Chromatin modifier HUSH co-operates with RNA decay factor NEXT to restrict transposable element expression

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Respective contributions of Arabidopsis DCL2 and DCL4 to RNA silencing

Dicer proteins are central to the different mechanisms involving RNA interference. Plants have evolved multiple DICER‐LIKE (DCL) copies, thus enabling functional diversification. In Arabidopsis, DCL2 and DCL4 process double‐stranded RNA into 22 and 21 nucleotide small interfering (si)RNAs, respectively, and have overlapping functions with regards to virus and transgene silencing. Nonetheless, some studies have reported that dcl2 or dcl4 single mutations are sometimes sufficient to hinder silencing. To better dissect the role of DCL2 and DCL4, we analyzed silencing kinetics and efficiencies using different transgenic systems in single and double mutant backgrounds. The results indicate that DCL2 stimulates transitivity and secondary siRNA production through DCL4 while being sufficient for silencing on its own. Notably, silencing of 35S‐driven transgenes functions more efficiently in dcl4 mutants, indicating that DCL4 mostly obscures DCL2 in wild‐type plants. Nonetheless, in a dcl4 mutant compromised in phloem‐originating silencing, ectopically expressed DCL2 allows restoration of silencing, suggesting that DCL2 is not, or poorly, expressed in phloem. Remarkably, this ectopic DCL2 contribution to phloem‐originating silencing is dependent on the activity of RNA‐DEPENDENT RNA POLYMERASE6. These results indicate that, despite differences in the silencing activity of their small RNA products, DCL2 and DCL4 mostly act redundantly yet hierarchically when present simultaneously.     

Conferring high‐temperature tolerance to nontransgenic tomato scions using graft transmission of RNA silencing of the fatty acid desaturase gene

We investigated graft transmission of high‐temperature tolerance in tomato scions to nontransgenic scions from transgenic rootstocks, where the fatty acid desaturase gene (LeFAD7) was RNA‐silenced. Tomato was transformed with a plasmid carrying an inverted repeat of LeFAD7 by Agrobacterium. Several transgenic lines showed the lower amounts of LeFAD7 RNA and unsaturated fatty acids, while nontransgenic control did not, and siRNA was detected in the transgenic lines, but not in control. These lines grew under conditions of high temperature, while nontransgenic control did not. Further, the nontransgenic plants were grafted onto the silenced transgenic plants. The scions showed less of the target gene RNA, and siRNA was detected. Under high‐temperature conditions, these grafted plants grew, while control grafted plants did not. Thus, it was shown that high‐temperature tolerance was conferred in the nontransgenic scions after grafting onto the silenced rootstocks.     

Host‐induced gene silencing inhibits the biotrophic pathogen causing downy mildew of lettuce

Host‐induced gene silencing (HIGS) is an RNA interference‐based approach in which small interfering RNAs (siRNAs) are produced in the host plant and subsequently move into the pathogen to silence pathogen genes. As a proof‐of‐concept, we generated stable transgenic lettuce plants expressing siRNAs targeting potentially vital genes of Bremia lactucae, a biotrophic oomycete that causes downy mildew, the most important disease of lettuce worldwide. Transgenic plants, expressing inverted repeats of fragments of either the Highly Abundant Message #34 (HAM34) or Cellulose Synthase (CES1) genes of B. lactucae, specifically suppressed expression of these genes, resulting in greatly reduced growth and inhibition of sporulation of B. lactucae. This demonstrates that HIGS can provide effective control of B. lactucae in lettuce; such control does not rely on ephemeral resistance conferred by major resistance genes and therefore offers new opportunities for durable control of diverse diseases in numerous crops.     

小RNA与蛋白质的相互作用

小分子调控RNA,包括siRNA（small interfering RNA）、miRNA（microRNA）和piRNA（piwiinteracting RNA）、hsRNA（heterochromatin associatedsmall RNA）等,是当前生命科学研究的前沿热点。越来越多的证据表明,这些小分子RNA存在于几乎所有较高等的真核生物细胞中,对生物体具有非常重要的调控功能。它们通过各种序列特异性的RNA基因沉默作用,包括RNA干扰（RNAi）、翻译抑制、异染色质形成等,调控诸如生长发育、应激反应、沉默转座子等各种各样的细胞进程。随着对这些小分子调控RNA的发现,一些RNascⅢ酶家族成员、Argonaute蛋白质家族成员及RNA结合蛋白质等先后被鉴定为小RNA的胞内蛋白质合作者,参与小RNA的加工成熟和在细胞内行使功能。本综述简介一些RNA沉默作用途径中重要组分的结构和功能的研究进展。     

