosaic virus 35S promoter. An artificial bipartite viral vector delivery system was created by separating the AltMV RNA-dependent RNA polymerase and Triple Gene Block (TGB)123-Coat protein (CP) coding regions into two constructs each bearing the AltMV 5 and 3 non-coding regions, which recombined in planta to generate a full-length AltMV genome. Substitution of TGB1 L(88)P, and equivalent changes in other potexvirus TGB1 proteins, affected RNA silencing suppression efficacy and suitability of the vectors from protein expression to VIGS.

Insights into Alternanthera mosaic virus TGB3 Functions: Interactions with Nicotiana benthamiana PsbO Correlate with Chloroplast Vesiculation and Veinal Necrosis Caused by TGB3 Over-Expression

Alternanthera mosaic virus (AltMV) triple gene block 3 (TGB3) protein is involved in viral movement. AltMV TGB3 subcellular localization was previously shown to be distinct from that of Potato virus X (PVX) TGB3, and a chloroplast binding domain identified; veinal necrosis and chloroplast vesiculation were observed in Nicotiana benthamiana when AltMV TGB3 was over-expressed from PVX. Plants with over-expressed TGB3 showed more lethal damage under dark conditions than under light. Yeast-two-hybrid analysis and bimolecular fluorescence complementation (BiFC) reveal that Arabidopsis thaliana PsbO1 has strong interactions with TGB3; N. benthamiana PsbO (NbPsbO) also showed obvious interaction signals with TGB3 through BiFC. These results demonstrate an important role for TGB3 in virus cell-to-cell movement and virus-host plant interactions. The Photosystem II oxygen-evolving complex protein PsbO interaction with TGB3 is presumed to have a crucial role in symptom development and lethal damage under dark conditions. In order to further examine interactions between AtPsbO1, NbPsbO, and TGB3, and to identify the binding domain(s) in TGB3 protein, BiFC assays were performed between AtPsbO1 or NbPsbO and various mutants of TGB3. Interactions with C-terminally deleted TGB3 were significantly weaker than those with wild-type TGB3, and both N-terminally deleted TGB3 and a TGB3 mutant previously shown to lose chloroplast interactions failed to interact detectably with PsbO in BiFC. To gain additional information about TGB3 interactions in AltMV-susceptible plants, we cloned 12 natural AltMV TGB3 sequence variants into a PVX expression vector to examine differences in symptom development in N. benthamiana. Symptom differences were observed on PVX over-expression, with all AltMV TGB3 variants showing more severe symptoms than the WT PVX control, but without obvious correlation to sequence differences.

Nucleotide sequence and genomic organization of a newly identified member of the genus Carmovirus, soybean yellow mottle mosaic virus, from soybean

The viral genome of soybean yellow mottle mosaic virus (SYMMV) from infected soybean (Glycine max) in Korea was cloned and sequenced. The complete monopartite single-stranded RNA genome of SYMMV consists of 4009 base pairs with six putative open reading frames and includes 5′- and 3′-untranslated regions of 39 and 229 nucleotides, respectively. The nucleotide and coat protein sequences of SYMMV share the highest sequence identity with those of cowpea mottle virus. Based on its genomic organization, its predicted amino acid sequence, and its phylogenetic relatedness to known carmoviruses, we report that SYMMV is a new member of the genus Carmovirus in the family Tombusviridae.

Arabidopsis TTR1 Causes LRR-Dependent Lethal Systemic Necrosis, rather than Systemic Acquired Resistance, to Tobacco Ringspot Virus

Most Arabidopsis ecotypes display tolerance to the Tobacco ringspot virus (TRSV), but a subset of Arabidopsis ecotypes, including Estland (Est), develop lethal systemic necrosis (LSN), which differs from the localized hypersensitive responses (HRs) or systemic acquired resistance (SAR) characteristic of incompatible reactions. Neither viral replication nor the systemic movement of TRSV was restricted in tolerant or sensitive Arabidopsis ecotypes; therefore, the LSN phenotype shown in the sensitive ecotypes might not be due to viral accumulation. In the present study, we identified the Est TTR1 gene (tolerance to Tobacco ringspot virus 1) encoding a TIR-NBS-LRR protein that controls the ecotype-dependent tolerant/sensitive phenotypes by a map-based cloning method. The tolerant Col-0 ecotype Arabidopsis transformed with the sensitive Est TTR1 allele developed an LSN phenotype upon TRSV infection, suggesting that the Est TTR1 allele is dominant over the tolerant ttr1 allele of Col-0. Multiple sequence alignments of 10 tolerant ecotypes from those of eight sensitive ecotypes showed that 10 LRR amino acid polymorphisms were consistently distributed across the TTR1/ttr1 alleles. Site-directed mutagenesis of these amino acids in the LRR region revealed that two sites, L956S and K1124Q, completely abolished the LSN phenotype. VIGS study revealed that TTR1 is dependent on SGT1, rather than EDS1. The LSN phenotype by TTR1 was shown to be transferred to Nicotiana benthamiana, demonstrating functional conservation of TTR1 across plant families, which are involved in SGT-dependent defense responses, rather than EDS1-dependent signaling pathways.

