Optimizing the use of twitter for research dissemination: The "Three Facts and a Story" randomized-controlled trial.

BACKGROUND & AIMS: Published research promoted on twitter reaches more readers. Tweets with graphics are more engaging than those without. However, data are limited regarding how to optimize multimedia tweets for engagement. METHODS: The "Three facts and a Story" trial is a randomized-controlled trial comparing a tweet featuring a graphical abstract to paired tweets featuring the personal motivations behind the research and a summary of the findings. Fifty-four studies published by the Journal of Hepatology were randomized at the time of online publication. The primary endpoint was assessed at 28-days from online publication with a primary outcome of full-text downloads from the website. Secondary outcomes included page views and twitter engagement including impressions, likes, and retweets. RESULTS: Overall, 31 studies received standard tweets and 23 received story tweets. Five studies were randomized to story tweets but crossed over to standard tweets for lack of author participation. Most papers tweeted were original articles (94% standard, 91% story) and clinical topics (55% standard, 61% story). Story tweets were associated with a significant increase in the number of full text downloads, 51 (34-71) vs. 25 (13-41), p = 0.002. There was also a non-significant increase in the number of page views. Story tweets generated an average of >1,000 more impressions than standard tweets (5,388 vs. 4,280, p = 0.002). Story tweets were associated with a similar number of retweets, and a non-significant increase in the number of likes. CONCLUSION: Tweets featuring the authors and their motivations may increase engagement with published research.

Forward genetic screens identify a role for the mitochondrial HER2 in E-2-hexenal responsiveness.

KEY MESSAGE: This work adds a new player, HER2, downstream of the perception of E-2-hexenal, a green leaf volatile, and shows that E-2-hexenal specifically changes the redox status of the mitochondria. It is widely accepted that plants produce and respond to green leaf volatiles (GLVs), but the molecular components involved in transducing their perception are largely unknown. The GLV E-2-hexenal inhibits root elongation in seedlings and, using this phenotype, we isolated E-2-hexenal response (her) Arabidopsis thaliana mutants. Using map-based cloning we positioned the her2 mutation to the At5g63620 locus, resulting in a phenylalanine instead of serine on position 223. Knockdown and overexpression lines of HER2 confirmed the role of HER2, which encodes an oxidoreductase, in the responsiveness to E-2-hexenal. Since E-2-hexenal is a reactive electrophile species, which are known to influence the redox status of cells, we utilized redox sensitive GFP2 (roGFP2) to determine the redox status of E-2-hexenal-treated root cells. Since the signal peptide of HER2 directed mCherry to the mitochondria, we targeted the expression of roGFP2 to this organelle besides the cytosol. E-2-hexenal specifically induced a change in the redox status in the mitochondria. We did not see a difference in the redox status in her2 compared to wild-type Arabidopsis. Still, the mitochondrial redox status did not change with Z-3-hexenol, another abundant GLV. These results indicate that HER2 is involved in transducing the perception of E-2-hexenal, which changes the redox status of the mitochondria.

WRKY40 and WRKY6 act downstream of the green leaf volatile E-2-hexenal in Arabidopsis.

Plants are known to be responsive to volatiles, but knowledge about the molecular players involved in transducing their perception remains scarce. We study the response of Arabidopsis thaliana to E-2-hexenal, one of the green leaf volatiles (GLV) that is produced upon wounding, herbivory or infection with pathogens. We have taken a transcriptomics approach to identify genes that are induced by E-2-hexenal, but not by defence hormones or other GLVs. Furthermore, by studying the promoters of early E-2-hexenal-induced genes we determined that the only statistically enriched cis-element was the W-box motif. Since members of the plant-specific family of WRKY transcription factors act in trans on this cis-element, we focused on WRKY6, 40 and 53 that were most strongly induced by E-2-hexenal. Root elongation of Arabidopsis seedlings of the wrky40 wrky6 double mutant was much less inhibited than in wt plants, similar to the E-2-hexenal-responsive mutant her1, which is perturbed in γ-amino butyric acid (GABA) metabolism. The induction of several of the E-2-hexenal-specific genes was much higher in the wrky40, wrky6 or wrky40 wrky6 mutants, including GAD4, a glutamate decarboxylase that catalyzes the formation of GABA from glutamate. In conclusion, WRKY6 and 40 seem to act as important players transducing E-2-hexenal perception.

Green leaf volatiles: a plant's multifunctional weapon against herbivores and pathogens.

Plants cannot avoid being attacked by an almost infinite number of microorganisms and insects. Consequently, they arm themselves with molecular weapons against their attackers. Plant defense responses are the result of a complex signaling network, in which the hormones jasmonic acid (JA), salicylic acid (SA) and ethylene (ET) are the usual suspects under the magnifying glass when researchers investigate host-pest interactions. However, Green Leaf Volatiles (GLVs), C6 molecules, which are very quickly produced and/or emitted upon herbivory or pathogen infection by almost every green plant, also play an important role in plant defenses. GLVs are semiochemicals used by insects to find their food or their conspecifics. They have also been reported to be fundamental in indirect defenses and to have a direct effect on pests, but these are not the only roles of GLVs. These volatiles, being probably one of the fastest weapons exploited, are also able to directly elicit or prime plant defense responses. Moreover, GLVs, via crosstalk with phytohormones, mostly JA, can influence the outcome of the plant's defense response against pathogens. For all these reasons GLVs should be considered as co-protagonists in the play between plants and their attackers.

