Infected Tissue Extracts Research Articles

Identification and Functional Characterization of Viroid Circular RNAs.

Viroids are relatively small, noncoding, plant circular RNAs. In contrast to other plant circular RNAs of endogenous origin, viroids are infectious agents able to replicate autonomously in the appropriate host. Because of their highly base-paired structures, they can be purified from infected tissue extracts using nonionic CF11 chromatography. Depending on the host plant species, viroid RNA preparation may also require polysaccharide removal by an extraction with 2-methoxyethanol followed by precipitation with cetyltrimethylammonium bromide. Electrophoretic analyses of this kind of preparations frequently show differential bands corresponding to the viroid circular molecules, which are absent in those from healthy plants. These RNA preparations can also be used for viroid transmission to new plants by mechanical inoculation.

Stability of the WSSV ORF94 VNTR genotype marker during passage in marine shrimp, freshwater crayfish and freshwater prawns.

The white spot syndrome virus (WSSV) genome contains 3 variable number tandem repeat (VNTR) regions, located in open reading frame (ORF) 75, ORF94 and ORF125, which have been employed for molecular epizootiological studies. A previous report suggested that the ORF 94 VNTR is highly unstable, varying in the number of tandem repeats during single passages from shrimp to other crustaceans. As such rapid variations would have profound implications for the interpretation of molecular epizootiological data, we re-examined the stability of the ORF94 VNTR. Two WSSV isolates with different ORF94 VNTR genotypes (TRS5 and TRS7) were obtained from disease outbreaks in farmed black tiger shrimp Penaeus monodon in Indonesia. High titre stocks of each virus were produced by injection in specific pathogen-free (SPF) Pacific white shrimp Litopenaeus vannamei with filtered infected tissue extracts, and the genotypes were confirmed. Each stock (macerated tissue) was then used to feed SPF Pacific white shrimp, freshwater crayfish (Cherax sp.) and freshwater prawns Macrobrachium rosenbergii through 3 successive passages involving alternative hosts at each level. Taqman real-time PCR was conducted on samples from each group to confirm infection and quantify viral genetic loads. ORF94 VNTR genotype analysis conducted on samples from each of the 43 passage groups indicated no variations in the VNTR number in either genotype TRS5 or genotype TRS7. This finding is contrary to the previous report and suggests that ORF94 VNTR are stable during multiple passages in these crustaceans.

Production of monoclonal antibodies for detection of Citrus leprosis virus C in enzyme-linked immuno-assays and immunocapture reverse transcription-polymerase chain reaction

Citrus leprosis virus C (CiLV-C) causes damage in citrus production in the South and Central America. Since closely related types of citrus viruses have recently been described monoclonal antibodies (MAbs) are needed for accurate and sensitive diagnosis of CiLV-C. In this study, MAbs to the expressed coat protein of CiLV-C were produced for serological detection of CiLV-C in crude extracts of infected tissues in double antibody sandwich enzyme-linked immunosorbent assays (DAS-ELISA), dot blot immunosorbent assays (DBIA) and immuonocapture-reverse transcription-polymerase chain reaction (IC-RT-PCR) procedures. Monoclonal antibodies were developed in mice to the purified expressed coat protein of CiLV-C. The published standard protocols of DAS-ELISA, DBIA and IC-RT-PCR were followed for the detection of coat protein p29 of CiLV-C in the crude extracts of CiLV-C infected tissues. Two monoclonal antibodies, designated G10 and C11, were identified from four potential candidates for the specific and sensitive detection of coat protein p29 of CiLV-C in the crude citrus extracts of CiLV-C infected tissues in DAS-ELISA, whereas G10 was also selected based on performance for use in the DBIA and IC-RT-PCR diagnostic assays. Sensitivity analysis comparing the three methods for detection of coat protein p29 of CiLV-C determined that IC-RT-PCR was more sensitive than DAS-ELISA and DBIA. The creation of MAbs to CiLV-C allows for the sensitive and accurate detection of the virus from CiLV-C infected citrus leaf tissues. Successful detection of the virus in three diagnostic assays formats provides flexibility to diagnosticians who can use either ELISA or DBIA for screening large numbers of samples, and IC-RT-PCR for rapid, sensitive confirmation testing.