Soybean mosaic virus Infection and Helper Component-protease Enhance Accumulation of Bean pod mottle virus-Specific siRNAs

Soybean plants infected with Bean pod mottle virus (BPMV) develop acute symptoms that usually decrease in severity over time. In other plant-virus interactions, this type of symptom recovery has been associated with degradation of viral RNAs by RNA silencing, which is accompanied by the accumulation of virus-derived small interfering RNAs (siRNAs). In this study, changes in the accumulation of BPMV siRNAs were investigated in soybean plants infected with BPMV alone, or infected with both BPMV and Soybean mosaic virus (SMV) and in transgenic soybean plants expressing SMV helper component-protease (HC-Pro). In many potyviruses, HC-Pro is a potent suppressor of RNA silencing. In plants infected with BPMV alone, accumulation of siRNAs was positively correlated with symptom severity and accumulation of BPMV genomic RNAs. Plants infected with both BPMV and SMV and BPMV-infected transgenic soybean plants expressing SMV HC-Pro exhibited severe symptoms characteristic of BPMVSMV synergism, and showed enhanced accumulation of BPMV RNAs and siRNAs compared to plants infected with BPMV alone and nontransgenic plants. Likewise, SMV HC-Pro enhanced the accumulation of siRNAs produced from a silenced green fluorescent protein gene in transient expression assays, while the P19 silencing suppressor of Tomato bushy stunt virus did not. Consistent with the modes of action of HC-Pro in other systems, which have shown that HC-Pro suppresses RNA silencing by preventing the unwinding of duplex siRNAs and inhibiting siRNA methylation, these studies showed that SMV HC-Pro interfered with the activities of RNA-induced silencing complexes, but not the activities of Dicer-like enzymes in antiviral defenses.

Biological and molecular characterization of Soybean yellow common mosaic virus, a new species in the genus Sobemovirus

A novel soybean-infecting sobemovirus termed Soybean yellow common mosaic virus (SYCMV) was characterized. The virus has a single, positive-strand RNA genome of 4152 nucleotides. The virus contains four putative open reading frames encoding P1 (78-566 nt), polyprotein ORF2a (524-2248 nt), polymerase domain ORF2b (1852-3417 nt), and CP (3227-4030 nt). The entire nucleotide sequence of SYCMV showed 31.2-71.3% nucleotide identity with the previously known eleven species of sobemovirus. In host range analysis of SYCMV, in which twenty one species and three different Nicotiana tabacum cultivars belonging to seven families were inoculated with the virus, SYCMV had a narrow host range, infecting only Glycine max and G. soja. Based on the obtained sequence, full-length clones of SYCMV were constructed. Symptoms produced by inoculation with clones were indistinguishable from those produced by inoculation with sap from symptomatic plants. Viral RNA accumulation of SYCMV was detected in the upper leaves by Northern blotting. This indicated that full-length clones of SYCMV were sufficient to produce disease symptoms. Genomic organization, the predicted amino acid sequence, and phylogenetic analyses with known sobemoviruses confirmed the assignment of SYCMV as a new member of the genus Sobemovirus.

