Luciferase Complementation Imaging Research Articles

GoSTR, a negative modulator of stem trichome formation in cotton.

Trichomes, the outward projection of plant epidermal tissue, provide an effective defense against stress and insect pests. Although numerous genes have been identified to be involved in trichome development, the molecular mechanism for trichome cell fate determination is not well enunciated. Here, we reported GoSTR functions as a master repressor for stem trichome formation, which was isolated by map-based cloning based on a large F2 segregating population derived from a cross between TM-1 (pubescent stem) and J220 (smooth stem). Sequence alignment revealed a critical G-to-T point mutation in GoSTR's coding region thatconverted codon 2 from GCA (Alanine) to TCA (Serine). This mutation occurred between the majority ofGossypium hirsutum with pubescent stem (GG-haplotype) and G. barbadense with glabrous stem (TT-haplotype). Silencing of GoSTR in J220 and Hai7124 via virus-induced gene silencing resulted in the pubescent stems but no visible change in leaf trichomes, suggesting stem trichomes and leaf trichomes are genetically distinct. Yeast two-hybrid assay and luciferase complementation imaging assay showed GoSTR interacts with GoHD1 and GoHOX3, two key regulators of trichome development. Comparative transcriptomic analysis further indicated that many transcription factors such as GhMYB109, GhTTG1, and GhMYC1/GhDEL65 which function as positive regulators of trichomes were significantly upregulated in the stem from the GoSTR-silencing plant. Taken together, these results indicate that GoSTR functions as an essential negative modulator of stem trichomes and its transcripts will greatly repress trichome cell differentiation and growth. This study provided valuable insights for plant epidermal hair initiation and differentiation research.

The glycoside hydrolase 28 member VdEPG1 is a virulence factor of Verticillium dahliae and interacts with the jasmonic acid pathway-related gene GhOPR9.

Glycoside hydrolase (GH) family members act as virulence factors and regulate plant immune responses during pathogen infection. Here, we characterized the GH28 family member endopolygalacturonase VdEPG1 in Verticillium dahliae. VdEPG1 acts as a virulence factor during V. dahliae infection. The expression level of VdEPG1 was greatly increased in V. dahliae inoculated on cotton roots. VdEPG1 suppressed VdNLP1-mediated cell death by modulating pathogenesis-related genes in Nicotiana benthamiana. Knocking out VdEPG1 led to a significant decrease in the pathogenicity of V. dahliae in cotton. The deletion strains were more susceptible to osmotic stress and the ability of V. dahliae to utilize carbon sources was deficient. In addition, the deletion strains lost the ability to penetrate cellophane membrane, with mycelia showing a disordered arrangement on the membrane, and spore development was affected. A jasmonic acid (JA) pathway-related gene, GhOPR9, was identified as interacting with VdEPG1 in the yeast two-hybrid system. The interaction was further confirmed by bimolecular fluorescence complementation and luciferase complementation imaging assays in N. benthamiana leaves. GhOPR9 plays a positive role in the resistance of cotton to V. dahliae by regulating JA biosynthesis. These results indicate that VdEPG1 may be able to regulate host immune responses as a virulence factor through modulating the GhOPR9-mediated JA biosynthesis.

Transcription factors TgbHLH95 and TgbZIP44 co-target terpene biosynthesis gene TgGPPS in Torreya grandis nuts.

Terpenes are volatile compounds responsible for aroma and the postharvest quality of commercially important xiangfei (Torreya grandis) nuts, and there is interest in understanding the regulation of their biosynthesis. Here, a transcriptomics analysis of xiangfei nuts after harvest identified 156 genes associated with the terpenoid metabolic pathway. A geranyl diphosphate (GPP) synthase (TgGPPS) involved in production of the monoterpene precursor GPP was targeted for functional characterization, and its transcript levels positively correlated with terpene levels. Furthermore, transient overexpression of TgGPPS in tobacco (Nicotiana tabacum) leaves or tomato (Solanum lycopersicum) fruit led to monoterpene accumulation. Analysis of differentially expressed transcription factors identified one basic helix-loop-helix protein (TgbHLH95) and one basic leucine zipper protein (TgbZIP44) as potential TgGPPS regulators. TgbHLH95 showed significant transactivation of the TgGPPS promoter, and its transient overexpression in tobacco leaves led to monoterpene accumulation, whereas TgbZIP44 directly bound to an ACGT-containing element in the TgGPPS promoter, as determined by yeast one-hybrid test and electrophoretic mobility shift assay. Bimolecular fluorescence complementation, firefly luciferase complementation imaging, co-immunoprecipitation and GST pull-down assays confirmed a direct protein-protein interaction between TgbHLH95 and TgbZIP44 in vivo and in vitro, and in combination these proteins induced the TgGPPS promoter up to 4.7-fold in transactivation assays. These results indicate that a TgbHLH95/TgbZIP44 complex activates the TgGPPS promoter and up-regulates terpene biosynthesis in xiangfei nuts after harvest, thereby contributing to its aroma.

