RNA Interference Construct Research Articles

A modified feeding RNAi method for simultaneous knock-down of more than one gene in Caenorhabditis elegans

RNA interference (RNAi) is a commonly used technique for reverse genetic approaches in Caenorhabditis elegans. Feeding RNAi is the most convenient and inexpensive method for performing genome-wide RNAi screens. However, it has been reported that knock-down of two genes (double RNAi) by feeding RNAi using a mixture of bacteria that each contained one dsRNA species produced poor results. To overcome this problem of inefficiency, we designed and tested a double feeding RNAi method using a single RNAi construct containing two gene fragments. From experiments with three different sets of genes, we found that the new double RNAi method consistently produced significantly enhanced double knock-down phenotypes. The double feeding RNAi approach described here provides a method to consistently examine phenotypes caused by depletion of more than one gene in C. elegans.

Simultaneous suppression of three genes related to brassinosteroid (BR) biosynthesis altered campesterol and BR contents, and led to a dwarf phenotype in Arabidopsis thaliana

We generated transgenic lines of Arabidopsis thaliana with an RNA interference construct that expressed hairpin double-stranded RNA for DET2:DWF4:SMT2 to induce sequence-specific RNA silencing. In transgenic plants, expressions of DET2, DWF4, and SMT2 were simultaneously reduced, and the campesterol content was increased by up to 420% compared to the level in the wild-type plant. Triple knock-down of the DET2, DWF4, and SMT2 enzymes also resulted in reduction of brassinosteroid (BR)-specific biosynthesis intermediates. Transgenic plants harboring the RNA interference construct displayed a semi-dwarf phenotype due to altered development. Our findings indicate that redesigning of plant architecture is possible through simultaneous suppression of multiple genes involved in BR biosynthesis.

Distinct Roles of NR2A and NR2B Cytoplasmic Tails in Long-Term Potentiation

NMDA receptors (NMDARs) are critical mediators of activity-dependent synaptic plasticity, but the differential roles of NR2A- versus NR2B-containing NMDARs have been controversial. Here, we investigate the roles of NR2A and NR2B in long-term potentiation (LTP) in organotypic hippocampal slice cultures using RNA interference (RNAi) and overexpression, to complement pharmacological approaches. In young slices, when NR2B is the predominant subunit expressed, LTP is blocked by the NR2B-selective antagonist Ro25-6981 [R-(R,S)-alpha-(4-hydroxyphenyl)-beta-methyl-4-(phenylmethyl)-1-piperidine propranol]. As slices mature and NR2A expression rises, activation of NR2B receptors became no longer necessary for LTP induction. LTP was blocked, however, by RNAi knockdown of NR2B, and this was rescued by coexpression of an RNAi-resistant NR2B (NR2B*) cDNA. Interestingly, a chimeric NR2B subunit in which the C-terminal cytoplasmic tail was replaced by that of NR2A failed to rescue LTP, whereas the reverse chimera, NR2A channel with NR2B tail, was able to restore LTP. Thus, expression of NR2B with its intact cytoplasmic tail is required for LTP induction, at an age when channel activity of NR2B-NMDARs is not required for LTP. Overexpression of wild-type NR2A failed to rescue LTP in neurons transfected with the NR2B-RNAi construct, despite restoring NMDA-EPSC amplitude to a similar level as NR2B*. Surprisingly, an NR2A construct lacking its entire C-terminal cytoplasmic tail regained its ability to restore LTP. Together, these data suggest that the NR2B subunit plays a critical role for LTP, presumably by recruiting relevant molecules important for LTP via its cytoplasmic tail. In contrast, NR2A is not essential for LTP, and its cytoplasmic tail seems to carry inhibitory factors for LTP.