Isolation and Characterization of Thaxtomin-Type Phytotoxins Associated with Streptomyces ipomoeae

Chromatographic investigations of Streptomyces ipomoeae infected potato tissue and sweetpotato tissue yielded three compounds which on aseptically cultured minitubers individually reproduced symptoms typical of the common scab disease. Spectroscopic examination of the two minor components elicited sufficient information to identify them as analogs of the major constituent which had been previously characterized as 12-N-methylcyclo-(L-4-nitrotryptophyl-L-phenylalanyl). Subsequent investigations of the infected tissue extracts identified a possible toxin precursor substance as 12-N-methylcyclo-(L-tryptophyl-L-phenylalanyl)

Kinetics of phytoalexin accumulation in potato tubers of different genotypes infected with Erwinia carotovora ssp Atroseptica

The time-courses of rotting and phytoalexin accumulation in tuber soft rot tissue of two cultivars of Solanum tuberosum subspecies tuberosum were examined from 1–7 days after inoculation with Erwinia carotovora ssp atroseptica . In extracts of infected tissue, rishitin, solavetivone, phytuberin, hydroxysolavetivone, phytuberol, dehydroxyphytuberol, solanascone and various fatty acid ethyl esters were detected. This paper describes a new and convenient method of extracting phytoalexins and analysing complex extracts with fused silica capillary gas chromatography. Low concentrations of phytoalexins were correlated with extensive rotting. Thus, tubers incubated anaerobically rotted rapidly and extensively and no phytoalexins were detected. When tubers were incubated aerobically, stored mature tubers accumulated considerable amounts of rishitin and smaller amounts of solavetivone and phytuberin, and exhibited less extensive rotting. In the tubers of cultivar Grata, rishitin accumulated more rapidly than in those of cv. Irmgard, but the differences in susceptibility were not significant. Aerobically incubated immature unstored tubers rotted extensively and no phytoalexins were found in the rot, which demonstrated the influence of physiological age on the resistance. The selected accessions of Solanum tuberosum spp andigena showed the most pronounced correlation between high concentrations of phytoalexins in the tissue and low susceptibility.

Monoclonal antibodies reactive with specific amino acid sequences of the 126 K protein of tobacco mosaic virus.

The 126 K protein of tobacco mosaic virus (TMV) is an NTP binding protein that has guanylyl transferase activity and is predicted to be an ATPase/helicase. In this paper we report the generation of monoclonal antibodies (Mabs) that react with specific amino acid sequences of the 126K protein. The Mabs were generated after immunizing mice with a partially purified preparation of the 126 K protein (native) obtained by centrifugal fractionation of the infected tissue extracts. The Mabs were assayed for specific reactivity by western blotting and by their reactivity with non-overlapping decapeptides corresponding to the entire amino acid sequence of the 126 K protein of TMV. A total of 11 Mabs reactive with specific peptides and three other Mabs that did not react with any peptide but reacted with the 126 K protein were identified.

Similar Structure and Reactivity of Satellite Tobacco Ringspot Virus RNA Obtained from Infected Tissue and by in Vitro Transcription

The 359 nucleotide residue (nt) satellite RNA of tobacco ringspot virus increases detectably only in association with replicating tobacco ringspot virus and becomes encapsidated in the virus coat protein. Results from previous reports are consistent with participation of rolling circle transcription in replication of the satellite RNA: (i) both the more abundant plus polarity satellite RNA, s(+)RNA and the complementary s(-)RNA occur in multimeric forms that self-cleave to release the unit length, 359 nt satellite RNA, (ii) circles of both s(+)RNA and s(-)RNA are present in extracts of infected tissue, and (iii) s(-)RNA, but not S(+)RNA, spontaneously and efficiently circularizes in vitro. Our analyses of RNA in tissue extracts suggest that s(+)RNA of all forms is about 100-fold more abundant than s(-)RNA. Nucleic acids were purified rapidly to minimize interconversion of linear and circular forms. For s(+)RNA and for s(-)RNA, the circular and the linear forms were detected in about equal amounts in tissue extracts. The linear s(-)RNA from tissue extracts was found to have the same 5′-terminal sequence as previously was found for s(-)RNA self-cleaved from in vitro transcripts. Like s(-)RNA synthesized in vitro, the circular and linear s(-)RNA from tissue extracts spontaneously and readily interconverted during incubation in vitro. In contrast, the bulk of the circular and linear forms of S(+)RNA were stable. The very limited interconversion of s(+)RNA forms in vitro suggests that circularization in vivo is enzymically catalyzed. Encapsidated satellite RNA was found to be composed of linear, unit length and multimeric forms, including previously undocumented s(-)RNA present in approximately the same relative abundance compared to s(+)RNA as was observed for RNA from tissue extracts. Circles were not detected in encapsidated RNA. We interpret our results in the context of a rolling circle model for satellite RNA replication.