A Combinatorial TIR1-Aux/IAA Co-Receptor System for Peach Fruit Softening

Fruit softening is an important characteristic of peach fruit ripening. The auxin receptor TIR1 (Transport Inhibitor Response 1) plays an important role in plant growth and fruit maturation. Still, little research has been conducted on the relation of TIR1 to the softening of peach fruits. In this study, the hardness of isolated peach fruits was reduced under exogenous NAA treatment at low concentrations. At the same time, the low concentration of NAA treatment reduced the transcription level of PpPG and Ppβ-GAL genes related to cell wall softening and PpACS1 genes related to ethylene synthesis. The transient overexpression of the PpTIR1 gene in peach fruit blocks caused significant down-regulation of the expression of early auxin-responsive genes, ethylene synthesis, and cell wall metabolic genes related to fruit firmness. Through yeast two-hybrid technology, bimolecular fluorescence complementary technology, and a firefly luciferase complementation imaging assay, we were able to unveil an interaction between PpTIR1 and PpIAA1/3/5/9/27 proteins. Furthermore, it was determined that the interaction depended on auxin and its type and concentration. These results show that the PpTIR1-Aux/IAA module has a possible regulatory effect on fruit ripening and softening.

GhSINA1, a SEVEN in ABSENTIA ubiquitin ligase, negatively regulates fiber development in Upland cotton

Protein ubiquitination is essential for plant growth and responses to the environment. The SEVEN IN ABSENTIA (SINA) ubiquitin ligases have been extensively studied in plants, but information on their roles in fiber development is limited. Here, we identified GhSINA1 in Upland cotton (Gossypium hirsutum), which has a conserved RING finger domain and SINA domain. Quantitative real-time PCR (qRT-PCR) analysis showed that GhSINA1 was preferentially expressed during fiber initiation and elongation, especially during initiation in the fuzzless-lintless cotton mutant. Subcellular localization experiments indicated that GhSINA1 localized to the nucleus. In vitro ubiquitination analysis revealed that GhSINA1 has E3 ubiquitin ligase activity. Ectopic overexpression of GhSINA1 in Arabidopsis thaliana reduced the number and length of root hairs and trichomes. Yeast two-hybrid (Y2H), firefly luciferase complementation imaging (LCI), and bimolecular fluorescence complementation (BiFC) assays demonstrated that the GhSINA1 proteins could interact with each other to form homodimers and heterodimers. Overall, these results suggest that GhSINA1 may act as a negative regulator in cotton fiber development through homodimerization and heterodimerization.

Identification of ApbHLH1 as a Partner Interacting with ApMYB1 to Promote Anthocyanin Biosynthesis during Autumnal Leaf Coloration in Acer palmatum

Anthocyanin biosynthesis determines the leaf color of Acer palmatum as a widely-planted landscape tree. Previously, ApMYB1 has been characterized as a positive regulator of anthocyanin biosynthesis. To further elucidate the mechanism of leaf coloration, the present study identified a basic helix-loop-helix (bHLH) transcription factor (ApbHLH1) through the phylogenetic analysis of 156 putative bHLH proteins in Acer palmatum and eight reference bHLHs which were known to be involved in the anthocyanin biosynthesis of selected plants. Protein structure comparison showed that ApbHLH1 has a conserved bHLH domain, and its N-terminal contains an MYB-interacting region. The expression of ApbHLH1 in leaves was found to not be correlated with anthocyanin contents either in green, semi-red leaves or during leaf autumnal senescence when anthocyanin content increased. ApbHLH1 expression in detached leaves was induced by exogenous senescence-promoting chemicals, including H2O2, SA, MeJA, ACC and ABA, with certain durations. In particular, either high light or low temperature induced ApbHLH1 expression significantly, and combination of high light and low temperatures seemed more effective in inducing ApbHLH1 expression. Luciferase complementation imaging assays confirmed the physical interaction between ApbHLH1 and ApMYB1, which could be abolished by either the truncating MYB-interacting region of ApbHLH1 or the deleting bHLH interacting domain of ApMYB1. The transient expression of ApbHLH1 could not induce anthocyanin production, while the co-expression of ApbHLH1 and ApMYB1 resulted in a higher accumulation of anthocyanins compared to the expression of ApMYB1 alone in tobacco leaves. Collectively, our results revealed that ApbHLH1 participated in leaf coloration through binding with ApMYB1 and enhancing the ApMYB1 function of promoting anthocyanin biosynthesis during leaf autumnal reddening in Acer palmatum. ApbHLH1 could have the potential for breeding color-leafed plants through co-transformation with ApMYB1.

