Identification of the Actin-Binding Region and Binding to Host Plant Apple Actin of Immunodominant Transmembrane Protein of 'Candidatus Phytoplasma mali'.

'Candidatus Phytoplasma mali' ('Ca. P. mali') has only one major membrane protein, the immunodominant membrane protein (Imp), which is regarded as being close to the ancestor of all phytoplasma immunodominant membrane proteins. Imp binds to actin and possibly facilitates its movement in the plant or insect host cells. However, protein sequences of Imp are quite diverse among phytoplasma species, thus resulting in difficulties in identifying conserved domains across species. In this work, we compare Imp protein sequences of 'Ca. P. mali' strain PM19 (Imp-PM19) with Imp of different strains of 'Ca. P. mali' and identify its actin-binding domain. Moreover, we show that Imp binds to the actin of apple (Malus x domestica), which is the host plant of 'Ca. P. mali'. Using molecular and scanning force spectroscopy analysis, we find that the actin-binding domain of Imp-PM19 contains a highly positively charged amino acid cluster. Our result could allow investigating a possible correlation between Imp variants and the infectivity of the corresponding 'Ca. P. mali' isolates.

Beyond Destabilizing Activity of SAP11-like Effector of Candidatus Phytoplasma mali Strain PM19.

It was shown that the SAP11 effector of different Candidatus Phytoplasma can destabilize some TEOSINE BRANCHES/CYCLOIDEA/PROLIFERATING CELL FACTORs (TCPs), resulting in plant phenotypes such as witches' broom and crinkled leaves. Some SAP11 exclusively localize in the nucleus, while the others localize in the cytoplasm and the nucleus. The SAP11-like effector of Candidatus Phytoplasma mali strain PM19 (SAP11PM19) localizes in both compartments of plant cells. We show here that SAP11PM19 can destabilize TCPs in both the nucleus and the cytoplasm. However, expression of SAP11PM19 exclusively in the nucleus resulted in the disappearance of leaf phenotypes while still showing the witches' broom phenotype. Moreover, we show that SAP11PM19 can not only destabilize TCPs but also relocalizes these proteins in the nucleus. Interestingly, three different transgenic Nicotiana species expressing SAP11PM19 show all the same witches' broom phenotype but different leaf phenotypes. A possible mechanism of SAP11-TCP interaction is discussed.

The Effect of the Anticipated Nuclear Localization Sequence of 'Candidatus Phytoplasma mali' SAP11-like Protein on Localization of the Protein and Destabilization of TCP Transcription Factor.

SAP11 is an effector protein that has been identified in various phytoplasma species. It localizes in the plant nucleus and can bind and destabilize TEOSINE BRANCHES/CYCLOIDEA/PROLIFERATING CELL FACTOR (TCP) transcription factors. Although SAP11 of different phytoplasma species share similar activities, their protein sequences differ greatly. Here, we demonstrate that the SAP11-like protein of 'Candidatus Phytoplasma mali' ('Ca. P. mali') strain PM19 localizes into the plant nucleus without requiring the anticipated nuclear localization sequence (NLS). We show that the protein induces crinkled leaves and siliques, and witches' broom symptoms, in transgenic Arabidopsis thaliana (A. thaliana) plants and binds to six members of class I and all members of class II TCP transcription factors of A. thaliana in yeast two-hybrid assays. We also identified a 17 amino acid stretch previously predicted to be a nuclear localization sequence that is important for the binding of some of the TCPs, which results in a crinkled leaf and silique phenotype in transgenic A. thaliana. Moreover, we provide evidence that the SAP11-like protein has a destabilizing effect on some TCPs in vivo.

'Candidatus Phytoplasma mali' Genome Encodes a Protein that Functions as an E3 Ubiquitin Ligase and Could Inhibit Plant Basal Defense.

Phytoplasmas are the causative agent of numerous diseases of plant species all over the world, including important food crops. The mode by which phytoplasmas multiply and behave in their host is poorly understood and often based on genomic data. We used yeast two-hybrid screening to find new protein-protein interactions between the causal agent of apple proliferation 'Candidatus Phytoplasma mali' and its host plant. Here, we report that the 'Ca. P. mali' strain PM19 genome encodes a protein PM19_00185 that interacts with at least six different ubiquitin-conjugating enzymes (UBC; E2) of Arabidopsis thaliana. An in vitro ubiquitination assay showed that PM19_00185 is enzymatically active as E3 ligase with A. thaliana E2 UBC09 and Malus domestica E2 UBC10. We show that a nonhost bacteria (Pseudomonas syringae pv. tabaci) can grow in transgenic A. thaliana plant lines expressing PM19_00185. A connection of phytoplasma effector proteins with the proteasome proteolytic pathway has been reported before. However, this is, to our knowledge, the first time that a phytoplasma effector protein with E3 ligase activity has been reported.

Electrostatic conditions define the 2D self-assembly of tomato bushy stunt viruses on solid surfaces.

Plant viruses which are self-assembled on a substrate are interesting building blocks in nanobiotechnology, in particular, for the creation of 2D ordered structures. In this article, the self-assembly of different genetically modified types of the tomato bushy stunt virus spin-coated on pristine silicon was investigated by scanning force and scanning electron microscopy. Amino acid side chains were integrated in the capsids of the viruses by extending the coat protein with different charged amino acid clusters (tetra-aspartate-hexa-histidine, hexa-aspartate, or tetra-arginine-tags). The influence of the resulting electrostatic forces based on virus-virus and virus-surface interactions on the formation of self-assembled monolayers will be presented and discussed in the context of differences in surface coverage for different pH values. It could be shown that the largest surface coverage can be achieved when there is an attraction between the whole virus and the surface and only a minor repulsion between the viruses at a given pH.