Development of a Nested Polymerase Chain Reaction Assay for Detection of Colletotrichum acutatum on Symptomless Strawberry Leaves

A nested polymerase chain reaction (PCR) assay was developed for detection of Colletotrichum acutatum on symptomless strawberry leaves. In pure culture, the assay detected as little as 1.0 fg of DNA extracted from mycelium and as few as 1.5 conidia ml-1 when conidial suspensions were sonicated. On detached inoculated leaves, three alternative protocols to dislodge the pathogen were assessed: (i) immersion of whole leaves in 0.05% Tween 20 and manual agitation in plastic bags for 1 min (A); (ii) immersion in Tween 20, sonication for 30 min, then agitation for 1 min (SA); and (iii) freezing for 3 h, incubation for 2 days at 27°C, immersion in Tween 20, then sonication for 30 min and agitation for 1 min (FISA). Each method removed significantly (P ≤ 0.05) more conidia from leaves than the nontreated control; however, removal of appressoria did not vary among assays. In composite samples of noninoculated and inoculated (1.5 ×103 conidia ml-1) strawberry leaves, the nested PCR assay using the FISA protocol detected C. acutatum in as few as 1 infested leaf in 50 noninfested leaves. In a strawberry field, the assay detected the presence of C. acutatum in samples of asymptomatic strawberry leaves, showing potential as a powerful tool for reliable diagnosis of the pathogen in the field.

Virus Disease Incidences and Transmission Ecology of Oriental Melons in Seongju Area

Throughout the years 2008 to 2010, we analyzed approximately two thousand oriental melon samples collected from Seongju, using electron microscopy and testing by RT-PCR using primers specific for eight cucurbit-infecting viruses. Data from RT-PCR indicated that Cucumber green mottle mosaic virus (CGMMV), Watermelon mosaic virus 2 (WMV2) and Zucchini yellow mosaic virus (ZYMV) were present and the other viruses were not detected. Among them, CGMMV and WMV2 were the most prevalent pathogens. CGMMV was thought to infect oriental melon from the early growing season, and reached nearly 100% in the later of growing period. Otherwise, WMV2 emerged from June, several months later compared to CGMMV. CGMMV was detected from all aerial parts of the oriental melon including seeds, but not from the roots of the grafted pumpkin rootstock. Seed of two out of five commercial varieties were shown to be CGMMV positive. Nine varieties of pumpkins used as rootstocks were not infected with CGMMV. When the seedlings of grafted oriental melon were transplanted into pots mixed with the oriental melon debris infected with CGMMV, they were not infected by CGMMV. Cutting of pruning shear and the contact of tendrils contributed 48% and 30% to the transmission of the virus, respectively.

The coat protein of Alternanthera mosaic virus is the elicitor of a temperature-sensitive systemic necrosis in Nicotiana benthamiana, and interacts with a host boron transporter protein

Different isolates of Alternanthera mosaic virus (AltMV; Potexvirus), including four infectious clones derived from AltMV-SP, induce distinct systemic symptoms in Nicotiana benthamiana. Virus accumulation was enhanced at 15 °C compared to 25 °C; severe clone AltMV 3-7 induced systemic necrosis (SN) and plant death at 15 °C. No interaction with potexvirus resistance gene Rx was detected, although SN was ablated by silencing of SGT1, as for other cases of potexvirus-induced necrosis. Substitution of AltMV 3-7 coat protein (CPSP) with that from AltMV-Po (CP(Po)) eliminated SN at 15 °C, and ameliorated symptoms in Alternanthera dentata and soybean. Substitution of only two residues from CP(Po) [either MN(13,14)ID or LA(76,77)IS] efficiently ablated SN in N. benthamiana. CPSP but not CP(Po) interacted with Arabidopsis boron transporter protein AtBOR1 by yeast two-hybrid assay; N. benthamiana homolog NbBOR1 interacted more strongly with CPSP than CP(Po) in bimolecular fluorescence complementation, and may affect recognition of CP as an elicitor of SN.

Control Efficacy of Milk Concentration Against Powdery Mildew of Strawberry

The aim of this study was to determine the effect of milk as one of the environmental friendly materials that substitute chemical fungicides for control powdery mildew (Sphaerotheca aphanis) of strawberries (Fragaria × ananassa Duch.). ``Maehyang`` and ``Akihime`` varieties planted in greenhouses were evaluated for the control of powdery mildew. Applications of 5%, 10% and 20% milk had much better effects on controlling powdery mildew. In particular, 10% milk showed a higher efficacy than other concentrations applied onto strawberry in greenhouse experiments. Foliar spray application of 10% milk was effective for powdery mildew,whereas drench application was not. Also, foliar spray of 10% milk was able to accelerate more firmness and calcium contents of strawberry fruits than the nontreated.The 10% milk applied with fertilizer salts (Ca(H2PO4)2·H2O and KH2PO4) showed there was a similar efficacy to 10% milk alone in greenhouse experiments. White crystals and cracks on strawberry fruits appeared by 20% milk. This result indicated that 10% milk was a useful substitute for fungicides to control powdery mildew of strawberry.