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In JoVE (1)
Other Publications (11)
- Zeitschrift Für Naturforschung. C, Journal of Biosciences
- Journal of Plant Physiology
- Journal of Plant Physiology
- Molecular Plant-microbe Interactions : MPMI
- Planta
- Molecular Plant-microbe Interactions : MPMI
- Journal of Agricultural and Food Chemistry
- Molecular Plant-microbe Interactions : MPMI
- Proceedings of the National Academy of Sciences of the United States of America
- The Plant Journal : for Cell and Molecular Biology
- Science (New York, N.Y.)
Articles by Xiquan Gao in JoVE
JoVE General
Agrobacterium-Mediated Virus-Induced Gene Silencing Assay In Cotton
1Department of Biochemistry and Biophysics, Institute of Plant Genomics and Biotechnology, Texas A&M University, 2Department of Plant Pathology and Microbiology, Institute of Plant Genomics and Biotechnology, Texas A&M University
We present the detailed protocol for Agrobacterium-mediated virus-induced gene silencing (VIGS) assay in cotton. The tobacco rattle virus (TRV)-derived VIGS vectors were deployed to induce RNA silencing of cotton GrCLA1, Cloroplastos alterados 1 gene. The albino phenotype caused by silencing GrCLA1 was observed at the seedling stage within 2 weeks after inoculation.
Other articles by Xiquan Gao on PubMed
Effects of Theobroxide, a Natural Product, on the Level of Endogenous Jasmonoids
The natural potato microtuber inducing substance, theobroxide, strongly induces the formation of tuber of potato (Solanum tuberosum L.) and flower bud of morning glory (Pharbitis nil) plants under non-inducing conditions (long days) (Yoshihara et al., 2000). In the present study, theobroxide was evaluated for its effect on the level of endogenous jasmonoids in different tissues of such two plants. An in vitro bioassay using cultures of single-node segments of potato stems was performed with the supplement of theobroxide in the medium. The endogenous jasmonic acid (JA) and its analogue tuberonic acid (TA, 12-hydroxyjasmonic acid) in segments and microtubers were quantitatively analyzed. The increase in the endogenous JA level caused by theobroxide was observed in both segments and microtubers. Endogenous TA was only detected in segments, and the content increased with the concentration of theobroxide. As for morning glory, the whole plant was sprayed with theobroxide for 1 approximately 5 weeks under different photoperiods and endogenous JA in the leaves was quantitatively analyzed. Theobroxide spraying increased the level of endogenous JA in the leaves of the plants grown under both long and short days.
Inhibitory Role of Gibberellins in Theobroxide-induced Flowering of Pharbitis Nil
Theobroxide, a novel active compound isolated from a fungus, has been reported previously to induce potato tuberization and flower bud formation in Pharbitis nil under non-inductive long-day conditions. Up to date, the action mechanism of theobroxide on flower-bud induction of P. nil, however, is still unknown. In the present study, we observed a reduction of the stem length, along with the induction of flower buds, in theobroxide-treated and short-day-grown P. nil plants. Also, the results showed that flower bud formation was delayed markedly in P. nil seedlings with removal of cotyledons or exposure to night break. The suppression effect of night-break and cotyledon-removal, however, was abolished completely by spraying theobroxide. Endogenous gibberellin(1/3) contents in P. nil plants treated with theobroxide or grown under short-day conditions were relatively lower, suggesting that gibberellins probably are negatively involved in theobroxide- and short-day-induced flower-bud formation of P. nil.
Inhibition of Stem Elongation in Spinach by Theobroxide
In the current study, we investigated the influences of theobroxide on stem elongation in spinach (Spinacia oleracea). Our results showed that stem elongation and flower formation were inhibited by spraying spinach plants with theobroxide under inductive, long day conditions (16 h light/8 h dark), while application of exogenous applied GA3 prevented the effect of theobroxide. Quantitative analysis showed that theobroxide suppressed GA1 biosynthesis, whereas the endogenous content of jasmonic acid was unchanged. However, under short day conditions (10 h light/14 h dark), there were no differences in stem length between treated and untreated plants. These results suggest that the inhibition of stem elongation by theobroxide is probably due to the suppression of gibberellin biosynthesis.
