An intriguing new paradigm in plant biology is that systemically mobile mRNAs play a role in coordinating development. In this process, specific mRNAs are loaded into the phloem transport stream for translocation to distant tissues, where they may impact on developmental processes. However, despite its potential significance for plant growth regulation, mRNA trafficking remains poorly understood and challenging to study. Here, we show that phloem-mobile mRNAs can also traffic between widely divergent species from a host to the plant parasite lespedeza dodder (Cuscuta pentagona Engelm.). Reverse transcriptionpolymerase chain reaction and microarray analysis were used to detect specific tomato (Lycopersicon esculentum Mill.) transcripts in dodder grown on tomato that were not present in control dodder grown on other host species. Foreign transcripts included LeGAI, which has previously been shown to be translocated in the phloem, as well as nine other transcripts not reported to be mobile. Dodders are parasitic plants that obtain resources by drawing from the phloem of a host plant and have joint plasmodesmata with host cortical cells. Although viruses are known to move between dodder and its hosts, translocation of endogenous plant mRNA has not been reported. These results point to a potentially new level of interspecies communication, and raise questions about the ability of parasites to recognize, use, and respond to transcripts acquired from their hosts.Dodders are obligate stem parasitic plants that have close physical linkages with their hosts. Because of their limited photosynthetic ability and dependence upon the host plant for water and nutrients, a dodder seedling must form connections with a host within several days after germination. Once established on the host, the dodder root system senesces and the mature vegetative plant consists entirely of a yelloworange stem that twines around host stems and leaves.

BackgroundReduced yields of ethanol due to bacterial contamination in fermentation cultures weaken the economics of biofuel production. Lactic acid bacteria are considered the most problematic, and surveys of commercial fuel ethanol facilities have found that species of Lactobacillus are predominant. Bacteriophage lytic enzymes are peptidoglycan hydrolases that can degrade the Gram positive cell wall when exposed externally and provide a novel source of antimicrobials that are highly refractory to resistance development.ResultsThe streptococcal phage LambdaSa2 (λSa2) endolysin demonstrated strong lytic activity towards 17 of 22 strains of lactobacilli, staphylococci or streptococci and maintained an optimal specific activity at pH 5.5 and in the presence of ≤ 5% ethanol (fermentation conditions) toward L. fermentum. Lactobacillus bacteriophage endolysins LysA, LysA2 and LysgaY showed exolytic activity towards 60% of the lactobacilli tested including four L. fermentum isolates from fuel ethanol fermentations. In turbidity reduction assays LysA was able to reduce optical density >75% for 50% of the sensitive strains and >50% for the remaining strains. LysA2 and LysgaY were only able to decrease cellular turbidity by <50%. Optimal specific activities were achieved for LysA, LysA2, and LysgaY at pH 5.5. The presence of ethanol (≤5%) did not reduce the lytic activity. Lysins were able to reduce both L. fermentum (BR0315-1) (λSa2 endolysin) and L. reuteri (B-14171) (LysA) contaminants in mock fermentations of corn fiber hydrolysates.ConclusionBacteriophage lytic enzymes are strong candidates for application as antimicrobials to control lactic acid bacterial contamination in fuel ethanol fermentations.

