One-Enzyme RTX-PCR for the Detection of RNA Viruses from Multiple Virus Genera and Crop Plants.

Reverse transcription PCR (RT-PCR) is a popular method for detecting RNA viruses in plants. RT-PCR is usually performed in a classical two-step procedure: in the first step, cDNA is synthesized by reverse transcriptase (RT), followed by PCR amplification by a thermostable polymerase in a separate tube in the second step. However, one-step kits containing multiple enzymes optimized for RT and PCR amplification in a single tube can also be used. Here, we describe an RT-PCR single-enzyme assay based on an RTX DNA polymerase that has both RT and polymerase activities. The expression plasmid pET_RTX_(exo-) was transferred to various E. coli genotypes that either compensated for codon bias (Rosetta-gami 2) or contained additional chaperones to promote solubility (BL21 (DE3) with plasmids pKJE8 or pTf2). The RTX enzyme was then purified and used for the RT-PCR assay. Several purified plant viruses (TMV, PVX, and PVY) were used to determine the efficiency of the assay compared to a commercial one-step RT-PCR kit. The RT-PCR assay with the RTX enzyme was validated for the detection of viruses from different genera using both total RNA and crude sap from infected plants. The detection endpoint of RTX-PCR for purified TMV was estimated to be approximately 0.01 pg of the whole virus per 25 µL reaction, corresponding to 6 virus particles/µL. Interestingly, the endpoint for detection of TMV from crude sap was also 0.01 pg per reaction in simulated crude plant extracts. The longest RNA fragment that could be amplified in a one-tube arrangement was 2379 bp long. The longest DNA fragment that could be amplified during a 10s extension was 6899 bp long. In total, we were able to detect 13 viruses from 11 genera using RTX-PCR. For each virus, two to three specific fragments were amplified. The RT-PCR assay using the RTX enzyme described here is a very robust, inexpensive, rapid, easy to perform, and sensitive single-enzyme assay for the detection of plant viruses.

Dynamics of the Pollen Sequestrome Defined by Subcellular Coupled Omics.

Reproduction success in angiosperm plants depends on robust pollen tube growth through the female pistil tissues to ensure successful fertilization. Accordingly, there is an apparent evolutionary trend to accumulate significant reserves during pollen maturation, including a population of stored mRNAs, that are utilized later for a massive translation of various proteins in growing pollen tubes. Here, we performed a thorough transcriptomic and proteomic analysis of stored and translated transcripts in three subcellular compartments of tobacco (Nicotiana tabacum), long-term storage EDTA/puromycin-resistant particles, translating polysomes, and free ribonuclear particles, throughout tobacco pollen development and in in vitro-growing pollen tubes. We demonstrated that the composition of the aforementioned complexes is not rigid and that numerous transcripts were redistributed among these complexes during pollen development, which may represent an important mechanism of translational regulation. Therefore, we defined the pollen sequestrome as a distinct and highly dynamic compartment for the storage of stable, translationally repressed transcripts and demonstrated its dynamics. We propose that EDTA/puromycin-resistant particle complexes represent aggregated nontranslating monosomes as the primary mediators of messenger RNA sequestration. Such organization is extremely useful in fast tip-growing pollen tubes, where rapid and orchestrated protein synthesis must take place in specific regions.

Characterization of pollen-expressed bZIP protein interactions and the role of ATbZIP18 in the male gametophyte.

KEY MESSAGE : bZIP TF network in pollen. Transcriptional control of gene expression represents an important mechanism guiding organisms through developmental processes and providing plasticity towards environmental stimuli. Because of their sessile nature, plants require effective gene regulation for rapid response to variation in environmental and developmental conditions. Transcription factors (TFs) provide such control ensuring correct gene expression in spatial and temporal manner. Our work reports the interaction network of six bZIP TFs expressed in Arabidopsis thaliana pollen and highlights the potential functional role for AtbZIP18 in pollen. AtbZIP18 was shown to interact with three other pollen-expressed bZIP TFs-AtbZIP34, AtbZIP52, and AtbZIP61 in yeast two-hybrid assays. AtbZIP18 transcripts are highly expressed in pollen, and at the subcellular level, an AtbZIP18-GFP fusion protein was located in the nucleus and cytoplasm/ER. To address the role of AtbZIP18 in the male gametophyte, we performed phenotypic analysis of a T-DNA knockout allele, which showed slightly reduced transmission through the male gametophyte. Some of the phenotype defects in atbzip18 pollen, although observed at low penetrance, were similar to those seen at higher frequency in the T-DNA knockout of the interacting partner, AtbZIP34. To gain deeper insight into the regulatory role of AtbZIP18, we analysed atbzip18/- pollen microarray data. Our results point towards a potential repressive role for AtbZIP18 and its functional redundancy with AtbZIP34 in pollen.

Tobacco susceptibility to Potato virus Y(NTN) infection is affected by grafting and endogenous cytokinin content.

Faster or stronger response to pathogen occurs if plants undergo prior priming. Cytokinins seem to be also involved in plant priming and in response to pathogens. Susceptibility to Potato virus Y(NTN) (PVY(NTN)) was studied in transgenic cytokinin overproducing (Pssu-ipt) tobacco and compared with nontransgenic plants. Since cytokinin overproduction inhibits development of plant roots and grafting overcomes this limitation, both types were grown as rooted and/or grafted plants to check also the effect of grafting. Control rooted tobacco (C), the most susceptible to PVY(NTN), showed always symptoms during the infection together with the rising virus content and a systemic response, such as accumulation of H2O2, salicylic acid (SA) and other phenolic acids, and stress-induced enzyme activities. In transgenic and grafted plants, the response to PVY(NTN) was dependent on protective mechanisms activated prior to the inoculation. In Pssu-ipt tobacco, cytokinin active forms and SA contents exceeded manifold their content in C. Grafting promoted the accumulation of phenolics, but SA, and stimulated peroxidase activities. Thus, the pre-infection barrier established in both transgenic and grafted plants helped to suppress partly the virus multiplication and resulted in milder symptom development. However, only the synergic effect of both grafting and the high cytokinins led to PVY(NTN) tolerance in transgenic grafts. Possible mechanisms were discussed.

Transcription of atp1 is influenced by both genomic configuration and nuclear background in the highly rearranged mitochondrial genomes of Silene vulgaris.

An extraordinary variation in mitochondrial DNA sequence exists in angiosperm Silene vulgaris. The atp1 gene is flanked by very variable regions, as deduced from four completely sequenced mitochondrial genomes of this species. This diversity contributed to a highly variable transcript profile of this gene observed across S. vulgaris populations. We examined the atp1 transcript in the KOV mitochondrial genome and found three 5' ends, created most likely by the combination of transcription initiation and RNA processing. Most atp1 transcripts terminated about 70 bp upstream of the translation stop codon, which was present in only 10 % of them. Controlled crosses between a KOV mother and a geographically distant pollen donor (Krasnoyarsk, Russia) showed that nuclear background also affected atp1 transcription. The distant pollen donor introduced the factor(s) preventing the formation of a long 2,100 nt-transcript, because this long atp1 transcript reappeared in the progeny from self-crosses. The highly rearranged mitochondrial genomes with a variation in gene flanking regions make S. vulgaris an excellent model for the study of mitochondrial gene expression in plants.