Disruption of a Maize 9-lipoxygenase Results in Increased Resistance to Fungal Pathogens and Reduced Levels of Contamination with Mycotoxin Fumonisin
Plant oxylipins, produced via the lipoxygenase (LOX) pathway, function as signals in defense and development. In fungi, oxylipins are potent regulators of mycotoxin biosynthesis and sporogenesis. Previous studies showed that plant 9-LOX-derived fatty acid hydroperoxides induce conidiation and mycotoxin production. Here, we tested the hypothesis that oxylipins produced by the maize 9-LOX pathway are required by pathogens to produce spores and mycotoxins and to successfully colonize the host. Maize mutants were generated in which the function of a 9-LOX gene, ZmLOX3, was abolished by an insertion of a Mutator transposon in its coding sequence, which resulted in reduced levels of several 9-LOX-derived hydroperoxides. Supporting our hypothesis, conidiation and production of the mycotoxin fumonisin B1 by Fusarium verticillioides were drastically reduced in kernels of the lox3 mutants compared with near-isogenic wild types. Similarly, conidia production and disease severity of anthracnose leaf blight caused by Colletotrichum graminicola were significantly reduced in the lox3 mutants. Moreover, lox3 mutants displayed increased resistance to southern leaf blight caused by Cochliobolus heterostrophus and stalk rots caused by both F. verticillioides and C. graminicola. These data strongly suggest that oxylipin metabolism mediated by a specific plant 9-LOX isoform is required for fungal pathogenesis, including disease development and production of spores and mycotoxins.
A Novel Plastidial Lipoxygenase of Maize (Zea Mays) ZmLOX6 Encodes for a Fatty Acid Hydroperoxide Lyase and is Uniquely Regulated by Phytohormones and Pathogen Infection
Lipoxygenases (LOXs) are members of a large enzyme family that catalyze oxygenation of free polyunsaturated fatty acids into diverse hydroperoxide compounds, collectively called oxylipins. Although LOXs have been well studied in dicot species, reports of the genes encoding these enzymes are scarce for monocots, especially maize. Herein, we reported the cloning, characterization and molecular functional analysis of a novel maize LOX gene, ZmLOX6. The ZmLOX6 nucleotide sequence encodes a deduced translation product of 892 amino acids. Phylogenetic analysis showed that ZmLOX6 is distantly related to previously reported 9- or 13-LOXs from maize and other plant species, including rice and Arabidopsis. Although sequence prediction suggested cytoplasmic localization of this protein, ZmLOX6 protein has been reportedly isolated from mesophyll cell chloroplasts, emphasizing the unique features of this protein. Plastidial localization was confirmed by chloroplast uptake experiments with the in vitro translated protein. Analysis of recombinant protein revealed that ZmLOX6 has lost fatty acid hydroperoxide forming activity but 13-LOX-derived fatty acid hydroperoxides were cleaved into odd-chain omega-oxo fatty acids and as yet not identified C5-compound. In line with its reported abundance in mesophyll cells, ZmLOX6 was predominantly expressed in leaf tissue. Northern blot analysis demonstrated that ZmLOX6 was induced by jasmonic acid, but repressed by abscisic acid, salicylic acid and ethylene and was not responsive to wounding or insects. Further, this gene was strongly induced by the fungal pathogen Cochliobolus carbonum during compatible interactions, suggesting that ZmLOX6 may contribute to susceptibility to this pathogen. The potential involvement of ZmLOX6 in maize interactions with pathogens is discussed.