The ability to hydrolyze microcrystalline cellulose is an uncommon feature in the microbial world, but one that can be exploited for conversion of lignocellulosic feedstocks into bio-based fuels and chemicals. Understanding the physiological and biochemical mechanisms by which microorganisms deconstruct cellulosic material is key to achieving this objective. The Glucan Degradation Locus (GDL) in the genomes of extremely thermophilic species encodes polysaccharide lyases (PLs), unique cellulose binding proteins (tāpirins), and putative post-translational modifying enzymes, in addition to multi-domain, multi-functional glycoside hydrolases (GHs), thereby representing an alternative paradigm for plant biomass degradation, as compared to fungal or cellulosomal systems. To examine the individual and collective roles of the glycolytic enzymes, the six GHs in the GDL of were systematically deleted, and the extent to which the resulting mutant strains could solubilize microcrystalline cellulose (Avicel) and plant biomasses (switchgrass or poplar) was examined. Three of the GDL enzymes, Athe_1867 (CelA) (GH9-CBM3-CBM3-CBM3-GH48), Athe_1859 (GH5-CBM3-CBM3-GH44), and Athe_1857 (GH10-CBM3-CBM3-GH48), acted synergistically and accounted for 92% of naked microcellulose (Avicel) degradation. However, the relative importance of the GDL GHs varied for the plant biomass substrates tested. Furthermore, mixed cultures of mutant strains showed switchgrass solubilization depended on the secretome-bound enzymes collectively produced by the culture and not on the specific strain from which they came. These results demonstrate that certain GDL GHs are primarily responsible for the degradation of microcrystalline-containing substrates by and provide new insights into the workings of a novel microbial mechanism for lignocellulose utilization. The efficient and extensive degradation of complex polysaccharides in lignocellulosic biomass, particularly microcrystalline cellulose, remains a major barrier to its use as a renewable feedstock for the production of fuels and chemicals. Extremely thermophilic bacteria from the genus rapidly degrade plant biomass to fermentable sugars at temperatures between 70-78°C, although the specific mechanism by which this occurs is not clear. Previous comparative genomic studies identified a genomic locus found only in certain species that was hypothesized to be mainly responsible for microcrystalline cellulose degradation. By systematically deleting genes in this locus in , the nuanced, substrate-specific, roles of glycolytic enzymes in deconstructing crystalline cellulose and plant biomasses could be discerned. The results here point to synergism of three multi-domain cellulases in , working in conjunction with the aggregate, secreted enzyme inventory, as the key to the plant biomass degradation ability by this extreme thermophile.

Recent research indicates that RNA translocation occurs between certain parasitic plant species and their hosts. The movement of at least 27 mRNAs has been demonstrated between hosts and Cuscuta pentagona Engelm., with the largest proportion of these being regulatory genes. Movement of RNAi signals has been documented from hosts to the parasites Triphysaria versicolor (Frisch & CA Mey) and Orobanche aegyptiaca (Pers.), demonstrating that the regulation of genes in one species can be influenced by transfer of RNA signals through a parasitic association. This review considers the implications of these findings in light of present understanding of host-parasite connections and the growing body of evidence that RNAs are able to act as signal molecules that convey regulatory information in a cell- and tissue-specific manner. Together, this suggests that parasitic plants can exchange RNAs with their hosts, and that this may be part of the coordinated growth and development that occurs during the process of parasitism. This phenomenon offers promise for new insights into parasitic plants, and new opportunities for the control of parasitic weeds.

The trichothecene mycotoxin deoxynivalenol (DON) is a common contaminant of small grains, such as wheat and barley, in the United States. New strategies to mitigate the threat of DON need to be developed and implemented. TRI101 and TRI201 are trichothecene 3-O-acetyltransferases that are able to modify DON and reduce its toxicity. Recent work has highlighted differences in the activities of TRI101 from two different species of Fusarium (F. graminearum and F. sporotrichioides), but little is known about the relative activities of TRI101/TRI201 enzymes produced by other species of Fusarium. We cloned TRI101 or TRI201 genes from seven different species of Fusarium and found genetic identity between sequences ranging from 66% to 98%. In vitro feeding studies using transformed yeast showed that all of the TRI101/TRI201 enzymes tested were able to acetylate DON; conversion of DON to 3-acetyl-deoxynivalenol (3ADON) ranged from 50.5% to 100.0%, depending on the Fusarium species from which the gene originated. A time course assay showed that the rate of acetylation varied from species to species, with the gene from F. sporotrichioides having the lowest rate. Steady-state kinetic assays using seven purified enzymes produced catalytic efficiencies for DON acetylation ranging from 6.8 ؋ 10 4 M ؊1 ⅐ s ؊1 to 4.7 ؋ 10 6 M ؊1 ⅐ s ؊1. Thermostability measurements for the seven orthologs ranged from 37.1°C to 43.2°C. Extended sequence analysis of portions of TRI101/TRI201 from 31 species of Fusarium (including known trichothecene producers and nonproducers) suggested that other members of the genus may contain functional TRI101/TRI201 genes, some with the potential to outperform those evaluated in the present study.

