Transient Gene Expression Assays Research Articles

Transcription Factor CmWRKY13 Regulates Cucurbitacin B Biosynthesis Leading to Bitterness in Oriental Melon Fruit (Cucumis melo var. Makuwa Makino).

Bitterness, caused by cucurbitacin B (CuB), is one of the important traits that affects melon fruit quality and consumer acceptance. Therefore, the detailed mechanism behind the regulation of CuB biosynthesis on melon fruit needs to be further explored. This study investigated CuB content and transcriptomes of "YMR" melon fruit treated by 5 and 20 mg L-1 CPPU. The content of CuB reaches its peak in 5 days and then decreases. WGCNA identified the WRKY transcription factor (TF), CmWRKY13, coexpressed with CuB biosynthetic genes (Cm180, Cm170, Cm160, and CmACT). Yeast one-hybrid, dual-luciferase, and transient gene expression assays were conducted and suggested that the nucleus-localized CmWRKY13 transactivated the promoters of CuB biosynthetic genes and participated in the regulation of CuB biosynthesis. Furthermore, CmWRKY13 could interact with CmBt, the fruit bitterness-specific TF, which synergistically activated CuB biosynthetic gene expression. These findings provide a novel mechanistic insight for CuB biosynthesis and regulation in melon cultivation.

A single dominant GLOBOSA allele accounts for repeated origins of hose-in-hose flowers in Sinningia (Gesneriaceae).

Plants bearing double flowers have long been cultivated as ornamental plants. Hose-in-hose flowers, bearing 2-whorled corolla tubes in whorls 1 and 2, are uncommon but recur in Sinningia (Gesnerioideae, Gesneriaceae). In this study, we selected 15 hose-in-hose cultivars as materials to explore the underlying molecular and genetic mechanisms of this floral architecture. We found that they originated from different hybridization events within the Dircaea clade. Three B-class MADS-box genes were globally expressed in all floral whorls, but only GLOBOSA1 (GLO1) has accumulated a dominant mutation, i.e., the insertion of a hAT-like miniature inverted-repeat transposable element (MITE) into its promoter, that co-segregated with the hose-in-hose phenotype. In addition, all 15 hose-in-hose cultivars contained the same dominant GLO1 allele. Transient gene expression assays confirmed the role of this MITE insertion in up-regulating the promoter activity of GLO1 by providing several cis-regulatory elements. Genetic transformation in heterologous Chirita pumila (Didymocarpoideae, Gesneriaceae) verified that this dominant GLO1 allele is sufficient to confer the hose-in-hose phenotype. We further demonstrated that both the GLO1 allele and the hAT-like MITE descended from wild S. cardinalis with single flowers. This study highlights the significance of wide hybridization in frequent gains of the dominant GLO1 allele and thereafter repeated occurrence of hose-in-hose flowers in Sinningia.

A protoplast-based transient gene expression assay for the identification of heat and oxidative stress-regulatory genes in perennial ryegrass

BackgroundWith the accumulating omics data, an efficient and time-saving transient assay to express target genes is desired. Mesophyll protoplasts, maintaining most stress-physiological responses and cellular activities as intact plants, offer an alternative transient assay to study target genes’ effects on heat and oxidative stress responses.ResultsIn this study, a perennial ryegrass (Loliumperenne L.) mesophyll protoplast-based assay was established to effectively over- or down-regulate target genes. The relative expression levels of the target genes could be quantified using RT-qPCR, and the effects of heat and H2O2-induced oxidative stress on protoplasts’ viability could be quantitatively measured. The practicality of the assay was demonstrated by identifying the potential thermos-sensor genes LpTT3.1/LpTT3.2 in ryegrass that over-expressing these genes significantly altered protoplasts’ viability rates after heat stress.ConclusionThis protoplast-based rapid stress regulatory gene identification assay was briefed as ‘PRIDA’ that will complement the stable genetic transformation studies to rapidly identify candidate stress-regulatory genes in perennial ryegrass and other grass species.

