Exploring the resilience of wheat crops grown in short rotations through minimising the build-up of an important soil-borne fungal pathogen.

Given the increasing demand for wheat which is forecast, cropping of wheat in short rotations will likely remain a common practice. However, in temperate wheat growing regions the soil-borne fungal pathogen Gaeumannomyces tritici becomes a major constraint on productivity. In cultivar rotation field experiments on the Rothamsted Farm (Hertfordshire, UK) we demonstrated a substantial reduction in take-all disease and grain yield increases of up to 2.4 tonnes/ha when a low take-all inoculum building wheat cultivar was grown in the first year of wheat cropping. Phenotyping of 71 modern elite wheat cultivars for the take-all inoculum build-up trait across six diverse trial sites identified a few cultivars which exhibited a consistent lowering of take-all inoculum build-up. However, there was also evidence of a significant interaction effect between trial site and cultivar when a pooled Residual Maximum Likelihood (REML) procedure was conducted. There was no evidence of an unusual rooting phenotype associated with take-all inoculum build-up in two independent field experiments and a sand column experiment. Together our results highlight the complex interactions between wheat genotype, environmental conditions and take-all inoculum build-up. Further work is required to determine the underlying genetic and mechanistic basis of this important phenomenon.

Structure-based virtual screening of hypothetical inhibitors of the enzyme longiborneol synthase-a potential target to reduce Fusarium head blight disease.

Fusarium head blight (FHB) is one of the most destructive diseases of wheat and other cereals worldwide. During infection, the Fusarium fungi produce mycotoxins that represent a high risk to human and animal health. Developing small-molecule inhibitors to specifically reduce mycotoxin levels would be highly beneficial since current treatments unspecifically target the Fusarium pathogen. Culmorin possesses a well-known important synergistically virulence role among mycotoxins, and longiborneol synthase appears to be a key enzyme for its synthesis, thus making longiborneol synthase a particularly interesting target. This study aims to discover potent and less toxic agrochemicals against FHB. These compounds would hamper culmorin synthesis by inhibiting longiborneol synthase. In order to select starting molecules for further investigation, we have conducted a structure-based virtual screening investigation. A longiborneol synthase structural model is first built using homology modeling, followed by molecular dynamics simulations that provided the required input for a protein-ligand ensemble docking procedure. From this strategy, the three most interesting compounds (hits) were selected among the 25 top-ranked docked compounds from a library of 15,000 drug-like compounds. These putative inhibitors of longiborneol synthase provide a sound starting point for further studies involving molecular modeling coupled to biochemical experiments. This process could eventually lead to the development of novel approaches to reduce mycotoxin contamination in harvested grain.

An analysis of Pseudomonas genomic diversity in take-all infected wheat fields reveals the lasting impact of wheat cultivars on the soil microbiota.

Manipulation of the soil microbiota associated with crop plants has huge promise for the control of crop pathogens. However, to fully realize this potential we need a better understanding of the relationship between the soil environment and the genes and phenotypes that enable microbes to colonize plants and contribute to biocontrol. A recent 2 years of investigation into the effect of wheat variety on second year crop yield in the context of take-all fungal infection presented the opportunity to examine soil microbiomes under closely defined field conditions. Amplicon sequencing of second year soil samples showed that Pseudomonas spp. were particularly affected by the wheat cultivar grown in year one. Consequently, 318 rhizosphere-associated Pseudomonas fluorescens strains were isolated and characterized across a variety of genetic and phenotypic traits. Again, the wheat variety grown in the first year of the study was shown to exert considerable selective pressure on both the extent and nature of Pseudomonas genomic diversity. Furthermore, multiple significant correlations were identified within the phenotypic/genetic structure of the Pseudomonas population, and between individual genotypes and the external wheat field environment. The approach outlined here has considerable future potential for our understanding of plant-microbe interactions, and for the broader analysis of complex microbial communities.

Inactivation of plant infecting fungal and viral pathogens to achieve biological containment in drainage water using UV treatment.