Background: One of the challenges facing the fuel ethanol industry is the management of bacterial contamination during fermentation. Lactobacillus species are the predominant contaminants that decrease the profitability of biofuel production by reducing ethanol yields and causing "stuck" fermentations, which incur additional economic losses via expensive antibiotic treatments and disinfection costs. The current use of antibiotic treatments has led to the emergence of drug-resistant bacterial strains, and antibiotic residues in distillers dried grains with solubles (DDGS) are a concern for the feed and food industries. This underscores the need for new, non-antibiotic, eco-friendly mitigation strategies for bacterial contamination. The specific objectives of this work were to (1) express genes encoding bacteriophage lytic enzymes (endolysins) in Saccharomyces cerevisiae, (2) assess the lytic activity of the yeast-expressed enzymes against different species of Lactobacillus that commonly contaminate fuel ethanol fermentations, and (3) test the ability of yeast expressing lytic enzymes to reduce Lactobacillus fermentum during fermentation. Implementing antibiotic-free strategies to reduce fermentation contaminants will enable more cost-effective fuel ethanol production and will impact both producers and consumers in the farm-to-fork continuum. Results: Two genes encoding the lytic enzymes LysA and LysA2 were individually expressed in S. cerevisiae on multi-copy plasmids under the control of a galactose-inducible promoter. The enzymes purified from yeast were lytic against Lactobacillus isolates collected from fermentors at a commercial dry grind ethanol facility including Lactobacillus fermentum, Lactobacillus brevis, and Lactobacillus mucosae. Reductions of L. fermentum in experimentally infected fermentations with yeast expressing LysA or LysA2 ranged from 0.5 log 10 colony-forming units per mL (CFU/mL) to 1.8 log 10 (CFU/mL) over 72 h and fermentations treated with transformed yeast lysate showed reductions that ranged from 0.9 log 10 (CFU/mL) to 3.3 log 10 (CFU/mL). Likewise, lactic acid and acetic acid levels were reduced in all experimentally infected fermentations containing transformed yeast (harboring endolysin expressing plasmids) relative to the corresponding fermentations with untransformed yeast.

BackgroundThe trichothecene mycotoxin deoxynivalenol (DON) may be concentrated in distillers dried grains with solubles (DDGS; a co-product of fuel ethanol fermentation) when grain containing DON is used to produce fuel ethanol. Even low levels of DON (≤ 5 ppm) in DDGS sold as feed pose a significant threat to the health of monogastric animals. New and improved strategies to reduce DON in DDGS need to be developed and implemented to address this problem. Enzymes known as trichothecene 3-O-acetyltransferases convert DON to 3-acetyldeoxynivalenol (3ADON), and may reduce its toxicity in plants and animals.ResultsTwo Fusarium trichothecene 3-O-acetyltransferases (FgTRI101 and FfTRI201) were cloned and expressed in yeast (Saccharomyces cerevisiae) during a series of small-scale ethanol fermentations using barley (Hordeum vulgare). DON was concentrated 1.6 to 8.2 times in DDGS compared with the starting ground grain. During the fermentation process, FgTRI101 converted 9.2% to 55.3% of the DON to 3ADON, resulting in DDGS with reductions in DON and increases in 3ADON in the Virginia winter barley cultivars Eve, Thoroughbred and Price, and the experimental line VA06H-25. Analysis of barley mashes prepared from the barley line VA04B-125 showed that yeast expressing FfTRI201 were more effective at acetylating DON than those expressing FgTRI101; DON conversion for FfTRI201 ranged from 26.1% to 28.3%, whereas DON conversion for FgTRI101 ranged from 18.3% to 21.8% in VA04B-125 mashes. Ethanol yields were highest with the industrial yeast strain Ethanol Red®, which also consumed galactose when present in the mash.ConclusionsThis study demonstrates the potential of using yeast expressing a trichothecene 3-O-acetyltransferase to modify DON during commercial fuel ethanol fermentation.

Microbial fermentation of lignocellulosic biomass to produce industrial chemicals is exacerbated by the recalcitrant network of lignin, cellulose and hemicelluloses comprising the plant secondary cell wall. In this study, we show that transgenic poplar ( Populus trichocarpa ) lines can be solubilized without any pretreatment by the extreme thermophile Caldicellulosiruptor bescii that has been metabolically engineered to shift its fermentation products away from inhibitory organic acids to ethanol. Carbohydrate solubilization and conversion of unpretreated milled biomass is nearly 90% for two transgenic lines, compared to only 25% for wild-type poplar. Unexpectedly, unpretreated intact poplar stems achieved nearly 70% of the fermentation production observed with milled poplar as the substrate. The nearly quantitative microbial conversion of the carbohydrate content of unpretreated transgenic lignocellulosic biomass bodes well for full utilization of renewable biomass feedstocks.