Quantitative Estimation of Promoter Activity in Cannabis sativa Using Agroinfiltration-Based Transient Gene Expression.

To properly assess promoter activity, which is critical for understanding biosynthetic pathways in different plant species, we use agroinfiltration-based transient gene expression assay. We compare the activity of several known promoters in Nicotiana benthamiana with their activity in Cannabis sativa (both hemp and medicinal cannabis), which has attracted much attention in recent years for its industrial, medicinal, and recreational properties. Here we describe an optimized protocol for transient expression in Cannabis combined with a ratiometric GUS reporter system that allows more accurate evaluation of promoter activity and reduces the effects of variable infiltration efficiency.

Simplified Protocol to Demonstrate Gene Expression in Nicotiana benthamiana Using an Agrobacterium-Mediated Transient Assay.

Agrobacterium-mediated transient gene expression in Nicotiana benthamiana is widely used to study gene function in plants. One dramatic phenotype that is frequently screened for is cell death. Here, we present a simplified protocol for Agrobacterium-mediated transient gene expression by infiltration. Compared with current methods, the novel protocol can be done without a centrifuge or spectrometer, thereby suitable for K-12 outreach programs as well as rapidly identifying genes that induce cell death. Key features • The protocol simplifies the widely used Agrobacterium-mediated transient gene expression assay [1] and can be completed within one week when plants are available. • Rice XB3 gene can induce a dramatic and easily identifiable cell death phenotype in Nicotiana benthamiana. • Allows identification of cell death-inducing genes and is suitable for teaching. • Compared to the currently used methods, our protocol omits the use of agroinfiltration buffer, pH meter, temperature-controlled growth chamber, centrifuge, and spectrophotometer. Graphical overview Agrobacterium infiltration (agroinfiltration) of Nicotiana benthamiana. The photo demonstrates the method of agroinfiltration into the abaxial side of leaves using a needleless syringe.

NAC domain transcription factors VNI2 and ATAF2 form protein complexes and regulate leaf senescence.

The NAM, ATAF1/2, and CUC2 (NAC) domain transcription factor VND-INTERACTING2 (VNI2) negatively regulates xylem vessel formation by interacting with another NAC domain transcription factor, VASCULAR-RELATED NAC-DOMAIN7 (VND7), a master regulator of xylem vessel formation. Here, we screened interacting proteins with VNI2 using yeast two-hybrid assay and isolated two NAC domain transcription factors, Arabidopsis thaliana ACTIVATION FACTOR 2 (ATAF2) and NAC DOMAIN CONTAINING PROTEIN 102 (ANAC102). A transient gene expression assay showed that ATAF2 upregulates the expression of genes involved in leaf senescence, and VNI2 effectively inhibits the transcriptional activation activity of ATAF2. vni2 mutants accelerate leaf senescence, whereas ataf2 mutants delay leaf senescence. In addition, the accelerated leaf senescence phenotype of the vni2 mutant is recovered by simultaneous mutation of ATAF2. Our findings strongly suggest that VNI2 interacts with and inhibits ATAF2, resulting in negatively regulating leaf senescence.

Optimization of gene editing in cowpea through protoplast transformation and agroinfiltration by targeting the phytoene desaturase gene.