A simple method to estimate the isoelectric point of modified Tomato bushy stunt virus (TBSV) particles.

We present a simple method to estimate the isoelectric point (pI) of Tomato Bushy Stunt particles. We demonstrate that the combination of agarose gels with different pH buffers can be used to electrophorese the virus particles and their migration patterns can be compared. This method allows us to estimate the pI of the virus particles (wild type, wt, and genetically modified particles) and to monitor the effect of the pI of modified peptide side chains of the viral capsid subunit on the pI of the whole virus particle.

Identification of Highly Specific scFvs against Total Adiponectin for Diagnostic Purposes.

Adiponectin is one of the most abundant adipokines secreted from adipose tissue. It acts as an endogenous insulin sensitizer and plasma concentrations are inversely correlated with obesity and metabolic syndrome. A decrease in plasma adiponectin levels normally indicates increased hormonal activity of the visceral lipid tissue, which is associated with decreased insulin sensitivity. It may therefore be considered a valuable biomarker for elucidating the underlying deteriorations resulting in type 2 diabetes and macrovascular disease. Here we present the use of phage display technology to identify highly specific antibody fragments (scFvs) against adiponectin. The selected scFvs showed highly specific binding to globular and native adiponectin in ELISA tests. By using our phage display technology, we were able to obtain monoclonal antibodies with specific high affinity binding to the target protein in an effective and easy to upscale manner. The selected scFvs against adiponectin can be used for developing immunoassays suitable for use in metabolic syndrome diagnosis and monitoring.

Immunodominant membrane proteins of phytoplasmas.

Phytoplasmas are plant-pathogenic, phloem-colonizing, cell wall-less microorganisms that are primarily dependent on insect transmission for their spread and survival. The life cycle of phytoplasmas involves replication in insects and host plants. Until recently, phytoplasmas have resisted all attempts at cultivation in cell-free media, making these pathogens poorly characterized on a physiological and biochemical basis. However, host-pathogen relationships can be studied by investigating immunodominant membrane proteins (IDPs), which are located on the exterior surfaces of phytoplasma cells and are the most abundant proteins of the cell membrane. These membrane proteins come in direct contact with both insect and plant hosts and are thought to play a crucial role in phytoplasma spread both within the plant and by insect vectors. Therefore, there is great interest in studying this class of proteins. We summarize and discuss important investigations about these membrane proteins, which have already provided a better understanding of the host-phytoplasma relationship.

Binding and processing of small dsRNA molecules by the class 1 RNase III protein encoded by sweet potato chlorotic stunt virus.

Sweet potato chlorotic stunt virus (SPCSV; genus Crinivirus, family Closteroviridae) causes heavy yield losses in sweet potato plants co-infected with other viruses. The dsRNA-specific class 1 RNase III-like endoribonuclease (RNase3) encoded by SPCSV suppresses post-transcriptional gene silencing and eliminates antiviral defence in sweet potato plants in an endoribonuclease activity-dependent manner. RNase3 can cleave long dsRNA molecules, synthetic small interfering RNAs (siRNAs), and plant- and virus-derived siRNAs extracted from sweet potato plants. In this study, conditions for efficient expression and purification of enzymically active recombinant RNase3 were established. Similar to bacterial class 1 RNase III enzymes, RNase3-Ala (a dsRNA cleavage-deficient mutant) bound to and processed double-stranded siRNA (ds-siRNA) as a dimer. The results support the classification of SPCSV RNase3 as a class 1 RNase III enzyme. There is little information about the specificity of RNase III enzymes on small dsRNAs. In vitro assays indicated that ds-siRNAs and microRNAs (miRNAs) with a regular A-form conformation were cleaved by RNase3, but asymmetrical bulges, extensive mismatches and 2'-O-methylation of ds-siRNA and miRNA interfered with processing. Whereas Mg(2+) was the cation that best supported the catalytic activity of RNase3, binding of 21 nt small dsRNA molecules was most efficient in the presence of Mn(2+). Processing of long dsRNA by RNase3 was efficient at pH 7.5 and 8.5, whereas ds-siRNA was processed more efficiently at pH 8.5. The results revealed factors that influence binding and processing of small dsRNA substrates by class 1 RNase III in vitro or make them unsuitable for processing by the enzyme.

Improvement of PCR reaction conditions for site-directed mutagenesis of big plasmids.

QuickChange mutagenesis is the method of choice for site-directed mutagenesis (SDM) of target sequences in a plasmid. It can be applied successfully to small plasmids (up to 10 kb). However, this method cannot efficiently mutate bigger plasmids. Using KOD Hot Start polymerase in combination with high performance liquid chromatography (HPLC) purified primers, we were able to achieve SDM in big plasmids (up to 16 kb) involving not only a single base change but also multiple base changes. Moreover, only six polymerase chain reaction (PCR) cycles and 0.5 µl of polymerase (instead of 18 PCR cycles and 1.0 µl of enzyme in the standard protocol) were sufficient for the reaction.

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.

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.

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.

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.

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.

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.

Homemade site directed mutagenesis of whole plasmids.

Site directed mutagenesis of whole plasmids is a simple way to create slightly different variations of an original plasmid. With this method the cloned target gene can be altered by substitution, deletion or insertion of a few bases directly into a plasmid. It works by simply amplifying the whole plasmid, in a non PCR-based thermocycling reaction. During the reaction mutagenic primers, carrying the desired mutation, are integrated into the newly synthesized plasmid. In this video tutorial we demonstrate an easy and cost effective way to introduce base substitutions into a plasmid. The protocol works with standard reagents and is independent from commercial kits, which often are very expensive. Applying this protocol can reduce the total cost of a reaction to an eighth of what it costs using some of the commercial kits. In this video we also comment on critical steps during the process and give detailed instructions on how to design the mutagenic primers.

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.

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.