Development of a rapid diagnostic assay for the detection of tomato chlorotic dwarf viroid based on isothermal reverse-transcription-recombinase polymerase amplification.

A molecular diagnostic assay utilizing reverse transcription-recombinase polymerase amplification (RT-RPA) at an isothermal constant temperature of 39°C and target-specific primers and probe were developed for the rapid, sensitive, and specific detection of tomato chlorotic dwarf viroid (TCDVd) in infected leaf and seed tissues. The performance of the AmplifyRP(®) Acceler8™ RT-RPA diagnostic assay, utilizing a lateral flow strip contained within an amplicon detection chamber, was evaluated and the results were compared with a standard RT-PCR assay. The AmplifyRP(®) Acceler8™ assay was specific for TCDVd in leaf and seed tissues, its sensitivity was comparable to conventional RT-PCR in leaf tissues, and it does not require extensive sample purification, specialized equipment, or technical expertise. This is the first report utilizing an RT-RPA assay to detect viroids and the assay can be used both in the laboratory and in the field for TCDVd detection.

Rapid diagnostic detection of plum pox virus in Prunus plants by isothermal AmplifyRP(®) using reverse transcription-recombinase polymerase amplification.

Plum pox virus (PPV) causes the most destructive viral disease known as plum pox or Sharka disease in stone fruit trees. As an important regulated pathogen, detection of PPV is thus of critical importance to quarantine and eradication of the spreading disease. In this study, the innovative development of two AmplifyRP(®) tests is reported for a rapid isothermal detection of PPV using reverse transcription-recombinase polymerase amplification. In an AmplifyRP(®) test, all specific recombination and amplification reactions occur at a constant temperature without thermal cycling and the test results are either recorded in real-time with a portable fluorescence reader or displayed using a lateral flow strip contained inside an amplicon detection chamber. The major improvement of this assay is that the entire test from sample preparation to result can be completed in as little as 20min and can be performed easily both in laboratories and in the field. The results from this study demonstrated the ability of the AmplifyRP(®) technique to detect all nine PPV strains (An, C, CR, D, EA, M, Rec, T, or W). Among the economic benefits to pathogen surveys is the higher sensitivity of the AmplifyRP(®) to detect PPV when compared to the conventional ELISA and ImmunoStrip(®) assays. This is the first report describing the use of such an innovative technique to detect rapidly plant viruses affecting perennial crops.

Rapid and sensitive detection of Little cherry virus 2 using isothermal reverse transcription-recombinase polymerase amplification.

Little cherry virus 2 (LChV2) (genus Ampelovirus) is the primary causal agent of little cherry disease (LCD) in sweet cherry (Prunus avium) in North America and other parts of the world. This mealybug-transmitted virus does not induce significant foliar symptoms in most sweet cherry cultivars, but does cause virus-infected trees to yield unevenly ripened small fruits with poor flavor. Most fruits from infected trees are unmarketable. In the present study, an isothermal reverse transcription-recombinase polymerase amplification (RT-RPA) technique was developed using LChV2 coat protein specific primers and probe. Detection of terminally labeled amplicons was achieved with a high affinity lateral flow strip. The RT-RPA is confirmed to be simple, fast, and specific. In comparison, although it retains the sensitivity of RT-PCR, it is a more cost-effective procedure. RT-RPA will be a very useful tool for detecting LChV2 from crude extracts in any growth stage of sweet cherry from field samples.

The Sll0606 protein is required for photosystem II assembly/stability in the cyanobacterium Synechocystis sp. PCC 6803.