Cowpea (Vigna unguiculata) is a legume staple widely grown across Sub-Saharan Africa and other tropical and sub-tropical regions. Considering projected climate change and global population increases, cowpea's adaptation to hot climates, resistance to drought, and nitrogen-fixing capabilities make it an especially attractive crop for facing future challenges. Despite these beneficial traits, efficient varietal improvement is challenging in cowpea due to its recalcitrance to transformation and long regeneration times. Transient gene expression assays can provide solutions to alleviate these issues as they allow researchers to test gene editing constructs before investing in the time and resource- intensive process of transformation. In this study, we developed an improved cowpea protoplast isolation protocol, a transient protoplast assay, and an agroinfiltration assay to be used for initial testing and validation of gene editing constructs and for gene expression studies. To test these protocols, we assessed the efficacy of a CRISPR-Cas9 construct containing four multiplexed single-guide RNA (sgRNA) sequences using polyethylene glycol (PEG)-mediated transformation and agroinfiltration with phytoene desaturase (PDS) as the target gene. Sanger sequencing of DNA from transformed protoplasts and agroinfiltrated cowpea leaves revealed several large deletions in the target sequences. The protoplast system and agroinfiltration protocol developed in this study provide versatile tools to test gene editing components before initiating plant transformation, thus improving the chance of using active sgRNAs and attaining the desired edits and target phenotype.

Pathogen Resistance Depending on Jacalin-Dirigent Chimeric Proteins Is Common among Poaceae but Absent in the Dicot Arabidopsis as Evidenced by Analysis of Homologous Single-Domain Proteins.

MonocotJRLs are Poaceae-specific two-domain proteins that consist of a jacalin-related lectin (JRL) and a dirigent (DIR) domain which participate in multiple developmental processes, including disease resistance. For OsJAC1, a monocotJRL from rice, it has been confirmed that constitutive expression in transgenic rice or barley plants facilitates broad-spectrum disease resistance. In this process, both domains of OsJAC1 act cooperatively, as evidenced from experiments with artificially separated JRL- or DIR-domain-containing proteins. Interestingly, these chimeric proteins did not evolve in dicotyledonous plants. Instead, proteins with a single JRL domain, multiple JRL domains or JRL domains fused to domains other than DIR domains are present. In this study, we wanted to test if the cooperative function of JRL and DIR proteins leading to pathogen resistance was conserved in the dicotyledonous plant Arabidopsis thaliana. In Arabidopsis, we identified 50 JRL and 24 DIR proteins, respectively, from which seven single-domain JRL and two single-domain DIR candidates were selected. A single-cell transient gene expression assay in barley revealed that specific combinations of the Arabidopsis JRL and DIR candidates reduced the penetration success of barley powdery mildew. Strikingly, one of these pairs, AtJAX1 and AtDIR19, is encoded by genes located next to each other on chromosome one. However, when using natural variation and analyzing Arabidopsis ecotypes that express full-length or truncated versions of AtJAX1, the presence/absence of the full-length AtJAX1 protein could not be correlated with resistance to the powdery mildew fungus Golovinomyces orontii. Furthermore, an analysis of the additional JRL and DIR candidates in a bi-fluorescence complementation assay in Nicotiana benthamiana revealed no direct interaction of these JRL/DIR pairs. Since transgenic Arabidopsis plants expressing OsJAC1-GFP also did not show increased resistance to G. orontii, it was concluded that the resistance mediated by the synergistic activities of DIR and JRL proteins is specific for members of the Poaceae, at least regarding the resistance against powdery mildew. Arabidopsis lacks the essential components of the DIR-JRL-dependent resistance pathway.

Developing a robust in vivo hairy root system for assessing transgene expression and genome editing efficiency in papaya

