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Cecropin A-Derived Peptides Are Potent Inhibitors of Fungal Plant Pathogens

Cecropins are naturally occurring peptides that play an important role in the immune response of insects. Cecropin A-derived and cecropin A-melittin hybrid peptides, all smaller than the natural compound cecropin A, were synthesized and tested for their ability to inhibit growth of several agronomically important fungal pathogens. We found that an 11-amino-acid sequence, corresponding to the N-terminal amphipathic alpha-helix domain of cecropin A, exhibited antifungal activity. Differences in susceptibility of the various pathogens were observed, Phytophthora infestans being particularly sensitive to the shortened cecropin A peptides (IC50 = 2 x 10(-6) M). Biotoxicity of the shortest cecropin A-derived peptide was variously affected by the presence of proteins extracted from leaves of tobacco and tomato plants, either total extracts or intercellular fluids (ICFs). Overall, there was a greater tolerance to tomato protein extracts than to tobacco extracts. These findings suggest that tobacco should not be used as a model for testing the possible protective effects of transgenically expressed, cecropin-based genes. The feasibility of tailoring cecropin A genes to enhance crop protection in particular plant/fungus combinations is discussed.

AtCPK1 calcium-dependent protein kinase mediates pathogen resistance in Arabidopsis.

In mammals, lipid bodies play a key role during pathological and infectious diseases. However, our knowledge on the function of plant lipid bodies, apart from their role as the major site of lipid storage in seed tissues, remains limited. Here, we provide evidence that a calcium-dependent protein kinase (CPK) mediates pathogen resistance in Arabidopsis. AtCPK1 expression is rapidly induced by fungal elicitors. Loss-of-function mutants of AtCPK1 exhibit higher susceptibility to pathogen infection compared to wild-type plants. Conversely, over-expression of AtCPK1 leads to accumulation of salicylic acid (SA) and constitutive expression of SA-regulated defence and disease resistance genes, which, in turn, results in broad-spectrum protection against pathogen infection. Expression studies in mutants affected in SA-mediated defence responses revealed an interlocked feedback loop governing AtCPK1 expression and components of the SA-dependent signalling pathway. Moreover, we demonstrate the dual localization of AtCPK1 in lipid bodies and peroxisomes. Overall, our findings identify AtCPK1 as a component of the innate immune system of Arabidopsis plants.

Sucrose-Mediated Priming of Plant Defense Responses and Broad-Spectrum Disease Resistance by Overexpression of the Maize Pathogenesis-Related PRms Protein in Rice Plants

Expression of pathogenesis-related (PR) genes is part of the plants natural defense response against pathogen attack. The PRms gene encodes a fungal-inducible PR protein from maize. Here, we demonstrate that expression of PRms in transgenic rice confers broad-spectrum protection against pathogens, including fungal (Magnaporthe oryzae, Fusarium verticillioides, and Helminthosporium oryzae) and bacterial (Erwinia chrysanthemi) pathogens. The PRms-mediated disease resistance in rice plants is associated with an enhanced capacity to express and activate the natural plant defense mechanisms. Thus, PRms rice plants display a basal level of expression of endogenous defense genes in the absence of the pathogen. PRms plants also exhibit stronger and quicker defense responses during pathogen infection. We also have found that sucrose accumulates at higher levels in leaves of PRms plants. Sucrose responsiveness of rice defense genes correlates with the pathogen-responsive priming of their expression in PRms rice plants. Moreover, pretreatment of rice plants with sucrose enhances resistance to M. oryzae infection. Together, these results support a sucrose-mediated priming of defense responses in PRms rice plants which results in broad-spectrum disease resistance.

Transgenic rice plants expressing the antifungal AFP protein from Aspergillus giganteus show enhanced resistance to the rice blast fungus Magnaporthe grisea.

The Aspergillus giganteus antifungal protein (AFP), encoded by the afp gene, has been reported to possess in vitro antifungal activity against various economically important fungal pathogens, including the rice blast fungus Magnaporthe grisea. In this study, transgenic rice (Oryza sativa) constitutively expressing the afp gene was generated by Agrobacterium-mediated transformation. Two different DNA constructs containing either the afp cDNA sequence from Aspergillus or a chemically synthesized codon-optimized afp gene were introduced into rice plants. In both cases, the DNA region encoding the signal sequence from the tobacco AP24 gene was N-terminally fused to the coding sequence of the mature AFP protein. Transgenic rice plants showed stable integration and inheritance of the transgene. No effect on plant morphology was observed in the afp-expressing rice lines. The inhibitory activity of protein extracts prepared from leaves of afp plants on the in vitro growth of M. grisea indicated that the AFP protein produced by the trangenic rice plants was biologically active. Several of the T2 homozygous afp lines were challenged with M. grisea in a detached leaf infection assay. Transformants exhibited resistance to rice blast at various levels. Altogether, the results presented here indicate that AFP can be functionally expressed in rice plants for protection against the rice blast fungus M. grisea.