E-2-hexenal promotes susceptibility to Pseudomonas syringae by activating jasmonic acid pathways in Arabidopsis.

Green leaf volatiles (GLVs) are C6-molecules - alcohols, aldehydes, and esters - produced by plants upon herbivory or during pathogen infection. Exposure to this blend of volatiles induces defense-related responses in neighboring undamaged plants, thus assigning a role to GLVs in regulating plant defenses. Here we compared Arabidopsis thaliana ecotype Landsberg erecta (Ler) with a hydroperoxide lyase line, hpl1, unable to synthesize GLVs, for susceptibility to Pseudomonas syringae pv. tomato (DC3000). We found that the growth of DC3000 was significantly reduced in the hpl1 mutant. This phenomenon correlated with lower jasmonic acid (JA) levels and higher salicylic acid levels in the hpl1 mutant. Furthermore, upon infection, the JA-responsive genes VSP2 and LEC were only slightly or not induced, respectively, in hpl1. This suggests that the reduced growth of DC3000 in hpl1 plants is due to the constraint of JA-dependent responses. Treatment of hpl1 plants with E-2-hexenal, one of the more reactive GLVs, prior to infection with DC3000, resulted in increased growth of DC3000 in hpl1, thus complementing this mutant. Interestingly, the growth of DC3000 also increased in Ler plants treated with E-2-hexenal. This stronger growth was not dependent on the JA-signaling component MYC2, but on ORA59, an integrator of JA and ethylene signaling pathways, and on the production of coronatine by DC3000. GLVs may have multiple effects on plant-pathogen interactions, in this case reducing resistance to Pseudomonas syringae via JA and ORA59.

Improved herbivore resistance in cultivated tomato with the sesquiterpene biosynthetic pathway from a wild relative.

Tomato breeding has been tremendously efficient in increasing fruit quality and quantity but did not focus on improving herbivore resistance. The biosynthetic pathway for the production of 7-epizingiberene in a wild tomato was introduced into a cultivated greenhouse variety with the aim to obtain herbivore resistance. 7-Epizingiberene is a specific sesquiterpene with toxic and repellent properties that is produced and stored in glandular trichomes. We identified 7-epizingiberene synthase (ShZIS) that belongs to a new class of sesquiterpene synthases, exclusively using Z-Z-farnesyl-diphosphate (zFPP) in plastids, probably arisen through neo-functionalization of a common ancestor. Expression of the ShZIS and zFPP synthases in the glandular trichomes of cultivated tomato resulted in the production of 7-epizingiberene. These tomatoes gained resistance to several herbivores that are pests of tomato. Hence, introduction of this sesquiterpene biosynthetic pathway into cultivated tomatoes resulted in improved herbivore resistance.

Mutations in γ-aminobutyric acid (GABA) transaminase genes in plants or Pseudomonas syringae reduce bacterial virulence.

Pseudomonas syringae pv. tomato DC3000 is a bacterial pathogen of Arabidopsis and tomato that grows in the apoplast. The non-protein amino acid γ-amino butyric acid (GABA) is produced by Arabidopsis and tomato and is the most abundant amino acid in the apoplastic fluid of tomato. The DC3000 genome harbors three genes annotated as gabT GABA transaminases. A DC3000 mutant lacking all three gabT genes was constructed and found to be unable to utilize GABA as a sole carbon and nitrogen source. In complete minimal media supplemented with GABA, the mutant grew less well than wild-type DC3000 and showed strongly reduced expression of hrpL and avrPto, which encode an alternative sigma factor and effector, respectively, associated with the type III secretion system. The growth of the gabT triple mutant was weakly reduced in Arabidopsis ecotype Landberg erecta (Ler) and strongly reduced in the Ler pop2-1 GABA transaminase-deficient mutant that accumulates higher levels of GABA. Much of the ability to grow on GABA-amended minimal media or in Arabidopsis pop2-1 leaves could be restored to the gabT triple mutant by expression in trans of just gabT2. The ability of DC3000 to elicit the hypersensitive response (HR) in tobacco leaves is dependent upon deployment of the type III secretion system, and the gabT triple mutant was less able than wild-type DC3000 to elicit this HR when bacteria were infiltrated along with GABA at levels of 1 mm or more. GABA may have multiple effects on P. syringae-plant interactions, with elevated levels increasing disease resistance.

The Arabidopsis her1 mutant implicates GABA in E-2-hexenal responsiveness.