Papaya is one of the most important fruits in tropical and subtropical countries. However, genetic improvement has had limited success to date due to time-consuming and complex transformation and regeneration technologies, as well as a lack of reproducible and efficient transient gene expression assays. Here, we report the development of a highly efficient Rhizobium rhizogenes-based in vivo hairy root system for evaluating transgene expression and activity including CRISPR/Cas gene editing reagents in the Vietnamese papaya cultivar Linhan. To optimize the papaya transformation parameters, we introduced the R. rhizogenes strain K599 into papaya hypocotyls at 1-, 5- and 10-mm below the cotyledon nodes by a needle using 5-, 7- and 10-day old seedlings and then monitored the frequency of hairy root formation at 18 days post infection. We found that the age of the seedlings and the distance of the infection site from the cotyledon node were inversely correlated with the efficacy of hairy root induction, being 5-day-old plants and 1-mm distance the best parameters. The etablished protocol was then employed to investigate transformation frequency using the gus reporter gene. Of the tested hairy roots, 47.22% were positive for GUS staining, which indicates a high level of transgene transfer and stability. Finally, we introduced a dual guide RNA CRISPR/Cas9 cassette targeting eukaryotic translation initiation factor isoform 4E (eIF(iso)4E) gene into papaya by R. rhizogenes and then screened for gene editing events by heteroduplex analysis and Sanger sequencing. Our analysis revealed that 50% of induced roots contained the expected mutations in the eIF(iso)4E gene, which makes our system ideal for testing transgene activity prior making stable transgenic papaya lines.

A multi-omics framework reveals strawberry flavor genes and their regulatory elements.

Flavor is essential to consumer preference of foods and is an increasing focus of plant breeding programs. In fruit crops, identifying genes underlying volatile organic compounds has great promise to accelerate flavor improvement, but polyploidy and heterozygosity in many species have slowed progress. Here we use octoploid cultivated strawberry to demonstrate how genomic heterozygosity, transcriptomic intricacy and fruit metabolomic diversity can be treated as strengths and leveraged to uncover fruit flavor genes and their regulatory elements. Multi-omics datasets were generated including an expression quantitative trait loci map with 196 diverse breeding lines, haplotype-phased genomes of a highly-flavored breeding selection, a genome-wide structural variant map using five haplotypes, and volatile genome-wide association study (GWAS) with > 300 individuals. Overlaying regulatory elements, structural variants and GWAS-linked allele-specific expression of numerous genes to variation in volatile compounds important to flavor. In one example, the functional role of anthranilate synthase alpha subunit 1 in methyl anthranilate biosynthesis was supported via fruit transient gene expression assays. These results demonstrate a framework for flavor gene discovery in fruit crops and a pathway to molecular breeding of cultivars with complex and desirable flavor.

MiR390-tasiRNA3-ARF4 pathway is involved in regulating flowering time in woodland strawberry.

miR390-tasiRNA3-ARF4 pathway was identified in woodland strawberry. FvemiR390 was involved in the regulation of flowering time, and miR390-tasiRNA3-ARF4 regulated flowering time through FveAP1/FveFUL in woodland strawberry. miRNA is an important type of regulator, and widely involved in plant growth, development and stress response. As a conserved miRNA family, the function of miR390 has been studied in many species, but poorly understood in woodland strawberry. In this study, we found that the members of miR390 family were highly conservative, and FvemiR390a and FvemiR390b have the same mature sequence. Therefore, we chose FveMIR390a to generate FvemiR390 mature sequence for functional studies. Subsequently, the result of transient gene expression assay proved that FvemiR390 negatively regulates FveARF4 through miR390-tasiRNA3-ARF4 pathway. Using transgenic plants, we discovered that the overexpression of FveMIR390a delayed flowering in woodland strawberry. Further studies revealed that the expressions of FveAP1 and FveFUL were lower in transgenic plants, which indicates miR390-tasiRNA3-ARF4 pathway delays flowering time through the FveAP1/FveFUL in woodland strawberry. Moreover, the expression of FvemiR390 responded to exogenous hormones, which also provides a reference for the application of exogenous hormones in regulating the flowering time of woodland strawberry.

Mitochondrial GFP-Tagged Protein Localization Using Transient Transformations in Arabidopsis thaliana.

Transient transformation assays for the analysis of protein localization are routinely used as rapid and convenient alternatives to stable transformation. In this chapter, we describe two transient gene expression assays (e.g., isolation and transformation of protoplasts, and agroinfiltration of leaves) optimized for Arabidopsis thaliana, and we combine them with fluorescence microscopy, with the final aim to investigate in vivo the subcellular localization of a mitochondrial protein of interest fused to a fluorescent reporter.