Resistance to ErbB2 Tyrosine Kinase Inhibitors in Breast Cancer Is Mediated by Calcium-Dependent Activation of RelA

The widespread clinical use of therapies targeting the ErbB2 receptor tyrosine kinase oncogene represents a significant advance in breast cancer treatment. However, the development of therapeutic resistance represents a dilemma limiting their clinical efficacy, particularly small-molecule tyrosine kinase inhibitors that block ErbB2 autophosphorylation and activation. Here, we show that lapatinib (GW572016), a highly selective, small-molecule inhibitor of the ErbB2 and epidermal growth factor receptor tyrosine kinases, which was recently approved for the treatment of advanced-stage ErbB2(+) breast cancer, unexpectedly triggered a cytoprotective stress response in ErbB2(+) breast cancer cell lines, which was mediated by the calcium-dependent activation of RelA, the prosurvival subunit of NF-kappaB. Abrogation of lapatinib-induced RelA activation using either small interfering RNA constructs or an intracellular calcium chelator enhanced the apoptotic effects of lapatinib in parental ErbB2(+) breast cancer cells and overcame therapeutic resistance to lapatinib in ErbB2(+) breast cancer lines that had been rendered resistant to lapatinib through chronic exposure to the drug, mimicking the clinical setting. In addition, analysis of changes in phospho-RelA expression in sequential clinical biopsies from ErbB2(+) breast cancers treated with lapatinib monotherapy revealed marginally statistically significant differences between responders and nonresponders, which was consistent with our preclinical findings. Elucidating the regulation of RelA by lapatinib in ErbB2(+) breast cancers, and showing its role in the development of therapeutic resistance to lapatinib, identifies another therapeutic target to overcome or prevent the onset of resistance to lapatinib in some women with ErbB2(+) breast cancers.

Down-regulation of the CSLF6 gene results in decreased (1,3;1,4)-beta-D-glucan in endosperm of wheat.

(1,3;1,4)-beta-d-Glucan (beta-glucan) accounts for 20% of the total cell walls in the starchy endosperm of wheat (Triticum aestivum) and is an important source of dietary fiber for human nutrition with potential health benefits. Bioinformatic and array analyses of gene expression profiles in developing caryopses identified the CELLULOSE SYNTHASE-LIKE F6 (CSLF6) gene as encoding a putative beta-glucan synthase. RNA interference constructs were therefore designed to down-regulate CSLF6 gene expression and expressed in transgenic wheat under the control of a starchy endosperm-specific HMW subunit gene promoter. Analysis of wholemeal flours using an enzyme-based kit and by high-performance anion-exchange chromatography after digestion with lichenase showed decreases in total beta-glucan of between 30% and 52% and between 36% and 53%, respectively, in five transgenic lines compared to three control lines. The content of water-extractable beta-glucan was also reduced by about 50% in the transgenic lines, and the M(r) distribution of the fraction was decreased from an average of 79 to 85 x 10(4) g/mol in the controls and 36 to 57 x 10(4) g/mol in the transgenics. Immunolocalization of beta-glucan in semithin sections of mature and developing grains confirmed that the impact of the transgene was confined to the starchy endosperm with little or no effect on the aleurone or outer layers of the grain. The results confirm that the CSLF6 gene of wheat encodes a beta-glucan synthase and indicate that transgenic manipulation can be used to enhance the health benefits of wheat products.

Down-regulation of four putative arabinoxylan feruloyl transferase genes from family PF02458 reduces ester-linked ferulate content in rice cell walls.