AIM: To explore whether ultraviolet (UV) light treatment within a closed circulating and filtered water drainage system can kill plant pathogenic species. METHODS AND RESULTS: Ultraviolet experiments at 254 nm were conducted to determine the inactivation coefficients for seven plant pathogenic species. At 200 mJ cm(-2), the individual species log reductions obtained for six Ascomycete fungi and a cereal virus were as follows: Leptosphaeria maculans (9·9-log), Leptosphaeria biglobosa (7·1-log), Barley stripe mosaic virus (BSMV) (4·1-log), Mycosphaerella graminicola (2·9-log), Fusarium culmorum (1·2-log), Fusarium graminearum (0·6-log) and Magnaporthe oryzae (0·3-log). Dilution experiments showed that BSMV was rendered noninfectious when diluted to >1/512. Follow-up large-scale experiments using up to 400 l of microbiologically contaminated waste water revealed that the filtration of drainage water followed by UV treatment could successfully be used to inactivate several plant pathogens. CONCLUSIONS: By combining sedimentation, filtration and UV irradiation within a closed system, plant pathogens can be successfully removed from collected drainage water. SIGNIFICANCE AND IMPACT OF THE STUDY: Ultraviolet irradiation is a relatively low cost, energy efficient and labour nonintensive method to decontaminate water arising from a suite of higher biological containment level laboratories and plant growth rooms where genetically modified and/or quarantine fungal and viral plant pathogenic organisms are being used for research purposes.

The Sbm1 locus conferring resistance to Soil-borne cereal mosaic virus maps to a gene-rich region on 5DL in wheat.

A mosaic disease caused by Soil-borne cereal mosaic virus (SBCMV) is becoming increasingly important, particularly in winter wheat in Europe. As there are currently no effective cultural practices or practical environmentally friendly chemicals for disease control, host plant resistance is an important objective in breeding programs. However, development of resistant cultivars is slow owing to difficulties in germplasm screening for resistance. Therefore, there is a need to identify molecular markers linked to SBCMV-resistance gene(s), so that quick and accurate laboratory-based marker-assisted selection rather than prolonged field-based screens for resistance can be used in developing resistant cultivars. We previously demonstrated that resistance to SBCMV in Triticum aestivum 'Cadenza' is controlled by a single locus. In this work, we used AFLP and microsatellite technology to map this resistance locus, with the proposed name Sbm1, to the distal end of chromosome 5DL. Interestingly, several expressed disease-resistance gene analogues also map to this gene-rich region on 5DL. Closely linked (approximately 17 cM interval) markers, BARC110 and WMC765, RRES01 and BARC144, that flank Sbm1 will be very useful in breeding for selection of germplasm carrying Sbm1.

Developmental control of promoter activity is not responsible for mature onset of Cf-9B-mediated resistance to leaf mold in tomato.

Cf-9 confers resistance to tomato seedlings and mature plants against Cladosporium fulvum races expressing the Avr9 elicitor. It is the central member of a cluster of five paralogous genes in an introgressed segment of chromosome 1 derived from Lycopersicon pimpinellifolium. The other four genes have been named Hcr9-9A, Hcr9-9B, Hcr9-9D, and Hcr9-9E. Hcr9-9B, here designated Cf-9B, encodes weaker resistance than Cf-9, recognizes a different elicitor, and protects only mature plants from infection. The onset of Cf-9B-mediated resistance and the molecular basis for its developmental control were investigated in this study. Fungal inoculation of tomato plants containing reciprocal Cf-9/Cf-9B promoter-coding region swaps, analysis of tomato plants containing promoter-gusA fusions, and a reverse transcriptase-polymerase chain reaction study of Cf-9 and Cf-9B transcripts in tomato plants suggested that transcriptional control of Cf-9B did not account for the late onset of Cf-9B-mediated resistance. Alternative explanations for the onset of Cf-9B-mediated resistance in mature plants are discussed.

Salicylic acid is not required for Cf-2- and Cf-9-dependent resistance of tomato to Cladosporium fulvum.

Tomato leaves or cotyledons expressing the Cf-2 or Cf-9 Cladosporium fulvum resistance genes induce salicylic acid (SA) synthesis following infiltration with intercellular washing fluid (IF) containing the fungal peptide elicitors Avr2 and Avr9. We investigated whether SA was required for Cf gene-dependent resistance. Tomato plants expressing the bacterial gene nahG, encoding salicylate hydroxylase, did not accumulate SA in response to IF infiltration but remained fully resistant to C. fulvum. NahG Cf0 plants were as susceptible to C. fulvum as wild-type Cf0. Neither free nor conjugated salicylic acid accumulated in IF-infiltrated Cf2 and Cf9 NahG leaves and cotyledons but conjugated catechol did accumulate. The Cf-9-dependent necrotic response to IF was prevented in NahG plants and replaced by a chlorotic Cf-2-like response. SA also potentiated Cf-9-mediated necrosis in IF-infiltrated wild-type leaves. In contrast, the Cf-2-dependent IF response was retained in NahG leaves and chlorosis was more pronounced than in the wild-type. The distribution of cell death between different cell types was altered in both Cf2 and Cf9 NahG leaves after IF injection. IF-induced accumulation of three SA-inducible defence-related genes was delayed and reduced but not abolished in NahG Cf2 and Cf9 leaves and cotyledons. NahG Tm-22 tomato showed increased hypersensitive response (HR) lesion size upon TMV infection, as observed in TMV-inoculated N gene-containing NahG tobacco plants.

cDNA-AFLP reveals a striking overlap in race-specific resistance and wound response gene expression profiles.