Efficient isolation of protoplasts from rice calli with pause points and its application in transient gene expression and genome editing assays

BackgroundAn efficient in vivo transient transfection system using protoplasts is an important tool to study gene expression, metabolic pathways, and multiple mutagenesis parameters in plants. Although rice protoplasts can be isolated from germinated seedlings or cell suspension culture, preparation of those donor tissues can be inefficient, time-consuming, and laborious. Additionally, the lengthy process of protoplast isolation and transfection needs to be completed in a single day.ResultsHere we report a protocol for the isolation of protoplasts directly from rice calli, without using seedlings or suspension culture. The method is developed to employ discretionary pause points during protoplast isolation and before transfection. Protoplasts maintained within a sucrose cushion partway through isolation, for completion on a subsequent day, per the first pause point, are referred to as S protoplasts. Fully isolated protoplasts maintained in MMG solution for transfection on a subsequent day, per the second pause point, are referred to as M protoplasts. Both S and M protoplasts, 1 day after initiation of protoplast isolation, had minimal loss of viability and transfection efficiency compared to protoplasts 0 days after isolation. S protoplast viability decreases at a lower rate over time than that of M protoplasts and can be used with added flexibility for transient transfection assays and time-course experiments. The protoplasts produced by this method are competent for transfection of both plasmids and ribonucleoproteins (RNPs). Cas9 RNPs were used to demonstrate the utility of these protoplasts to assay genome editing in vivo.ConclusionThe current study describes a highly effective and accessible method to isolate protoplasts from callus tissue induced from rice seeds. This method utilizes donor materials that are resource-efficient and easy to propagate, permits convenience via pause points, and allows for flexible transfection days after protoplast isolation. It provides an advantageous and useful platform for a variety of in vivo transient transfection studies in rice.

Identification of Natural Resistance Mediated by Recognition of Phytophthora infestans Effector Gene Avr3aEM in Potato

Late blight is considered the most renowned devastating potato disease worldwide. Resistance gene (R)-based resistance to late blight is the most effective method to inhibit infection by the causal agent Phytophthora infestans. However, the limited availability of resistant potato varieties and the rapid loss of R resistance, caused by P. infestans virulence variability, make disease control rely on fungicide application. We employed an Agrobacterium tumefaciens-mediated transient gene expression assay and effector biology approach to understand late blight resistance of Chinese varieties that showed years of promising field performance. We are particularly interested in PiAvr3aEM, the most common virulent allele of PiAvr3aKI that triggers a R3a-mediated hypersensitive response (HR) and late blight resistance. Through our significantly improved A. tumefaciens-mediated transient gene expression assay in potato using cultured seedlings, we characterized two dominant potato varieties, Qingshu9 and Longshu7, in China by transient expression of P. infestans effector genes. Transient expression of 10 known avirulence genes showed that PiAvr4 and PiAvr8 (PiAvrsmira2) could induce HR in Qingshu9, and PiAvrvnt1.1 in Longshu7, respectively. Our study also indicated that PiAvr3aEM is recognized by these two potato varieties, and is likely involved in their significant field performance of late blight resistance. The identification of natural resistance mediated by PiAvr3aEM recognition in Qingshu9 and Longshu7 will facilitate breeding for improved potato resistance against P. infestans.

The Genetics of Differential Gene Expression Related to Fruit Traits in Strawberry (Fragaria ×ananassa).