The Arabidopsis AtNPR1 Inversely Modulates Defense Responses Against Fungal, Bacterial, or Viral Pathogens While Conferring Hypersensitivity to Abiotic Stresses in Transgenic Rice

The nonexpressor of pathogenesis-related (PR) genes (NPR1) protein plays an important role in mediating defense responses activated by pathogens in Arabidopsis. In rice, a disease-resistance pathway similar to the Arabidopsis NPR1-mediated signaling pathway one has been described. Here, we show that constitutive expression of the Arabidopsis NPR1 (AtNPR1) gene in rice confers resistance against fungal and bacterial pathogens. AtNPR1 exerts its protective effects against fungal pathogens by priming the expression of salicylic acid (SA)-responsive endogenous genes, such as the PR1b, TLP (PR5), PR10, and PBZ1. However, expression of AtNPR1 in rice has negative effects on viral infections. The AtNPR1-expressing rice plants showed a higher susceptibility to infection by the Rice yellow mottle virus (RYMV) which correlated well with a misregulation of RYMV-responsive genes, including expression of the SA-regulated RNA-dependent RNA polymerase 1 gene (OsRDR1). Moreover, AtNPR1 negatively regulates the expression of genes playing a role in the plant response to salt and drought stress (rab21, salT, and dip1), which results in a higher sensitivity of AtNPR1 rice to the two types of abiotic stress. These observations suggest that AtNPR1 has both positive and negative regulatory roles in mediating defense responses against biotic and abiotic stresses.

Identification of a novel microRNA (miRNA) from rice that targets an alternatively spliced transcript of the Nramp6 (Natural resistance‐associated macrophage protein 6) gene involved in pathogen resistance

Plants have evolved efficient defence mechanisms to defend themselves from pathogen attack. Although many studies have focused on the transcriptional regulation of defence responses, less is known about the involvement of microRNAs (miRNAs) as post-transcriptional regulators of gene expression in plant immunity. This work investigates miRNAs that are regulated by elicitors from the blast fungus Magnaporthe oryzae in rice (Oryza sativa). Small RNA libraries were constructed from rice tissues and subjected to high-throughput sequencing for the identification of elicitor-responsive miRNAs. Target gene expression was examined by microarray analysis. Transgenic lines were used for the analysis of miRNA functioning in disease resistance. Elicitor treatment is accompanied by dynamic alterations in the expression of a significant number of miRNAs, including new members of annotated miRNAs. Novel miRNAs from rice are proposed. We report a new rice miRNA, osa-miR7695, which negatively regulates an alternatively spliced transcript of OsNramp6 (Natural resistance-associated macrophage protein 6). This novel miRNA experienced natural and domestication selection events during evolution, and its overexpression in rice confers pathogen resistance. This study highlights an miRNA-mediated regulation of OsNramp6 in disease resistance, whilst illustrating the existence of a novel regulatory network that integrates miRNA function and mRNA processing in plant immunity.

Enhanced resistance to the rice blast fungus Magnaporthe grisea conferred by expression of a cecropin A gene in transgenic rice

Cecropins are a family of antimicrobial peptides, which constitute an important key component of the immune response in insects. Here, we demonstrate that transgenic rice (Oryza sativa L.) plants expressing the cecropin A gene from the giant silk moth Hyalophora cecropia show enhanced resistance to Magnaporthegrisea, the causal agent of the rice blast disease. Two plant codon-optimized synthetic cecropin A genes, which were designed either to retain the cecropin A peptide in the endoplasmic reticulum, the ER-CecA gene, or to secrete cecropin A to the extracellular space, the Ap-CecA gene, were prepared. Both cecropin A genes were efficiently expressed in transgenic rice. The inhibitory activity of protein extracts prepared from leaves of cecropin A-expressing plants on the in vitro growth of M. grisea indicated that the cecropin A protein produced by the transgenic rice plants was biologically active. Whereas no effect on plant phenotype was observed in ER-CecA plants, most of the rice lines expressing the Ap-CecA gene were non-fertile. Cecropin A rice plants exhibited resistance to rice blast at various levels. Transgene expression of cecropin A genes was not accompanied by an induction of pathogenesis-related (PR) gene expression supporting that the transgene product itself is directly active against the pathogen. Taken together, the results presented in this study suggest that the cecropin A gene, when designed for retention of cecropin A into the endoplasmic reticulum, could be a useful candidate for protection of rice plants against the rice blast fungus M. grisea.

