Patrick Schweizer

Salicylic Acid in Rice (Biosynthesis, Conjugation, and Possible Role)

Salicylic acid (SA) is a natural inducer of disease resistance in some dicotyledonous plants. Rice seedlings (Oryza sativa L.) had the highest levels of SA among all plants tested for SA content (between 0.01 and 37.19 [mu]g/g fresh weight). The second leaf of rice seedlings had slightly lower SA levels than any younger leaves. To investigate the role of SA in rice disease resistance, we examined the levels of SA in rice (cv M-201) after inoculation with bacterial and fungal pathogens. SA levels did not increase after inoculation with either the avirulent pathogen Pseudomonas syringae D20 or with the rice pathogens Magnaporthe grisea, the causal agent of rice blast, and Rhizoctonia solani, the causal agent of sheath blight. However, leaf SA levels in 28 rice varieties showed a correlation with generalized blast resistance, indicating that SA may play a role as a constitutive defense compound. Biosynthesis and metabolism of SA in rice was studied and compared to that of tobacco. Rice shoots converted [14C]cinnamic acid to SA and the lignin precursors p-coumaric and ferulic acids, whereas [14C]benzoic acid was readily converted to SA. The data suggest that in rice, as in tobacco, SA is synthesized from cinnamic acid via benzoic acid. In rice shoots, SA is largely present as a free acid; however, exogenously supplied SA was converted to [beta]-O-D-glucosylSA by an SA-inducible glucosyltransferase (SA-GTase). A 7-fold induction of SA-GTase activity was observed after 6 h of feeding 1 mM SA. Both rice roots and shoots showed similar patterns of SA-GTase induction by SA, with maximal induction after feeding with 1 mM SA.

HIGS: Host-Induced Gene Silencing in the Obligate Biotrophic Fungal Pathogen Blumeria graminis

This work examines the effects of RNA interference constructs expressed in host cells on target RNAs in Blumeria graminis, an obligate biotrophic fungal pathogen of barley, and finds that RNAs in the host can affect pathogen transcript levels and pathogen development, thereby providing both a useful research tool and a potentially important means for engineering plant disease resistance. Powdery mildew fungi are obligate biotrophic pathogens that only grow on living hosts and cause damage in thousands of plant species. Despite their agronomical importance, little direct functional evidence for genes of pathogenicity and virulence is currently available because mutagenesis and transformation protocols are lacking. Here, we show that the accumulation in barley (Hordeum vulgare) and wheat (Triticum aestivum) of double-stranded or antisense RNA targeting fungal transcripts affects the development of the powdery mildew fungus Blumeria graminis. Proof of concept for host-induced gene silencing was obtained by silencing the effector gene Avra10, which resulted in reduced fungal development in the absence, but not in the presence, of the matching resistance gene Mla10. The fungus could be rescued from the silencing of Avra10 by the transient expression of a synthetic gene that was resistant to RNA interference (RNAi) due to silent point mutations. The results suggest traffic of RNA molecules from host plants into B. graminis and may lead to an RNAi-based crop protection strategy against fungal pathogens.

A High-Throughput Gene-Silencing System for the Functional Assessment of Defense-Related Genes in Barley Epidermal Cells

Large-scale gene silencing by RNA interference (RNAi) offers the possibility to address gene function in eukaryotic organisms at a depth unprecedented until recently. Although genome-wide RNAi approaches are being carried out in organisms like Caenorhabditis elegans, Drosophila spp. or human after the corresponding tools had been developed, knock-down of only single or a few genes by RNAi has been reported in plants thus far. Here, we present a method for high-throughput, transient-induced gene silencing (TIGS) by RNAi in barley epidermal cells that is based on biolistic transgene delivery. This method will be useful to address gene function of shoot epidermis resulting in cell-autonomous phenotypes such as resistance or susceptibility to the powdery-mildew fungus Blumeria graminis f. sp. hordei. Gene function in epidermal cell elongation, stomata regulation, or UV resistance might be addressed as well. Libraries of RNAi constructs can be built up by a new, cost-efficient method that combines highly efficient ligation and recombination by the Gateway cloning system. This method allows cloning of any blunt-ended DNA fragment without the need of adaptor sequences. The final RNAi destination vector was found to direct highly efficient RNAi, as reflected by complete knock-down of a cotransformed green fluorescent protein reporter gene as well as by complete phenolcopy of the recessive loss-of-function mlo resistance gene. By using this method, a role of the t-SNARE protein HvSNAP34 in three types of durable, race-nonspecific resistance was observed.

