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.

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.

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 ten selected genes. The Nr, Swiss-Prot, GO, COG and 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.

Whitefly-transmitted begomoviruses are economically important plant pathogens that cause severe problems in many crop plants, such as tomato, papaya, cotton, and tobacco. Tomato yellow leaf curl virus (TYLCV) is a typical monopartite begomovirus that has been extensively studied, but methods that can efficiently control begomoviruses are still scarce. In this study, we combined artificial microRNA (amiRNA)-mediated silencing technology and clay nanosheet-mediated delivery by spraying and developed a method for efficiently preventing TYLCV infection in tomato plants. We designed three amiRNAs that target different regions of TYLCV to silence virus-produced transcripts. Three plant expression vectors expressing pre-amiRNAs were constructed, and recombinant plasmid DNAs (pDNAs) were loaded onto nontoxic and degradable layered double hydroxide (LDH) clay nanosheets. LDH nanosheets containing multiple pDNAs were sprayed onto plant leaves. We found that the designed amiRNAs were significantly accumulated in leaves 7 days after spraying, while the pDNAs were sustainably detected for 35 days after the spray, suggesting that the LDH nanosheets released pDNAs in a sustained manner, protected pDNAs from degradation and efficiently delivered pDNAs into plant cells. Importantly, when the LDH nanosheets coated with pDNAs were sprayed onto plants infected by TYLCV, both the disease severity and TYLCV viral concentration in sprayed plants were significantly decreased during the 35 days, while the levels of H₂O₂ were significantly increased in those plants. Taken together, these results indicate that LDH nanosheets loaded with pDNAs expressing amiRNAs can be a sustainable and promising tool for begomovirus control.

Transcription factors of the AP2/ERF family play important roles in plant growth, development, and responses to biotic and abiotic stresses. In this study, a physic nut AP2/ERF gene, JcDREB2, was functionally characterized. Real-time PCR analysis revealed that JcDREB2 was expressed mainly in the leaf and could be induced by abscisic acid (ABA) but suppressed by gibberellin (GA) and salt. Transient expression of a JcDREB2-YFP fusion protein in Arabidopsis protoplasts cells suggested that JcDREB2 is localized in the nucleus. Rice plants overexpressing JcDREB2 exhibited dwarf and GA-deficient phenotypes with shorter shoots and roots than those of wild-type plants. The dwarfism phenotype could be rescued by the application of exogenous GA3. The expression levels of GA biosynthetic genes including OsGA20ox1, OsGA20ox2, OsGA20ox4, OsGA3ox2, OsCPS1, OsKO2 and OsKAO were significantly reduced in plants overexpressing JcDREB2. Overexpression of JcDREB2 in rice increased sensitivity to salt stress. Increases in the expression levels of several salt-tolerance-related genes in response to salt stress were impaired in JcDREB2-overexpressing plants. These results demonstrated not only that JcDREB2 influences GA metabolism, but also that it can participate in the regulation of the salt stress response in rice.