Overexpression of a Calcium-Dependent Protein Kinase Confers Salt and Drought Tolerance in Rice by Preventing Membrane Lipid Peroxidation

A calcium-dependent protein kinase at the plasma membrane prevents membrane lipid peroxidation and confers tolerance to salt and drought stress in rice plants. The OsCPK4 gene is a member of the complex gene family of calcium-dependent protein kinases in rice (Oryza sativa). Here, we report that OsCPK4 expression is induced by high salinity, drought, and the phytohormone abscisic acid. Moreover, a plasma membrane localization of OsCPK4 was observed by transient expression assays of green fluorescent protein-tagged OsCPK4 in onion (Allium cepa) epidermal cells. Overexpression of OsCPK4 in rice plants significantly enhances tolerance to salt and drought stress. Knockdown rice plants, however, are severely impaired in growth and development. Compared with control plants, OsCPK4 overexpressor plants exhibit stronger water-holding capability and reduced levels of membrane lipid peroxidation and electrolyte leakage under drought or salt stress conditions. Also, salt-treated OsCPK4 seedlings accumulate less Na+ in their roots. We carried out microarray analysis of transgenic rice overexpressing OsCPK4 and found that overexpression of OsCPK4 has a low impact on the rice transcriptome. Moreover, no genes were found to be commonly regulated by OsCPK4 in roots and leaves of rice plants. A significant number of genes involved in lipid metabolism and protection against oxidative stress appear to be up-regulated by OsCPK4 in roots of overexpressor plants. Meanwhile, OsCPK4 overexpression has no effect on the expression of well-characterized abiotic stress-associated transcriptional regulatory networks (i.e. ORYZA SATIVA DEHYDRATION-RESPONSIVE ELEMENT BINDING PROTEIN1 and ORYZA SATIVA No Apical Meristem, Arabidopsis Transcription Activation Factor1-2, Cup-Shaped Cotyledon6 genes) and LATE EMBRYOGENESIS ABUNDANT genes in their roots. Taken together, our data show that OsCPK4 functions as a positive regulator of the salt and drought stress responses in rice via the protection of cellular membranes from stress-induced oxidative damage.

Expression of a Solanum tuberosum cyclophilin gene is regulated by fungal infection and abiotic stress conditions

Abstract Cyclophilins (CyPs) are ubiquitous proteins with an intrinsic enzymatic activity of peptidyl-prolyl cis-trans isomerase that catalyzes the rotation of X-Pro peptide bonds. These enzymes are believed to play a role in the folding of certain proteins. In addition, CyPs might be important in signal transduction processes. A cDNA library was prepared from potato (Solanum tuberosusm) tubers infected with the fungus Fusarium solani f. sp eumartii. Using a PCR-amplified subtracted cDNA probe, a clone encoding a cytosolic form of CyP, called StCyP (Solanum ṯuberosum CyP), was isolated. Except in tubers, StCyP is expressed at high levels in tissues of healthy potato plants. Northern blot analyses revealed that both wounding and fungal infection increased the level of StCyP mRNA in tubers. However, whereas wounding causes a transient accumulation of StCyP mRNA, fungal infection results in a maintained accumulation of this transcript. StCyP mRNA accumulation is also stimulated by the application of absicic acid (ABA) and methyl jasmonate (MeJA) in tubers. Treatment with fungal elicitor or salicilic acid (SA) has no effect on the level of StCyP mRNA accumulation. Together these results indicate that the observed accumulation of StCyP mRNAs in fungal-infected potato tubers might be a response to the wound produced by the penetration and colonization of the tissue by the pathogen. Furthermore, accumulation of StCyP transcripts was also detected when the potato tubers were exposed to heat-shock treatment. These findings support a role for cyclophilins in the plant response to environmental stresses.

Diurnal oscillation in the accumulation of Arabidopsis microRNAs, miR167, miR168, miR171 and miR398

MicroRNAs (miRNAs) are small RNAs acting as regulators of eukaryotic gene expression at the post‐transcriptional level. Plant miRNAs have been implicated in developmental processes and adaptation to the environment. We show that the accumulation of four Arabidopsis miRNAs (miR171, miR398, miR168 and miR167) oscillates during the diurnal cycle, their accumulation increasing during the light period of the daytime and decreasing in darkness. This oscillatory pattern of miRNA accumulation is not governed by the circadian clock. These results suggest a potential role of light in controlling miRNA accumulation while defining a new level of regulation of miRNA gene expression in Arabidopsis.