When wounded or attacked by herbivores or pathogens, plants produce a blend of six-carbon alcohols, aldehydes and esters, known as C6-volatiles. Undamaged plants, when exposed to C6-volatiles, respond by inducing defense-related genes and secondary metabolites, suggesting that C6-volatiles can act as signaling molecules regulating plant defense responses. However, to date, the molecular mechanisms by which plants perceive and respond to these volatiles are unknown. To elucidate such mechanisms, we decided to isolate Arabidopsis thaliana mutants in which responses to C6-volatiles were altered. We observed that treatment of Arabidopsis seedlings with the C6-volatile E-2-hexenal inhibits root elongation. Among C6-volatiles this response is specific to E-2-hexenal, and is not dependent on ethylene, jasmonic and salicylic acid. Using this bioassay, we isolated 18 E-2-hexenal-response (her) mutants that showed sustained root growth after E-2-hexenal treatment. Here, we focused on the molecular characterization of one of these mutants, her1. Microarray and map-based cloning revealed that her1 encodes a gamma-amino butyric acid transaminase (GABA-TP), an enzyme that degrades GABA. As a consequence of the mutation, her1 plants accumulate high GABA levels in all their organs. Based on the observation that E-2-hexenal treatment induces GABA accumulation, and that high GABA levels confer resistance to E-2-hexenal, we propose a role for GABA in mediating E-2-hexenal responses.

The Medicago truncatula lysin [corrected] motif-receptor-like kinase gene family includes NFP and new nodule-expressed genes.

Rhizobial Nod factors are key symbiotic signals responsible for starting the nodulation process in host legume plants. Of the six Medicago truncatula genes controlling a Nod factor signaling pathway, Nod Factor Perception (NFP) was reported as a candidate Nod factor receptor gene. Here, we provide further evidence for this by showing that NFP is a lysin [corrected] motif (LysM)-receptor-like kinase (RLK). NFP was shown both to be expressed in association with infection thread development and to be involved in the infection process. Consistent with deviations from conserved kinase domain sequences, NFP did not show autophosphorylation activity, suggesting that NFP needs to associate with an active kinase or has unusual functional characteristics different from classical kinases. Identification of nine new M. truncatula LysM-RLK genes revealed a larger family than in the nonlegumes Arabidopsis (Arabidopsis thaliana) or rice (Oryza sativa) of at least 17 members that can be divided into three subfamilies. Three LysM domains could be structurally predicted for all M. truncatula LysM-RLK proteins, whereas one subfamily, which includes NFP, was characterized by deviations from conserved kinase sequences. Most of the newly identified genes were found to be expressed in roots and nodules, suggesting this class of receptors may be more extensively involved in nodulation than was previously known.

Formation of organelle-like N2-fixing symbiosomes in legume root nodules is controlled by DMI2.

In most legume nodules, the N2-fixing rhizobia are present as organelle-like structures inside their host cells. These structures, named symbiosomes, contain one or a few rhizobia surrounded by a plant membrane. Symbiosome formation requires the release of bacteria from cell-wall-bound infection threads. In primitive legumes, rhizobia are hosted in intracellular infection threads that, in contrast to symbiosomes, are bound by a cell wall. The formation of symbiosomes is presumed to represent a major step in the evolution of legume-nodule symbiosis, because symbiosomes facilitate the exchange of metabolites between the two symbionts. Here, we show that the genes, which are essential for initiating nodule formation, are also actively transcribed in mature Medicago truncatula nodules in the region where symbiosome formation occurs. At least one of these genes, encoding the receptor kinase DOES NOT MAKE INFECTIONS 2 (DMI2) is essential for symbiosome formation. The protein locates to the host cell plasma membrane and to the membrane surrounding the infection threads. A partial reduction of DMI2 expression causes a phenotype that resembles the infection structures found in primitive legume nodules, because infected cells are occupied by large intracellular infection threads instead of by organelle-like symbiosomes.

Use of the fluorescent timer DsRED-E5 as reporter to monitor dynamics of gene activity in plants.

Fluorescent proteins, such as green fluorescent protein and red fluorescent protein (DsRED), have become frequently used reporters in plant biology. However, their potential to monitor dynamic gene regulation is limited by their high stability. The recently made DsRED-E5 variant overcame this problem. DsRED-E5 changes its emission spectrum over time from green to red in a concentration independent manner. Therefore, the green to red fluorescence ratio indicates the age of the protein and can be used as a fluorescent timer to monitor dynamics of gene expression. Here, we analyzed the potential of DsRED-E5 as reporter in plant cells. We showed that in cowpea (Vigna unguiculata) mesophyll protoplasts, DsRED-E5 changes its fluorescence in a way similar to animal cells. Moreover, the timing of this shift is suitable to study developmental processes in plants. To test whether DsRed-E5 can be used to monitor gene regulation in plant organs, we placed DsRED-E5 under the control of promoters that are either up- or down-regulated (MtACT4 and LeEXT1 promoters) or constitutively expressed (MtACT2 promoter) during root hair development in Medicago truncatula. Analysis of the fluorescence ratios clearly provided more accurate insight into the timing of promoter activity.