Host‐induced gene silencing of an essential chitin synthase gene confers durable resistance to Fusarium head blight and seedling blight in wheat

Fusarium head blight (FHB) and Fusarium seedling blight (FSB) of wheat, caused by Fusarium pathogens, are devastating diseases worldwide. We report the expression of RNA interference (RNAi) sequences derived from an essential Fusarium graminearum (Fg) virulence gene, chitin synthase (Chs) 3b, as a method to enhance resistance of wheat plants to fungal pathogens. Deletion of Chs3b was lethal to Fg; disruption of the other Chs gene family members generated knockout mutants with diverse impacts on Fg. Comparative expression analyses revealed that among the Chs gene family members, Chs3b had the highest expression levels during Fg colonization of wheat. Three hairpin RNAi constructs corresponding to the different regions of Chs3b were found to silence Chs3b in transgenic Fg strains. Co‐expression of these three RNAi constructs in two independent elite wheat cultivar transgenic lines conferred high levels of stable, consistent resistance (combined type I and II resistance) to both FHB and FSB throughout the T₃ to T₅ generations. Confocal microscopy revealed profoundly restricted mycelia in Fg‐infected transgenic wheat plants. Presence of the three specific short interfering RNAs in transgenic wheat plants was confirmed by Northern blotting, and these RNAs efficiently down‐regulated Chs3b in the colonizing Fusarium pathogens on wheat seedlings and spikes. Our results demonstrate that host‐induced gene silencing of an essential fungal chitin synthase gene is an effective strategy for enhancing resistance in crop plants under field test conditions.     

Anoxia can increase the rate of decay for cnidarian tissue: Using Actinia equina to understand the early fossil record

An experimental decay methodology is developed for a cnidarian model organism to serve as a comparison to the many previous such studies on bilaterians. This allows an examination of inherent bias against the fossilisation of cnidarian tissue and their diagnostic characters, under what conditions these occur, and in what way. The decay sequence of Actinia equina was examined under a series of controlled conditions. These experiments show that cnidarian decay begins with an initial rupturing of the epidermis, followed by rapid loss of recognisable internal morphological characters. This suggests that bacteria work quicker on the epidermis than autolysis does on the internal anatomy. The data also show that diploblastic tissue is not universally decayed more slowly under anoxic or reducing conditions than under oxic conditions. Indeed, some cnidarian characters decay more rapidly under anoxic conditions than they do under oxic conditions. This suggests the decay pathways acting may be different to those affecting soft bilaterian tissue such as soft epidermis and internal organs. What is most important in the decay of soft polyp anatomy is the microbial community, which can be dominated by oxic or anoxic bacteria. Different Lagerstätte, even of the same type, will inevitably have subtle difference in their bacterial communities, which among other factors, could be a control on soft polyp preservation leading to either an absence of compelling soft anthozoans (Burgess Shale) or an astonishing abundance (Qingjiang biota).     

Carvacrol and eugenol effectively inhibit Rhizopus stolonifer and control postharvest soft rot decay in peaches

Aims

This study aimed to investigate the antifungal mechanism of carvacrol and eugenol to inhibit Rhizopus stolonifer and the control of postharvest soft rot decay in peaches.

Methods and Results

To investigate the antifungal mechanism, the effects of carvacrol and eugenol on the mycelium growth, leakages of cytoplasmic contents, mycelium morphology, cell membrane and membrane composition of R. stolonifer were studied. Carvacrol and eugenol both exhibited dose‐dependent antifungal activity against R. stolonifer, carvacrol at a concentration of 2 μl per plant and eugenol at a concentration of 4 μl per plant inhibited fungal growth completely. The two essential oils (EOs) increased cell membrane penetrability and caused the leakage of cytoplasm, nucleic acid and protein content. The observation using scanning electron microscopy and fluorescent microscopy showed modification of the hyphal morphology and breakage of the cell plasma membrane. Decreased ergosterol contents confirmed that the two EOs could destroy the membrane of R. stolonifer. For the in vivo test, the inhibition of soft rot disease and the induction of defence‐related enzymes were investigated. Carvacrol and eugenol significantly reduced the incidence and severity of soft rot decay in inoculated peaches. The best treatments for controlling soft rot decay were obtained at 0·5 μl l^?1 for carvacrol and 1 μl l^?1 for eugenol. The activities of defence‐related enzymes in peaches were also enhanced by fumigation with two EOs.