Sub-cellular Localization of the 25-kDa Protein Encoded in the Triple Gene Block of Potato Virus X

The 25-kDa protein of potato virus X (PVX) is encoded by the 5′ open reading frame of the overlapping triple gene block, has a GKS sequence motif characteristic of nucleotide binding proteins, and is thought to play a role in movement between cells. We have produced a polyclonal antibody to the 25-kDa protein to investigate its synthesis and intracellular location. On Western blots of infected tissue extracts this antibody detected a single major protein of the expected mobility, produced earlier than the viral coat protein in infected protoplasts and located predominantly in the 1000 g pellet fraction of infected cells. Immunogold labeling of thin sections of infected tissue revealed that the 25-kDa protein is associated with the cytoplasmic inclusion bodies characteristic of PVX infections, but not with the cell wall or plasmodesmata. Therefore, although mutational studies indicate a role for the 25-kDa protein in the cell to cell movement of viral infection it is unlikely to act in precisely the same manner as the other well-characterized movement proteins of tobacco mosaic and cowpea mosaic viruses.

ENZYMATIC CDNA AMPLIFICATION OF HOP STUNT VIROID VARIANTS FROM NATURALLY INFECTED FRUIT CROPS

Hop stunt viroid (HSV) cDNA reverse transcribed in vitro from total nucleic acid extracts of infected tissue was amplified using Taq I DNA polymerase and HSV group-specific DNA primers in a reverse transcription-polymerase chain reaction (RT-PCR). The RT-PCR assay is a rapid and specific method to determine HSV infection in fruit crops without viroid purification or the need for large amounts of infected tissue.

Effect of actinomycin D on replication of satellite tobacco ringspot virus RNA in plant protoplasts

We have developed a three-component system of host, tobacco ringspot virus (TobRV), and satellite tobacco ringspot virus RNA (sTobRV RNA) for investigating the specific contributions of host components or TobRV gene products to the propagative cycle of satellite RNA. Cowpea ( Vigna unguiculata) protoplasts were inoculated with sTobRV and TobRV genomic RNAs by electroporation. An increase in sTobRV RNA was detected both by blot hybridization and by incorporation of [ 14C]uridine into material with the electrophoretic mobility of sTobRV RNA. DNA-dependent RNA synthesis in uninoculated protoplasts was effectively inhibited by 50 μg/ml actinomycin D (Act D) in the medium. Addition of Act D to protoplasts 24 or 48 hr after coinoculation with sTobRV RNA and TobRV genomic RNAs had little effect on accumulation of sTobRV RNA, whereas addition at 24 hr prior to coinoculation prevented any detected accumulation of sTobRV RNA of either polarity. Our results and previous findings of RNA complementary to encapsidated satellite RNA in extracts of infected tissue suggest that an RNA-dependent RNA polymerase is responsible for the synthesis of sTobRV RNA. The strongly inhibitory effect of Act D when added early implies a role for a host factor in the early phase of sTobRV RNA replication.