Lysine malonylation of DgnsLIPID TRANSFER PROTEIN1 at the K81 site improves cold resistance in chrysanthemum.

Lysine malonylation (Kmal) is a recently discovered posttranslational modification, and its role in the response to abiotic stress has not been reported in plants. In this study, we isolated a nonspecific lipid transfer protein, DgnsLTP1, from chrysanthemum (Dendranthema grandiflorum var. Jinba). Overexpression and CRISPR-Cas9-mediated gene editing of DgnsLTP1 demonstrated that the protein endows chrysanthemum with cold tolerance. Yeast 2-hybrid, bimolecular fluorescence complementation, luciferase complementation imaging, and coimmunoprecipitation experimental results showed that DgnsLTP1 interacts with a plasma membrane intrinsic protein (PIP) DgPIP. Overexpressing DgPIP boosted the expression of DgGPX (glutathione peroxidase), increased the activity of GPX, and decreased the accumulation of reactive oxygen species (ROS), thereby enhancing the low-temperature stress tolerance of chrysanthemum, while the CRISPR-Cas9-mediated mutant dgpip inhibited this process. Transgenic analyses in chrysanthemum showed that DgnsLTP1 improves the cold resistance of chrysanthemum in a DgPIP-dependent manner. Moreover, Kmal of DgnsLTP1 at the K81 site prevented the degradation of DgPIP in Nicotiana benthamiana and chrysanthemum, further promoted DgGPX expression, enhanced GPX activity, and scavenged excess ROS produced by cold stress, thereby further enhancing the cold resistance of chrysanthemum.

PHB3 Is Required for the Assembly and Activity of Mitochondrial ATP Synthase in Arabidopsis.

Mitochondrial ATP synthase is a multiprotein complex, which consists of a matrix-localized F1 domain (F1-ATPase) and an inner membrane-embedded Fo domain (Fo-ATPase). The assembly process of mitochondrial ATP synthase is complex and requires the function of many assembly factors. Although extensive studies on mitochondrial ATP synthase assembly have been conducted on yeast, much less study has been performed on plants. Here, we revealed the function of Arabidopsis prohibitin 3 (PHB3) in mitochondrial ATP synthase assembly by characterizing the phb3 mutant. The blue native PAGE (BN-PAGE) and in-gel activity staining assays showed that the activities of ATP synthase and F1-ATPase were significantly decreased in the phb3 mutant. The absence of PHB3 resulted in the accumulation of the Fo-ATPase and F1-ATPase intermediates, whereas the abundance of the Fo-ATPase subunit a was decreased in the ATP synthase monomer. Furthermore, we showed that PHB3 could interact with the F1-ATPase subunits β and δ in the yeast two-hybrid system (Y2H) and luciferase complementation imaging (LCI) assay and with Fo-ATPase subunit c in the LCI assay. These results indicate that PHB3 acts as an assembly factor required for the assembly and activity of mitochondrial ATP synthase.

ALA reverses ABA-induced stomatal closure by modulating PP2AC and SnRK2.6 activity in apple leaves.