Maize 9-lipoxygenase ZmLOX3 Controls Development, Root-specific Expression of Defense Genes, and Resistance to Root-knot Nematodes
Root-knot nematodes (RKN) are severe pests of maize. Although lipoxygenase (LOX) pathways and their oxylipin products have been implicated in plant-nematode interactions, prior to this report there was no conclusive genetic evidence for the function of any plant LOX gene in such interactions. We showed that expression of a maize 9-LOX gene, ZmLOX3, increased steadily and peaked at 7 days after inoculation with Meloidogyne incognita RKN. Mu-insertional lox3-4 mutants displayed increased attractiveness to RKN and an increased number of juveniles and eggs. A set of jasmonic acid (JA)- and ethylene (ET)-responsive and biosynthetic genes as well as salicylic acid (SA)-dependent genes were overexpressed specifically in the roots of lox3-4 mutants. Consistent with this, levels of JA, SA, and ET were elevated in lox3-4 mutant roots, but not in leaves. Unlike wild types, in lox3-4 mutant roots, a phenylalanine ammonia lyase (PAL) gene was not RKN-inducible, suggesting a role for PAL-mediated metabolism in nematode resistance. In addition to these alterations in the defense status of roots, lox3-4 knockout mutants displayed precocious senescence and reduced root length and plant height compared with the wild type, suggesting that ZmLOX3 is required for normal plant development. Taken together, our data indicate that the ZmLOX3-mediated pathway may act as a root-specific suppressor of all three major defense signaling pathways to channel plant energy into growth processes, but is required for normal levels of resistance against nematodes.
Considerations Regarding the Use of Hyperspectral Imaging Data in Classifications of Food Products, Exemplified by Analysis of Maize Kernels
Development of robust analytical procedures is critical when using hyperspectral imaging technology in food technology and agriculture. This study used near-isogenic inbred corn lines to address two basic questions: (1) To what extent is classification accuracy increased by grinding maize kernels? (2) Can the classification accuracy of two near-isogenic inbred lines be increased by using a spectral filter to classify only certain hyperspectral profiles from each image cube? Whole kernels and ground kernels in two particle intervals, 0.250-0.354 mm (size 1) and 0.354-0.841 mm (size 2), were examined. Spectral profiles acquired from ground kernels had higher spectral repeatability than data collected from whole kernels. The classification error of discriminant functions from whole kernels was >3 times lower than that of size 1 ground particles. Applying a spectral filter to input data had negligible effect on classifications of hyperspectral profiles from whole kernels and size 2 ground particles, but for size 1 ground particles a considerable increase in accuracy was observed. Independent validation confirmed that distinction between wild type and mutant inbred maize lines could be conducted with >80% accuracy after the proposed spectral filter had been applied to hyperspectral profiles of size 1 ground particles. A combination of discriminant analysis and regression analysis could be used to accurately predict mixture ratios of the two inbred lines. The use of spectral filtering to increase the level of spectral repeatability and the use of hyperspectral imaging technology in large-scale commercial operations are discussed.
Inactivation of the Lipoxygenase ZmLOX3 Increases Susceptibility of Maize to Aspergillus Spp
Plant and fungal lipoxygenases (LOX) catalyze the oxidation of polyunsaturated fatty acids, creating fatty-acid hydroperoxides (oxylipins). Fungal oxylipins are required for normal fungal development and secondary metabolism, and plant host-derived oxylipins interfere with these processes in fungi, presumably by signal mimicry. The maize LOX gene ZmLOX3 has been implicated previously in seed-Aspergillus interactions, so we tested the interactions of a mutant maize line (lox3-4, in which ZmLOX3 is disrupted) with the mycotoxigenic seed-infecting fungi Aspergillus flavus and Aspergillus nidulans. The lox3-4 mutant was more susceptible than wild-type maize to both Aspergillus species. All strains of A. flavus and A. nidulans produced more conidia and aflatoxin (or the precursor sterigmatocystin) on lox3-4 kernels than on wild-type kernels, in vitro and under field conditions. Although oxylipins did not differ detectably between A. flavus-infected kernels of the lox3-4 and wild-type (WT) maize, oxylipin precursors (free fatty acids) and a downstream metabolite (jasmonic acid) accumulated to greater levels in lox3-4 than in WT kernels. The increased resistance of the lox3-4 mutant to other fungal pathogens (Fusarium, Colletotrichum, Cochliobolus, and Exserohilum spp.) is in sharp contrast to results described herein for Aspergillus spp., suggesting that outcomes of LOX-governed host-pathogen interactions are pathogen-specific.