Industrial processes to produce ethanol from lignocellulosic materials are available, but improved efficiency is necessary to make them economically viable. One of the limitations for lignocellulosic conversion to ethanol is the inaccessibility of the cellulose and hemicelluloses within the tight cell wall matrix. Ferulates (FA) can cross-link different arabinoxylan molecules in the cell wall of grasses via diferulate and oligoferulate bridges. This complex cross-linking is thought to be a key factor in limiting the biodegradability of grass cell walls and, therefore, the reduction in FA is an attractive target to improve enzyme accessibility to cellulose and hemicelluloses. Unfortunately, our knowledge of the genes responsible for the incorporation of FA to the cell wall is limited. A bioinformatics prediction based on the gene similarities and higher transcript abundance in grasses relative to dicot species suggested that genes from the pfam family PF02458 may act as arabinoxylan feruloyl transferases. We show here that the FA content in the cell walls and the transcript levels of rice genes Os05g08640, Os06g39470, Os01g09010 and Os06g39390, are both higher in the stems than in the leaves. In addition, an RNA interference (RNAi) construct that simultaneously down-regulates transcript levels of these four genes is associated with a significant reduction in FA of the cell walls from the leaves of the transgenic plants relative to the control (19% reduction, P < 0.0001). Therefore, our experimental results in rice support the bioinformatics prediction that members of family PF02458 are involved in the incorporation of FA into the cell wall in grasses.Electronic supplementary materialThe online version of this article (doi:10.1007/s00425-009-1077-1) contains supplementary material, which is available to authorized users.

Lighting Up Neuronal Pathways: The Development of a Novel Transgenic Rat that Identifies Fos-Activated Neurons Using a Red Fluorescent Protein

Lighting Up Neuronal Pathways: The Development of a Novel Transgenic Rat that Identifies Fos-Activated Neurons Using a Red Fluorescent Protein

Improved Expression and Reactivity of Transduced Tumor-Specific TCRs in Human Lymphocytes by Specific Silencing of Endogenous TCR

Adoptive T-cell therapy using lymphocytes genetically engineered to express tumor antigen-specific TCRs is an attractive strategy for treating patients with malignancies. However, there are potential drawbacks to this strategy: mispairing of the introduced TCR alpha/beta chains with the endogenous TCR subunits and competition of CD3 molecules between the introduced and endogenous TCRs can impair cell surface expression of the transduced TCR, resulting in insufficient function and potential generation of autoreactive T cells. In addition, the risk of tumor development following the infusion of cells with aberrant vector insertion sites increases with the vector copy number in the transduced cells. In this study, we developed retroviral vectors encoding both small interfering RNA constructs that specifically down-regulate endogenous TCR and a codon-optimized, small interfering RNA-resistant TCR specific for the human tumor antigens MAGE-A4 or WT1. At low copy numbers of the integrated vector, the transduced human lymphocytes exhibited high surface expression of the introduced tumor-specific TCR and reduced expression of endogenous TCRs. In consequence, the vector-transduced lymphocytes showed enhanced cytotoxic activity against antigen-expressing tumor cells. Therefore, our novel TCR gene therapy may open a new gate for effective immunotherapy in cancer patients.

Caenorhabditis elegans PI3K mutants reveal novel genes underlying exceptional stress resistance and lifespan.

Two age-1 nonsense mutants, truncating the class-I phosphatidylinositol 3-kinase catalytic subunit (PI3K(CS)) before its kinase domain, confer extraordinary longevity and stress-resistance to Caenorhabditis elegans. These traits, unique to second-generation homozygotes, are blunted at the first generation and are largely reversed by additional mutations to DAF-16/FOXO, a transcription factor downstream of AGE-1 in insulin-like signaling. The strong age-1 alleles (mg44, m333) were compared with the weaker hx546 allele on expression microarrays, testing four independent cohorts of each allele. Among 276 genes with significantly differential expression, 92% showed fewer transcripts in adults carrying strong age-1 alleles rather than hx546. This proportion is significantly greater than the slight bias observed when contrasting age-1 alleles to wild-type worms. Thus, transcriptional changes peculiar to nonsense alleles primarily involve either gene silencing or failure of transcriptional activation. A subset of genes responding preferentially to age-1-nonsense alleles was reassessed by real-time polymerase chain reaction, in worms bearing strong or weak age-1 alleles; nearly all of these were significantly more responsive to the age-1(mg44) allele than to age-1(hx546). Additional mutation of daf-16 reverted the majority of altered mg44-F2 expression levels to approximately wild-type values, although a substantial number of genes remained significantly distinct from wild-type, implying that age-1(mg44) modulates transcription through both DAF-16/FOXO-dependent and -independent channels. When age-1-inhibited genes were targeted by RNA interference (RNAi) in wild-type or age-1(hx546) adults, most conferred significant oxidative-stress protection. RNAi constructs targeting two of those genes were shown previously to extend life, and RNAi's targeting five novel genes were found here to increase lifespan. PI3K-null mutants may thus implicate novel mechanisms of life extension.