The tomato Cf-9 gene confers resistance to races of the fungal pathogen Cladosporium fulvum expressing the Avr9 gene. cDNA amplified fragment length polymorphism analysis was used to display transcripts whose expression is rapidly altered during the Avr9- and Cf-9-mediated defense response in tobacco cell cultures. Diphenyleneiodonium was used to abolish the production of active oxygen species during gene induction. Of 30,000 fragments inspected, 290 showed altered abundance, of which 263 were induced independently of active oxygen species. cDNA clones were obtained for 13 ACRE (for Avr9/Cf-9 rapidly elicited) genes. ACRE gene induction occurred in the presence of cycloheximide. Avr9 induced ACRE gene expression in leaves. Surprisingly, ACRE genes were also rapidly but transiently induced in leaves in response to other stresses. The amino acid sequences of some ACRE proteins are homologous to sequences of known proteins such as ethylene response element binding protein transcription factors, the N resistance protein, a calcium binding protein, 13-lipoxygenase, and a RING-H2 zinc finger protein. Rapid induction of ACRE genes suggests that they play a pivotal role during plant defense responses.

Comparison of the hypersensitive response induced by the tomato Cf-4 and Cf-9 genes in Nicotiana spp.

We have previously shown that tomato Cf-9 induces an Avr9-dependent hypersensitive response (HR) in Nicotiana tabacum and potato. We show here that Cf-4 also induces an Avr4-dependent HR in two tobacco species (N. tabacum and N. benthamiana). The HR induced by Cf-4 and Cf-9 was compared in stable tobacco transgenics by a seedling lethal assay and resistance to recombinant Potato virus X expressing Avr4 or Avr9. We also compared HR induction with Agrobacterium-mediated transient expression. The Cf-4/Avr4 combination induced a more rapid HR than Cf-9/Avr9. Sensitive assays for Cf-9 and Cf-4 function should prove useful for structure/function analyses of these resistance proteins in tobacco.

Early signalling events in the Avr9/Cf-9-dependent plant defence response.

Abstract Resistance of tomato to the leaf mould fungus Cladosporium fulvum is controlled by the interaction between a plant-encoded resistance gene (Cf-9) and pathogen-encoded avirulence (Avr9) gene. Our objective is to understand the underlying molecular mechanisms that transmit the Cf-9/Avr9-dependent pathogen perception event and activate the plant defence response. Our approach toward the understanding of Cf-function is based on the analysis of early Cf-9/Avr9-mediated responses and signalling events. Because Cf-9 transgenically expressed in tobacco retains its specificity and activity to the Avr9 elicitor, signalling experiments were conducted in the heterologous system using these transgenic lines or derived Cf9 tobacco cell cultures. Among the earliest responses to the Avr9/Cf-9 elicitation event were rapid changes in ion-fluxes, the synthesis of active oxygen species (AOS), probably catalysed by a plant NADPH-oxidase, and the transient activation of two MAP kinases. These kinases were identified as WIPK (wounding-induced protein kinase) and SIPK (salicylic-acid induced kinase) from tobacco. Studies with pharmacological inhibitors suggested that the MAP kinases are located in an independent signalling pathway from the Avr9/Cf-9-dependent synthesis of AOS. SIPK and WIPK were involved in pathogen-related elicitation processes as well as in abiotic stress responses. This indicates that the plant defence is triggered via a signalling network that shares components with pathways originating from abiotic environmental stress stimuli.

K+ channels of Cf-9 transgenic tobacco guard cells as targets for Cladosporium fulvum Avr9 elicitor-dependent signal transduction.