Octoploid strawberry (Fragaria ×ananassa) is a major specialty crop under intense annual selection for traits relating to plant vigor and fruit quality. Most functional validation experiments rely on transgenic or transient gene expression assays in the mature receptacle. These findings are not typically translatable to breeding without identifying a natural genetic source of transcript level variation, and developing reliable markers for selection in octoploids. Expression QTL (eQTL) analysis is a genetic/transcriptomic association approach for identifying sequence variants predicting differential expression. This eQTL study analyzed a wide array of mature receptacle-expressed genes, encompassing the majority of total mature receptacle transcript accumulation and almost all strawberry genes described in the literature. These results identified segregating genetic variants associated with the differential expression of hundreds of strawberry genes, many with known interest to breeders. Several of these eQTL pertain to published genes whose expression levels have been demonstrated to influence mature receptacle phenotypes. Many include key genes of the phenylpropanoid pathway, vitamin C, carotenoid, pectin, and receptacle carbohydrate/sugar metabolism. These subgenome-specific genetic markers may allow breeders to select for desired ranges of target gene expression. These results may also guide basic research efforts and facilitate the identification of causal genes underlying trait QTL.

A modified transient gene expression protocol for subcellular protein localization analysis in rice

Transient protein expression is a useful technique for investigating the protein localization and functional analysis of stress responses in rice plants. Currently, available methods for in planta transient expression analysis include Agrobacterium-mediated transformation, protoplast transformation using polyethylene glycol or electroporation, and biolistic bombardment expression which have several disadvantages and are not well suited for the rice. Therefore, development of a method for rapid and efficient analysis of protein expression, subcellular localization, pathogen effector screening, and protein–protein interaction in rice is required. We developed a protocol for in planta gene expression analysis in sliced rice sheath cells by modifying and optimizing the biolistic particle bombardment technique. By obtaining thin sections (~400 μm) of rice sheath cells, auto-fluorescence from chlorophyll was eliminated. This system was validated through the localization of marker genes specifically expressed in nuclei, plasma membranes, and tonoplast. In addition, high transformation efficiency of 30% was achieved. Therefore, this protocol provides a new and rapid method for transient gene expression assay in rice. Protein secretion was examined in rice sheath cells using predicted secretory proteins from rice blast fungus, indicating that this method is applicable to plant–microbe interaction studies. The transient expression protocol established here is well optimized for protein localization, secretion, and host–pathogen protein interaction studies in rice. A typical experiment can be completed in three days.

CRISPR/Cas9-mediated knockout of the RDR6 gene in Nicotiana benthamiana for efficient transient expression of recombinant proteins.

RDR6 gene knockout Nicotiana benthamiana plant was successfully produced using CRISPR/Cas9 technology. The production of recombinant proteins in plants has many advantages, such as safety and reduced costs. However, there are several problems with this technology, especially low levels of protein production. The dysfunction of the RNA silencing mechanism in plant cells would be effective to improve recombinant protein production because the RNA silencing mechanism efficiently degrades transgene-derived mRNAs. Therefore, to overcome this problem, clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 technology was used to develop RNA silencing-related gene knockout transgenic Nicotiana benthamiana. We successfully produced RNA-dependent RNA polymerase 6 (RDR6), one of the most important components of the RNA silencing mechanism-knockout N. benthamiana (ΔRDR6 plants). The ΔRDR6 plants had abnormal flowers and were sterile, as with the Arabidopsis RDR6 mutants. However, a transient gene expression assay showed that the ΔRDR6 plants accumulated larger amounts of green fluorescent protein (GFP) and GFP mRNA than the wild-type (WT) plants. Small RNA sequencing analysis revealed that levels of small interfering RNA against the GFP gene were greatly reduced in the ΔRDR6 plants, as compared to that of the WT plants. These findings demonstrate that the ΔRDR6 plants can express larger amounts of recombinant proteins than WT plants and, therefore, would be useful for recombinant protein production and understanding the contributions of RDR6 to genetic and physiological events in plants.

LBD13 positively regulates lateral root formation in Arabidopsis.