Metabolic Engineering of Lignan Biosynthesis in Forsythia Cell Culture

Lignans are a large class of secondary metabolites in plants, with numerous biological effects in mammals, including antitumor and antioxidant activities. Sesamin, the most abundant furofuran-class lignan in sesame seeds (Sesamum plants), is produced by the cytochrome P450 enzyme CYP81Q1 from the precursor lignan, pinoresinol. In contrast, Forsythia plants produce dibenzylbutyrolactone-class lignans, such as matairesinol, from pinoresinol via the catalysis of pinoresinol/lariciresinol reductase (PLR) and secoisolariciresinol dehydrogenase. Here we present the engineering of lignan biosynthesis in Forsythia cell suspension cultures for the development of an efficient production method of beneficial lignans. A suspension cell culture prepared from leaves of Forsythia koreana produced lignans, mainly pinoresinol and matairesinol glucosides, at levels comparable with that obtained from the leaves. In an attempt to increase the pinoresinol content in Forsythia, we generated a transgenic cell line overexpressing an RNA interference (RNAi) construct of PLR (PLR-RNAi). Down-regulation of PLR expression led to a complete loss of matairesinol and an accumulation of approximately 20-fold pinoresinol in its glucoside form in comparison with the non-transformant. Moreover, the Forsythia transgenic cells co-expressing CYP81Q1 and PLR-RNAi exhibited production of sesamin as well as accumulation of pinoresinol glucoside. These data suggest Forsythia cell suspension to be a promising tool for the engineering of lignan production. To the best of our knowledge, this is the first report on transgenic production of an exogenous lignan in a plant species.

The evolutionarily conserved RNA binding protein SMOOTH is essential for maintaining normal muscle function

The Drosophila smooth gene encodes an RNA binding protein which has been well conserved through evolution. To investigate the pleiotropic functions mediated by the smooth gene, we have selected and characterized two sm mutants which are viable as adults yet display robust phenotypes (including a significant decrease in lifespan). Utilizing these mutants, we have made the novel observation that disruption of the smooth/CG9218 locus leads to age-dependent muscle degeneration, and motor dysfunction. Histological characterization of adult sm mutants revealed marked abnormalities in the major thoracic tubular muscle: the tergal depressor of the trochanter (TDT). Corresponding defects include extensive loss/disruption of striations and nuclei. These pathological changes are recapitulated in flies that express a smooth RNA interference construct (sm RNAi) in the mesoderm. In contrast, targeting sm RNAi constructs to motor neurons does not alter muscle morphology. In addition to examining the TDT phenotype, we explored whether other muscular abnormalities were evident. Utilizing physiological assays developed in the laboratory, we have found that the thoracic muscle defect is preceded by dysmotility of the gastrointestinal tract. SMOOTH thus joins a growing list of hnRNPs that have previously been linked to muscle physiology/ pathophysiology. Our findings in Drosophila set the stage for investigating the role of the corresponding mammalian homolog, hnRNP L, in muscle function.