An insertional transposon mutation in the sll0606 gene was found to lead to a loss of photoautotrophy but not photoheterotrophy in the cyanobacterium Synechocystis sp. PCC 6803. Complementation analysis of this mutant (Tsll0606) indicated that an intact sll0606 gene could fully restore photoautotrophic growth. Gene organization in the vicinity of sll0606 indicates that it is not contained in an operon. No electron transport activity was detected in Tsll0606 using water as an electron donor and 2,6-dichlorobenzoquinone as an electron acceptor, indicating that Photosystem II (PS II) was defective. Electron transport activity using dichlorophenol indolephenol plus ascorbate as an electron donor to methyl viologen, however, was the same as observed in the control strain. This indicated that electron flow through Photosystem I was normal. Fluorescence induction and decay parameters verified that Photosystem II was highly compromised. The quantum yield for energy trapping by Photosystem II (F(V)/F(M)) in the mutant was less than 10% of that observed in the control strain. The small variable fluorescence yield observed after a single saturating flash exhibited aberrant Q(A)(-) reoxidation kinetics that were insensitive to dichloromethylurea. Immunological analysis indicated that whereas the D2 and CP47 proteins were modestly affected, the D1 and CP43 components were dramatically reduced. Analysis of two-dimensional blue native/lithium dodecyl sulfate-polyacrylamide gels indicated that no intact PS II monomer or dimers were observed in the mutant. The CP43-less PS II monomer did accumulate to detectable levels. Our results indicate that the Sll0606 protein is required for the assembly/stability of a functionally competent Photosystem II.

Identification of two genes, sll0804 and slr1306, as putative components of the CO2-concentrating mechanism in the cyanobacterium Synechocystis sp. strain PCC 6803.

Insertional transposon mutations in the sll0804 and slr1306 genes were found to lead to a loss of optimal photoautotrophy in the cyanobacterium Synechocystis sp. strain PCC 6803 grown under ambient CO(2) concentrations (350 ppm). Mutants containing these insertions (4BA2 and 3ZA12, respectively) could grow photoheterotrophically on glucose or photoautotrophically at elevated CO(2) concentrations (50,000 ppm). Both of these mutants exhibited an impaired affinity for inorganic carbon. Consequently, the Sll0804 and Slr1306 proteins appear to be putative components of the carbon-concentrating mechanism in Synechocystis sp. strain PCC 6803.

The malic enzyme is required for optimal photoautotrophic growth of Synechocystis sp. strain PCC 6803 under continuous light but not under a diurnal light regimen.

A mutation was recovered in the slr0721 gene, which encodes the decarboxylating NADP(+)-dependent malic enzyme in the cyanobacterium Synechocystis sp. strain PCC 6803, yielding the mutant 3WEZ. Under continuous light, 3WEZ exhibits poor photoautotrophic growth while growing photoheterotrophically on glucose at rates nearly indistinguishable from wild-type rates. Interestingly, under diurnal light conditions (12 h of light and 12 h of dark), normal photoautotrophic growth of the mutant is completely restored.

Four novel genes required for optimal photoautotrophic growth of the cyanobacterium Synechocystis sp. strain PCC 6803 identified by in vitro transposon mutagenesis.

Four novel Synechocystis sp. strain PCC 6803 genes (sll1495, sll0804, slr1306, and slr1125) which encode hypothetical proteins were determined by transposon mutagenesis to be required for optimal photoautotrophic growth. Mutations were also recovered in ccmK4, a carboxysome coat protein homologue, and me, the decarboxylating NADP(+)-dependent malic enzyme. This is the first report that these known genes are required for optimal photoautotrophy.

Proliferating Floral Organs (Pfo), a Lotus japonicus gene required for specifying floral meristem determinacy and organ identity, encodes an F-box protein.

To study flower development in the model legume Lotus japonicus, a population of transgenic plants containing a maize transposable element (Ac) in their genome was screened for floral mutants. One mutation named proliferating floral organs (pfo) causes plants to produce a large number of sepal-like organs instead of normal flowers. It segregates as a single recessive Mendelian locus, and causes sterility. Scanning electron microscopy revealed that pfo affects the identity, number and arrangement of floral organs. Sepal-like organs form in the first whorl, and secondary floral meristems are produced in the next whorl. These in turn produce sepal-like organs in the first whorl and floral meristems in the second whorl, and the process is reiterated. Petals and stamens are absent while carpels are either absent or reduced. The pfo phenotype was correlated with the presence of an Ac insertion yielding a 1.6-kb HindIII restriction fragment on Southern blots. Both the mutant phenotype and this Ac element are unstable. Using the transposon as a tag, the Pfo gene was isolated. Conceptual translation of Pfo predicts a protein containing an F-box, with high overall similarity to the Antirrhinum FIMBRIATA, Arabidopsis UNUSUAL FLORAL ORGANS and Pisum sativum Stamina pistilloida proteins. This suggests that Pfo may regulate floral organ identity and meristem determinacy by targeting proteins for ubiquitination.