RNA interference (RNAi) is a sequence-specific, posttranscriptional gene silencing (PTGS) process in plants that is mediated by dsRNA homologous to the silenced gene(s). In this study, we report an efficient method to produce dsRNA using a bacterial expression system. Two fragments of the Sugarcane Mosaic Virus (SCMV) CP (coat protein) gene were amplified by RT-PCR, and cloned into the inverted-repeat cloning vector pUCCRNAi. The two recombinant plasmids were transformed individually into E. coli HT115, an RNase-III deficient strain, and dsRNA was induced by isopropyl-β-D: -thiogalactopyranoside (IPTG). The crude extracts of E. coli HT115 containing large amounts of dsRNA were applied to plants as a spray and the experiment confirmed a preventative efficacy. Our findings demonstrated that spraying crude dsRNA-containing extracts inhibited SCMV infection, and the dsRNA derived from an upstream region (CP1) was more effective than was dsRNA derived from a downstream region (CP2) of the SCMV CP gene. The results provide a valuable tool for plant viral control using dsRNA and the PTGS approach.

No abstract

Cyanobacteria are globally important primary producers that have an exceptionally large iron requirement for photosynthesis. In many aquatic ecosystems, the levels of dissolved iron are so low and some of the chemical species so unreactive that growth of cyanobacteria is impaired. Pathways of iron uptake through cyanobacterial membranes are now being elucidated, but the molecular details are still largely unknown. Here we report that the non-siderophore-producing cyanobacterium Synechocystis sp. PCC 6803 contains three exbB-exbD gene clusters that are obligatorily required for growth and are involved in iron acquisition. The three exbB-exbDs are redundant, but single and double mutants have reduced rates of iron uptake compared with wild-type cells, and the triple mutant appeared to be lethal. Short-term measurements in chemically well-defined medium show that iron uptake by Synechocystis depends on inorganic iron (Fe 0 ) concentration and ExbB-ExbD complexes are essentially required for the Fe 0 transport process. Although transport of iron bound to a model siderophore, ferrioxamine B, is also reduced in the exbB-exbD mutants, the rate of uptake at similar total [Fe] is about 800-fold slower than Fe 0 , suggesting that hydroxamate siderophore iron uptake may be less ecologically relevant than free iron. These results provide the first evidence that ExbB-ExbD is involved in inorganic iron uptake and is an essential part of the iron acquisition pathway in cyanobacteria. The involvement of an ExbB-ExbD system for inorganic iron uptake may allow cyanobacteria to more tightly maintain iron homeostasis, particularly in variable environments where iron concentrations range from limiting to sufficient.

Cyanobacteria are foundational drivers of global nutrient cycling, with high intracellular iron (Fe) requirements. Fe is found at extremely low concentrations in aquatic systems, however, and the ways in which cyanobacteria take up Fe are largely unknown, especially the initial step in Fe transport across the outer membrane. Here, we identified one TonB protein and four TonB-dependent transporters (TBDTs) of the energy-requiring Fe acquisition system and six porins of the passive diffusion Fe uptake system in the model cyanobacterium sp. strain PCC 6803. The results experimentally demonstrated that TBDTs not only participated in organic ferri-siderophore uptake but also in inorganic free Fe (Fe') acquisition.Fe uptake rate measurements showed that a TBDT quadruple mutant acquired Fe at a lower rate than the wild type and lost nearly all ability to take up ferri-siderophores, indicating that TBDTs are critical for siderophore uptake. However, the mutant retained the ability to take up Fe' at 42% of the wild-type Fe' uptake rate, suggesting additional pathways of Fe' acquisition besides TBDTs, likely by porins. Mutations in four of the six porin-encoding genes produced a low-Fe-sensitive phenotype, while a mutation in all six genes was lethal to cell survival. These diverse outer membrane Fe uptake pathways reflect cyanobacterial evolution and adaptation under a range of Fe regimes across aquatic systems. Cyanobacteria are globally important primary producers and contribute about 25% of global CO fixation. Low Fe bioavailability in surface waters is thought to limit the primary productivity in as much as 40% of the global ocean. The Fe acquisition strategies that cyanobacteria have evolved to overcome Fe deficiency remain poorly characterized. We experimentally characterized the key players and the cooperative work mode of two Fe uptake pathways, including an active uptake pathway and a passive diffusion pathway in the model cyanobacterium sp. PCC 6803. Our finding proved that cyanobacteria use ferri-siderophore transporters to take up Fe', and they shed light on the adaptive mechanisms of cyanobacteria to cope with widespread Fe deficiency across aquatic environments.

Cyanobacteria are known to survive in iron-deficient environments, but the ways in which they acquire Fe and acclimate are not completely understood. Here we report a novel gene sll1263 that is required for Synechocystis sp. strain PCC 6803 to grow under iron-deficient conditions. sll1263 encodes a putative cation diffusion facilitator protein (CDF) that shows 50% amino acid similarity with ferrous iron efflux protein (FieF) of heterotrophic bacteria. In bacteria, the gene product is involved in metal export from the cell, but in Synechocystis sll1263 plays a role in iron uptake. The results show that expression of sll1263 was induced by iron-deficient conditions and its inactivation significantly decreased the growth rate of an sll1263(-) mutant. Other genes known to be required for Fe acquisition were also strongly up-regulated in the mutant even in the presence of high Fe. Overexpression of sll1263 increased growth under iron deficiency but reduced growth under high-iron stress, suggesting that the gene product was involved in iron uptake rather than detoxification. Expression of FieF in the sll1263(-) mutant was unable to rescue the Fe-deficient phenotype, but Sll1263 completely restored it. Measurements of cellular iron content and the iron uptake rate showed that they were significantly less in the sll1263(-) mutant than in the wild type, consistent with a role for sll1263 in iron uptake. We hypothesize that the low-iron habitats and high-iron requirements of cyanobacteria may be the reason why cyanobacterial CDF protein functions in Fe uptake and not efflux as in non-photosynthetic bacteria.