Actin Cytoskeleton and Golgi Involvement in Barley stripe mosaic virus Movement and Cell Wall Localization of Triple Gene Block Proteins.

Barley stripe mosaic virus (BSMV) induces massive actin filament thickening at the infection front of infected Nicotiana benthamiana leaves. To determine the mechanisms leading to actin remodeling, fluorescent protein fusions of the BSMV triple gene block (TGB) proteins were coexpressed in cells with the actin marker DsRed: Talin. TGB ectopic expression experiments revealed that TGB3 is a major elicitor of filament thickening, that TGB2 resulted in formation of intermediate DsRed:Talin filaments, and that TGB1 alone had no obvious effects on actin filament structure. Latrunculin B (LatB) treatments retarded BSMV cell-to-cell movement, disrupted actin filament organization, and dramatically decreased the proportion of paired TGB3 foci appearing at the cell wall (CW). BSMV infection of transgenic plants tagged with GFP-KDEL exhibited membrane proliferation and vesicle formation that were especially evident around the nucleus. Similar membrane proliferation occurred in plants expressing TGB2 and/or TGB3, and DsRed: Talin fluorescence in these plants colocalized with the ER vesicles. TGB3 also associated with the Golgi apparatus and overlapped with cortical vesicles appearing at the cell periphery. Brefeldin A treatments disrupted Golgi and also altered vesicles at the CW, but failed to interfere with TGB CW localization. Our results indicate that actin cytoskeleton interactions are important in BSMV cell-to-cell movement and for CW localization of TGB3

Characterization of Developmental- and Stress-Mediated Expression of Cinnamoyl-CoA Reductase in Kenaf ( Hibiscus cannabinus

Cinnamoyl-CoA reductase (CCR) is an important enzyme for lignin biosynthesis as it catalyzes the first specific committed step in monolignol biosynthesis. We have cloned a full length coding sequence of CCR from kenaf (Hibiscus cannabinus L.), which contains a 1,020-bp open reading frame (ORF), encoding 339 amino acids of 37.37 kDa, with an isoelectric point (pI) of 6.27 (JX524276, HcCCR2). BLAST result found that it has high homology with other plant CCR orthologs. Multiple alignment with other plant CCR sequences showed that it contains two highly conserved motifs: NAD(P) binding domain (VTGAGGFIASWMVKLLLEKGY) at N-terminal and probable catalytic domain (NWYCYGK). According to phylogenetic analysis, it was closely related to CCR sequences of Gossypium hirsutum (ACQ59094) and Populus trichocarpa (CAC07424). HcCCR2 showed ubiquitous expression in various kenaf tissues and the highest expression was detected in mature flower. HcCCR2 was expressed differentially in response to various stresses, and the highest expression was observed by drought and NaCl treatments

Comparative transcriptional analysis of caffeoyl-coenzyme A 3-O-methyltransferase from Hibiscus cannabinus L., during developmental stages in various tissues and stress regulation,”

Abstract We have cloned a full-length gene, putatively encoding for caffeoyl-coenzyme A 3-O-methyltransferase (CCoAOMT), an important enzyme involved in lignin biosynthesis, from kenaf (Hibiscus cannabinus L.). Herein, we investigated the expression pattern of a CCoAOMT orthologue from various tissues and organs during development, and in response to different environmental cues. The full-length CCoAOMT orthologue of kenaf consists of a 744 bp open reading frame (ORF), encoding for 247 amino acids of 27.91 kDa and an isoelectric point (pI) of 5.43. The deduced amino acids of CCoAOMT evidenced a high degree of identity (up to 84%) with other plant CCoAOMT sequences. Phylogenetic analysis demonstrated its close relationship with the CCoAOMT of Gossypium hirsutum (ACQ59096). Kenaf CCoAOMT harbors eight highly conserved motifs: A, B, and C are putative S-adenosylmethioine (SAM)-binding motifs and D, E, F, G, and H are CCoAOMT signature motifs. According to quantitative real-time reverse transcriptase polymerase chain reaction (q-PCR) analysis, the kenaf CCoAOMT transcript was detected in all plant tissues and organs, whereas the highest expression was noted in mature flower tissues, which indicates that it might be involved in the flower development or in the biosynthesis of flower specific compound. All the treatments highly induced the expression of CCoAOMT transcripts in the stems of 3-week-old kenaf, which indicates that it might have a role in stress regulatory pathway. Among the treatments, the cold and H 2 O 2 -treated samples evidenced the highest levels of expression at 6 and 24 h after treatment, respectively, whereas the wounded and NaCl-treated samples evidenced lower expression levels, which suggest that different signaling networks are involved for stress mediated up regulation of HcCCoAOMT transcripts. The highest transcript level of CCoAOMT was detected at either early (within 12 h of treatments) or intermediate (24 h after treatments) time points of treatments, except drought treated sample. Early induction was observed in the case of H 2 O 2 and SA (salicylic acid), and intermediate induction occurring as the result of wounding, NaCl, cold and ABA (abscisic acid). Whereas drought treated sample showed highest expression at seven days after treatment. MeJA (methyl jasmonic acid) treatment showed a complex biphasic expression which is different from others. In summary, we have cloned and characterized a full-length gene putatively encoding for CCoAOMT, which also showed stress responsive differential expression

Characterization of Developmental-and Stress-Mediated Expression of Cinnamoyl-CoA Reductase in Kenaf (Hibiscus cannabinus L.)