The Cf-9 gene encodes an extracytosolic leucine-rich repeat (LRR) protein that is membrane anchored near its C-terminus. The protein confers resistance in tomato to races of the fungus Cladosporium fulvum expressing the corresponding avirulence gene Avr9. In Nicotiana tabacum the Cf-9 transgene confers sensitivity to the Avr9 elicitor, and leads on elicitation to a subset of defence responses qualitatively similar to those normally seen in the tomato host. One of the earliest responses, both in the native and transgenic hosts, results in K+ salt loss from the infected tissues. However, the mechanism(s) underlying this solute flux and its control is poorly understood. We have explored the actions of Avr9 on Cf-9 transgenic Nicotiana using guard cells as a model. Much detail of guard cell ion channels and their regulation is already known. Measurements were carried out on intact guard cells in epidermal peels, and the currents carried by inward- (IK,in) and outward-rectifying (IK,out) K+ channels were characterized under voltage clamp. Exposures to Avr9-containing extracts resulted in a 2.5- to 3-fold stimulation of IK,out and almost complete suppression of IK,in within 3-5 min. The K+ channel responses were irreversible. They were specific for the Avr9 elicitor, were not observed in guard cells of Nicotiana lacking the Cf-9 transgene and, from kinetic analyses, could be ascribed to changes in channel gating. Both K+ channel responses were found to be saturable functions of Avr9 concentration and were completely blocked in the presence of 0.5 microM staurosporine and 100 microM H7, both broad-range protein kinase antagonists. These results demonstrate the ability of the Cf-9 transgene to couple Avr9 elicitation specifically to a concerted action on two discrete K+ channels and they indicate a role for protein phosphorylation in Avr9/Cf-9 signal transduction leading to transport control.

Six Arabidopsis thaliana homologues of the human respiratory burst oxidase (gp91phox).

An NADPH oxidase analogous to that in mammalian phagocytes has been hypothesized to produce reactive oxygen species (ROS) in the plant defence response. A. thaliana contains at least six gp91phox homologues, designated AtrbohA-F (A. thaliana Respiratory Burst Oxidase Homologues), which map to different positions. Transcripts of three of these genes can be detected in healthy plants by RNA gel blot analyses. The Atrboh gene products are closely related to gp91phox and the intron locations suggest a common evolutionary origin. A putative EF-hand Ca(2+)-binding motif in the extended N-terminal region of the Atrboh proteins suggests a direct regulatory effect of Ca2+ on the activity of the NADPH oxidase in plants.

Molecular, genetic and physiological analysis of Cladosporium resistance gene function in tomato.

Characterization of the DNA sequence of 4 tomato leaf mould disease resistance genes (Cf-2, Cf-4, Cf-5 and Cf-9) leads to the prediction that they encode C-terminally membrane anchored glycopeptides with many extracytoplasmic leucine rich repeats (LRRs). The N terminal LRRs are variable between the Cf-genes, suggesting a role in specificity, and the C terminal LRRs are more conserved, suggesting a role in signal transduction. Genetic analysis has revealed several Rcr genes that are required for Cf-gene function; their isolation will help us understand how Cf-genes work. Cf-9 confers responsiveness to pathogen-encoded Avr9 peptide on introduction to tobacco. Tobacco suspension cultures carrying the Cf-9 gene produce reactive oxygen species in response to Avr9 peptide, whereas untransformed cultures do not. The significance of these observations is discussed.

Novel disease resistance specificities result from sequence exchange between tandemly repeated genes at the Cf-4/9 locus of tomato.

Tomato Cf genes confer resistance to C. fulvum, reside in complex loci carrying multiple genes, and encode predicted membrane-bound proteins with extracytoplasmic leucine-rich repeats. At least two Cf-9 homologs confer novel C. fulvum resistance specificities. Comparison of 11 genes revealed 7 hypervariable amino acid positions in a motif of the leucine-rich repeats predicted to form a beta-strand/beta-turn in which the hypervariable residues are solvent exposed and potentially contribute to recognition specificity. Higher nonsynonymous than synonymous substitution rates in this region imply selection for sequence diversification. We propose that the level of polymorphism between intergenic regions determines the frequency of sequence exchange between the tandemly repeated genes. This permits sufficient exchange to generate sequence diversity but prevents sequence homogenization.

rbohA, a rice homologue of the mammalian gp91phox respiratory burst oxidase gene.

It has been hypothesized that plants contain respiratory burst oxidase which, upon activation, oxidize NADPH and generate extracellular superoxide, O2.-. These proteins are proposed to play a central role in defence against pathogens. However, plant DNA sequences that encode proteins with similarity to components of respiratory burst oxidase have not previously been reported. This paper describes the complete cDNA and genomic DNA sequence of the rice rbohA (for respiratory burst oxidase homologue) gene. The predicted RbohA product is most similar to the main catalytic subunit, gp91phox, of the respiratory burst oxidase of neutrophils. Reverse transcriptase PCR detects rbohA transcripts in both roots and shoots of healthy rice plants.