Cherry leafroll virus infections are affected by a satellite RNA that the virus does not support

Previous research showed that tobacco ringspot virus (TobRV), a member of the nepovirus group, acts as a supporting virus for the 359-nucleotide residue satellite tobacco ringspot virus RNA (STobRV RNA), resulting in STobRV RNA replication and its encapsidation in TobRV coat protein. In some hosts STobRV RNA decreases the yield of TobRV and the severity of TobRV-induced symptoms. We report here that inclusion of STobRV RNA in an inoculum of cherry leafroll virus (CLRV), another nepovirus, prevented the accumulation of CLRV in the inoculated leaves of cowpea ( Vigna unguiculata) and caused the symptoms to be less severe than those induced when CLRV was inoculated alone. CLRV spread to, and increased in, uninoculated, developing leaves whether or not it was coinoculated with STobRV RNA. STobRV RNA was not detected in CLRV particles or in extracts of infected tissue from the coinoculated plants, indicating that CLRV does not support STobRV RNA. STobRV RNA strongly interfered with the in vitro translation of the RNAs of CLRV and of cowpea mosaic virus (CPMV). Coinoculation of STobRV RNA and CPMV had no detected effect on infections by CPMV, so inhibition of translation in vitro and of replication in vivo were correlated only for CLRV. Results from a new in vitro assay for proteolytic processing of CLRV polyproteins gave no indication of an effect of STobRV RNA on this reaction.

Gene I products of cauliflower mosaic virus detected in extracts of infected tissue

The product of cauliflower mosaic virus (CaMV) gene I has been characterized from extracts of infected plants. Two size classes of this protein can be identified by the use of specific antiserum. The antiserum was induced against a chimeric protein produced in E. coli from a gene fusion between a fragment of the CaMV genome and the,β-galactosidase gene. A 36-kDa form of the gene I product is associated with virus particles. A 45-kDa form of this product was found only in the insoluble fraction of tissue homogenates. This insoluble fraction, the only fraction found to contain the product of viral gene VI, contains the virally induced inclusion bodies.

Detection of a mycoplasma‐like organism in peanut plants with witches' broom using indirect enzyme‐linked immunosorbent assay (ELISA)

The mycoplasma‐like organism (MLO) associated with peanut (groundnut) witches’ broom (PWB) from India was partially purified and an antiserum produced against it. Using a protein A indirect enzyme‐linked immunosorbent assay (ELISA) procedure, PWB MLO was detected in crude extracts of leaves. stems and pegs of infected peanut plants, although stems were a better source than leaves and pegs. Extracts of infected tissues of three diseases of assumed MLO etiology in India, little leaf of brinjal (eggplant). I med rosed witches “broom, and Daturd sp. witches’ broom, failed to react with the PWB MLO antiserum.

Lethality of intestinal tissue extracts from Eimeria-infected chickens.

Evidence exists that lethal activity is associated with extracts from Eimeria tenella-infected caecal tissues. We examined tissues of the small intestine infected with other species of Eimeria for a similar lethal activity. Extracts were prepared from intestinal tissues of chickens infected with either E. acervulina, E. brunetti, E. maxima, E. mitis or E. necatrix. Appropriate control extracts from tissues of non-infected chickens were also prepared. Intravenous injection of the extracts into healthy chickens showed that lethality of infected tissue extracts was higher than control extracts and was independent of the site of infection within the intestine. Comparison of the lethality of caecal extracts from chickens infected with embryo-adapted or parent lines of E. tenella showed that lethality was independent of the recognized pathogenic characteristics of the lines. Also, it was demonstrated that lethal activity is associated with extracts of tissues containing massive numbers of arrested and non-arrested sporozoites and early trophozoites.

Isolation and partial characterization of the amorphous cytoplasmic inclusions associated with infections caused by two potyviruses

The potyviruses pepper mottle (PeMV) and watermelon mosaic virus-1, a strain of papaya ringspot (PRSV-W), induce the formation of a second type of cytoplasmic inclusion in addition to cylindrical (pinwheel) inclusions. The conspicuous aggregates of electron-dense material with imperfect spherical shapes have been called amorphous inclusions (AI). The AI were isolated from extracts of infected tissue using a combination of clarification with Triton X-100 and low-speed centrifugations through sucrose cushions. Electrophoresis, in sodium dodecyl sulfate-permeated polyacrylamide gels (SDS-PAGE), of the SDS-dissociated AI revealed a single constituent protein with a molecular weight of 51,000. SDS-PAGE-purified AI proteins were immunogenic and serologically unrelated to proteins of host, capsid, cylindrical inclusions, and tobacco etch virus nuclear inclusions. The AI protein of PeMV was distinct from the AI protein of PRSV-W on the basis of peptide analysis and serological tests. The antisera prepared to SDS-PAGE-purified AI proteins reacted specifically with Al in situ in immunofluorescence tests. The characteristics of the AI indicate that they are of viral origin.

