Ustilago maydis is a biotrophic pathogen causing maize (Zea mays) smut disease. Transcriptome profiling of infected maize plants indicated that a gene encoding a putative cystatin (CC9) is induced upon penetration by U. maydis wild type. By contrast, cc9 is not induced after infection with the U. maydis effector mutant Dpep1, which elicits massive plant defenses. Silencing of cc9 resulted in a strongly induced maize defense gene expression and a hypersensitive response to U. maydis wild-type infection. Consequently, fungal colonization was strongly reduced in cc9-silenced plants, while recombinant CC9 prevented salicylic acid (SA)-induced defenses. Protease activity profiling revealed a strong induction of maize Cys proteases in SA-treated leaves, which could be inhibited by addition of CC9. Transgenic maize plants overexpressing cc9-mCherry showed an apoplastic localization of CC9. The transgenic plants showed a block in Cys protease activity and SA-dependent gene expression. Moreover, activated apoplastic Cys proteases induced SA-associated defense gene expression in naïve plants, which could be suppressed by CC9. We show that apoplastic Cys proteases play a pivotal role in maize defense signaling. Moreover, we identified cystatin CC9 as a novel compatibility factor that suppresses Cys protease activity to allow biotrophic interaction of maize with the fungal pathogen U. maydis.

The development of powerful “omics” technologies has enabled researchers to identify many genes of interest for which comprehensive functional analyses are highly desirable. However, the production of lines which ectopically express recombinant genes, or those in which endogenous genes are knocked down via stable transformation, remains a major bottleneck for the association between genetics and gene function in monocotyledonous crops. Methods of effective DNA transfer into regenerable cells of immature embryos from cereals by means of Agrobacterium tumefaciens have been modified in a stepwise manner. The effect of particular improvement measures has often not been significantly evident, whereas their combined implementation has resulted in meaningful advances. Here, we provide updated protocols for the Agrobacterium-mediated generation of stably transgenic barley, wheat, triticale and maize. Based upon these methods, several hundred independent transgenic lines have been delivered, with efficiencies of inoculated embryos leading to stably transgenic plants reaching 86% in barley, 10% in wheat, 4% in triticale, and 24% in maize.

HighlightWheat plants transiently or stably accumulating antisense or double-stranded RNA, which are directed against essential genes of the Fusarium head blight fungus F. culmorum, were more resistant against the disease.

Summary• Infection of maize (Zea mays) plants with the corn smut fungus Ustilago maydis leads to the formation of large tumors on the stem, leaves and inflorescences. In this biotrophic interaction, plant defense responses are actively suppressed by the pathogen, and previous transcriptome analyses of infected maize plants showed massive and stage-specific changes in host gene expression during disease progression.• To identify maize genes that are functionally involved in the interaction with U. maydis, we adapted a virus-induced gene silencing (VIGS) system based on the brome mosaic virus (BMV) for maize. Conditions were established that allowed successful U. maydis infection of BMV-preinfected maize plants. This set-up enabled quantification of VIGS and its impact on U. maydis infection using a quantitative real-time PCR (qRT-PCR)-based readout.• In proof-of-principle experiments, an U. maydis-induced terpene synthase was shown to negatively regulate disease development while a protein involved in cell death inhibition was required for full virulence of U. maydis.• The results suggest that this system is a versatile tool for the rapid identification of maize genes that determine compatibility with U. maydis.

SummaryMaize (corn) is one of the most widely grown cereal crops globally. Fungal diseases of maize cause significant economic damage by reducing maize yields and by increasing input costs for disease management. The most sustainable control of maize diseases is through the release and planting of maize cultivars with durable disease resistance. The wheat gene Lr34 provides durable and partial field resistance against multiple fungal diseases of wheat, including three wheat rust pathogens and wheat powdery mildew. Because of its unique qualities, Lr34 became a cornerstone in many wheat disease resistance programmes. The Lr34 resistance is encoded by a rare variant of an ATP‐binding cassette (ABC) transporter that evolved after wheat domestication. An Lr34‐like disease resistance phenotype has not been reported in other cereal species, including maize. Here, we transformed the Lr34 resistance gene into the maize hybrid Hi‐II. Lr34‐expressing maize plants showed increased resistance against the biotrophic fungal disease common rust and the hemi‐biotrophic disease northern corn leaf blight. Furthermore, the Lr34‐expressing maize plants developed a late leaf tip necrosis phenotype, without negative impact on plant growth. With this and previous reports, it could be shown that Lr34 is effective against various biotrophic and hemi‐biotrophic diseases that collectively parasitize all major cereal crop species.

Stable genetic transformation of maize is of vital importance to enable detailed functional analyses of genes involved in plant-microbe interaction. However, efficient maize transformation is still difficult to perform since only a few exotic genotypes are well amenable to this method and published protocols are only hardly reproducible in other labs. The goal of this work is to develop a reliable method of Agrobacteriummediated gene transfer to highly embryogenic Hi II hybrid maize immature zygotic embryos in order to provide a pow-A C H T U N G T R E N N U N G erful technical platform within the DFG-Forschergruppe 666. This consortium collectively aims to elucidate compatibility mechanisms of plants and fungal microbes. The induction of regenerable callus represents an essential step in the transformation process. Several media components were varied with the main focus on growth regulators. As a result, N6 basal medium supplemented with L-proline, casein hydrolysate and dicamba turned out to be most suitable for the efficient formation of somatic embryos at the callus surface. For the subsequent plant regeneration, a simple MS-based medium was utilised. Gene transfer was conducted using the Agrobacterium tumefaciens strain EHA105 harbouring a standard binary vector with the bar gene as selectable marker and the gus-intron reporter gene under the control of a doubled enhanced CaMV35S promoter. Several factors of the co-culture were examined to optimize transient GUS-expression of immature embryos. An elevation of acetosyringone concentration, the addition of the reducing agent dithiothreitol, the substitution of sucrose by maltose as osmoticum and carbohydrate source, and an increase of the Agrobacterium population density proved to be the most effective measures to improve the transformation protocol. First transformation experiments based on the newly developed N6-based medium resulted in 33 GUS expressing calli. The highest proportion of GUS-expressing calli (13 %) was observed in an experiment using embryos of Hi II A x Hi II B with a size of 1.9 mm. So far, eight independent transgenic lines were confirmed by PCR. An analysis of gene integration patterns by Southern blot is under way.