Tobacco rattle virus (TRV)-mediated virus-induced gene silencing (VIGS) has been frequently used in dicots. Here we show that it can also be used in monocots, by presenting a system involving use of a novel infiltration solution (containing acetosyringone, cysteine, and Tween 20) that enables whole-plant level VIGS of (germinated) seeds in wheat and maize. Using the established system, phytoene desaturase (PDS) genes were successfully silenced, resulting in typical photo-bleaching symptoms in the leaves of treated wheat and maize. In addition, three wheat homoeoalleles of MLO, a key gene repressing defense responses to powdery mildew in wheat, were simultaneously silenced in susceptible wheat with this system, resulting in it becoming resistant to powdery mildew. The system has the advantages generally associated with TRV-mediated VIGS systems (e.g., high-efficiency, mild virus infection symptoms, and effectiveness in different organs). However, it also has the following further advantages: (germinated) seed-stage agroinfiltration; greater rapidity and convenience; whole-plant level gene silencing; adequately stable transformation; and suitability for studying functions of genes involved in seed germination and early plant development stages.

Transient transformation is simpler, more efficient and economical in analyzing protein subcellular localization than stable transformation. Fluorescent fusion proteins were often used in transient transformation to follow the in vivo behavior of proteins. Onion epidermis, which has large, living and transparent cells in a monolayer, is suitable to visualize fluorescent fusion proteins. The often used transient transformation methods included particle bombardment, protoplast transfection and Agrobacterium-mediated transformation. Particle bombardment in onion epidermis was successfully established, however, it was expensive, biolistic equipment dependent and with low transformation efficiency. We developed a highly efficient in planta transient transformation method in onion epidermis by using a special agroinfiltration method, which could be fulfilled within 5 days from the pretreatment of onion bulb to the best time-point for analyzing gene expression. The transformation conditions were optimized to achieve 43.87% transformation efficiency in living onion epidermis. The developed method has advantages in cost, time-consuming, equipment dependency and transformation efficiency in contrast with those methods of particle bombardment in onion epidermal cells, protoplast transfection and Agrobacterium-mediated transient transformation in leaf epidermal cells of other plants. It will facilitate the analysis of protein subcellular localization on a large scale.

Effectively achieving wound healing is a great challenge. Herein, we facilely prepared temperature-responsive MXene nanobelt fibers (T-RMFs) carrying vitamin E with a controllable release ability for wound healing. These T-RMFs were composed of MXene nanosheets spread along polyacrylonitrile and polyvinylpyrrolidone composite nanobelts together with a thermosensitive PAAV- coating layer. The high mass loading and high surface area of the MXene nanosheets endow the T-RMFs with excellent photothermal properties. The temperature could be easily controlled by near-infrared (NIR) irradiation exposure, and then the thermoresponsive polymeric coating layer relaxed the interface to dissolve vitamin E and promote vitamin E release. The T-RMFs demonstrated excellent biocompatibility and wound-healing functions in cellular and animal tests. The facile method, high mass loading, high surface area, excellent wound-healing functions, interesting nanosheet/nanobelt structure, mass production potential, and NIR responsive properties of these T-RMFs indicate the great potential of our nanobelts for wound healing, tissue engineering, and much broader application areas. This facile nanosheet/nanobelt preparation strategy paves a new way for nanomaterial fabrication and applications.

Tomato Verticillium wilt is a soil-borne vascular disease caused by the necrotrophic fungus Verticillium dahliae. Although some understanding of plant defense mechanisms against V. dahliae infection has been gained for incompatible interactions, including identification of inducible resistant genes and defense signaling pathways, the genes and signaling pathways involved in the compatible interaction remain unclear. To investigate the molecular basis of the compatible interaction between tomato and V. dahliae, transcriptomes of V. dahliae infected tomatoes were compared to those of a control group. A total of approximately 25 million high-quality reads were generated by means of the RNA sequencing (RNA-seq) method. The sequence reads were aligned to the tomato reference genome and analyzed to measure gene expression levels, and to identify alternative splicing events. Comparative analysis between the two samples revealed 1,953 significantly differentially expressed genes (DEGs), including 1,281 up-regulated and 672 down-regulated genes. The RNA-Seq output was confirmed using RT-qPCR for 10 selected genes. The Nr, Swiss-Prot, Gene Ontology (GO), Clusters of Orthologous Groups (COG), and Kyoto Encyclopedia of Genes and Genomes (KEGG) databases were used to annotate DEG functions. Of the 1,953 DEGs identified, 1,953, 1,579, 1,739, 862, and 380 were assigned by Nr, Swiss-Prot, GO, COG, and KEGG, respectively. The important functional groups identified via GO and COG enrichment were those responsible for fundamental biological regulation, secondary metabolism, and signal transduction. Of DEGs assigned to 87 KEGG pathways, most were associated with phenylpropanoid metabolism and plant–pathogen interaction pathways. Most of the DEGs involved in these two pathways were up-regulated, and may be involved in regulating the tomato-V. dahliae compatible interaction. The results will help to identify key susceptible genes and contribute to a better understanding of the mechanisms of tomato susceptible response to V. dahliae.