Cinnamoyl-CoA reductase (CCR) is an important enzyme for lignin biosynthesis as it catalyzes the first specific committed step in monolignol biosynthesis. We have cloned a full length coding sequence of CCR from kenaf (Hibiscus cannabinus L.), which contains a 1,020-bp open reading frame (ORF), encoding 339 amino acids of 37.37 kDa, with an isoelectric point (pI) of 6.27 (JX524276, HcCCR2). BLAST result found that it has high homology with other plant CCR orthologs. Multiple alignment with other plant CCR sequences showed that it contains two highly conserved motifs: NAD(P) binding domain (VTGAGGFIASWMVKLLLEKGY) at N-terminal and probable catalytic domain (NWYCYGK). According to phylogenetic analysis, it was closely related to CCR sequences of Gossypium hirsutum (ACQ59094) and Populus trichocarpa (CAC07424). HcCCR2 showed ubiquitous expression in various kenaf tissues and the highest expression was detected in mature flower. HcCCR2 was expressed differentially in response to various stresses, and the highest expression was observed by drought and NaCl treatments

Soybean Mosaic Virus Hc-Pro Enhances Viral Symptoms, Bean Pod Mottle Virus and Transgene RNA Stability, siRNA Accumulation, and Reduces Soybean Seed Production

149 p.Thesis (Ph.D.)--University of Illinois at Urbana-Champaign, 2004.The interactions between plant viruses and their hosts are constantly evolving. When plants produce a defense system against viral infection, viruses develop means to overcome this host response. Soybean [Glycine max (L.) Merr.] plants infected with Bean pod mottle virus (BPMV) alone develop acute symptoms that nearly disappear as plants grow. If BPMV infection is accompanied with Soybean mosaic virus (SMV), then severe symptoms develop. Plant recovery from a single infection by BPMV has been linked to degradation of viral RNA by post-transcriptional gene silencing (PTGS), which is characterized by the accumulation of small interfering RNAs (siRNAs). In soybean plants infected with BPMV alone, siRNA accumulation was correlated with accumulation of BPMV genomic RNAs, but siRNA were not detected in plants that recovered from symptom. Soybean plants infected with BPMV and SMV accumulated high levels of BPMV RNA and BPMV siRNAs. Similarly, BPMV-infected transgenic plants expressing SMV helper component protease (HC-Pro) at high levels had severe symptoms and enhanced accumulation of BPMV RNA and siRNAs. Transgenic lines expressing high levels of SMV HC-Pro mRNA had altered unifoliate leaf morphologies and reduced seed production. When inoculated with three SMV strains, the severity of initial symptoms was directly related to the levels of HC-Pro mRNA accumulation in the transgenic plants. i.e, plants expressing the lowest level of HC-Pro mRNA showed mild initial symptoms, and plants accumulating the highest level of SMV HC-Pro initially showed very severe symptoms. Later, symptoms disappeared and SMV titers were greatly reduced. The accumulations of SMV-specific siRNAs were related to the levels of viral genomic RNA accumulation, and siRNA diminished as symptoms disappeared in SMV infected transgenic lines. Tobacco ring spot virus (TRSV) induced a hypersensitive reaction when inoculated on transgenic plants that expressed G2 HC-Pro, but not in transgenic plants expressing G5 HC-Pro. SMV HC-Pro enhanced the recovery of hygromycin-resistant somatic embryos from immature soybean cotyledonary explants co-cultured with Agrobacterium . Expression of SMV HC-Pro in transgenic plants partially stabilized the expression of a beta-glucuronidase (GUS) transgene that was susceptible to PTGS. SMV infection stabilized GUS gene expression, enzyme activity, and enhanced GUS siRNA accumulation.U of I OnlyRestricted to the U of I community idenfinitely during batch ingest of legacy ETD

Soybean Mosaic Virus Hc-Pro Enhances Viral Symptoms, Bean Pod Mottle Virus and Transgene RNA Stability, siRNA Accumulation, and Reduces Soybean Seed Production