Identification of RNA Helicase A as a New Host Factor in the Replication Cycle of Foot-and-Mouth Disease Virus

Foot-and-mouth disease virus (FMDV), as with other RNA viruses, recruits various host cell factors to assist in the translation and replication of the virus genome. In this study, we investigated the role of RNA helicase A (RHA) in the life cycle of FMDV. Immunofluorescent microscopy (IFM) showed a change in the subcellular distribution of RHA from the nucleus to the cytoplasm in FMDV-infected cells as infection progressed. Unlike nuclear RHA, the RHA detected in the cytoplasm reacted with an antibody that recognizes only the nonmethylated form of RHA. In contrast to alterations in the subcellular distribution of nuclear factors observed during infection with the related cardioviruses, cytoplasmic accumulation of RHA did not require the activity of the FMDV leader protein. Using IFM, we have found cytoplasmic RHA in proximity to the viral 2C and 3A proteins, which promotes the assembly of the replication complexes, as well as cellular poly(A) binding protein (PABP). Coimmunoprecipitation assays confirmed that these proteins are complexed with RHA. We have also identified a novel interaction between RHA and the S fragment in the FMDV 5' nontranslated region. Moreover, a reduction in the expression of RHA, using RHA-specific small interfering RNA constructs, inhibited FMDV replication. These results indicate that RHA plays an essential role in the replication of FMDV and potentially other picornaviruses through ribonucleoprotein complex formation at the 5' end of the genome and by interactions with 2C, 3A, and PABP.

WDR34 is a novel TAK1-associated suppressor of the IL-1R/TLR3/TLR4-induced NF-kappaB activation pathway.

Toll-like receptors (TLRs) act as sensors of microbial components and elicit innate immune responses. All TLR signaling pathways activate the nuclear factor-kappaB (NF-kappaB), which controls the expression of inflammatory cytokine genes. Transforming growth factor-beta-activated kinase 1 (TAK1) is a serine/threonine protein kinase that is critically involved in the activation of NF-kappaB by tumor necrosis factor (TNFalpha), interleukin-1beta (IL-1beta) and TLR ligands. In this study, we identified a novel protein, WD40 domain repeat protein 34 (WDR34) as a TAK1-interacting protein in yeast two-hybrid screens. WDR34 interacted with TAK1, TAK1-binding protein 2 (TAB2), TAK1-binding protein 3 (TAB3) and tumor necrosis factor receptor-associated factor 6 (TRAF6) in overexpression and under physiological conditions. Overexpression of WDR34 inhibited IL-1beta-, polyI:C- and lipopolysaccharide (LPS)-induced but not TNFalpha-induced NF-kappaB activation, whereas knockdown of WDR34 by a RNA-interference construct potentiated NF-kappaB activation by these ligands. Our findings suggest that WDR34 is a TAK1-associated inhibitor of the IL-1R/TLR3/TLR4-induced NF-kappaB activation pathway.

Dok-4 Is a Novel Negative Regulator of T Cell Activation

Dok-4 (downstream of tyrosine kinase-4) is a recently identified member of the Dok family of adaptor proteins, which are characterized by an amino-terminal pleckstrin homology domain, a phosphotyrosine-binding domain, and a carboxyl-terminal region containing several tyrosines and poly-proline-rich motifs. Two members of the Dok family, Dok-1 and Dok-2, have already been described as negative regulators in T cells. However, the function of Dok-4, which is also expressed in T cells, remains unknown. In this study, we report that Dok-4 is phosphorylated after TCR engagement and shuttled within the cytoplasm of T cells before being recruited to the polarized microtubule organizing center after the formation of the immunological synapse. Loss-of-function experiments using RNA interference constructs show that Dok-4 is a negative regulator of ERK phosphorylation, IL-2 promoter activity, and T cell proliferation. Exogenous expression of wild-type Dok-4 induces a significant activation of Rap1, which is involved in the regulation of ERK. The pleckstrin homology domain of Dok-4 is required both for its cytoplasmic shuttling and relocalization as well as for its inhibitory properties on T cell activation. Thus, Dok-4 represents a novel negative regulator of T cells.

Functional characterization of the Arabidopsis beta-ketoacyl-coenzyme A reductase candidates of the fatty acid elongase.