Conclusion

This study showed that carvacrol and eugenol could effectively inhibit the growth of R. stolonifer in vitro and successfully control the incidence of soft rot decay in honey peaches.

Significance and Impact of the Study

The above findings may be the main antifungal mechanism of carvacrol and eugenol on R. stolonifer. Furthermore, carvacrol and eugenol are helpful for their commercial application on the preservation of fresh fruit.     

CRISPR/SpCas9-mediated double knockout of barley Microrchidia MORC1 and MORC6a reveals their strong involvement in plant immunity,transcriptional gene silencing and plant growth

The Microrchidia (MORC) family proteins are important nuclear regulators in both animals and plants with critical roles in epigenetic gene silencing and genome stabilization. In the crop plant barley (Hordeum vulgare), seven MORC gene family members have been described. While barley HvMORC1 has been functionally characterized, very little information is available about other HvMORC paralogs. In this study, we elucidate the role of HvMORC6a and its potential interactors in regulating plant immunity via analysis of CRISPR/SpCas9-mediated single and double knockout (dKO) mutants, hvmorc1 (previously generated and characterized by our group), hvmorc6a, and hvmorc1/6a. For generation of hvmorc1/6a, we utilized two different strategies: (i) successive Agrobacterium-mediated transformation of homozygous single mutants, hvmorc1 and hvmorc6a, with the respective second construct, and (ii) simultaneous transformation with both hvmorc1 and hvmorc6a CRISPR/SpCas9 constructs. Total mutation efficiency in transformed homozygous single mutants ranged from 80 to 90%, while upon simultaneous transformation, SpCas9-induced mutation in both HvMORC1 and HvMORC6a genes was observed in 58% of T0 plants. Subsequent infection assays showed that HvMORC6a covers a key role in resistance to biotrophic (Blumeria graminis) and necrotrophic (Fusarium graminearum) plant pathogenic fungi, where the dKO hvmorc1/6a showed the strongest resistant phenotype. Consistent with this, the dKO showed highest levels of basal PR gene expression and derepression of TEs. Finally, we demonstrate that HvMORC1 and HvMORC6a form distinct nucleocytoplasmic homo-/heteromers with other HvMORCs and interact with components of the RNA-directed DNA methylation (RdDM) pathway, further substantiating that MORC proteins are involved in the regulation of TEs in barley.     

Enhanced resistance to soybean cyst nematode Heterodera glycines in transgenic soybean by silencing putative CLE receptors

CLE peptides are small extracellular proteins important in regulating plant meristematic activity through the CLE‐receptor kinase‐WOX signalling module. Stem cell pools in the SAM (shoot apical meristem), RAM (root apical meristem) and vascular cambium are controlled by CLE signalling pathways. Interestingly, plant‐parasitic cyst nematodes secrete CLE‐like effector proteins, which act as ligand mimics of plant CLE peptides and are required for successful parasitism. Recently, we demonstrated that Arabidopsis CLE receptors CLAVATA1 (CLV1), the CLAVATA2 (CLV2)/CORYNE (CRN) heterodimer receptor complex and RECEPTOR‐LIKE PROTEIN KINASE 2 (RPK2), which transmit the CLV3 signal in the SAM, are required for perception of beet cyst nematode Heterodera schachtii CLEs. Reduction in nematode infection was observed in clv1, clv2, crn, rpk2 and combined double and triple mutants. In an effort to develop nematode resistance in an agriculturally important crop, orthologues of Arabidopsis receptors including CLV1, CLV2, CRN and RPK2 were identified from soybean, a host for the soybean cyst nematode Heterodera glycines. For each of the receptors, there are at least two paralogues in the soybean genome. Localization studies showed that most receptors are expressed in the root, but vary in their level of expression and spatial expression patterns. Expression in nematode‐induced feeding cells was also confirmed. In vitro direct binding of the soybean receptors with the HgCLE peptide was analysed. Knock‐down of the receptors in soybean hairy roots showed enhanced resistance to SCN. Our findings suggest that targeted disruption of nematode CLE signalling may be a potential means to engineer nematode resistance in crop plants.