A Transient Assay System for the Functional Assessment of Defense-Related Genes in Wheat

The generation and characterization of transgenic wheat plants is a tedious and time-consuming process that limits the number of putatively important transgenes that can be tested. We therefore established a transient assay system based on wheat leaves to study the effect of transiently expressed genes on the interaction with the wheat powdery mildew fungus Erysiphe (syn. Blumeria) graminis f. sp. tritici. Young wheat leaves were bombarded with tungsten particles coated with a mixture of plasmids carrying the β-glucuronidase (GUS) reporter gene and a test gene. Leaves were subsequently challenge inoculated with E. graminis and the fungus was allowed to develop for 40 h. After being stained for GUS enzymatic activity as well as for epiphytic fungal structures, the phenotype of transformed epidermal cells was evaluated by bright-field microscopy. The fungus was routinely found to penetrate cells transiently expressing GUS with an efficiency of approximately 35%, which should suffice to detect putative trans...

The Multigene Family Encoding Germin-Like Proteins of Barley. Regulation and Function in Basal Host Resistance

Germin-like proteins (GLPs) have been shown to be encoded by multigene families in several plant species and a role of some subfamily members in defense against pathogen attack has been proposed based on gene regulation studies and transgenic approaches. We studied the function of six GLP subfamilies of barley (Hordeum vulgare) by selecting single mRNAs for gene expression studies as well as overexpression and gene-silencing experiments in barley and Arabidopsis (Arabidopsis thaliana). Expression of all six subfamilies was high in very young seedlings, including roots. The expression pattern gradually changed from developmental to conditional with increasing plant age, whereby pathogen attack and exogenous hydrogen peroxide application were found to be the strongest signals for induction of several GLP subfamilies. Transcripts of four of five GLP subfamilies that are expressed in shoots were predominantly accumulating in the leaf epidermis. Transient overexpression of HvGER4 or HvGER5 as well as transient silencing by RNA interference of HvGER3 or HvGER5 protected barley epidermal cells from attack by the appropriate powdery mildew fungus Blumeria graminis f. sp. hordei. Silencing of HvGER4 induced hypersusceptibility. Transient and stable expression of subfamily members revealed HvGER5 as a new extracellular superoxide dismutase, and protection by overexpression could be demonstrated to be dependent on superoxide dismutase activity of the encoded protein. Data suggest a complex interplay of HvGER proteins in fine regulation of basal resistance against B. graminis.

The Germinlike Protein GLP4 Exhibits Superoxide Dismutase Activity and Is an Important Component of Quantitative Resistance in Wheat and Barley

Germinlike proteins (GLP) are encoded in plants by a gene family with proposed functions in plant development and defense. Genes of GLP subfamily 4 of barley (HvGLP4, formerly referred to as HvOxOLP) and the wheat orthologue TaGLP4 (formerly referred to as TaGLP2a) were previously found to be expressed in pathogen-attacked epidermal tissue of barley and wheat leaves, and the corresponding proteins are proposed to accumulate in the apoplast. Here, the role of HvGLP4 and TaGLP4 in the defense of barley and wheat against Blumeria graminis (DC.) E. O. Speer, the cereal powdery mildew fungus, was examined in an epidermal transient expression system and in transgenic Arabidopsis thaliana plants overexpressing His-tagged HvGLP4. Leaf extracts of transgenic Arabidopsis overexpressing HvGLP4 contained a novel His-tagged protein with superoxide dismutase activity and HvGLP4 epitopes. Transient overexpression of TaGLP4 and HvGLP4 enhanced resistance against B. graminis in wheat and barley, whereas transient silencing by RNA interference reduced basal resistance in both cereals. The effect of GLP4 overexpression or silencing was strongly influenced by the genotype of the plant. The data suggest that members of GLP subfamily 4 are components of quantitative resistance in both barley and wheat, acting together with other, as yet unknown, plant components.

Jasmonate-Inducible Genes Are Activated in Rice by Pathogen Attack without a Concomitant Increase in Endogenous Jasmonic Acid Levels.

