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In JoVE (1)
Other Publications (8)
Articles by Kajohn Boonrod in JoVE
JoVE General
Homemade Site Directed Mutagenesis of Whole Plasmids
1Department of Biology, Johannes Gutenberg-University Mainz, Germany, 2Proteomics division, AlPlanta, Neustadt an der Weinstrasse, Germany
Site directed mutagenesis of whole plasmids is a simple way to create slightly different variations of an original plasmid. Here we demonstrate an easy and cost effective way to introduce base substitutions into a plasmid using standard reagents.
Other articles by Kajohn Boonrod on PubMed
Single-chain Antibodies Against a Plant Viral RNA-dependent RNA Polymerase Confer Virus Resistance
Crop loss due to viral diseases is still a major problem for agriculture today. We present a strategy to achieve virus resistance based on the expression of single-chain Fv fragments (scFvs) against a conserved domain in a plant viral RNA-dependent RNA polymerase (RdRp), a key enzyme in virus replication. The selected scFvs inhibited complementary RNA synthesis of different plant virus RdRps in vitro and virus replication in planta. Moreover, the scFvs also bound to the RdRp of the distantly related hepatitis C virus. T(1) and T(2) progeny of transgenic lines of Nicotiana benthamiana expressing different scFvs either in the cytosol or in the endoplasmic reticulum showed varying degrees of resistance against four plant viruses from different genera, three of which belong to the Tombusviridae family. Virus resistance based on antibodies to RdRps adds another tool to the repertoire for combating plant viruses.
Prevalence and Molecular Characterization of Human and Bovine Cryptosporidium Isolates in Thailand
This study was performed to determine the prevalence and genotypes of Cryptosporidium species among HIV patients and cattle in Thailand. Stool specimens were collected from 46 HIV patients from Prabat Nampu Temple, Lop Buri Province in central Thailand. Two hundred fecal samples from dairy cattle were collected from seven farms in Chon Buri Province, the eastern part of Thailand. Each sample was concentrated by Sheather's sucrose flotation technique and stained by acid fast stain (AFS) for the identification of oocysts by microscopy. All HIV stool samples and 83 fecal specimens from cattle were further tested using nested polymerase chain reaction (PCR) targeting the 18S SSUrRNA gene to characterize the detected species. In HIV patient samples, the detection rate was 28.7% by AFS and 4.35% by nested PCR. In cattle samples, the detection rate was 13% by AFS and 9.63% by nested PCR. After DNA sequencing results, we identified the genotypes of the Cryptosporidium from seven of the PCR positive samples. All were found to be C. parvum. The findings presented here represent the first genetic identification of Cryptosporidium species in cattle in Thailand.
Analysis of an Autoproteolytic Activity of Rice Yellow Mottle Virus Silencing Suppressor P1
Ectopically expressed rice yellow mottle virus P1 fusion proteins were found to be cleaved in planta and in Escherichia coli. Cleavage takes place in the absence of bacterial protease activity, indicating that the P1 fusion is autocatalytically processed independently of host factors. N-terminal sequencing of the C-terminal cleavage product of transiently expressed P1/GFP (green fluorescence protein) in Nicotiana benthamiana showed that the cleavage site is located between the first two amino acids (aa) downstream of the P1 sequence. Mutagenesis experiments revealed that a phenylalanine to valine substitution at position 157 of the P1 aa sequence impairs proper cleavage, which is nearly unaffected by replacement of phenylalanine with tyrosine. Deletion of methionine(159) (first GFP aa residue) appeared to not affect P1/GFP cleavage. N-terminal P1-tagging with GFP turned out to impair autocleavage, whereas a small His-tag could not fully prevent cleavage. Additionally, a modified P1/GFP carrying an N-terminal deletion of 81 aa was not cleaved. These findings indicate that this region is involved in the proteolysis mechanism and that large N-terminal fusion partners might affect correct folding of the P1 necessary for self-catalysis.
First Description of Cryptosporidium Bovis in Japan and Diagnosis and Genotyping of Cryptosporidium Spp. in Diarrheic Pre-weaned Calves in Hokkaido
Eighty fecal samples from pre-weaned calves with diarrhea were collected in the Tokachi area in Northern Japan to investigate the prevalence of Cryptosporidium species in such animals. Oocysts from fecal samples collected from each animal were concentrated using sucrose gradient centrifugation. Genomic DNA was extracted from each sample and processed by nested PCR to amplify the partial SSU rRNA gene of Cryptosporidium. Cryptosporidium infections were detected in 75% of the samples. Sequence analysis was performed on all positive samples. Phylogenetic analysis of 33 successfully sequenced isolates of the SSUrRNA PCR products revealed all but one were Cryptosporidium parvum infections. The remaining single case was Cryptosporidium bovis. These findings suggest that C. parvum is prevalent in diarrheic pre-weaned calves and can be a source of cryptosporidial infections for humans and animals in Hokkaido.