Involvement of Reactive Oxygen Species, Glutathione Metabolism, and Lipid Peroxidation in the Cf-Gene-Dependent Defense Response of Tomato Cotyledons Induced by Race-Specific Elicitors of Cladosporium fulvum.

The chronological order of responses to Cladosporium fulvum (Cooke) (Cf) race-specific elicitors was assessed in cotyledons of three near-isogenic tomato (Lycopersicon esculentum Mill.) lines carrying either Cf-9 or Cf-2 or no Cf gene. The responses observed were dependent on the presence of a Cf gene, Avr-gene product dose injected, and the relative humidity (RH) of the growth chamber. At ambient RH, superoxide formation and lipid peroxidation occurred after 2 h (Cf9) and 4 h (Cf2). At elevated RH (98%) and at lower avirulence elicitor dose, Cf-Avr-dependent lipid peroxidation was considerably attenuated. Significant electrolyte leakage occurred by 18 h but only at the lower RH. Total glutathione levels began to increase 2 to 4 h and 4 to 8 h after challenge of Cf9 and Cf2 cells, respectively, and by 48 h reached 665 and 570% of initial levels. A large proportion of this accumulation (87%) was as oxidized glutathione. When the RH was increased to 98%, increases in glutathione levels were strongly attenuated. Increased lipoxygenase enzyme activity was detected 8 h postchallenge in either incompatible interaction. These results indicate that the activation of the Cf-Avr-mediated defense response results in severe oxidative stress.

Race-Specific Elicitors of Cladosporium fulvum Induce Changes in Cell Morphology and the Synthesis of Ethylene and Salicylic Acid in Tomato Plants Carrying the Corresponding Cf Disease Resistance Gene.

Defense responses mediated by the genetically unlinked Cf-9 and Cf-2 genes were compared with those involving no Cf gene (Cf0). Compatible tomato (Lycopersicon esculentum)-Cladosporium fulvum intercellular washing fluids were injected into tomato cotyledons, and the kinetics of responses was monitored under conditions of 70 and 98% relative humidity. The latter conditions suppressed the normal macroscopic responses. For the Cf-9-Avr9 interaction, stomatal opening was induced within 3 to 4 h and after 9 h mesophyll cell death commenced. A burst of ethylene production occurred between 9 and 12.5 h and remained elevated. Free salicylic acid levels increased after 12 h, peaked at 24 h, and thereafter declined. For the Cf-2-Avr2 interaction, stomata became plugged after 8 h, and salicylic acid and ethylene levels increased by 12 and 18 h, respectively, and thereafter declined. Host cell death commenced around vascular tissue by 24 h. Cell death in both incompatible interactions was frequently preceded by cell enlargement. For Cf0-injected plants, no significant responses were detected. High humidity delayed and reduced the Cf-Avr-gene-dependent cell death and ethylene synthesis, whereas induced salicylic acid levels were unaffected for Cf-2-Avr2 and reduced in magnitude only for Cf-9-Avr9.

Isolation of the tomato Cf-9 gene for resistance to Cladosporium fulvum by transposon tagging.

The tomato Cf-9 gene confers resistance to infection by races of the fungus Cladosporium fulvum that carry the avirulence gene Avr9. The Cf-9 gene was isolated by transposon tagging with the maize transposable element Dissociation. The DNA sequence of Cf-9 encodes a putative membrane-anchored extracytoplasmic glycoprotein. The predicted protein shows homology to the receptor domain of several receptor-like protein kinases in Arabidopsis, to antifungal polygalacturonase-inhibiting proteins in plants, and to other members of the leucine-rich repeat family of proteins. This structure is consistent with that of a receptor that could bind Avr9 peptide and activate plant defense.

Developmentally regulated cell death on expression of the fungal avirulence gene Avr9 in tomato seedlings carrying the disease-resistance gene Cf-9.

Plant defense responses are induced when the products of disease-resistance genes and pathogen avirulence genes interact. We report here the effects of expressing the Cladosporium fulvum avirulence Avr9 gene product in a tomato line containing the Cf-9 disease-resistance gene. A synthetic Avr9 gene was constructed to produce constitutive high-level expression of AVR9 peptide in the plant apoplast. Avr9 expression in Cf-9-containing tomato lines is lethal, but cell death is developmentally regulated, in that necrosis is not visible until 10 days after planting seed. Plant lines lacking Cf-9 and expressing Avr9 remain healthy. The synthetic Avr9 gene exhibited the same specificity of action as the authentic C. fulvum Avr9 gene. Our results have significant implications for strategies using the gene combination Avr9/Cf-9 to engineer plants with enhanced disease resistance.