In plants, very-long-chain fatty acids (VLCFAs; >18 carbon) are precursors of sphingolipids, triacylglycerols, cuticular waxes, and suberin. VLCFAs are synthesized by a multiprotein membrane-bound fatty acid elongation system that catalyzes four successive enzymatic reactions: condensation, reduction, dehydration, and a second reduction. A bioinformatics survey of the Arabidopsis (Arabidopsis thaliana) genome has revealed two sequences homologous to YBR159w encoding a Saccharomyces cerevisiae beta-ketoacyl reductase (KCR), which catalyzes the first reduction during VLCFA elongation. Expression analyses showed that both AtKCR1 and AtKCR2 genes were transcribed in siliques, flowers, inflorescence stems, leaves, as well as developing embryos, but only AtKCR1 transcript was detected in roots. Fluorescent protein-tagged AtKCR1 and AtKCR2 were localized to the endoplasmic reticulum, the site of fatty acid elongation. Complementation of the yeast ybr159Delta mutant demonstrated that the two KCR proteins are divergent and that only AtKCR1 can restore heterologous elongase activity similar to the native yeast KCR gene. Analyses of insertional mutants in AtKCR1 and AtKCR2 revealed that loss of AtKCR1 function results in embryo lethality, which cannot be rescued by AtKCR2 expression using the AtKCR1 promoter. In contrast, a disruption of the AtKCR2 gene had no obvious phenotypic effect. Taken together, these results indicate that only AtKCR1 is a functional KCR isoform involved in microsomal fatty acid elongation. To investigate the roles of AtKCR1 in postembryonic development, transgenic lines expressing RNA interference and overexpression constructs targeted against AtKCR1 were generated. Morphological and biochemical characterization of these lines confirmed that suppressed KCR activity results in a reduction of cuticular wax load and affects VLCFA composition of sphingolipids, seed triacylglycerols, and root glycerolipids, demonstrating in planta that KCR is involved in elongation reactions supplying VLCFA for all these diverse classes of lipids.

Simple construction of plant RNAi vectors using long oligonucleotides

RNA interference (RNAi) is one of the most important technologies currently available for the analysis of gene function. However, despite the development of various methods, it is still difficult to construct RNAi vectors for plants with the appropriate inverted repeat fragments to produce double-stranded RNA for knockdown experiments. To solve this problem we have developed an easy and simple method to make RNAi constructs using two long oligonucleotides consisting of partially complementary sequences without the need for PCR amplification and multiple cloning steps. CHS RNAi plants generated using this method showed yellow seed color and a decrease in antocyanin content--phenotypes typically observed in CHS loss-of-function mutants. Moreover, we demonstrated specific knockdown of both the PHYA and PHYB genes using a tandem RNAi construct. This method thus represents a powerful tool for gene knockdown in plants.

Induction of apoptosis by Hax‐1 siRNA in melanoma cells

HS1-associated protein X-1 (Hax-1) is a novel intracellular protein and recent studies suggested that it is an anti-apoptotic factor in different tumors. Hax-1 expression was upregulated in various metastatic tumors and cancer cell lines, including melanoma. To understand the role of Hax-1 in melanoma development and progression, we constructed Hax-1 short interfering RNA (siRNA) expression vectors to downregulate Hax-1 expression in a human melanoma A375 cell line. One of the two Hax-1 RNA interference (RNAi) constructs significantly reduced melanoma cell viability, which was due to induction of apoptosis in A375 cells. Molecularly, the induced apoptosis through downregulation of Hax-1 expression was mediated by activation of caspase-3 and poly-ADP-ribose polymerase (PARP) enzymatic activity in A375 cells. The data indicate that Hax-1 plays a role in suppression of apoptosis and promotion of melanoma cell growth, suggesting that this Hax-1 siRNA has a therapeutic indication in control of melanoma.