5-Aminolevulinic acid (ALA), known as a new natural plant growth regulator, can reverse abscisic acid (ABA)-induced stomatal closure. The protein phosphatase 2A (PP2A) played an important role in regulation of stomatal movement by ALA and ABA; however, the underlying molecular mechanisms remain unclear. Here, we report that ALA promotes MdPP2A activity and gene expression in the leaf epidermis of apple (Malus × domestica Borkh.), and expression of the catalytic subunit MdPP2AC was most significantly correlated with stomatal aperture. Western blotting showed that ALA enhanced MdPP2AC protein abundance and phosphorylation. Y2H (yeast two hybrid), FLC (firefly luciferase complementation imaging) and BiFC (Bimolecular fluorescence complementation) assays showed that MdPP2AC interacted with several other MdPP2A subunits as well as MdSnRK2.6 (Sucrose non-fermenting 1-related protein kinase 2.6), and the latter interaction was further verified by pull-down and MST (microscale thermophoresis) assays. ALA downregulated ABA-induced MdSnRK2.6 gene expression, kinase activity, and protein phosphorylation. In transiently transgenic apple leaves, OE-MdPP2AC promoted stomatal aperture by reducing Ca2+ and H2O2 levels but increasing flavonol levels in guard cells. Conversely, OE-MdSnRK2.6 induced stomatal closure by increasing Ca2+ and H2O2 but reducing flavonols. Partial silencing of these genes had opposite effects on Ca2+, H2O2, flavonols, and stomatal movement. Application of exogenous ALA stimulated PP2A activity, which promoted SnRK2.6 dephosphorylation and lower kinase activity in wild-type and transgenic apple leaves. We therefore propose that PP2AC, which dephosphorylates SnRK2.6 and represses its enzyme activity, mediates ALA signaling to inhibit ABA-induced stomatal closure in apple leaves.

Acetylation of GhCaM7 enhances cotton resistance to Verticillium dahliae.

Protein lysine acetylation is an important post-translational modification mechanism involved in cellular regulation in eukaryotes. Calmodulin (CaM) is a ubiquitous Ca2+ sensor in eukaryotes and is crucial for plant immunity, but it is so far unclear whether acetylation is involved in CaM-mediated plant immunity. Here, we found that GhCaM7 is acetylated upon Verticillium dahliae (V. dahliae) infection and a positive regulator of V. dahliae resistance. Overexpressing GhCaM7 in cotton and Arabidopsis enhances V. dahliae resistance and knocking-down GhCaM7 makes cotton more susceptible to V. dahliae. Transgenic Arabidopsis plants overexpressing GhCaM7 with mutation at the acetylation site are more susceptible to V. dahliae than transgenics overexpressing the wild-type GhCaM7, implying the importance of the acetylated GhCaM7 in response to V. dahliae infection. Yeast two-hybrid, bimolecular fluorescent complementation, luciferase complementation imaging, and coimmunoprecipitation assays demonstrated interaction between GhCaM7 and an osmotin protein GhOSM34 that was shown to have a positive role in V. dahliae resistance. GhCaM7 and GhOSM34 are co-localized in the cell membrane. Upon V. dahliae infection, the Ca2+ content reduces almost instantly in plants with downregulated GhCaM7 or GhOSM34. Down regulating GhOSM34 enhances accumulation of Na+ and increases cell osmotic pressure. Comparative transcriptomic analyses between cotton plants with an increased or reduced expression level of GhCaM7 and wild-type plants indicate the involvement of jasmonic acid signaling pathways and reactive oxygen species in GhCaM7-enabled disease resistance. Together, these results demonstrate the involvement of CaM protein in the interaction between cotton and V. dahliae, and more importantly, the involvement of the acetylated CaM in the interaction.

Functional characterization of the GhNRT2.1e gene reveals its significant role in improving nitrogen use efficiency in Gossypium hirsutum.

Nitrate is the primary type of nitrogen available to plants, which is absorbed and transported by nitrate transporter 2 (NRT2) at low nitrate conditions. Genome-wide identification of NRT2 genes in G. hirsutum was performed. Gene expression patterns were revealed using RNA-seq and qRT-PCR. Gene functions were characterized using overexpression in A. thaliana and silencing in G. hirsutum. Protein interactions were verified by yeast two-hybrid and luciferase complementation imaging (LCI) assays. We identified 14, 14, seven, and seven NRT2 proteins in G. hirsutum, G. barbadense, G. raimondii, and G. arboreum. Most NRT2 proteins were predicted in the plasma membrane. The NRT2 genes were classified into four distinct groups through evolutionary relationships, with members of the same group similar in conserved motifs and gene structure. The promoter regions of NRT2 genes included many elements related to growth regulation, phytohormones, and abiotic stresses. Tissue expression pattern results revealed that most GhNRT2 genes were specifically expressed in roots. Under low nitrate conditions, GhNRT2 genes exhibited different expression levels, with GhNRT2.1e being the most up-regulated. Arabidopsis plants overexpressing GhNRT2.1e exhibited increased biomass, nitrogen and nitrate accumulation, nitrogen uptake and utilization efficiency, nitrogen-metabolizing enzyme activity, and amino acid content under low nitrate conditions. In addition, GhNRT2.1e-silenced plants exhibited suppressed nitrate uptake and accumulation, hampered plant growth, affected nitrogen metabolism processes, and reduced tolerance to low nitrate. The results showed that GhNRT2.1e could promote nitrate uptake and transport under low nitrate conditions, thus effectively increasing nitrogen use efficiency (NUE). We found that GhNRT2.1e interacts with GhNAR2.1 by yeast two-hybrid and LCI assays. Our research lays the foundation to increase NUE and cultivate new cotton varieties with efficient nitrogen use.