A Receptor-like Cytoplasmic Kinase, BIK1, Associates with a Flagellin Receptor Complex to Initiate Plant Innate Immunity
Plants and animals rely on innate immunity to prevent infections by detection of microbe-associated molecular patterns (MAMPs) through pattern-recognition receptors (PRRs). The plant PRR FLS2, a leucine-rich repeat-receptor kinase, recognizes bacterial flagellin and initiates immune signaling by association with another leucine-rich repeat-receptor-like kinase, BAK1. It remains unknown how the FLS2/BAK1 receptor complex activates intracellular signaling cascades. Here we identified the receptor-like cytoplasmic kinase BIK1 that is rapidly phosphorylated upon flagellin perception, depending on both FLS2 and BAK1. BIK1 associates with FLS2 and BAK1 in vivo and in vitro. BIK1 is phosphorylated by BAK1, and BIK1 also directly phosphorylates BAK1 and FLS2 in vitro. The flagellin phosphorylation site Thr(237) of BIK1 is required for its phosphorylation on BAK1 and FLS2, suggesting that BIK1 is likely first phosphorylated upon flagellin perception and subsequently transphosphorylates FLS2/BAK1 to propagate flagellin signaling. Importantly, bik1 mutants are compromised in diverse flagellin-mediated responses and immunity to the nonpathogenic bacterial infection. Thus, BIK1 is an essential component in MAMP signal transduction, which links the MAMP receptor complex to downstream intracellular signaling.
Silencing GhNDR1 and GhMKK2 Compromises Cotton Resistance to Verticillium Wilt
Cotton is an important cash crop worldwide, and is a significant source of fiber, feed, foodstuff, oil and biofuel products. Considerable effort has been expended to increase sustainable yield and quality through molecular breeding and genetic engineering of new cotton cultivars. Given the recent availability of the whole-genome sequence of cotton, it is necessary to develop molecular tools and resources for large-scale analysis of gene functions at the genome-wide level. We have successfully developed an Agrobacterium-mediated virus-induced gene silencing (VIGS) assay in several cotton cultivars with various genetic backgrounds. The genes of interest were potently and readily silenced within 2 weeks after inoculation at the seedling stage. Importantly, we showed that silencing GhNDR1 and GhMKK2 compromised cotton resistance to the infection by Verticillium dahliae, a fungal pathogen causing Verticillium wilt. Furthermore, we developed a cotton protoplast system for transient gene expression to study gene functions by a gain-of-function approach. The viable protoplasts were isolated from green cotyledons, etiolated cotyledons and true leaves, and responded to a wide range of pathogen elicitors and phytohormones. Remarkably, cotton plants possess conserved, but also distinct, MAP kinase activation with Arabidopsis upon bacterial elicitor flagellin perception. Thus, using gene silencing assays, we have shown that GhNDR1 and GhMKK2 are required for Verticillium resistance in cotton, and have developed high throughput loss-of-function and gain-of-function assays for functional genomic studies in cotton.
Direct Ubiquitination of Pattern Recognition Receptor FLS2 Attenuates Plant Innate Immunity
Innate immune responses are triggered by the activation of pattern-recognition receptors (PRRs). The Arabidopsis PRR FLAGELLIN-SENSING 2 (FLS2) senses bacterial flagellin and initiates immune signaling through association with BAK1. The molecular mechanisms underlying the attenuation of FLS2 activation are largely unknown. We report that flagellin induces recruitment of two closely related U-box E3 ubiquitin ligases, PUB12 and PUB13, to FLS2 receptor complex in Arabidopsis. BAK1 phosphorylates PUB12 and PUB13 and is required for FLS2-PUB12/13 association. PUB12 and PUB13 polyubiquitinate FLS2 and promote flagellin-induced FLS2 degradation, and the pub12 and pub13 mutants displayed elevated immune responses to flagellin treatment. Our study has revealed a unique regulatory circuit of direct ubiquitination and turnover of FLS2 by BAK1-mediated phosphorylation and recruitment of specific E3 ligases for attenuation of immune signaling.