Lateral Organ Boundaries Domain 13 (LBD13), which is expressed in emerged lateral roots and encodes a transcriptional activator, plays an important role in lateral root formation in Arabidopsis. Lateral roots (LRs) are major determinants of root system architecture, contributing to the survival strategies of plants. Members of the LBD gene family encode plant-specific transcription factors that play key roles in plant organ development. Several LBD genes, such as LBD14, 16, 18, 29, and 33, have been shown to play important roles in regulating LR development in Arabidopsis. In the present study, we show that LBD13 is expressed in emerged LRs and LR meristems of elongated LRs and regulates LR formation in Arabidopsis. Transient gene expression assays with Arabidopsis protoplasts showed that LBD13 is localized to the nucleus and harbors transcription-activating potential. Knock-down of LBD13 expression by RNA interference resulted in reduced LR formation, whereas overexpression of LBD13 enhanced LR formation in transgenic Arabidopsis. Analysis of β-glucuronidase (GUS) expression under the control of the LBD13 promoter showed that GUS staining was detected in LRs emerged from the primary root, but not in LR primordia. Moreover, both the distribution of LR primordium number and developmental kinetics of LR primordia were not affected either by knock-down or by overexpression of LBD13. Taken together, these results suggest that LBD13 is a nuclear-localized transcriptional activator and controls LR formation during or after LR emergence.

Molecular cloning and expression analysis of three ThERFs involved in the response to waterlogging stress of Taxodium 'Zhongshanshan406', and subcellular localization of the gene products.

As a subfamily of the APETALA 2/ethylene response element binding protein (AP2/EREBP) transcription factor superfamily, the ethylene response factor (ERF) is widely involved in the regulation of growth and response to various abiotic stresses in plants, and has been shown to be the main transcription factor regulating transcription of the genes related to hypoxia and waterlogging stress. In this study, three ThERF genes, with significant differences in expression profile in response to flooding stress, were identified from the transcriptomics data acquired from Taxodium hybrid ‘Zhongshanshan 406’ (T. mucronatum Tenore × T. distichum (L.) Rich) under waterlogging stress: ThERF15, ThERF39 and ThRAP2.3 (GenBank ID: KY463467, KY463468 and KY463470, respectively).The full-length cDNA of each of the three ERFs was obtained using the RACE (rapid amplification cDNA ends) method, and all three were intron-free. Multiple protein sequence alignments indicated that ThERF15, ThERF39 and ThRAP2.3 proteins all had only one AP2-ERF domain and belonged to the ERF subfamily. A transient gene expression assay demonstrated that ThERF15, ThERF39 and ThRAP2.3 were all localized to the nucleus. Real-time quantitative PCR (qPCR) revealed that the expression of ThERF15, ThERF39 and ThRAP2.3 exhibited significant differences, compared with the control, in response to two levels of flooding treatment (half-flooding or total-submergence) of ‘Zhongshanshan 406’. Quantification of ethylene concentration revealed that ethylene was more relevant to the level of expression than the period of flooding treatment. Based on the experimental results above, ThERF15, ThERF39 and ThRAP2.3 were identified as being related to the regulation of downstream flooding- responsive gene expression in ‘Zhongshanshan 406’. ThRAP2.3 is most likely to be a key downstream-response ERF gene to respond to the output of the ethylene signal generated by flooding stress.

Transient Expression Assay in NahG Arabidopsis Plants Using Agrobacterium tumefaciens.

Agrobacterium-mediated transient expression has greatly contributed to research in molecular plant biology but has low efficiency and inconsistency in Arabidopsis thaliana (Arabidopsis). Here, we describe a simple, efficient and fast protocol to make transient gene expression in NahG Arabidopsis plants using Agrobacterium tumefaciens. This protocol has been successfully used to assess protein sub-cellular localization and accumulation, enzyme activity, and protein-protein interaction. In addition, this assay overcomes the use of Nicotiana benthamiana plants as a surrogate system for transient gene expression assays. Finally, the use of this protocol does not require complex inoculation methods or specific growth conditions, and can be used with different Agrobacterium strains with similar results.