Expression, Purification and Functional Characterization of Recombinant Zucchini Yellow Mosaic Virus HC-Pro
HC-Pro is a helper component-proteinase which acts as a multifunctional protein in the potyviral life cycle. Apart from its proteolytic activity, HC-Pro has the capacity to bind duplex small RNAs (sRNAs). To investigate HC-Pro-mediated sRNA binding in vitro, high amounts of purified protein are required. For this purpose, the Zucchini yellow mosaic virus (ZYMV) HC-Pro was expressed as a fusion with hexa-histidine (6xHis) or maltose-binding protein (MBP) in Escherichia coli. The expressed fusion proteins were purified by affinity chromatography. 6xHis:HC-Pro and MBP:HC-Pro were partially soluble. Electrophoretic mobility-shift assays demonstrated that only MBP:HC-Pro exhibits the sRNA binding activity. The recombinant HC-Pro bound 21 bp siRNAs as well as 19 bp and 24 bp siRNAs. A point mutation in the highly conserved FRNK box produced the HC-Pro(FINK) protein, previously shown to be associated with reduced viral symptoms and weak sRNA binding. In this study, sRNA binding of the MBP:HA-HC-Pro(FINK) was not detectable. The high yield of purified HC-Pro offers the possibility to study the biochemistry of the protein in detail.
The Helper Component-proteinase of the Zucchini Yellow Mosaic Virus Inhibits the Hua Enhancer 1 Methyltransferase Activity in Vitro
The helper component-proteinase (HC-Pro) is a multifunctional protein found among potyviruses. With respect to its silencing suppressor function, small RNA binding appears to be the major activity of HC-Pro. HC-Pro could also exhibit other suppressor activities. HC-Pro may inhibit the Hua Enhancer 1 (HEN1) activity. There is indirect evidence showing that either transient or stable expression of HC-Pro in plants results in an increase of non-methylated small RNAs. Here, we demonstrated that recombinant Zucchini yellow mosaic virus (ZYMV) HC-Pro inhibited the methyltransferase activity of HEN1 in vitro. Moreover, we found that the HC-Pro(FINK) mutant, which has lost small RNA-binding activity, inhibited HEN1 activity, while the truncated proteins and total soluble bacterial proteins did not. Using the ELISA-binding assay, we provided evidence that the HC-Pro(FRNK) wild-type and HC-Pro(FINK) both bound to HEN1, with HC-Pro(FRNK) binding stronger than HC-Pro(FINK). Motif mapping analysis revealed that the amino acids located between positions 139 and 320 of ZYMV HC-Pro were associated with HEN1 interaction.
Diverse Spontaneous Silencing of a Transgene Among Two Nicotiana Species
In plants, transgenes frequently become spontaneously silenced for unknown reasons. Typically, transgene silencing involves the generation of small interfering RNAs (siRNAs) that directly or indirectly target cognate DNA and mRNA sequences for methylation and degradation, respectively. In this report, we compared spontaneous silencing of a transgene in Nicotiana benthamiana and Nicotiana tabacum. In both species, abundant siRNAs were produced. In N. benthamiana, the self-silencing process involved mRNA degradation and dense DNA methylation of the homologous coding region. In N. tabacum, self-silencing occurred without complete mRNA degradation and with low methylation of the cognate coding region. Our data indicated that in plants, siRNA-mediated spontaneous silencing is, in addition to mRNA degradation, based on translational inhibition. Differences in the initiation and establishment of self-silencing together with marked differences in the degree of de novo DNA methylation showed that the mechanistic details of RNA silencing, although largely conserved, may vary also in genetically close plant species.
Analysis of the Autoproteolytic Activity of the Recombinant Helper Component Proteinase from Zucchini Yellow Mosaic Virus
The multifunctional helper component proteinase (HC-Pro) of potyviruses contains an autoproteolytic function that, together with the protein 1 (P1) and NIa proteinase, processes the polyprotein into mature proteins. In this study, we analysed the autoproteolytic active domain of zucchini yellow mosaic virus (ZYMV) HC-Pro. Several Escherichia coli-expressed MBP:HC-Pro:GFP mutants containing deletions or point mutations at either the N- or C-terminus of the HC-Pro protein were examined. Our results showed that amino acids essential for the proteolytic activity of ZYMV HC-Pro are distinct from those of the tobacco etch virus HC-Pro, although the amino acid sequences in the proteolytic active domain are conserved among potyviruses.