The whitebacked planthopper (WBPH), Sogatella furcifera, and brown planthopper (BPH) Nilaparvata lugens Stå l are important sucking insects of rice (Oryza sativa L.) crops throughout the world. Rice 'B5', which has derived its resistance genes from the wild rice O. officinalis Wall ex Watt, is a line that is highly resistant to both WBPH and BPH. Previously, two resistance genes against BPH, Qbp1, and Qbp2 in 'B5' had been mapped onto chromosome 3 and chromosome 4, respectively. In this study, we employed a mapping population composed of 187 recombinant inbred lines (RILs), produced from a cross between 'B5' and susceptible variety 'Minghui63', to locate the WBPH and BPH resistance genes. A RFLP survey of the bulked extremes from the RIL population identified two genomic regions, one on chromosome 3 and the other on chromosome 4, likely containing the resistance genes to planthoppers. QTL analysis of the RILs further confirmed that two WBPH resistance genes were mapped on the same loci as Qbp1 and Qbp2, using a linkage map with 242 molecular markers distributed on 12 rice chromosomes. Of the two WBPH resistance genes, one designated Wbph7(t) was located within a 1.1-cM region between R1925 and G1318 on chromosome 3, the other designated Wbph8(t) was within a 0.3-cM region flanked by R288 and S11182 on chromosome 4. A two-way analysis of variance showed that two loci acted independently with each other in determining WBPH resistance. The results have significant implications in studying the interactions between sucking insects and plants and in breeding programs of resistance to rice planthoppers.

A new protocol was established for the regeneration of Solanum nigrum by frog egg-like bodies (FELBs), which are novel somatic embryogenesis (SE) structures induced from the root, stem, and leaf explants. The root, stem, and leaf explants (93.33%, 85.10%, and 100.00%, respectively) were induced to form special embryonic calli on Murashige and Skoog (MS) medium containing 1.0 mg/L 2,4-dichlorophenoxyacetic acid, under dark condition. Further, special embryonic calli from the root, stem, and leaf explants (86.97%, 83.30%, and 99.47%, respectively) were developed into FELBs. Plantlets of FELBs from the three explants were induced in vitro on MS medium supplemented with 5.0 mg/L 6-benzylaminopurine and 0.1 mg/L gibberellic acid, and 100.00% plantlet induction rates were noted. However, plantlet induction in vivo on MS medium supplemented with 20 mg/L thidiazuron showed rates of 38.63%, 15.63%, and 61.30% for the root, stem, and leaf explants, respectively, which were lower than those of the in vitro culture. Morphological and histological analyses of FELBs at different development stages revealed that they are a novel type of SE structure that developed from the mesophyll (leaf) or cortex (stem and root) cells of S. nigrum.

An optimized regeneration and Agrobacterium-mediated transformation protocol based on whole cotyledonary node explants was developed in soybean (Glycine max) cultivar Zhong Huang 13. Adding 6-benzylaminopurine (BAP) in a germinating medium could significantly increase regeneration efficiency; the optimal BAP concentration for shoot formation was 0.5 mg/L. The concentrations of plant growth regulators in a shoot induction medium were optimized by the orthogonal test [L9 (3(3))]. The best combination for shoot regeneration was a medium of Murashige & Skoog salts with B5 vitamins (MSB) supplemented with 3.5 mg/L BAP, 0.2 mg/L indole-3-butyric acid (IBA), and 0.2 mg/L kinetin (KT). Under this favorable condition, one node could regenerate 28-30 shoots. Soybean whole cotyledonary nodes were transformed by inoculation with A. tumefaciens strain EHA105 harboring a vector pBI121 containing a β-glucuronidase gene (gus). GUS assay, polymerase chain reaction, and Southern blot analysis indicated that the gus gene was transformed into soybean plants with 23.1% transformation efficiency. Transgenic plants could be obtained within 5-6 weeks, which was about 4 weeks less than that of a traditional single cotyledonary node method.

Interspecific hybridization and polyploidization may involve programmed genetic and epigenetic changes. In this study, we used the methylation-sensitive amplified polymorphism (MSAP) method to survey cytosine methylation alterations that occurred in F(1) hybrid and BC(1) progeny following interspecific hybridization between Oryza sativa and O. officinalis. Across all 316 parental methylated sites, 25 (7.9%) cytosine methylation alterations were detected in the F(1) and/or BC(1) progeny. Thirty additional cytosine methylation alterations were detected at parental non-methylated or novel sites. In total, 55 cytosine methylation alterations (90.9% of all alterations) were detected in the F(1) hybrid, which were maintained in the BC(1) progeny. The alterations in cytosine methylation were biased toward the O. officinalis parent and were in some cases repeatable in independent hybridizations between O. sativa and O. officinalis. Twelve fragments showing cytosine methylation alterations were isolated, sequenced and subsequently validated by methylation-sensitive Southern blot analysis. Where possible, we designed species-specific primers to amplify the polymorphic transcripts from either the O. sativa or the O. officinalis parent using reverse transcription (RT)-PCR in combination with single-strand conformation polymorphism (SSCP) analysis. In four of five cases, modified gene expression could be correlated with the altered cytosine methylation pattern. Our results demonstrated cytosine methylation alterations induced by interspecific hybridization between a rice cultivar and its wild relative, and indicated a direct relationship between cytosine methylation alteration and gene expression variation.