Comprehensive study of rice YABBY gene family: evolution, expression and interacting proteins analysis.

As plant-specific transcription regulators, YABBYs are involved in plant growth, development and stress responses. However, little information is available about genome-wide screening and identification of OsYABBY-interacting proteins. In this study, phylogenetic relationship, gene structure, protein structure and gene expression profile of eight OsYABBYs were carried out, all of which indicated that OsYABBYs were involved in different developmental processes and had functional differentiation. More importantly, PPI (protein-protein interaction) analysis and molecular docking simulation predicted that WUSCHEL-related homeobox (WOX) proteins might be interacting proteins of OsYABBYs. Yeast two-hybrid (Y2H) and luciferase complementation imaging assays (LCI) further confirmed that OsYABBYs (except for OsYABBY7) could interact with OsWOX3A in vitro and in vivo. In addition, OsYABBY3 and OsYABBY5 also could interact with OsWUS. Taken together, our results provided valuable information for further elucidating OsYABBYs regulation mechanism in improving rice performance.

Transcription factors NAC20 and NAC26 interact with RPBF to activate albumin accumulations in rice endosperm.

Transcription factors NAC20 and NAC26 interact with RPBF to activate albumin accumulations in rice endosperm.

NtPIN3 positively regulates low phosphorus tolerance by changing root elongation, Pi concentration and antioxidant capacity in tobacco

Phosphorus (P) is a critical nutrient for plants, and inorganic orthophosphate (Pi) deficiency results in declining crop quality and yields. Auxin plays a key regulatory effect on plant growth in response to Pi deficiency, yet the regulatory mechanisms remain poorly understood. In this study, we cloned and identified an auxin efflux transporter, NtPIN3, involved in the low phosphate response of tobacco (Nicotiana tabacum). NtPIN3 is located on the cytoplasmic membrane, and high expression levels of NtPIN3 were observed under phosphorus deficiency conditions. Moreover, NtPIN3 overexpression in tobacco could dramatically enhance low Pi tolerance by increasing the root length, which was consistent with the GUS activity and the auxin concentration in the root. Notably, Pi concentration and antioxidant capacity in overexpression transgenic tobacco were significantly increased compared with the wild type (WT) under low Pi stress conditions due to Pi uptake (NtPT1 and NtPT2) and antioxidant capacity (superoxide dismutase, peroxidase, and catalase). In contrast, compared with WT, the ntpin3 mutant significantly reduced root length, auxin concentration, Pi concentration, and antioxidant capacity under low Pi stress conditions, suggesting that NtPIN3 is a positive regulator of low phosphate in tobacco. In addition, the yeast-two-hybrid and luciferase complementation imaging assay confirmed that NtPIN3 interacted with NtPIN2. Altogether, the NtPIN3 involved in low Pi response and overexpression of NtPIN3 contributed to enhanced tolerance to low Pi stress, which proved an underlying gene for breeding low Pi-stress tolerant plants.