Genome-wide analysis of Notch signalling in Drosophila by transgenic RNAi

Genome-wide RNA interference (RNAi) screens have identified near-complete sets of genes involved in cellular processes. However, this methodology has not yet been used to study complex developmental processes in a tissue-specific manner. Here we report the use of a library of Drosophila strains expressing inducible hairpin RNAi constructs to study the Notch signalling pathway during external sensory organ development. We assigned putative loss-of-function phenotypes to 21.2% of the protein-coding Drosophila genes. Using secondary assays, we identified 6 new genes involved in asymmetric cell division and 23 novel genes regulating the Notch signalling pathway. By integrating our phenotypic results with protein interaction data, we constructed a genome-wide, functionally validated interaction network governing Notch signalling and asymmetric cell division. We used clustering algorithms to identify nuclear import pathways and the COP9 signallosome as Notch regulators. Our results show that complex developmental processes can be analysed on a genome-wide level and provide a unique resource for functional annotation of the Drosophila genome.

Suppression of a Phospholipase D Gene,OsPLDβ1, Activates Defense Responses and Increases Disease Resistance in Rice

Phospholipase D (PLD) plays an important role in plants, including responses to abiotic as well as biotic stresses. A survey of the rice (Oryza sativa) genome database indicated the presence of 17 PLD genes in the genome, among which OsPLDalpha1, OsPLDalpha5, and OsPLDbeta1 were highly expressed in most tissues studied. To examine the physiological function of PLD in rice, we made knockdown plants for each PLD isoform by introducing gene-specific RNA interference constructs. One of them, OsPLDbeta1-knockdown plants, showed the accumulation of reactive oxygen species in the absence of pathogen infection. Reverse transcription-polymerase chain reaction and DNA microarray analyses revealed that the knockdown of OsPLDbeta1 resulted in the up-/down-regulation of more than 1,400 genes, including the induction of defense-related genes such as pathogenesis-related protein genes and WRKY/ERF family transcription factor genes. Hypersensitive response-like cell death and phytoalexin production were also observed at a later phase of growth in the OsPLDbeta1-knockdown plants. These results indicated that the OsPLDbeta1-knockdown plants spontaneously activated the defense responses in the absence of pathogen infection. Furthermore, the OsPLDbeta1-knockdown plants exhibited increased resistance to the infection of major pathogens of rice, Pyricularia grisea and Xanthomonas oryzae pv oryzae. These results suggested that OsPLDbeta1 functions as a negative regulator of defense responses and disease resistance in rice.

PAMP‐induced defense responses in potato require both salicylic acid and jasmonic acid

To elucidate the molecular mechanisms underlying pathogen-associated molecular pattern (PAMP)-induced defense responses in potato (Solanum tuberosum), the role of the signaling compounds salicylic acid (SA) and jasmonic acid (JA) was analyzed. Pep-13, a PAMP from Phytophthora, induces the accumulation of SA, JA and hydrogen peroxide, as well as the activation of defense genes and hypersensitive-like cell death. We have previously shown that SA is required for Pep-13-induced defense responses. To assess the importance of JA, RNA interference constructs targeted at the JA biosynthetic genes, allene oxide cyclase and 12-oxophytodienoic acid reductase, were expressed in transgenic potato plants. In addition, expression of the F-box protein COI1 was reduced by RNA interference. Plants expressing the RNA interference constructs failed to accumulate the respective transcripts in response to wounding or Pep-13 treatment, neither did they contain significant amounts of JA after elicitation. In response to infiltration of Pep-13, the transgenic plants exhibited a highly reduced accumulation of reactive oxygen species as well as reduced hypersensitive cell death. The ability of the JA-deficient plants to accumulate SA suggests that SA accumulation is independent or upstream of JA accumulation. These data show that PAMP responses in potato require both SA and JA and that, in contrast to Arabidopsis, these compounds act in the same signal transduction pathway. Despite their inability to fully respond to PAMP treatment, the transgenic RNA interference plants are not altered in their basal defense against Phytophthora infestans.