149 p.Thesis (Ph.D.)--University of Illinois at Urbana-Champaign, 2004.The interactions between plant viruses and their hosts are constantly evolving. When plants produce a defense system against viral infection, viruses develop means to overcome this host response. Soybean [Glycine max (L.) Merr.] plants infected with Bean pod mottle virus (BPMV) alone develop acute symptoms that nearly disappear as plants grow. If BPMV infection is accompanied with Soybean mosaic virus (SMV), then severe symptoms develop. Plant recovery from a single infection by BPMV has been linked to degradation of viral RNA by post-transcriptional gene silencing (PTGS), which is characterized by the accumulation of small interfering RNAs (siRNAs). In soybean plants infected with BPMV alone, siRNA accumulation was correlated with accumulation of BPMV genomic RNAs, but siRNA were not detected in plants that recovered from symptom. Soybean plants infected with BPMV and SMV accumulated high levels of BPMV RNA and BPMV siRNAs. Similarly, BPMV-infected transgenic plants expressing SMV helper component protease (HC-Pro) at high levels had severe symptoms and enhanced accumulation of BPMV RNA and siRNAs. Transgenic lines expressing high levels of SMV HC-Pro mRNA had altered unifoliate leaf morphologies and reduced seed production. When inoculated with three SMV strains, the severity of initial symptoms was directly related to the levels of HC-Pro mRNA accumulation in the transgenic plants. i.e, plants expressing the lowest level of HC-Pro mRNA showed mild initial symptoms, and plants accumulating the highest level of SMV HC-Pro initially showed very severe symptoms. Later, symptoms disappeared and SMV titers were greatly reduced. The accumulations of SMV-specific siRNAs were related to the levels of viral genomic RNA accumulation, and siRNA diminished as symptoms disappeared in SMV infected transgenic lines. Tobacco ring spot virus (TRSV) induced a hypersensitive reaction when inoculated on transgenic plants that expressed G2 HC-Pro, but not in transgenic plants expressing G5 HC-Pro. SMV HC-Pro enhanced the recovery of hygromycin-resistant somatic embryos from immature soybean cotyledonary explants co-cultured with Agrobacterium . Expression of SMV HC-Pro in transgenic plants partially stabilized the expression of a beta-glucuronidase (GUS) transgene that was susceptible to PTGS. SMV infection stabilized GUS gene expression, enzyme activity, and enhanced GUS siRNA accumulation.U of I OnlyRestricted to the U of I community idenfinitely during batch ingest of legacy ETD

Deep Sequencing Analysis of Apple Infecting Viruses in Korea

Deep sequencing has generated 52 contigs derived from five viruses; Apple chlorotic leaf spot virus (ACLSV), Apple stem grooving virus (ASGV), Apple stem pitting virus (ASPV), Apple green crinkle associated virus (AGCaV), and Apricot latent virus (ApLV) were identified from eight apple samples showing small leaves and/or growth retardation. Nucleotide (nt) sequence identity of the assembled contigs was from 68% to 99% compared to the reference sequences of the five respective viral genomes. Sequences of ASPV and ASGV were the most abundantly represented by the 52 contigs assembled. The presence of the five viruses in the samples was confirmed by RT-PCR using specific primers based on the sequences of each assembled contig. All five viruses were detected in three of the samples, whereas all samples had mixed infections with at least two viruses. The most frequently detected virus was ASPV, followed by ASGV, ApLV, ACLSV, and AGCaV which were withal found in mixed infections in the tested samples. AGCaV was identified in assembled contigs ID 1012480 and 93549, which showed 82% and 78% nt sequence identity with ORF1 of AGCaV isolate Aurora-1. ApLV was identified in three assembled contigs, ID 65587, 1802365, and 116777, which showed 77%, 78%, and 76% nt sequence identity respectively with ORF1 of ApLV isolate LA2. Deep sequencing assay was shown to be a valuable and powerful tool for detection and identification of known and unknown virome in infected apple trees, here identifying ApLV and AGCaV in commercial orchards in Korea for the first time

Characteristics of a Lettuce mosaic virus Isolate Infecting Lettuce in Korea

Lettuce mosaic virus (LMV) causes disease of plants in the family Asteraceae, especially lettuce crops. LMV isolates have previously been clustered in three main groups, LMV-Yar, LMV-Greek and LMVRoW. The first two groups, LMV-Yar and LMV-Greek, have similar characteristics such as no seed-borne transmission and non-resistance-breaking. The latter one, LMV-RoW, comprising a large percentage of the LMV isolates contains two large subgroups, LMV-Common and LMV-Most. To date, however, no Korean LMV isolate has been classified and characterized. In this study, LMV-Muju, the Korean LMV isolate, was isolated from lettuce showing pale green and mottle symptoms, and its complete genome sequence was determined. Classification method of LMV isolates based on nucleotide sequence divergence of the NIb-CP junction showed that LMV-Muju was categorized as LMV-Common. LMV-Muju was more similar to LMV-O (LMV-Common subgroup) than to LMV-E (LMV-RoW group but not LMV-Common subgroup) even in the amino acid domains of HC-Pro associated with pathogenicity, and in the CI and VPg regions related to ability to overcome resistance. Taken together, LMV-Muju belongs to the LMV-Common subgroup, and is expected to be a seed-borne, non-resistance-breaking isolate. According to our analysis, all other LMV isolates not previously assigned to a subgroup were also included in the LMV-RoW group