DORN1 and GORK regulate stomatal closure in Arabidopsis mediated by volatile organic compound ethyl vinyl ketone

Evk (ethyl vinyl ketone) is a signal substance for plant defense, but little is known about how evk mediates stomatal closure. Through stomatal biology experiments, we found that evk can mediate stomatal closure, and stomatal closure is weakened when DORN1 (DOES NOT RESPOND TO NUCLEOTIDES 1) and GORK (GATED OUTWARDLY-RECTIFYING K+ CHANNEL) are mutated. In addition, it was found by non-invasive micro-test technology (NMT) that the K+ efflux mediated by evk was significantly weakened when DORN and GORK were mutated. Yeast two-hybrid (Y2H), firefly luciferase complementation imaging (LCI), and in vitro pull-down assays demonstrated that DORN1 and GORK could interact in vitro and in vivo. It was found by molecular docking that evk could combine with MRP (Multidrug Resistance-associated Protein), thus affecting ATP transport, promoting eATP (extracellular ATP) concentration increase and realizing downstream signal transduction. Through inoculation of botrytis cinerea, it was found that evk improved the antibacterial activity of Arabidopsis thaliana. As revealed by reverse transcription quantitative PCR (RT-qPCR), the expression of defense related genes was enhanced by evk treatment. Evk is a potential green antibacterial drug.

The involvement of Ein3-binding F-box protein PbrEBF3 in regulating ethylene signaling during Cuiguan pear fruit ripening

Ein3-binding F-box (EBF) proteins have been determined to modulate ethylene response processes by regulating EIN3/EIL protein degradation in Arabidopsis and tomato. However, the function of pear PbrEBFs in ethylene-dependent responses during fruit ripening remains unclear. In this study, PbrEBF1, PbrEBF2, and PbrEBF3 display contrasting expression patterns in response to ethylene and 1-MCP treatment. PbrEBF3 displayed potential fruit ripening-associated function in a transient expression experiment. Yeast two-hybrid (Y2H) and Firefly luciferase complementation imaging (LCI) assays indicated that PbrEBF3 interacts with PbrEIL1, PbrEIL2, and PbrEIL3 proteins. In turn, the transcription of PbrEBF3 is directly regulated by PbrEILs via a feedback loop. PbrEILs trigger a transcriptional cascade of PbrERF24 and finally affect ethylene synthesis. Overall, PbrEBF3 plays a central role in pear fruit ripening through mediation of the ethylene signaling pathway.

Genome-wide identification and functional analysis of the TCP gene family in rye (Secale cereale L.)

TEOSINTE BRANCHED1/CYCLOIDEA/PCF (TCP) proteins are plant-specific transcription factors that play significant roles in plant growth, development, and stress response. Rye is a high-value crop with strong resistance to adverse environments. However, the functions of TCP proteins in rye are rarely reported. Based on a genome-wide analysis, the present study identified 26 TCP genes (ScTCPs) in rye. Mapping showed an uneven distribution of the ScTCP genes on the seven rye chromosomes and detected three pairs of tandem duplication genes. Phylogenetic analysis divided these genes into PCF (Proliferrating Cell Factors), CIN (CINCINNATA), and CYC (CYCLOIDEA)/TB1 (Teosinte Branched1) classes, which showed the highest homology between rye and wheat genes. Analysis of miRNA targeting sites indicated that five ScTCP genes were identified as potential targets of miRNA319. Promoter cis-acting elements analysis indicated that ScTCPs were regulated by light signals. Further analysis of the gene expression patterns and functional annotations suggested the role of a few ScTCPs in grain development and stress response. In addition, two TB1 homologous genes (ScTCP9 and ScTCP10) were identified in the ScTCP family. Synteny analysis showed that TB1 orthologous gene pairs existed before the ancestral divergence. Finally, the yeast two-hybrid assay and luciferase complementation imaging assay proved that ScTCP9, localized in the nucleus, interacts with ScFT (Flowering locus T), indicating their role in regulating flowering time. Taken together, this comprehensive study of ScTCPs provides important information for further research on gene function and crop improvement.

PM2b, a CC-NBS-LRR protein, interacts with TaWRKY76-D to regulate powdery mildew resistance in common wheat.