A Rapid Procedure for the Purification of Avian Encephalomyelitis Viruses

A rapid procedure for the purification of egg-grown or field preparations of avian encephalomyelitis virus (AEV) of neural origin is described. Extracts of infected tissues were clarified and then partly purified with trichlorotrifluorethane (Freon TF), and the virus present was concentrated with polyethylene glycol. The concentrates were then re-extracted with Freon, and a portion was labeled with 125iodine. During subsequent purification steps, virus could be readily detected by monitoring for radioactivity, thus eliminating the need to determine the infectivity in individual fractions or to examine for the presence of virions by electron microscopy. Final purification was achieved by cesium-chloride equilibrium or sucrose-velocity-gradient centrifugation. Virus purified in this manner was shown to be free of tissue debris, to be specific for AEV by immune electron microscopy, and to possess structural proteins characteristic of picornaviruses.

Detection of double-stranded RNA by serologically specific electron microscopy

Details of a procedure for detecting double-stranded RNA (dsRNA) in virus and viroid infected tissue extracts using serologically specific electron microscopy are given. A method for staining dsRNA, based on in situ formation of uranyl phosphate, that consistently permits the examination of dsRNA by electron microscopy without shadowing with heavy metals, is described. The method provides for routine assays for dsRNA in crude extracts without the variable results associated with shadowing procedures. DsRNA was found to accumulate in older leaves of sorghum systemically infected with sugarcane mosaic virus. In contrast, dsRNA was not detected in older cowpea leaves systemically infected with cowpea mosaic virus but was readily found in inoculated leaves 4 days post inoculation and young systemically infected leaves.

RNA sequences complementary to citrus exocortis viroid in nucleic acid preparations from infected Gynura aurantiaca

Molecular hybridization with (125)I-labeled citrus exocortis viroid RNA has been used to survey nucleic acid preparations from Gynura aurantiaca for viroid complementary molecules. A differential hybridization effect was detected between nucleic acid extracts from healthy and infected tissue in which significant RNase-resistant (125)I-labeled citrus exocortis viroid resulted in hybridization studies with the infected tissue extracts. Subsequent characterization indicated that RNA from infected tissue was involved in the formation of a duplex molecule with citrus exocortis viroid RNA and had properties of an RNA.RNA hybrid. Subcellular fractionation of infected tissue indicates that the complementary RNA is present in nuclear and soluble RNA fractions. This RNA may represent an intermediate molecule in the replication of the viroid or a pathogenic expression and may have a regulatory role in the host cell.

A rôle for β-glucanases in the parasitism of cereals by Claviceps purpurea

Histological investigation of parasitism of Claviceps purpurea on rye and wheat revealed the close proximity of the fungus to host vascular elements at the host-parasite interface. Extracts of infected tissue and developing ergots possessed glucanase activity, and honeydew, which exudes from infected florets, showed about ten-fold greater activity. Callose, a β-1–3-glucan which occurs normally in sieve tubes and is deposited by wounded cells in response to injury, fluoresces bright yellow when stained with aniline blue and irradiated with u.v. light. Infected tissue extracts, and honeydew, hydrolysed wound and phloem callose, as shown by an almost total elimination of fluorescence due to this material in wounded ovary sections. It is suggested that the glucanases of Claviceps purpurea play a rôle in maintaining a nutrient supply to the pathogen by hydrolysing callose and preventing its further deposition since none is found in infected ovaries.

A messenger RNA for capsid protein isolated from tobacco mosaic virus-infected tissue

A low molecular weight virus-related RNA component (LMC) was isolated from extracts of TMV-infected leaf tissue by employing a combination of the techniques of sucrose density gradient sedimentation and polyacrylamide gel electrophoresis. It was found that LMC has an estimated molecular weight of 2.5 × 10 5 and acts as a template for the synthesis of TMV capsid protein in a wheat germ-derived cell-free protein synthesizing system. Evidence is presented for the possible existence of other unique virus-related RNA species in extracts of infected tissue.