The possible role of the octadecanoid signaling pathway with jasmonic acid (JA) as the central component in defense-gene regulation of pathogen-attacked rice was studied. Rice (Oryza sativa L.) seedlings were treated with JA or inoculated with the rice blast fungus Magnaporthe grisea (Hebert) Barr., and gene-expression patterns were compared between the two treatments. JA application induced the accumulation of a number of pathogenesis-related (PR) gene products at the mRNA and protein levels, but pathogen attack did not enhance the levels of (-)-JA during the time required for PR gene expression. Pathogen-induced accumulation of PR1-like proteins was reduced in plants treated with tetcyclacis, a novel inhibitor of jasmonate biosynthesis. There was an additive and negative interaction between JA and an elicitor from M. grisea with respect to induction of PR1-like proteins and of an abundant JA-and wound-induced protein of 26 kD, respectively. Finally, activation of the octadecanoid signaling pathway and induction of a number of PR genes by exogenous application of JA did not confer local acquired resistance to rice. The data suggest that accumulation of nonconjugated (-)-JA is not necessary for induction of PR genes and that JA does not orchestrate localized defense responses in pathogen-attacked rice. Instead, JA appears to be embedded in a signaling network with another pathogen-induced pathway(s) and may be required at a certain minimal level for induction of some PR genes.

A Set of Modular Binary Vectors for Transformation of Cereals

Genetic transformation of crop plants offers the possibility of testing hypotheses about the function of individual genes as well as the exploitation of transgenes for targeted trait improvement. However, in most cereals, this option has long been compromised by tedious and low-efficiency transformation protocols, as well as by the lack of versatile vector systems. After having adopted and further improved the protocols for Agrobacterium-mediated stable transformation of barley (Hordeum vulgare) and wheat (Triticum aestivum), we now present a versatile set of binary vectors for transgene overexpression, as well as for gene silencing by double-stranded RNA interference. The vector set is offered with a series of functionally validated promoters and allows for rapid integration of the desired genes or gene fragments by GATEWAY-based recombination. Additional in-built flexibility lies in the choice of plant selectable markers, cassette orientation, and simple integration of further promoters to drive specific expression of genes of interest. Functionality of the cereal vector set has been demonstrated by transient as well as stable transformation experiments for transgene overexpression, as well as for targeted gene silencing in barley.

Gene-Expression Patterns and Levels of Jasmonic Acid in Rice Treated with the Resistance Inducer 2,6-Dichloroisonicotinic Acid

Acquired disease resistance can be induced in rice (Oryza sativa) by a number of synthetic or natural compounds, but the molecular mechanisms behind the phenomenon are poorly understood. One of the synthetic inducers of resistance, 2,6-dichloroisonicotinic acid (INA), efficiently protected rice leaves from infection by the rice blast fungus Magnaporthe grisea (Hebert) Barr. A comparison of gene-expression patterns in plants treated with INA versus plants inoculated with the compatible pathogen M. grisea or the incompatible pathogen Pseudomonas syringae pv syringae revealed only a marginal overlap: 6 gene products, including pathogenesis-related proteins (PR1-PR9), accumulated in both INA-treated and pathogen-attacked leaves, whereas 26 other gene products accumulated only in INA-treated or only in pathogen-attacked leaves. Lipoxygenase enzyme activity and levels of nonconjugated jasmonic acid (JA) were enhanced in leaves of plants treated with a high dose of INA (100 ppm). Exogenously applied JA enhanced the gene induction and plant protection caused by lower doses of INA (0.1 to 10 ppm) that by themselves did not give rise to enhanced levels of endogenous (-)-JA. These data suggest that INA, aside from activating a pathogen-induced signaling pathway, also induces events that are not related to pathogenesis. JA acts as an enhancer of both types of INA-induced reactions in rice.

Structure, expression and localization of a germin-like protein in barley (Hordeum vulgare L.) that is insolubilized in stressed leaves

The primary leaves of young barley seedlings contain two major, extracellular, acid-soluble proteins of ca. 22 and 23 kDa apparent molecular mass. These proteins disappeared from the intercellular washing fluid upon stress treatments that enhanced H2O2 levels and that induced resistance to subsequent challenge by the powdery mildew fungus Erysiphe graminis f. sp. hordei. A partial peptide sequence of the 22 kDa protein was determined, and a cDNA clone was isolated. The 22 kDa protein belongs the the group of germin-like proteins (GLPs) and was designated HvGLP1. Despite its similarity to germin, i.e. oxalate oxidase, no oxalate oxidase activity of HvGLP1 could be detected. The RNA and soluble protein of HvGLP1 was highly abundant in young leaves, less abundant in older leaves and absent in roots. HvGLP1 RNA oscillated with a circadian rhythm, the minimum and maximum of RNA abundance being at the end of the dark and light periods, respectively. Heat and H2O2 treatment as well as pathogen infection caused disappearance of HvGLP1 protein from the fraction of soluble proteins of the intercellular space. HvGLP1 protein could be re-solubilized from cell walls of heat- or H2O2-treated leaves by boiling in SDS suggesting non-covalent cross linking. Although a physiological role of HvGLP1 insolubilization is still open, the protein may serve as marker for oxidative stress in cereals.