Powdery mildew caused by Blumeria graminis f. sp. tritici (Bgt) is a destructive disease of wheat throughout the world. Host resistance is considered the most sustainable way to control this disease. Powdery mildew resistance gene Pm2b was mapped to the same genetic interval with Pm2a and PmCH1357 cloned previously, but showed different resistance spectra from them, indicating that they might be caused by different resistance genes or alleles. In this study, Pm2b was delimited to a 1.64 Mb physical interval using a large segregating population containing 4,354 F2:3 families of resistant parent KM2939 and susceptible cultivar Shimai 15. In this interval, TraesCS5D03G0111700 encoding the coiled-coil nucleotide-binding site leucine-rich repeat protein (CC-NBS-LRR) was determined as the candidate gene of Pm2b. Silencing by barley stripe mosaic virus-induced gene silencing (BSMV-VIGS) technology and two independent mutants analysis in KM2939 confirmed the candidate gene TraesCS5D03G0111700 was Pm2b. The sequence of Pm2b was consistent with Pm2a/PmCH1357. Subcellular localization showed Pm2b was located on the cell nucleus and plasma membrane. Pm2b had the highest expression level in leaves and was rapidly up-regulated after inoculating with Bgt isolate E09. The yeast two-hybrid (Y2H) and luciferase complementation imaging assays (LCI) showed that PM2b could self-associate through the NB domain. Notably, we identified PM2b interacting with the transcription factor TaWRKY76-D, which depended on the NB domain of PM2b and WRKY domain of TaWRKY76-D. TaWRKY76-D negatively regulated the resistance to powdery mildew in wheat. The specific KASP marker K529 could take the advantage of high-throughput and high-efficiency for detecting Pm2b and be useful in molecular marker assisted-selection breeding. In conclusion, cloning and disease resistance mechanism analysis of Pm2b provided an example to emphasize a need of the molecular isolation of resistance genes, which has implications in marker assisted wheat breeding.

ALA Upregulates MdPTPA Expression to Increase the PP2A Activity and Promote Stomatal Opening in Apple Leaves

5-Aminolevulinic acid (ALA) is a new natural plant growth regulator that inhibits abscisic acid (ABA)-induced stomatal closure. Studies have shown that protein phosphatase 2 A (PP2A) is involved in ALA-ABA antagonistically regulating stomatal movement; however, the molecular mechanisms underlying remain unclear. Here, we report that ALA promoted MdPP2A activity and the MdPP2AC expression in the epidermis of apple (Malus × domestica Borkh. cv. Fuji) leaves. Y2H (Yeast two hybrid), BiFC (Bimolecular fluorescence complement), and FLC (Firefly luciferase complementation imaging assay) analysis showed that MdPP2AC interacted with MdPTPA, a phosphortyrosyl phosphatase activator. Furthermore, the transient overexpression or interference-expression of MdPTPA transgenic apple leaves were developed. The results showed that overexpression of MdPTPA promoted stomatal opening by reducing Ca2+ and H2O2 but increasing flavonols in guard cells. Conversely, when the MdPTPA was silenced in transient transgenic apple leaves, the Ca2+, H2O2 and flavonols in guard cells and stomatal movement were completely conversed. In the transgenic apple leaves, exogenous ALA stimulated PP2A but repressed SnRK2.6 activity, while the responses are the same as that in the wild type. Therefore, we propose that MdPTPA, which increases the PP2A activity, mediates ALA signaling to promote stomatal opening in apple leaves.

Functional characterization of the soybean cyst nematode effector SCN-27D09 using the model plant pathogenic fungus Magnaporthe oryzae-mediated delivery system

Soybean cyst nematode (SCN, Heterodera glycines) is widely considered as the model plant-parasitic nematode, which secretes effector proteins to manipulate host responses. In this study, we cloned a dorsal gland-expressed effector protein SCN-27D09 that belongs to the same family as Hg10A07 in SCN. We used the model plant pathogen rice blast fungus (Magnaporthe oryzae) to quickly predict and characterize the functions of SCN-27D09. By using M. oryzae secretion system in barley, we confirmed that the signal peptide of SCN-27D09 has secretory activity and can guide the protein into the host cells. Heterologous expression of SCN-27D09 in M. oryzae significantly enhanced the susceptibility of barley to M. oryzae. SCN-27D09 can inhibit Bax-triggered cell death when expressed in Nicotiana benthamiana. Overexpression of SCN-27D09 in soybean hairy root also increased the susceptibility of soybean plants to SCN. Moreover, yeast two-hybrid and firefly luciferase complementation imaging assays showed that SCN-27D09 interacts with a soybean plant kinase GmIPK-2. Functional characterization of GmIPK-2 revealed its positive role in soybean resistance, indicating that SCN-27D09 might compromise the function of GmIPK-2 to facilitate nematode infection. Our results not only uncover the biological role of SCN-27D09 in suppressing plant defense responses and therefore promoting nematode parasitism, but also reaffirm the potential application of the model plant pathogenic fungus M. oryzae in investigating the pathogenic roles of candidate effectors of phytonematodes.